- Email: [email protected]
Association of Tobacco Use, Alcohol Consumption and HER2 Polymorphisms With Response to Trastuzumab in HER2-Positive Breast Cancer Patients
Accepted Manuscript Association between tobacco and alcohol consumption, HER2 polymorphisms and response to trastuzumab in HER2-positive breast cancer patients Daniela Furrer, Simon Jacob, Annick Michaud, Louise Provencher, Julie Lemieux, Caroline Diorio PII:
S1526-8209(17)30318-X
DOI:
10.1016/j.clbc.2017.11.012
Reference:
CLBC 719
To appear in:
Clinical Breast Cancer
Received Date: 25 May 2017 Revised Date:
12 October 2017
Accepted Date: 20 November 2017
Please cite this article as: Furrer D, Jacob S, Michaud A, Provencher L, Lemieux J, Diorio C, Association between tobacco and alcohol consumption, HER2 polymorphisms and response to trastuzumab in HER2-positive breast cancer patients, Clinical Breast Cancer (2017), doi: 10.1016/j.clbc.2017.11.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Association between tobacco and alcohol consumption, HER2 polymorphisms and response to trastuzumab in HER2-positive breast cancer patients Daniela Furrer a,b,d, Simon Jacob Lemieux a,b,c,d, Caroline Diorio a,b,c,d
a,b,c,d
, Annick Michaud
, Louise Provencher
a,b,c,d
, Julie
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a
a,b
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Centre de Recherche sur le cancer de l'Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada ; b Axe Oncologie, Centre de Recherche du CHU de Québec-Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada; c Centre des maladies du sein Deschênes-Fabia, Hôpital du Saint-Sacrement, 1050, chemin Ste-Foy, Québec, QC, G1S 4L8, Canada; d Faculté de Médecine, Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada Corresponding author :
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Caroline Diorio Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Hôpital du Saint-Sacrement, 1050 chemin Ste-Foy, Quebec City, QC G1S 4L8, Canada Phone: +1-418-682-7511 ext. 84726 Fax: +1-418-682-7949 e-mail: [email protected]
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MicroAbstract Trastuzumab resistance in HER2-positive breast cancer patients remains a major clinical problem. We evaluated the association between HER2 polymorphisms, tobacco and alcohol
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consumption and disease-free survival in HER2-positive breast cancer patients. Our observations suggest that tobacco and alcohol consumption as well as one HER2 polymorphism could influence trastuzumab response. These results need to be confirmed in a larger cohort study.
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Abstract
Purpose. Although the administration of trastuzumab has improved the survival of HER2-positive
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breast cancer patients, resistance remains a major clinical obstacle. We retrospectively evaluated the association between HER2 polymorphisms, tobacco and alcohol consumption and diseasefree survival (DFS) in HER2-positive breast cancer patients. Patients and Methods. Clinicopathological and survival data (median follow-up time: 7.4 years) were collected from medical records for 236 non-metastatic HER2-positive trastuzumab-treated breast cancer patients. Tobacco and alcohol consumption were assessed using validated questionnaires and
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HER2 polymorphisms (Ile655Val and Ala1170Pro) were determined using TaqMan assay. Multivariate Cox proportional hazards models were used to analyse DFS. Results. Compared to non-smokers, patients who smoked before breast cancer diagnosis showed a worse DFS (hazard ratio (HR): 2.63, p=0.001), and this association was stronger among patients who smoked > 20
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cigarettes/day or who spent > two decades smoking before their diagnosis (HR: 3.65, p=0.01, and HR: 3.19, p=0.002, respectively). Smoking during trastuzumab treatment was associated with
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DFS, but only among patients with estrogen receptor-negative tumors (HR: 4.49, p=0.02). Compared to non-drinkers, patients who consumed alcohol before breast cancer diagnosis had a significantly better DFS (HR: 0.56, p=0.03). No association was observed between alcohol consumption during trastuzumab treatment and DFS. Concerning HER2 polymorphisms, patients with Ile/Val or Val/Val genotype had a significantly worse DFS than those with the Ile/Ile genotype (HR: 4.96, p=0.01). Conclusions. Our results suggest that tobacco and alcohol consumption as well as HER2 Ile655Val polymorphism could influence trastuzumab response. These results need to be confirmed in a larger cohort study. 2
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Key words : SNP; tobacco use; NNK; alcohol use; resveratrol; HER2 inhibitors Introduction
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The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that activates several growth-promoting signaling pathways including PI3K/AKT and ERK/MAPK 1. HER2 gene amplification and receptor overexpression occur in 15 to 20% of breast cancer patients and are associated with worse prognosis 2. In addition, HER2-positive status is regarded as a
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predictive marker of response to anti-HER2 therapies 3. The administration of trastuzumab has led to significant improvement in disease-free and overall survival of HER2-positive breast cancer patients when combined with standard chemotherapy in the adjuvant and metastatic 4, 5
. Despite this achievement, primary and acquired resistance to trastuzumab represents
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settings
a major obstacle in the clinical management of HER2-positive breast cancer patients 6. Therefore, there is an urgent need to identify factors that could influence trastuzumab response in HER2positive breast cancer patients.
There is increasing evidence that lifestyle factors including tobacco and alcohol consumption 7-9
. Notably, associations between
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might have an impact on survival of breast cancer patients
tobacco and ethanol exposure and HER2 expression have been reported in the literature. It has been shown that 4-(methylnitrosamino)-1-3-(3-pyridyl)-1-butanon (NNK), a potent tobaccospecific carcinogen
10
, activates the ERK/MAPK signaling pathway (a growth-promoting 11
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signaling pathway activated by HER2) in human normal mammary epithelial cells
Furthermore, a study that examined the association between tobacco consumption at time of
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breast cancer diagnosis and risk of recurrence in a cohort of 3,340 breast cancer patients showed that recurrence risk was significantly increased in trastuzumab-naïve HER2-positive breast cancer patients (n=177) who smoked at time of diagnosis, suggesting that tobacco effect might be particularly strong in this molecular subtype of breast cancer 12. The only study that analysed the association between tobacco use and response to trastuzumab in a cohort of 248 metastatic trastuzumab-treated HER2-positive breast cancer patients reported that response rate in smokers (former and active) was not statistically different from that of never smokers
13
. Concerning
alcohol consumption, a recent study performed in a cohort of 105,972 women highlighted that 3
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alcohol consumption might represent a risk factor for HER2-positive breast cancer subtype
14
.
Furthermore, it has been reported that HER2 overexpression was associated with an increased response to ethanol-stimulated cell proliferation, suggesting that HER2 overexpression may amplify ethanol-induced signaling and enhance invasion capacity of breast cancer cells promoted
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by alcohol 15. No study has evaluated the impact of alcohol consumption on trastuzumab response in HER2-positive breast cancer patients yet.
In addition to lifestyle factors, genetic factors may also influence the efficacy of antineoplastic
polymorphisms (SNPs)
18
19, 20
. The Ile655Val polymorphism leads to the substitution of
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20
. The HER2 gene contains several single nucleotide
. Two of the most investigated HER2 SNPs are Ile655Val (rs1136201)
and Ala1170Pro (rs1058808) Isoleucine for Valine
16, 17
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drugs in breast cancer patients
. A preclinical study reported that the Val substitution might increase
tumorigenic potential of breast cancer cells as it predisposes the HER2 receptor to assume an active conformation leading to enhanced activity of the tyrosine kinase domain Ala1170Pro polymorphism results in the substitution of Alanine for Proline relevance of this polymorphism, however, remains undefined
22
22
21
. The
. The biological
. To date, only two studies have
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investigated the association between HER2 Ile655Val SNP and the response to trastuzumab 23, 24. Whereas Beauclair and collaborators did not observe an association between HER2 genotype and response to trastuzumab in a cohort of 61 patients with advanced HER2-positive breast cancer 23, Han et al. found that Ile655Val SNP was associated with better disease-free survival (DFS)
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among 212 HER2-positive breast cancer patients who received adjuvant trastuzumab treatment 24
Thus, we hypothesized that the risk of recurrence among trastuzumab-treated HER2-positive breast cancer patients is increased by tobacco and alcohol consumption and affected by HER2 polymorphisms. The goal of our study was to evaluate the association between tobacco and alcohol consumption and HER2 polymorphisms with the risk of recurrence in a cohort of nonmetastatic HER2-positive breast cancer patients treated with trastuzumab.
Material and Methods 4
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Study population and data collection. The study population consisted of 237 women with nonmetastatic HER2-positive breast cancer diagnosed (or chemotherapy completed) between July 1st,
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2005 and January 1st, 2010 and treated with trastuzumab. One patient was excluded due to loss of follow-up after breast cancer diagnosis. Therefore, the study population consisted of 236 patients. This hospital-based study took place at the Centre des Maladies du Sein Deschênes-Fabia (CMSDF), a specialized breast center in Quebec City, Canada. All patients underwent mastectomy or segmental resection. Clinicopathological data and information on treatment
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received and follow-up were retrospectively extracted from a registry held by CMSDF. The CMSDF conducts active follow-up of all breast cancer patients to determine vital status. Vital
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status was updated by linkage of the CMSDF register with the database of beneficiaries of the Quebec universal health insurance system (Régie de l’assurance maladie du Québec) and with the Quebec mortality database held by the Institut de la statistique de Québec. Follow-up phone calls were conducted by the research nurse (08/2016) for six patients for whom follow-up information was not available in the registry. Patients were followed until August 18, 2016, which was the end of the study period.
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Informed written consent was obtained from all individual participants included in the study. Ethical approval for the main study and substudy was obtained from the Research Committee of the CHU of Quebec (#DR-002-1227 and DR-002-1265).
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Current smoking status, including the average number of cigarettes smoked per day and the number of years spent smoking, was assessed using a validated self-administered questionnaire at
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two time periods: at the time of the breast cancer diagnosis (n=236) and during trastuzumab treatment (n=131). For 211 out of 236 patients, current alcohol consumption (yes/no) was documented using a validated self-administered questionnaire at the time of the breast cancer diagnosis. Moreover, 128 patients completed a validated graduated beverage-specific frequency questionnaire
25
that investigated the type (wine, beer and spirits) and quantity of alcohol intake
per week at two time periods: at the time of breast cancer diagnosis and during trastuzumab treatment.
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Polymorphism substudy. Seventy-three patients who consented to participate in the tissue banking activities of the CMSDF were included in the substudy. Ile655Val and Ala1170Pro polymorphisms were measured in breast tumor tissues as previously reported
26
. Briefly, DNA
Qiagen DNA Mini Kit (Qiagen, Mississauga, Ontario, Canada)
27
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was extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissues using the . DNA samples were blindly
genotyped for two SNPs [Ile655Val (rs1136201) and Ala1170Pro (rs1058808)] located in HER2 gene by TaqMan SNP Genotyping Assays (Life Technologies, Burlington, Ontario, Canada). Each batch also included negative (no DNA) and positive controls to ensure genotyping
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accuracy. Deviation from Hardy-Weinberg equilibrium was evaluated for each SNP; p-value was 0.39 for Ile655Val SNP and 0.03 for Ala1170Pro SNP. The linkage disequilibrium strength was
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assessed with Lewontin's D' statistic for pairwise SNPs and was 0.02, which is similar to another study 28.
Study end points. For all analyses, the primary end point was disease-free survival (DFS), defined as time from date of mastectomy to first recurrence (locoregional or distant). Patients who were alive at the last follow-up were censored at the last follow-up date (August 18, 2016), and
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patients who died without experiencing breast cancer recurrence were censored at the time of death. Six patients died from causes other than breast cancer.
Statistical analysis. Descriptive statistics were used for baseline characteristics. Univariate and
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multivariate Cox proportional hazard regressions were used to estimate hazard ratios (HR) and corresponding 95% confidence intervals (CI) for the effect of tobacco use, alcohol consumption
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or HER2 SNPs on survival (DFS). The Cox proportional hazards assumption was tested both numerically and graphically. Prognostic factors associated with DFS with a p-value <0.20 in univariate analyses were included in multivariate analyses to account for potential confounders. Adjustment variables included age at diagnosis (≤ 55 (reference), > 55 years), body mass index (< 20, ≥ 20 and < 25 (reference), ≥ 25 kg/m2), stage (I (reference), II, III), adjuvant endocrine therapy (yes (reference), no), and radiotherapy (yes (reference), no). One patient for whom adjustment variables were unknown was excluded from the multivariate analysis. Given that MAPK signaling is reportedly significantly hyperactivated in ER-negative tumors overexpressing 6
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HER2
29
, we performed stratified analysis according to ER status. All statistical tests were two-
sided, and p-values <0.05 were considered statistically significant. All analyses were performed
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using the SAS software (version 9.1.3; SAS Institute Inc., Cary, NC, USA).
Results
Over a median follow-up period of 7.4 years, 66 patients out of 236 (28.0%) experienced a recurrence. Baseline characteristics of the study population are presented in Table 1. Approximately 35.0% of patients were younger than 50 years and 55.1% had a body mass index
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higher than 25 kg/m2. Almost two-thirds (63.1%) of patients had grade III tumors, 53.4% had positive lymph node status, and 29.7% had stage III tumors. Positive ER and PR status was
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observed in 66.1% and 47.0% of patients, respectively. Most of the patients had radiotherapy (85.2%) and 63.6% of patients received adjuvant endocrine therapy. At the time of their diagnosis, 16% of patients were current smokers and 58% consumed alcohol.
Association between tobacco use and DFS is presented in Table 2. Compared to non-smokers, the adjusted hazard ratio (HR) for DFS was 2.63 (95% CI, 1.48 to 4.68, p=0.001) for patients who
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smoked before breast cancer diagnosis. Average number of cigarettes smoked per day and number of years spent smoking before breast cancer diagnosis were associated with breast cancer recurrence, but only in the extreme category (HR: 3.65, 95% CI, 1.35 to 9.89, p=0.01 for > 20 cigarettes per day, and HR: 3.19, 95% CI, 1.55 to 6.56, p=0.002 for > 20 years spent smoking,
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respectively). For the 131 patients for whom we had information about smoking status during trastuzumab treatment, smoking during trastuzumab treatment was not significantly associated
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with breast cancer recurrence (HR: 2.15, 95% CI, 0.92 to 5.04, p=0.08).
When stratified according to ER status, an increased risk of recurrence was observed in the ERnegative subgroup (Table 3). Among patients with ER-negative tumors, the adjusted hazard ratio for DFS was 3.73 (95% CI, 1.56 to 8.89, p=0.003) for patients who smoked before breast cancer diagnosis, and 4.49 (95% CI, 1.26 to 16.00, p=0.02) for patients who smoked during trastuzumab treatment when compared to non-smokers. In the ER-positive subgroup, neither smoking before
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breast cancer diagnosis nor during trastuzumab treatment was associated with DFS (HR: 1.91; 95% CI, 0.84 to 4.31, p = 0.12, and HR: 1.33; 95% CI, 0.27 to 6.47, p=0.72, respectively).
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Association between alcohol consumption and DFS is presented in Table 4. Compared to nondrinkers, the adjusted hazard ratio for DFS was 0.56 (95% CI, 0.33 to 0.94, p=0.03) for patients who consumed alcohol before breast cancer diagnosis. Further analyses by type of alcohol consumed revealed that wine consumption before breast cancer diagnosis was associated with reduced risk of breast cancer recurrence compared to non-wine drinkers (adjusted HR: 0.42; 95%
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CI, 0.18 to 0.95, p=0.04). However, beer consumption before breast cancer diagnosis was not significantly associated with a higher risk of breast cancer recurrence compared to non-beer
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drinkers (adjusted HR: 1.60; 95% CI, 0.47 to 5.47, p=0.46). For the 128 patients for whom we had information about alcohol use during trastuzumab treatment, we did not observe a significant association between DFS and consumption of alcohol (adjusted HR: 0.68, 95% CI, 0.30 to 1.56, p=0.36), wine (HR: 0.55; 95% CI, 0.23 to 1.33, p=0.18) or beer (HR: 1.98; 95% CI, 0.53 to 7.33, p=0.31). We could not generate results regarding the association between spirits consumption neither before breast cancer diagnostic nor during trastuzumab treatment and risk of cancer
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recurrence since only a few women consumed spirits. Moreover, there is no data on the association between alcohol consumption and DFS according to ER status due to an insufficient number of patients.
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HER2 polymorphism substudy
HER2 genotype was determined in breast cancer tissue of 73 breast cancer patients. As
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previously reported, Ile655Val and Ala1170Pro genotypes measured in breast cancer tissues were available for 71 and 69 breast cancer patients, respectively 26. Briefly, 77.5% (50/71) of patients were homozygous for the Ile/Ile genotype, 7.0% (5/71) were heterozygous for the Ile/Val genotype, and 15.5% (11/71) were homozygous for the Val/Val genotype. Regarding the Ala1170Pro polymorphism, 50.7% (35/69) of patients were homozygous for the Ala/Ala genotype, 13.1% (9/69) were heterozygous for the Ala/Pro genotype, and 36.2% (25/69) were homozygous for the Pro/Pro genotype.
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Association between Ile655Val polymorphism and DFS is presented in Table 5. Compared to patients with Ile/Ile genotype, those with the Val/Ile or the Val/Val genotypes showed worse DFS after adjustment for potential confounders (HR 4.96, 95% CI 1.45 to 17.02, p = 0.01). Regarding
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the Ala1170Pro polymorphism, compared to patients with the Ala/Ala genotype, those with the Ala/Pro or the Pro/Pro genotypes showed non-significant better DFS even after adjustment for confounding factors (HR 0.70, 95% CI 0.23 – 2.09, p = 0.77).
Discussion
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In a cohort of non-metastatic HER2-positive breast cancer patients, we observed that tobacco use before breast cancer diagnosis or during trastuzumab treatment was associated with breast cancer
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recurrence. Moreover, our results suggest that heavy tobacco exposure, in amount and duration, before breast cancer diagnosis increases the risk of recurrence. To the best of our knowledge, only one other study has investigated the association between tobacco consumption and trastuzumab response 13. In contrast to our results, Santini and collaborators reported that tobacco exposure before breast cancer diagnosis was not associated with targeted treatment response
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among 248 metastatic trastuzumab-treated HER2-positive breast cancer patients. Furthermore,
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they reported that the number of cigarettes smoked before breast cancer diagnosis was not associated with patient outcome. This inconsistency in results could be explained by the differences in study population (early HER2-positive vs. metastatic HER2-positive breast cancer patients) and in the analyses performed (multivariate vs. univariate). Moreover, Santini et al. did
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not consider the numbers of years spent smoking before breast cancer diagnosis, nor did they perform stratified analysis according to ER status. MAPK signaling is reportedly significantly 29
. In agreement with these
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hyperactivated in ER-negative tumors overexpressing HER2
preclinical observations, we observed that tobacco exposure before diagnosis and during trastuzumab treatment was not only associated with worse DFS but also had a stronger effect in the ER-negative subgroup. In addition, a recent study observed that the effect of tobacco consumption before breast cancer diagnosis differed according to molecular tumor subtype. Similar to our results, the authors reported that breast cancer recurrence risk was significantly higher in breast cancer patients harbouring the HER2 tumor subtype (therefore HER2-positive and ER-negative) 12. 9
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In our study, alcohol consumption before breast cancer diagnosis was associated with better treatment response. However, we did not observe an association between alcohol consumption
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during trastuzumab treatment and breast cancer recurrence. To date, no other study evaluated the association between alcohol intake and response to trastuzumab. Given that our results contrasted with our initial hypothesis that alcohol exposure augmented the risk of relapse in HER2-positive trastuzumab-treated breast cancer patients, we evaluated the effect of alcohol type on DFS. Although our sample size was small, our results suggest that not all types of alcohol, but wine
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consumption before breast cancer diagnosis may reduce the risk of recurrence. Some preclinical data suggest that resveratrol, a polyphenol contained in the skin of grapes and in red wine 30, may
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be protective against some types of cancers including breast cancer 31-33. These results are in line with another recent preclinical study that showed that the addition of resveratrol to HER2positive breast cancer cell lines treated with trastuzumab increased the cytotoxicity of the antiHER2 agent compared to HER2-positive breast cancer cell lines treated with trastuzumab alone 34
.
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In our substudy, patients carrying the Ile/Val or Val/Val genotypes had significantly worse DFS compared to patients carrying the Ile/Ile genotype, after adjustment for potential confounders. Two previous studies examining the association between HER2 Ile655Val polymorphism and trastuzumab response in HER2-positive breast cancer patients reported conflicting results
23, 24
.
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Beauclair and collaborators showed that Ile655Val polymorphism measured in blood was not associated with trastuzumab treatment response among 61 patients with advanced HER2-positive
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breast cancer patients 23. On the other hand, Han and collaborators observed, in their study of 212 trastuzumab-treated HER2-positive breast cancer patients, that patients carrying the Ile/Val or Val/Val genotypes measured in blood showed significantly better DFS compared to those carrying Ile/Ile genotype 24. Several elements could explain the discrepancies between our results and those reported in these two studies. These include the tissue type used for genotype determination (FFPE breast cancer tissues (our study) vs. blood
23, 24
), the characteristics of the
study population (early (our study) vs. advanced breast cancer patients performed (multivariate (our study) vs. univariate models
23, 24
23
), and the analyses
). In our opinion, our results are 10
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concordant with HER2 biology. It can be argued that since Val substitution leads to enhanced activity of the tyrosine kinase domain
21
, and that trastuzumab does not block dimerization with
other HER family members 35, 36, HER2-positive breast cancer cells carrying the Val allele might
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be more prone to developing trastuzumab resistance in the presence of alternative signaling pathways.
Our study adds knowledge to the few others that have examined the association between tobacco consumption and HER2 polymorphisms with trastuzumab response, in addition to providing
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information on the effects of alcohol use on treatment response. However, our study presents some weaknesses, including the small sample size and its retrospective design. Moreover,
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although we performed multivariate analysis, we cannot completely exclude the possibility of residual confounding. In addition, it is well known that self-reported tobacco and alcohol exposure is subject to error, but if any, it would result in non-differential misclassification and would have underestimated the true associations. Furthermore, categorization of alcohol intake may have affected results given that patients who consumed alcohol in modest amounts were classified as non-drinkers in the analysis examining alcohol consumption and breast cancer
Conclusion
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recurrence during trastuzumab treatment. This might have underestimated the true association.
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Our results suggest that lifestyle factors such as tobacco and alcohol consumption and genetic factors like HER2 polymorphism could influence efficacy of trastuzumab treatment in HER2-
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positive breast cancer patients. These results need to be confirmed in a larger cohort, using detailed questionnaires about tobacco use and type of alcohol consumed at several times following a diagnosis of breast cancer. The findings in our study, if confirmed, might contribute to developing lifestyle habits recommendations for trastuzumab-treated HER2-positive breast cancer patients. Another important aspect that still needs to be evaluated is the relative predictive importance of tobacco and alcohol consumption regarding the response to trastuzumab. This new knowledge will help physicians provide better recommendations to their breast cancer patients on
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which lifestyle habits they should preferentially modify in order to maximize their response to targeted anti-HER2 treatment.
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Clinical practice points Resistance toward trastuzumab in HER2-positive breast cancer patients remains a major clinical problem. Therefore, there is an urgent need to identify factors that could influence trastuzumab response in HER2-positive breast cancer patients. We evaluated the association between HER2 polymorphisms, tobacco and alcohol consumption and survival in HER2-positive breast cancer
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patients. Our observations suggest that tobacco and alcohol consumption as well as one HER2 polymorphism could influence trastuzumab response. The findings in our study, if confirmed in a
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larger cohort, might contribute to developing lifestyle habits recommendations for trastuzumabtreated HER2-positive breast cancer patients.
Acknowledgments
The authors acknowledge Hoffmann-La Roche Limited for their support. The funding source had
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no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Clinical specimens were provided by the Fondation du cancer du sein du Québec and the Banque de tissus et de données of the Réseau de recherche sur le cancer of the FRQS, which is affiliated with the Canadian Tumour Repository Network. DF received doctoral fellowships
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from the Fonds de recherche du Québec - Santé and the Laval University Cancer Research Center. CD is a recipient of the Canadian Breast Cancer Foundation-Canadian Cancer Society Capacity Development award (award #703003) and the FRQS Research Scholar. JL was a
study.
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Clinical Research Scholar from the Fonds de la recherche en santé du Québec at the time of the
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Conflict of interest
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The authors declare that they have no conflict of interest.
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Ma C, Lin H, Leonard SS, Shi X, Ye J, Luo J. Overexpression of ErbB2 enhances ethanol-stimulated intracellular signaling and invasion of human mammary epithelial and breast cancer cells in vitro. Oncogene. 2003;22:5281-5290. Caskey CT. Using genetic diagnosis to determine individual therapeutic utility. Annual review of medicine. 2010;61:1-15. Moreno-Munoz D, de la Haba-Rodriguez JR, Conde F, et al. Genetic variants in the reninangiotensin system predict response to bevacizumab in cancer patients. European journal of clinical investigation. 2015;45:1325-1332. Alaoui-Jamali MA, Morand GB, da Silva SD. ErbB polymorphisms: insights and implications for response to targeted cancer therapeutics. Frontiers in genetics. 2015;6:17. Han W, Kang D, Lee JE, et al. A haplotype analysis of HER-2 gene polymorphisms: association with breast cancer risk, HER-2 protein expression in the tumor, and disease recurrence in Korea. Clinical cancer research : an official journal of the American Association for Cancer Research. 2005;11:4775-4778. Papewalis J, Nikitin AY, Rajewsky MF. G to A polymorphism at amino acid codon 655 of the human erbB-2/HER2 gene. Nucleic acids research. 1991;19:5452. Fleishman SJ, Schlessinger J, Ben-Tal N. A putative molecular-activation switch in the transmembrane domain of erbB2. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:15937-15940. Tommasi S, Fedele V, Lacalamita R, et al. 655Val and 1170Pro ERBB2 SNPs in familial breast cancer risk and BRCA1 alterations. Cellular oncology : the official journal of the International Society for Cellular Oncology. 2007;29:241-248. Beauclair S, Formento P, Fischel JL, et al. Role of the HER2 [Ile655Val] genetic polymorphism in tumorogenesis and in the risk of trastuzumab-related cardiotoxicity. Annals of oncology : official journal of the European Society for Medical Oncology. 2007;18:1335-1341. Han X, Diao L, Xu Y, et al. Association between the HER2 Ile655Val polymorphism and response to trastuzumab in women with operable primary breast cancer. Annals of oncology : official journal of the European Society for Medical Oncology. 2014;25:11581164. Dawson DA. Methodological issues in measuring alcohol use. Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism. 2003;27:18-29. Furrer D, Lemieux J, Cote MA, et al. Evaluation of human epidermal growth factor receptor 2 (HER2) single nucleotide polymorphisms (SNPs) in normal and breast tumor tissues and their link with breast cancer prognostic factors. Breast. 2016;30:191-196. Gagnon JF, Sanschagrin F, Jacob S, et al. Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues. Experimental and molecular pathology. 2010;88:184-189. Benusiglio PR, Lesueur F, Luccarini C, et al. Common ERBB2 polymorphisms and risk of breast cancer in a white British population: A case control study. Breast cancer research : BCR. 2005;7:204-2099. Creighton CJ, Hilger AM, Murthy S, Rae JM, Chinnaiyan AM, El-Ashry D. Activation of mitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha-negative human breast tumors. Cancer research. 2006;66:3903-3911. 15
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35. 36.
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33.
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32.
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Brakenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2001;15:1798-1800. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer research. 2004;24:2783-2840. Athar M, Back JH, Tang X, et al. Resveratrol: a review of preclinical studies for human cancer prevention. Toxicology and applied pharmacology. 2007;224:274-283. Stewart JR, Artime MC, O'Brian CA. Resveratrol: a candidate nutritional substance for prostate cancer prevention. The Journal of nutrition. 2003;133:2440S-2443S. Abdel-Latif GA, Al-Abd AM, Tadros MG, Al-Abbasi FA, Khalifa AE, Abdel-Naim AB. The chemomodulatory effects of resveratrol and didox on herceptin cytotoxicity in breast cancer cell lines. Scientific reports. 2015;5:12054. Agus DB, Akita RW, Fox WD, et al. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer cell. 2002;2:127-137. Ghosh R, Narasanna A, Wang SE, et al. Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers. Cancer research. 2011;71:1871-1882.
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%
82 154
34.8 65.2
18 88 130
7.6 37.3 55.1
86 149 1
34.4 63.1 2.5
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46.6 53.4
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110 126
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N
60 106 70
25.4 44.9 29.7
79 156 1
33.5 66.1 0.4
124 111 1
52.6 47.0 0.4
85 150 1
36.0 63.6 0.4
35 201
14.8 85.2
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Age (years) ≤50 >50 Body mass index (kg/m2) >15-<20 ≥20-<25 ≥25 Grade I/II III Unknown Lymph node status Negative Positive Stage I II III Estrogen receptor status Negative Positive Unknown Progesterone receptor status Negative Positive Unknown Adjuvant endocrine therapy No Yes Unknown Radiotherapy No Yes
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Table 1. Baseline characteristics of the study population (n=236).
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Table 2. Unadjusted and adjusted hazard ratios for disease-free survival according to tobacco consumption before breast cancer diagnosis and during trastuzumab treatment.
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Factor Without recurrence With recurrence Univariate HR p-value Adjusted HR Adjusted Smoking (95% CI) (95% CI)* p-value* Before breast cancer (n=170) (n=66) diagnosis No 150 (88.2%) 48 (72.7%)** 1.00 (reference) 1.00 (reference) Yes 20 (11.8%) 18 (27.3%) 2.58 (1.46 – 4.55) 2.63 (1.48 – 4.68) 0.001 0.001 Cigarettes/day (n=154) (n=57) Non-smokers 134 (87.0%) 44 (77.2%)** 1.00 (reference) 1.00 (reference) 1-20 17 (11.0%) 8 (14.0%) 1.28 (0.60 – 2.73) 0.52 1.32 (0.62 – 2.85) 0.47 > 20 3 (2.0%) 5 (8.8%) 4.27 (1.67 – 10.95) 0.003 3.65 (1.35 – 9.89) 0.01 Smoking duration (n=137) (n=52) (years) Non-smokers 117 (85.4%) 35 (67.3%)** 1.00 (reference) 1.00 (reference) 1-20 9 (6.6%) 5 (9.6%) 2.04 (0.80 – 5.20) 0.14 2.15 (0.81 – 5.67) 0.13 > 20 11 (8.0%) 12 (23.1%) 2.86 (1.48 – 5.54) 3.19 (1.55 – 6.56) 0.002 0.002 During trastuzumab (n=96) (n=35) treatment No 86 (89.6%) 28 (80.0%) 1.00 (reference) 1.00 (reference) Yes 10 (10.4%) 7 (20.0%) 2.03 (0.88 – 4.68) 0.09 2.15 (0.92 – 5.04) 0.08 HR: hazard ratio; CI: confidence interval. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy; ** One patient was excluded from the multivariate analysis due to missing value for adjuvant endocrine therapy.
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Table 3. Unadjusted and adjusted hazard ratios for disease-free survival according to tobacco consumption before breast cancer diagnosis and during trastuzumab treatment, stratified by ER status. With recurrence
(n=50)
(n=29)
46 (92.0%) 4 (8.0%) (n=33)
20 (69.0%) 9 (31.0%) (n=16)
30 (91.0%) 3 (9.0%)
11 (68.8%) 5 (31.2%)
Univariate HR (95% CI)
p-value
Adjusted HR (95% CI)*
Adjusted p-value*
0.002
1.00 (reference) 3.73 (1.56 – 8.89)
0.003
0.01
1.00 (reference) 4.49 (1.26 – 16.00)
0.02
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Without recurrence
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1.00 (reference) 3.73 (1.64 – 8.48)
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Factor Smoking ER-negative patients: Before breast cancer diagnosis No Yes During trastuzumab treatment No Yes
1.00 (reference) 3.92 (1.29 – 11.85)
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ER-positive patients: Before breast cancer (n=120) (n=36) diagnosis No 104 (87.0%) 27 (75.0%) 1.00 (reference) 1.00 (reference) Yes 16 (13.0%) 9 (25.0%) 1.90 (0.89 – 4.04) 0.09 1.91 (0.84 – 4.31) 0.12 During trastuzumab (n=63) (n=19) treatment No 56 (89.0%) 17 (89.5%) 1.00 (reference) 1.00 (reference) Yes 7 (11.0%) 2 (10.5%) 0.96 (0.22 – 4.14) 0.95 1.33 (0.27 – 6.47) 0.72 HR: hazard ratio; CI: confidence interval. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy.
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Table 4. Unadjusted and adjusted hazard ratios for disease-free survival according to alcohol consumption before breast cancer diagnosis and during trastuzumab treatment. With recurrence
56 (37.3%) 94 (62.7%)
33 (54.1%)** 28 (45.9%)
(n=96) 62 (64.6%) 34 (35.4%) (n=96) 90 (93.7%) 6 (6.3%) (n=96) 92 (95.8%) 4 (4.2%) (n=95)
(n=35) 27 (77.2%) 8 (22.8%) (n=35) 32 (91.4%) 3 (8.6%) (n=34) 34 (100.0%) 0 (0.0%) (n=33)
64 (67.4%) 31 (32.6%)
23 (69.7%) 10 (30.3%)
(n=96) 72 (75.0%) 24 (25.0%) (n=96) 91 (94.8%) 5 (5.2%)
Univariate HR (95% CI)
Adjusted HR (95% CI)*
Adjusted p-value*
0.02
1.00 (reference) 0.56 (0.33 – 0.94)
0.03
1.00 (reference) 0.52 (0.24 – 1.15)
0.11
1.00 (reference) 0.42 (0.18 – 0.95)
0.04
1.00 (reference) 1.45 (0.44 – 4.75)
0.54
1.00 (reference) 1.60 (0.47 – 5.47)
0.46
1.00 (reference) N/A
N/A
1.00 (reference) N/A
N/A
1.00 (reference) 0.91 (0.43 – 1.91)
0.79
1.00 (reference) 0.68 (0.30 – 1.56)
0.36
1.00 (reference) 0.72 (0.31 – 1.65)
0.43
1.00 (reference) 0.55 (0.23 – 1.33)
0.18
1.00 (reference) 1.62 (0.49 – 5.32)
0.43
1.00 (reference) 1.98 (0.53 – 7.33)
0.31
(n=61)
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1.00 (reference) 0.55 (0.33 – 0.92)
(N=36) 28 (77.8%) 8 (22.2%) (n=34) 31 (91,2%) 3 (8.8%)
p-value
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Without recurrence (n=150)
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Factor Alcohol Before breast cancer diagnosis No Yes According to type of alcohol consumed Wine 0-2 drinks/week > 2 drinks/week Beer 0-2 drinks/week > 2 drinks/week Spirits 0-2 drinks/week > 2 drinks/week During trastuzumab treatment 0-2 drinks per week > 2 drinks per week According to type of alcohol consumed Wine 0-2 drinks/week > 2 drinks/week Beer 0-2 drinks/week > 2 drinks/week
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Spirits (n=95) (N=34) 0-2 drinks/week 93 (97.9%) 34 (100.0%) 1.00 (reference) 1.00 (reference) > 2 drinks/week 2 (2.1%) 0 (0.0%) N/A N/A N/A N/A HR: hazard ratio; CI: confidence interval; N/A: not applicable. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy; ** One patient was excluded from the multivariate analysis due to missing value for adjuvant endocrine therapy.
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Table 5. Unadjusted and adjusted hazard ratios for disease-free survival according to HER2 polymorphisms.
Ile655Val Ile/Ile Ile/Val or Val/Val
Without recurrence (n=53) 42 (79.3%) 11 (20.7%)
With recurrence (n=18) 13 (72.2%) 5 (27.6%)
Univariate HR (95% CI) 1.00 (reference) 2.00 (0.70 – 5.81)
p-value
Adjusted HR (95% CI)*
Adjusted p-value*
1.00 (reference) 4.96 (1.45 – 17.02)
0.01
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Polymorphism
0.20
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Ala1170Pro (N=51) (n=18) Ala/Ala 26 (51.0%) 10 (55.5%) 1.00 (reference) 1.00 (reference) Ala/Pro or Pro/Pro 25 (49.0%) 8 (44.5%) 0.84 (0.33 – 2.14) 0.71 0.70 (0.23 – 2.09) 0.77 HR: hazard ratio; CI: confidence interval. *Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy.
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S1526-8209(17)30318-X
DOI:
10.1016/j.clbc.2017.11.012
Reference:
CLBC 719
To appear in:
Clinical Breast Cancer
Received Date: 25 May 2017 Revised Date:
12 October 2017
Accepted Date: 20 November 2017
Please cite this article as: Furrer D, Jacob S, Michaud A, Provencher L, Lemieux J, Diorio C, Association between tobacco and alcohol consumption, HER2 polymorphisms and response to trastuzumab in HER2-positive breast cancer patients, Clinical Breast Cancer (2017), doi: 10.1016/j.clbc.2017.11.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Association between tobacco and alcohol consumption, HER2 polymorphisms and response to trastuzumab in HER2-positive breast cancer patients Daniela Furrer a,b,d, Simon Jacob Lemieux a,b,c,d, Caroline Diorio a,b,c,d
a,b,c,d
, Annick Michaud
, Louise Provencher
a,b,c,d
, Julie
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a
a,b
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Centre de Recherche sur le cancer de l'Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada ; b Axe Oncologie, Centre de Recherche du CHU de Québec-Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada; c Centre des maladies du sein Deschênes-Fabia, Hôpital du Saint-Sacrement, 1050, chemin Ste-Foy, Québec, QC, G1S 4L8, Canada; d Faculté de Médecine, Université Laval, 1050 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada Corresponding author :
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Caroline Diorio Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Hôpital du Saint-Sacrement, 1050 chemin Ste-Foy, Quebec City, QC G1S 4L8, Canada Phone: +1-418-682-7511 ext. 84726 Fax: +1-418-682-7949 e-mail: [email protected]
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MicroAbstract Trastuzumab resistance in HER2-positive breast cancer patients remains a major clinical problem. We evaluated the association between HER2 polymorphisms, tobacco and alcohol
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consumption and disease-free survival in HER2-positive breast cancer patients. Our observations suggest that tobacco and alcohol consumption as well as one HER2 polymorphism could influence trastuzumab response. These results need to be confirmed in a larger cohort study.
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Abstract
Purpose. Although the administration of trastuzumab has improved the survival of HER2-positive
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breast cancer patients, resistance remains a major clinical obstacle. We retrospectively evaluated the association between HER2 polymorphisms, tobacco and alcohol consumption and diseasefree survival (DFS) in HER2-positive breast cancer patients. Patients and Methods. Clinicopathological and survival data (median follow-up time: 7.4 years) were collected from medical records for 236 non-metastatic HER2-positive trastuzumab-treated breast cancer patients. Tobacco and alcohol consumption were assessed using validated questionnaires and
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HER2 polymorphisms (Ile655Val and Ala1170Pro) were determined using TaqMan assay. Multivariate Cox proportional hazards models were used to analyse DFS. Results. Compared to non-smokers, patients who smoked before breast cancer diagnosis showed a worse DFS (hazard ratio (HR): 2.63, p=0.001), and this association was stronger among patients who smoked > 20
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cigarettes/day or who spent > two decades smoking before their diagnosis (HR: 3.65, p=0.01, and HR: 3.19, p=0.002, respectively). Smoking during trastuzumab treatment was associated with
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DFS, but only among patients with estrogen receptor-negative tumors (HR: 4.49, p=0.02). Compared to non-drinkers, patients who consumed alcohol before breast cancer diagnosis had a significantly better DFS (HR: 0.56, p=0.03). No association was observed between alcohol consumption during trastuzumab treatment and DFS. Concerning HER2 polymorphisms, patients with Ile/Val or Val/Val genotype had a significantly worse DFS than those with the Ile/Ile genotype (HR: 4.96, p=0.01). Conclusions. Our results suggest that tobacco and alcohol consumption as well as HER2 Ile655Val polymorphism could influence trastuzumab response. These results need to be confirmed in a larger cohort study. 2
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Key words : SNP; tobacco use; NNK; alcohol use; resveratrol; HER2 inhibitors Introduction
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The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that activates several growth-promoting signaling pathways including PI3K/AKT and ERK/MAPK 1. HER2 gene amplification and receptor overexpression occur in 15 to 20% of breast cancer patients and are associated with worse prognosis 2. In addition, HER2-positive status is regarded as a
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predictive marker of response to anti-HER2 therapies 3. The administration of trastuzumab has led to significant improvement in disease-free and overall survival of HER2-positive breast cancer patients when combined with standard chemotherapy in the adjuvant and metastatic 4, 5
. Despite this achievement, primary and acquired resistance to trastuzumab represents
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settings
a major obstacle in the clinical management of HER2-positive breast cancer patients 6. Therefore, there is an urgent need to identify factors that could influence trastuzumab response in HER2positive breast cancer patients.
There is increasing evidence that lifestyle factors including tobacco and alcohol consumption 7-9
. Notably, associations between
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might have an impact on survival of breast cancer patients
tobacco and ethanol exposure and HER2 expression have been reported in the literature. It has been shown that 4-(methylnitrosamino)-1-3-(3-pyridyl)-1-butanon (NNK), a potent tobaccospecific carcinogen
10
, activates the ERK/MAPK signaling pathway (a growth-promoting 11
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signaling pathway activated by HER2) in human normal mammary epithelial cells
Furthermore, a study that examined the association between tobacco consumption at time of
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breast cancer diagnosis and risk of recurrence in a cohort of 3,340 breast cancer patients showed that recurrence risk was significantly increased in trastuzumab-naïve HER2-positive breast cancer patients (n=177) who smoked at time of diagnosis, suggesting that tobacco effect might be particularly strong in this molecular subtype of breast cancer 12. The only study that analysed the association between tobacco use and response to trastuzumab in a cohort of 248 metastatic trastuzumab-treated HER2-positive breast cancer patients reported that response rate in smokers (former and active) was not statistically different from that of never smokers
13
. Concerning
alcohol consumption, a recent study performed in a cohort of 105,972 women highlighted that 3
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alcohol consumption might represent a risk factor for HER2-positive breast cancer subtype
14
.
Furthermore, it has been reported that HER2 overexpression was associated with an increased response to ethanol-stimulated cell proliferation, suggesting that HER2 overexpression may amplify ethanol-induced signaling and enhance invasion capacity of breast cancer cells promoted
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by alcohol 15. No study has evaluated the impact of alcohol consumption on trastuzumab response in HER2-positive breast cancer patients yet.
In addition to lifestyle factors, genetic factors may also influence the efficacy of antineoplastic
polymorphisms (SNPs)
18
19, 20
. The Ile655Val polymorphism leads to the substitution of
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20
. The HER2 gene contains several single nucleotide
. Two of the most investigated HER2 SNPs are Ile655Val (rs1136201)
and Ala1170Pro (rs1058808) Isoleucine for Valine
16, 17
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drugs in breast cancer patients
. A preclinical study reported that the Val substitution might increase
tumorigenic potential of breast cancer cells as it predisposes the HER2 receptor to assume an active conformation leading to enhanced activity of the tyrosine kinase domain Ala1170Pro polymorphism results in the substitution of Alanine for Proline relevance of this polymorphism, however, remains undefined
22
22
21
. The
. The biological
. To date, only two studies have
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investigated the association between HER2 Ile655Val SNP and the response to trastuzumab 23, 24. Whereas Beauclair and collaborators did not observe an association between HER2 genotype and response to trastuzumab in a cohort of 61 patients with advanced HER2-positive breast cancer 23, Han et al. found that Ile655Val SNP was associated with better disease-free survival (DFS)
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among 212 HER2-positive breast cancer patients who received adjuvant trastuzumab treatment 24
Thus, we hypothesized that the risk of recurrence among trastuzumab-treated HER2-positive breast cancer patients is increased by tobacco and alcohol consumption and affected by HER2 polymorphisms. The goal of our study was to evaluate the association between tobacco and alcohol consumption and HER2 polymorphisms with the risk of recurrence in a cohort of nonmetastatic HER2-positive breast cancer patients treated with trastuzumab.
Material and Methods 4
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Study population and data collection. The study population consisted of 237 women with nonmetastatic HER2-positive breast cancer diagnosed (or chemotherapy completed) between July 1st,
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2005 and January 1st, 2010 and treated with trastuzumab. One patient was excluded due to loss of follow-up after breast cancer diagnosis. Therefore, the study population consisted of 236 patients. This hospital-based study took place at the Centre des Maladies du Sein Deschênes-Fabia (CMSDF), a specialized breast center in Quebec City, Canada. All patients underwent mastectomy or segmental resection. Clinicopathological data and information on treatment
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received and follow-up were retrospectively extracted from a registry held by CMSDF. The CMSDF conducts active follow-up of all breast cancer patients to determine vital status. Vital
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status was updated by linkage of the CMSDF register with the database of beneficiaries of the Quebec universal health insurance system (Régie de l’assurance maladie du Québec) and with the Quebec mortality database held by the Institut de la statistique de Québec. Follow-up phone calls were conducted by the research nurse (08/2016) for six patients for whom follow-up information was not available in the registry. Patients were followed until August 18, 2016, which was the end of the study period.
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Informed written consent was obtained from all individual participants included in the study. Ethical approval for the main study and substudy was obtained from the Research Committee of the CHU of Quebec (#DR-002-1227 and DR-002-1265).
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Current smoking status, including the average number of cigarettes smoked per day and the number of years spent smoking, was assessed using a validated self-administered questionnaire at
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two time periods: at the time of the breast cancer diagnosis (n=236) and during trastuzumab treatment (n=131). For 211 out of 236 patients, current alcohol consumption (yes/no) was documented using a validated self-administered questionnaire at the time of the breast cancer diagnosis. Moreover, 128 patients completed a validated graduated beverage-specific frequency questionnaire
25
that investigated the type (wine, beer and spirits) and quantity of alcohol intake
per week at two time periods: at the time of breast cancer diagnosis and during trastuzumab treatment.
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Polymorphism substudy. Seventy-three patients who consented to participate in the tissue banking activities of the CMSDF were included in the substudy. Ile655Val and Ala1170Pro polymorphisms were measured in breast tumor tissues as previously reported
26
. Briefly, DNA
Qiagen DNA Mini Kit (Qiagen, Mississauga, Ontario, Canada)
27
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was extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissues using the . DNA samples were blindly
genotyped for two SNPs [Ile655Val (rs1136201) and Ala1170Pro (rs1058808)] located in HER2 gene by TaqMan SNP Genotyping Assays (Life Technologies, Burlington, Ontario, Canada). Each batch also included negative (no DNA) and positive controls to ensure genotyping
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accuracy. Deviation from Hardy-Weinberg equilibrium was evaluated for each SNP; p-value was 0.39 for Ile655Val SNP and 0.03 for Ala1170Pro SNP. The linkage disequilibrium strength was
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assessed with Lewontin's D' statistic for pairwise SNPs and was 0.02, which is similar to another study 28.
Study end points. For all analyses, the primary end point was disease-free survival (DFS), defined as time from date of mastectomy to first recurrence (locoregional or distant). Patients who were alive at the last follow-up were censored at the last follow-up date (August 18, 2016), and
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patients who died without experiencing breast cancer recurrence were censored at the time of death. Six patients died from causes other than breast cancer.
Statistical analysis. Descriptive statistics were used for baseline characteristics. Univariate and
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multivariate Cox proportional hazard regressions were used to estimate hazard ratios (HR) and corresponding 95% confidence intervals (CI) for the effect of tobacco use, alcohol consumption
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or HER2 SNPs on survival (DFS). The Cox proportional hazards assumption was tested both numerically and graphically. Prognostic factors associated with DFS with a p-value <0.20 in univariate analyses were included in multivariate analyses to account for potential confounders. Adjustment variables included age at diagnosis (≤ 55 (reference), > 55 years), body mass index (< 20, ≥ 20 and < 25 (reference), ≥ 25 kg/m2), stage (I (reference), II, III), adjuvant endocrine therapy (yes (reference), no), and radiotherapy (yes (reference), no). One patient for whom adjustment variables were unknown was excluded from the multivariate analysis. Given that MAPK signaling is reportedly significantly hyperactivated in ER-negative tumors overexpressing 6
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HER2
29
, we performed stratified analysis according to ER status. All statistical tests were two-
sided, and p-values <0.05 were considered statistically significant. All analyses were performed
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using the SAS software (version 9.1.3; SAS Institute Inc., Cary, NC, USA).
Results
Over a median follow-up period of 7.4 years, 66 patients out of 236 (28.0%) experienced a recurrence. Baseline characteristics of the study population are presented in Table 1. Approximately 35.0% of patients were younger than 50 years and 55.1% had a body mass index
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higher than 25 kg/m2. Almost two-thirds (63.1%) of patients had grade III tumors, 53.4% had positive lymph node status, and 29.7% had stage III tumors. Positive ER and PR status was
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observed in 66.1% and 47.0% of patients, respectively. Most of the patients had radiotherapy (85.2%) and 63.6% of patients received adjuvant endocrine therapy. At the time of their diagnosis, 16% of patients were current smokers and 58% consumed alcohol.
Association between tobacco use and DFS is presented in Table 2. Compared to non-smokers, the adjusted hazard ratio (HR) for DFS was 2.63 (95% CI, 1.48 to 4.68, p=0.001) for patients who
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smoked before breast cancer diagnosis. Average number of cigarettes smoked per day and number of years spent smoking before breast cancer diagnosis were associated with breast cancer recurrence, but only in the extreme category (HR: 3.65, 95% CI, 1.35 to 9.89, p=0.01 for > 20 cigarettes per day, and HR: 3.19, 95% CI, 1.55 to 6.56, p=0.002 for > 20 years spent smoking,
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respectively). For the 131 patients for whom we had information about smoking status during trastuzumab treatment, smoking during trastuzumab treatment was not significantly associated
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with breast cancer recurrence (HR: 2.15, 95% CI, 0.92 to 5.04, p=0.08).
When stratified according to ER status, an increased risk of recurrence was observed in the ERnegative subgroup (Table 3). Among patients with ER-negative tumors, the adjusted hazard ratio for DFS was 3.73 (95% CI, 1.56 to 8.89, p=0.003) for patients who smoked before breast cancer diagnosis, and 4.49 (95% CI, 1.26 to 16.00, p=0.02) for patients who smoked during trastuzumab treatment when compared to non-smokers. In the ER-positive subgroup, neither smoking before
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breast cancer diagnosis nor during trastuzumab treatment was associated with DFS (HR: 1.91; 95% CI, 0.84 to 4.31, p = 0.12, and HR: 1.33; 95% CI, 0.27 to 6.47, p=0.72, respectively).
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Association between alcohol consumption and DFS is presented in Table 4. Compared to nondrinkers, the adjusted hazard ratio for DFS was 0.56 (95% CI, 0.33 to 0.94, p=0.03) for patients who consumed alcohol before breast cancer diagnosis. Further analyses by type of alcohol consumed revealed that wine consumption before breast cancer diagnosis was associated with reduced risk of breast cancer recurrence compared to non-wine drinkers (adjusted HR: 0.42; 95%
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CI, 0.18 to 0.95, p=0.04). However, beer consumption before breast cancer diagnosis was not significantly associated with a higher risk of breast cancer recurrence compared to non-beer
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drinkers (adjusted HR: 1.60; 95% CI, 0.47 to 5.47, p=0.46). For the 128 patients for whom we had information about alcohol use during trastuzumab treatment, we did not observe a significant association between DFS and consumption of alcohol (adjusted HR: 0.68, 95% CI, 0.30 to 1.56, p=0.36), wine (HR: 0.55; 95% CI, 0.23 to 1.33, p=0.18) or beer (HR: 1.98; 95% CI, 0.53 to 7.33, p=0.31). We could not generate results regarding the association between spirits consumption neither before breast cancer diagnostic nor during trastuzumab treatment and risk of cancer
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recurrence since only a few women consumed spirits. Moreover, there is no data on the association between alcohol consumption and DFS according to ER status due to an insufficient number of patients.
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HER2 polymorphism substudy
HER2 genotype was determined in breast cancer tissue of 73 breast cancer patients. As
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previously reported, Ile655Val and Ala1170Pro genotypes measured in breast cancer tissues were available for 71 and 69 breast cancer patients, respectively 26. Briefly, 77.5% (50/71) of patients were homozygous for the Ile/Ile genotype, 7.0% (5/71) were heterozygous for the Ile/Val genotype, and 15.5% (11/71) were homozygous for the Val/Val genotype. Regarding the Ala1170Pro polymorphism, 50.7% (35/69) of patients were homozygous for the Ala/Ala genotype, 13.1% (9/69) were heterozygous for the Ala/Pro genotype, and 36.2% (25/69) were homozygous for the Pro/Pro genotype.
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Association between Ile655Val polymorphism and DFS is presented in Table 5. Compared to patients with Ile/Ile genotype, those with the Val/Ile or the Val/Val genotypes showed worse DFS after adjustment for potential confounders (HR 4.96, 95% CI 1.45 to 17.02, p = 0.01). Regarding
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the Ala1170Pro polymorphism, compared to patients with the Ala/Ala genotype, those with the Ala/Pro or the Pro/Pro genotypes showed non-significant better DFS even after adjustment for confounding factors (HR 0.70, 95% CI 0.23 – 2.09, p = 0.77).
Discussion
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In a cohort of non-metastatic HER2-positive breast cancer patients, we observed that tobacco use before breast cancer diagnosis or during trastuzumab treatment was associated with breast cancer
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recurrence. Moreover, our results suggest that heavy tobacco exposure, in amount and duration, before breast cancer diagnosis increases the risk of recurrence. To the best of our knowledge, only one other study has investigated the association between tobacco consumption and trastuzumab response 13. In contrast to our results, Santini and collaborators reported that tobacco exposure before breast cancer diagnosis was not associated with targeted treatment response
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among 248 metastatic trastuzumab-treated HER2-positive breast cancer patients. Furthermore,
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they reported that the number of cigarettes smoked before breast cancer diagnosis was not associated with patient outcome. This inconsistency in results could be explained by the differences in study population (early HER2-positive vs. metastatic HER2-positive breast cancer patients) and in the analyses performed (multivariate vs. univariate). Moreover, Santini et al. did
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not consider the numbers of years spent smoking before breast cancer diagnosis, nor did they perform stratified analysis according to ER status. MAPK signaling is reportedly significantly 29
. In agreement with these
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hyperactivated in ER-negative tumors overexpressing HER2
preclinical observations, we observed that tobacco exposure before diagnosis and during trastuzumab treatment was not only associated with worse DFS but also had a stronger effect in the ER-negative subgroup. In addition, a recent study observed that the effect of tobacco consumption before breast cancer diagnosis differed according to molecular tumor subtype. Similar to our results, the authors reported that breast cancer recurrence risk was significantly higher in breast cancer patients harbouring the HER2 tumor subtype (therefore HER2-positive and ER-negative) 12. 9
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In our study, alcohol consumption before breast cancer diagnosis was associated with better treatment response. However, we did not observe an association between alcohol consumption
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during trastuzumab treatment and breast cancer recurrence. To date, no other study evaluated the association between alcohol intake and response to trastuzumab. Given that our results contrasted with our initial hypothesis that alcohol exposure augmented the risk of relapse in HER2-positive trastuzumab-treated breast cancer patients, we evaluated the effect of alcohol type on DFS. Although our sample size was small, our results suggest that not all types of alcohol, but wine
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consumption before breast cancer diagnosis may reduce the risk of recurrence. Some preclinical data suggest that resveratrol, a polyphenol contained in the skin of grapes and in red wine 30, may
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be protective against some types of cancers including breast cancer 31-33. These results are in line with another recent preclinical study that showed that the addition of resveratrol to HER2positive breast cancer cell lines treated with trastuzumab increased the cytotoxicity of the antiHER2 agent compared to HER2-positive breast cancer cell lines treated with trastuzumab alone 34
.
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In our substudy, patients carrying the Ile/Val or Val/Val genotypes had significantly worse DFS compared to patients carrying the Ile/Ile genotype, after adjustment for potential confounders. Two previous studies examining the association between HER2 Ile655Val polymorphism and trastuzumab response in HER2-positive breast cancer patients reported conflicting results
23, 24
.
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Beauclair and collaborators showed that Ile655Val polymorphism measured in blood was not associated with trastuzumab treatment response among 61 patients with advanced HER2-positive
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breast cancer patients 23. On the other hand, Han and collaborators observed, in their study of 212 trastuzumab-treated HER2-positive breast cancer patients, that patients carrying the Ile/Val or Val/Val genotypes measured in blood showed significantly better DFS compared to those carrying Ile/Ile genotype 24. Several elements could explain the discrepancies between our results and those reported in these two studies. These include the tissue type used for genotype determination (FFPE breast cancer tissues (our study) vs. blood
23, 24
), the characteristics of the
study population (early (our study) vs. advanced breast cancer patients performed (multivariate (our study) vs. univariate models
23, 24
23
), and the analyses
). In our opinion, our results are 10
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concordant with HER2 biology. It can be argued that since Val substitution leads to enhanced activity of the tyrosine kinase domain
21
, and that trastuzumab does not block dimerization with
other HER family members 35, 36, HER2-positive breast cancer cells carrying the Val allele might
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be more prone to developing trastuzumab resistance in the presence of alternative signaling pathways.
Our study adds knowledge to the few others that have examined the association between tobacco consumption and HER2 polymorphisms with trastuzumab response, in addition to providing
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information on the effects of alcohol use on treatment response. However, our study presents some weaknesses, including the small sample size and its retrospective design. Moreover,
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although we performed multivariate analysis, we cannot completely exclude the possibility of residual confounding. In addition, it is well known that self-reported tobacco and alcohol exposure is subject to error, but if any, it would result in non-differential misclassification and would have underestimated the true associations. Furthermore, categorization of alcohol intake may have affected results given that patients who consumed alcohol in modest amounts were classified as non-drinkers in the analysis examining alcohol consumption and breast cancer
Conclusion
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recurrence during trastuzumab treatment. This might have underestimated the true association.
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Our results suggest that lifestyle factors such as tobacco and alcohol consumption and genetic factors like HER2 polymorphism could influence efficacy of trastuzumab treatment in HER2-
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positive breast cancer patients. These results need to be confirmed in a larger cohort, using detailed questionnaires about tobacco use and type of alcohol consumed at several times following a diagnosis of breast cancer. The findings in our study, if confirmed, might contribute to developing lifestyle habits recommendations for trastuzumab-treated HER2-positive breast cancer patients. Another important aspect that still needs to be evaluated is the relative predictive importance of tobacco and alcohol consumption regarding the response to trastuzumab. This new knowledge will help physicians provide better recommendations to their breast cancer patients on
11
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which lifestyle habits they should preferentially modify in order to maximize their response to targeted anti-HER2 treatment.
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Clinical practice points Resistance toward trastuzumab in HER2-positive breast cancer patients remains a major clinical problem. Therefore, there is an urgent need to identify factors that could influence trastuzumab response in HER2-positive breast cancer patients. We evaluated the association between HER2 polymorphisms, tobacco and alcohol consumption and survival in HER2-positive breast cancer
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patients. Our observations suggest that tobacco and alcohol consumption as well as one HER2 polymorphism could influence trastuzumab response. The findings in our study, if confirmed in a
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larger cohort, might contribute to developing lifestyle habits recommendations for trastuzumabtreated HER2-positive breast cancer patients.
Acknowledgments
The authors acknowledge Hoffmann-La Roche Limited for their support. The funding source had
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no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Clinical specimens were provided by the Fondation du cancer du sein du Québec and the Banque de tissus et de données of the Réseau de recherche sur le cancer of the FRQS, which is affiliated with the Canadian Tumour Repository Network. DF received doctoral fellowships
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from the Fonds de recherche du Québec - Santé and the Laval University Cancer Research Center. CD is a recipient of the Canadian Breast Cancer Foundation-Canadian Cancer Society Capacity Development award (award #703003) and the FRQS Research Scholar. JL was a
study.
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Clinical Research Scholar from the Fonds de la recherche en santé du Québec at the time of the
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Conflict of interest
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The authors declare that they have no conflict of interest.
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%
82 154
34.8 65.2
18 88 130
7.6 37.3 55.1
86 149 1
34.4 63.1 2.5
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46.6 53.4
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110 126
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N
60 106 70
25.4 44.9 29.7
79 156 1
33.5 66.1 0.4
124 111 1
52.6 47.0 0.4
85 150 1
36.0 63.6 0.4
35 201
14.8 85.2
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Age (years) ≤50 >50 Body mass index (kg/m2) >15-<20 ≥20-<25 ≥25 Grade I/II III Unknown Lymph node status Negative Positive Stage I II III Estrogen receptor status Negative Positive Unknown Progesterone receptor status Negative Positive Unknown Adjuvant endocrine therapy No Yes Unknown Radiotherapy No Yes
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Table 1. Baseline characteristics of the study population (n=236).
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Table 2. Unadjusted and adjusted hazard ratios for disease-free survival according to tobacco consumption before breast cancer diagnosis and during trastuzumab treatment.
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Factor Without recurrence With recurrence Univariate HR p-value Adjusted HR Adjusted Smoking (95% CI) (95% CI)* p-value* Before breast cancer (n=170) (n=66) diagnosis No 150 (88.2%) 48 (72.7%)** 1.00 (reference) 1.00 (reference) Yes 20 (11.8%) 18 (27.3%) 2.58 (1.46 – 4.55) 2.63 (1.48 – 4.68) 0.001 0.001 Cigarettes/day (n=154) (n=57) Non-smokers 134 (87.0%) 44 (77.2%)** 1.00 (reference) 1.00 (reference) 1-20 17 (11.0%) 8 (14.0%) 1.28 (0.60 – 2.73) 0.52 1.32 (0.62 – 2.85) 0.47 > 20 3 (2.0%) 5 (8.8%) 4.27 (1.67 – 10.95) 0.003 3.65 (1.35 – 9.89) 0.01 Smoking duration (n=137) (n=52) (years) Non-smokers 117 (85.4%) 35 (67.3%)** 1.00 (reference) 1.00 (reference) 1-20 9 (6.6%) 5 (9.6%) 2.04 (0.80 – 5.20) 0.14 2.15 (0.81 – 5.67) 0.13 > 20 11 (8.0%) 12 (23.1%) 2.86 (1.48 – 5.54) 3.19 (1.55 – 6.56) 0.002 0.002 During trastuzumab (n=96) (n=35) treatment No 86 (89.6%) 28 (80.0%) 1.00 (reference) 1.00 (reference) Yes 10 (10.4%) 7 (20.0%) 2.03 (0.88 – 4.68) 0.09 2.15 (0.92 – 5.04) 0.08 HR: hazard ratio; CI: confidence interval. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy; ** One patient was excluded from the multivariate analysis due to missing value for adjuvant endocrine therapy.
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Table 3. Unadjusted and adjusted hazard ratios for disease-free survival according to tobacco consumption before breast cancer diagnosis and during trastuzumab treatment, stratified by ER status. With recurrence
(n=50)
(n=29)
46 (92.0%) 4 (8.0%) (n=33)
20 (69.0%) 9 (31.0%) (n=16)
30 (91.0%) 3 (9.0%)
11 (68.8%) 5 (31.2%)
Univariate HR (95% CI)
p-value
Adjusted HR (95% CI)*
Adjusted p-value*
0.002
1.00 (reference) 3.73 (1.56 – 8.89)
0.003
0.01
1.00 (reference) 4.49 (1.26 – 16.00)
0.02
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Without recurrence
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1.00 (reference) 3.73 (1.64 – 8.48)
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Factor Smoking ER-negative patients: Before breast cancer diagnosis No Yes During trastuzumab treatment No Yes
1.00 (reference) 3.92 (1.29 – 11.85)
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ER-positive patients: Before breast cancer (n=120) (n=36) diagnosis No 104 (87.0%) 27 (75.0%) 1.00 (reference) 1.00 (reference) Yes 16 (13.0%) 9 (25.0%) 1.90 (0.89 – 4.04) 0.09 1.91 (0.84 – 4.31) 0.12 During trastuzumab (n=63) (n=19) treatment No 56 (89.0%) 17 (89.5%) 1.00 (reference) 1.00 (reference) Yes 7 (11.0%) 2 (10.5%) 0.96 (0.22 – 4.14) 0.95 1.33 (0.27 – 6.47) 0.72 HR: hazard ratio; CI: confidence interval. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy.
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Table 4. Unadjusted and adjusted hazard ratios for disease-free survival according to alcohol consumption before breast cancer diagnosis and during trastuzumab treatment. With recurrence
56 (37.3%) 94 (62.7%)
33 (54.1%)** 28 (45.9%)
(n=96) 62 (64.6%) 34 (35.4%) (n=96) 90 (93.7%) 6 (6.3%) (n=96) 92 (95.8%) 4 (4.2%) (n=95)
(n=35) 27 (77.2%) 8 (22.8%) (n=35) 32 (91.4%) 3 (8.6%) (n=34) 34 (100.0%) 0 (0.0%) (n=33)
64 (67.4%) 31 (32.6%)
23 (69.7%) 10 (30.3%)
(n=96) 72 (75.0%) 24 (25.0%) (n=96) 91 (94.8%) 5 (5.2%)
Univariate HR (95% CI)
Adjusted HR (95% CI)*
Adjusted p-value*
0.02
1.00 (reference) 0.56 (0.33 – 0.94)
0.03
1.00 (reference) 0.52 (0.24 – 1.15)
0.11
1.00 (reference) 0.42 (0.18 – 0.95)
0.04
1.00 (reference) 1.45 (0.44 – 4.75)
0.54
1.00 (reference) 1.60 (0.47 – 5.47)
0.46
1.00 (reference) N/A
N/A
1.00 (reference) N/A
N/A
1.00 (reference) 0.91 (0.43 – 1.91)
0.79
1.00 (reference) 0.68 (0.30 – 1.56)
0.36
1.00 (reference) 0.72 (0.31 – 1.65)
0.43
1.00 (reference) 0.55 (0.23 – 1.33)
0.18
1.00 (reference) 1.62 (0.49 – 5.32)
0.43
1.00 (reference) 1.98 (0.53 – 7.33)
0.31
(n=61)
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1.00 (reference) 0.55 (0.33 – 0.92)
(N=36) 28 (77.8%) 8 (22.2%) (n=34) 31 (91,2%) 3 (8.8%)
p-value
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Without recurrence (n=150)
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Factor Alcohol Before breast cancer diagnosis No Yes According to type of alcohol consumed Wine 0-2 drinks/week > 2 drinks/week Beer 0-2 drinks/week > 2 drinks/week Spirits 0-2 drinks/week > 2 drinks/week During trastuzumab treatment 0-2 drinks per week > 2 drinks per week According to type of alcohol consumed Wine 0-2 drinks/week > 2 drinks/week Beer 0-2 drinks/week > 2 drinks/week
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Spirits (n=95) (N=34) 0-2 drinks/week 93 (97.9%) 34 (100.0%) 1.00 (reference) 1.00 (reference) > 2 drinks/week 2 (2.1%) 0 (0.0%) N/A N/A N/A N/A HR: hazard ratio; CI: confidence interval; N/A: not applicable. * Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy; ** One patient was excluded from the multivariate analysis due to missing value for adjuvant endocrine therapy.
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Table 5. Unadjusted and adjusted hazard ratios for disease-free survival according to HER2 polymorphisms.
Ile655Val Ile/Ile Ile/Val or Val/Val
Without recurrence (n=53) 42 (79.3%) 11 (20.7%)
With recurrence (n=18) 13 (72.2%) 5 (27.6%)
Univariate HR (95% CI) 1.00 (reference) 2.00 (0.70 – 5.81)
p-value
Adjusted HR (95% CI)*
Adjusted p-value*
1.00 (reference) 4.96 (1.45 – 17.02)
0.01
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Polymorphism
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Ala1170Pro (N=51) (n=18) Ala/Ala 26 (51.0%) 10 (55.5%) 1.00 (reference) 1.00 (reference) Ala/Pro or Pro/Pro 25 (49.0%) 8 (44.5%) 0.84 (0.33 – 2.14) 0.71 0.70 (0.23 – 2.09) 0.77 HR: hazard ratio; CI: confidence interval. *Multivariable HRs and 95% CIs are adjusted for age at diagnosis, body mass index, stage, adjuvant endocrine therapy and radiotherapy.
22