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The dog as a naturally-occurring model for insulin-like growth factor type 1 receptor-overexpressing breast cancer: an observational cohort study

BMC Cancer volume 15, Article number: 664 (2015) Cite this article

Abstract

Background

Dogs spontaneously develop invasive mammary carcinoma with a high prevalence of the triple-negative (TN) subtype (lack of ER-Estrogen Receptor and PR-Progesterone Receptor expression, lack of HER2-Human Epidermal Growth Factor Receptor 2 overexpression), making this animal model relevant for investigating new therapeutic pathways. Insulin-like growth factor Type-1 receptor (IGF1R) is frequently overexpressed in primary human breast cancers, with a growing role in the TN phenotype. The purpose of this study was to investigate the Dog as a candidate model for IGF1R-overexpressing mammary carcinoma.

Methods

150 bitches with canine mammary carcinoma (CMC) and a known 2-year follow-up were retrospectively included. IGF1R expression was assessed by immunohistochemistry (IHC) using a similar scoring system as for HER2 in breast cancer. The prognostic value of the IGF1R expression was assessed in terms of overall and specific survival as well as disease-free interval (DFI).

Results

47 CMC (31 %) were classified as luminal and 103 (69 %) as triple-negative (TN-CMC). 41 % of CMC overexpressed IGF1R (IHC score 3+) of which 76 % were TN-CMC and 62 % grade III. IGF1R overexpression was associated with aggressive features including lymphovascular invasion, histological grade III, low ER expression and the TN phenotype. Univariate and multivariate analyses revealed that IGF1R overexpression was associated with shorter overall and specific survivals and shorter DFI in TN-CMC.

Conclusions

IGF1R overexpression is common and related to a poor outcome in canine invasive mammary carcinoma, particularly in the triple negative subtype, as in human breast cancer. Preclinical studies using the Dog as a spontaneous animal model could be considered to investigate new therapies targeting IGF1R in triple-negative breast cancer.

Peer Review reports

Background

The identification of relevant naturally-occurring animal models is of particular interest in oncology in order to accelerate the development of effective diagnostic and therapeutic innovations for human patients. The Dog is a really good candidate as its physiology [1] and genome [2] are very similar to that of humans. Dogs share the same environment as humans with highly comparable nutritional needs, and naturally develop various cancers with a shorter natural history [3]. This spontaneous animal model could be highly beneficial to translational breast cancer research as the human classification of breast cancers is relevant to canine mammary carcinomas [4, 5], even if some histological entities (particularly complex mammary carcinoma) are quite different between human and dog [6]. Interestingly, the triple negative (TN) immunophenotype, one of the most aggressive breast cancer subtypes defined by the lack of ER (Estrogen Receptor), PR (Progesterone Receptor) and HER2 (Epidermal Growth factor Receptor type 2) overexpression, is well recognized in dogs [7, 8].

In various human cancers including breast cancer, the Insulin-like Growth Factor (IGF) family is closely related to oncogenesis [9, 10], in situ tumor growth [11], invasion and metastasis [11], with IGF1R (Insulin-like Growth Factor Type 1-Receptor) acting as a real oncogene and being overexpressed in more than 50 % of primary breast cancers [12]. This is particularly true for the TN breast cancer cells (estrogen-unresponsive), in which IGF1R is largely expressed and IGF-1 stimulates proliferation and survival, making them responsive in vitro to anti-IGF1R therapies [13, 14]. An ongoing phase I clinical trial of the IGF1R inhibitor OSI-906 in humans affected by advanced solid tumors showed few adverse effects and no unexpected toxicities [15]. Even if a phase II clinical trial using ganitumab (an anti-IGF1R antibody) did not show any improvement for women with hormone-receptor positive and advanced breast cancer [16], a phase I trial using another anti-IGF1R antibody (cixutumumab) showed promising results by prolonging stable diseases [17]. IGF1R expression is highly related to prognosis in breast cancer, with a prognostic value dependent on the ER status of the tumors: in ER-positive breast cancer, IGF1R overexpression is related to favorable outcome [18] as opposed to ER-negative carcinomas, in which IGF1R overexression is associated with a poor outcome [19].

In canine mammary carcinoma, tissue GH (Growth Hormone) and IGF-1 have been positively correlated with tumor malignancy, as well as with tissue levels of progesterone and 17β-estradiol [20]. IGF1R expression has also been reported to be higher in histologic types of worse prognosis [21] although some studies did not show any significant association between IGF1R expression in mammary carcinomas and the clinical outcome in canine patients [22]. In addition, IGF-1 and IGF1R have been implicated in other canine cancers including osteosarcoma [23, 24], malignant melanoma [25] and testis tumors [26], suggesting a major role of the IGF system in canine oncology.

In this study, IGF1R expression was retrospectively investigated by immunohistochemistry (IHC) in a large cohort of canine invasive mammary carcinomas in order to determine the extent of similarities between canine and human mammary carcinomas, with respect to the role of IGF1R in tumor biology and natural history.

Methods

Patients and samples

Invasive mammary carcinomas surgically removed from 150 bitches, formalin-fixed and sent to two laboratories of veterinary histopathology (Laboratoire d’Histopathologie Animale, Oniris, Nantes, France and Laboratoire d’Anatomie Pathologique Vétérinaire d’Amboise, Amboise, France) between 2007 and 2010 were retrospectively selected. The owners’ written consent and approval from the Oniris College of Veterinary Medicine local Animal Welfare Committee were obtained prior to inclusion.

Dogs were eligible for inclusion when a diagnosis of invasive mammary ductal carcinoma was established by histological analysis and confirmed by an absent layer of p63-positive myoepithelial cells (anti-p63 antibody, clone ab111449, abcam plc) by immunohistochemistry (IHC) that differentiates invasive from in situ breast ductal carcinoma [27, 28]. All female dogs that had received any adjuvant chemotherapy and/or for which follow-up was not available for at least 2 years after mastectomy, were excluded from the study.

Breed, age and reproductive status (including age of neutering) at time of mastectomy, as well as the number and location of mammary carcinoma(s), were recorded for each bitch. Two-year follow-up was obtained through telephone interviews with referral veterinarians with particular emphasis on the occurrence of recurrence (i.e. the occurrence of an another mammary tumor on the same mammary gland) and/or of a new primary mammary tumor, and the animal’s outcome (alive or dead and cause of death, i.e., unrelated or related to the mammary carcinoma whether the animals died naturally or were euthanatized because of metastases). Overall Survival (OS) was defined as the time between surgery (mastectomy) and death from any cause; uncensored cases corresponded to dead animals; censored cases were still alive at least two years post-diagnosis. Specific Survival (SS) was defined as the time between surgery and death attributable to the mammary carcinoma; censored cases corresponded to dogs still alive, dogs that died from unknown cause, and dogs that died from another cause than the mammary carcinoma. The interval from surgery to the first local recurrence, new primary tumor, lymph node metastasis and/or distant metastasis was also assessed, and defined the disease-free interval (DFI).

Histopathology and immunohistochemistry (IHC)

All tumors were paraffin-embedded immediately after reception. 4 μm-thick serial sections were performed onto positively charged slides (Superfrost plus, Menzel-Glaser, Germany). After Hematoxylin and Eosin (HE) staining, mammary carcinomas were classified by five independent pathologists (one human breast pathologist and four veterinary pathologists) according to the WHO’s classification system of canine mammary tumors [28, 29, 30], and graded according to the criteria of Elston and Ellis [31] as well-differentiated (grade I), moderately differentiated (grade II) or poorly differentiated (grade III) carcinomas. The histologically assessed size of mammary carcinoma(s) with 2 cm chosen as a threshold according to the American Joint Committee on Cancer (AJCC), lymphovascular invasion, completeness of surgical excision, dermal infiltration, cutaneous ulceration, muscle invasion, squamous differentiation, inflammation and central necrosis were recorded for each case. In case of multifocal or multicentric carcinomas, the tumor with the highest pathologic size and/or highest histological grade was included in the study.

Automated IHC (Benchmark XT Ventana, Roche Diagnostics) was performed using antibodies against ERα (Estrogen Receptor alpha, clone C311, Santa Cruz), PR (Progesterone Receptor, clone 1E2, Ventana), HER2 (Human Epidermal Growth Factor Receptor 2 clone 4B5, Ventana), Ki-67 (clone MIB1, Dako), CK5/6 (Cytokeratin 5/6, clone D5/16B4, Dako), EGFR (Epidermal Growth Factor Receptor Type 1 clone 31G7, Invitrogen) and IGF1R (Insulin-like Growth Factor type 1-Receptor clone G11, Ventana). IHC protocols are detailed in Additional file 1: Table S1.

Scoring of the immunohistochemical staining was performed by the five independent pathologists. ER, PR and Ki-67 were assessed based on the number of positive nuclei among 500 counted cells (manual image analysis involving the use of the image J software, Research Service Branch, National Institute of Health, Bethesda, Maryland, USA). ER and PR were considered positive if nuclear staining was observed in more than 10 % of the cells [32] and Ki-67 in more than 20 % of the cells [33]. HER2 [32, 34] was scored as follow: 0 for no staining at all or incomplete, faint/barely perceptible membrane staining in less than 10 % of the cells; score 1+ for incomplete and faint/barely perceptible membrane staining in more than 10 % of the cells; 2+ for circumferential and incomplete and/or weak/moderate membrane staining in more than 10 % of the cells; and 3+ for circumferential and complete and intense membrane staining in more than 10 % of the cells. Carcinomas were considered positive for HER2 only for a 3+ IHC score [32]. IGF1R was scored in accordance with the HER2 expression scoring system [19, 35]: a negative result was defined as the complete absence of membrane staining (score 0) or the presence of weak membrane staining in less than 10 % of the cells or incomplete membrane staining in more than 10 % of the cells (score 1+) in any portion of the tumor; a score 2+ was applied for complete and weak to moderate membrane staining in more than 10 % of the cells; and a score 3+ for complete and intense membrane staining in more than 10 % of the tumor cells [34]. EGFR [36] was considered positive if membrane staining was observed in more than 10 % of the cells. Positivity to cytokeratins 5/6 (CK5/6) was defined with a threshold of 10 % [37].

Negative controls for IHC were included in each run, and consisted in replacing the primary antibody with normal mouse or rabbit serum (prediluted reagents, Roche Diagnostics). The positive controls were internal controls in most cases (i.e., skin epidermis and hair follicles for Ki-67, CK 5/6, EGFR and IGF1R; mammary gland surrounding the carcinoma for ER and PR), as stated in Table 1. For HER2 IHC, the pathway HER2 4-in-1 control slides (Roche Diagnostics) were chosen because they allow the quality of staining for each HER2 score (0, 1+, 2+, 3+) to be assessed.

Table 1 Characteristics of the dogs and their invasive mammary carcinomas
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Photographs of slides were taken using an Eclipse 50i microscope and a Nikon DS Fi-1 digital camera (Nikon Instruments Europe B.V.).

Statistical analysis

The Statview (Statview 5 SAS Institute Inc.) and R (R 3.1.1 GUI 1.65) softwares were used for statistical analyses. Results are given as median and range unless otherwise indicated. Non-parametric tests were used after checking for normality and independence of the data by Kolmogorov-Smirnov test and graphic assessment. The correlation between IGF1R expression and categorical variables (age groups, histological grade, clinical stage, nodal stage, hormone receptor status, and immunophenotype) was analyzed using the Pearson chi-square test or the Fisher exact test. Correlations between numeric variables were determined by Spearman’s test. The Kaplan-Meier non-parametric method was used for univariate survival analysis and the log-rank test was used to assess differences among groups. Cox proportional-hazard regression model was used to examine all factors found to be predictive of survival in univariate analysis simultaneously. A p-value of less than 0.05 was considered significant.

Results

Clinicopathological findings

The study population consisted in 117 intact and 33 spayed female dogs. Age at surgery ranged from 5.1 to 16.3 years (median 10.9 years). The 150 invasive carcinomas were classified as Luminal and Triple Negative according to ER, PR and HER2 expressions [4, 5]: 47 (31.3 %) were of Luminal subtype (ERα ≥ 10 % and/or PR ≥ 10 %), of which 17 were Luminal-A (Ki-67 < 20 %) and 30 were Luminal-B (Ki-67 ≥ 20 %), and 103 (68.7 %) were classified as Triple Negative (ERα < 10 %, PR < 10 %, HER2 score other than 3+), of which 70 were basal-like (Cytokeratin-CK 5/6 and/or Epidermal Growth Factor Receptor-EGFR positive), and 33 were non-basal-like (CK 5/6 and EGFR negative). No carcinoma was HER2 overexpressing, although immunohistochemical scores 3+ were obtained with the positive controls (human breast cancer lines, control slides provided by Roche Diagnostics). The main clinicopathological findings are summarized in Table 1.

The median follow-up period was 36.3 months. In total, 130 dogs (86.7 %) died. The median time between the date of diagnosis and the date of death was 8.4 months [2 days–60.3 months]. The median DFI was 22.5 months with a 2-year recurrence and/or metastasis rate of 42 %. The median SS was 28.1 months with a 2-year cancer-related mortality rate of 39.3 %. The median OS was 11.0 months with a 2-year mortality rate of 68.7 %.

IGF1R expression

The IGF1R staining was exclusively observed in the plasma membrane with cytoplasmic blush only observed when IGF1R was strongly expressed. Only membrane immunoreactivity was taken into account for scoring IGF1R expression. IGF1R was strongly expressed in epithelial cells of the hair follicles, hyperplastic and dysplastic mammary tissues adjacent to the tumors (Fig. 1). The number of cases with IGF1R score 0-1+ was 34 (22.7 %, of which 11 (7.3 %) score 0 and 23 (15.4 %) score 1+), 54 cases (36.0 %) were IGF1R score 2+ and 62 (41.3 %) were IGF1R score 3+ (Fig. 1). Considering the luminal and triple negative immunophenotypes separately, the IGF1R 0–1+, 2+ and 3+ scores occurred in 17 (36.2 %), 14 (29.8 %) and 16 (34.0 %) luminal canine mammary carcinomas and in 17 (16.5 %), 40 (38.8 %) and 46 (44.7 %) triple-negative canine mammary carcinomas respectively.

Fig. 1
figure 1

Immunohistochemical staining of IGF1R expression in normal and neoplastic canine mammary glands. IGF1R (Insulin-like growth factor type 1 receptor) expression was scored according to the intensity of the membrane staining in accordance with the HER-2 scoring system. a Hair follicle positive for IGF1R expression, b Normal mammary gland with a score 2+ for IGF1R, c Invasive ductal mammary carcinoma with a score 0 for IGF1R, d Invasive ductal mammary carcinoma with a score 1+ for IGF1R, (E) Invasive ductal mammary carcinoma with a score 2+ for IGF1R, f Invasive ductal mammary carcinoma with a score 3+ for IGF1R (Immunohistochemical staining, original magnification × 400). Bar = 50 micrometers

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Association of IGF1R expression and clinicopathological features

IGF1R overexpression (IHC score 3+) was significantly associated with aggressive features including lymphovascular invasion, histological grade III, absent or low ER and PR expression, and the TN immunophenotype (Table 2). In the Luminal subtype, IGF1R overexpression was also significantly correlated with aggressive features including high histological grade (OR = 7.78 [1.71–45.30], p = 0.01) and lymphovascular invasion (OR = 5.42 [1.27–27.20], p = 0.03), except for dermal infiltration for which IGF1R score 2+ (OR = 0.07 [0.03–0.46], p = 0.02) and 3+ (OR = 0.13 [0.02–0.64], p = 0.02) were associated with an absence of dermal infiltration (Additional file 2: Table S2). In the TN subtype, IGF1R overexpression was only significantly related to a high histological grade (OR = 5.54 [1.67–22.25], p = 0.02).

Table 2 Significant associations between IGF1R expression and clinicopathological features of the 150 canine mammary carcinomas
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Prognostic value of IGF1R expression

By univariate analysis, IGF1R overexpression was associated with a poor outcome in terms of disease-free interval (p = 0.04), overall (p < 0.001) and specific (p = 0.001) survival (Fig. 2). Univariate analyses revealed that other factors were associated with a poor prognosis (DFI, OS and SS), including multifocality of the mammary carcinoma, nodal stage at diagnosis, histological grade, surgical margin status, lymphovascular invasion, ER expression and immunophenotype (Tables 3, 4 and 5). Multivariate analysis using Cox proportional-hazard regression was then carried out. When several significant prognostic factors were overlapping (for example nodal stage at mastectomy and lymphovascular invasion or immunophenotype and ER/PR expression), only one was selected as a covariate in the model.

Fig. 2
figure 2

Kaplan-Meier analysis of OS, SS and DFI in 150 canine invasive mammary carcinomas according to IGF1R expression. IGF1R: Insulin-like growth factor type 1 receptor

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Table 3 Factors associated with overall survival (OS) in canine invasive mammary carcinomas (n = 150)
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Table 4 Factors associated with specific survival (SS) in canine invasive mammary carcinomas (n = 150)
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Table 5 Factors associated with disease-free interval (DFI) in canine invasive mammary carcinomas (n = 150)
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For overall survival, IGF1R overexpression appeared to be a strong and independent prognostic factor associated with a poor outcome, as well as an age of more than 11 years, lymphovascular invasion, positive margin status of the surgical sample and the presence of a peritumoral inflammation (Table 3). With regard to specific survival, IGF1R overexpression, lymphovascular invasion, and the presence of central necrosis showed a significant independent prognostic value (Table 4). By multivariate analysis for disease-free interval, IGF1R overexpression was no longer significantly associated with an earlier recurrence, new primary tumor and/or lymph node and distant metastasis (p = 0.13) (Table 5).

The prognostic impact of IGF1R was also assessed separately in the luminal and the TN immunophenotypes.

In the luminal subtype (n = 47), IGF1R overexpression was associated with a shorter OS (HR = 3.13 [1.41–6.96]; p = 0.005) and SS (HR = 4.72 [1.42–15.77]; p = 0.01) by univariate analysis (Additional file 3: Tables S3 and Additional file 4: Table S4). By multivariate analysis, IGF1R overexpression was also a significant strong and independent prognostic factor associated with a poor outcome in terms of OS and SS, as well as an age of more than 11 years.

In the TN subtype (n = 103), IGF1R overexpression was also associated with a shorter OS (HR = 2.24 [1.23–4.10]; p = 0.009) and SS (HR = 2.49 [1.07–5.81]; p = 0.03) by univariate analysis. IGF1R expression retained a significant and independent prognostic value for OS by multivariate analysis, as well as the age of the dog at neutering, occurrence of a new primary mammary tumor, histological grade, surgical margin status and presence of central necrosis (Additional file 5: Table S5). Finally, IGF1R was also a significant and independent prognostic factor for SS in the TN immunophenotype, with lymphovascular invasion and central necrosis (HR = 0.47 [0.24–0.93]; p = 0.03) as covariates (Additional file 6: Table S6).

IGF1R expression did not show any prognostic value in terms of DFI either in the luminal or TN subgroup.

Discussion

The objective of this study was to investigate IGF1R expression in a large cohort of canine invasive carcinomas, focusing on its relationship with the clinicopathological features and prognosis, in terms of overall, specific and disease-free survivals, in order to evaluate the similarities between the role of IGF1R in the canine species and those previously reported in human breast cancer. We found that IGF1R was frequently expressed in canine invasive mammary carcinoma, as more than 90 % showed at least a weak membrane staining for IGF1R. This result is in accordance with the previous human studies, as usually more than 80 % of the invasive breast cancer cells are positive for IGF1R [18, 35, 38]. In human breast cancer, few studies take into account both membrane and cytoplasmic IGF1R expression [18, 39, 40]. We only considered membrane staining for scoring IGF1R expression, as cytoplasmic blush was only observed when IGF1R was strongly expressed. Methods used for IGF1R scoring depend on the study, but most of the published results consider that a score of 3+ by immunohistochemistry (mostly defined as complete and intense membrane staining in more than 10 % of the cells, as for HER2 scoring) defined IGF1R overexpression [19, 35]. Thus, we chose to score IGF1R in accordance with the scoring of HER2 in breast cancer and then grouped the negative scores (complete absence of membrane staining or the presence of weak membrane staining in less than 10 % of the cells) and 1+ (incomplete membrane staining in more than 10 % of the cells), compared with the positive scores 2+ (complete and weak to moderate membrane staining in more than 10 % of the cells) and 3+ (complete and intense membrane staining in more than 10 % of the cells) as Shin et al. previously did in human breast cancer [19]. However, the grouping of the score 0 and 1+ is questionable, as the normal canine mammary gland [22] (Fig. 1), like the human breast [41, 42], naturally shows a weak (1+) to moderate (2+) IGF1R expression, implying that the absence of expression is abnormal and not necessarily a good prognostic factor. Indeed, some studies showed that IGF1R negativity and down-regulation was associated with a worse prognosis [43] in tamoxifen-treated postmenopausal breast cancer and correlated with aggressive features such as poor differentiation and high proliferation [44]. The number of cases in the present study with a score 0 for IGF1R expression was too small (n = 11) to analyze this group separately, implying that this is a rare condition that requires more cases for definitive conclusions.

When luminal and triple-negative subtypes were assessed separately, IGF1R overexpression (score 3+) was comparable in frequency to that reported in human breast cancer in which more than 45 % of the triple-negative breast carcinomas show strong expression of IGF1R [18, 19, 40, 41]. In human breast cancer and canine mammary carcinoma, several studies have shown that IGF1R expression parallels ER expression [18, 20, 39, 41], but we found that IGF1R overexpression was correlated with the negativity for ER and PR in the total cohort as Law et al. showed for phosphorylated IGF1R/IR expression in human breast cancer [45]. This contradictory result could be due to a biological difference concerning IGF1R and ER between dogs and humans. The fact that IGF1R parallels ER expression in canine mammary carcinoma in the study of Queiroga et al. [20] is also controversial: the cohort was small (40 mammary carcinomas) and unlike the present study, the invasive nature of the mammary carcinomas was not assessed. In our luminal subgroup, no correlation was found between hormonal receptor (ER and PR) and IGF1R expression. Nevertheless, this result has to be confirmed on a larger cohort of luminal canine mammary carcinomas.

IGF1R expression was also correlated with other aggressive features in both luminal and TN subtypes (such as high histological grades or presence of lymphovascular invasion). These results are in accordance with previously published studies in canine mammary carcinoma, as IGF-1 and IGF1R expression were respectively related to tumor malignancy [20] and histological types with worse prognosis [21]. This finding is in line with the fact that IGF1R is considered as a real oncogene closely involved in survival, proliferation, tumor growth, invasion and metastasis as it was demonstrated in canine osteosarcoma-derived cell lines [23]. In human breast cancer, results are controversial and generally depend on the ER status of the carcinomas. Indeed, extensive crosstalk between ER and IGF1R is now well-established from several in vitro studies, which demonstrate a synergistic effect of IGF1R and ER on the proliferation of human breast cancer cells [46, 47]. Even if some studies did not find any significant results [35, 48, 49], IGF1R positivity was generally related to favorable prognostic features in ER-positive breast cancer, including low histological grade [19]. On the contrary, strong IGF1R expression was associated with aggressive features in triple negative breast cancer, such as high histological grade [40]. However, no study to date has investigated the crosstalk between IGF1R and ER in canine mammary cell lines. A difference of receptor biology between Human and Dog cannot be excluded and should thus be further investigated.

Some studies show that the complete negativity or low expression of IGF1R is related to a worse prognosis [43, 44]. On the contrary, rare studies reveal that high IGF1R mRNA [50] and phosphorylated IGF1R/IR [45] are associated with a poor prognosis, whatever the molecular subtype of breast cancer. In addition, even if human studies show contradictory results, it seems that the IGF1R prognostic value also depends on the tumor ER status: in ER-positive mammary carcinomas, IGF1R overexpression is related to a favorable prognosis [18, 19] as opposed to the triple-negative subtype, in which IGF1R overexpression is associated with a poor outcome [18, 19, 40]. In the present study, no difference was found between the luminal and triple-negative subtypes of canine mammary carcinoma according to the prognostic value of IGF1R expression: IGF1R overexpression was associated with a poor prognosis in both luminal and triple-negative canine mammary carcinomas. The fact that none of the dogs of this study received adjuvant endocrine therapy is however a major difference between humans and dogs after a diagnosis of luminal mammary carcinoma, and this difference is likely to interfere with prognosis. Furthermore, only 47 luminal mammary carcinomas were included in this study and further investigations with a higher number of luminal mammary carcinomas are needed to confirm this result. Nonetheless, the expression and prognostic value of IGF1R overexpression is of particular interest in the triple negative subtype since it is associated with a poor prognosis, particularly in young women for which this type is more frequent [51]. Indeed, there is a lack of effective treatment for triple negative breast cancer and the search for relevant therapeutic targets is of major concern [52]. IGF1R could be a good candidate [53] with a translational approach based on clinical trials in dogs.

Conclusions

IGF1R overexpression is common in canine mammary carcinoma and related to a poor clinical outcome, particularly in the triple negative subtype. The Dog appears to be a relevant naturally-occurring model of IGF1R overexpressing triple-negative breast cancer, opening the way for possible translational perspectives in the search for new therapeutic opportunities, including anti-IGF1R therapies.

Abbreviations

CK5/6:

Cytokeratin 5/6

CMC:

Canine mammary carcinoma

DFI:

Disease-free interval

EGFR:

Epidermal growth factor receptor

ER:

Estrogen receptor

HE:

Hematoxylin and eosin

HER2:

Human epidermal growth factor receptor 2

IGF:

Insulin-like growth factor

IGF1R:

Insulin like growth factor type 1 receptor

IHC:

Immunohistochemistry

IR:

Insulin receptor

OS:

Overall survival

PR:

Progesterone receptor

SS:

Specific survival

TN:

Triple negative

References

  1. Ranieri G, Gadaleta CD, Patruno R, Zizzo N, Daidone MG, Hansson MG, et al. A model of study for human cancer: spontaneous occurring tumors in dogs. Biological features and translation for new anticancer therapies. Crit Rev Oncol Hematol. 2013;88:187–97.

    Article  CAS  PubMed  Google Scholar 

  2. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal M, et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature. 2005;438:803–19.

    Article  CAS  PubMed  Google Scholar 

  3. Pinho SS, Carvalho S, Cabral J, Reis CA, Gärtner F. Canine tumors: a spontaneous animal model of human carcinogenesis. Transl Res. 2012;159:165–72.

    Article  PubMed  Google Scholar 

  4. Gama A, Alves A, Schmitt F. Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: application of the human classification. Virchows Arch. 2008;453:123–32.

    Article  CAS  PubMed  Google Scholar 

  5. Sassi F, Benazzi C, Castellani G, Sarli G. Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry. BMC Vet Res. 2010;6:5.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Liu D, Xiong H, Ellis AE, Northrup NC, Rodriguez CO, O’Regan RM, et al. Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer. Cancer Res. 2014;74:5045–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Abadie J, Nguyen F, Loussouarn D, Bemelmans I, Catherine C, Albaric O, et al. Spontaneous canine mammary carcinoma as a model of human triple-negative breast cancer. J Comp Pathol. 2012;146:79.

    Article  Google Scholar 

  8. Kim NH, Lim HY, Im KS, Kim JH, Sur J-H. Identification of triple-negative and basal-like canine mammary carcinomas using four basal markers. J Comp Pathol. 2013;148:298–306.

    Article  CAS  PubMed  Google Scholar 

  9. Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev. 2000;21:215–44.

    Article  CAS  PubMed  Google Scholar 

  10. Kleinberg DL, Wood TL, Furth PA, Lee AV. Growth hormone and insulin-like growth factor-I in the transition from normal mammary development to preneoplastic mammary lesions. Endocr Rev. 2009;30:51–74.

    Article  CAS  PubMed  Google Scholar 

  11. Samani AA, Yakar S, LeRoith D, Brodt P. The role of the IGF system in cancer growth and metastasis: overview and recent insights. Endocr Rev. 2007;28:20–47.

    Article  CAS  PubMed  Google Scholar 

  12. Werner H, Bruchim I. The insulin-like growth factor-I receptor as an oncogene. Arch Physiol Biochem. 2009;115:58–71.

    Article  CAS  PubMed  Google Scholar 

  13. Davison Z, de Blacquière GE, Westley BR, May FEB. Insulin-like growth factor-dependent proliferation and survival of triple-negative breast cancer cells: implications for therapy. Neoplasia. 2011;13:504–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Litzenburger BC, Creighton CJ, Tsimelzon A, Chan BT, Hilsenbeck SG, Wang T, et al. High IGF-IR activity in triple-negative breast cancer cell lines and tumorgrafts correlates with sensitivity to anti-IGF-IR therapy. Clin Cancer Res. 2011;17:2314–27.

    Article  CAS  PubMed  Google Scholar 

  15. Jones RL, Kim ES, Nava-Parada P, Alam S, Johnson FM, Stephens AW, et al. Phase I study of intermittent oral dosing of the insulin-like growth factor-1 and insulin receptors inhibitor OSI-906 in patients with advanced solid tumors. Clin Cancer Res. 2014;21:693–700.

    Article  PubMed  Google Scholar 

  16. Robertson JF, Ferrero J-M, Bourgeois H, Kennecke H, de Boer RH, Jacot W, et al. Ganitumab with either exemestane or fulvestrant for postmenopausal women with advanced, hormone-receptor-positive breast cancer: a randomised, controlled, double-blind, phase 2 trial. Lancet Oncol. 2013;14:228–35.

    Article  CAS  PubMed  Google Scholar 

  17. Ma CX, Suman VJ, Goetz M, Haluska P, Moynihan T, Nanda R, et al. A phase I trial of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer. Breast Cancer Res Treat. 2013;139:145–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hartog H, Horlings HM, Van Der Vegt B, Kreike B, Ajouaou A, Van De Vijver MJ, et al. Divergent effects of insulin-like growth factor-1 receptor expression on prognosis of estrogen receptor positive versus triple negative invasive ductal breast carcinoma. Breast Cancer Res Treat. 2011;129:725–36.

    Article  CAS  PubMed  Google Scholar 

  19. Shin S-J, Gong G, Lee HJ, Kang J, Bae YK, Lee A, et al. Positive expression of insulin-like growth factor-1 receptor is associated with a positive hormone receptor status and a favorable prognosis in breast cancer. J Breast Cancer. 2014;17:113–20.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Queiroga FL, Pérez-Alenza MD, Silvan G, Peña L, Lopes CS, Illera JC. Crosstalk between GH/IGF-I axis and steroid hormones (progesterone, 17beta-estradiol) in canine mammary tumours. J Steroid Biochem Mol Biol. 2008;110:76–82.

    Article  CAS  PubMed  Google Scholar 

  21. Dolka I, Motyl T, Malicka E, Sapierzynski R, Fabisiak M. Relationship between receptors for insulin-like growth factor - I, steroid hormones and apoptosis-associated proteins in canine mammary tumors. Pol J Vet Sci. 2011;14:245–51.

    CAS  PubMed  Google Scholar 

  22. Klopfleisch R, Hvid H, Klose P, da Costa A, Gruber AD. Insulin receptor is expressed in normal canine mammary gland and benign adenomas but decreased in metastatic canine mammary carcinomas similar to human breast cancer. Vet Comp Oncol. 2010;8:293–301.

    Article  CAS  PubMed  Google Scholar 

  23. MacEwen EG, Pastor J, Kutzke J, Tsan R, Kurzman ID, Thamm DH, et al. IGF-1 receptor contributes to the malignant phenotype in human and canine osteosarcoma. J Cell Biochem. 2004;92:77–91.

    Article  CAS  PubMed  Google Scholar 

  24. Maniscalco L, Iussich S, Morello E, Martano M, Gattino F, Miretti S, et al. Increased expression of insulin-like growth factor-1 receptor is correlated with worse survival in canine appendicular osteosarcoma. Vet J. 2014.

  25. Thamm DH, Huelsmeyer MK, Mitzey AM, Qurollo B, Rose BJ, Kurzman ID. RT-PCR-based tyrosine kinase display profiling of canine melanoma: IGF-1 receptor as a potential therapeutic target. Melanoma Res. 2010;20:35–42.

    Article  CAS  PubMed  Google Scholar 

  26. Peters MAJ, Mol JA, van Wolferen ME, Oosterlaken-Dijksterhuis MA, Teerds KJ, van Sluijs FJ. Expression of the insulin-like growth factor (IGF) system and steroidogenic enzymes in canine testis tumors. Reprod Biol Endocrinol. 2003;1:22.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Shamloula MM, El-Shorbagy SH, Saied EME. P63 and cytokeratin8/18 expression in breast, atypical ductal hyperplasia, ductal carcinoma in situ and invasive duct carcinoma. J Egypt Natl Canc Inst. 2007;19:202–10.

    PubMed  Google Scholar 

  28. Moriya T, Kanomata N, Kozuka Y, Fukumoto M, Iwachido N, Hata S, et al. Usefulness of immunohistochemistry for differential diagnosis between benign and malignant breast lesions. Breast Cancer. 2009;16:173–8.

    Article  PubMed  Google Scholar 

  29. Misdrop W, Else RW, Hellmen E LT. Histological classification of mammary tumors of the dog and the cat. In World Health Organization International Histological Classification of Tumors of Domestic Animals. 2nd edition. Edited by Armed Forces Institute of Pathology. Washington DC; 1999:1–59

  30. Goldschmidt M, Peña L, Rasotto R, Zappulli V. Classification and grading of canine mammary tumors. Vet Pathol. 2011;48:117–31.

    Article  CAS  PubMed  Google Scholar 

  31. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19:403–10.

    Article  CAS  PubMed  Google Scholar 

  32. Peña L, Gama A, Goldschmidt MH, Abadie J, Benazzi C, Castagnaro M, et al. Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry. Vet Pathol. 2014;51:127–45.

    Article  PubMed  Google Scholar 

  33. Senkus E, Kyriakides S, Penault-Llorca F, Poortmans P, Thompson A, Zackrisson S, et al. Primary breast cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013;24 Suppl 6:vi7–23.

    Article  PubMed  Google Scholar 

  34. Wolff AC, Hammond MEH, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American society of clinical oncology/College of American Pathologists Clinical Practice Guideline Update. J Clin Oncol. 2013;31:3997–4013.

    Article  PubMed  Google Scholar 

  35. Shimizu C, Hasegawa T, Tani Y, Takahashi F, Takeuchi M, Watanabe T, et al. Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis. Hum Pathol. 2004;35:1537–42.

    Article  CAS  PubMed  Google Scholar 

  36. Gama A, Gärtner F, Alves A, Schmitt F. Immunohistochemical expression of Epidermal Growth Factor Receptor (EGFR) in canine mammary tissues. Res Vet Sci. 2009;87:432–7.

    Article  CAS  PubMed  Google Scholar 

  37. Rakha EA, El-Sayed ME, Green AR, Paish EC, Lee AHS, Ellis IO. Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression. Histopathology. 2007;50:434–8.

    Article  CAS  PubMed  Google Scholar 

  38. Papa V, Gliozzo B, Clark GM, McGuire WL, Moore D, Fujita-Yamaguchi Y, et al. Insulin-like growth factor-I receptors are overexpressed and predict a low risk in human breast cancer. Cancer Res. 1993;53:3736–40.

    CAS  PubMed  Google Scholar 

  39. Happerfield LC, Miles DW, Barnes DM, Thomsen LL, Smith P, Hanby A. The localization of the insulin-like growth factor receptor 1 (IGFR-1) in benign and malignant breast tissue. J Pathol. 1997;183:412–7.

    Article  CAS  PubMed  Google Scholar 

  40. Iqbal J, Thike AA, Cheok PY, Tse GM-K, Tan PH. Insulin growth factor receptor-1 expression and loss of PTEN protein predict early recurrence in triple-negative breast cancer. Histopathology. 2012;61:652–9.

    PubMed  Google Scholar 

  41. Bhargava R, Beriwal S, McManus K, Dabbs DJ. Insulin-like growth factor receptor-1 (IGF-1R) expression in normal breast, proliferative breast lesions, and breast carcinoma. Appl Immunohistochem Mol Morphol AIMM Off Publ Soc Appl Immunohistochem. 2011;19:218–25.

    CAS  Google Scholar 

  42. Tamimi RM, Colditz GA, Wang Y, Collins LC, Hu R, Rosner B, et al. Expression of IGF1R in normal breast tissue and subsequent risk of breast cancer. Breast Cancer Res Treat. 2011;128:243–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Aaltonen KE, Rosendahl AH, Olsson H, Malmström P, Hartman L, Fernö M. Association between insulin-like growth factor-1 receptor (IGF1R) negativity and poor prognosis in a cohort of women with primary breast cancer. BMC Cancer. 2014;14:794.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Schnarr B, Strunz K, Ohsam J, Benner A, Wacker J, Mayer D. Down-regulation of insulin-like growth factor-I receptor and insulin receptor substrate-1 expression in advanced human breast cancer. Int J Cancer. 2000;89:506–13.

    Article  CAS  PubMed  Google Scholar 

  45. Law JH, Habibi G, Hu K, Masoudi H, Wang MYC, Stratford AL, et al. Phosphorylated insulin-like growth factor-i/insulin receptor is present in all breast cancer subtypes and is related to poor survival. Cancer Res. 2008;68:10238–46.

    Article  CAS  PubMed  Google Scholar 

  46. Hamelers IHL, Steenbergh PH. Interactions between estrogen and insulin-like growth factor signaling pathways in human breast tumor cells. Endocr Relat Cancer. 2003;10:331–45.

    Article  CAS  PubMed  Google Scholar 

  47. Surmacz E, Bartucci M. Role of estrogen receptor alpha in modulating IGF-I receptor signaling and function in breast cancer. J Exp Clin Cancer Res. 2004;23:385–94.

    CAS  PubMed  Google Scholar 

  48. Chong KYM, Subramanian A, Mokbel K, Sharma AK. The prognostic significance of the insulin-like growth factor-1 ligand and receptor expression in breast cancer tissue. J Surg Oncol. 2011;104:228–35.

    Article  CAS  PubMed  Google Scholar 

  49. Maor S, Yosepovich A, Papa MZ, Yarden RI, Mayer D, Friedman E, et al. Elevated insulin-like growth factor-I receptor (IGF-IR) levels in primary breast tumors associated with BRCA1 mutations. Cancer Lett. 2007;257:236–43.

    Article  CAS  PubMed  Google Scholar 

  50. Peiró G, Adrover E, Sánchez-Tejada L, Lerma E, Planelles M, Sánchez-Payá J, et al. Increased insulin-like growth factor-1 receptor mRNA expression predicts poor survival in immunophenotypes of early breast carcinoma. Mod Pathol an Off J United States Can Acad Pathol Inc. 2011;24:201–8.

    Article  Google Scholar 

  51. Boyle P. Triple-negative breast cancer: epidemiological considerations and recommendations. Ann Oncol. 2012;23 Suppl 6:vi7–12.

    Article  PubMed  Google Scholar 

  52. Engebraaten O, Vollan HKM, Børresen-Dale A-L. Triple-negative breast cancer and the need for new therapeutic targets. Am J Pathol. 2013;183:1064–74.

    Article  CAS  PubMed  Google Scholar 

  53. Beckwith H, Yee D. Insulin-like growth factors, insulin, and growth hormone signaling in breast cancer: implications for targeted therapy. Endocr Pract. 2014;1–18.

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Acknowledgements

The authors thank Dr Claire Hanzenne, Dr Ingrid Bemelmans, Dr Catherine Ibisch, Dr Floriane Morio, and Dr Clotilde de Brito, who helped in the collection of clinical and follow-up data of the dogs. We are deeply indebted to Pr Laura Pena (Veterinary School, University of Madrid, Spain) and Pr Adelina Gama (University of Vila Real, Portugal) for their expertise in canine mammary carcinomas and their involvement in the classification, grading, and determination of immunophenotypes. The authors also thank the veterinary pathologists (Dr Jean-Loïc Le Net, Dr Virginie Théau, Dr Pierre Lagourette, Dr Olivier Albaric and Dr Sophie Labrut) who performed the initial diagnoses, as well as the technicians in histopathology (Mr. Bernard Fernandez, Mrs Florence Lezin, and Catherine Guéreaud). Finally, we thank the referring veterinarians and the owners of the dogs included in this study, who gave us the clinical and follow-up data.

Financial support

This work was supported by the French National Cancer Institute (INCa, Institut National du Cancer) with a grant for PhD students on translational research (Grant N°201108; 2011). This work was partly financially sustained by Roche diagnostics for the immunophenotype of the carcinomas.

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Authors and Affiliations

  1. Oniris, Université Nantes-Angers-Le Mans, Department of Human Health, Biomedical Research and Animal Models, AMaROC Unit and LDHvet laboratory, Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering, Site de la Chantrerie, Route de Gachet, Nantes, F-44307, France

    Laetitia Jaillardon, Jérome Abadie, Tiffanie Godard, Brigitte Siliart & Frédérique Nguyen

  2. Institut de Cancérologie de l’Ouest, Boulevard Jacques Monod Saint Herblain-Nantes cedex, Centre de Recherche du Cancer Nantes-Angers, UMR-INSERM U892/CNRS 6299, Nantes, F-44805, France

    Mario Campone

  3. Hopital G&R Laënnec, Boulevard Jacques Monod, Saint Herblain-Nantes cedex, Nantes, F-44093, France

    Delphine Loussouarn

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  1. Laetitia Jaillardon
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  3. Tiffanie Godard
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  7. Frédérique Nguyen
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Correspondence to Laetitia Jaillardon.

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Competing interests

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

Authors’ contributions

LJ carried out the design of the study, the analysis and interpretation of the data, participated in the immunophenotype of the mammary carcinomas, drafted the work and wrote the manuscript. JA carried out the histological analysis, the immunophenotype of the mammary carcinomas and revised the manuscript. TG contributed to the acquisition of the data, participated in the follow-up of the dogs and contributed to the survival study. DL participated in the histological and immunophenotype analysis of the mammary carcinomas in relation to breast cancer classification. MC and BS participated in the design of the study, drafted and revised the manuscript. FN carried out the histological analysis and complete immunophenotype of the mammary carcinomas, participated in the design of the study, contributed to the analysis and interpretation of the data and help to draft the work. All authors read and approved the final manuscript.

Additional files

Additional file 1: Table S1.

Primary antibodies and immunohistochemical protocols (Benchmark XT Ventana, Roche Diagnostics). All dilutions were performed using a commercially available diluent (Ventana Medical Systems). aUltraview and bOptiview Universal DAB detection kit: multimer-technology based detection system. cIView Universal DAB detection kit: biotin streptavidin system. ERα: Estrogen Receptor alpha, PR: Progesterone Receptor, HER2: Epidermal Growth Factor type 2 Receptor, CK5/6: Cytokeratin 5/6, EGFR: Epidermal Growth Factor type 1 Receptor, IGF1R: Insulin-like growth factor type 1 receptor. CC1: Cell Conditioning 1. (DOC 33 kb)

Additional file 2: Table S2.

Significant associations between IGF1R expression and clinicopathological features of 47 luminal canine mammary carcinomas. IGF1R score 0–1+ is considered as the reference for each parameter. IGF1R Insulin-like Growth Factor type 1 Receptor. LVI: Lymphovascular Invasion. OR: Odd Ratio. 95 % CI: 95 % Confidence Interval. (DOC 30 kb)

Additional file 3: Table S3.

Factors associated with overall survival (OS) in 47 Luminal canine invasive mammary carcinomas. Univariate (log rank test) and multivariate survival analyses (Cox proportional hazard regression). HR: Hazard Ratio, 95 % CI: 95 % Confidence Interval, HER2: Epidermal Growth Factor type 2 Receptor, CK5/6: Cytokeratin 5/6, EGFR: Epidermal Growth Factor type 1 Receptor, IGF1R: Insulin-like Growth Factor type 1 Receptor. (DOC 35 kb)

Additional file 4: Table S4.

Factors associated with specific survival (SS) in 47 Luminal canine invasive mammary carcinomas. Univariate (log rank test) and multivariate survival analyses (Cox proportional hazard regression). HR: Hazard Ratio, 95 % CI: 95 % Confidence Interval, HER2: Epidermal Growth Factor type 2 Receptor, IGF1R: Insulin-like Growth Factor type 1 Receptor, LVI: Lymphovascular Invasion. (DOC 33 kb)

Additional file 5: Table S5.

Factors associated with overall survival (OS) in 103 Triple-negative canine invasive mammary carcinomas. Univariate (log rank test) and multivariate survival analyses (Cox proportional hazard regression). HR: Hazard Ratio, 95 % CI: 95 % Confidence Interval, IGF1R: Insulin-like Growth Factor type 1 Receptor, LVI: Lymphovascular Invasion. When several significant prognostic factors overlapped, only one was selected for the multivariate analysis (LVI was chosen between lymph node status and LVI because it could have been determined in all cases). (DOC 38 kb)

Additional file 6: Table S6.

Factors associated with specific survival (SS) in 103 Triple-negative canine invasive mammary carcinomas. Univariate (log rank test) and multivariate survival analyses (Cox proportional hazard regression). HR: Hazard Ratio, 95 % CI: 95 % Confidence Interval, IGF1R: Insulin-like Growth Factor type 1 Receptor, LVI: Lymphovascular Invasion. When several significant prognostic factors overlapped, only one was selected for the multivariate analysis (LVI was chosen between lymph node status and LVI because it could have been determined in all cases). (DOC 35 kb)

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Jaillardon, L., Abadie, J., Godard, T. et al. The dog as a naturally-occurring model for insulin-like growth factor type 1 receptor-overexpressing breast cancer: an observational cohort study. BMC Cancer 15, 664 (2015). https://doi.org/10.1186/s12885-015-1670-6

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Keywords

  • Spontaneous animal model
  • Canine mammary carcinoma
  • IGF1R
  • Triple-negative
  • Comparative oncology

BMC Cancer

ISSN: 1471-2407

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