The aim of this study was to evaluate the association between insulin-like growth factor 1 receptor (IGF-1R) expression in breast cancer tissue and mammographic density and the clinical significance of IGF-1R overexpression. A total of 167 patients with primary invasive breast cancer were analyzed. Mammographic breast density and IGF-1R overexpression were correlated with clinicopathological parameters and analyzed by overall survival (OS) and disease-free survival (DFS). Increased breast tissue density was significantly associated with age, body mass index, menopausal status, histological grade and IGF-1R overexpression in the univariate analysis and with age (P=0.001), histological grade (P=0.045) and IGF-1R overexpression (P=0.021) in the multivariate analysis. IGF-1R overexpression was significantly associated with dense breast tissue in patients aged >40 years (P=0.002). IGF-1R overexpression in breast cancer in premenopausal women was associated with human epidermal growth factor receptor 2 (HER-2) positivity (P=0.016) and worse DFS (P=0.0414). There was no significant difference in OS and DFS between dense and non-dense breast tissue. IGF-1R expression in breast cancer tissue was significantly associated with mammographic breast tissue density in patients aged >40 years. It appears that IGF-1R expression in breast cancer tissue plays an important role in breast cancer in patients with dense breast tissue. In premenopausal women, IGF-1R overexpression in breast cancer tissue was significantly associated with HER-2 positivity and poor DFS.
Due to the growing public interest in breast cancer and the widespread use of mammographic screening, the early detection rate of breast cancer has increased. Increased breast tissue density, however, may make mammograms more difficult to interpret. Dense breast tissue is considered one of the major risk factors of breast cancer, although the underlying mechanism has yet to be elucidated (
As a factor associated with mammographic breast density, the insulin-like growth factor (IGF) system has been well documented and reported to be of more relevance to premenopausal women (
In terms of its structure, IGF-1R is similar to insulin receptors and consists of an α chain that has two extracellular binding domains and two transmembrane β chains, forming a heterodimer. IGF-1R is a transmembrane tyrosine kinase receptor and its expression is known to be involved in mitosis, advancement and metastasis of breast cancer (
The present study aimed to investigate the correlations between IGF-1R expression in breast cancer tissues, mammographic density and other clinicopathological factors and determine the clinical significance of IGF-1R overexpression.
A total of 167 breast cancer patients (median age 47years; range, 20–81 years) who were operated at the Department of Surgery of Severance Hospital between January, 2000 and December, 2001 were analyzed. Patient information, including age, height, weight, menopausal status, medication history, survival and recurrence status and clinicopathological data, including tumour size, nuclear grade, histological grade and lymph node metastasis, were obtained from electronic medical records. The mean follow-up was 91 months (range, 5–115 months). Mammographic breast density and IGF-1R overexpression were correlated with clinicopathological parameters and analyzed by overall survival (OS) and disease-free survival (DFS). This study conformed to the guidelines of the local Ethics Committee.
Serial 4-µm sections of the tissue microarray block, containing breast cancer tissue with a diameter of 3 mm, were mounted on electrostatic slides, heat-dried at 56°C for 30 min, deparaffinized in xylene and rehydrated through graded concentrations of ethanol. The slides were incubated in a solution of 3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. The slides were incubated in 0.3% bovine serum albumin/1X Tris-buffered saline (TBS) for 20 min to reduce non-specific background staining. A primary antibody was applied for 30 min at room temperature. After a series of TBS rinses, the bound antibody was detected using a polymer secondary antibody from the Dako EnVision+ system (Dako, Carpinteria, CA, USA). The slides were rinsed with a TBS series and visualized after a 10-min incubation of liquid 3,3′-diaminobenzidine (DAB) in buffered substrate (Dako) for 10 min. The slides were counterstained with hematoxylin. IHC analysis for rabbit anti-ER antibody (RM-9101; SP1, 1:100; Thermo Scientific, San Diego, CA, USA), mouse anti-progesterone receptor (PR) antibody (M3569; PgR, 1:50; DakoCytomation, Glostrup, Denmark), rabbit human epidermal growth factor receptor 2 (HER-2) antibody (A0485; polyclonal, 1:1,500; DakoCytomation), mouse epidermal growth factor receptor (EGFR) antibody (NCL-EGFR-384; EGFR 25, 1:50; Novocastra, Newcastle, UK), mouse cytokeratins (CK) 5/6 antibody (M7237; D5/16B4, 1:100; DakoCytomation), mouse Ki-67 antibody (M7240; MIB-1, 1:10; DakoCytomation). For IHC staining of rabbit IGF-1R antibody (3027), tissue sections were cut and placed on Superfrost Plus microscope slides (Fisher Scientific, San Diego, CA, USA). Using the Benchmark XT automated IHC stainer (IRβ, 1:10; Cell Signaling Technology, Inc., Danvers, MA, USA), the slides were stained. The sections were deparaffinized using EZ Prep solution. CC1 standard (a pH 8.4 buffer containing Tris/Borate/EDTA) was used for antigen retrieval. DAB inhibitor (3% H2O2; endogenous peroxidase) was blocked for 4 min at 37°C temperature. The slides were incubated with antibodies for 40 min at 37°C and a secondary antibody of Univeral HRP Multimer for 8 min at 37°C. The slides were then treated with DAB+H2O2 substrate for 8 min, followed by hematoxylin and bluing reagent counterstain at 37°C. Reaction buffer (pH 7.6 Tris buffer) was used as a washing solution. Detection was performed using the Ventana Ultraview DAB kit (Ventana Medical Systems, Inc., Tucson, AZ, USA).
All IHC markers were evaluated by light microscopy. The immunostained slides were scored according to the percentage of tumour cells exhibiting nuclear (ER, PR), cytoplasmic (CK 5/6) and membranous (HER-2, EGFR) staining. A cut-off value of ≥1% positively stained nuclei was used to define ER and PR positivity. HER-2 staining was analyzed as follows: 0, no immunostaining; 1+, weak incomplete membranous staining, <10% of the tumour cells; 2+, complete membranous staining, either uniform or weak in ≥10% of the tumour cells; and 3+, uniform intense membranous staining in ≥30% of the tumour cells. HER-2 immunostaining was considered to be positive when strong (3+) membranous staining was observed, whereas cases scored as 0 or 1+ were considered to be negative. The cases exhibiting 2+ HER-2 expression were evaluated for HER-2 amplification by fluorescent
Breast cancer subtypes were classified according to the IHC and FISH results for ER, PR and HER-2 as follows: Luminal A type, ER- or/and PR-positive and HER-2-negative; luminal B type, ER- or/and PR-positive and HER-2 overexpressed or/and amplified; HER-2 type, ER- and PR-negative and HER-2 overexpressed or/and amplified; triple-negative breast cancer type, ER-, PR-and HER-2 negative.
The mammographic images taken at the time of operation or within 1 year after the operation were evaluated. All the mammograms were performed in the same clinic with the Lorad M-III unit (Lorad Medical Systems, Danbury, CT, USA) and were reviewed by one radiologist and one breast surgeon with experience in the assessment of breast density by visual grading system. On the basis of American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) breast composition, four density patterns were designated as grades 1–4, with dense breast tissue graded as 3 and 4 (
Data were processed using SPSS software for Windows, version 12.0 (SPSS Inc., Chicago, IL, USA). The Chi-square test for univariate analysis and the logistic regression analysis for multivariate analysis were used to assess the correlation among IGF-1R expression, mammographic breast density, clinicopathological variables and breast cancer-related biomarkers. Cohen's Kappa test was used to assess intra- and inter-observer variability in the evaluation of mammographic breast density. Kaplan-Meier survival curves were employed to evaluate OS and DFS. The Chi-square test was used for univariate analysis and multivariate regression analysis was performed using the Cox proportional hazards model, with variables including mammographic breast density, expression of IGF-1R, nuclear grade, histological grade, tumour size, lymph node metastasis, ER, PR and HER-2 status, EGFR, CK 5/6 and Ki-67 for survival analysis. P<0.05 was considered to indicate a statistically significant difference.
The clinicopathological characteristics of the patients and the correlation among IGF-1R expression, mammographic breast density, clinicopathological variables and breast cancer-related biomarkers are shown in
The density patterns on mammography were non-dense breast tissue (grades 1 and 2) in 97 patients (58.1%) and dense breast tissue in 70 patients (41.9%). The frequency of dense breast tissue according to age was 100% (20–29 years), 70% (30–39 years), 40.3% (40–49 years), 43.8% (50–59 years), 10.6% (60–69 years) and 0% (70–89 years) (data not shown). Dense breast tissue was significantly associated with age, body mass index (BMI), menopausal status, histological grade and IGF-1R overexpression in the univariate analysis and with age (P=0.001), histological grade (P=0.045) and IGF-1R overexpression (P=0.021) in the multivariate analysis (
IGF-1R expression was scored as 0 in 34 patients (20.4%), 1 in 70 patients (41.9%), 2 in 60 patients (35.9%) and 3 in 3 patients (1.8%). The frequency of IGF-1R overexpression according to age was 0% (20–29 years), 34.5% (30–39 years), 45.2% (40–49 years), 37.8% (50–59 years), 31.6% (60–69 years), 16.7% (70–79 years) and 0% (80–89 years) (data not shown). IGF-1R overexpression was significantly associated with dense breast tissue at ages >40 years (P=0.002). Overexpression of IGF-1R was identified in 63 patients (37.7%), but exhibited no correlation with any clinicopathological parameters, such as age, BMI, primary tumour size, nuclear grade, histological grade, lymph node metastasis, ER, PR, HER-2, EGFR and Ki-67 (
ER expression (P=0.010, odds ratio=0.516) was associated with DFS and nuclear grade (P=0.029, odds ratio=0.068), histologic grade (P=0.019, odds ratio=16.318), lymph node metastasis (P=0.014, odds ratio=3.140) and ER expression (P=0.016, odds ratio=0.041) were associated with OS (
The earlier detection rate of breast cancer is continuously increasing, as a result of the growing public interest in breast cancer and the wider application of selective mammographic screening. However, increased breast density makes it more difficult to detect tumours in women exhibiting dense breast tissue on mammography; in addition, the presence of dense breast tissue
In this study, the BI-RADS classification, which is widely in use today, was adopted for analyzing mammographic density. Although this is a semi-quantitative method, rather than a quantitative measurement, similar to the computer-assisted breast density assessment, it has been found to be close to a quantitative method, exhibiting high consistency rates in terms of intra- and inter-observer variability. According to comparative studies on different mammographic readings, no significant difference was found between the BI-RADS-based group and the other group using computer-based calculation of absolute areas in terms of several clinicopathological factors (
The univariate analysis identified factors such as age, BMI, menopausal status and histological grade as relevant to dense breast tissue, whereas the multivariate analysis demonstrated the relevance of age and histological grade. In this study, the univariate analysis demonstrated that dense breast tissue was a more frequent finding in pre- rather than postmenopausal women, whereas the multivariate analysis found no such relevance, suggesting that age rather than menopausal status is more significant. In terms of BMI, the univariate analysis identified an inverse correlation between increased BMI and frequency of dense breast tissue, whereas the multivariate analysis demonstrated no significant correlation. Age was found to be inversely correlated with dense breast tissue. In an autopsy study by Li
IGF-1 in the blood is considered to be an important factor affecting breast tissue density. Anti-estrogens administered to patients with high IGF-1 in the blood reduced IGF-1 levels and breast tissue density, indicating that IGF-1 affects breast density (
IGF-1R is associated with several types of cancer, including breast and prostate cancer. The signaling stages of IGF-1R are crucial for the normal development of mammary tissues and play an important role in mitosis and anti-apoptosis. The self-phosphorylation of IGF-1R, the phosphorylation of insulin receptor substrates (IRS) 1–4 by tyrosine kinase and the activation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling system play important roles in the differentiation and survival of tumour cells (
As regards the expression of IGF-1R as a prognostic factor, Kim
The overexpression of IGF-1R in premenopausal women was found to be relevant to HER-2 positivity with low DFS, indicating that IGF-1R overexpression in premenopausal women may represent an unfavorable prognostic factor. In the subgroup analysis based on the overexpression of IGF-1R and the presence/absence of dense breast tissue, the concomitant occurrence of IGF-1R overexpression and dense breast tissue in premenopausal women was associated with a lower DFS (P=0.0154) and may be considered as an unfavorable prognostic factor compared to those with no such concomitant characteristics. Several recent studies reported IGF-1R overexpression to be an unfavorable prognostic factor, but the results were inconsistent, which warrants further investigation (
In conclusion, IGF-1R expression in breast cancer tissue was found to be significantly associated with mammographic breast density in patients aged >40 years. It Appears that IGF-1R expression in breast cancer plays an important role in cases with dense breast tissue. In premenopausal women, IGF-1R overexpression in breast cancer tissue was significantly associated with HER-2 positivity and poor DFS. However, IGF-1R overexpression in this study exhibited no correlation with other clinicopathological parameters. Dense breast tissue was found to be associated with age and histological grade. There was no difference in DFS and OS according to breast density.
Immunohistochemical findings of insulin-like growth factor 1 receptor (IGF-1R) expression. IGF-1R expression was scored according to area and intensity of membranous staining. (A) score 0; (B) score 1; (C) score 2; and (D) score 3. Magnification, x100.
Mammographic breast density. Mammographic breast density is an estimate of the extent of fibroglandular tissue in relation to fat. (A) 0–25%; (B) 26–50%; (C) 51–75%; (D) 76–100%.
(A) Overall survival (OS), (B) disease-free survival (DFS) in premenopausal women and (C) OS, (D) DFS in postmenopausal women according to the breast density.
(A) Overall survival (OS), (B) disease-free survival (DFS) in premenopausal women and (C) OS, (D) DFS in postmenopausal women according to insulin-like growth factor 1 receptor (IGF-1R) overexpression.
Correlation of insulin-like growth factor 1 receptor (IGF-1R) expression and mammographic breast density with clinicopathological variables in primary breast cancer.
IGF-1R expression | Dense breast tissue | ||||||
---|---|---|---|---|---|---|---|
Low | High | Negative | Positive | ||||
Variables | Total (%) | No. (%) | No. (%) | P-value | No. (%) | No. (%) | P-value |
Age, years | |||||||
Median, 47 (range, 20–81) | 167 | 104 (62.3) | 63 (37.7) | 0.207 | 97 (58.1) | 70 (41.9) | 0.001 |
Menopausal status | |||||||
Premenopausal | 98 (58.7) | 59 (56.7) | 39 (61.9) | 0.338 | 50 (51.5) | 48 (68.6) | 0.889 |
Postmenopausal | 69 (41.3) | 45 (43.3) | 24 (38.1) | 47 (48.5) | 22 (31.4) | ||
BMI | |||||||
<23 | 70 (41.9) | 44 (42.3) | 26 (41.3) | 0.312 | 33 (34.0) | 37 (52.9) | 0.093 |
≥23 | 97 (58.1) | 60 (57.7) | 37 (58.7) | 64 (66.0) | 33 (47.1) | ||
Nuclear grade | |||||||
1 | 10 (6.0) | 6 (5.8) | 4 (6.3) | 0.147 | 6 (6.2) | 4 (5.7) | 0.218 |
2 | 99 (59.3) | 65 (62.5) | 34 (54.0) | 63 (64.9) | 36 (51.4) | ||
3 | 58 (34.7) | 33 (31.7) | 25 (39.7) | 28 (28.9) | 30 (42.9) | ||
Histological grade | |||||||
1 | 28 (16.8) | 18 (17.3) | 10 (15.9) | 0.357 | 23 (23.8) | 5 (7.1) | 0.045 |
2 | 88 (52.7) | 53 (51.0) | 35 (55.5) | 50 (51.5) | 38 (54.3) | ||
3 | 51 (30.5) | 33 (31.7) | 18 (28.6) | 24 (24.7) | 27 (38.6) | ||
Tumor size | |||||||
T1 | 66 (39.5) | 37 (35.6) | 29 (46.0) | 0.145 | 43 (44.3) | 23 (32.8) | 0.828 |
T2 | 97 (58.1) | 65 (62.5) | 32 (50.8) | 52 (53.6) | 45 (64.3) | ||
T3 | 4 (2.4) | 2 (1.9) | 2 (3.2) | 2 (2.1) | 2 (2.9) | ||
Lymph node metastasis | |||||||
N0 | 92 (55.1) | 58 (55.7) | 34 (54.0) | 0.782 | 60 (61.9) | 32 (45.7) | 0.193 |
N1 | 44 (26.3) | 29 (27.9) | 15 (23.8) | 23 (23.7) | 21 (30.0) | ||
N2 | 20 (12.0) | 11 (10.6) | 9 (14.3) | 10 (10.3) | 10 (14.3) | ||
N3 | 11 (6.6) | 6 (5.8) | 5 (7.9) | 4 (4.1) | 7 (10.0) | ||
Subtype | |||||||
Luminal A | 86 (51.5) | 50 (48.1) | 36 (57.1) | 0.670 | 51 (52.6) | 35 (50.0) | 0.842 |
Luminal B | 21 (12.6) | 12 (11.5) | 9 (14.3) | 14 (14.4) | 7 (10.0) | ||
HER-2 | 24 (14.4) | 19 (18.3) | 5 (7.9 | 11 (11.3) | 13 (18.6) | ||
TNBC | 36 (21.5) | 23 (22.1) | 13 (20.7 | 21 (21.7) | 15 (21.4) | ||
IGF-1R expression | |||||||
Low (0,1) | 104 (62.3) | 104 (100.0) | 0 (0.0) | 68 (70.1) | 36 (51.4) | 0.021 | |
High (2,3) | 63 (37.7) | 0 (0.0) | 63 (100.0) | 29 (29.9) | 34 (48.6) | ||
Dense breast tissue | |||||||
Negative | 97 (58.1) | 68 (65.4) | 29 (46.0) | 0.021 | 97 (100.0) | 0 (0.0) | |
Positive | 70 (41.9) | 36 (34.6) | 34 (54.0) | 0 (0.0) | 70 (100.0) |
BMI, body mass index; HER-2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer.
Correlation of insulin-like growth factor 1 receptor (IGF-1R) expression and mammographic breast density with breast cancer-related biomarkers.
IGF-1R expression | Dense breast tissue | ||||||
---|---|---|---|---|---|---|---|
Low | High | Negative | Positive | ||||
Biomarkers | Total (%) (n=167) | No. (%) (n=104) | No. (%) (n=63) | P-value | No. (%) (n=97) | No. (%) (n=70) | P-value |
ER | |||||||
Negative | 63 (37.7) | 44 (42.3) | 19 (30.2) | 0.301 | 34 (35.1) | 29 (41.4) | 0.855 |
Positive | 104 (62.3) | 60 (57.7) | 44 (69.8) | 63 (64.9) | 41 (58.6) | ||
PR | |||||||
Negative | 95 (56.9) | 62 (59.6) | 33 (52.4) | 0.845 | 49 (50.5) | 46 (65.7) | 0.059 |
Positive | 72 (43.1) | 42 (40.4) | 30 (47.6) | 48 (49.5) | 24 (34.3) | ||
HER-2 | |||||||
Negative | 122 (73.1) | 73 (70.2) | 49 (77.8) | 0.215 | 72 (74.2) | 50 (71.4) | 0.405 |
Positive | 45 (26.9) | 31 (29.8) | 14 (22.2) | 25 (25.8) | 20 (28.6) | ||
EGFR | |||||||
Negative | 141 (84.4) | 86 (82.7) | 55 (87.3) | 0.980 | 83 (85.6) | 58 (82.9) | 0.958 |
Positive | 26 (15.6) | 18 (17.3) | 8 (12.7) | 14 (14.4) | 12 (17.1) | ||
CK 5/6 | |||||||
Negative | 156 (93.4) | 97 (93.3) | 59 (93.7) | 0.812 | 90 (92.8) | 66 (94.3) | 0.194 |
Positive | 11 (6.6) | 7 (6.7) | 4 (6.3) | 7 (7.2) | 4 (5.7) | ||
Ki-67 | |||||||
Low, <10% | 123 (73.7) | 72 (69.2) | 51 (81.0) | 0.160 | 73 (75.3) | 50 (71.4) | 0.947 |
High, ≥10% | 44 (26.3) | 32 (30.8) | 12 (19.0) | 24 (24.7) | 20 (28.6) |
ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; CK, cytokeratin.
Cox's proportional hazards regression models for overall survival (OS) and disease-free survival (DFS).
OS | DFS | ||||||
---|---|---|---|---|---|---|---|
Variables | No. | RR | 95% CI | P-value | RR | 95% CI | P-value |
Dense breast tissue | |||||||
Negative | 97 | 0.647 | 0.138–3.424 | 0.647 | 1.175 | 0.816–1.692 | 0.387 |
Positive | 70 | ||||||
Nuclear grade | |||||||
1 | 10 | 0.068 | 0.006–0.759 | 0.029 | 1.086 | 0.694–1.699 | 0.719 |
2 | 99 | ||||||
3 | 58 | ||||||
HistologicAL grade | |||||||
1 | 28 | 16.318 | 1.571–169.479 | 0.019 | 0.774 | 0.539–1.113 | 0.167 |
2 | 88 | ||||||
3 | 51 | ||||||
Tumor size | |||||||
T1 | 66 | 0.327 | 0.327–6.331 | 0.630 | 0.799 | 0.571–1.117 | 0.190 |
T2 | 97 | ||||||
T3 | 4 | ||||||
LN metastasis | |||||||
N0 | 92 | 3.140 | 1.255–7.857 | 0.014 | 1.218 | 0.986–1.505 | 0.068 |
N1 | 44 | ||||||
N2 | 20 | ||||||
N3 | 11 | ||||||
ER | |||||||
Negative | 63 | 0.041 | 0.003–0.549 | 0.016 | 0.516 | 0.312–0.854 | 0.010 |
Positive | 104 | ||||||
PR | |||||||
Negative | 95 | 6.908 | 0.552–86.498 | 0.134 | 0.820 | 0.526–1.277 | 0.379 |
Positive | 72 | ||||||
HER-2 | |||||||
Negative | 122 | 0.940 | 0.177–4.995 | 0.940 | 0.882 | 0.581–1.340 | 0.557 |
Positive | 45 | ||||||
EGFR | |||||||
Negative | 141 | 0.000 | 0.000 | 0.983 | 0.663 | 0.382–1.151 | 0.144 |
Positive | 26 | ||||||
CK 5/6 | |||||||
Negative | 156 | 0.001 | 0.000 | 0.993 | 1.476 | 0.672–3.242 | 0.333 |
Positive | 11 | ||||||
Ki-67, % | |||||||
Low (<10) | 123 | 7.922 | 0.460–136.514 | 0.154 | 1.345 | 0.891–2.030 | 0.158 |
High (≥10) | 44 | ||||||
IGF-1R | |||||||
Low (0,1) | 104 | 1.093 | 0.176–6.795 | 0.924 | 0.928 | 0.637–1.353 | 0.698 |
High (2,3) | 63 |
RR, relative risk; CI, confidence interval; LN, lymph node; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; CK, cytokeratin; IGF-1R, insulin-like growth factor 1 receptor.