The expression of insulin-like growth factor-1 receptor (IGF-1R), which is involved in the genesis and progression of breast cancer, is thought to be associated with the overall survival (OS) of patients. However, the predictive and prognostic significance of the IGF-1R expression in breast cancer remains controversial. The present study aimed to identify the factors associated with the levels of phosphorylated (p)-IGF-1R in breast cancer, their impact on the outcomes of breast cancer patients, and the prognostic value of alterations of p-IGF-1R during neoadjuvant chemotherapy (NAC). The present study included 348 female breast cancer patients whose paraffin-embedded tumor tissue sections had been collected by biopsy and/or resection, among which the pre-NAC and post-NAC sections were available from 40 patients. Human epidermal growth factor receptor 2 (HER2) positivity and molecular subtype were significantly associated with the presence of p-IGF-1R in the tumor tissue (P<0.05). Patients with p-IGF-1R present in the tumor tissue had a shorter OS (P=0.003). The p-IGF-1R levels in the tumor after NAC differed significantly from those prior to NAC (P=0.005); however, this alteration in p-IGF-1R levels was not associated with a shorter OS. In parallel with HER2, p-IGF-1R appears to be a promising indicator for predicting clinical outcomes and may be an attractive target for improving the efficacy of antitumor therapy, particularly for patients with HER2-negative, estrogen receptor-positive and luminal B tumors.
Breast cancer is one of the most common malignancies and the leading cause of cancer-associated mortality in females worldwide (
The role of insulin-like growth factor-1 receptor (IGF-1R) in breast carcinogenesis has attracted increasing attention over the last two decades (
Considering IGF-1R phosphorylation as an essential step for the function of IGF-1R (
A total of 348 female patients admitted to the Department of Breast Surgery of the Second Hospital of Shandong University (Jinan, China) from January 2010 to December 2014 were enrolled in the present study. All patients included in the final analysis were newly diagnosed and histologically confirmed to have breast cancer. The complete clinical data of each patient were recorded, and their breast cancer tissue samples were collected. All records were collected on the basis of patient written informed consent for the use of clinical data, their enrollment in the present study and for the analysis of their tissues. None of them had received any prior cancer treatment, including surgery, chemotherapy, radiotherapy and endocrine therapy. After enrollment, all treatments given by our department were based on NCCN guideline (
The clinical information of all patients was retrieved from electronic and paper-based medical records available from the Second Hospital of Shandong University (Jinan, China), which had been obtained from patients or their family members upon admission. All records were collected on the basis of complete informed consent. The patient data comprised of seven aspects, including i) demographic characteristics: Gender, age, ethnicity, marital status, occupation, height and weight; ii) gynecological details: Age of menarche, duration of menstruation, menstrual cycle and menopausal status; iii) fertility conditions: Number of children, age at first birth and breastfeeding conditions; iv) personal medical history (diagnosed by doctors prior to enrollment in the present study): Hypertension, diabetes mellitus, heart disease, viral hepatitis, benign breast disorders, uterine fibroids and ovarian cysts; v) family history: Breast cancer, other cancer, all types of cancer; vi) physical breast examination: Size, position, texture, mobility and smoothness of tumor and palpation of lymph nodes; vii) treatment-associated information: Time and methods of operation, details regarding lymph node dissection, regimen of chemotherapy, neoadjuvant chemotherapy and endocrine therapy; and viii) pathological characteristics: Pathological types, tumor stage, ER, progesterone receptor (PR), HER2, Ki-67 and lymph node metastasis status.
In total, immunohistochemical analysis of the expression of p-IGF-1R was performed in 388 breast cancer tissue sections as previously reported (
Either membranous or cytoplasm staining for p-IGF-1R was defined as positive. The results were assessed by an experienced pathologist using the immunoreactive scoring (IRS) criteria (
OS was defined as the time from diagnosis (date of biopsy) until the time-point of mortality or the last follow-up for patients. Disease-free survival (DFS) was defined as the time from diagnosis (date of biopsy) to events, including local relapse or distant metastases, the occurrence of a new primary tumor or death without evidence of cancer.
SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis of the data. For descriptive analysis, the values of continuous variables were expressed as the mean ± the standard deviation, and those of categorical variables were presented as the frequency. To describe the distribution of p-IGF-1R and to test whether p-IGF-1R was correlated with any clinicopathological parameters, Pearson's Chi-Square tests were performed for categorical variables. The Wilcoxon rank sum test was performed for comparing p-IGF-1R alterations during neoadjuvant chemotherapy (NAC). Logistic regression was performed to identify factors associated with p-IGF-1R levels. Correlations between p-IGF-1R and OS were analyzed by Kaplan-Meier survival analysis and multivariable Cox regression, and differences between subgroups were calculated using the log-rank test. All tests were two-sided, and P<0.05 was considered to indicate a statistically significant difference.
Of the 348 female breast cancer patients enrolled in the present study, 80 received NAC. Among those 80 patients, paraffin-embedded tumor tissue sections from the pre-NAC and post-NAC time-points were available for 40 patients. These tumor tissues had been collected by biopsy and resection, respectively. In total, 388 tumor tissue sections were analyzed by immunohistochemistry to detect p-IGF-1R. The demographic characteristics and medical history of the patients are summarized in
The age of these 348 patients ranged from 22 to 84 years, and the average age was 51.73±11.85 years. The cohort included 144 underweight or normal weight patients (BMI<24 kg/m2, accounting for 41.3%) and 191 overweight or obese patients (BMI≥24 kg/m2, accounting for 54.9%), while BMI data were missing for 13 patients.
Baseline p-IGF-1R was determined by immunohistochemical analysis of p-IGF-1R in tissue collected by resection for patients who did not receive NAC and in the biopsy specimens for patients who were to receive NAC. The baseline expression of p-IGF-1R was assessed in all 348 cases of breast cancer. The results demonstrated that 238 cases (68.4%) had a p-IGF-1R negative status, while 110 cases (31.6%) had a positive status, among whom weak (+), moderate (++) and strong (+++) p-IGF-1R staining was observed in 74 (21.3%), 32 (9.2%) and 4 (1.1%) cases, respectively. Representative immunohistochemical images are displayed in
To investigate whether p-IGF-1R may affect the clinicopathological characteristics of breast cancer patients, the association between the p-IGF-1R status and clinicopathological features was assessed (
To examine whether the level of p-IGF-1R changes during NAC, p-IGF-1R was measured in breast cancer patients prior to and after NAC (
To examine the association between multiple factors and p-IGF-1R expression in breast cancer, a univariate analysis was performed. The results are presented in
The follow-up survival data were available for 276 patients. A total of 259 patients survived at the last time-point of follow-up and the mean duration of follow-up was 3 years, ranging from 1–6 years. The mean OS time was 39.8±11.6 months (range, 10.1–74.7 months). The mean OS time for patients with p-IGF-1R-negative and -positive tumors was 42.0±10.8 months and 35.6±12.0 months, respectively. The p-IGF-1R levels at baseline were significantly associated with OS (log-rank test, P=0.003;
Considering the alteration of p-IGF-1R during NAC mentioned above, the present study further assessed the association of alterations of p-IGF-1R levels with OS and DFS (
In the present study, factors reported by others to be associated with the baseline IGF-1R expression were assessed (
IGF-1R is a heterotetrameric transmembrane receptor tyrosine kinase, which is widely expressed in normal human tissues and is frequently upregulated in breast cancer (
In the present study, an association between baseline p-IGF-1R expression and a wide range of factors was identified. To date, several studies have investigated the association of clinicopathological features with p-IGF-1R expression in breast cancer (
Breast cancer is classified into four major subtypes based on its ER, PR, HER2 and Ki-67 status: Luminal A, luminal B, HER2-enriched and TNBC. One study has demonstrated that high IGF-1R expression levels were more frequently seen in luminal A (52%), luminal B (57.5%) and luminal/HER2 (44.8%) patients, whereas the HER2-enriched (90.3%) and BL (77.5%) tumors had lower IGF-1R expression (
At present, the prognostic significance of IGF-1R is controversial. One study reported that IGF-1R expression is not associated with any clinical outcomes (
In order to further explore the prognostic efficacy of p-IGF-1R in different clinicopathological variables, subgroup analyses were performed. In the subgroups of HER2-negative, ER-positive and luminal B tumors, patients with a p-IGF-1R-positive status had a shorter OS. As is known, unlike that of luminal types, which may be treated with endocrine therapy, the prognosis of TNBC patients is almost always poor due to the lack of molecular treatment targets (
To date, only a few clinical studies have indicated that chemotherapy may induce alterations in IGF-1R. Heskamp
In conclusion, the presence of p-IGF-1R determined by immunohistochemistry was indicated to be associated with several clinical and pathological variables, and is paralleled with certain biomarkers linked with poor outcome, including HER2. Based on the Cox regression result, p-IGF-1R is an independent prognostic factor, which indicates that p-IGF-1R may be a promising indicator for predicting clinical outcomes and an attractive target for improving the efficiency of anti-tumor therapy, particularly for patients with HER2-negative, ER-positive and luminal B tumors. Further studies with larger sample sizes of patients receiving NAC will elucidate whether the changes of p-IGF-1R expression caused by NAC may be associated with survival.
The authors would like to thank Dr Alejandro Fernandez-Escobar from the Faculty of Medicine of CES University (Medellin, Colombia). for his contribution in revising the manuscript, all patients involved in the present study for their participation and the Department of Pathology (The Second Hospital of Shandong University, Shandong, China) for their collaboration and cooperation.
The authors are grateful for the support from the Key Project of the Natural Science Foundation of Shandong Province, China (grant no. ZR2014HZ004), the Key Research and Development Program of Shandong Province (grant no. 2016GSF201130) and the Youth Fund of the Second Hospital of Shandong University (grant no. Y2015010026).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZY and LiL conceived and designed the experiments; SL, LYL, ZM, MF, CY, WZ, YW, LuL, FW, LY, FZ, YX, SH, QF, QZ and DG performed the experiments; SL, LYL and ZM analyzed the data; SL and LYL wrote and revised the manuscript; ZY supplied suggestions on study design and prepared the manuscript. The final version of the manuscript has been read and approved by all authors, and each author believes that the manuscript represents honest work.
All procedures were in accordance with the guidelines set by the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Second Hospital of Shandong University (Jinan, China). All records were collected on the basis of patient written informed consent for the use of clinical data, their enrollment in the present study and for the analysis of their tissues
Patients consent for publication of the histology images.
The authors declare that there are no competing interests regarding the publication of this article.
Immunohistochemical analysis of p-IGF-1R in tumor tissues. (A) Representative image of breast cancer tissue with p-IGF-1R-negative status (magnification, ×100). (B) Breast cancer tissue with immunostaining for p-IGF-1R (magnification, ×100). (C) Breast cancer tissue with p-IGF-1R-negative status (magnification, ×200). (D) Breast cancer tissue with immunostaining for p-IGF-1R (magnification, ×200). p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor.
(A) Overall survival and (B) disease-free survival according to baseline p-IGF-1R levels in tumor tissues. p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor.
Influence of the expression of various tumor markers in tumor tissues on the overall survival of breast cancer patients stratified by their p-IGF-1R status. Overall survival of breast cancer patients with (A) HER2-negative and (B) HER2-positive status, (C) ER-negative and (D) ER-positive status, and (E) luminal B expression. p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2.
Effect of alterations in p-IGF-1R expression in the tumor tissues during neo-adjuvant chemotherapy on (A) overall survival and (B) disease-free survival of breast cancer patients. p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor.
Basic demographic characteristics and medical history of the breast cancer patients stratified by p-IGF-1R status.
p-IGF-1R | |||||
---|---|---|---|---|---|
Variables | N (%) | Negative | Positive | χ2 | P-value |
Age (years; range, 22–84) | 2.893 | 0.411 | |||
20–35 | 24 (6.9) | 19 (8.0) | 5 (4.5) | ||
36–50 | 151 (43.4) | 99 (41.6) | 52 (47.3) | ||
51–65 | 132 (37.9) | 89 (37.4) | 43 (39.1) | ||
>65 | 41 (11.8) | 31 (13.0) | 10 (9.1) | ||
Occupation | 17.101 | 0.004 | |||
Farmer | 85 (24.4) | 53 (22.3) | 32 (29.1) | ||
Worker |
85 (24.4) | 67 (28.2) | 18 (16.4) | ||
Retiree | 62 (17.8) | 43 (18.1) | 19 (17.3) | ||
Unemployed | 51 (14.7) | 39 (16.4) | 12 (10.9) | ||
Teacher, public servant, accountant or medical staff | 17 (4.9) | 13 (5.5) | 4 (3.6) | ||
Other | 48 (13.8) | 23 (9.7) | 25 (22.7) | ||
Ethnicity | 0.775 | 0.681 | |||
Han | 345 (99.1) | 236 (99.2) | 109 (99.1) | ||
Hui | 2 (0.6) | 1 (0.4) | 1 (0.9) | ||
Manchu | 1 (0.3) | 1 (0.4) | 0 (0) | ||
Marital status | 0.028 | 1.000 | |||
Married | 339 (97.4) | 232 (97.5) | 107 (97.3) | ||
Single | 7 (2.0) | 5 (2.1) | 2 (1.8) | ||
Unknown | 2 (0.6) | 1 (0.4) | 1 (0.9) | ||
BMI (kg/m2) | 1.982 | 0.555 | |||
<18.5 | 5 (1.4) | 3 (1.3) | 2 (1.8) | ||
18.5–23.9 | 139 (39.9) | 101 (42.4) | 38 (34.5) | ||
24.0–27.9 | 138 (39.7) | 90 (37.8) | 48 (43.6) | ||
≥28 | 53 (15.2) | 36 (15.1) | 17 (15.5) | ||
Unknown | 13 (3.7) | 8 (3.4) | 5 (4.5) | ||
Family history of breast cancer |
0.530 | 0.467 | |||
Yes | 31 (8.9) | 23 (9.7) | 8 (7.3) | ||
No | 317 (91.1) | 215 (90.3) | 102 (92.7) | ||
Family history of other cancer |
2.178 | 0.140 | |||
Yes | 52 (14.9) | 31 (13.0) | 21 (19.1) | ||
No | 296 (85.1) | 207 (87.0) | 89 (80.9) | ||
Family history of cancer |
0.319 | 0.572 | |||
Yes | 82 (23.6) | 54 (22.7) | 28 (25.5) | ||
No | 266 (76.4) | 184 (77.3) | 82 (74.5) | ||
Hypertension |
2.099 | 0.147 | |||
Yes | 80 (23.0) | 60 (25.2) | 20 (18.2) | ||
No | 268 (77.0) | 178 (74.8) | 90 (81.8) | ||
Diabetes mellitus |
0.915 | 0.339 | |||
Yes | 33 (9.5) | 25 (10.5) | 8 (7.3) | ||
No | 315 (90.5) | 213 (89.5) | 102 (92.7) | ||
Heart disease |
2.117 | 0.146 | |||
Yes | 34 (9.8) | 27 (11.3) | 7 (6.4) | ||
No | 314 (90.2) | 211 (88.7) | 103 (93.6) | ||
Breast benign tumor |
1.322 | 0.361 | |||
Yes | 13 (3.7) | 7 (2.9) | 6 (5.5) | ||
No | 335 (96.3) | 231 (97.1) | 104 (94.5) | ||
Uterine fibroid |
6.150 | 0.013 | |||
Yes | 28 (8.0) | 25 (10.5) | 3 (2.7) | ||
No | 320 (92.0) | 213 (89.5) | 107 (97.3) | ||
Ovarian cyst |
0.131 | 0.711 | |||
Yes | 8 (2.3) | 5 (2.1) | 3 (2.7) | ||
No | 340 (97.7) | 233 (97.9) | 107 (97.3) |
Selected parameters refer to
non-agricultural manual laborer
family history
all cancer types
personal history as diagnosed by doctors prior to enrollment. Values are expressed as n (%). BMI, body mass index; p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor. Unknown data was not included in statistical analyses.
Association of p-IGF-1R status with various clinicopathological characteristics.
p-IGF-1R | |||||
---|---|---|---|---|---|
Characteristics | N (%) | Negative | Positive | χ2 | P-value |
NAC | 4.466 | 0.035 | |||
Yes | 80 (23.0) | 47 (19.7) | 33 (30.0) | ||
No | 268 (77.0) | 191 (80.3) | 77 (70.0) | ||
Pathological type | 0.424 | 0.935 | |||
IDC | 296 (85.1) | 202 (84.9) | 94 (85.5) | ||
DCIS | 29 (8.3) | 20 (8.4) | 9 (8.2) | ||
ILC | 5 (1.4) | 4 (1.7) | 1 (0.9) | ||
Other type | 17 (4.9) | 11 (4.6) | 6 (5.5) | ||
Unknown | 1 (0.3) | 1 (0.4) | 0 (0) | ||
pT stage |
5.195 | 0.073 | |||
T1 | 131 (37.6) | 93 (39.1) | 38 (34.5) | ||
T2 | 99 (28.4) | 61 (25.6) | 38 (34.5) | ||
T3 | 6 (1.7) | 6 (2.5) | 0 (0) | ||
Unknown | 112 (32.2) | 78 (32.8) | 34 (30.9) | ||
pTNM stage |
0.142 | 0.931 | |||
I | 112 (32.2) | 77 (32.4) | 35 (31.8) | ||
II | 140 (40.2) | 99 (41.6) | 41 (37.3) | ||
III | 64 (18.4) | 44 (18.5) | 20 (18.2) | ||
Unknown | 32 (9.2) | 18 (7.6) | 14 (12.7) | ||
Molecular subtype | 9.598 | 0.022 | |||
Luminal A | 60 (17.4) | 47 (19.9) | 13 (11.9) | ||
Luminal B | 204 (59.1) | 132 (55.9) | 72 (66.1) | ||
HER2-enriched | 17 (4.9) | 8 (3.4) | 9 (8.3) | ||
TNBC | 23 (6.7) | 19 (8.1) | 4 (3.7) | ||
Unknown | 41 (11.9) | 30 (12.7) | 11 (10.1) | ||
ER status | 0.005 | 0.945 | |||
Positive | 288 (82.8) | 197 (82.8) | 91 (82.7) | ||
Negative | 53 (15.2) | 36 (15.1) | 17 (15.5) | ||
Unknown | 7 (2.0) | 5 (2.1) | 2 (1.8) | ||
PR status | 0.753 | 0.385 | |||
Positive | 232 (66.7) | 162 (68.1) | 70 (63.6) | ||
Negative | 106 (30.5) | 69 (29.0) | 37 (33.6) | ||
Unknown | 10 (2.9) | 7 (2.9) | 3 (2.7) | ||
HER2 status | 15.173 | <0.001 | |||
Positive | 67 (19.3) | 34 (14.3) | 33 (30.0) | ||
Negative | 211 (60.6) | 160 (67.2) | 51 (46.4) | ||
Unknown | 70 (20.1) | 44 (18.5) | 26 (23.6) | ||
Ki-67 status | 1.074 | 0.300 | |||
0–14% | 102 (29.3) | 73 (30.7) | 29 (26.4) | ||
>14 | 228 (65.5) | 150 (63.0) | 78 (70.9) | ||
Unknown | 18 (5.2) | 15 (6.3) | 3 (2.7) | ||
Lymph node status |
6.364 | 0.095 | |||
0 | 207 (59.5) | 140 (58.8) | 67 (60.9) | ||
1–3 | 61 (17.5) | 48 (20.2) | 13 (11.8) | ||
4–9 | 24 (6.9) | 13 (5.5) | 11 (10.0) | ||
>9 | 31 (8.9) | 24 (10.1) | 7 (6.4) | ||
Unknown | 25 (7.2) | 13 (5.5) | 12 (10.9) |
This study did not include any patients with pT4 tumors. Only 6 patients with pT3 tumors with negative p-IGF-1R
this study did not include any patients with stage IV tumors
Numbers of affected lymph nodes. NAC, neoadjuvant chemotherapy; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma
Changes in p-IGF-1R levels in patients receiving NAC.
p-IGF-1R level | |||||||
---|---|---|---|---|---|---|---|
Group | N (%) | 0 | + | ++ | +++ | W | P-value |
Biopsy (pre-NAC) | 40 (100) | 22 (55.0) | 12 (30.0) | 4 (10.0) | 2 (5.0) | 1,386.000 | 0.005 |
Resection (post-NAC) | 40 (100) | 34 (85.0) | 3 (7.5) | 3 (7.5) | 0 (0) |
p-IGF-1R, phosphorylated insulin-like growth factor 1 receptor; NAC, neoadjuvant chemotherapy.
Association of clinicopathological characteristics of breast cancer patients with p-IGF-1R positivity determined by univariate logistic regression.
Overall survival | |||
---|---|---|---|
Variables | OR | 95%CI | P-value |
NAC (yes vs. no) | 1.742 | 1.038–2.923 | 0.036 |
pT stage (pT2 and pT3 vs. pT1) | 1.156 | 0.704–1.897 | 0.567 |
pT1 | 1.000 | ||
pT2 | 1.525 | 0.876–2.652 | 0.135 |
pT3 | 0.000 | 0.000 | 0.999 |
pTNM stage (III/IV vs. I/II) | 0.988 | 0.712–1.372 | 0.944 |
Molecular subtype (Luminal B, HER2-enriched and TNBC vs. Luminal A) | 1.085 | 0.788–1.492 | 0.618 |
Luminal A | 1.000 | 0.027 | |
Luminal B | 1.972 | 1.001–3.885 | 0.050 |
HER2-enriched | 4.067 | 1.310–12.632 | 0.015 |
TNBC | 0.761 | 0.220–2.632 | 0.666 |
ER (positive vs. negative) | 0.978 | 0.522–1.833 | 0.945 |
PR (positive vs. negative) | 0.806 | 0.495–1.313 | 0.386 |
HER2 (positive vs. negative) | 3.045 | 1.716–5.402 | <0.001 |
Ki-67 (≥14 vs. <14%) | 1.309 | 0.786–2.179 | 0.301 |
OR, odds ratio; CI, confidence interval; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; pTNM, pathological tumor-nodes-metastasis; ER, estrogen receptor; PR, progesterone receptor; NAC, neoadjuvant chemotherapy.
Association of the NAC and HER2 status of breast cancer patients with p-IGF-1R positivity determined by multivariate logistic regression.
Overall survival | |||
---|---|---|---|
Variables | OR | 95%CI | P-value |
NAC (yes vs. no) | 2.326 | 1.018–5.318 | 0.045 |
HER2 (yes vs. no) | 2.093 | 0.982–4.462 | 0.056 |
OR, odds ratio; CI, confidence interval; HER2, human epidermal growth factor receptor 2; NAC, neoadjuvant chemotherapy.
Association between p-IGF-1R status and overall survival, as determined by Cox regression analysis.
Overall survival | |||
---|---|---|---|
Variable | OR | 95%CI | P-value |
IGF-1R (positive vs. negative) | 3.640 | 1.246–10.630 | 0.018 |
IGF-1R, insulin-like growth factor-1 receptor; OR, odds ratio; CI, confidence interval.