J Cancer Res Clin Oncol DOI 10.1007/s00432-014-1693-4
Original Article – Clinical Oncology
Axitinib alone or in combination with chemotherapeutic drugs exerts potent antitumor activity against human gastric cancer cells in vitro and in vivo Qiong He · Jing Gao · Sai Ge · Tingting Wang · Yanyan Li · Zhi Peng · Yilin Li · Lin Shen
Received: 21 January 2014 / Accepted: 22 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Objective As the new oral selective VEGFR tyrosine kinase inhibitor, axitinib (AG-013736) exerts powerful antitumor activity in multiple solid tumors, while its’ effect was unclear in gastric cancer. We aimed to investigate the antitumor activity of axitinib alone or combined with chemotherapeutic drugs against human gastric cancer cells in vitro and in vivo. Methods The IC50 values of drugs were determined by MTS assay. The median effect of Chou-Talalay was used to assess the synergistic effect of two drugs. Flow cytometry was employed to analyze cell cycle and cell apoptosis. Cell senescence and microvessel density were evaluated by SA-β-gal staining and CD34 staining, respectively. BGC823-derived xenografts in nude mice were established to investigate the effects of drugs in vivo. Results Axitinib alone could inhibit cell proliferation and retard tumor growth through inducing cell cycle arrest at G2/M phase, cell senescence, cell apoptosis, and Qiong He, Jing Gao, and Sai Ge have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1693-4) contains supplementary material, which is available to authorized users. Q. He · J. Gao · S. Ge · T. Wang · Y. Li · Z. Peng · Y. Li · L. Shen (*) Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, Fu‑Cheng Road 52, Hai‑Dian District, Beijing 100142, China e-mail:
[email protected] Q. He Department of Chemotherapy, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310022, China
antiangiogenesis in vitro and in vivo. Axitinib combined with 5-fluorouracil (5-FU) had synergistic inhibitory effect compared to axitinib or 5-FU alone. However, the highest inhibitory effect was found between axitinib and cisplatin (inhibitory ratio >80 % compared to control), which was significantly higher than any single drug (inhibitory ratio for single 5-FU, cisplatin, and axitinib >10, >40, and >40 %, respectively, compared to control) or axitinib combined with 5-FU (inhibitory ratio >50 % compared to control). Conclusion We highlighted for the first time that axitinib alone or in combination with 5-fluorouracil or cisplatin has potent antitumor activity against human gastric cancer in vitro and in vivo, which provided solid evidence for future clinical trial. Keywords Axitinib · Tyrosine kinase inhibitor · Gastric cancer · Antitumor activity
Introduction Axitinib (AG-013736) is an oral, potent, and selective small-molecule tyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3 extensively studied in the last few years. In nonclinical and clinical studies, axitinib has the effectiveness on inhibition of angiogenesis, vascular permeability, and blood flow (Li et al. 2005; Liu et al. 2005). Compared to other similar tyrosine kinase inhibitors, axitinib seems to be a more potent and selective VEGFR inhibitor leading to its effective activity (Hu-Lowe et al. 2008). Axitinib alone showed antitumor activity in multiple tumors containing thyroid cancer, non-small-cell lung cancer, melanoma, and so on (Cohen et al. 2008; Schiller et al. 2009; Fruehauf et al. 2011). Recently, axitinib was
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approved by the US Food and Drug Administration (FDA) to be used as the second-line therapy of advanced renal cancer following the failure of IFN-α or TKIs therapy (Rini et al. 2011). Also, the antitumor activity was increased when axitinib in combination with other chemotherapeutic drugs in advanced pancreatic adenocarcinoma, metastatic breast cancer, and so on (Kindler et al. 2011; Rugo et al. 2011; Sharma et al. 2010). Gastric cancer is the third leading cause in male and ranks the fourth leading cause in female of cancer death in developing countries (Jemal et al. 2011). About 70 % gastric cancer patients have a locally advanced or metastatic disease at the time of initial diagnosis, and for patients with advanced disease, the prognosis is very poor (Yu et al. 2012). Combinative regimens containing fluorouracil or cisplatin were most commonly used in clinical practice for advanced gastric cancer with a modest but unsatisfactory improvement for prognosis (Koizumi et al. 2008; Ajani et al. 2010). Nowadays, no tyrosine kinase inhibitors were successfully used in the treatment of gastric cancer, despite the significant antitumor effects in animal models. In consideration of the potent antitumor activity of axitinib alone or in combination of other drugs in multiple tumors and the requirement of new drugs to be helpful for gastric cancer treatment, we aimed to evaluate the antitumor activity of axitinib in human gastric cancer cells in vitro and in vivo in this study. Moreover, we investigated the possible mechanisms of axitinib and the synergistic effects of axitinib combined with 5-fluorouracil or cisplatin.
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from Shanghai Institutes for Biological Sciences, CAS. Cells were cultured in RMPI-1640 medium (Gibco BRL, Rockville, MD), and Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented with 10 % fetal bovine serum (Gibco BRL) and incubated in a humidified 37 °C incubator supplemented with 5 % CO2. MTS cell proliferation assay BGC-823 and HGC-27 cells were seeded at about 3,0005,000 cells per well in a 96-well plate and incubated overnight in complete medium. Cells were treated with axitinib, 5-FU, DDP alone, or axitinib combined with 5-FU or DDP. Cell viability was determined after 48 h of drug exposure using MTS tetrazolium substrate (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) according to the manufacturer’s instructions. The absorbance was measured at 490 nm using a spectrophotometer. All experiments were repeated three times with at least triplicates for each concentration. Assessment of synergetic effect of axitinib and 5‑FU or DDP
Materials and methods
Assessment of synergetic effect of two drugs was performed according to our previous description (Wang et al. 2013). Briefly, the median effect method of Chou-Talalay was used, and combination index (CI) was calculated using the described formula. CI < 1, CI = 1, and CI > 1 indicated synergism, additive effect, and antagonism, respectively. Fa-CI plots (Fa: fraction affected) were drawn to present the effect of combination.
Drugs
In vivo xenograft model studies
Axitinib (AG-013736) was kindly provided by Pfizer (La Jolla, CA) as a white powder and stored at 4 °C away from light. For in vitro studies, axitinib was dissolved in dimethylsulfoxide as the stock concentration of 10 mmol/L and stored at −20 °C. For in vivo animal experiments, axitinib was formulated in 0.5 % carboxymethylcellulose (0.5 % CMC) as a homogeneous suspension (5 mg/mL) and stored at 4 °C away from light. 5-fluorouracil (5-FU, 250 mg/10 mL) injection was purchased from Tianjin Jinyao Amino Acid Co., Ltd., China. Cisplatin lyophilized powder (DDP, 10 mg) was purchased from Qilu Pharmaceutical Co., Ltd., China and was formulated in 0.9 % NaCl.
BGC-823 cells were suspended in phosphate-buffered saline (PBS) at a concentration of 1 × 107/mL, and 100 µL cell suspension was subcutaneously injected into the right oxter of 18–20 g female BALB/c athymic nu/nu mice (Vital River, China). When tumor volume was about 100 mm3, mice were randomized into six groups (5 mice/group): control group (physiological saline, once a day for 2 weeks); axitinib group (25 mg/kg body weight, twice a day for 2 weeks); 5-FU group (10 mg/kg body weight, once every 2 days for 2 weeks); DDP group (3 mg/kg body weight, once a week for 2 weeks); axitinib combined with 5-FU group (same as the single drug); and axitinib combined with DDP group (same as the single drug). Tumors were measured twice a week, and tumor volume was calculated by the formula V = L × W2 × 1/2 (V, volume; L, length of tumor; W, width of tumor). All animal experiments were performed in accordance with the animal experimental guidelines of Peking University Cancer Hospital.
Cell lines and cell culture Human gastric cancer cells BGC-823 was kindly provided by Professor Youyong Lv (Peking University Cancer Hospital and Institute), and HGC-27 was purchased
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Cell cycle analysis After treated with axitinib for 48 h, cells were harvested and fixed in 70 % cold ethanol for at least 12 h in 4 °C. Cells were stained with 50 μg/mL propidium iodide (BD Biosciences) at room temperature for 30 min in the dark, and cell cycle was performed by a FACS Aria Calibur (BD Biosciences) and analyzed by ModFit 3.0 software (BD Biosciences). All experiments were performed in triplicate. Cell apoptosis assay Cell apoptosis was conducted using an Annexin V-FITC/ PI apoptosis detection kit (KeyGEN BioTECH, China; Cat: KGA108). After cells were treated with axitinib for 3, 6, and 12 h, cells were harvested and analyzed by flow cytometry (BD Biosciences). Cell apoptosis was analyzed by WinMDI 2.9 software (BD Biosciences). Immunocytochemistry After cells were treated with axitinib for 48 h, cells were washed with PBS twice and fixed with 4 % paraformaldehyde for 10 min at room temperature. Cells were permeabilized with 0.1 % Triton X-100 for 10 min at room temperature and blocked with 0.5 % bovine serum albumin for 30 min at 37 °C. The cells were exposed to anti-cyclin B1 antibody (Abgent Corporation, dilution: 1:300) and then stained with a FITC-conjugated secondary antibody. After overlaying coverslip, the slides were imaged using a fluorescence microscopy (Leica TCS SP5, Germany). The positive cells were counted from three random microscopic fields.
Biotechnology, China) according to the manufacture’s instructions. Briefly, after cells were treated with axitinib for 48 h, cells were fixed with fixing solution for 15 min at room temperature and then stained with SA-β-gal solution at 37 °C overnight. The positive cells were counted from three random microscopic fields. Immunohistochemistry staining After mice were killed, the xenografts were isolated and FFPE (Formalin Fixed Paraffin Embedded) tissue blocks were made. Immunohistochemistry staining was performed as previous described (Gao et al. 2011). Briefly, FFPE tumor sections with 4 um thick were deparaffinized xylene and hydrated in graded alcohols, followed by antigen retrieval and endogenous peroxidase treatment. Sections were then incubated with CD34 (Abcam, Hong Kong) and p16 (Santa Cruz Biotechnology, China) antibodies, respectively. Signal production employed general type IgG-HRP polymer (Beijing CoWin Biotech Co., Ltd.) and diaminobenzidine substrate. Sections were scored as following: intensity of staining (1, weak; 2, moderate; 3, strong) and percentage of cell stained (1, 0–10 %; 2, 11–50 %; 3, 51–100 %). Statistical analysis We used SPSS 18.0 software to perform statistical analysis. Repeated measures analysis of variance was used to compare the difference of tumor growth between different groups in vitro study. One-way ANOVA was used in vivo study. P < 0.05 was considered statistically significant.
Western blot
Results
Total protein before and after axitinib treatment was extracted from cell pellets using CytoBuster Protein Extraction Reagent (Merck Millipore, Darmstadt, Germany). After measurement of protein concentration using the DC protein assay method of Bradford (Bio-Rad, Hercules, CA), about 20 micrograms of protein were separated on 12 % SDS-PAGE. After transfer, the nitrocellulose membrane (GE Healthcare, Piscataway, NJ) was incubated with cyclin B1 (Abgent Corporation, dilution: 1:1,000) and BCL2 (Cell Signaling, dilution: 1:1,000) antibodies at 4 °C overnight and secondary antibody at room temperature for 1 h. Proteins were visualized using ECL Plus Western Blotting Detection Reagents (GE Healthcare).
Axitinib alone or combined with 5‑FU or DDP inhibits growth of gastric cancer cells in a dose‑dependent manner
SA‑β‑gal assay
BGC-823 HGC-27
Cell senescence was determined by Senescence β-Galactosidase Staining Kit (Beyotime Institute of
IC50, the 50 % inhibitory concentration; 5-FU, 5-fluorouracil; DDP, cisplatin
IC50 values for axitinib, 5-FU, and DDP in BGC-823 and HGC-27 cell lines were determined by MTS assay and shown in Table 1. Axitinib alone inhibited cell growth in a dose-dependent manner, as well as 5-FU and DDP (Fig. 1). Compared to any individual drug, combination of axitinib Table 1 IC50 of BGC-823 and HGC-27 for axitinib, 5-FU and DDP Cell line
IC50 (µmol/L) axitinib
5-FU
DDP
4.27 ± 0.75
161.25 ± 20.93
2.49 ± 0.39
12.50 ± 2.05
213.00 ± 27.70
2.34 ± 1.02
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Fig. 1 Inhibition of axitinib, 5-FU, and DDP on BGC-823 and HGC27 cell lines. Cells were exposed to various concentrations of axitinib, 5-FU, and DDP alone or in combination for 48 h. Cell viability was
determined by MTS assay. Axitinib alone or in combination with 5-FU or DDP inhibited growth of BGC-823 (a, b) and HGC-27 (c, d) cells in a dose-dependent manner (n = 3, mean ± SD, *P < 0.05)
with 5-FU or DDP exerted a stronger inhibition of cell growth with a dose-dependent manner (Fig. 1).
Compared to control group (tumor volume: 1,973.0 mm3; tumor weight: 1.65 g), axitinib alone (tumor volume: 1,093.7 mm3; tumor weight: 0.74 g; P < 0.01) had significant inhibitory effect on BGC-823 xenograft. When axitinib combined with 5-FU (tumor volume: 910.8 mm3; tumor weight: 0.71 g), the inhibitory effect was significantly higher than 5-FU alone (tumor volume: 1,680.3 mm3; tumor weight: 1.42 g; P < 0.01), but no significant difference with axitinib alone (P > 0.05). However, combination of axitinib and DDP (tumor volume: 369.2 mm3; tumor weight: 0.31 g) had a significant synergistic inhibitory effect on BGC-823 xenografts compared to axitinib alone or DDP alone (tumor volume: 1,075.4 mm3; tumor weight: 0.95 g) (Fig. 3). Also, the synergism between axitinib and DDP was significantly better than that between axitinib and 5-FU (P < 0.01).
Analysis of synergistic effects between axitinib and 5‑FU or DDP in vitro In BGC-823 cells, axitinib combined with 5-FU had a synergistic inhibitory effect (CI < 1) when Fa value was ≤0.8, while synergism between axitinib and DDP (CI < 1) was observed when Fa value was >0.4 (Fig. 2a, b). For HGC-27 cells, axitinib combined with 5-FU or DDP induced significant synergistic growth inhibition (CI < 1, Fig. 2c, d). The detailed CI values and Fa for axitinib combined with 5-FU or DDP were shown in Supplementary Table 1. Inhibitory effect of axitinib alone or combined with 5‑FU or DDP on growth of BGC‑823 xenografts
Axitinib induced cell cycle arrest at G2/M phase
By the 18th day after the initial treatment, all animals were alive and tumors were isolated from mice and weighed.
To explore the potential mechanisms responsible for the inhibitory effect of axitinib in gastric cancer cells, cell
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J Cancer Res Clin Oncol Fig. 2 Synergistic effects between axitinib and 5-FU or DDP in vitro. Fa-CI plots were drawn to show the synergistic effects between axitinib and 5-FU or DDP in BGC-823 and HGC-27 cell lines. In BGC823 cell line, when Fa value was ≤0.8 or >0.4, a synergistic inhibitory effect (CI < 1) was seen between axitinib and 5-FU a or DDP b. Axitinib combined with 5-FU c or DDP d induced significant synergistic growth inhibition on HGC-27 cells
cycle analysis was performed. The percentage of cells in G2/M phase was significantly increased in BGC-823 (44.03 vs. 19.26 %) and HGC-27 (38.07 vs. 17.34 %) cells after axitinib treatment compared to control (P < 0.05, Fig. 4a). Concomitant with cell cycle arrest at G2 phase, the expression of cell metaphase-specific protein cyclin B1 was upregulated by cell immunofluorescence assay and Western blot. The percentage of cyclin B1-positive cells after axitinib treatment was significantly higher than control group (P < 0.01), which was validated by Western blot (Fig. 4B). Evaluation of cell apoptosis induced by axitinib In order to determine whether the inhibitory effect by axitinib in gastric cancer cells was due to an induction of apoptosis, cellular apoptotic rate was evaluated using Annexin V-FITC and PI staining by flow cytometry. As shown in Fig. 4c, compared to control, the proportion of apoptotic cells treated by axitinib in BGC-823 and HGC-27 cell lines was significantly increased (P < 0.05), companied with the downregulation of BCL2 (Fig. 4c).
using SA-β-gal staining method in vitro. After axitinib treatment, the percentage of positive staining cells (blue) was significantly more than control (P < 0.05, Fig. 4d). To investigate whether cell senescence occurred after axitinib treatment in vivo, the expression of a senescence-associated protein p16 was detected in tumor tissues by immunohistochemistry. As shown in Fig. 4e, the expression level of p16 after axitinib treatment was significantly increased than control (Fig. 4e). Axitinib inhibited the expression of CD34 after axitinib treatment Axitinib was reported to play its antitumor activity by inhibiting angiogenesis in other solid tumors. In order to determine whether axitinib had antiangiogenesis activity in gastric cancer cells, tumor tissues from mice xenografts after axitinib treatment were stained with CD34 antibody by immunohistochemistry. The results indicated that CD34 staining after axitinib treatment was significantly weaker than control (Fig. 4f). This result demonstrated that axitinib could inhibit tumor vascularization.
Analysis of cell senescence induced by axitinib In this study, we found that the morphology of gastric cancer cells after axitinib treatment was larger than control, which suggested that there might be cell senescence. To validate this hypothesis, cell senescence was determined
Discussion Axitinib alone exerted potent antitumor activity in several solid tumors (Cohen et al. 2008; Schiller et al. 2009;
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Fig. 3 Inhibitory effects of axitinib alone or in combination with 5-FU or DDP on BGC-823 xenografts. a The growth curves of xenografts treated with different drugs (5 mice/group, mean ± SD). b
After treatment, tumor xenografts were isolated and photographed. c Tumor weight of each group (5 mice/group, mean ± SD)
Fruehauf et al. 2011), which was unclear in gastric cancer. In this study, we demonstrated that axitinib alone inhibited the growth of BGC-823 and HGC-27 gastric cancer cells in a dose-dependent manner in vitro (Fig. 1). Based on the in vivo results, the inhibitory effect of axitinib on mice xenografts is better than 5-FU and not worse than DDP (Fig. 3). These results suggested that axitinib had promising antitumor activity in gastric cancer. Combination regimens of two or more drugs for gastric cancer were commonly used in clinical practice, and we investigated the specific effects between axitinib and 5-FU or DDP (5-FU and DDP are most commonly used drugs for gastric cancer). Figure 2 shows when Fa value was ≤0.8, a synergistic inhibitory effect (CI < 1) was seen between axitinib and 5-FU, and when Fa > 0.4, axitinib enhanced the antitumor effect of DDP in BGC-823 cells. However, axitinib combined with 5-FU or DDP induced significant synergistic growth inhibition on HGC-27 cells (data shown in Supplementary Table 1). This might due to the diversity
of genetic characteristics of different cells. For the in vivo studies, the inhibitory effect of axitinib combined with 5-FU was significantly higher than 5-FU alone, but similar with axitinib alone (Fig. 3). But an exciting synergistic inhibitory effect on the growth of xenografts was observed between axitinib and DDP, which was obviously greater than axitinib or DDP alone (P < 0.01, Fig. 3). In supporting our results, studies have demonstrated that axitinib had synergistic inhibitory effect with platinum in non-smallcell lung cancer (Kozloff et al. 2012; Ulahannan and Brahmer 2011). We then explored the possible mechanisms through which axitinib played its activity. It was reported that axitinib could induce cell cycle arrest at G2/M phase and cell apoptosis, as well as antiangiogenesis (Stehle et al. 2013). The possible mechanisms of axitinib were therefore analyzed in BGC-823 and HGC-27 cells in this study. Our results showed that axitinib could induce cell cycle arrest at G2/M phase, which was further validated according to
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Fig. 4 The possible mechanisms of axitinib in gastric cancer cell lines. Axitinib inhibited the growth of gastric cancer cells through inducing cell cycle arrest at G2/M phase, cell apoptosis, cell senescence, and antiangiogenesis. a Axitinib induced cell cycle arrest at G2/M phase in BGC-823 and HGC-27 cell lines. b After axitinib treatment, cyclin B1 was significantly upregulated in BGC-823 and HGC-27 cell lines by immunocytochemistry (n = 3, mean ± SD, *P < 0.05) and Western blot. c Cells were exposed to axitinib (dose: 1/2 IC50 or IC50) for 3, 6, and 12 h. The percentage of apoptotic cells
after axitinib treatment was significantly higher than control (n = 3, mean ± SD, *P < 0.05) companied with the downregulation of BCL2. d After axitinib treatment, the dimension of cells was larger than control, and the positive cells of SA-β-gal staining were also more than control group in vitro (n = 3, mean ± SD, *P < 0.05). e The expression level of p16 was upregulated after axitinib treatment compared to control group in vivo. f Tumor vascularization was significantly suppressed by axitinib through detecting the expression of CD34
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the upregulation of cyclin B1 (Fig. 4a, b). Moreover, axitinib could also induce cell apoptosis in a dose- and timedependent manner (Fig. 4c). In the course of axitinib treatment, we found some changes of morphological characteristics of cells, such as cell swelling, increase in intracellular solid particles, and so on, which suggested some features of cell senescence. Results from SA-β-gal staining confirmed our hypothesis that axitinib induced cell senescence in vitro and in vivo (Fig. 4d, e). Antiangiogenesis is an important mechanism of axitinib in other tumors (HuLowe et al. 2008; Rössler et al. 2011), which was also confirmed in this study. After axitinib treatment, the microvessel density of tumors represented by CD34 staining was significantly lower than control group (Fig. 4f). From our in vivo study, the highest inhibitory effect on xenografts was observed between axitinib and DDP (Fig. 3) with no obvious toxicity on mice. The volume of xenografts treated with axitinib combined with DDP was almost the same with initial treatment. This result provided a solid evidence for the future clinical trial. Although axitinib had synergism with 5-FU, the inhibitory effect between axitinib and 5-FU was significantly lower than that between axitinib and DDP. It is well known that 5-FU, DDP, and paclitaxel are the most common drugs for gastric cancer. In this study, we also investigated whether axitinib had synergism with paclitaxel, and the results demonstrated that antagonism not synergism was found between axitinib and paclitaxel in vitro and in vivo (data not shown). The possible reasons were not explored in this study, and the functional mechanisms of axitinib alone or combined with chemotherapy drugs remain to be further investigated. Up to now, only one study reported the mechanism of synergism between axitinib and SN-38 (the active metabolite of irinotecan) in dermal microvascular endothelial cells, which suggested that axitinib combined with SN-38 greatly inhibited the expression of ATP7A and ABCG2 genes and increased the SN-38 intracellular concentration (Canu et al. 2011). In the following days, we will further validate the inhibitory effect of axitinib in gastric cancer patient-derived xenografts and investigate the definite functional mechanisms of axitinib in gastric cancer. In conclusion, axitinib alone or combined with 5-FU or DDP exerts potent antitumor activity against human gastric cancer in vitro and in vivo, especially axitinib combined with DDP, suggesting a potential therapeutic application in gastric cancer. Our results will provide solid evidence for future clinical trials. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 81172110), National High Technology Research and Development Program (No. 2012AA 02A 504), and Beijing Municipal Science and Technology Commission Program (No. Z11110706730000). The authors thank Pfizer for the providing
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J Cancer Res Clin Oncol of axitinib and Dr. Zhongwu Li (Department of pathology, Peking University Cancer Hospital and Institute) for the IHC scoring. Conflict of interest The authors declare that they have no conflict of interest.
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