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Receptor activator for nuclear factor κ B expression predicts poor prognosis in breast cancer patients with bone metastasis but not in patients with visceral metastasis
  1. Lingyun Zhang1,
  2. Yuee Teng1,
  3. Ye Zhang1,
  4. Jing Liu1,
  5. Ling Xu1,
  6. Jinglei Qu1,
  7. Kezuo Hou1,
  8. Xianghong Yang2,
  9. Yunpeng Liu1,
  10. Xiujuan Qu1
  1. 1Department of Medical Oncology, The First Hospital of China Medical University, Shenyang, China
  2. 2Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, China
  1. Correspondence to Professor Yunpeng Liu, Department of Medical Oncology, The First Hospital of China Medical University, No.155, North Nanjing Street, Heping District, Shenyang 110001, China; cmuliuyunpeng{at}yahoo.cn and Professor Xiujuan Qu, Department of Medical Oncology, The First Hospital of China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang 110001, China; xiujuanqu{at}yahoo.com

Abstract

Background It was recently reported that receptor activator for nuclear factor κ B ligand (RANKL)/receptor activator for nuclear factor κ B (RANK) pathway is critical for RANK-expressing cancer cells to home to bone and associates with disease progression of cancer. The present study was aimed to evaluate the effect of RANK on prognosis in breast cancer patients with bone metastasis and patients with visceral metastasis.

Methods Immunohistochemical staining for RANK was carried out on paraffin-embedded primary tumour tissue sections from 102 patients with metastatic breast cancer. Survival analysis and Cox proportional hazards model were used to explore the prognostic value of RANK expression in breast cancer.

Results The RANK expression rates were 47.1% in metastatic breast cancer. Patients with RANK expression showed significantly poor progression-free survival and disease-specific survival. Subgroup analysis demonstrated that the significant difference in prognosis completely resulted from the occurrence of bone metastasis. Multivariate analysis demonstrated that RANK expression was an independent predictor of bone metastasis-free survival and disease-specific survival in patients with bone metastasis.

Conclusions RANK expression might be an independent predictor of poor prognosis in breast cancer patients with bone metastasis, and RANK expression does not associate with the prognosis in patients with visceral metastasis.

  • Receptor activator for nuclear factor κ B
  • breast cancer
  • bone metastasis
  • prognosis
  • oncology
  • bone
  • haem-oncology
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Background

Advanced breast cancer commonly spreads to the bones, lungs, liver or brain, and bone is the most common site of breast cancer metastasis. Nearly all patients with advanced breast cancer finally develop bone metastasis and suffer from the serious bone metastases-associated complications, including chronic bone pain, fracture, spinal cord compression and hypercalcaemia. Thus, the elucidation of the mechanism of breast cancer distant metastasis is important, in particular bone metastasis.

For the past decade, it was thought that chemokine pathways such as SDF-1/CXCR4 and CCL21/CCR7 played an important role in breast cancer distant metastasis.1 2 However, it has been shown that inhibition of these pathways in vivo only partially blocks metastatic behaviour,1–3 indicating that the existence of other factors regulate the metastasis of breast cancer cells. RANKL (receptor activator for nuclear factor κ B ligand) is a member of the tumour necrosis factor family of cytokines, and its receptor RANK (receptor activator for nuclear factor κ B) is primarily expressed on osteoclasts. Previous studies have shown that RANKL binds to RANK or the decoy receptor osteoprotegerin to control osteoclast differentiation, activation and survival.4–9 It has recently been reported that RANK is also expressed on cancer cells, and the RANKL/RANK pathway induces migration of prostate cancer cells, renal cancer cells, lung cancer cells and human breast cancer cells.3 10–13 Since RANKL is typically expressed on osteoblasts and bone stromal cells, it is possible that the RANKL/RANK pathway triggers the bone-specific migration of RANK-expressing cancer cells. This has been recently confirmed in vivo in malignant melanoma and prostate cancer animal models3 11 and in patients with renal cancer.10 Recently, it was reported that RANK expression is associated with disease progression and bone metastasis in patients with primary breast cancer,13 renal cancer10 and prostate cancer.14 However, the effect of RANK expression on bone metastasis, disease progression and sequential prognosis still remains unclear in patients with metastatic breast cancer. Moreover, since RANKL is also expressed in the liver, lung, lymph nodes and brain,4 5 15–17 it has not been previously reported whether the RANKL/RANK pathway is associated with metastasis to these organs.

In the present study, RANK expression and the effect on prognosis in patients with metastatic breast cancer were evaluated, and the role of RANK expression in bone metastasis and visceral metastasis was explored by subgroup analysis. The results revealed that RANK expression is associated with poor prognosis in patients with bone metastasis but not in patients with visceral metastasis.

Methods

Patients and tissue samples

A total of 102 patients with metastatic breast cancer (including lung, brain, bone, liver and distant lymph node metastasis) were retrospectively analysed during the median follow-up of 40 (range 7–143) months from diagnosis, including 62 patients with bone metastases. The patients were histologically diagnosed and/or received surgery between July 1997 and May 2008 in the First Hospital of China Medical University. Primary tumour tissues from these patients were collected. Clinicopathological parameters were evaluated by reviewing medical charts and pathology records. This study was approved by the Human Ethics Review Committee of the First Hospital of China Medical University. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki and its later revision. All the patients had been treated according to the modern guidelines, including the use of adjuvant chemotherapy, irradiation and endocrine therapy for oestrogen receptor-positive/progesterone receptor-positive patients and others.

Progression-free survival (PFS) was defined as the time between the date of diagnosis and the date of distant metastases and/or cancer-related death. Disease-specific survival (DSS) was defined as the time between the date of diagnosis and cancer-related death. Bone metastasis-free survival (BFS) was defined as the time between the date of diagnosis and the date of bone metastasis diagnosis.

Immunohistochemistry analysis

Formalin-fixed paraffin-embedded specimens of tumours were collected from the Department of Pathology at the First Hospital of China Medical University. Immunohistochemical staining for RANK (anti-RANK, SC-52951, dilution 1:500; Santa Cruz, USA) was performed using the biotin–streptavidin–peroxidase procedure (UltraSensitive S-P kit; Maixin-Bio, Shanghai, China) as described previously.18 Briefly, each slide was deparaffinised in xylene and rehydrated in graded alcohol to Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4). After antigen retrieval by 10 mM sodium citrate (pH 6.0), blocking of endogenous peroxidase activity and application of the primary antibody (4°C, overnight), samples were incubated with biotinylated goat anti-mouse immunoglobulin (room temperature, 10 min) and streptavidin conjugated to horseradish peroxidase (room temperature, 10 min). A 3,3′-diaminobenzidine' (DAB) chromogen solution and a substrate buffer containing hydrogen peroxide served as the substrate system (DAB kit; Maixin-Bio). Tissue sections were counterstained with haematoxylin and permanently mounted. The evaluation of immunohistochemistry results was performed independently by two observers (KH and XY) who had no prior knowledge of the clinical information or pathological parameters. The immunoreactivity was scored based on both intensity of staining (negative=0, weak=1, moderate=2, strong=3) and percentage of positive tumour cells (<10%=0, 10%–50%=1, >50%=2) as reported previously.19 The final score was calculated by multiplication of the single scores, giving intensity and percentage of positive cells (range=0–6). A score of at least 2 was considered positive.

Statistics

The association of staining intensity with clinicopathological patterns was assessed using Fisher's exact test or the χ2 test. The log-rank test and the Kaplan–Meier method were used for the patient survival analysis. Univariate and multivariate analysis was performed according to the Cox proportional hazards model. p Value <0.05 was considered significant. Statistical analyses were carried out using SPSS V.16.0 for Windows (SPSS, Inc.).

Results

RANK expression in human breast cancer

A total of 102 histologically confirmed breast cancer samples were obtained, including 87 (85.3%) invasive ductal carcinomas, six (5.9%) invasive lobular carcinomas and nine (8.8%) carcinomas of another type. The median age of patients was 52 (range 26–76) years, and the median follow-up time was 40 (range 7–143) months. RANK expression was evident upon immunostaining (figure 1); RANK-positive expression was observed in 48 (47.1%) breast cancer tissue samples. The RANK-positive expression rate was significantly higher in women younger than 35 years and in women with PFS <3 years. No difference in RANK expression was evident among other clinicopathological parameters, such as primary tumour size, histology grade, and so forth (table 1).

Figure 1

Representative images of receptor activator for nuclear factor κ B (RANK) immunohistochemical staining. (A) RANK-negative staining (−). (B) RANK-positive staining (+) in cell membranes and cytoplasmy (in brown). Magnification ×200.

Table 1

Correlation of RANK expression with clinicopathological parameters in patients with breast cancer (n=102)

The prognostic value of RANK expression in patients with metastatic breast cancer

To determine the prognostic value of RANK expression, survival analysis was performed. The result showed that subjects exhibiting positive RANK expression demonstrated a significantly poorer prognosis than those negative for RANK expression in terms of both PFS (p=0.023) and DSS (p=0.001) (figure 2A). In patients with bone metastasis, the subjects exhibiting positive RANK expression showed significantly poorer prognosis than those negative for RANK expression in terms of both BFS (p=0.003) and DSS (p<0.001). The median BFS of RANK-positive and RANK-negative patients was 15 and 26 months, respectively (HR=2.266; 95% CI 1.289 to 3.984), and the median DSS was 34 and 56 months, respectively (HR=3.079; 95% CI 1.545 to 6.135). However, in patients with non-bone metastasis, no difference in PFS (p=0.949) and DSS (p=0.214) was shown between RANK-positive and RANK-negative patients (figure 2C).

Figure 2

Kaplan–Meier curves for receptor activator for nuclear factor κ B (RANK) expression in patients with breast cancer. (A) In the total metastatic breast cancer population, RANK-positive patients (n=48) had a significant reduction in progression-free survival (PFS) and disease-specific survival (DSS) compared with RANK-negative patients (n=54). (B) Subgroup analysis in patients with bone metastasis, RANK-positive patients (n=26) had a significant reduction in bone metastasis-free survival (BFS) and DSS compared with RANK-negative patients (n=35). (C) Subgroup analysis in patients with non-bone metastasis, no significant difference was shown in PFS and DSS between RANK-negative patients (n=19) and RANK-positive patients (n=22).

Furthermore, univariate analysis in 61 patients with bone metastasis showed that RANK-positive expression, age <35 years and non-lymph node metastasis predicted poor BFS; RANK-positive expression and age <35 years predicted poor DSS. Multivariate analysis showed that RANK expression was an independent predictor for BFS. RANK expression, age <35 years and histology grade 3–4, respectively, were independent predictors for DSS (table 2).

Table 2

Cox univariate and multivariate analysis of (A) bone metastasis-free survival and (B) disease-specific survival in breast cancer patients with bone metastasis (n=61)

Taken together, these data indicated that RANK expression has potential value in assessing the clinical outcome for breast cancer patients with bone metastasis.

Discussion

The RANKL/RANK pathway has previously emerged as the key pathway in the regulation of osteolysis in skeletal metastasis. However, recent studies have shown that RANK is also expressed on the surface of some cancer cells and that RANKL functions as a chemokine and directs cancer cells to preferentially migrate into bone, which is the crucial and initial step for bone metastasis. About 38% of prostate cancers,14 100% of oesophageal adenocarcinomas20 and 82% of clear cell renal cell carcinomas10 express RANK. Additionally, RANK expression correlates with the more aggressive, advanced and metastatic tumour in renal cell carcinomas,10 breast cancers13 and primary prostate cancers.14 In the present study, RANK was expressed in around 47% of patients with metastatic breast cancers. Moreover, RANK expression significantly correlated with young age and short PFS in breast cancer, implying that RANK expression accelerated the disease progression of patients with breast cancer.

RANKL was expressed in bone, liver, lung, lymph node and brain,4 5 15–17 indicating that the RANKL/RANK pathway may be associated not only with metastasis to bone but also with metastasis to other organs. The survival analysis results showed that patients who exhibit RANK expression have significantly shorter PFS and DSS compared with RANK-negative patients. To investigate whether the difference arises from bone metastasis alone or from all distant metastasis, subgroup analysis was performed. The results demonstrated that for patients with bone metastasis, RANK-positive patients showed much poorer BFS and DSS, whereas in patients with non-bone metastasis, no significant difference in PFS and DSS was found. The results indicated that the significant difference in prognosis was completely due to patients with bone metastasis. Additionally, multivariate analysis showed that RANK expression was an independent predictor for DSS in patients with bone metastasis.

Although the sample size of this retrospective analysis was comparable to works within the field, larger multi-institutional prospective studies are needed to confirm our observations and to determine whether other prognostic markers exist that may have been missed in smaller studies.

To our knowledge, this is the first study showing that RANK expression associates with the poor prognosis in patients with bone metastasis rather than those with visceral metastasis.

Take-home messages

  • RANK expression is significantly associated with poor PFS and DSS in metastatic breast cancer.

  • Subgroup analysis showed that RANK expression predicts poor prognosis in breast cancer patients with bone metastasis but not in patients with visceral metastasis.

Acknowledgments

We thank our clinical and laboratory colleagues who have contributed to our research.

References

View Abstract

Footnotes

  • Funding This work was supported by Chinese National Foundation of National Sciences grants (grant number 30700807); Science and Technology project of Liaoning Province (grant number 2010225032). The funder had an important role in the design and conduct of the study.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Human Ethics Review Committee of the First Hospital of China Medical University.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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