CXCR4 and RANK Combination as a Predictor of Breast Cancer Bone Metastasis in Indonesia

Background: Breast cancer has the highest prevalence and incidence among cancers in women. Moreover, it is the most prevalent among all other cancers in Indonesia. Bone metastasis results in higher incidence, better overall survival, but requires higher-cost treatments than visceral metastasis. Therefore, an early predictor of bone metastasis is needed to make early interventions to be effi ciently given. Instead of other biomarkers of bone metastasis, CXCR4 and RANK are highly expressed in breast cancer and correlate with bone metastasis. Hence this study is expected to prove the potential of CXCR4 and RANK combination as a predictor of breast cancer bone metastasis. Methods: In a case-control study, CXCR4 and RANK immunohistochemistry tests were done in 58 stage I-IV breast cancer Indonesian subjects. Association between marker combinations and incidence of bone metastasis was analyzed. Then, the diagnosis accuracy values were calculated. Moreover, its associations with clinicopathological factors were also examined. Results: There was a signifi cant association of the highly expressed CXCR4 and RANK combination with bone metastasis (P <0.01). Besides, the combination of CXCR4 and RANK detections resulted in 100% sensitivity and 66% specifi city in predicting bone metastasis. Breast cancer's stage was signifi cantly associated with CXCR4 and RANK expressions combination (P<0.01). Conclusions: A combination of CXCR4 and RANK expressions could act as a screening method to predict the bone metastasis in breast cancer.


Introduction
Breast cancer has the highest prevalence and incidence among any other cancers in women [1]. In Indonesia, breast cancer is the most prevalent among all cancer [1,2]. Based on Global Burden of Cancer 2018, the global incidence of breast cancer was around 2,093,876 and the mortality was around 1,761,007. In Indonesia, the incidence was estimated at around 58,256 and the mortality was around 22,692 [1].
Breast cancer frequently metastasizes to bone approximately in 70% of advanced breast cancer patients [3].
Citation: Yulian  Even though bone metastasis results in a better overall survival rather than visceral metastasis, it gives an enormous burden to the treatment cost. Hence, tumor markers are expected to early predict the bone metastasis [3][4][5]. This may help early intervention to enhance the quality of life and reduce the treatment cost of the patient.
Many biomarkers were hypothesized to be implicated in  [6][7][8][9]. Among those markers, CXCR4 and RANK are known to have the greatest association with breast cancer bone metastasis, and both are involved in cancer cell's homing to bones [10]. Moreover, CXCR4 and RANK act in the earliest cascade of the bone metastasis process [11]. Hence, this study aims to prove the potential of combined CXCR4 and RANK detections in predicting the bone metastasis occurrence of a breast cancer patient. All Immunohistochemical (IHC) stains were then examined and scored by experienced breast pathologists. Cells from fi ve random different fi elds were counted using high magnifi cation.

Methods
Positive and negative control specimens for each marker analysis were used in this study for the comparation. Positive controls were breast cancer specimen samples showing both positive marker expressions. Besides, negative controls were specimens prepared through the same procedure but without using the primary antibody.
Besides, the associations of marker expressions with metastasis, menopausal status, cancer stadium, lymph node involvement, hormonal receptor, and HER-2 amplifi cation were also analyzed.
Shapiro-Wilk test was used for normality test, if P>0.05 the data distribution was normal and the data would be presented as mean±deviation standard. Otherwise, the data distribution was considered not normal and the data would be presented as median (minimum-maximum). Chi-Square/Fischer-Exact was used for analytical test for comparing BM and NBM group depended on the normality test.

Results
A total of 58 women breast cancer subjects participated in this study. Each of BM and NBM group included 29 subjects. In BM group, 51.7% had menopause, 100% had late-stage breast cancer, 100% had high tumor grade, 65.5% had No Special Type (NST), 72.4% had lymph node involvement, 86.2% were Estrogen Receptor/ Progesterone Receptor (ER/PR) positive, and 62.1% had HER-2 amplifi cation. In NBM Group, 51.7% had menopause, 27.6% had late-stage breast cancer, 93.1% had high tumor grade, 58.6% had NST type, 44.8% had lymph node involvement, 82.6% were ER/PR positive, and 48.3% had HER-2 amplifi cation. The detailed data on the characteristics of subjects are provided in Table 1. The majority of subjects with bone metastasis had highly-expressed CXCR4 and RANK. In contrast, most of the non-bone metastasis subjects showed low expression of both markers. The representative images of immunohistochemistry results are provided in Figures 1,2.

Association of CXCR4 and RANK expressions with breast cancer bone metastasis
High CXCR4, RANK, and CXCR4+RANK expressions were signifi cantly associated with breast cancer bone metastasis (P <0.01). Breast cancer subjects with high CXCR4 expressions Citation: Yulian Table 2. The authors assessed the diagnostic accuracy of CXCR4, RANK, and CXCR4+RANK expression in breast cancer bone metastasis based on sensitivity, specifi city, positive predictive value, and negative predictive value. The results of the calculation on diagnosis accuracy are provided in Table 3. The sensitivity and negative predictive value of the CXCR4+RANK combination was 100% and higher than the values got from the single marker usage. However, the specifi city and the positive predictive values of the marker combination were lower than the single marker usage.

Association of clinicopathological features with CXCR4 expression, RANK expression and bone metastasis
The relationship between clinicopathological features and breast cancer bone metastasis was assessed. By using bivariate analysis as shown in Table 4, the authors found a signifi cant relationship between breast cancer staging (P <0.01), lymph node involvement (P =0.03), CXCR4 expression (P <0.01), and RANK expression (P <0.01) with breast cancer bone metastasis.
Moreover, the authors analyzed the markers association with clinicopathological factors as provided in Table 5. CXCR4 and RANK were only signifi cantly associated with the stage of breast cancer.

Discussion
In the BM group of this study, we found that CXCR4 was highly expressed in 28 (96.6%) subjects and lowly expressed in 1 (3.4%) patient. RANK was highly expressed in 28 (96.6%)   Citation: Yulian [3]. These molecules are active in primary cancer cells including in their bone metastasis of breast, hepatocellular, and prostate carcinoma [18].
RANK is a surface receptor commonly found on mature osteoclasts and their progenitors [19]. It induces osteoclastogenesis and controls calcium metabolism. RANKL is a polypeptide which found on the surface of osteoblast and bone stromal cells [10]. It binds to RANK on preosteoclast and mature osteoclast. Signaling through RANK would activate transcription factors, which leads to the differentiation of osteoclast progenitors and limits apoptosis of mature osteoclast [20]. On the other hand, OPG (Osteoprotegerin) prevents the RANK-RANKL interaction on the osteoclast cell membrane [21]. If OPG binds to RANKL, osteoclastogenesis will be inhibited, resulting in bone resorption cessation [21]. However, the RANKL-RANK axis promotes the activation, proliferation, and survival of osteoclast. This stimulates tumorigenesis and metastasis in the bone. Studies reported that breast cancer cell expresses RANK protein on their surface, while RANKL acts as a chemotactic factor of cancer cells [19,20].     (SDF-1) is present [22,23]. The overexpression of CXCR4 was found in around 30% of primary breast cancer cells, which is proved to mediate cancer cells migrating to target organs, especially where SDF-1 is abundant such as bone [23].
A recent study noted the CXCR4 expression in primary breast cancer was signifi cantly associated with the development of bone metastases [10,24]. Besides, RANK is highly expressed in primary breast cancer [25]. High expression of RANK in breast cancer stimulates migration to the bone which has a high expression of RANKL. Researchers found that predictive accuracy can be further increased with RANK expression [10].

Conclusions
In conclusion, a combination of CXCR4 high expression and RANK high expression can act as a predictor for breast cancer bone metastasis. Cancer staging plays an important role in determining CXCR4 and RANK expressions in breast cancer bone metastasis. Therefore, CXCR4 and RANK combination can be used as a screening method to predict bone metastasis in early-stage breast cancer in the future.