Cite this asYulian ED, Panigoro SS, Sudijono B, Gautama W, Rustamadji P, et al. (2021) CXCR4 and RANK Combination as a Predictor of Breast Cancer Bone Metastasis in Indonesia. J Surg Surgical Res 7(1): 020-025. DOI: 10.17352/2455-2968.000130
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 efficiently 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 significant 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% specificity in predicting bone metastasis. Breast cancer's stage was significantly 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.
IL-8: Interleukin-8; RANK: Receptor Activator of Nuclear Factor Kappa-B; RANKL: Receptor Activator of Nuclear Factor Kappa-B Ligand; CXCR4: C-X-C Chemokine Receptor Type 4; PTHrP: Parathyroid Hormone-related Protein; B-CTx : Beta-C-Terminal Telopeptide; BM: Bone Metastasis; NBM: Non-Bone Metastasis; IHC: Immunohistochemical; CSLEX-1: Sialyl Lewis X-1 (CSLEX-1) with Cancer Antigen 15-3 (CA 15-3); CA 15-3: Cancer Antigen 15-3; NTx: N-Telopeptide of type I collagen.
Breast cancer has the highest prevalence and incidence among any other cancers in women . 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 .
Breast cancer frequently metastasizes to bone approximately in 70% of advanced breast cancer patients . 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 [3-5]. 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 breast cancer bone metastasis such as Osteopontin, Osteonectin, Interleukin-8 (IL-8), Receptor Activator of Nuclear Factor Kappa-B (RANK), Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), C-X-C Chemokine Receptor Type 4 (CXCR4), bone sialoprotein, tumor cell surface’s integrin, Fas/Fas ligand, Parathyroid Hormone-related Protein (PTHrP), and Beta-C-Terminal Telopeptide (B-CTx) [6-9]. 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 . Moreover, CXCR4 and RANK act in the earliest cascade of the bone metastasis process . 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.
A retrospective case-control study was conducted in a national general hospital. This study had been approved by medical ethics committee of the university. Fifty-eight breast cancer specimens were obtained from an Anatomical Pathology Laboratory between January 2015 to October 2016. Moreover, the consecutive sampling method was used. Paraffin-embedded tissue blocks from stage I-IV breast cancer subjects were divided into two groups based on the presence of bone metastasis (which were confirmed by radiological examinations) within 5 years after initial breast cancer diagnosis (based on data stated in medical record): Bone Metastasis (BM) vs Non-Bone Metastasis (NBM). Paraffin blocks that were damaged in the process or taken from patients with any previous treatment were excluded.
The paraffin-embedded tissue blocks were cut into four μm sections, one tissue section was then stained with Hematoxylin and Eosin to confirm the breast cancer. Two other tissue sections were used for CXCR4 and RANK immunohistochemistry staining. Immunohistochemistry staining was done using an automatic immunohistochemical stainer. Ventana Roche BenchMark XT. CXCR4 GTX22090 (1:100 dilution) and RANK GTX31188 (1:200 dilution) were used as the immunohistochemical markers. Antigen-antibody reactions were visualized by Ultraview Brown Counterstain DAB Detection Kit (Ventana Medical Systems).
All Immunohistochemical (IHC) stains were then examined and scored by experienced breast pathologists. Cells from five random different fields were counted using high magnification.
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.
The following IHC staining scoring is taken and modified from the previous method used in a study by Sun, et al. . The percentage of CXCR4 positive tumor was determined semi-quantitatively as follows: 0 (0%), 1 (1-10% positive cells), 2 (11-50% positive cells), 3 (51-80% positive cells), and 4 (81-100% positive cells). Cytoplasmic staining intensity was classified into 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Then, the positive cell percentage and staining intensity were multiplied to define a composite score. Composite score was classified into 0 (negative), + (1-4), ++ (5-8), and +++ (9-12). CXCR4 was considered highly expressed if the composite score was ++ or +++ (5-12 score). The intensity of RANK staining was classified into four groups based on a method by Li, et al. including absent (0), positive but less intense (1), same intense (2), and more intense (3) compared to the positive control . A more than 1 staining intensity score in more than 50% of cancer cells was considered as highly expressed. The combination of CXCR4 and RANK expressions was considered high if one or both of the markers were highly expressed.
Besides, the associations of marker expressions with metastasis, menopausal status, cancer stadium, lymph node involvement, hormonal receptor, and HER-2 amplification 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.
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 amplification. 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 amplification. 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.
High CXCR4, RANK, and CXCR4+RANK expressions were significantly associated with breast cancer bone metastasis (P <0.01). Breast cancer subjects with high CXCR4 expressions would have 134.4 folds of risk for bone metastasis than low CXCR4 expressions. Meanwhile, breast cancer subjects with high-RANK expressions would have 88 folds of risk for bone metastasis than low-RANK expressions. The results of the analysis of the associations can be seen in Table 2. The authors assessed the diagnostic accuracy of CXCR4, RANK, and CXCR4+RANK expression in breast cancer bone metastasis based on sensitivity, specificity, 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 specificity and the positive predictive values of the marker combination were lower than the single marker usage.
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 significant 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 significantly associated with the stage of breast cancer.