Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Risk association of RANKL and OPG gene polymorphism with breast cancer to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan

Abstract

Introduction

The receptor activator NF-κB ligand (RANKL) and Osteoprotegrin (OPG) single nucleotide polymorphisms (SNPs) have been associated with the risk of breast cancer to bone metastasis. This study was designed to investigate the association of RANKL and OPG gene polymorphisms with breast to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan.

Materials and methods

A total of 215 participants were enrolled containing 106 breast cancer patients, 58 breast to bone metastasis and 51 age and gender matched healthy controls. RANKL (rs9533156) and OPG (rs2073618, rs3102735) polymorphisms were genotyped in genomic DNA, using Tetra-ARMS PCR protocol. The results were analyzed among the three groups and P-value less then 0.05 were considered statistically significant.

Results

Our results displayed significant association of OPG (rs3102735) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis as a disease risk. However, there was no association observed for OPG (rs2073618) risk allele and corresponding genotypes with the diseases risk. Similarly, RANKL (rs9533156) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis exhibited significant association except for the risk allele carrying genotypes in breast to bone metastasis.

Conclusion

OPG (rs3102735) and RANKL (rs9533156) exhibited significant association with breast to bone metastasis while OPG (rs2073618) didn’t show significant association with breast to bone metastasis in Pashtun population of Pakistan. However, this study unlocks more questions to investigate the exact scenario of genetic predisposition of breast to bone metastasis.

Citation: Hayat F, Khan NU, Khan AU, Ahmad I, Alamri AM, Iftikhar B (2022) Risk association of RANKL and OPG gene polymorphism with breast cancer to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan. PLoS ONE 17(11): e0276813. https://doi.org/10.1371/journal.pone.0276813

Editor: Filomena de Nigris, Universita degli Studi della Campania Luigi Vanvitelli, ITALY

Received: August 24, 2022; Accepted: October 14, 2022; Published: November 8, 2022

Copyright: © 2022 Hayat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting information files.

Funding: The authors appreciate the support of the Research Center for Advanced Materials Science (RCAMS) at King Khalid University Abha, Saudi Arabia through a project number RCAMS/KKU/22. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Breast cancer is the most frequently diagnosed cancer in women worldwide [1] and one of the leading cancers in Asian countries, including Pakistan [2]. Breast cancer is regularly metastasizing to various organs such as bone (60–80%), lung (57–77%), liver (5–12%) and brain (10–30%) [3–6]. The process of metastasis is a multi-step sequential cascade starting primary tumor local invasion, progression towards lymphogenic spread and colonization at distant sites [7]. Genetic factors may predispose the probability of breast cancer metastasize to bone [8]. The discovery of the apoptosis regulator receptor activator NF-κB ligand (RANKL) and osteoclastogenesis inhibitor OPG signaling pathway exhibited major regulatory system for osteoclast formation and also OPG/RANKL alliance controlled the bone remodeling [9, 10], contributed significantly to the physiological bone turnover and also involved in osteoclast differentiation and activation [11].

RANKL-OPG pathway contributes to the primary tumorigenesis, bone metastasis and regulated by factors including progesterone and prolactin. Skeletal diseases caused by excessive osteoclastic activity including breast cancer to bone metastasis, rheumatoid arthritis and osteoporosis were triggered by imbalance between RANKL and OPG genes [12–15]. In addition, the RANKL and OPG also involved in proliferation of malignant and benign breast tumors [9]. Over expression of OPG by cancerous cells, increases tumor growth and cell proliferation [16]. OPG genetic variants are associated with bone mineral density, bone turnover and osteonecrosis [17–19].

RANKL polymorphism located in the proximal promoter interacts with transcription factors such as heat shock proteins, E2F1 (E2F transcription factor 1), vitamin D3 and core-binding factor a1 (Cbfa1), which affect the expression of RANKL gene [20, 21]. RANKL rs9533156 (T>C) causes DNA hypermethylation leading to gene silencing and transcriptional inactivation, that affects the binding of transcriptional factors [22, 23]. OPG rs2073618 influence secretions from the cells and drastically affect the secretory kinetics [10].

RANKL/OPG pathway suggests an important effect on the pathogenesis of breast cancer due to functional properties of genes. Genetic variation within OPG (rs3102735) promoter region and RANKL (rs9533156) near 5’ region could have an effect on the expression of genes and may have an influence on the development of tumors [10, 12]. Various studies conducted have elucidated the association of RANKL and OPG polymorphism with the breast cancer to bone metastasis risk in different ethnicities [24]. However, unfortunately, there is no such work done on Pakistani population practically the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Here this study aimed to investigate the association of above-mentioned polymorphisms with breast cancer to bone metastasis in the patients of Pashtun population of Khyber Pakhtunkhwa, Pakistan.

Materials and methods

Study design, ethical approval and sampling

This case-control study was conducted at the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar (UAP), Pakistan. Patients were recruited from the Institute of Radiotherapy and Nuclear Medicine (IRNUM) Peshawar, Pakistan. The study protocol and informed consent (patients proforma) were approved (IBGE/UAP/2020/003) by the Ethical Committee of IBGE, UAP. Written informed consent was taken from all the participants while explaining the aim and objectives of the study. A total of 215 participants were enrolled, with age ranging from 25–80 years, containing (106 breast cancer patients only, 58 breast cancer to bone metastasis, and 51 healthy controls) from January 2021–December 2021. The demographic, clinical parameters and blood samples were collected from all the subjects through venipuncture procedure using sterile syringe. The blood was stored at– 20ºC in EDTA tubes at IBGE until further process.

DNA isolation, SNP genotyping and gel electrophoresis

DNA was extracted from the whole blood using salting out (non-enzymatic) method and SNP genotyping was confirmed via T-ARMS PCR, previously adopted in our lab [25, 26]. Specific primers were designed using primer blast software for each SNP (Table 1). The PCR mixture (10μL) was prepared, consisting of 5μL master mix (Thermo Fisher Scientific, DreamTaq Master Mix (2X), 0.5μL of each forward and reverse primer, 3μL of ddH2O and 1μL of template DNA. The PCR condition were, initial denaturation at 95°C for 5min. followed by 35 cycles, denaturation at 95°C for 30 sec. annealing for 30 sec. at 61°C, 63°C and 60°C for OPG rs2073618, rs3102735 and RANKL rs9533156 respectively, extension 72°C for 30 sec. and final extension at 72°C for 7min. The amplified PCR products were run and confirmed using 1.5% gel electrophoresis with 1KB DNA ladder (Thermo Fisher Scientific).

thumbnail
Download:
Table 1. Specific primer sequences used for genotyping.

https://doi.org/10.1371/journal.pone.0276813.t001

Statistical analysis

SPSS software (version 2.0) was used for statistical analysis. Demographic data was analyzed via descriptive statistical method and p<0.05 was considered significant. Chi-square test was performed for categorical data, while t-test and one-way ANOVA was applied for analysis of quantitative data. The genotypic and allelic frequency was calculated by using chi-square and Fisher exact test. Online software named Medcalc was used for obtaining odd ratios and 95% CI.

Results

Demographic and clinicopathological parameters association with diseases risk

A total of 215 participants were recruited in this study, consisting of (breast cancer patients, BC = 106, breast to bone metastasis, BM = 58, and healthy controls, HC = 51). The demographics and clinical characteristics association was checked among the study groups (Table 2). The results showed no significant association among the ages (p = 0.811), tumor size (p = 0.268), stage (p = 0.067), HER2 status (p = 0.284), PR status (p = 1.00), ER status (p = 0.940) and Luminal subtypes (p = 0.639). However, significant association was observed in menstrual status (p = 0.0001), nulliparous (p = 0.0001), family history (p = 0.0001), localization (p = 0.0001), Ki67 (p = 0.0001), histology (p = 0.0001), nodal status (p = 0.0001) and IHC subtypes (p = 0.0001).

thumbnail
Download:
Table 2. Demographics, clinical and pathological parameters and their association with the disease risk.

https://doi.org/10.1371/journal.pone.0276813.t002

OPG (rs3102735) and RANKL (rs9533156) exhibited significant association with breast cancer risk while OPG (rs2073618) didn’t show association compared with healthy controls

All the samples were analyzed for SNPs genotyping using T-ARMS-PCR, run and confirmed on 2% agarose gel with 1KB DNA ladder (Fig 1). The results indicated that OPG (rs2073618) risk allele C (OR: 1.2293 CI: 0.7651–1.9751, p = 0.3934) and risk allele containing genotypes CC (OR: 1.7262 CI: 0.6016–4.9532, p = 0.3101) and GC (OR: 1.5517 CI: 0.6165–3.9056, p = 0.3508) were statistically not significantly associated with breast cancer risk. In contrast, the risk allele of OPG (rs3102735) C (OR: 0.4882 CI: 0.2959–0.8057, p = 0.005) and respective genotypes CC (OR: 0.2365 CI: 0.0696–0.8032, p = 0.0208) and TC (OR: 0.4138 CI: 0.1991–0.8599, p = 0.0181) were statistically significant. Similarly, in case of RANKL (rs9533156), the frequency of risk allele C (OR: 2.5887 CI: 1.5924–4.2082, p = 0.0001) and respective genotypes CC (OR: 8.1818 CI: 2.7796–24.0836, p = 0.0001) and TC (OR: 3.7500 CI: 1.4028–10.0243, p = 0.0084) were significantly associated with disease risk (Table 3).

thumbnail
Download:
Fig 1. Electrophoretogram showing T-ARMS PCR based amplification of random samples (a) OPG (rs2073618), (b) OPG (rs3102735), (c) RANKL (rs9533156), L, 1KB DNA ladder (Thermo Fishier Scientific).

https://doi.org/10.1371/journal.pone.0276813.g001

thumbnail
Download:
Table 3. Allelic and genotypic frequencies of OPG and RANKL in breast cancer vs healthy controls.

https://doi.org/10.1371/journal.pone.0276813.t003

OPG (rs3102735) and RANKL (rs9533156) exhibited risk association with breast cancer to bone metastasis while OPG (rs2073618) didn’t show association compared with healthy controls

The genotypic and allelic frequencies of OPG and RANKL in bone metastasis and healthy controls were investigated (Table 4). The results indicated that OPG (rs2073618), risk allele C (OR: 1.0990 CI: 0.6448–1.8731, p = 0.7282) and risk allele containing genotypes CC (OR: 1.2963 CI: 0.3961–4.2421, p = 0.6679) and GC (OR: 1.3410 CI: 0.4805–3.7422, p = 0.5752) didn’t show statistically significant association. Unlikely, OPG (rs3102735) indicated that risk allele C (OR: 1.8857 CI: 1.1000–3.2327, p = 0.0211) and genotypes CC (OR: 4.5714 CI: 1.3382–15.6164, p = 0.0153) and TC (OR: 2.4286 CI: 0.9068–6.5041, p = 0.0775) were significantly associated with bone metastasis. Similarly, the RANKL (rs9533156) risk allele C (OR: 5.000 CI: 2.7280–9.1642, p = 0.0001) and homozygous mutant genotype CC (OR: 11.1818 CI: 3.3299–37.5486, p = 0.0001) were significantly associated with high risk of breast to bone metastasis but the heterozygous genotype TC (OR: 1.4400 CI: 0.4234–4.8972, p = 0.5593) was not associated with disease risk.

thumbnail
Download:
Table 4. Frequencies of OPG and RANKL polymorphisms in bone metastasis vs healthy controls.

https://doi.org/10.1371/journal.pone.0276813.t004

OPG (rs3102735) and RANKL (rs9533156) exhibited association with breast to bone metastasis compared with breast cancer patients

The frequencies of OPG and RANKL in breast cancer and breast to bone metastasis were also checked (Table 5). The results displayed that OPG (rs2073618) risk allele C (OR: 1.1186 CI: 0.7094–1.7639, p = 0.6295) and risk allele containing genotypes CC (OR: 1.3316 CI: 0.4647–3.8155, p = 0.5938) and GC (OR: 1.1571 CI: 0.4548–2.9440, p = 0.7593) were not associated with breast to bone metastasis compared with breast cancer patients. Conversely, OPG (rs3102735) risk allele C (OR: 0.1704 CI: 0.0716–0.4053, p = 0.0001) and genotypes CC (OR: 0.0517 CI: 0.0157–0.1708, p = 0.0001) and TC (OR: 0.1704 CI: 0.0716–0.4053, p = 0.001) were were strongly associated with breast to bone metastasis. Likewise, RANKL (rs9533156) risk allele C (OR: 0.5177 CI: 0.2994–0.8953 p = 0.0185) was found significant with disease risk, while the genotypes CC (OR: 0.7317 CI: 0.2220–2.4114, p = 0.6077) and TC (OR: 2.6042 CI: 0.7221–9.3922, p = 0.1436) were not associated with diseases risk.

thumbnail
Download:
Table 5. OPG and RANKL frequencies in breast cancer and breast to bone metastasis.

https://doi.org/10.1371/journal.pone.0276813.t005

Discussion

Breast cancer is also one of the frequently diagnosed cancers in woman of Pashtun population of Khyber Pakhtunkhwa, Pakistan. Unfortunately, there is very little work conducted so far, to explore the genetic predisposition of breast cancer and associated metastasis in this region. Here, this study aimed to check the association of OPG (rs2073618, rs3102735) and RANKL (rs9533156) polymorphism with breast cancer to bone metastasis, as there is no report available for the said population. However, similar studies have been conducted in different ethnicities worldwide to confirm the association of OPG and RANKL polymorphism with breast cancer to bone metastasis, which displayed contradictory results [24, 27].

Interestingly, our results demonstrated that OPG (rs2073618), risk allele C (p = 0.3934) and corresponding genotypes GC (p = 0.3508 and CC (p = 0.3101) didn’t establish significant association with breast cancer risk and also no significant correlation was observed between risk allele C (p = 0.7282) and risk allele containing genotypes GC (p = 0.5752, CC (p = 0.6679) with breast to bone metastasis when compared to healthy controls (Tables 3 and 4), which is in contrast to the previous study conducted in Egyptians and Chinese Han population [10, 14, 28, 29]. Another study conducted among Caucasians (rs2073618), which revealed the minor allele to be protective against breast cancer pathogenesis [30]. However, in the present study individuals carrying homozygous mutant genotype (CC) were at increased risk of breast cancer (OR = 1.7262 95% CI = 0.6016–4.9532) and breast to bone metastasis (OR = 1.3316, 95% CI = 0.4647–3.8155) as compared to the individuals carrying heterozygous genotype. Regarding the OPG (rs3102735), the results were found significant between risk allele and risk allele containing genotypes with breast cancer and breast to bone metastasis. Our study result is similar to that of the Caucasian population, where the minor allele C and the corresponding genotypes CC and TC were more frequent in breast cancer patients and showed 1.5-fold increased risk [10]. However, more investigation will be required with large data sets to validate the association of OPG polymorphism with breast to bone metastasis.

Concerning the RANKL (rs9533156), significant association was observed between the risk allele and corresponding genotypes with breast cancer and breast to bone metastasis. Although, in the current study individuals carrying homozygous mutant genotype (CC) were at increased risk of breast cancer (OR = 8.1818 95% CI = 2.7796–24.0836) as compared to breast to bone metastasis (OR = 0.7317, 95% CI = 0.2220–2.4114). The mutant genotype frequency was found higher among breast cancer than bone metastasis patients. Previous study among Egyptians revealed T the major allele frequently among the breast cancer cases, while the present research found minor allele C repeatedly among breast cancer patients when compared to the healthy controls, which is in contrast to the study done in Caucasian and Egyptian population [10, 14]. A study expressed that minor allele as well as the genotype of the minor allele of the RANKL rs9533156 was strongly associated with a higher BMI (>/ = 28) patients with breast cancer group. Whether obese patients carrying the minor allele from one of the two RANKL SNPs have an additionally a higher risk of developing breast cancer remains open in this study.

Excluding the contradictory results of genetic polymorphism and risk of breast to bone metastasis in various ethnicities, both OPG and RANKL have important role in breast cancer to bone metastasis proliferation and progression [31]. However, more investigation will be required to see the exact mechanism of both OPG and RANKL polymorphism and their association with breast cancer to bone metastasis in various population.

Conclusion

Our study revealed that OPG (rs3102735) and RANKL (rs9533156) exhibited significant association with the risk of breast to bone metastasis in Pashtun population, while OPG (rs2073618) didn’t show significant association with disease risk. However, more investigation will be required while targeting the full gene sequencing or whole exome sequencing to find the exact situation of genetic predisposition of breast to bone metastasis in Pashtun Population.

References

  1. 1. Bray F, Ferlay J, Soerjomataram I. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 2018;394–424.
  2. 2. Menhas R, Umer S. Breast cancer among Pakistani women. Iran J Public Health. 2015;44:586–7. pmid:26056679
  3. 3. Parkes A, Clifton K, Al-Awadhi A, Oke O, Warneke CL, Litton JK, et al. Characterization of bone only metastasis patients with respect to tumor subtypes. npj Breast Cancer. Springer US; 2018;4:1–7.
  4. 4. Xiao W, Zheng S, Liu P, Zou Y, Xie X, Yu P, et al. Risk factors and survival outcomes in patients with breast cancer and lung metastasis: a population-based study. Cancer Med. 2018;7:922–30. pmid:29473333
  5. 5. Bale R, Putzer D, Schullian P. Local treatment of breast cancer liver metastasis. Cancers (Basel). 2019;11:1–15. pmid:31514362
  6. 6. Brosnan EM, Anders CK. Understanding patterns of brain metastasis in breast cancer and designing rational therapeutic strategies. Ann Transl Med. 2018;6:163–163. pmid:29911111
  7. 7. Van Zijl F, Krupitza G, Mikulits W. Initial steps of metastasis: Cell invasion and endothelial transmigration. Mutat Res—Rev Mutat Res. Elsevier B.V.; 2011;728:23–34. pmid:21605699
  8. 8. Soni A, Ren Z, Hameed O, Chanda D, Morgan CJ, Siegal GP, et al. Breast cancer subtypes predispose the site of distant metastases. Am J Clin Pathol. 2015; pmid:25779997
  9. 9. Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys. 2008;473:139–46. pmid:18395508
  10. 10. Ney JT, Juhasz-Boess I, Gruenhage F, Graeber S, Bohle RM, Pfreundschuh M, et al. Genetic polymorphism of the OPG gene associated with breast cancer. BMC Cancer. 2013;13. pmid:23369128
  11. 11. Weitzmann MN. The Role of Inflammatory Cytokines, the RANKL/OPG Axis, and the Immunoskeletal Interface in Physiological Bone Turnover and Osteoporosis. Scientifica (Cairo). 2013;2013:1–29.
  12. 12. Sisay M, Mengistu G, Edessa D. The RANK/RANKL/OPG system in tumorigenesis and metastasis of cancer stem cell: Potential targets for anticancer therapy. Onco Targets Ther. 2017;10:3801–10. pmid:28794644
  13. 13. Sarink D, Schock H, Johnson T, Overvad K, Holm M, Tjønneland A, et al. Circulating RANKL and RANKL/OPG and breast cancer risk by ER and PR subtype: Results from the EPIC cohort. Cancer Prev Res. 2017;10:525–34. pmid:28701332
  14. 14. Omar HS, Shaker OG, Nassar YH, Marzouk SA, ElMarzouky MS. The association between RANKL and Osteoprotegerin gene polymorphisms with breast cancer. Mol Cell Biochem. 2015;403:219–29. pmid:25724681
  15. 15. Santini D, Schiavon G, Vincenzi B, Gaeta L, Pantano F, Russo A, et al. Receptor activator of NF-kB (rank) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients. PLoS One. 2011;6. pmid:21559440
  16. 16. Fisher JL, Thomas-Mudge RJ, Elliott J, Hards DK, Sims NA, Slavin J, et al. Osteoprotegerin overexpression by breast cancer cells enhances orthotopic and osseous tumor growth and contrasts with that delivered therapeutically. Cancer Res. 2006;66:3620–8. pmid:16585187
  17. 17. Hsu YH, Niu T, Terwedow HA, Xu X, Feng Y, Li Z, et al. Variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are associated with bone mineral density at different skeletal sites in men. Hum Genet. 2006;118:568–77. pmid:16249885
  18. 18. Roshandel D, Holliday KL, Pye SR, Boonen S, Borghs H, Vanderschueren D, et al. Genetic variation in the RANKL/RANK/OPG signaling pathway is associated with bone turnover and bone mineral density in men. J Bone Miner Res. 2010;25:1830–8. pmid:20205168
  19. 19. Katz J, Gong Y, Salmasinia D, Hou W, Burkley B, Ferreira P, et al. Genetic polymorphisms and other risk factors associated with bisphosphonate induced osteonecrosis of the jaw. Int J Oral Maxillofac Surg. 2011;40:605–11. pmid:21396799
  20. 20. Kim K, Kim JH, Lee J, Jin HM, Lee SH, Fisher DE, et al. Nuclear factor of activated T cells c1 induces osteoclast-associated receptor gene expression during tumor necrosis factor-related activation-induced cytokine-mediated osteoclastogenesis. J Biol Chem. © 2005 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.; 2005;280:35209–16. pmid:16109714
  21. 21. Hu Y, Sun M, Nadiminty N, Lou W, Pinder E, Gao AC. Transcriptional regulation of human RANK ligand gene expression by E2F1. Biochem Biophys Res Commun. Elsevier Inc.; 2008;370:440–4. pmid:18381203
  22. 22. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology. Nature Publishing Group; 2013;38:23–38. pmid:22781841
  23. 23. Rasmussen KD, Helin K. Role of TET enzymes in DNA methylation, development, and cancer. Genes Dev. 2016;30:733–50. pmid:27036965
  24. 24. Geerts D, Chopra C, Connelly L. Osteoprotegerin: Relationship to Breast Cancer Risk and Prognosis. Front Oncol. 2020;10:1–7.
  25. 25. Shah M, Danish L, Khan NU, Zaman F, Ismail M, Hussain M, et al. Determination of mutations in iron regulating genes of beta thalassemia major patients of Khyber Pakhtunkhwa, Pakistan. Mol Genet Genomic Med. 2020;8:1–10. pmid:32588561
  26. 26. Adnan F, Khan NU, Iqbal A, Ali I, Petruzziello A, Sabatino R, et al. Interleukin-6 polymorphisms in HCC patients chronically infected with HCV. Infect Agent Cancer. Infectious Agents and Cancer; 2020;15:1–7.
  27. 27. Hein A, Bayer CM, Schrauder MG, Häberle L, Heusinger K, Strick R, et al. Polymorphisms in the RANK/RANKL genes and their effect on bone specific prognosis in breast cancer patients. Biomed Res Int. 2014;2014.
  28. 28. Shaker OG, Senousy MA. Association of SNP-SNP Interactions Between RANKL, OPG, CHI3L1, and VDR Genes With Breast Cancer Risk in Egyptian Women. Clin Breast Cancer [Internet]. Elsevier Inc.; 2019;19:e220–38. Available from: pmid:30309792
  29. 29. Wang J, Lu K, Song Y, Zhao S, Ma W, Xuan Q, et al. RANKL and OPG polymorphisms are associated with aromatase inhibitor-related musculoskeletal adverse events in Chinese Han breast cancer patients. PLoS One [Internet]. 2015;10:1–17. Available from: pmid:26218592
  30. 30. Assmann G, Juhasz-Boess I, Gruenhage F, Graeber S, Wieczorek S, Pfreundschuh M, et al. Genetic polymorphisms of the OPG gene associated with breast cancer. J Clin Oncol [Internet]. Wolters Kluwer; 2012;30:1523. Available from:
  31. 31. Infante M, Fabi A, Cognetti F, Gorini S, Caprio M, Fabbri A. RANKL/RANK/OPG system beyond bone remodeling: involvement in breast cancer and clinical perspectives. Available from: pmid:30621730