role of rank, rankl, opg, and cxcr4 tissue markers in predicting bone metastases in breast cancer...

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Original Study

Role of RANK, RANKL, OPG, and CXCR4Tissue Markers in Predicting Bone Metastases in

Breast Cancer PatientsToni Ibrahim,1 Emanuele Sacanna,1 Michele Gaudio,2 Laura Mercatali,1

Emanuela Scarpi,3 Wainer Zoli,4 Patrizia Serra,1 Rossana Ricci,1 Luigi Serra,2

Yibin Kang,5 Dino Amadori1

AbstractThis is a retrospective study on 40 breast cancer patients, of which 20 have bone metastases, 10 have visceralmetastases, and 10 have no evidence of disease, aimed at evaluating the role of CXCR4 and the RANK/RANKL/OPG system to predict bone metastases. CXCR4 expression, alone or in combination with RANK,identified patients destined to relapse to bone.Background: The RANK/RANKL/OPG system is active in primary cancers such as breast, prostate, and also in their bonemetastases. CXCR4 chemokine receptor is highly expressed in human breast cancer cells and is believed to facilitate thehoming of tumor cells to organs such as bone that express high levels of its ligand SDF1. Our study aimed to investigatewhether the analysis of these markers with an inexpensive and simple test can help to predict bone metastases in breastcancer patients. Patients and Methods: Marker expression was evaluated by immunohistochemical staining in paraffin-embedded tissue sections of primary breast cancers from 40 individuals: 20 patients with bone metastases (BM), 10 withvisceral metastases (VM; considered together as the relapsed group), and 10 with no evidence of disease (NED). Results:RANKL was not detected in tumor cells. OPG- and RANK-positive tumors are found with similar frequency in NED (20%)and in relapsed patients (23% and 17%, respectively). However, in the latter subgroup, only RANK positivity was alwaysassociated with bone relapse. The frequency of CXCR4-positive tumors was three-fold higher in relapsed (30%) than inNED (10%) patients and positivity was always linked to bone metastases. Considering NED and VM patients togetherversus BM patients, we observed that CXCR4 expression, alone (P � .008) or in combination with RANK (P � .001),identified patients destined to relapse to bone. Conclusion: Our results provide the first clinical evidence to support apivotal role of combined CXCR4 and RANK expression in predicting bone relapse.

Clinical Breast Cancer, Vol. 11, No. 6, 369-75 © 2011 Elsevier Inc. All rights reserved.
Keywords: Bone metastases, Breast cancer, CXCR4, OPG, RANK/RANKL
IntroductionMetastasis to distant organs is the primary cause of cancer-related

mortality in most malignant tumors. The development of bone metas-

1Osteoncology Center, Istituto Scientifico Romagnolo per lo Studio e la Cura deiumori (I.R.S.T.), Meldola, Italy

2Pathology Unit, Morgagni-Pierantoni Hospital, Forlì, Italy3Biostatistics and Clinical Trials Unit, I.R.S.T., Meldola, Italy4Biosciences Laboratory, I.R.S.T., Meldola, Italy5Department of Molecular Biology, Princeton University, Princeton, NJ

Submitted: Feb 25, 2011; Revised: May 10, 2011; Accepted: May 11, 2011

Address for correspondence: Toni Ibrahim, BSc, MSc, MD, PhD, OsteoncologyCenter, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.),via P. Maroncelli 40, 47014 Meldola (FC), Italy.
Tel: �39 0543 739251; Fax: �39 0543 739221; e-mail contact: [email protected]
1526-8209/$ - see frontmatter © 2011 Elsevier Inc. All rights reserved.doi: 10.1016/j.clbc.2011.05.001

tases is common in many types of advanced cancer, including prostate,lung, and breast. It is estimated that between 65% and 75% of breast

cancer patients who have relapsed disease develop bone metastasis.1,2

The interaction of tumor cells with the stroma at the metastatic site plays

a critical role in the establishment of metastases.3,4 Tumor cells reaching

the bone microenvironment exploit the normal process of bone remod-

eling by initiating bidirectional pathological interactions with bone cells

and other stromal cells. These interactions, which may occur via direct

cell-cell interaction or may be mediated by soluble factors produced by

tumor cells, stromal cells, or both, lead to the establishment of a “vicious

cycle” of bone metastasis development.5

RANK, the receptor activator of nuclear factor-kB, is expressed on
the surface of osteoclasts. Its binding to RANKL, expressed in osteo-
Clinical Breast Cancer December 2011 369

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pur

Tissue Markers as Bone Relapse Predictors

370

blasts and bone marrow stromal cells and secreted by activatedT-cells, promotes osteoclast differentiation and maturation, inhibitsosteoclast apoptosis, and consequently increases bone resorption.6

RANKL also stimulates the migration of RANK-expressing tumorcells, primary breast epithelial cells, and osteoclasts.7

Osteoprotegerin (OPG) is a member of the tumor necrosis factorreceptor family and a decoy receptor for RANKL; it is secreted byosteoblasts and other cell types and appears to prevent bone destruc-tion by blocking RANKL from binding to its receptor.8 The RANK/RANKL/OPG system, with its functional equilibrium, is involved inthe homeostasis of bone remodeling, and its dysregulation caused byneoplastic cells is responsible for osteolysis associated with malignanttumors and for the development of bone metastases. These moleculesare active in cells of primary cancers such as breast, prostate, andhepatocellular carcinoma, and also in their bone metastases.7,9-11

It is now generally accepted that chemokines and their receptorsplay an important role in the regulation of metastasis organ tropismfrom breast cancer. Although chemokines are known to induce cellmigration, especially of the immune system, there is evidence thattumor cells may use chemokine-mediated mechanisms to spread toother organs. In particular, it has been observed that CXCR4 chemo-kine receptor is highly expressed in human breast cancer cells andthat its ligand, CXCL12/SDF-1�, is highly expressed in organs to

hich breast cancer often metastasizes, eg, bone marrow, lung, liver,nd lymph nodes.12,13

Recently, results of a clinical trial in the adjuvant setting of breastcancer showed that not all patients benefit from bisphosphonatetreatment, highlighting the importance of identifying patients whohave a high probability of developing bone metastases that could betreated.14

Our retrospective case-control study aimed to investigate and de-fine the role of RANK, RANKL, OPG, and CXCR4 expression inpredicting bone metastases from primary breast cancers.

Patients and MethodsStudy Design

This was a retrospective observational case-control study con-ducted at the Istituto Scientifico Romagnolo per lo Studio e la Curadei Tumori (I.R.S.T.), in Meldola, Italy. Our primary objective wasto evaluate the predictive role of RANK, RANKL, OPG, andCXCR4, determined by immunohistochemistry in paraffin-embed-ded tissue sections, in the development of bone metastases frombreast cancer patients. Secondary objectives were to evaluate the cor-relation between markers and biological parameters of the primarytumor, ie, stage at diagnosis, grade, proliferative activity (Ki67), andestrogen-progesterone receptor (ER, PgR) and HER2 status.

The study was designed to focus on three groups of patients whowere operated on for breast cancer: the first group was composed ofpatients with radiologically confirmed bone metastases (BM) thatdeveloped within 10 years of surgery; the second group had radio-logically confirmed visceral metastases (VM) that developed within10 years of surgery; both groups formed the relapsed patient sub-group. The third group included patients with no evidence of disease(NED) at a minimum follow-up of 10 years. The protocol was re-
viewed and approved by the local ethics committee. s
Clinical Breast Cancer December 2011

Inclusion CriteriaFemales (� 18 years old) with histologically confirmed ductal

infiltrating breast cancer were eligible for enrolment. Forty womenwith resected breast cancer were selected from patients who wereobserved from 1997 to 2000 in Morgagni-Pierantoni Hospital inForlì, Italy. ER/PgR, HER2, and Ki67 status was required to beknown and patients could have received adjuvant therapy (chemo-therapy or hormone therapy). The three patient groups werematched for age classes (� 60 years, � 60 years). Surgical tumortissue had to be available for analysis.

Biologic DeterminationsMarker expression and biological and morphological characteris-

tics of tumors were determined on 3-�m sections of primary tumorsin the Pathology Unit of Morgagni-Pierantoni Hospital. Histologi-cal grade was determined by assessing tubule formation, nuclear pleo-morphism, and number of mitotic figures, according to the Elston andEllis classification.15 Cell proliferation was determined by scoring at least1000 cells and expressed as Ki-67 index using mouse monoclonal MIB-1antibody (Dako, Glostrup, Denmark); 20% of immunopositive cellswere considered the cut-off to distinguish between slowly and rapidlyproliferating tumors. Anti-ER6F11 and anti-PgR 16 monoclonal anti-bodies (Novocastra, Newcastle-upon-Tyne, UK) diluted 1:300 and1:600 for 30 minutes, respectively, were used for ER and PgR evalua-tion. A sample was considered positive when more than 10% of immu-nopositive cells were detected.

HER2 expression was determined by immunohistochemistry us-ing HerceptTest (Dako) according to the manufacturer’s instruc-tions. The immunoreaction was scored as follows: 3�, complete andintense membrane staining in � 10% tumor cells; 2�, complete butmoderate staining in � 10% cells, 1�, weak and incomplete stainingin � 10% cells; 0, no membrane staining or staining in � 10% cells.HER2 status was considered positive when the score was � 2�.

Expression of OPG, RANK, RANKL, and CXCR4 was also de-termined in 3-�m thick sections by immunohistochemistry (Table1). Heat-induced epitope retrieval was performed for 30 minutes at98.5°C in citrate buffer at pH 6. Sections were then treated with 3%hydrogen peroxide (H2O2) for 15 minutes to block endogenous

eroxidase and incubated with 1% normal goat serum for 20 min-tes at room temperature. Primary incubations were performed atoom temperature for 1 hour (2 hours for RANKL). The standard

Table 1 Antibody Characteristics

AntibodyAgainst Source Type Diluent Dilution

OPG Santa Cruz H-249 Rabbitpolyclonal PBS-T 1:50

RANK Santa Cruz N-20 Goatpolyclonal PBS-T 1:50

RANKL Abcam Ab45039 Mousemonoclonal

Dako Background-reducing 1:100

CXCR4 Abcam Ab58176 Mousemonoclonal

Dako Background-reducing 1:200

Abbreviation: PBS-T � phosphate buffered saline 0.05% Tween 20.

treptavidin-biotin-peroxidase complex method was used to bind the

sl1(mad

atsmasct

Toni Ibrahim et al

primary antibody (LSAB System Universal Kit, Dako). Reactionproducts were visualized using 0.3% diaminobenzidine (Dako)olution and counterstained with Harris hematoxylin (Bio-Optica, Mi-an, Italy). In the OPG and RANK determinations, an additional0-minute rinse with phosphate buffered saline 0.05% Tween 20PBS-T), pH 7.0 was performed before incubating with 1% nor-al goat serum. An internal positive and negative control for each

ntibody was routinely used for all immunohistochemistryeterminations.The slides were read in a blinded fashion by a pathologist (M.G.)

nd an independent observer (E.S.) at magnification �40 on infil-rating tumor areas. Patterns of cytoplasmic staining, intensity, andtained-cell percentage were assessed. Intensity was scored as low,oderate, or high with respect to background staining. The percent-

ge of positive cells was estimated by calculating the ratio of positivelytained invasive tumor cells to the total number of invasive tumorells. Tumor staining patterns for CXCR4 were defined as high cy-oplasmic expression using the criteria of Cabioglu et al.16-19 Briefly,

moderate staining in � 50% of cells or any strong staining wasconsidered positive. It was possible for patients to have both nuclearand cytoplasmic staining. For other markers, however, staining wascytoplasmic and a sample was considered positive in the presence ofstrong staining in � 30% of cells or moderate staining in � 60% of

Figure 1 Cytoplasmic Positivity for OPG, RANK, and CXCR4 inPrimary Breast Cancer

OPG

RANK

CXCR4

cells (Figure 1).

Breast cancer samples that showed marked positivity for each an-tibody were routinely included in each experiment as positive con-trols. Negative controls were prepared using the same procedure butwithout the primary antibody.

Statistical AnalysisRecent literature data have reported CXCR4 expression values

ranging from 40%16,17 to 60%18,19 in primary breast cancer. There-fore, we hypothesized a CXCR4 value of approximately 40% forNED and VM patients, and of 80% for the BM group. The �2 test,at the fixed significance level of 5%, had 80% power; 10 patients forNED and VM patients and 20 patients for the BM group wererecruited to evaluate this difference. Descriptive statistics were re-ported as proportions and median values. The �2 test was applied todetermine the strength of the association between subgroup of pa-tients and markers. All statistical analyses were performed using theStatistical Package for Social Science (SPSS, version 17.0) and statis-tical significance was defined as P � .05. All P values were two-sided.Multiple logistic regression analysis was performed to analyze therelative risk (RR) and 95% confidence intervals (CI) for bone metas-tases as a response variable, and CXCR4 expression, stage, or hor-mone receptor status as prognostic variables.

ResultsCase Series

The overall series consisted of 40 patients: 10 NED with a medianfollow-up of 10.5 years (range, 10.1 to 11.8 years) and 30 relapsedpatients, of whom 10 had VM and 20 had BM. The median timebetween primary breast cancer diagnosis and detection of the firstmetastasis was 2.5 years (range, 0.1 to 9.0, 95% CI 1.4-4.3). Medianage was 62 years (range, 48 to 78 years) for NED patients, 66 years(range, 52 to 86 years) for VM, and 69 years (range, 42 to 87 years)for BM patients. In all three groups, 50% of patients were � 60 yearsold. No patient enrolled in the present study had received neoadju-vant chemotherapy. Fifty percent of NED patients received chemo-therapy after surgery followed by hormone therapy, 30% receivedchemotherapy alone, and 20% received hormone therapy alone. Tenpercent of VM patients underwent chemotherapy plus hormonetherapy as adjuvant treatment, 70% received chemotherapy, and10% received hormone therapy, whereas 10% did not receive anytreatment. Finally, 45% of BM patients received adjuvant chemo-therapy followed by hormone therapy, 30% received chemotherapy,and 20% received hormone therapy, whereas 5% of patients did notundergo any form of treatment.

Biological and Pathological CharacteristicsTumor characteristics of the overall series are reported in Table 2.

Stage III disease at diagnosis was more frequent in patients whorelapsed (43%), especially with bone lesions (55%), than in NED(10%). Moreover, the frequency of grade 3 tumors was two-foldhigher in VM (60%) and BM (65%) patients than in NED patients(30%). Conversely, Ki-67 index was not strongly associated withrelapse even though a higher, albeit not statistically significant, fre-quency of visceral metastases was observed in patients with rapidlyrather than slowly proliferating tumors (70% versus 30%,
respectively).

.g

e; PgRgroup o


372

Although ER and PgR status of the primary tumor in NED pa-tients and relapsed patients were not substantially different, a break-down analysis revealed that the highest frequency of BM (80%)occurred in patients with ER-positive primary tumors and the high-est frequency of VM (80%) in those with PgR-negative primarytumors. Ninety-five percent of patients relapsed to bone had negativeHER2 status. None of the parameters evaluated were statisticallysignificant.

CXCR4, RANK/RANKL and OPG ExpressionRANKL was never expressed in breast cancer cells in our case series

but only in rare stromal cells. With regard to the other markers,positivity was always evaluated at the cytoplasmic level, varying as afunction of specific markers and patient subgroup. In particular,OPG-positive tumors were similar in NED (20%) and relapsed(23%) patients, independently of site of relapse. Likewise, the fre-quency of RANK-positive tumors was similar in NED (20%) andrelapsed (17%) patients, although positivity in the latter subgroupwas always associated with bone relapse. The frequency of CXCR4-positive tumors was three-fold higher in relapsed (30%) than inNED (10%) patients and marker positivity was always associatedwith the presence of bone lesions (Table 3). Furthermore, normal

Table 2 Biologic and Pathologic Tumor Characteristicsa

NED Patients(n � 10) Overall (n

n % n

Stage

I 3 30 4

II 6 60 13

III 1 10 13

Grade

1 1 10 1

2 6 60 10

3 3 30 19

Ki67

Low 6 60 13

High 4 40 17

ER status

Positive 7 70 20

Negative 3 30 10

PgR status

Positive 4 40 14

Negative 6 60 16

HER-2 status

Positive 1 10 7

Negative 9 90 23

Abbreviations: BM � bone metastasis; ER � estrogen receptor; NED � no evidence of diseasa No correlation was found between clinicopathologic characteristics of primary tumors and sub

tissue in cancer patients was negative for all of the study markers.


Taking into consideration a possible prospective use of the differ-ent markers in predicting bone metastases, we compared NED-VMpatients with the BM group and observed that CXCR4 expression,alone (P � .008) or in combination with RANK (P � .001), iden-tified those who subsequently relapsed to bone. Furthermore, weperformed logistic regression analysis to evaluate the predictive roleof the combination of the two markers with respect to stage and ERstatus. The CXCR4�RANK combination was an independent pre-dictive marker of relapse to bone, increasing the RR of bone relapse9.3-fold in the BM group with respect to NED-VM patients (P �

008). Considering only patients who relapsed to viscera as controlroup, the RR of bone relapse increased 16.1-fold.

DiscussionTo the best of our knowledge, our results are the first to show that

expression of CXCR4 and RANK is the best combination to identifybreast cancer patients whose disease has a high probability to metas-tasize to bone. OPG is similarly expressed in primary breast cancersof NED or relapsed patients regardless of site of metastases. Althoughour data confirmed that breast cancer cells can produce OPG, therewas no evidence to support a predictive role of OPG expression inorgan-specific selection of metastases from primary breast cancer,

Relapsed Patients

) VM (n � 10) BM (n � 20)

n % n %

4 2 20 2 10

3 6 60 7 35

3 2 20 11 55

0 0 1 5

3 4 40 6 30

3 6 60 13 65

3 3 30 10 50

7 7 70 10 50

7 4 40 16 80

3 6 60 4 20

7 2 20 12 60

3 8 80 8 40

3 6 60 1 5

7 4 40 19 95

� progesterone receptor; VM � visceral metastasis.f patients.

� 30

%

1

4

4

4

3

6

4

5

6

3

4

5

2

7

although it is known that OPG is likely to increase survival of breast

fttw

it

le

brs

aa

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att

e; VM �

Toni Ibrahim et al

cancer cells that metastasize to the bone microenvironment.20 In ourcase series, we did not observe any expression of RANKL in primarybreast cancers, in agreement with results reported by Trinkaus et al 21

but in contrast to others.22,23 The difference in our findings may beattributed to the different antibodies used. This, in addition to thefact that RANKL is present in stromal cells, indicates that the role ofthis marker in primary breast cancer requires further investigation.

Recent studies have reported a generally high RANK expression inprimary breast cancer and preclinical data have shown that tumorcells with high RANK levels are stimulated by RANKL to migrate tobone.7 Furthermore, concordance has been found between RANKexpression in the primary and the matched bone metastases fromseveral solid tumors, including breast.24 In our study, RANK wasequally expressed in patients with no evidence of disease and in re-lapsed patients. However, in the latter subgroup, positivity in theprimary tumor was always associated with bone relapse, stronglysuggesting an involvement of this marker in organ selection.

We have already suggested a pivotal role of CXCR4 expression asa predictor of bone metastases in primary breast cancer.25 Althoughit has been suggested that CXCR4 expression may be a key determi-nant in organ selection by tumor cells during metastatic progression,such a hypothesis has yet to be extensively confirmed in clinicalstudies. In agreement with previous studies on breast and other can-cer types, we observed that CXCR4 expression was either nuclear orcytoplasmic,25-27 whereas no or minimal CXCR4 expression wasound in normal breast tissue and in normal tissue adjacent to theumor.28 We had also observed this double staining, but decided inhe present study to use cytoplasmic staining for positivity because itould ensure better assay specificity.25

Shim et al showed that, in cultured cells, CXCL12 binding toCXCR4 induced translocation of CXCR4 to the cytoplasm and nu-cleus of cells.29 The translocation of CXCR4 to the nucleus may benvolved in biological processes and function as a transcription fac-or, as has been described in human lung cancer cells.30

A number of studies have reported that CXCR4 expression in pri-mary breast tumors correlates with the presence of nodal metastases orwith bone marrow involvement.17,18,31 Although the role of the variousocalization patterns of chemokine receptors is not fully understood,ach would seem to have a different biological significance.

Our findings are consistent with those previously reported onreast cancer, prostate cancer, and hepatocarcinoma in which a cor-elation was seen between CXCR4 expression and bone metasta-

Table 3 Frequency of Marker-expressing Tumors

Relaps

NED Patients(n � 10)

Overall(n � 30) (n

% (95% CI) % (95% CI) %

OPG 20 (6-52) 23 (12-41) 20

RANK 20 (6-52) 17 (7-34) 0

CXCR4 10 (0-29) 30 (14-46) 0

Abbreviations: BM � bone metastasis; CI � confidence interval; NED � no evidence of diseas

es.31-33 Recently, CXCR4 expression in primary breast cancer was

lso described as an indicator of the development of bone metastasis,lbeit with low predictive accuracy.32 Importantly, although our cur-

rent study is based on a limited number of patients, our results indi-cate that high expression of CXCR4 can identify patients at high riskof relapse to bone. We also show that the predictive accuracy can befurther increased when this marker is considered together withRANK expression. The combination of the two markers in multi-variate analysis was found to be an independent predictive factor inthe evaluation of bone relapse. It has been reported previously thatthe expression of OPG, RANK, and RANKL in prostate cancer andof CXCR4 in breast cancer are significant and independent predic-tors of a poor prognosis.9,11,33 However, we did not find any corre-lation between any of the studied markers and reduced survival inpatients with breast cancer, possibly because of the small number ofpatients included in the study.

Current research into cancer metastases is focusing on the intrinsicfactors that regulate the homing of tumor cells to the metastatic site.The microenvironment of hematopoietic stem cells (HSC) or nicheregulates HSC fate decisions. The complexity of the niche, with itscellular and non cellular components, has recently been acknowl-edged. There is also growing evidence to suggest that the niche isinvolved in the development of leukemia stem cells and in metastaticbone disease from solid tumors such as breast cancer.34

In animal models, high bone turnover favors tumor cell coloniza-tion in the bone35 and the inhibition of bone turnover with bispho-sphonates has been seen to affect the development and growth pro-gression of experimental bone disease.36 In clinical practice, targetingsteoclastic bone resorption with bisphosphonates has proven to be aseful strategy for improving outcome in an adjuvant breast canceretting37,38 and for preventing skeletal-related events in patients with

metastatic bone disease.39 Recently, a new humanized monoclonalntibody, anti-RANKL (Denosumab), which interferes with the ac-ivity of the RANK/RANKL/OPG axis, has shown to be effective inhe treatment of bone metastases from breast cancer.40 Moreover,

results of the AZURE study, one of the largest phase III studies ofadjuvant bisphosphonates, did not support the routine use of adju-vant zoledronic acid in the management of early breast cancer, al-though subgroup analysis of postmenopausal women, independentlyfrom endocrine receptor status suggested benefit of bisphosphonateon disease-free survival and overall survival.14

These results underline the importance of identifying patientswith a high risk of bone metastases who could benefit from an ap-

atientsP

BM vs NEDP

BM vs VM0)BM

(n � 20)

% CI) % (95% CI) Patients Patients

6-52) 25 (11-47) 1.000 1.000

0 25 (11-47) 1.000 .140

0 45 (23-67) .101 .013

visceral metastasis.

ed P

VM� 1

(95

(

propriate bone-targeted adjuvant therapy.41 In the present retrospec-


mmp

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

2

2

3

3

3

3


374

tive case-control study we analyzed the expression of proteins knownto be involved in bone metastasis, and provided evidence supportingthe use of CXCR4 and RANK in identifying patients with a high riskof bone relapse.

We observed, in agreement with other authors, a relation be-tween ER expression and HER2-negativity of the primary tumorand bone relapse,42 but with low specificity with respect to our

arker. Other studies have reported that the incidence of bonearrow metastases is significantly higher in patients with HER2-

ositive primary tumors.43,44

ConclusionIn conclusion, in our case series, ER-positive and HER2-negative

tumors were observed in a high percentage of patients who relapsedto bone, but also in women who did not develop metastases. On theother hand, CXCR4 expression alone and especially in combinationwith RANK would appear to be a statistically significant predictor ofbone relapse. Such evidence could represent an important tool toidentify patients with a high probability of developing bone metas-tases that could be treated with adjuvant therapy, bisphosphonates,or other bone-targeted therapy, such as Denosumab. We are cur-rently performing studies in a larger series of breast cancer patients tovalidate these findings.

Clinical Practice Points● It has been suggested that CXCR4 expression may be a key deter-

minant in organ selection by tumor cells during metastatic pro-gression, but such a hypothesis has yet to be extensively confirmedin clinical studies.

● Recently, CXCR4 expression in primary breast cancer was alsodescribed as an indicator of the development of bone metastasis,albeit with low predictive accuracy.

● The results from our study are in agreement with these previousstudies. To the best of our knowledge, our results are the first toshow that CXCR4 and RANK is the best combination to identifybreast cancer patients with high probability to metastasize to bone.

● Such evidence could represent an important tool to identify pa-tients with a high probability of developing bone metastases thatcould be treated with adjuvant therapy, bisphosphonates, or otherbone-targeted therapy, such as Denosumab. We are currently per-forming studies in a larger series of breast cancer patients to vali-date these findings.

AcknowledgmentsThe authors thank Prof. Rosella Silvestrini for her invaluable sci-

entific contribution and Gráinne Tierney and Ian Seymour for edit-ing the manuscript.

DisclosureAll authors state that they have no conflicts of interest.

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role of rank, rankl, opg, and cxcr4 tissue markers in predicting bone metastases in breast cancer...

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