First prospective data on breast cancer patients from the multicentre italian bone metastasis database

Bone metastases (BM) are still the main cause of morbidity in cancer patients because of skeletal-related events (SREs) that reduce quality of life. They have also led to increased social and healthcare costs. At present, data available on BM are insufficient. This was a multicentre prospective observational study of patients with BM from breast cancer (BC) with at least 6 months’ follow-up. Information on patients at the first diagnosis of BM, including demographics and characteristics of the primary tumor and BM. Data were periodically updated by participating centres and reviewed by the coordinator centre. From October 2014 to July 2019, 618 patients with BM from solid tumors were enrolled and 220 were eligible for the present study. Median age was 62 years (range 26–86). Median follow-up was 34 months (range 6–149). At the time of enrolment, 109 (50%) had only BM (BOM) and 109 (50%) had concomitant visceral lesions and BM (BVM). Median time-to-first BM was 47 months (range 0–312) in BOM and 78.6 months in BVM patients. Disease-free interval differed on the basis of BC molecular subtype and stage. Ninety-eight BM patients had at least on SRE. Zoledronate was used in 69.1% of cases and denosumab in 28.3%. First-line treatment was hormone-based (50.7%), chemotherapy-based (38.7%) or chemotherapy- + hormone therapy-based (9.7%). Median progression-free and overall survival were 15.1 months (95% CI 12.6–18.4) and 66.8 months (95% CI 52.1–79.2), respectively. Our prospective study could substantially help to better understand the natural history of BM from BC.

There is still limited information available on BM clinical presentation, the difference in disease response between bone and visceral sites, and the difference in prognosis between solitary, oligometastatic and multiple sites or axial and trunk bone metastases 10 . A clearer understanding of their natural evolution would thus help us to identify new strategies capable of reducing both BM incidence and morbidity.
The risk of SREs in BC patients with BM has been the focus of numerous studies [11][12][13] . However, their findings are of limited value because of their poor generalizability with respect to current clinical practice. In the retrospective studies, authors usually considered a lengthy time period during which available therapies and clinical practice may have changed substantially. In the prospective studies, patients were followed for a short period (24 months) and data were extrapolated from a BC database rather than from a database dedicated to BM. Furthermore, in recent years, new therapeutic options have become available. There has also been growing interest in BM since dedicated multidisciplinary groups began to emerge 14 .
The main aims of this prospective multicenter study were to evaluate the evolution of skeletal disease in BC patients, assess the impact of BM on disease outcome, examine the role of a number of clinical-pathological parameters in predicting survival, and further our understanding of the natural history of patients with BM from BC.

Materials and methods
This was a multicentre prospective observational study of patients with BM from BC with at least 6 months follow-up, enrolled into the prospective Italian Bone Metastases Data Base (BMDB). The study was approved by the Local Ethics Committee of each participating centre and carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. List of participating centers was provided in Supplementary Table 1. Written informed consent was obtained from all patients. Information on data source is provided in Appendix 1.
Data extraction and measure definition. The evolution of skeletal disease in BC patients was evaluated by extracting data from the BMDB for patients who had a first diagnosis of BC with a synchronous (within 2 months) or metachronous diagnosis of bone metastasis and were followed up for at least 6 months after the BM diagnosis.
Time to event outcomes were defined as follows: disease-free interval (DFI) was the time from primary BC disease to the appearance of the first metastasis (bone or visceral), and bone disease-free interval (bDFI) was the time between diagnosis of primary BC and first diagnosis of BM. Overall survival (OS) was calculated as the time from the date of the diagnosis of primary BC to the date of death. OS from metastatic disease (metOS) was calculated as the time from the diagnosis of metastasis (either bone or visceral) to death. Progression-free survival (PFS) was the time between the date of the first diagnosis of bone metastasis and date of the first documented evidence of disease progression (bone or visceral) and death. Bone PFS (bPFS) was the time between the date of the first diagnosis of BM and first progression to bone and death. Time-to-first SRE was the time between the first diagnosis of BM and the first SRE event. Patients without events of interest were censored at the date of the last follow-up visit.
Statistical analysis. Descriptive statistics are used to summarize baseline patient characteristics, BM characteristics and treatment patterns. Continuous variables are presented using median and range or interquartile range. The Wilcoxon rank-sum test was used for continuous variables, together with the chi-squared test or Fisher's exact test, as appropriate. McNemar's test was used in cases of paired data. Time-to-event measures were analysed using the Kaplan-Meier method, and the nonparametric log-rank test was used to evaluate the role of stratification factor. We used the Cox proportional hazards regression model to estimate hazard ratios (HRs) and relative 95% confidence intervals (CI) of potential clinical prognostic factors for time-to-event outcomes.
All statistical analyses were performed using STATA/MP 15.0 for Windows (StataCorp LLP, College Station, TX, USA).

Results
Patient characteristics. From October 1st 2014 to June 30th 2018, 618 patients with BM from any solid tumor were registered in the Italian BMDB. Three hundred and nine had BC as the primary site of disease and 220/309 with at least 6 months' follow up were included in the present analysis (Fig. 1 Tables 1  and 2. Luminal A and B tumors were more frequently associated with BOM, whereas basal-like or HER2enriched BC subtypes more often showed BVM (p = 0.012). A higher, albeit not significant, Ki-67 value was observed for patients with BVM (p = 0.074). The majority of patients had T0-T2 (n = 159, 85%) and node-positive tumors (n = 138, 74.6%) at diagnosis (Table 3), the former associated with a higher rate of metachronous BM than synchronous BM (83.2% and 16.8%, respectively) (p < 0.001) ( Table 4). Patients with N0 primary tumors had a higher incidence of metachronous BM than synchronous (95.8% and 4.2%, respectively) (p = 0.001). No difference between BOM or BVM according to node status (node negative vs. node positive tumors) was observed. Both node-negative and node-positive patients showed a high rate of metachronous BM (95.8% and 75.5%, respectively), even if in node-negative patients there is a significantly higher proportion of patients with metachronous BM. Time to event outcomes. Disease-free interval. Disease free-interval was calculated excluding patients with synchronous disease at bone (n = 49) and visceral (n = 2). The disease-free interval (DFI) differed slightly according to molecular subtype. The univariate hazard ratio (HR) for visceral or bone metastasis was higher in luminal B tumors (1.66, 95% confidence interval [CI] 1.1-2.5) (p = 0.023), basal-like tumors (3.92, 95% CI 1.6-9.7) (p = 0.003), and HER2-enriched tumors (1.28, 95% CI 0.7-2.4) (p = 0.442).
Bone disease-free interval. For this analysis, were excluded all patients with synchronous bone metastasis (n = 49). Median time to BM appearance was 78.2 months (95% confidence interval [CI] 63.6-87.9) for all patients.
Time to first SRE. Ninety-eight (44.5%) patients had at least one SRE during the course of their metastatic disease. Patients treated with zoledronic acid or pamidronate had a similar HR for SREs with respect to untreated patients (HR 1.32, 95% CI 0.74-2.38), while those taking denosumab had a HR of 0.20 (95% CI 0.04-0.87), indicating a reduced risk of SRE ( Supplementary Fig. 4). Supplementary data are reported in Appendix 2.

Discussion
Bone metastases represent a common complication of cancer, their incidence reaching around 65% in BC 2 . There are still aspects of bone metastatic disease that need to be further investigated 15,16 .
As reported in previous studies, our case series showed a majority of lytic bone metastases 17,18 . The nature (lytic or not) of BM would not appear to impact patient outcome. Small BC tumors (T0-T2) were associated with metachronous BM, with a time to bone involvement of 65.1 months after the primary diagnosis of BC, indicating that the information collected also regarded patients with latent BM. This subgroup possesses the clinical phenotype of bone metastatic cells characterized by dormancy in which adjuvant BTT could prove useful to prevent BM formation 19 . The mOS from the diagnosis of distant disease (5.5 years) was similar to that reported in other studies, whereas mOS from the primary BC diagnosis differed [20][21][22] . Such findings reflect the good prognosis of the primary BC patients included in our study, representing a real-world population.
Recent studies on a population-based cancer registry and a National Cancer Database observed that patients aged < 60 years now show better survival than those reported in previous studies in which younger age and premenopausal status were associated with poorer survival [22][23][24] . Our study had similar findings, with improved  25,26 .
The initial stage of disease at BC diagnosis represented an important prognostic factor for survival and is consistent with the literature on this topic 27,28 . Some preclinical and clinical studies have reported promising results for concurrent ENDO + CH in postmenopausal patients with metastatic hormone-positive BC 29,30 . In line with these findings, our patients undergoing the ENDO + CH combination showed a slight benefit in terms of mPFS with respect to those treated with ENDO + CH ± BIO. These fascinating suggestions warrant further exploration and validation prospective clinical trials. www.nature.com/scientificreports/ Previous studies have shown a better prognosis for BOM patients than for those with BVM. However, in our study BOM and BVM groups had a similar mOS which may be attributable to a conditioning effect of the molecular BC subtype and also to new treatments available.
A recently published study on BOM reported no correlation between bone pain and survival. Our results are in line with these findings in both BOM and BVM groups. BM localization proved to have a prognostic value in our case series, an appendicular site negatively impacting mOS more than axial or mixed localizations. In a retrospective study conducted by Parkes et al., BOM patients with axial localisation had a mOS from the diagnosis of distant disease of 5.62 years compared to 6.78 years for those with appendicular BM and 4.58 years the appendicular + axial BM group 31 . A possible explanation for this could be the higher incidence of axial bone metastases in luminal BC.
Another interesting finding of our study was that patients with single or oligo-metastases had a better prognosis than those with multiple bone lesions. Although there is already evidence of this for the former 32 , interest in oligo-metastatic disease has recently come to the fore because of its challenging management 33,34 .
It has been seen that changes in BC cell biology occur between primary tumors and metastases 35,36 . In our study a significant difference in PgR expression was observed between primary BC and metastatic bone lesions. www.nature.com/scientificreports/ Clinicians should take this into account in cases of disease recurrence more than 5 years after the primary diagnosis.
Age was found to have a significant impact on OS. This is in line with retrospective works published on BC and BM patients in which age was found to be an independent prognostic factor 30 .
Finally, our data confirm the protective effect of denosumab in preventing SREs, which leveled off over time. Albeit with an incidence reducing over time.
One of the limitations of our study is the patient sample even though it's consistent with other studies in which patients were followed prospectively in a dedicated database on BM and not extrapolated from large registries 28,32 . Another limit is the lack of information available on specific treatments, a result of opting not to enter a large amount of data into the database. To overcome this we grouped treatments into specific categories.
In contrast, the strengths of our study are its collection of prospective data on BM and their clinical evolution and the fact that it constitutes a national representative study population for this disease setting. www.nature.com/scientificreports/ In conclusion, the Italian BMDB represents an invaluable tool to better understand the natural history of bone metastases from breast cancer and improve their management. The Italian BMDB continues to enroll patients also in other solid tumors to increase the case series and give more answer to clinician questions.

Data availability
The datasets gathered and analyzed during the current study are available from the corresponding author on reasonable request.