Short summary

It is well known that breast cancer-induced bone disease often continues to progress although patients are given anti-resorptive treatment. Up to 50% of breast cancer patients receiving monthly zoledronic acid treatment have new skeletal related events within 1 year of starting treatment and up to 65% within 2 years. Very similar effects are observed during treatment with the novel drug, denosumab. This clearly suggests that the suppression of bone resorption is far less potent than what could theoretically be expected.

The reasons for this incomplete suppression are without a doubt multi-factorial, but one of the reasons may be that although patients are treated as if they are alike they are most certainly not. With our protocol we would like to put our focus on this issue with a clinical and an experimental approach. In the clinical approach we will address the resorptive behavior of osteoclasts in response to cancer disease and treatment in each individual as detected by the levels of bone biomarkers. In the experimental approach we will test the sensitivity of osteoclasts (generated from different individuals) to zoledronic and see if variations amongst the participants explain this variance in sensitivity.

Rationale

Hypotheses of direct patient-related relevance

• The use of bone biomarkers, CTX and PINP, can predict if patients respond to the anti-resorptive treatment earlier than the present routine methods.
• The use of bone biomarkers, CTX and PINP, can predict if patients' cancer-induced bone disease progress earlier than the present routine methods.
• SNPs and/or epigenetic differences can explain the variance in sensitivity to zoledronic acid amongst patients.

• Osteoclasts from different individuals will display a different sensitivity to zoledronic acid in vitro.
• The acidic environment and cytokines generated by growing tumor cells in the bone marrow will directly influence the sensitivity to anti-resorptive treatment and the resorptive behavior of osteoclast in vitro.
• SNPs and/or epigenetic differences can explain the variance in sensitivity to zoledronic acid observed in vitro. 

Description of the cohort

Inclusion criteria:

• 50 female breast cancer patients with newly identified bone metastases that are young than 80 years of age (under 80) (recruitment is expected to take place within the first 2 years of the protocol).
• 50 healthy female blood donors ranging between 40 and 67 years of age (patients will be recruited evenly throughout the first 2 to 3 years).

Exclusion criteria:

• Breast cancer patients that are 80 years or older (?80 years)
• Blood donors that are younger than 40 (<40) or older than 67 years (>67)
• Recent or existing fracture that is NOT related to the metastatic cancer disease
• Blood donors: Prior bisphosphonate treatment.
• Breast cancer patients: Prior bisphosphonate treatment due to osteoporosis

Data and biological material

Samples Day 0:

Breast cancer patients:

• Collect 3 ml blood for SNP/DNA methylation analyses.
• Collect "fasting" blood sample (2x 4 ml) prior to treatment with an anti-resorptive agent to measure CTX (bone resorption marker) and PINP (bone formation marker).

Blood donors:

• Obtain buffy coat.
• Collect additional "fasting" blood sample (2x 4 ml) within 1 or 2 weeks from the time of the blood donation to measure CTX and P1NP.
• Collect additional 3 ml blood sample within 1 or 2 weeks from the time of the blood donation for SNP/DNA methylation analyses.

Data collection Day 0:

Breast cancer patients (data will be obtained by project nurse or by questions from MD to the patient):

• Sex, age, pre-/postmenopausal, start of the last menstruation cycle, weight, height, prior or existing treatment.
• Any previous fractures? When?
• Smoker/non-smoker.
• Medical history.
• Month and year of primary diagnosis
• Date of identification of bone metastasis along with the images used for the diagnosis (X-ray, CT and/or scintigraphy).
• Evaluation of the extent of bone metastasis (early, intermediate, progressed or similar). 

Blood donors (data will be obtained by questionnaire handed out to donor):

• Sex, age, pre-/postmenopausal, start of the last menstruation cycle, weight, height, prior complications? any previous fractures? when?
• Smoker/non-smoker

Follow-up of BC pts ONLY:                     

• Collect "fasting" blood samples (2x 4 ml) every month for the first 12 months and every 3 months for the following two years to measure CTX (bone resorption marker) and P1NP (bone formation marker).
• Patients will be routinely checked for new SRE every 9 to 12 weeks by CT-scan and/or scintigraphy depending on the treatment strategy or by decision of the responsible MD.
• Definition of SRE: new osteolysis/-es identified by CT-scanning, pathologic fractures, the requirement for surgery/radiotherapy and spinal cord compression.

Collaborating researchers and departments

Department of Clinical Cell Biology, Vejle Hospital:

• Associate Professor Kent Søe, PhD, MSc
• Professor Jean-Marie Delaissé, PhD, MSc
• PhD-student Anaïs Møller, MSc
• Research technician Jacob Bastholm Olesen 

Departments of Oncology at Esbjerg Hospital and Vejle Hospitals:

• Consultant Troels Bechmann, PhD, MD
• Consultant Erik H. Jakobsen, MD

Department of Immunology and Biochemistry, Vejle Hospital:

• Consultant/head of department Jonna Skov Madsen, PhD, MD

Department of Genetics, Vejle Hospital:

• Professor Silvia Rogatto, PhD, MSc

Department of Clinical Diagnostics, Esbjerg Hospital:

• Consultant/head of department Anna-Maria Bloch Münster, PhD, MD