Circulating miRNAs.

Primary Objectives:

• To identify a panel of miRNAs, detectable in the circulation, which are altered in breast cancer patients
• To identify specific combinations of miRNAs ('signatures') which associate with breast cancer intrinsic subtypes, and thereby could aid in prognostication and treatment planning on an individual patient basis.

Secondary Objective:

• To determine if systemic miRNA analysis can be used as a biomarker for monitoring response to chemotherapy, in the neoadjuvant setting and in patients who present with breast cancer recurrence and are treated with upfront chemotherapy and/or hormonal therapy.

This is a prospective cohort studies, involving three study cohorts:

Cohort 1: All newly diagnosed breast cancer patients who are scheduled to undergo neoadjuvant chemotherapy.

Cohort 2: All breast cancer patients who present with metastatic disease, disease recurrence or progression, who are commencing up-front chemotherapy +/- hormonal therapy.

Cohort 3: All breast cancer patients who present with metastatic disease who are commencing hormonal therapy only.

Blood Sample

Blood Sampling - Cohort 1:

• Blood sample 1: at presentation before commencing neoadjuvant treatment.

• Blood sample 2: midway through their chemotherapy treatment (after 2nd cycle if they are enrolled in a 4 cycle regimen, or after 4th cycle if they are prescribed an 8 week regimen).

• Blood sample 3: post-chemotherapy (before surgery as applicable).

• Blood sample 4: 2 to 4 weeks after surgery, or 2 to 4 weeks post 3rd blood sampling if patients do not undergo surgery.

• Blood sample 5: once during follow-up of 12 to 18 months after surgery or 12 to 18 months post 3rd blood sampling if patients do not undergo surgery.

Blood Sampling - Cohort 2:

• Pre-treatment blood sample: at presentation before commencing treatment.

• On study blood samples: taken at monthly (± 1 week) intervals for a period of 6 months from date of pre-treatment blood sample, despite whether the patient is on treatment or completed treatment.

On study blood sample 1: 1 month (± 1 week) from date of pre-treatment blood sample

On study blood sample 2: 2 months (± 1 week) from date of pre- treatment blood sample

On study blood sample 3: 3 months (± 1 week) from date of pre-treatment blood sample

On study blood sample 4: 4 months (± 1 week) from date of pre-treatment blood sample

On study blood sample 5: 5 months (± 1 week) from date of pre-treatment blood sample

On study blood sample 6: 6 months (± 1 week) from date of pre-treatment blood sample

• End of study blood sample: once during follow-up of 12 to 18 months from date of pre-treatment blood sample or at the time of disease progression.

Blood Sampling - Cohort 3:

• Pre-treatment blood sample: at presentation before commencing treatment.

• On study blood samples: taken at the following time points despite whether the patient is on treatment or completed treatment.

On study blood sample 1: 3 months (± 2 weeks) from date of pre-treatment blood sample On study blood sample 2: 6 months (± 2 weeks) from date of pre-treatment blood sample On study blood sample 3: 12 months (± 2 weeks) from date of pre-treatment blood sample

• End of study blood sample: 18 months (± 2 weeks) from date of pre-treatment blood sample or at the time of disease progression.

Blood samples will be processed for miRNA analysis, which involves:

• Lysis using Trizol 2. RNA isolation 3. Assessing concentration and integrity of RNA using Nanodrop spectrophotometry 4. cDNA synthesis (using miRNA specific stem loop primers) 5. PCR amplification and relative quantification (using miRNA specific probes)

Inclusion criteria

• Patient must meet the criteria for either:

Cohort 1: All patients with a new diagnosis of breast cancer, who are destined to undergo neoadjuvant chemotherapy.

OR Cohort 2: All breast cancer patients who present with metastatic disease, disease recurrence or progression who will receive up-front chemotherapy ± hormonal therapy.

OR Cohort 3: All breast cancer patient who present with metastatic disease who are commencing hormonal therapy only.

• Patients must be aged 18 years or over.
• Patients must be able to give written informed consent.

Exclusion criteria

All patients, who do not fulfil the inclusion criteria mentioned above.