MicroRNAs Methylation and Expression Profiling for Identification of Breast Cancer Patients at High Risk to Develop Distant Metastases
Conditions: Breast Cancer Female, Metastases Breast
Sponsor: Casa Sollievo della Sofferenza IRCCS

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Study Title: BREMIR MicroRNAs methylation and expression profiling for 
identification of breast cancer patients at high risk to develop distant 
metastases. 
 
   
CLINICAL PROTOCOL and STATISTICAL PLAN 
Protocol for the Collection, Preservation, and Use of Biological Material from Breast 
Cancer Patients to Identify Epigenetic Alterations Associated with Prognosis. 
 
 
English translation from the original document in Italian approved by the Ethical Commettee 
on October 26th 2018. 
 
 

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Rationale 
Breast cancer is the most common malignancy and the second leading cause of death after lung 
cancer in women. The mortality associated with breast cancer is primarily due to metastatic spread 
to other organs, making the ability to predict, identify, and eliminate metastases one of the major 
challenges in the treatment of this disease (1). Recent studies have shown that the propensity of 
tumors to develop metastases is driven by genetic and epigenetic alterations that occur early during 
carcinogenesis (2). Among these alterations, an important role appears to be played by a class of 
small non-coding RNAs known as microRNAs (miRNAs) (3-9). 
Study Objective 
The primary objective of this study is to identify and validate a panel of miRNAs whose 
expression/methylation can predict the development of distant metastases (Metastasis-Free 
Survival, MFS). Secondary objectives include: a) evaluating the association between miRNA 
expression/methylation and overall survival (OS); b) assessing the use of non-invasive methods to 
monitor minimal residual disease during follow-up; c) evaluating methodologies that allow the 
implementation of miRNA expression/methylation analysis in clinical practice. 
 
Experimental Design 
The study consists of four experimental phases to be conducted over three years: 
Phase I: Identification in a retrospective cohort of patients (training cohort) of a panel of miRNAs 
with an expression/methylation profile associated with the development of distant metastases. The 
study will focus on 21 miRNAs for expression alterations and 22 miRNAs for methylation state 
alterations. The retrospective cohort will consist of tumor and normal tissue obtained from 352 breast 
cancer patients, stored at -80°C in the Oncology Laboratory. Expression analysis will be performed 
on RNA extracted from normal and pathological tissues using RT-qPCR at the Oncology Laboratory 
of the IRCCS Casa Sollievo della Sofferenza. Methylation analysis will be conducted using 
pyrosequencing in collaboration with the Bellvitge Biomedical Research Institute (IDIBELL), 
Barcelona, Spain.  
The molecular analysis results will be correlated with pathological data provided by the Pathology 
Unit and clinical data provided by the Breast Surgery Unit and the Oncology Unit. The primary 
endpoint of the study is the identification of miRNAs whose expression/methylation is correlated with 
Metastasis-Free Survival (MFS), while the assessment of associations with Overall Survival (OS) 
and treatment response will be secondary endpoints. RT-qPCR and pyrosequencing data will be 
compared between groups using the non-parametric Wilcoxon rank test. The association between 
endpoints will be evaluated using univariate and multivariate COX regression models. Model 

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calibration, i.e., the evaluation of the agreement between observed outcomes and predictions, will 
be determined using the Hosmer-Lemeshow survival test. The models' ability to discriminate 
between subjects who will develop an event and those who will not will be assessed using the 
modified C-statistic method for censored data. The added prognostic value of the miRNA panel to 
already defined biomarkers will be measured using Integrated Discrimination Improvement (IDI) and 
the increase in C-statistic. The ability of the miRNA panel to improve glioma classification will be 
quantified using the survival-based Net Reclassification Index (NRI), followed by the application of a 
method based on the Kaplan-Meier test (10-13). 
Phase II: Validation of the prognostic and predictive value of the miRNA panel identified in Phase I 
in a prospective cohort (validation cohort). In this phase, the association between the miRNA 
expression profiles and the clinical course of the disease identified in Phase I will be confirmed in an 
independent prospective cohort. Both normal and tumor tissues of the patients, as well as plasma 
collected before surgery and at each follow-up time point, will be analyzed. 
Based on the annual number of surgeries performed at the Breast Surgery Unit, we estimate that 
the prospective cohort will consist of about 500 patients recruited over three years. To participate in 
the study, patients will need to confirm their willingness to donate biological material for molecular 
studies by signing an informed consent form. During the pre-operative visit, 2.5 ml of whole blood in 
Sodium Citrate will be drawn and processed within 1 hour for plasma recovery. After surgical removal 
of the lesion, the reference pathologist will evaluate the biological sample and, if it is adequate in 
quantity and quality to ensure a pathological diagnosis, will proceed with the identification and 
collection of normal and tumor tissue, which will be immediately frozen in liquid nitrogen and stored 
at -80°C. For each patient, 2.5 ml of whole blood in Sodium Citrate will be collected for plasma 
recovery at four subsequent time points: I) at the time of study enrollment; II) at the first outpatient 
visit; III) at the end of treatment; IV) at any disease recurrence. After surgical resection, the Breast 
Surgery staff will send the fresh surgical specimen to Pathology, where the pathologist will assess 
the possibility of sampling, and if positive, will proceed with the isolation of tumor tissue and normal 
tissue at least 2 cm from the neoplasm. The materials will be immediately collected in cryogenic 
tubes labeled with a unique reference code, immersed in liquid nitrogen, and stored at -80°C until 
use. The pathological and clinical data of the patients will be collected respectively by the Pathology 
Unit, the Breast Surgery Unit, and the Oncology Unit and stored in a database. Statistical analyses 
will be conducted as described in Phase I of the project. 
Phase III: Analysis of miRNA expression/methylation in Circulating Tumor Cells (CTCs). 
Approximately 40 patients undergoing treatment at the Oncology Unit of our institution with a 
diagnosis of metastatic breast cancer will be recruited. Before the start of therapy and at each follow-
up time point, 20 ml of blood will be drawn in Sodium Citrate. DNA and RNA obtained from tumor 

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tissue and CTCs will be analyzed for the expression/methylation of the microRNAs selected in 
Phases I and II of the project. 
Phase IV: Evaluation of new methodologies for miRNA expression analysis in routine clinical 
practice. The possibility of applying the in situ hybridization (ISH) method as an alternative to real-
time RT-qPCR to analyze miRNA expression in paraffin-embedded tissues will be evaluated. 
Paraffin-embedded material from a subgroup of 40 patients from the retrospective cohort available 
at our institution's Oncology Laboratory will be provided by the Pathology Unit. Paraffin-embedded 
tissues will be analyzed using the ISH method to evaluate the expression of miRNAs selected in 
Phase I of the study. For each case, the concordance between RT-qPCR performed on fresh tissue 
and ISH performed on paraffin-embedded tissue will be assessed.