A Study of Biological, Genetic, and Constitutional Factors and Non-invasive Monitoring to Assess Personal Cancer Risks

A Platform for Assessing Personal Risk of Developing or Recurring of Cancer: Study of Biological, Genetic, and Constitutional Factors and Non-invasive Monitoring of Subclinical Recurrences With Therapeutic Impact

The PRO-ACTIVE study aims to develop a clinical-translational program in the field of cancer prevention in all its phases (primary, secondary, and tertiary) to intervene before the clinical and radiological manifestation of the disease. It starts with risk prediction and leads to early diagnosis of the disease or recurrence in the subclinical phase.

The PRO-ACTIVE study includes the following activities:

• WP1: Integrated DNA-RNA approach for the identification of hereditary markers of predisposition to tumors
• WP2: Global biological and molecular analysis of the host and tumor for the prevention and monitoring of recurrences
• WP3: Analysis of the immunological status for the diagnosis of primary prevention and relapses in correlation to genetic and environmental factors
• WP4: Study of the tumor microenvironment for recurrence prediction

The observational study consists of a retrospective and a prospective part.

For the retrospective part 400 patients with breast tumors operated between 2000 and 2015 will be selected, of which 200 with hereditary breast tumors and 200 with non-hereditary breast tumors and with the following clinical, morphological and molecular class characteristics (luminal A and B, triple negatives) and stackable staging.

The tumor tissue will be subjected to immunocytochemical investigations to study the following parameters: angiogenesis (CD31), fibroblasts (CD34 and vimentin), macrophages (CD68) and tumor associated macrophages (M1: CD11c; M2: CD163); plasma cells (CD138), T lymphocytes (CD8); Mast cells (CD117).

The data will be correlated with the prognosis and with the risk of developing hereditary breast cancer.

The prospective part, on the other hand, envisages the enrollment of different cohorts of patients who are initially screened in WP1.

600 patients known for breast cancer (N=200), ovarian cancer (N=200) and colorectal cancer (N=200) candidates for germline genetic testing in the context of an oncogenic consultation will be evaluated. Patients will be selected before surgery and, if they agree to participate in the study, they will sign the informed consent in an oncogenetics consultancy context.

The selected patients will undergo blood sampling (5 ml in EDTA tubes) for the extraction of nucleic acids (DNA and RNA).

Based on the result of germline genetic testing, patients will be classified into 3 cohorts:

• COHORT 2A, subjects with hereditary inheritance (probands): the identification of about 10% (N=60) of probands out of 600 subjects examined (20 for pathology) is assumed.
• COHORT 2B, subjects identified by genetic counseling as being at risk of being carriers of a hereditary neoplastic syndrome, not proven by genetic tests: a population of 60 subjects, 20 for each type of tumour, with similar clinical characteristics of age and phenotype compared to cohort 2A.
• COHORT 2C, a further subgroup of patients (N=150), 50 for each type of tumor, negative results in diagnostic genetic analysis. In selected patients in cohort 2C, DNA/RNA will be analyzed for the identification of causative genetic variants in genes that have escaped routine diagnostic investigation.

Moreover, a further cohort of patients will be selected, COHORT 3 which includes 250 patients undergoing radical surgical treatment and with the following characteristics:

• locally advanced breast cancer with triple negative phenotype or with lobular histology (N=50);
• high-grade serous ovarian carcinoma (N=50);
• stage III melanoma (N=50);
• stage IIB and IIIA non-small cell lung cancer (N=50);
• colon cancer with lymph node involvement and vascular invasion (N=50).

Patients enrolled in COHORTS 2A, 2B and 3 will undergo the following blood draws for WP2:

• 20 ml in Cell-Free DNA BCT® CE tubes (Streck) for ctDNA analysis. Samples are centrifuged at 1600 (±150) g for 10 minutes at room temperature. After centrifugation, the plasma is subjected to further centrifugation for 10 minutes at 3000 (±150) g.
• 20 ml in Cell Save tubes for the isolation of CTCs.
• 30 ml for the study of circulating immune populations (WP3). Furthermore, the tumor tissue taken during the surgical phase will be collected and analyzed in WP4 and for the study of the microenvironment with "Next Generation Sequencing" techniques applied to the tumor as a whole and at the single cell level and to the immune populations (TILs).

Inclusion Criteria:

• Age >18 years;
• Patients with breast cancer, including patients who meet the AIOM criteria for eligibility for BRCA testing and patients with lobular breast cancer;
• Patients with radically resected colon cancer, including patients with stage III colon cancer and vascular invasion;
• Patients with ovarian carcinomas;
• Patients with metastatic melanoma;
• Patients with stage IIB and IIIA non-small cell lung cancer.

Exclusion Criteria:

• Age <18 years;
• Unwillingness or inability to give informed consent