• BRCA1
• DNA repair
• neoadjuvant therapy
• Next Generation Sequencing
• Triple Negative Breast Cancer

Abstract
Triple Negative Breast Cancer (TNBC), negative for estrogen, progesterone and Her2 receptor, constitute 10–20% of all breast cancers and more frequently affect younger patients. TNBC tumors are generally large in size, higher grade cancers with lymph node involvement at diagnosis, and are biologically more aggressive than other subtypes. Treatment of TNBC patients has been challenging due to the heterogeneity of the disease and the absence of well-defined molecular targets. TNBCs share many phenotypic features with BRCA1-related and basal-like tumors, such as receptors negativity, expression of basal cytokeratins CK5/6, and a similar gene expression profile. This similarity is defined BRCAness and suggests that TNBC and BRCA-related cancer may also share molecular and genetic features and common deficiency in DNA repair genes. In addition to BRCA1 and BRCA2, mutations in other genes involved in DNA repair may predispose to breast and ovarian cancer and the therapy response can be determined by germline or somatic alterations in these genes. So, the aim of this thesis is to investigate which genes involved in DNA repair are mutated in TNBC and to correlate a genetic signature with the response to neoadjuvant or adjuvant therapy.
The entire coding sequences and intron-exon junctions of 24 genes involved in the main DNA repair pathways (BRCA1, BRCA2, CDH1, MRE11A, MSH2, PARP1, CHEK2, MSH6, RAD52, PTEN, STK11, ERCC1, TP53, PMS1, PMS2, NBN, RAD50, BRIP1, BARD1, MLH1, MUTYH, RAD51C, TP53BP1 and PALB2) were screened using PGM Ion Torrent platform. Some of these are considered breast and ovarian cancer susceptibility genes, while others are associated with breast tumorigenesis. Moreover a set of genes was analyzed for the presence of large rearrangements by MLPA. Two cohort of patients diagnosed of TNBC have been analyzed: 19 patients, with and without family history of breast cancer, treated with neoadjuvant chemotherapy including taxane and anthracycline for germline mutations and 37 unselected TNBC patients with a follow up >5 years for somatic and germline mutations.
The mutational screening in the neoadjuvant setting, have identified 5 patients with clearly pathogenetic mutation in BRCA1, RAD51c and PALB2 accounting for a 20% of the total. Other 15 predicted pathogenetic variants in DNA repair genes were found. Data suggest a potential correlation between the presence of germline pathogenetic mutations, indicative of DNA repair defects, and a positive outcome after neoadjuvant therapy. Furthermore, this approach allowed the identification of 3 novel mutations predicted deleterious by in silico analysis and never reported in literature. From the second set of patients, BRCA1 and BRCA2 mutations were found in 7 patients and one patient, respectively. Also germline mutation in BRIP1 and CDH1 were found. Overall in 56 TNBC patients screened, this gene panel has evidenced that around 25% of them harbour a germline mutation in DNA repair genes. In addition to that, many interesting somatic variants were found in 8 genes (BRCA2, BRIP1, CDH1, MLH1, MSH2, NBN, PMS1, TP53) in this set of TNBC tissues. In particular TP53 was mutated in 68% of patients, and this condition was associated with a worse outcome. The large rearrangement analysis confirmed that PTEN and TP53 loss, along with EGFR gain, are very frequent events in TNBC.
This gene panel approach has proved useful to identify a large number of germline mutations in TNBC patients. In the next future, the genetic screening of TNBC patients, even without family history of breast cancer, may be a real option in order to design personalized therapy for individuals with a “BRCAness phenotype. Moreover, these results may provide new insights into TNBC pathogenesis and may help to further improve the accuracy of treatments.