Genetic Profile of DNA Repair Genes in Triple Negative Breast Cancer

University of Pisa

BIOS Research Doctorate School in BIOmolecular Sciences SSD BIO/11

Doctorate Course in Experimental and Molecular Oncology 2012-2014

Title of the Thesis:

Genetic profile of DNA repair genes in Triple Negative Breast Cancer

President of the Doctorate Course

Prof. Generoso Bevilacqua

Candidate Tutor

Laura Spugnesi Dr. Maria Adelaide Caligo

Summary

Abstract ...... 1

Introduction ...... 3 1. BREAST CANCER ...... 4 1.1. Histopathological features of Breast Cancer ...... 6 1.2. Gene expression profile define new classes of breast cancer ...... 7 1.3. Genomic landscape of breast cancer ...... 9

2. HEREDITARY BREAST CANCER ...... 11 2.1. BRCA genes ...... 11 2.2. Other breast cancer susceptibility genes ...... 14 2.2.1. High penetrant genes (low frequency) ...... 14 2.2.2. Moderate penetrant genes ...... 16

3. TRIPLE NEGATIVE BREAST CANCER ...... 20 3.1. Molecular characterization of TNBC ...... 21 3.2. Genomic features of TNBC ...... 24 3.3. TN and basal like: one doesn’t fit to another ...... 27

4. BRCA1 AND TNBC: THE BRCAness PHENOTYPE ...... 30 4.1. Other Susceptibility loci for TNBC ...... 32

5. DNA REPAIR AND BREAST CANCER ...... 34 5.1. DNA Repair Mechanisms ...... 37 5.1.1. Mismatch repair (MMR) ...... 37 5.1.2. Nucleotide Excision Repair (NER) ...... 38 5.1.3. Base excision repair (BER) ...... 39 5.1.4. Homologous recombination (HR) ...... 39 5.1.5. Non Homologous End Joining (NHEJ) ...... 40 5.1.6. Fanconi Anemia (FA) pathway...... 41

5.1.7. DNA-damage checkpoints ...... 41 5.2. Targeting DNA repair ...... 42 5.3. Biomarkers for DNA repair deficiency ...... 46 5.4. TNBC and DNA repair ...... 48

6. TNBC AND THERAPY ...... 49 6.1. Standard therapies for TNBC ...... 50 6.1.1. Anthracyclines ...... 51 6.1.2. Taxanes ...... 51 6.1.3. Platinum compounds ...... 52 6.1.4. Alkylating agents ...... 53 6.2. Target therapy: PARP1 inhibitors ...... 54 6.3. Neoadjuvant therapy as a tool to develop predictive biomarkers ...... 55

7. AIM OF THE STUDY ...... 59

Materials and Methods ...... 61 1. Germline screening: patients ...... 62 2. Tumor Tissues Screening: patients ...... 62 3. DNA extraction ...... 63 4. MLPA Analysis ...... 63 5. Libraries preparation and ION PGM sequencing : the ION torrent protocol ...... 64 6. Data processing and analysis ...... 67 7. Sanger sequencing ...... 69 8. High Resolution Melting (HRM) in healthy controls ...... 69 9. Loss Of Heterozygosity (LOH) analysis in tumour tissues ...... 70 10. Statistical Analysis ...... 71

Results ...... 72 1. GERMLINE MUTATIONAL ANALYSIS IN TNBC NEOADJUVANT PATIENTS ...... 73 1.1. Neoadjuvant patients histopathological features ...... 73 1.2. Mutational screening in neadiuvant patients and selection of variants ...... 74 1.3. Variant distribution among genes and the most interested pathways ...... 75

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1.4. Neoadjuvant germine screening: mutation overview ...... 76 1.5. Characterization of novel variants ...... 85 1.6. Pathogenetic variants carriers tend to have better clinical outcome ...... 86 2. MUTATIONAL SCREENING IN TNBC TISSUES ...... 88 2.1. TNBC features ...... 88 2.2. Selection of interesting variants in tumor tissues ...... 90 2.3. Overview of the rare and predicted pathogenetic variants in TNBC tissues ...... 91 2.4. Pathways and genes mutated in TNBC ...... 108 2.4.1. Homologous recombination genes ...... 108 2.4.2. Mismatch Repair genes ...... 109 2.4.3. Non Homologous End Joining genes ...... 110 2.4.4. Other pathways ...... 110 2.5. TNBC are enriched in genomic rearrangement involving DNA repair genes ...... 112 2.6. Correlation of genomic status of tumor tissue and disease free survival ...... 114

Discussion ...... 118

Bibliography...... 127

...alle radici e ai frutti…

Genetic profile of DNA repair genes in TNBC Abstract

Abstract

Triple Negative Breast Cancer (TNBC), negative for estrogen, progesterone and Her2 receptor, constitutes 10–20% of all breast cancers and more frequently affect young 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 a 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 4 novel mutations predicted deleterious by in silico analysis and never reported in literature. In the second set of patients, BRCA1 and BRCA2 mutations were found in 7 patients and one patient, respectively. Also germline

Genetic profile of DNA repair genes in TNBC Abstract

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 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.

Introduction

Genetic profile of DNA repair genes in TNBC Introduction

1. BREAST CANCER

Breast cancer is the most common cancer worldwide for females, and the 2nd most common cancer overall, with more than 1,676,000 new cases diagnosed in 2012 (25% of female cases and 13% of the total) (Ferlay et al. (2013)). The major risk factors for the development of breast cancer are hormonal and genetic (family history). Breast carcinomas can, therefore, be divided into sporadic cases, possibly related to hormonal exposure, and hereditary cases, associated with family history or germ-line mutations. The development of sporadic breast cancer is mainly related to hormone exposure: gender, age at menarche and menopause, reproductive history, breast-feeding, and exogenous estrogens. The majority of these cancers occur in postmenopausal women and overexpress estrogen receptor (ER). Estrogen itself has at least two major roles in the development of breast cancer. Metabolites of estrogen can cause mutations or generate DNA-damaging free radicals (Miller, 2003). Via its hormonal actions, estrogens drive the proliferation of premalignant lesions. However, other mechanisms play a role, as a significant subset of breast carcinomas are ER-negative or occur in women without increased estrogen exposure. Pathways to carcinogenesis are complex and variable. Indeed, not one common genetic or functional change can be found in every breast cancer. Most reported changes occur in only a subset of carcinomas and usually in highly variable combinations with other changes. A general model for carcinogenesis postulates that a normal cell must achieve seven new capabilities, including genetic instability, to become malignant (Hahn & Weinberg, 2002). In hereditary carcinoma, one or more of these alterations is facilitated by the inheritance of germ-line mutations. Each of the new capabilities can be achieved by a change in one of many genes. For example, changes in ER, EGF-R, RAS, or HER2/neu may result in self- sufficiency in growth signals. On the other hand, one cellular alteration (e.g., a change in a genes such as p53 that has a central role in controlling the cell cycle, DNA repair, and apoptosis) can affect more than one of these capabilities. Population of cells that harbour mutations give rise to morphologically recognizable breast lesions and are associated with an increased risk of progression to cancer. Loss of heterozygosity is a rare event in proliferative lesions but becomes frequent in atypical hyperplasia and is almost universally present in carcinoma in situ. During the progression, also due to increased genomic instability, the malignant cells become immortalized and acquire the ability to drive neo angiogenesis.

Genetic profile of DNA repair genes in TNBC Introduction

The majority of breast malignant lesions are adenocarcinomas, which are divided in in situ carcinoma and invasive carcinomas. Carcinoma in situ describes a neoplastic proliferation that is limited to ducts and lobules by the basement membrane. Invasive carcinoma has penetrated through the basement membrane into stroma: here the cells have the potential to invade the vasculature and reach lymph nodes or distant sites. By current convention, “lobular” refers to carcinoma of a specific type (not expressing E-cadherine), and “ductal” is used more generally for carcinomas with no other designation. The morphologic and biologic features of carcinomas are usually established at the in situ stage, since in the majority of cases the in situ lesion closely resembles the subsequent invasive carcinoma. Finding the cell of origin of breast cancers is a field of great interest: the cancer stem cell hypothesis proposes that malignant changes occur in a stem cell population and that only the malignant stem cells would contribute to tumor progression or recurrence (Campbell & Polyak, 2014),(Stingl & Caldas, 2007). The most likely cell type of origin of carcinomas is the ER-expressing luminal cell, since the majority of breast cancer are ER positive. ER-negative tumors may arise from ER- negative myoephielial cells or from a ER positive precursor that loses ER expression (Gauthier et al., 2007). Even this view of oncogenesis is focused on the malignant epithelial cell, is important to take into account the other tissue components. The structure and function of the normal breast require complex interactions between luminal cells, myoepithelial cells, and stromal cells. The same functions that allow for normal formation of new ductal branch points and lobules during puberty and pregnancy—abrogation of the basement membrane, increased proliferation, escape from growth inhibition, angiogenesis, and invasion of stroma—can be co-opted during carcinogenesis by abnormal epithelial cells, stromal cells, or both (Wiseman & Werb, 2002). While the changes described above are accumulating in the luminal cells or myoepithelial cells, parallel changes also occur due to mutation or epigenetic changes (e.g. DNA methylation) or via abnormal signaling pathways in these other cell types, resulting in the loss of normal cellular interactions and tissue structure. Loss of these normal functions also occurs with age, and this loss might contribute to the increased risk of breast cancer in older women. The final step of carcinogenesis, the transition of carcinoma limited by the basement membrane to ducts and lobules (carcinoma in situ) to invasive carcinoma, is the most important and unfortunately the least understood (Fig 1). There are many path that can lead to the development of breast cancer, this recognition had led to the introduction of molecular classification systems.

Genetic profile of DNA repair genes in TNBC Introduction

Fig 1. Tumor initiation and progression events (from Robbins, VIII edition)

1.1. Histopathological features of Breast Cancer

Breast cancer is a heterogeneous disease, and therefore, no golden standard therapy exists suitable for all tumours. For many years, tumours of the breast were characterized by tumour size only. Later on, a histological classification system was developed, dividing breast cancer into subgroups distinguished by the histological appearance of the tumour but even this subdivision failed to form homogeneous breast cancer subgroups (Weigelt et al., 2008), (Page, 2003). Currently, the most widely used classification system of breast cancer combines histo- morphological information (such as histological subtype and grading) as well as TNM staging information, i.e. tumour size (T) together with lymph node (N) and distant metastasis occurrence (M) (Elston & Ellis, 1991) (Sobin & Compton, 2010).

The outcome for women with breast cancer varies widely and is determined by many genetic, molecular and histopathological factors. From a clinical point of view the major prognostic factors are incorporated into the American Joint Committee on Cancer (AJCC) staging system, which divided patient into five stages (0 to IV) that are correlated with survival: - Invasive carcinoma (infiltrating the stroma tissue) versus in situ disease (confined to the ductal system)

Genetic profile of DNA repair genes in TNBC Introduction

- Distant Metastases - Lymph node metastases: axillary lymph node involvement is the most important prognostic factor, as with no nodal involvement, the 10-year disease free survival (DFS)is close to 80% and decrease to 10% when more than 10 nodes are positive. - Tumor size: this feature is the second most important prognostic factor - Locally advanced disease (carcinomas invading into skin or skeletal muscle at diagnosis) - Inflammatory carcinoma Many other minor prognostic factors are very important to define therapy strategy such as histological subtype, histological grade, estrogen and progesterone receptor, Her2 expression, lymphovascular invasion, proliferative rate, DNA content and gene expression profile.

1.2. Gene expression profile define new classes of breast cancer

Recognition of the importance of the hormone receptors and the human epidermal growth factor receptor 2 in breast cancer, and the large scale use of immunohistochemistry (IHC), enabled almost every cancer center in the world to differentiate breast cancer patients into three major groups: the hormone receptor positive group (which expresses ER and/or PgR), the HER2-positive group, and the ‘‘triple negative’’ group (which is negative for ER, PgR, and HER2). A more recent approach to classify breast cancer subgroups is gene expression profiling, which allows simultaneous assessment of the contribution of thousands of genes in a single tumor sample. This approach revealed a biological diversity in breast cancer that mirrors the clinical diversity in outcomes (Perou et al., 2000). In the first years of 2000, many studies have identified several subtypes of breast cancer that differ from one another and that may be considered as individual diseases, under the general term of “breast cancer disease”. Initial evidence for molecular subtypes of breast cancer came from a cDNA-microarray study of gene expression among a small number of tumor samples and several benign controls. In 2000, Perou and colleagues compared gene expression among 42 subjects of a group of over 8000 genes: a subset of 1753 was selected based on expression levels that differed by at least four-fold across all samples. Based on these genes, computer-assisted hierarchical clustering was performed to group samples with similar patterns of expression obtaining 4 main subgroups.

Genetic profile of DNA repair genes in TNBC Introduction

Two of the subgroups had expression patterns similar to normal breast epithelium, specifically basal and luminal epithelial cells. These two groups correspond to tumors that had previously been clinically classified as ER-positive or ER-negative. The third group was characterized by strong HER2 expression and low expression of ER and its related genes. The other group displayed a genetic expression profile most similar to normal breast tissue and remains enigmatic. Based on these findings, Perou proposed a novel molecular classification of breast cancer into basal-like, luminal, HER2-positive, and normal breast subgroups. In subsequent work by the same group, this classification was refined and expanded to distinguish between luminal A and luminal B tumor subtypes on the basis of their proliferation rate (Sorlie et al., 2001). The five molecular subtypes identified by Perou and Sorlie have been confirmed in independent data sets from other investigators (Sorlie et al., 2003) and among different ethnic groups of patients (Yu, Lee, Tan, & Tan, 2004):

Luminal A (40-55%): this is the largest group and consists of cancers that are ER and PgR positive and HER2/neu negative. The gene signature is dominated by genes under control of ER and characteristic of normal luminal cells. The majority are well differentiated and most occur in postmenopausal women. They are usually slowly growing and well respond to hormone therapy. Luminal B (15-20%) This group also express ER but is generally of higher grade, has a higher proliferative rate and often express HER2. They more likely have lymph node metastases and may respond to therapy. Normal breast like (6-10%): this is a small group of well-differentiated ER positive HER2 negative cancers and display no specific tumor expression pattern. Basal like (13-25%): These cancers are notable for the absence of ER, PgR and HER2 and the expression markers of myoepithelial cells (basal keratins, P-cadherin, p65, laminin), progenitor cells or putative stem cells (cytokeratins 5-6). This group include also medullary carcinoma, a subtype with a good prognosis, and metaplastic carcinoma, that instead show worse prognosis. Basal like cancers are of particular interest because of their distinct genetic and epidemiologic features. They generally have high grade and high proliferation rate, are associated with visceral metastasis and brain metastases. However 15-20% of tumors have a pathological complete response to chemotherapy. HER2/neu positive (7-12%): This group comprise ER negative carcinomas that overexpress HER2/neu protein. In 90% of cases overexpression is due to amplification of HER2 genomic locus, which includes also other genes. These cancers are poorly differentiated, have high proliferation rate and are associated with poor prognosis.

Genetic profile of DNA repair genes in TNBC Introduction

After these two pilot studies, the molecular classification has been enriched with larger studies. In 2009, Parker et al. derived a minimal gene set (PAM50) for classifying “intrinsic” subtypes of breast cancer (Parker et al., 2009). The PAM50 gene set has high agreement in classification with larger “intrinsic” gene sets previously used for subtyping, and is now commonly employed (Ellis et al., 2011), (Esserman et al., 2012), (Gonzalez- Angulo et al., 2012). The intrinsic subtypes are found to be highly conserved across different microarray platforms and across tumors from distinct ethnic populations (Sorlie et al., 2003), (Yu et al., 2004);(Parker et al., 2009). In a study of high-throughput protein expression analysis (Abd El-Rehim et al., 2005) immunohistochemical markers were assessed for 1076 tumor samples of invasive breast cancer and hierarchical clustering analysis was conducted to group tumors based on IHC characteristics. Six clusters of tumors were identified, roughly corresponding to the five subtypes previously identified, and one small new group consisting of only four tumor samples. Key histological markers that differed significantly between tumor types included androgen receptor, HER2, cytokeratin 18, MUC1, Cytokeratin 5/6, p53, nuclear BRCA1, ER, and E-cadherin. This large study gives additional support to the identification of luminal A, luminal B, normal-like, HER2-positive, and basal-like cancers as discrete biological entities.

1.3. Genomic landscape of breast cancer

The latest level at which breast cancer complexity has been dissected is the genomic level. In the last few years, have been published pivotal studies in which breast cancer tumours were clustered on the basis of their mutation profiles or the presence of other genomic aberrations (Curtis et al., 2012); (Cancer Genome Atlas, 2012b). Importantly, a study of The Cancer Genome Atlas (2011) revealed that breast and ovarian cancers are dominated by genomic alterations more than by somatic mutations in a list of selected genes. Using the largest sample collection with extensive genomic, transcriptomic and clinical annotation in existence, in 2012 Curtis and colleagues described a scheme for classifying breast tumors into 10 subtypes based on the pattern of copy number alterations (CNA). This classification was named IntClust since the clustering of tumors is based on the integration of genomic and transcriptomic data to find probable driver events. Around 1000 genes, whose alterations was correlated with a differential expression level, were considered to divide 2000 breast cancer specimen in different clusters.

Genetic profile of DNA repair genes in TNBC Introduction

An ER-positive subgroup composed by luminal tumours with alterations in chr 11 (IntClust2) was described. One subgroup (IntClust3) with low genomic instability was composed predominantly of luminal A cases, and was enriched for histotypes that typically have good prognosis, including invasive lobular and tubular carcinomas. Another subgroup (IntClust 4) was also composed of favorable outcome cases, but included both ER-positive and ER-negative cases and varied intrinsic subtypes, and had an essentially flat copy number landscape. A significant proportion of cases within this subgroup exhibit extensive lymphocytic infiltration. Several intermediate prognosis groups of predominantly ER-positive cancers were identified (Clusters 1, 6, 9). They also noted that the majority of basal-like tumours formed a stable, mostly high- genomic instability subgroup (IntClust10, n = 96). This subgroup had relatively good long- term outcomes after 5 years, and characteristic cis-acting alterations (5 loss/8q gain/10p gain/12p gain). The ERBB2-amplified cancers composed of HER2-enriched (ER- negative) cases and luminal (ER-positive) cases appear as IntClust 5. In 2012, another integrative and collaborative huge study leaded by The Cancer Genome Atlas Network was performed. Different sets of primary breast cancers were analyzed by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. This study demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity (luminal, basal-like, Her2 enriched, normal-like). To a great extent, the four major integrated clustering subdivisions correlated well with the previously-published mRNA-subtypes. The mutational screening by exome sequencing of 510 breast tumours identified 30,626 somatic mutations. The MuSiC package (Dees et al., 2012), which determines the significance of the observed mutation rate of each gene based on the background mutation rate, identified 35 Significantly Mutated Genes. In addition to identifying nearly all genes previously implicated in breast cancer (PIK3CA, PTEN, AKT1, TP53, GATA3, CDH1, RB1, MLL3, MAP3K1 and CDKN1B), a number of novel genes were identified including TBX3, RUNX1, CBFB, AFF2, PIK3R1, PTPN22, PTPRD, NF1, SF3B1, and CCND3. Unsupervised hierarchical clustering analysis of 525 tumors and 22 tumor adjacent normal tissues using the top 3,662 variably expressed genes identified 12 classes. MicroRNA expression levels were assayed via Illumina sequencing, using 1222 miRBase27 v16 mature and star strands as the reference database of microRNA transcripts/genes. Seven subtypes were identified. These subtypes correlated with mRNA-subtypes, ER, PR and HER2 clinical status but not with mutation status. Methylation profiling for a common set of

Genetic profile of DNA repair genes in TNBC Introduction

574 probes used in an unsupervised clustering analysis, identified five distinct DNA methylation groups. Group 3 showed a hyper-methylated phenotype and was significantly enriched for Luminal B mRNA-subtype and under-represented for PIK3CA and MAP3K1/MAP2K4 mutations. Group 5 showed the lowest levels of DNA methylation, overlapped with the Basal-like mRNA-subtype, and showed a high frequency of TP53 mutations. Quantified expression of 171 cancer-related proteins and phospho-proteins by RPPA (Reverse Phase Protein Array) was performed on 403 breast tumors. Unsupervised hierarchical clustering analyses identified seven subtypes. These protein subtypes were highly concordant with the mRNA-subtypes, particularly with Basal-like and HER2E mRNA subtypes. This study also performed analysis on a selected set of genes (Walsh et al., 2010) using the normal tissue DNA data and detected a number of germline predisposing variants. These analysis identified 47/507 patients with deleterious germline variants, representing nine different genes (ATM, BRCA1, BRCA2, BRIP1, CHEK2, NBN, PTEN, RAD51C, and TP53) supporting the hypothesis that ~10% of sporadic breast cancers may have a strong germline contribution. These data confirmed the association between the presence of germline BRCA1 mutations and Basal-like breast cancers (Sorlie et al., 2003); (Foulkes, 2003). The landscape of somatic alterations in breast cancer is complex and heterogeneous. This variety is reflected in the diverse clinical behavior of breast tumors and provides critical insight for the development of rational therapies.

2. HEREDITARY BREAST CANCER

In the middle of the 19th century, first reports described familial aggregation of breast cancers. Today, positive family history is one of the most important risk factors for developing breast cancer. It is currently estimated that approximately 5–10% of all breast cancers have a hereditary background. These families show an apparently dominant inheritance pattern and are often characterized by an early age of onset, overrepresentation of ovarian cancers, bilateral breast cancers, and male breast cancers (Honrado, Benitez, & Palacios, 2004).

2.1. BRCA genes

Many reports suggested that germline mutations in the BRCA1 and BRCA2 genes were responsible for the majority of hereditary breast cancers, although more recent studies

Genetic profile of DNA repair genes in TNBC Introduction

have demonstrated that mutations in the two genes only account for 25–28% of the family risk (Melchor & Benitez, 2013). However, it is expected that additional BRCA1/2 mutations remain undetected by the screening methods used today or the hereditary may be due to mutations in other genes. Women carrying a BRCA1 or BRCA2 germline mutation also have increased risk of developing ovarian cancer and fallopian tube cancer. In addition, BRCA2 mutation carriers also have increased risk of other cancer types such as male breast cancer, prostate cancer, pancreas cancer, gastrointestinal cancers and melanoma (Breast Cancer Linkage, 1999);(Thompson & Easton, 2004). In a large study by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), the median age of diagnosis was found be to be 40 years among BRCA1 and 43 years among BRCA2 mutation carriers (Mavaddat et al., 2012). Even though germline mutations in BRCA1 and BRCA2 confer high risk of breast and ovarian cancers, the penetrance of these genes is incomplete. The risk in BRCA1 and BRCA2 mutation carriers of developing breast cancer by the age of 70 is 45–87%. For ovarian cancer, the risk is 45–60% among BRCA1 mutation carriers and 11–35% among BRCA2 mutation carriers (A. Antoniou et al., 2003); (S. Chen & Parmigiani, 2007). However, the penetrance depends on several different factors, including the type of mutation and exogenous factors. Lifestyle factors such as physical exercise and lack of obesity in adolescence have been associated with significant delay in breast cancer onset (King, Marks, Mandell, & New York Breast Cancer Study, 2003). BRCA1 and BRCA2 function as tumor suppressor genes and are important in maintenance of genomic stability through their role in DNA damage signaling and DNA repair. Both BRCA1 and BRCA2 are implicated in mediating repair of double strand breaks by homologous recombination (HR) by interactions with RAD51. Upon DNA damage, BRCA1 will associate with RAD51 and localize to the damaged region by which BRCA1 becomes phosphorylated. BRCA2 functions downstream of BRCA1 by complex- formation with RAD51. The primary function of BRCA2 is to facilitate HR (Roy, Chun, & Powell, 2012). Cells deficient for BRCA1 or BRCA2 are unable to repair double strand breaks by the error-free HR, resulting in repair by the error-prone non-homologous end- joining (NHEJ) pathway introducing chromosomal instability. During S-phase, the expression levels of BRCA1 and BRCA2 increase, indicating a function in maintaining genomic stability during the DNA replication process. Besides its role in HR, BRCA1 appears to have additional functions in DNA repair. BRCA1 is also part of the BRCA1- associated genome-surveillance complex (BASC), which includes ATM, RAD50, MRE11, and NBS1 and the mismatch repair proteins MLH1, PMS2, MSH2, and MSH6. BRCA1 has also been demonstrated to be involved in transcription-coupled excision repair,

Genetic profile of DNA repair genes in TNBC Introduction

chromatin remodeling, and together with BARD1 in the ubiquitination process, by which proteins are tagged for degradation by the proteasome (Narod & Foulkes, 2004). In all, 1,790 distinct mutations, polymorphisms, and variants in the BRCA1 gene and 2,000 in BRCA2 have been reported to the Breast Cancer Information Core (BIC) database, respectively (July 2014, research.nhgri.nih.gov/bic). Approximately 53–55% of these are private mutations, are only detected in single families. Mutations are distributed across the entire coding sequences. The most common types of pathogenic mutations are small deletions or insertions or nonsense mutations resulting in protein truncation leading to non-functional protein. Mutations affecting splice-sites as well as large genomic rearrangements are also observed in both genes (Thomassen, Gerdes, Cruger, Jensen, & Kruse, 2006). Missense mutations, silent mutations, and polymorphisms are also frequently identified; however, the clinical interpretation of their pathogenic potential is often difficult. Also, variants such as small in-frame insertions and deletions and possible splice-site alterations are problematic for precise cancer-risk estimation. Almost 1,800 distinct sequence variants found in BRCA1 and BRCA2 are classified as having unknown clinical significance (variants of unknown significance, VUS). To assess the clinical significance of individually rare sequence variants is challenging, as existing methods require a high number of occurrences of the specific variant. In 2009, the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium was established with the purpose of evaluating the clinical significance of rare sequence variants by pooling genetic and associated clinical and histopathological information from a world-wide network of laboratories to gather sufficient data and resources to facilitate the classification of UVs (Spurdle et al., 2012). Recently, ENIGMA group have reported a useful tool to facilitate VUS analysis (Jhuraney et al., 2015). The BRCA1 circos is a visualization resource that compiles and displays functional data on all documented BRCA1 missense variants (http://research.nhgri.nih.gov/bic/circos/). It aggregates data from all published BRCA1 missense variants for functional studies, from yeast assay to Embryonic Stem cells in vitro assays, offering various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. A germline mutation in BRCA1 or BRCA2 only represents the first hit in the classical Knudson’s two-hit hypothesis; the second inactivating somatic mutation often involves deletion of the wild-type allele, known as loss of heterozygosity (LOH). LOH has been reported to be present in the majority (80%) of tumors arising from mutation carriers (N. Collins et al., 1995). Another somatic inactivation mechanism, epigenetic silencing by promoter methylation of BRCA1, has been reported in 9–13% of sporadic breast tumors, an up to 42% in non-BRCA1/2 hereditary breast tumors leading to reduced BRCA1

Genetic profile of DNA repair genes in TNBC Introduction

expression (Tapia et al., 2008);(Larsen et al., 2014). In contrast, BRCA1 promoter methylations are rare in tumors from BRCA1 and BRCA2 mutation carriers, and BRCA2 promoter methylation in general is seldom observed in both sporadic and hereditary breast cancers (Dworkin, Spearman, Tseng, Sweet, & Toland, 2009); (N. Collins, Wooster, & Stratton, 1997).

2.2. Other breast cancer susceptibility genes

Several rare gene variants have been described to confer an increased risk of breast cancer, involving high-penetrance such as TP53, CDH1, PTEN, STK11, RAD51C, and RAD51D and the low/moderate-penetrance genes such as ATM, CHEK2, BRIP1, and PALB2, among others. In general, most of these genes are involved in the maintenance of genomic integrity and DNA repair mechanisms, and many are associated with multiple cancer syndromes such as Li–Fraumeni syndrome (TP53), Cowden syndrome (PTEN), and Peutz–Jeghers syndrome (STK11/ LKB1) (Turnbull & Rahman, 2008);(Walsh & King, 2007). Furthermore, a number of common low-penetrance breast cancer alleles have recently been identified by genome-wide association studies (GWAS), including 10q26, 16q12, 2q35, 8q24, 5p12, 11p15, 5q11, and 2q33 (Easton et al., 2007).

2.2.1. High penetrant genes (low frequency)

TP53: TP53 is a tumor suppressor gene located on chromosome 17p13.1 that plays a major role in the regulation of cell growth (Menendez, Inga, & Resnick, 2009). TP53 germline mutations give rise to Li–Fraumeni syndrome (LFS), a rare predisposition cancer syndrome associated with approximately 1% of breast cancer cases. This gene predisposes for a wide spectrum of malignancies and patients with LFS, are mutated in TP53 in approximately 70%. Mutations are most commonly missense and small deletions (Varley, 2003). BC is the most frequent malignancy among femaleTP53 mutation carriers and represents up to 1/3 of all cancers in LFS families. Overall, although LFS is responsible for a small fraction of breast cancer cases, a woman with LFS has a breast cancer risk of 56% by the age of 45 and greater than 90% by the age of 60 (Olivier et al., 2003). Furthermore, recent studies have shown the majority of LFS-associated breast cancer cases are HER2/neu positive and nodes positive (Melhem-Bertrandt et al., 2012). Moreover it is suggested that the likelihood of the mutation in women <30 years of age and no family history is approximately 5–8% (Masciari et al., 2012).

Genetic profile of DNA repair genes in TNBC Introduction

The latest NCCN guidelines issued in 2014 recommends testing of TP53 in all women with BC <35 years of age regardless of family history “either concurrently with BRCA1/2 testing or as a follow up test after negative BRCA1/2 testing”.

PTEN: PTEN is a phosphatase tensin homolog located on chromosome 10q23.3. PTEN is a major break for carcinogenesis due to its phosphatidylinositol-3-kinase (PI3K) phosphatase activity. PTEN precise function is not clear; however, dysfunctional PTEN leads to inability to activate cell cycle arrest and apoptosis, leading to abnormal cell survival (Shen et al., 2007). Germline mutations in PTEN are the cause of Cowden syndrome (CS) an autosomal dominant disorder with incomplete penetrance, which is characterized by increased risk of malignant transformation. Approximately 80% of affected individuals will have a detectable PTEN mutation that may include a missense, point, deletion, insertion, frame shift or nonsense mutation. Among the 20% of patients with no identifiable PTEN mutation, half may bear a mutation in PTEN promoter (Zhou et al., 2003). BC is the most common malignancy associated with CS. Although CS is responsible for <1% of BC cases, affected females have a lifetime risk of BC that reaches 50%, with an average age at diagnosis much younger than in sporadic BC (36–46 years old) (Liaw et al., 1997).

STK11: The SKT-11 gene is located on chromosome 19p13.3 and encodes for serine– threonine protein kinase11. It is designated as a tumor suppressor gene, participating in membrane bonding and apoptosis (S. P. Collins, Reoma, Gamm, & Uhler, 2000). Furthermore, it is a negative regulator of the mTOR pathway. Germline mutations in SKT- 11 gene are the cause of Peutz–Jeghers syndrome (PJS), a rare autosomal dominant disorder characterized by multiple gastrointestinal polyps and mucocutaneous pigmentations of the lips, buccal mucosa and digits. Affected individuals are at increased risk for colorectal, breast, small bowel, pancreatic, gastric and ovarian cancer. Mutations of SKT-11 are detected in approximately 70–80% of patients with PJS, with 15% of them being deletions (Volikos et al., 2006). Women with PJS present with an increased risk for BC that reaches 50%. BC incidence has been shown to rise up to 32% by the age of 60, whereas it was only 8% by the age of 40 and a 20% risk for ovarian cancer (Lim et al., 2004).

CDH1: The E-Cadherin gene is a calcium dependent cell–cell adhesion molecule expressed in junctions between epithelial cells (Graziano, Humar, & Guilford, 2003). CDH1 germline mutations have been associated with hereditary diffuse gastric cancer (HDGC). Approximately 30% of families with HDGC due to CDH1 mutations also

Genetic profile of DNA repair genes in TNBC Introduction

include women with lobular breast cancer (LBC). However, women with LBC without a family history of HDGC rarely harbor a CDH1 mutation. It has been estimated that the cumulative risk of LBC in female CDH1 mutation carriers from HDGC families reaches 39% at 80 years old (Pharoah, Guilford, Caldas, & International Gastric Cancer Linkage, 2001). In contrast to HDGC, missense mutations are the most frequent alterations, accounting for 60% of the cases described so far. Truncating mutations account for the remaining 40% (Kluijt et al., 2012); (Schrader et al., 2008). Theoretically, women with LBC diagnosed before the age of 45 or those who have a family history of LBC and lack BRCA1/2 mutations, are eligible for CDH1 testing. However, family history of HDGC appears to be an important component. In a recent study it has been reported that among 408 cases of LBC without history of HDGC screened for CDH1 mutations, only three germline CDH1 mutations have been described (Schrader et al., 2011).

2.2.2. Moderate penetrant genes

CHEK2: CHEK2 gene encodes for a serine threonine kinase, which is activated in response to DNA double strand breaks (DSBs), contributing to signal transduction to downstream repair proteins. It has been also found to phosphorylate BRCA1, facilitating its role in DNA repair (Stracker, Usui, & Petrini, 2009). Certain CHEK2 mutations have been associated with BC. Mutation 110delC has been shown to increase BC risk by 2–3- fold. This mutation is particularly frequent in Northern European population, where it carries a lifetime risk of BC as high as 37%. Homozygotes have a 6-fold increased risk of BC (Adank et al., 2011). Carriers of CHEK2 mutations have an increased risk of bilateral or recurrent BC and tend to have a worse outcome (Mellemkjaer et al., 2008). This mutation is also associated with male BC. Although it is responsible for less than 1% of hereditary breast cancer syndromes, CHEK2 is an important gene, since its mutations are detected in approximately 5% of BC patients of non-BRCA families. The majority of CHEK2-associated BC is ER positive and therefore, might be subjective to chemoprevention with tamoxifen.

ATM: ATM is a multifunctional gene that plays a pivotal role in DSB repair and in cell cycle progression. Homozygous ATM mutation carriers suffer from ataxia telangiectasia (AT), a disorder characterized by cerebral ataxia, immunodeficiency and increased risk of certain malignancies, including BC (Ahmed & Rahman, 2006). On the other hand, heterozygous carriers of ATM mutations have a 2-fold increased BC risk (Thompson et al., 2005) compared to general population. In women under the age of

Genetic profile of DNA repair genes in TNBC Introduction

50, this risk reaches 5-fold. However, clinical utility of ATM genetic testing in heterozygotes is difficult to assess and there are no specific guidelines. Of note, decreased expression of ATM protein has been associated with aggressive features in sporadic BC (Bueno et al., 2014).

PALB2: PALB2 has emerged a new BC susceptibility gene. It is often described as a “binding partner and localizer of BRCA2”, contributing to DNA repair mechanism homologous recombination and tumor suppression (Xia et al., 2006). Classification of PALB2 as a BC susceptibility gene was based on data showing that 1.08% of individuals with hereditary BC negative for BRCA1/2harbor a monoallelic mutation in PALB2, which confers a 2-fold high risk (Rahman et al., 2007). In a recent study by Fernandes et al. that included approximately 1500 patients with familial BC, the prevalence of PALB2 mutations was 0.8%, with the majority occurring in high risk patients (Fernandes et al., 2014). Although, based on the above studies, PALB2 is characterized as a rare, intermediate-risk gene with regards to inherited genetic susceptibility to BC, a recent study that included 154 families who had mutations in the gene demonstrated a breast cancer risk of approximately 35% (A. C. Antoniou et al., 2014).

BRIP1: BRIP1 encodes a protein that was identified as the binding partner of BRCA1. Similar to PALB2, truncating mutations of the gene have been detected in BRCA1/2 negative BC families and the relative BC risk has been estimated to be approximately 2-fold (Seal et al., 2006). Interestingly, BRIP1 missense mutations have been found in high risk Jewish women who are BRCA1/2 negative (Catucci et al., 2012).

RAD51c: RAD51C is an integral component of FA/BRCA pathway and plays an important role in DSB repair through Homologous Recombination (HR). Homozygous mutations in RAD51C are associated with a FA phenotype, whereas heterozygous mutations have been identified in breast and ovarian cancer (OC) families. Initially, RAD51C was studied as a possible BC/OC susceptibility gene in 1100 high risk BRCA1/2 negative German families. In this study, six monoallelic RAD51C mutations were found in 1.3% of families with BC and OC. Interestingly, no pathogenic mutations were identified in high risk families with BC only (Meindl et al., 2010). Subsequently, several studies confirmed the occurrence of RAD51C predominantly in BC and/or OC families (Blanco et al., 2014), (Osorio et al., 2012). The inclusion of RAD51C gene in routine clinical testing is a matter of debate. In a recent study, no RAD51C mutation was found in 410 patients from BRCA1/2 negative families with at least one case of OC who were referred for genetic testing (De Leeneer et al., 2012). It is therefore suggested that mutations

Genetic profile of DNA repair genes in TNBC Introduction

in RAD51C gene occur in a low frequency, with an exception of some founder populations. On the other hand, RAD51C carcinomas are HR deficient and might benefit from new targeted therapies, such as PARP inhibitors. In this context, further genome- wide studies in high-risk families are warranted for the identification of RAD51C mutation carriers.

RAD51d: RAD51D is also an important co-factor of DSB repair mechanism. RAD51D protein forms a complex with RAD51C, RAD51B and XXRC2, which binds to single-stranded DNA and plays an important role in response to DNA damage. RAD51D has emerged mainly as an OC susceptibility gene (Osher et al., 2012). Recently, a study that analyzed 841 BRCA negative patients either from BC/OC families or with early onset BC/OC identified three RAD51D mutations in four BC/OC families with only one OC case (Gutierrez-Enriquez et al., 2014).

NBS, MRE11, RAD50: The MRN complex, consisting of MRE11, RAD50 and NBS1 proteins plays multiple key roles in DSB repair and maintenance of genomic integrity. Germline homozygous mutations in NBS1 and MRE11 cause the rare autosomal disorders Nijmegen Breakage Syndrome (NBS) and Ataxia Telangiectasia-like disorder (A-TLD), respectively (Rupnik, Grenon, & Lowndes, 2008). Pathogenic mutations have been found in all MRN genes. In a recent meta-analysis that included 9 case control studies, NBS1 mutation 657del5 has been associated with significant BC risk. There is less supporting evidence that RAD50 and MRE11might act as BC susceptibility genes. (Heikkinen, Karppinen, Soini, Makinen, & Winqvist, 2003); (Bartkova et al., 2008). In an attempt to assess the role of rare MRN variants as intermediate-risk BC susceptibility alleles, a recent study has performed a mutation analysis of MRN genes in a large number of early-onset BC cases. Interestingly, this study confirmed that rare missense substitutions and less frequently, protein-truncating variants of MRN genes confer a 2–3-fold increased risk of BC. However, this study has limitations, mainly because the three genes have been evaluated as if they constitute a single gene (Damiola et al., 2014).

BARD1: BARD1 (BRCA1-associated RING domain 1) encodes a protein that shares structural and functional similarities with BRCA1. It was initially identified as a protein interacting with BRCA1 in DSB repair and apoptosis initiation. Initially, only a small number of missense mutations and variants with unclear biological effect were identified.

Genetic profile of DNA repair genes in TNBC Introduction

Subsequently, several studies have found deleterious mutations in high risk BRCA negative BC families. On the other hand, as a BRCA1 interactor, BARD1 has been implicated in modification of BRCA1/2 associated BC risk. Although a number of studies have been controversial, a recent study that included a large number of BRCA1/2 mutation carriers has failed to support a role for BARD1 variation as a modifier of BC risk in this particular population.

FANCM: As previously noted, FA is a rare recessive genetic disorder, characterized by a high incidence of leukemias and solid tumors. To date, there are 13 FA genes identified, each one corresponding to one complementation group. Several FA genes are well known high or intermediate risk BC susceptibility genes (BRCA2, PALB2, BRIP1), which makes all members of the FA pathway attractive BC candidate genes. FANCM is a FA gene that plays a crucial role in DNA repair process (Wang, 2007). In a recent study, the c.5101C > T FANCM nonsense mutation has been associated with BC risk in a Finnish population. Importantly, the highest frequency of the mutation was observed in triple-negative BC patients. Apparently, further studies in different populations are essential for the evaluation of BC risk associated with the mutation (Kiiski et al., 2014).

Low and moderate penetrant genes/loci can only explain a minor fraction of the remaining non-BRCA1/2 families that show high incidence of breast cancer. Despite intensive research, genetic linkage analysis, and most recently, next-generation sequencing (NGS) exome studies have failed to identify other common high-penetrance breast cancer susceptibility genes, such as BRCA1 and BRCA2. A Genome-wide association studies (GWAS) and the recent international collaborative analyses have confirmed 77 common polymorphisms individually associated with breast cancer risk, which add a further 14% (Melchor & Benitez, 2013), (Michailidou et al., 2013). Evidence from an Illumina collaborative oncological gene environment study (iCOGS) experiment suggests that further single nucleotide polymorphisms (SNPs) may contribute at least 14% to the heritability, leaving only approximately 50% of hereditary cases as unexplained. No single high-penetrance gene is likely to account for a larger fraction of the remaining familial aggregation (Snape et al., 2012);(Smith et al., 2006). Instead, the remaining predisposition is expected to be a mixture of rare high-risk variants and polygenic mechanisms involving more common and/or rare low-penetrance alleles or rare moderate- penetrance genes, acting in concert to confer a high breast cancer-risk (Turnbull & Rahman, 2008). Patients with breast cancer should be offered genetic testing, according to consensus guidelines, when they present with a suspicious family history, specific high-risk disease

Genetic profile of DNA repair genes in TNBC Introduction

features (triple negative or bilateral breast cancer) or at a young age (Khatcheressian et al., 2013). However, the implementation of multigene panel testing has originated new issues regarding patient eligibility for gene testing beyond BRCA. According to National Comprehensive Cancer Network (NCCN) guidelines, gene panels are aimed at individuals who are tested negative for BRCA1/2 and for those whose family pedigree is suggestive of more than one hereditary syndrome.

3. TRIPLE NEGATIVE BREAST CANCER

Despite of the molecular ad expression efforts to better characterize the heterogeneity of breast tumors, the hormone evaluation is still the major feature considered for the immediate classification of a breast tumours and guide clinical management. Triple-negative breast cancer (TNBC) is defined by a lack of significant expression of the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) (L. Carey, Winer, Viale, Cameron, & Gianni, 2010) and accounts for 15% to 20% of newly diagnosed breast cancer cases (Metzger-Filho et al., 2012). Patients with TNBC are generally younger than the overall population of breast cancer patients (K. R. Bauer, Brown, Cress, Parise, & Caggiano, 2007), and they more frequently have larger and higher-grade tumors (Rakha et al., 2007). Different population-based studies have demonstrated a higher prevalence of TNBC among women of African American or black ethnicity (Morris et al., 2007),(Lund et al., 2009). A clear correlation has been made between young age at diagnosis and TNBC. In a large population-based study involving 6,370 patients, women with TNBC were significantly more likely to be under the age of 40 years (K. R. Bauer et al., 2007). TNBC is known to have an early peak of recurrence between the first and third year after diagnosis followed by a marked decrease in the recurrence rate in subsequent years and less frequent relapse after 8 years (Dent et al., 2007). Worse survival outcomes have been reported for TNBC tumors when compared with hormone receptor–positive tumors in several studies (Dent et al., 2009). Moreover, TNBCs have a site-specific distribution of recurrence. In a retrospective analysis of 1,608 patients, a greater proportion of TNBCs had visceral metastasis as first site of disease recurrence when compared with other types of BC (Dent et al., 2009). Despite these common features recognized in TNBC, there is notable diversity within the group. Histologic variability provides one example of such, with invasive ductal, metaplastic and medullary breast cancers coexisting in this patient population (Reis-Filho & Tutt, 2008).

Genetic profile of DNA repair genes in TNBC Introduction

Furthermore, the TNBC subtype does not directly correspond to a single molecular breast cancer subgroup (Rakha et al., 2009). Though most fit into the category of basal-like cancers, these groups are overlapping rather than synonymous. Defining TNBC through the absence of predictive biologic markers is limitating and could explain the heterogeneity of behavior and clinical outcome of TN patients. Subclassifications of TNBC based on the presence of biomarkers, gene signatures, and BRCA dysfunction have been proposed.

3.1. Molecular characterization of TNBC

In 2011 Lehmann group (Lehmann et al., 2011) performed a comprehensive and fundamental molecular analysis to better understand the basis of TBNC and to explore the true heterogeneity of this subgroup. To identify global differences in gene expression (GE) between TNBC subtypes, they compared 14 publicly available breast cancer microarray datasets, all generated on Affimetrix microarray. After the selection of set of 2188 genes significantly enriched in expression to determine the top canonical pathways associated, 337 TNBC were classified in 6 stable clusters: basal-like1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M),mesenchymal stem–like (MSL); luminal androgen receptor (LAR). Other 49 tumours were classified into an “unstable” group. BL1 subtype are heavily enriched in cell cycle and cell division components and pathways (cell cycle, DNA replication reactome, G2 cell-cycle pathway, RNA polymerase, and G1 to S cell cycle). The annotations are supported by the expression of genes associated with proliferation, such as AURKA, AURKB, CENPA, CENPF, BUB1, TTK, CCNA2, PRC1, MYC, NRAS, PLK1, and BIRC5. Elevated DNA damage response (ATR/BRCA) pathways accompany the proliferation pathways in the BL1 subtype. Increased proliferation and cell- cycle checkpoint loss are consistent with the elevated expression of the DNA damage response genes observed (CHEK1, FANCA, FANCG, RAD54BP, RAD51, NBN, EXO1, MSH2, MCM10, RAD21, and MDC1). Moreover the proliferative nature of this subtype is supported by the finding of high Ki-67 mRNA expression and nuclear Ki-67 staining as assessed by IHC analysis. The basal like tumours are Ki67 positive for 70% of the cases, while other subtypes in around 40%. Enrichment of proliferation genes and increased Ki-67 expression in basal-like TNBC tumors suggest that this subtype would preferentially respond to antimitotic agents such as taxanes (paclitaxel or docetaxel) (J. A. Bauer et al., 2010). This is notable when comparing the percentage of patients achieving a pathologic complete response (pCR) in 42 TNBC patients treated with neoadjuvant taxane in 2 studies (J. A. Bauer et al., 2010; Juul et al., 2010). In these combined studies, TNBC

Genetic profile of DNA repair genes in TNBC Introduction

patients whose tumors correlated to the basal-like (BL1 and BL2) subtype had a significantly higher pCR (63%; P = 0.042) when treated with taxane-based therapies as compared with mesenchymal-like (31%) or LAR (14%) subtypes. The BL2 subtype displays unique gene ontologies involving growth factor signaling (EGF pathway, NGF pathway, MET pathway, Wnt/β-catenin, and IGF1R pathway) as well as glycolysis and gluconeogenesis. Likewise, the BL2 subtype is uniquely enriched in growth factor receptor such as EGFR, MET, and EPHA2. This subtype has features suggestive of basal/myoepithelial origin as demonstrated by higher expression levels of TP63 and MME (CD10). The IM subtype is enriched of immune cell processes gene expression. These processes include immune cell signaling (TH1/TH2 pathway, NK cell pathway, B cell receptor signaling pathway, DC pathway, and T cell receptor signaling), cytokine signaling (cytokine pathway, IL-12 pathway, and IL-7 pathway), antigen processing and presentation, and signaling through core immune signal transduction pathways (NFKB, TNF, and JAK/STAT signaling). Immune signaling genes within the IM subtype substantially overlap with a gene signature for medullary breast cancer, a rare, histologically distinct form of TNBC that despite its high-grade histology is associated with a favorable prognosis (Bertucci et al., 2006). The Mesenchymal (M) subtype is enriched in components and pathways involved in cell motility (regulation of actin by Rho), ECM receptor interaction, and cell differentiation pathways (Wnt pathway, anaplastic lymphoma kinase (ALK) pathway, and TGF-β signaling). The MSL subtype shares similar profile of the M subtype, but differ for enhanced processes linked to growth factor signaling pathways that include inositol phosphate metabolism, EGFR, PDGF, calcium signaling, G-protein coupled receptor, and ERK1/2 signaling as well as ABC transporter and adipocytokine signaling. The prevalence of cell differentiation and growth factor signaling pathways is illustrated by expression of TGF-β signaling pathway components, epithelial-mesenchymal transition– associated (EMT-associated) genes and decreased E-cadherin (CDH1) expression. The MSL subtype is also enriched in genes involved in angiogenesis, including VEGFR2. Moreover in part overlap with IM subtype for the implication of immune signaling. One interesting difference between the M and MSL subtypes is that the MSL subtype expresses low levels of proliferation genes and higher levels of stem cells genes (ABCA8, PROCR, ENG, ALDHA1, PER1, ABCB1, TERF2IP BCL2, BMP2, and THY1), numerous HOX genes (HOXA5, HOXA10, MEIS1, MEIS2, MEOX1, MEOX2, and MSX1), and mesenchymal stem cell–specific markers (BMP2, ENG, ITGAV, KDR, NGFR, NT5E, PDGFRB, THY1, and VCAM1).

Genetic profile of DNA repair genes in TNBC Introduction

The M and MSL groups share similar features with a highly dedifferentiated type of breast cancer called metaplastic breast cancer, which is characterized by mesenchymal/sarcomatoid or squamous features and is chemoresistant (Gibson, Qian, Ku, & Lai, 2005). The MSL subtype also displays low expression of claudins 3, 4, and 7, consistent with a recently identified claudin-low subtype of breast cancer (Prat et al., 2010). Finally, the gene expression in the LAR group is the most differential among TNBC subtypes. This subtype is ER negative, but gene ontologies are heavily enriched in hormonally regulated pathways including steroid synthesis, porphyrin metabolism, and androgen/estrogen metabolism. AR (androgen receptor) mRNA was highly expressed together with numerous downstream AR targets and coactivators. This subtype showed high IHC staining for AR also. Tumors in the LAR subtype display luminal GE patterns, with FOXA1, KRT18, and XBP1 among the most highly expressed genes. Breast cancers can be classified as luminal or basal-like, dependent on their expression of different cytokeratins. TNBC tumor subtypes display differential expression of both basal- like cytokeratins (KRT5, KRT6A, KRT6B, KRT14, KRT16, KRT17, KRT23, and KRT81) and luminal cytokeratins (KRT7, KRT8, KRT18, and KRT19) across the subtypes. Only the tumors within the LAR subtype lacked basal cytokeratin expression and expressed high levels of luminal cytokeratins and other luminal markers. In the same work, a TNBC cell line panel was used to assess differential response to several agents targeting different pathway; PARP, AR, Src, and PI3K/mTOR signaling (Neve et al., 2006). BL1/BL2 cell lines, when treated with agents targeting DNA repair, especially platinum compounds, have in general a good response. PARP inhibitors treatment (olaparib and veliparib) showed instead more variability in response among cell lines. Cell lines expressing high levels of AR mRNA and protein, are sensitive to the AR antagonist bicalutamide and to an Hsp90 inhibitor, since this chaperonine is essential for AR protein folding. Importantly, AR status in TNBC patients may represent a molecular marker for preselection of patients for antiandrogen therapy. GE analysis of the mesenchymal-like subtypes demonstrated enrichment in the expression of genes in EMT and cell motility pathways. Since the non-receptor tyrosine kinase Src plays critical roles in cell migration, the authors investigated the effect of the Src inhibitor dasatinib on the panel of TNBC lines. Cell lines belonging to the mesenchymal-like subtypes (M and MSL) were more sensitive to dasatinib. Activating mutations in PIK3CA are the most frequent genetic event in breast cancer (Samuels et al., 2004): when TNBC cell lines were treated with the dual PI3K/mTOR inhibitor NVP-BEZ235, mesenchymal-like and LAR TNBC cell lines were more sensitive to

Genetic profile of DNA repair genes in TNBC Introduction

NVP-BEZ235 compared with basal like cell lines suggesting that deregulation of the PI3K pathway is important for these subtype.

3.2. Genomic features of TNBC

In the most comprehensive integrated genomic study by the Cancer Genome Atlas Network, basal-like tumors showed a high frequency of TP53 mutations (80%) which, when combined with inferred TP53 pathway activity, suggests that loss of TP53 function occurs within most, if not all, basal-like cancers. In addition to loss of TP53, loss of RB1 and BRCA1 are Basal-like features (Herschkowitz, He, Fan, & Perou, 2008). PIK3CA was the next most commonly mutated gene (~9%); however, defective PI3K pathway activity, whether from gene (Saal et al., 2008), protein (Stemke-Hale et al., 2008) or high PI3K/AKT pathway activities, was highest in Basal-like cancers. Alternative means of activating the PI3K pathway in Basal-like cancers likely includes loss of PTEN and INPP4B and/or amplification of PIK3CA. A recent paper from Shah (Shah et al., 2012) and collaborators described exome sequencing of 102 TNBC, and analyzed the clonal spectrum of somatic mutations in this group. The author searched for mutation enrichment patterns in three ways; by single gene mutation frequency over multiple cases; by the mutation frequency over multiple members of a gene family and by correlating mutation status with expression networks. First, similar to other studies (Langerod et al., 2007) p53 is the most frequently mutated gene 62% of basal TNBC and 43% of non-basal TNBC cases harboring a validated somatic mutation. Frequent mutations in PIK3CA at 10.2%, USH2A (Ushers syndrome gene, implicated in actin cytoskeletal functions) at 9.2%, MYO3A at 9.2%, PTEN and RB1 at 7.7%, SYNE1/2 at 9% and BRCA2 (3%) were observed. Considering background mutation rates, TP53, PIK3CA, RB1, PTEN, MYO3A and GH1 showed evidence of single gene selection. Several other well-known oncogenes (BRAF, NRAS, ERBB2, and ERBB3) have rare mutations. Moreover, the impact of gene family involvement through sparse mutation patterns in functionally connected genes was evaluated. TP53 related pathways along with chromatin remodeling, PIK3 signaling, ERBB2 signaling, integrin signaling and focal adhesion, WNT/cadherin signaling, growth hormone and nuclear receptor co-activators, ATM/Rb related pathways were the most affected. Also MYO3A, a cytoskeleton motor protein involved in cell shape/ motility, was related to several pathways upstream and downstream of integrin signaling. The mutated genes stretch from extracellular matrix interactions (ECM) (laminins, collagens), ECM receptors (integrins), several proteins

Genetic profile of DNA repair genes in TNBC Introduction

regulating actin cytoskeleton dynamics (myosins) and microtubule motor proteins (kinesins). Interestingly, a comparison of the RNA-seq with genomic data revealed that only 36% of validated somatic SNVs were observed in transcriptome sequence. As expected, the proportion of low abundance somatic SNVs observed in RNA is reflected in the distribution of wildtype, heterozygous and homozygous expressed mutations, consistent with the notion that low abundance alleles may represent rarer clones in the primary tumour. The tumours exhibit a wide spectrum of modes of clonal frequencies, with some cases showing only one or two frequency modes indicating a smaller number of clonal genotypes, whereas other tumours exhibit multiple clonal frequency modes, indicating more extensive clonal evolution. Consistent with “early driver gene” status, mutations in known tumour suppressors such as p53 tend to occur in the highest clonal frequency group in most tumours. However in some cases p53 resides in lower abundance clonal frequency groups suggesting it was not the founding event. Overall, expected, mutations in TP53 and PIK3CA showed significantly higher clonal frequencies, consistent with their roles in early tumourigenesis while, for example, pathways with cytoskeletal genes such as myosins, laminins, collagens and integrins tend to have lower median clonal frequencies suggesting that somatic mutations in these genes are acquired much later. Moreover, Shah noted that basal TNBC have more clonal frequency modes than non- basal TNBC. These observation highlighted the fact that at the time of diagnosis TNBC already display a widely varying clonal evolution that mirrors the variation in mutational evolution.

Mutations in BRCA1, PTEN and RB1 gene are associated with high chromosomal instability (Hu et al., 2009); indeed basal TNBC group displays the most instable genome. Besides mutations, other genetic changes such as copy number alterations occur differentially between distinct subtypes. This work observed that globally, CNA occur more often in BASAL TNBC than in any other subtype. A gene that was found to be specifically amplified in basal TNBC is nuclear factor 1/B (NFIB), residing on the short arm of chromosome 9. The function of this gene in cancer biology has to be defined yet; however, it plays a known role in central nervous system development (Han et al., 2008). A structural deletion of 80 Mb on chromosome 5q13-14 that occurs more frequently in TNBC contains the RASA1 gene. This gene has a function in de-activating RAS, and loss of RASA1 results in an overactive RAS tyrosine kinase.

Genetic profile of DNA repair genes in TNBC Introduction

Since RAS is a stimulator of cell growth, RAS overactivation leads to increased proliferation activity (Hu et al., 2009). Several studies linked basl TNBC to epidermal growth factor receptor (EGFR) expression, and percentages ranging from 42 to 71% were found (Cheang et al., 2008; Nielsen et al., 2004). This receptor, like HER2, is a potent stimulating factor of cell-growth-activating pathways and thus stimulates tumour growth when activated (Burgess 2008). EGFR expression in breast cancer is associated with poor disease outcome (Viale et al., 2009) showed worse disease-free survival (DFS), overall survival (OS) and distant disease-free survival (DDFS) for EGFR expressing TNBC compared to tumours without EGFR expression. Also, response rates of EGFR-positive breast tumours to chemotherapeutic therapy proved to be lower (Nogi et al., 2009). EGFR expression could be one of the causes of the poor disease outcome of basal TNBC. Hyperactivated FOXM1 is a transcriptional driver of the enhanced proliferation signature of basal-like cancers; even hyperactivated cMYC and HIF1α/ARNT network acts as key regulatory features. Even though chromosome 8q24 is amplified across all subtypes, high cMYC activation appears to be a Basal-like characteristic (Chandriani et al., 2009). The most frequent somatic events include ATM mutations, BRCA1 and BRCA2 inactivation, RB1 loss and Cyclin E1 amplification. Interestingly, these same alteration were identified previously for Serous Ovarian cancers (Cancer Genome Atlas Research, 2011). Furthermore, the Basal-like (and TNBC) mutation spectrum was reminiscent of the spectrum seen in Serous Ovarian cancers with only one gene (i.e. TP53) at >10% mutation frequency. Comparing copy number landscapes, we observed several common features between Ovarian and Basal-like tumors including widespread genomic instability and common gains of 1q, 3q, 8q and 12p, and loss of 4q, 5q and 8p. BRCA1 inactivation, RB1 loss and Cyclin E1 amplification high expression of AKT3, cMYC amplification and high expression, and a high frequency of TP53 mutations are the common feature between basal like breast cancer tumours and aggressive serous ovarian cancer. Given that most Basal-like cancers are TNBC, finding new drug targets for this group is critical. Unfortunately, the somatic mutation landscape for Basal-like breast cancers has not provided a common target aside from BRCA1 and BRCA2. The copy number landscape of Basal-like cancers, however, showed multiple amplifications and deletions, some of which may provide therapeutic targets Potential targets include losses of PTEN and INPP4B while many of the components of the PI3K and RAS-RAF-MEK pathway are amplified (but typically not mutated): PIK3CA (49%),

Genetic profile of DNA repair genes in TNBC Introduction

KRAS (32%), BRAF (30%), and HER1/EGFR (23%), FGFR1, FGFR2, IGFR1, c-KIT, MET and PDGFRA. Basal like tumors also have high HIF1α/ARNT pathway activity, suggesting that these malignancies might be susceptible to angiogenesis inhibitors and/or bioreductive drugs that become activated under hypoxic conditions.

3.3. TN and basal like: one doesn’t fit to another

The relationship between TNBC and basal-like breast cancer remains controversial (Rakha et al., 2009). Though TNBC and basal-like breast cancers share many characteristics, the terms are not equivalent. While TNBC is defined by lack of hormone receptor and HER2 expression, basal-like breast cancer is characterized by a gene expression profile similar to that of normal basal cells (Metzger-Filho et al., 2012). Though testing for TNBC has become quite routine in clinical practice, the identification of the basal-like cancers remains difficult due to requirement of gene-expression profiling. The complexity and costs of gene expression profiling limit its use in clinical practice. Different research groups have proposed IHC based surrogates to diagnose the genomically defined Basal like Breast Cancer. The IHC variables most commonly were the triple negativity definition, basal CKs (i.e. CK5/6, CK 4, and CK17), epidermal growth factor receptor (EGFR), and C-kit (CD117). In a study of 21 BLBC cases identified by gene expression analysis, this immunohistochemical panel of markers showed a sensitivity of 76% and a specificity of 100% for the identification of these tumors (Nielsen et al., 2004). The presence of such a BLBC surrogate within a group of TNBCs was associated with shorter survival when compared with the remaining TNBCs.

A study comparing TNBC expressing cytokeratin 5,6,14 and EGFR versus a TNBC group negative for these markers, showed that cycle regulators and cell proliferation appeared to be different between both tumor groups, with p53 and p16 proteins having discriminating roles being expressed at high levels by basal TNBC (Rakha et al., 2009). Moreover has been reported expression of luminal cytokeratin (CK19) in basal cancers when compared with cyotokeratins-negative tumours. This capacity to coexpress basal and luminal cytokeratins could have biological and clinical implications, suggesting that BLBC may either have features of dual-lineage differentiation or a more stem-like phenotype than not basal-like, which may result in their increased aggressiveness. Consistent with these findings, it has recently been reported that BLBC also differs non basal like tumors in the expression of CD44+/CD24-; the proposed population of breast cancer cells enriched for cells with stem cell properties (Honeth et al., 2008). At present,

Genetic profile of DNA repair genes in TNBC Introduction

there is no standardization for a panel of IHC markers to identify BLBC cancers, limiting their applicability in clinical practice. Therefore, it has been decided that it is more practical and biologically acceptable to restrict identification of BLBC to within the ER- and HER-2- negative group of tumors. However, up to 35% of basal-like breast cancers are not TNBC, and up to 25% of TNBCs are not basal-like (Prat et al., 2013); (L. Carey et al., 2010). GE profile of 1510 genes (Prat et al., 2013) revealed only partial concordance between receptor status and PAM50 profile: TN/luminal, TN/basal and TN/Her2 groups were discovered. The last class, for example, resembled expression profile of Her2 enriched subgroup even not overexpressing the receptor. Only five genes found significantly downregulated in TN/Her2 compared to Her2/non-TN and were all found in the 17q11–13 amplicon (HER2/ERBB2, GRB7, MED1, SCGB2A2 and STARD3). Thus, apart from the genes on the HER2 amplicon, almost no differences existed between subtype matched TN vs non- TN tumors when tested on the mRNA level. Moreover TN/basal-like basal showed significant lower mean age at diagnosis than other TN groups. Prat has also re-analyzed the TCGA data in the light of these new subgroups finding that even within TN cancers, the mutation spectrum observed continued to follow molecular subtype instead of a common biology under the TN entity. TN/basal-like and TN/Her2 tumors showed the largest number of total somatic mutations (mean number of mutations). In terms of TP53 somatic mutations, 40% TN/luminal tumors had TP53 mutations versus 85% TP53 mutations within TN/basal-like tumors and 100% TP53 mutations in TN/Her2. Moreover, BRCA1/2 deleterious mutations (somatic and germline mutations combined) were found in 16 of 73 (22%) TN/basal-like tumors versus 1 of 5 (20%) in TN/luminal tumors (which was a BRCA2 germline mutation) and 0 of 5 (0%) in TN/Her2 tumors. Conversely, somatic mutations in PI3KCA, which is a frequent ER/luminal tumor mutation, were found mainly in TN/luminal tumors.

Genetic profile of DNA repair genes in TNBC Introduction

Fig 2. Concordance between PAM50 signature e IHC (Prat et al., 2013). Distribution of the intrinsic molecular (left) and pathology-based (right) subtypes within triple-negative and basal-like tumors.

Several are the possibilities regarding the discrepancy between gene expression and IHC- based assays. First of all, is the false positivity or false negativity of the IHC-based assays which differs among labs and methods; then is the possibility that pathology and gene expression data could have been obtained from two different areas of the same tumor even high concordance among analysis on different regions of the same primary tumour has been shown. However, gene expression measures a large number of related genes, compared with the three individual pathology-based biomarkers that define TN disease. This multigene expression data might better capture the true biological profile. Recent studies have revised the classification by Lehmann, since the immunomodulatory subtype exhibit a signature most likely coming from microenvironment (i.e., coming from fibroblasts and immune cells) and not from the actual tumor cells. The classification ultimately uses three main groups (mesenchymal, basal-like, and LAR) that showed different responses to cytotoxic and targeted therapies. This three subtype classification is very concordant with the three main groups previously identified by Prat group (claudin- low, basal-like and luminal/HER2E). TNBC do not form a homogeneous group when analyzed by gene expression profiling. In contrast, the basal-like subtype does form a homogeneous group of tumours with a similar gene expression profile related to prognosis and therapy response (Bertucci, Finetti, Cervera, & Birnbaum, 2008); (Rakha et al., 2009). This indicates that the prognosis of TNBC may actually refer to the high percentage of triple-negative tumours falling in the basal-like subtype (Cheang et al., 2008). Indeed, several studies reported a poor disease-specific survival for the basal-like subtype (Sorlie et al., 2003); (Nielsen et al., 2004); (L. A. Carey et al., 2006); (Morris et al., 2007). Furthermore, in cases in which metastasis occurred, the disease-free survival interval was found to be significantly shorter (Sorlie et al., 2003). Also, at primary diagnosis, basal-like

Genetic profile of DNA repair genes in TNBC Introduction

tumours show adverse characteristics, such as a high nuclear and mitotic grade and unfavorable histological features, like high mitotic index and poor differentiation (L. A. Carey et al., 2006). Therefore, even if the triple-negative group of breast cancer is not a homogeneous disease entity, a substantial fraction of these tumours belongs to the basal-like type, which does form a homogeneous group. Thus, the overall poor prognosis of TNBC may be a result of this estimate overlapping. There is increasing evidence that a basal TNBC subtype develops mainly through a BRCA1-related pathway. Somatic mutations that contribute to TN basal development are possibly not related to this pathway, but may occur randomly due to increased genomic instability in these tumours. An hypothesis has been proposed in which non-basal-like TNBC and non-triple-negative BC are possibly not the direct result of this pathway, but receive their distinctive genotype because of random mutations (de Ruijter, Veeck, de Hoon, van Engeland, & Tjan-Heijnen, 2011). A breast tumour that develops following disruption of the BRCA1-related pathway, it might turn into basal TNBC However, because of the instable genome, it is possible that genomic changes occur that are not BRCA1 pathway related. If these genomic changes involve, for example, HER2 gene amplification, the tumour will no longer be basal TNBC, but non-triple-negative BC. Since the tumour benefits from HER2 overexpression, there exists a selective pressure towards HER2 amplification. This could be the explanation for the approximately 20–30% of basal-like cancers that are not triple-negative. Likewise, in non-basal-like tumours, a selective pressure is present towards losing hormone receptors expression, since this enables the tumour to grow independently from the presence or absence of growth-stimulating factors. This may represent the cause of the approximately 20–30% of TNBC that are not of the basal-like subtype.

4. BRCA1 AND TNBC: THE BRCAness PHENOTYPE

In search of pathways that lead to the development of basal TNBC, several studies have found that BRCA1-related breast cancers are associated with the TNBC subtype (Foulkes, 2003); (Lakhani et al., 2005); (Diaz, Cryns, Symmans, & Sneige, 2007), and TNBC expression profiles resemble those of BRCA1- related breast cancers (Foulkes, 2003). Moreover, it has been observed that around 75% of BRCA1-associated tumors are basal like or TN (Waddell et al., 2010); (Foulkes, 2010).

Genetic profile of DNA repair genes in TNBC Introduction

The phenotype that some sporadic tumours share traits with familial-BRCA cancer is called BRCAness and is described in a landmark paper (N. Turner, Tutt, & Ashworth, 2004). The importance of defining such a group of tumours lies within the clinical management of these tumours. As BRCA1 and BRCA2 are involved in the repair of DNA double-strand breaks (DSBs), a process called homologous recombination, dysfunctional BRCA proteins could make a tumour extra sensitive for drugs inducing those DNA DSBs. What the best method is to measure BRCAness is still not known. Therefore, also the extent of BRCAness in TNBC is currently in definition. Is known that BRCA1-related abnormalities (aCGH BRCA1-like profile, BRCA1 promoter methylation and low BRCA1 mRNA expression) were predominantly observed in TN tumours, whereas a BRCA2-like profile was mainly observed in hormone receptor positive tumours (Lips, Laddach, et al., 2011); (Lips et al., 2013). In these studies a aCGH BRCA1-like profiles and BRCA1 promoter methylation status were used as indicators of BRCAness. 76% of the TNBC cases had a BRCA1-like aCGH profile. Of these 30% had BRCA1 promoter methylation, while 16% had a germline BRCA1 mutation. For the remaining cases, explanation of the BRCAness phenotype remained incomplete suggesting that other mechanisms may be involved. For example, other genes of the DNA damage response pathway, where BRCA1 and BRCA2 have a role (N. Turner et al., 2004). Recently, methylation of FANC genes has been described (N. Turner et al., 2004); (Cancer Genome Atlas, 2012b)). In genetic counselling, an aCGH BRCA1 or BRCA2-like genomic profile may be an extra indication of BRCA1/2 involvement, whereas its absence can help to rule out a hereditary component (Joosse et al., 2009). Also, it can help to classify variants of unknown significance. Moreover, it has been proposed that all TNBCs below 50 years should be screened for BRCA1 and 2 mutations (Kwon et al., 2010); (Robertson et al., 2012). The BRCA1 promoter methylation analysis in pre-screening assay, can help in exclude patients unlikely carriers of mutations. These similarities gave rise to the idea that BRCA1 mutations could play a role in the development of TNBC, especially of the basal-like group. The BRCAness phenotype is correlated with alterations in DNA repair pathways. Sporadic basal-like and BRCA1-associated tumors share the loss of the PTEN locus, which point to a deficit in the HR pathway (Saal et al., 2008); (Mendes-Pereira et al., 2009). The presence of TP53 mutations in BRCA1-associated tumors is due to deficiency in DNA repair mechanisms induced by BRCA1 loss of function (Holstege et al., 2009). TP53 mutational status has been analyzed by Holstege et al. (Holstege et al., 2010), who

Genetic profile of DNA repair genes in TNBC Introduction

observed that TP53 mutations occur in almost all BRCA1-related tumors and basal-like BC, compared with in 20%– 50% of luminal tumors. In basal-like BC, the loss of 17q21, where BRCA1 maps (Staff, Isola, & Tanner, 2003), and low expression of BRCA1 at transcriptional and protein levels has been reported. Moreover, in 11%–13% of Basal-like BC, BRCA1- promoter hypermethylation was observed (Esteller et al., 2000) and a high expression of ID4, a transcriptional repressor of BRCA1, has been measured. This implies that low BRCA1 expression could also be the result of gene regulatory mechanisms, such as ID4 overexpression or promoter methylation.

Copy number alterations (CNAs) associated with Basal-like BC including many aberrations are also found in BRCA1 mutation carriers. CNAs observed in Basal-like BC include the loss of 10p containing MAP3K8, ZEB1 and FAM107B and the deletion of 16q and of 4q, which contains INPP4B, a tumor suppressor involved in PI3K signaling (56) frequently deleted in TNBCs/Basal-like BC. BRCA1- related tumors and Basal-like BC exhibit frequent loss of 5q11-35 (Johannsdottir et al., 2006); (Hu et al., 2009), where important genes involved in DNA repair, such as RAD17, RAD50 and RAP80bmap. In fact, Weigman observed that in 80% of tumors with 5q11-35 (RAD17 + RAD50) loss, there was also RB1/13q14.2 deletion and in 60% 5q11- 35 and TP53/17p13.1 loss co-occurs. These data led authors to speculate that the loss of the RAD17 + RAD50 locus with deletions of other regions contributes to impaired homologous recombination and then to genomic instability. Moreover, abnormalities in the inactive X chromosome (Xi) that destabilize its silenced state and activate genes that are inactive in non-cancerous cells are associated with loss of BRCA1 function (Ganesan et al., 2002), and they are also associated with basal TNBC (Richardson et al., 2006); (N. C. Turner et al., 2007). Taken together, these findings support the hypothesis that loss of BRCA1 function may play a major role in TN basal-like cancer development (Richardson et al., 2006). Still, the fact that not all them show loss of BRCA1 expression suggests that further genes related to the BRCA1 pathway are likely to be deregulated in the process of basal TNBC development (Richardson et al., 2006).

4.1. Other Susceptibility loci for TNBC

Several studies of unselected triple-negative cases have shown that 9% to 14% overall and approximately 20% of cases diagnosed under the age of 50 years harbor germline BRCA1 mutations. Similarly, as many as 34% of triple-negative cases with a family history

Genetic profile of DNA repair genes in TNBC Introduction

of breast cancer and 30% of triple-negative cases from women of Ashkenazi Jewish ancestry are associated with germline BRCA1 mutations. To a lesser extent, BRCA2 mutations are also associated with TNBC in that 16% to 23% of breast tumors arising in BRCA2 mutation carriers display triple-negative properties. While few breast cancer susceptibility genes have been systematically evaluated for mutations in triple-negative cases, it is already clear that up to 15% of unselected triple-negative cases result from inherited mutations in the BRCA1 and BRCA2 high-risk susceptibility genes. As previously reported, a study of The Cancer Genome Atlas Network (TCGA) provides further insights into the distribution of mutations in high-risk genes by breast cancer subtypes. Among the 93 basal-like tumors in this group, mutations were identified in BRCA1, BRCA2, RAD51C, and TP53. Interestingly, no mutations in the remaining 5 well known susceptibility genes (ATM, BRIP1, CHEK2, NBN, and PTEN) were detected among basal tumors. Large-scale examination of the mutational spectrum of all known breast cancer susceptibility genes in women with TNBC, and the individual TNBC subtypes, will be necessary to fully understand the role of these genes in triple negative.

Recently, genome wide association studies (GWAS) international from the Collaborative Oncological Gene-environment Study (iCOGS) have proposed common variants in 72 loci implicated in breast cancer predisposition. Interestingly, these risk loci are heterogeneous with respect to ER status, with only a subset associated with risk of ER-negative breast cancer. In particular variants in 7 loci associated with risk of ER-negative disease were identified. These include variants in TERT, 20q11, 6p14, MDM4, LGR6. The rs10069690 variant in the TERT promoter have been associated with ER-negative breast cancer, but also with increased risk of TNBC; moreover a separated analysis between HER2 positive and negative tumors has suggested that this variant may be uniquely associated with TNBC. In a similar way, also the MDM4 and 20q11 loci are also associated with TNBC phenotype. Interestingly, 3 loci specific to TNBC contain genes (TERT, c19orf62, and MDM4) that encode proteins involved in DNA repair and the preservation of genomic stability. The TERT gene encodes the catalytic subunit of telomerase, which controls telomere maintenance, and has been associated with genomic instability and linked to tumorigenesis. MDM4 is a repressor of TP53 and TP73 transcription and is important for cell-cycle regulation and apoptosis in response to DNA damage. C19orf62 encodes the MERIT40 protein, which is integral to the localization of the BRCA1-A complex during DNA double-strand break (DSB) repair, through the recruitment and retention of the BRCA1-BARD1 ubiquitin ligase. Telomere maintenance, DSB repair, and DNA damage

Genetic profile of DNA repair genes in TNBC Introduction

checkpoints have been linked as coordinating factors in genomic integrity. Indeed, one proposed mechanism of spontaneous telomere loss in cancer cells is a deficiency in DSB repair combined with oncogene-mediated DNA replication stress. In addition, evidence suggests that DNA damage checkpoint and DNA repair proteins have an essential role in telomere maintenance, by controlling the processing of telomeric DNA and through other mechanisms that have yet to be delineated. An alternative hypothesis is that variants or even combinations of variants in the TNBC- associated risk loci may act to change existing malignant breast lesions to a triple- negative phenotype during the formation of the tumor. For example, a particular locus such as ESR1 may predispose breast epithelium to cancer in general while others such as TERT and 19p13.1 may act downstream after tumorigenesis has begun to direct tumors towards the triple negative phenotype. Thus, the identification of these TNBC genetic loci offer exciting opportunities to better understand how triple-negative tumors arise (Stevens, Vachon, & Couch, 2013).

5. DNA REPAIR AND BREAST CANCER

During the cell cycle, cells progress through a series of cell cycle checkpoints before mitotic cell division and distribution of the genomic material to the daughter cells. In response to genotoxic stress, cells activate these checkpoints to prevent further progression through the cell cycle and initiate DNA repair (Jackson & Bartek, 2009). If the extent of DNA damage is beyond repair capacity, additional signaling pathways leading to the induction of apoptosis are activated, eliminating potentially dangerous mutated cells (Reinhardt & Schumacher, 2012). This signaling network, which is commonly referred to as the DNA damage response (DDR), is tightly controlled and involves regulation at the transcriptional, post-transcriptional, and post-translational levels (Reinhardt, Cannell, Morandell, & Yaffe, 2011). The genome integrity is strictly correlated with cancer prevention as genes, which encode components of the DDR and specifically of DNA repair pathways, are among the most frequently mutated genes in cancer (Ciriello et al., 2013). It is thought that inactivating mutations in DDR core components contribute to a ‘mutator phenotype’, leading to the accumulation of additional cancer-driving genomic aberrations (Loeb, Bielas, & Beckman, 2008) (Lord & Ashworth, 2012). Silencing parts of the DDR signaling cascade is a prerequisite for malignant transformation. These observations explain why human tumors typically display defects in DDR signaling and why genomic instability is emerging as a hallmark of cancer (Negrini, Gorgoulis, & Halazonetis, 2010).

Genetic profile of DNA repair genes in TNBC Introduction

Breast cancers and neoplasms are intimately related to DNA damage repair defects or defects in cell-cycle checkpoints which allow damaged DNA to go unrepaired. Most often tumor suppressor genes are involved with maintenance of DNA fidelity as is the case for BRCA1 (DNA damage repair), TP53 (cell cycle checkpoint) and PTEN (blockage of cell-cycle progression in G1 and participation in DNA repair). The genes involved in heritable susceptibility to cancer often function as DNA damage response effectors or cell cycle control effectors (Guler et al., 2011). Inherited breast cancers occur early and in pre-menopausal years because of the increased risk of loss of heterozygosity, and thus loss of gene expression of a DNA damage response or cell cycle control effector gene product (Gonzalez-Angulo et al., 2011); (Jara et al., 2010). In sporadic breast cancer an acquired mutation or epigenetic inactivation occurs due to mechanisms other than inheritance of defective genetic material. Again many of these mutations or epigenetic inactivations occur within genes involved in DNA damage repair. Breast tissue has increased opportunity for DNA damage occurrence because of the extensive remodeling that occurs throughout a woman’s life. Breast is one of the few organs in the body that undergo precisely defined cell death and cellular proliferation on a moderate to large scale during in utero development, puberty, monthly pre-menopausal 28-d cycles, during pregnancy, lactation and involution (weaning-induced process of massive mammary remodeling) (Rijnkels et al., 2010); (McCready, Arendt, Rudnick, & Kuperwasser, 2010). Thus, throughout a woman’s lifetime her mammary tissue is undergoing proliferation, apoptosis and differentiation at a rate higher than most other tissues. Moreover, it remains to be determined which dietary and environmental exposures may present the most DNA damage in early life or adulthood, and whether this accumulation of DNA damage is directly related to breast cancer risk. DNA assaults during these times of mammary remodeling may be endogenous (replication stress, oxidative species, replication errors), environmental (chemical exposures, food contaminants, naturally occurring endocrine disruptors in foods) or simply due to the process of aging which in itself can produce increased susceptibility to chromosomal abnormalities because of reduced expression of telomerase. Endogenous assaults on DNA are common such as DNA copying errors, replication stress due to a requirement for high levels of proliferation and endogenously created reactive oxygen species. Some DNA damage occurs under the normal fluctuations of physiological conditions. However, the process of DNA repair itself can create mutations, insertions, deletions and base replacements. For example, mutations can occur due to inappropriately high levels of a repair mechanism which normally suppresses tumorigenesis, mitotic recombination

Genetic profile of DNA repair genes in TNBC Introduction

and hyper-recombination which has recently been linked to mutations in BRCA1 but is also likely to occur due to other defects in DNA damage repair genes (Moynahan & Jasin, 2010); (Dever et al., 2011). Gross DNA lesions and chromosomal abnormalities can be induced by DNA replication stress. Replication stress is a hall-mark of pre-cancerous cells and may occur when accelerated replication is required such as tissue regeneration or response to hormones or growth factors which stimulate replication. During this time chromosomal abnormalities can accumulate in the DNA synthesis phase of the cell cycle (S phase) and carry over to mitosis (M phase) (Negrini et al., 2010). Reactive oxygen species (ROS), mainly produced by metabolic products of energy production in mitochondria can also promote DNA damage. Some of these potent oxidants react with hydroxyl groups of DNA and can induce the breakage of DNA strands. Normally PARP-1 is activated in a situation of oxidative stress as a mechanism to protect DNA from further damage and initiate either DNA repair mechanisms or cell death. Environmental assaults occur almost daily in adult tissue and can be due to exposure to radiation such as gamma irradiation or various chemicals known as carcinogens. Chemical DNA damaging agents are present in the environment, water, air, and pollution. Gamma irradiation can cause single and double-strand breaks, normally repaired by homologous recombination. Carcinogens are very different in actions and DNA base-pair analogs, as the analog of thymidine 5-bromouracil, hydroxylating agents, alkylating agents which can also cause epigenetic silencing of gene expression, deaminating agents, and intercalating agents, which cause DNA bulges that can be repaired to insert or delete a random base-pairing.

In normal human somatic cells, senescence occurs after a certain time and is triggered by the activation of interdependent mechanisms including telomere shortening. Telomeres naturally shorten due to the problem of replicating the ends of chromosomes in the linear DNA synthesis process utilized by mammals. In order to prevent premature telomere shortening, the cell utilizes telomerase complexes which contain a telomerase reverse transcriptase (hTERT in humans) and a telomerase RNA. Together this complex can renew the repetitive end-repeats of chromosomes and thus extend the proliferative potential of the cell. In somatic cells, telomerase expression is normally very low, however, in highly proliferating cells and cancer cells, telomerase expression and function is exceptionally high (Cerni, 2000); (Karimi-Busheri, Rasouli-Nia, Mackey, & Weinfeld, 2010). Recent in vitro studies suggest that breast cancers might contain tumor initiating cells which have become resistant to the normal induction of senescence. One of the tumor

Genetic profile of DNA repair genes in TNBC Introduction

suppressing mechanisms of the gene BRCT repeat inhibitor of hTERT (BRIT-1), which was discovered through a genetic screen of telomerase inhibitors is directly linked to stopping the inappropriate maintenance of telomeres (Rai et al., 2006). This cellular immortalization then can allow for accumulation of mutations which confer to the cancer cell an even further survival advantage or metastatic potential.

5.1. DNA Repair Mechanisms

There are several types of physical DNA lesions acquired throughout life. The broad categories include DNA mutations (insertions, deletions, inappropriate repetitions), and DNA chromosomal abnormalities including single strand breaks (SSB), double strand breaks (DSB) and chromosome fusion.

Fig 3. DNA repair pathway. Schematic representation of DNA damage events and mechanisms of response. The most important genes for each pathway are reported.

5.1.1. Mismatch repair (MMR)

MMR recognizes erroneous insertions, deletions, and mis-incorporations of bases that have been inserted due to problems into the intrinsic proofreading activity of the DNA polymerases. These lesions are detected by the MutSa complex (detection of small

Genetic profile of DNA repair genes in TNBC Introduction

mismatches, formed by Msh2/Msh6) and the MutSb complex (detection of large mismatches and insertion loops, formed by Msh2/ Msh3) (Kunkel & Erie, 2005). In eukaryotes it was proposed that the strand containing nicks is coated by PCNA. The MutLa complex (formed by MLH1 and PMS2) connects MutS with the PCNA complex by a mechanism that is not fully understood. Upon binding, the exonuclease Exo1 is recruited to the MutS/ MutLa complex and the lesion gap is filled by DNA Pold (G. M. Li, 2008). One unique characteristic of mismatch repair is that it is strand specific, focusing on correcting mistakes in the daughter strand and in this type of DNA repair, the parental strand is utilized as a template for DNA repair. MMR defects are common in colon cancer (Yoshida et al., 2011), while enhanced mismatch repair in breast and ovarian cancer cells can confer resistance to chemotherapeutic drugs based on platinum structure such as cisplatin. Approximately 70–80% of germline mutations identified in Hereditary No Poliposys Colon Cancer (HNPCC) families are mutations in MLH1 or MSH2, whereas mutations in MSH6 are found in approximately 10% of HNPCC families. Germline mutations in other human MMR genes, including PMS1, PMS2, MLH3, and exonuclease 1 (EXO1), have also been found in HNPCC families; however, they occur at a much lower frequency. In addition, inactivation of MLH1 by mutations at the promoter or coding sequences, or by promoter methylation, has been identified in sporadic colorectal tumors (Vasen et al., 2014).

5.1.2. Nucleotide Excision Repair (NER)

NER is a rapid and efficient mechanism used by cells to repair distortions in the DNA double helix which may be recognized as bulky adducts, which are induced by UV irradiation and platinum-based chemotherapeutics (Cleaver, 2005); (Shuck, Short, & Turchi, 2008). NER can be initiated by global-genome NER (GG-NER), recruiting primarily XPC/RAD23B and RPA/XPA complexes to the damaged site or by transcription-coupled- NER (TC-NER), recruiting a complex of XPG and CSB to sites of RNA polymerase stalling GG-NER and TC-NER culminate in recruitment of TFIIH. This factor incorporates the helicases XPB and XPD and unwinds a _30 nt fragment around the damaged site. After DNA unwinding, the complexes XPF/ERCC1 and XPG are recruited, and these exhibit nuclease activity at the 5’ and 3’ ends of the lesion. Finally, the damaged site is resynthesized by complexes consisting of DNA-Pold/e, RFC, and PCNA or, alternatively, DNA-Pold/e and XRCC1. This mechanism of DNA repair is particularly important in maintenance of the skin, and loss of NER activity is related to heritable skin diseases such as Xeroderma pigmentosum

Genetic profile of DNA repair genes in TNBC Introduction

or Cockayne syndrome. NER mechanisms differ from BER in that BER requires specific glycosylases which will recognize only certain types of DNA damage.

5.1.3. Base excision repair (BER)

Single-stranded breaks and non-helix-distorting base modifications are repaired by BER (David, O'Shea, & Kundu, 2007). Modified bases are detected and excised by DNA glycosylases such as Ogg1 and Mutyh. PARP1 and PARP2 are important sensors for SSBs and facilitate recruitment of additional BER factors such as XRCC1 to the site of injury (Campalans et al., 2013). Subsequently, XRCC1 recruits the APE1 nuclease, which cleaves the abasic site at the 5’ position. Next, RFC recruits a complex of PCNA and DNA-Pold/e to the damaged site, and this displaces and resynthesizes 2–8 nt from the damaged site. Finally, the endonuclease FEN1 cleaves the dislodged oligonucleotide and the SSB is resealed by ligase. An important protein involved in BER and single strand break repair is PARP1, and this particular target is under intense clinical investigation as a fruitful treatment for TNBC patients. For a period of time, little evidence existed to support the involvement of BER in human cancer or any other disorders. However, recent studies have demonstrated the existence of a human disorder linked to defective BER. This autosomal recessive disorder, referred to as MUTYH-associated polyposis (MAP), is associated with biallelic germline mutations of the human MUTYH, and is characterized by multiple colorectal adenomas and carcinomas (Cheadle & Sampson, 2007).

5.1.4. Homologous recombination (HR)

HR is a very flexible mechanism for repairing SSB, DSB, interstrand crosslinks, stalled or collapsed replication forks or simply for creating genetic diversity (Moynahan & Jasin, 2010). HR is an error-free DSB repair pathway that is largely restricted to the S- and G2- phases of the cell cycle, when an intact sister chromatid is available as a template (Chapman, Taylor, & Boulton, 2012). Upon initiation of the HR process, the DSB is resected to create a single-stranded (ss) 3’-overhang, which becomes rapidly coated with the ssDNA-binding replication protein A (RPA). This ssDNA overhang ultimately invades the sister chromatid. Once an RPA-coated ssDNA filament is generated, RPA is replaced by Rad51 in an ATM/CHK2/BRCA1/BRCA2/PALB2-dependent way (Krejci, Altmannova, Spirek, & Zhao, 2012). Rad51 lies at the heart of the HR mechanism and mediates homology search, strand exchange, and Holliday junction formation (Chapman et al., 2012). Although it accounts only for the repair of 10% of DSBs in mammalian cells, HR defects can have

Genetic profile of DNA repair genes in TNBC Introduction

severe consequences. The enormous importance of the HR pathway for genome maintenance and cancer prevention is underscored by the observation that patients with heterozygous germline mutations in different HR genes, such as BRCA1, BRCA2, and RAD51C, display a dramatically elevated risk for the development of cancer. Furthermore, disabling mutations in BRCA1, BRCA2, ATM, CHEK2, RAD50, RAD51C, and others have been recurrently identified in numerous cancers, including lung cancer, ovarian carcinomas, pancreatic ductal adenocarcinoma, and chronic lymphocytic leukemia (Cancer Genome Atlas Research, 2011); (Puente et al., 2011); (Hartlerode & Scully, 2009).

5.1.5. Non Homologous End Joining (NHEJ)

Mammalian cells also employ the NHEJ pathway to resolve DSBs. NHEJ-mediated DSB repair does not require the presence of an intact template. Thus, NHEJ is preferentially activated during the G1-phase of the cell cycle. Upon initiation of NHEJ, the non-catalytic subunits Ku70 and Ku80 form a heterodimer that detects and binds to the free DNA ends (Lees-Miller & Meek, 2003); (Deriano & Roth, 2013). The Ku70 and Ku80 complex subsequently recruits the catalytic subunit DNA-PKcs. DNA-PKcs kinase activity is essential for XRCC4- and Lig4-mediated rejoining of the broken DNA ends during NHEJ. NHEJ is an intrinsically error-prone mechanism because the original sequence can only be preserved when the two DNA ends can be ligated without prior resection. The predisposition to the AT-like disorder (ATDL) has been linked to mutations in the MRN complex, which is important for the resection of DSBs either in HR or in NHEJ (Thoms, Kuschal, & Emmert, 2007). The Nijmegen breakage syndrome is a rare human autosomal recessive disorder caused by hypomorphic NBS1 (NIBRIN) mutations (Thoms et al., 2007). This disorder is characterized by growth retardation, immunodeficiency, microcephaly, and cancer predispositions, particularly lymphomas. Cellular characteristics of NBS include radiosensitivity, increased levels of spontaneous and IR-induced chromosome breakage, and defective cell-cycle checkpoints. NHEJ is also a mechanism utilized by immune cells to create genetic diversity which in turn aids in the probability of certain types of immune cell recognition of diverse antigens to which humans are exposed. Initial studies suggest that breast cancer cells deficient in BRCA1/2 may utilize NHEJ in order to obtain a survival advantage (Keimling et al., 2008).

Genetic profile of DNA repair genes in TNBC Introduction

5.1.6. Fanconi Anemia (FA) pathway

The FA pathway is employed to remove interstrand crosslinks (ICLs) and shares components, such as BRCA2 and PALB2, with the HR and NER pathways (Kim & D'Andrea, 2012); (Kottemann & Smogorzewska, 2013). The proteins in the FA pathway can be grouped into three functional subgroups – the FA core complex, the FA-ID complex, and downstream FA proteins. The FA-ID complex facilitates ICL repair through the downstream FA proteins FANCD1 (BRCA2), FANCJ (BRIP1/BACH1), FANCN (PALB2), FANCO (SLX4), and FANCP (RAD51C) (Kim & D'Andrea, 2012). These downstream FA proteins include classic familial breast cancer predisposition genes, indicative of substantial overlap with the HR pathway. A crucial step in ICL repair is the removal of the ICL, which is mediated by the concerted activity of multiple enzymes.

5.1.7. DNA-damage checkpoints

The activation of DNA-damage checkpoints enforces the growth arrest of damaged cells and allows the DNA-repair mechanisms to mend the damaged DNA. The mechanisms of DNA-damage checkpoints are best understood during their responses to double-strand breaks (DSBs). Initiation of these checkpoints is dependent on the transient recruitment of the MRE11/RAD50/NBS1 (MRN) complex at DSB sites, followed by the recruitment/activation of ATM (Su, 2006). In addition, two other of phosphoinositide-3-kinase-related kinases, DNA–PK e ATR are also activated and involved in the response to DSBs. ATM, ATR, and DNA–PK phosphorylate various targets that contribute to the overall DNA damage response. Therefore, within minutes of DSB formation, active ATM phosphorylates different proteins that are essential for DNA-damage response and repair. An example includes the histone H2AX that, recruits other proteins and initiates the chromatin-remodeling process that is essential for the repair of damaged DNA. Other proteins recruited to sites of DSBs include TP53BP1 and BRCA1, all of which are ATM substrates and mediators in DNA-damage response. ATM and ATR are essential for the G1/S, intra-S-phase, and G2/M DNA-damage checkpoints, and are critical for the maintenance of genomic integrity. Defects in either ATM or ATR have been associated with human syndromes. ATM mutations are associated with the human ataxia–telangiectasia (AT), an autosomal recessive disorder characterized by cerebellar ataxia, progressive mental retardation, impaired immune functions, neurological problems, and malignancies. At the cellular level, AT phenotypes include chromosomal breakage and IR sensitivity. Defects of DNA-damage checkpoints,

Genetic profile of DNA repair genes in TNBC Introduction

similar to impaired DNA-damage repair, promote genomic instability and predispose individuals to immunodeficiency, neurological defects, and cancer (Niida & Nakanishi, 2006). Several recent studies applied next-generation sequencing to uncover the frequency of mutations and copy-number alterations across different cancer entities, including colorectal, breast, ovarian, endometrial, and prostate cancer, as well as leukemia. These studies revealed that different tumor types display an accumulation of mutations in distinct signaling pathways (Cancer Genome Atlas, 2012a); (Puente et al., 2011). For example, mutations in HR genes appear to be enriched in breast and ovarian cancer as whereas colorectal cancer exhibits alterations in MMR and HR, and prostate cancer accumulates inactivating mutations within the HR and NER pathways. Thus, alterations of DNA repair mechanisms are common events in carcinogenesis, but cancer entities show different inactivated pathways. To identify for each cancer entity the subcohort of DNA repair-defective tumors, it may therefore be most effective to search primarily for genetic alterations in the pathways that are predominantly altered for the respective tumor type.

5.2. Targeting DNA repair

In contrast to normal cells, cancer cells frequently fail to activate damage-sensor proteins and DNA-repair pathways are often dysfunctional. This relative DNA-repair deficiency stimulates mutagenesis and enhance tumorigenesis but, at the same time, may make tumor cells prone to the effects of DNA-damaging chemotherapy. From a clinical perspective, defects in DNA-repair mechanisms are often associated with a bad prognosis as they likely enhance the progression of disease, but they may predict a better outcome after treatment as they may predispose cells to sensitivity to DNA-damaging chemotherapy (Hakem, 2008). Many studies identified the possibility to directly target cell cycle and mitotic phases, or defective DNA damage response especially in BRCA1/2 related breast cancers. Platinum compounds produce covalent cross links between bases on the two opposing strands of DNA thereby causing replication fork stalling. These alterations may degenerate into DSBs that in BRCA deficient tumours cannot be repaired by an error-free mechanism (HR). In vitro studies with platinum compounds such as cisplatin or carboplatin have confirmed this hypothesis (Bhattacharyya, Ear, Koller, Weichselbaum, & Bishop, 2000) and in basal-like tumours, promising results with the administration of platinum agents have been achieved clinically (Chew et al., 2009). In contrast, evidence

Genetic profile of DNA repair genes in TNBC Introduction

has suggested that BRCA1-defective cells may show resistance to microtubule poisons, such as paclitaxel or docetaxel (Quinn et al., 2003). A promising approach comes from the employment of the PARP1 inhibitors in BRCA1/2 deficient cells according to the synthetic lethality. Synthetic lethality classically describes a state where two mutations, each having a viable phenotype, when combined, produce a lethal phenotype. PARP1 plays an essential role in cell survival in response to DNA damage. With low to moderate levels of DNA damage, PARP1 promotes cell cycle arrest and DNA repair. In the presence of extensive DNA damage, PARP1 meditates p53-regulated apoptosis and initiate cell death through necrosis (Bouchard, Rouleau, & Poirier, 2003). Activation of PARP1 is involved in very early DNA damage response, and its catalytic activity is rapidly increased by greater than 100-fold in response to DNA SSBs and DSBs. NAD+- dependent PARP1 activation results in the synthesis of long branched polymers of ADP- ribose (PAR) onto itself and other protein acceptors 15 to 30 seconds after DNA damage (Haince et al., 2008). PAR functions as a post-translational modification, a protein-binding matrix or a steric block. A variety of proteins involved in DNA repair or chromatin regulation including PARPs, topoisomerases, DNA-PK, XRCC1, p53, were found to bind PAR through PAR-binding motifs, indicating that dynamic and transient function of PAR may regulate activity of DNA repair proteins and other proteins or alter chromatin confirmation by PAR binding (Krishnakumar & Kraus, 2010). Moreover, depletion of PARP1 or PARP2 results in reduced recruitment of MRE11, RPA, and RAD51 to collapsed replication forks after damage, and this lead to a less effective fork restart. BRCA1 is also recruited to stalled replication forks during S-phase, and interacts with CtIP and the MRE11/Rad50/NBS complex to regulate end resection activity (L. Chen, Nievera, Lee, & Wu, 2008). These data taken together suggest there may be a functional interaction between BRCA1 and PARP1/2 in regulating DNA end-processing activity at stalled replication forks. BRCA1/2 mutant cells are sensitive to PARP inhibitors (McCabe et al., 2006); (Bryant et al., 2005); (Helleday, Bryant, & Schultz, 2005). The DSB repair defects of BRCA1/2 deficient cells are more dependent on PARP and BER to maintain genomic integrity, and loss of functions of both BRCA and PARP results in cell death (Farmer et al., 2005); (Fong et al., 2009). Four alternative distinct mechanisms have been proposed to explain this synthetic lethality (De Lorenzo, Patel, Hurley, & Kaufmann, 2013). Initial models suggested that selective PARP1 inhibition leads to the accumulation of SSBs, which are converted into DSBs during S-phase. According to this model, HR-proficient cells are capable of repairing these DSBs, whereas HR-defective cells fail to do so. Another model suggests that PARP1 inhibitors trap PARP1 on the damaged DNA, thereby preventing

Genetic profile of DNA repair genes in TNBC Introduction

access of other DNA repair factors. A further model proposes that BRCA1 is initially recruited to damaged DNA through its interaction partner BARD1, which in turn binds to a poly (ADP-ribose) polymer in a BRCT domain-dependent manner. PARP1 inhibition would thereby diminish the initial BRCA1 recruitment to damaged DNA. In addition, HR-defective BRCA1 mutants, which fail to be recruited to DSBs in a BRCT domain-dependent manner, might depend on BARD1/poly (ADP-ribose) polymer-driven recruitment Lastly, PARP1 inhibitors have been shown to lead to NHEJ activation in HR-defective cells. Error-prone NHEJ, in turn, has been suggested to be a major driver of PARP1 inhibitor-induced cytotoxicity. A more recent application of synthetic lethality looks at MSH3 factor, involved in MMR but also in HR and as damage sensor, and DNA-PK inhibitors. Analysis of the DSB repair kinetics in MSH3-proficient or deficient cells revealed that MSH3 deficiency was associated with a DSB repair defect similar to that observed in HR- defective BRCA1- deficient cells (Dietlein, Thelen, & Reinhardt, 2014). Both gH2AX- and Rad51 foci showed that MSH3- deficient cells failed to repair genotoxic lesions when DNA-PKcs was inhibited. Such a dual function in multiple distinct DNA repair pathways is a property that Msh3 shares with numerous other proteins, including ERCC1, RAD50, ATM,, XRCC1, PCNA, RFC, and RPA (Rouleau, Patel, Hendzel, Kaufmann, & Poirier, 2010); (Hoeijmakers, 2009).

Acquired resistance against PARP inhibitors represents a potential risk: recent reports revealed that PARP1 inhibitor-exposed BRCA2-deficient cells developed resistance through secondary intragenic deletions in BRCA2, resulting in the (re-)expression of HR- competent isoforms (Ashworth, 2008); (Wang & Figg, 2008). Furthermore, different groups showed that loss of 53BP1 partially rescues the HR defect in BRCA1- deficient cells and reverts their hypersensitivity to platinum-based drugs and PARP inhibitors (Bouwman et al., 2010); (Bunting et al., 2010); (Aly & Ganesan, 2011). The BRCT protein 53BP1 (p53 binding protein 1), which associates with Mre11, BRCA1 and H2AX, is important in HR and NHEJ to repair DSBs and is also required for DDR (Lowndes, 2010). Recent studies show a new role for 53BP1 as an inhibitor for HR. Intriguingly, loss of 53BP1 is significantly enriched in BRCA1/2-associated and triple- negative breast cancer (Bouwman et al., 2010), (Kass & Jasin, 2010), suggesting that 53BP1 status should be evaluated before PARP1 inhibitor treatment. These studies show that loss of 53BP1 is ‘synthetically viable’ with BRCA1 loss in ES cells. Loss of 53BP1 function can rescue the severe proliferation defect of BRCA1-mutant cells and restore functional capacity to perform HR mediated DNA repair.

Genetic profile of DNA repair genes in TNBC Introduction

Importantly, tumor cells from sporadic cancers with BRCAness phenotype are also sensitive to PARP inhibitors (N. Turner et al., 2004). A recent study identified a 60- gene signature profile for BRCAness in familial and sporadic ovarian cancers that correlated with platinum and PARP inhibitor responsiveness (Konstantinopoulos et al., 2010). FANCF promoter methylation has been detected in several types of sporadic cancer as a BRCAness phenotype, including ovarian, breast, head and neck, non-small cell lung and cervical carcinomas (Taniguchi et al., 2003). Mutations causing loss of PTEN expression are common in a range of hereditary and sporadic cancers (Salmena, Carracedo, & Pandolfi, 2008). Similar to BRCA1/2 mutant cells, PTEN mutant cells are also characterized by an increase in DSBs. The impact of PTEN deficiency on sensitivity to drugs targeting is still unclear (Mendes-Pereira et al., 2009). The PALB2 promoter may be hypermethylated, and downregulation of PALB2 expression is found in both hereditary and sporadic breast cancers (Turnbull & Rahman, 2008). PALB2 directly functions in HR repair and is required for the assembly of BRCA2 and RAD51 nuclear foci (Sy, Huen, & Chen, 2009). PALB2 deficiency also results in hypersensitivity of cancer cells in response to PARP inhibitors treatment (Buisson et al., 2010). Deficiency in other known HR pathway proteins may also show similar synthetic lethal relationship with PARP inhibition.

The observation that deficits in PALB2, PTEN, ATM, Mre11/NBS1, ATR, Chk1 or Chk2 (McCabe et al., 2006); (Bryant & Helleday, 2006) resulted in sensitivity to PARP inhibition suggests that PARP inhibitors would be beneficial for a wider range of cancers with BRCAness phenotype such as dysfunction of genes involved in HR and DDR pathways. The phenomena of BRCAness are recently being identified in an expanding list of cancers. Notably, BRCAness occurs not only in triple negative breast cancer but also in epithelial ovarian cancer and other types of cancer such as non-small cell lung cancer, head and neck cancer, prostate cancer and cervical carcinomas (N. Turner et al., 2004). The BRCAness phenotypic characterization is emerging as a novel and attractive strategy for treating cancer patients with the targeted PARP inhibitors therapies.

Genetic profile of DNA repair genes in TNBC Introduction

Fig 4. Synthetic lethality of PARP inhibitors in DNA repair defective cells. The DNA damage that occurs after inhibition of PARP activity cannot be adequately repaired BRCA deficient cells and eventually results in chromosomal instability, cell-cycle arrest, and subsequent apoptotic cell death. As DNA-repair processes remain intact in noncancerous cells, which generally retain at least one functional copy of both BRCA1 and BRCA2, PARP inhibition is hypothesized to selectively kill cancer cells, sparing normal tissue.

5.3. Biomarkers for DNA repair deficiency

In order to select patients who may benefit from DNA repair targeted therapy is crucial to develop molecular biomarkers to assess DNA repair deficiency. Current technologies such as high-throughput DNA microarrays, Real Time qPCR, protein microarrays followed by mass spectrometry, immunohistochemistry, immunofluorescence, are powerful tools to develop DNA repair protein expression profiling of patients’ tumors that may be sensitive to PARP inhibitors and others rugs. Predictive biomarkers are measured at baseline to identify patients who are likely or unlikely to benefit from a specific treatment, while a prognostic biomarker provides information about the patients prognosis in the absence of treatment or in the presence of standard treatment. Assessment of the activity of DNA repair pathways may influence treatment response and predict clinical outcome in tumor cells, identify new therapeutic targets and influence clinical decision making. It has been shown that DNA repair proteins are frequently changed in human cancers, indicated by measurements of DNA, RNA, protein determinations of biopsies.

Genetic profile of DNA repair genes in TNBC Introduction

An increasing number of studies on DNA repair pathways including DNA repair gene expression profiling, mutation status of DNA repair genes, expression levels of DNA repair proteins, nuclear foci status of DNA repair proteins, and DNA repair capacity have been demonstrated to have a predictive value for treatment outcome or the response to therapies in different types of cancer. H2AX has been considered as a DNA DSBs marker to evaluate the efficacy of various DSB inducing compounds and radiation, and its foci are known to be involved in the repair of DSBs by HR and NHEJ pathways (Lowndes & Toh, 2005). Similarly, the assessment of Rad51, 53BP1, and RPA foci is being investigated as they also indicate that recombination and repair machinery is accumulating at specific sites in the genome, likely in the vicinity of DNA damage. Evaluation of tumour tissues at protein expression level remain one of the most effective approach to get insights of DNA repair machinery. Proteins carry more information than nucleic acids due to alternative splicing and post-translational modifications of species of protein from each gene. Moreover, many physiologic changes are mediated post- transcriptionally and will not be revealed at the nucleic acid level. Therefore, protein biomarkers have a significant impact in cancer diagnostics and therapies. IHC on Tissue MacroArrays TMAs analysis can measure multiple factors in many samples at the same time. Quantitative immunofluorescence (IF) labeling on FFPE tissue has the capability for multiple labeling and is of higher resolution. For example ERCC1 is probably the most-studied protein as both a prognostic and predictive marker for the survival benefit from adjuvant platinum-based chemotherapy (Olaussen, Mountzios, & Soria, 2007), although all NER-deficient cells display sensitivity to cisplatin. DNA repair is also the focus of predicting responses to other DNA-damaging agents. For example, both MMR- and BER-related protein levels are thought to correlate with clinical response to alkylating agents. In a breast cancer study, complete pathologic response to neoadjuvant chemotherapy was associated with lower Rad51 foci in tumor biopsies obtained after chemotherapy (Graeser et al., 2010). Evaluation of PARP1 expression has been largely considered to predict PARP1 inhibitors response. Due to high variability of PARP1 protein depending of the cell cycle phase, rather than total PARP1 levels, a functional assay for the assessment of PAR (poly(ADP)ribose) polymer levels (Yang et al., 2009) may potentially assay HR function and possibly be a biomarker of BRCAness. Genetic-based assays have been used as surrogates to evaluate DNA repair capacity via quantitative real-time PCR of gene expression (Jalal, Earley, & Turchi, 2011) and single- nucleotide polymorphism analyses of DNA repair genes. Multiple studies have correlated

Genetic profile of DNA repair genes in TNBC Introduction

a variety of polymorphisms with the risk of development of different solid organ malignancies, and numerous DNA repair gene SNPs have been shown to be prognostic in patients with cancer. SNPs are an important genetic tool, but the interpretation of studies evaluating individual SNPs as they relate to variables, such as therapeutic efficacy, cancer risk, or prognosis, has numerous limitations, including linkage disequilibrium and inadequate statistical analyses (e.g., small sample size, multiple testing, and reproducibility). Potential signatures of BRCAness have been reported based on the differences in gene expression between hereditary BRCA1/2 related cancers and sporadic cancers. These gene expression signatures may enrich for a group of ‘hereditary-like’ sporadic cancers, and these cancers are more responsive to DNA damaging chemotherapy (Konstantinopoulos et al., 2010); (Rodriguez et al., 2010). In a way analogous to microsatellite instability in mismatch repair deficient cancers, there is likely to be a genomic mutational pattern that predicts for underlying HR deficiency. In part this may be a characteristic degree of gross genomic instability, but there is also some indication that certain complex mutations may be a marker of HR deficiency. For example, BRCA1 mutant breast cancers frequently have characteristic mutations in TP53 that are infrequent in sporadic cancer (Holstege et al., 2009). The presence of these complex mutations in sporadic triple negative breast cancers may predict benefit to neoadjuvant cisplatin (Silver et al., 2010), providing some support for their use as a biomarkers of HR deficiency.

5.4. TNBC and DNA repair

TNBC have been shown to harbour DNA repair deficiencies, including BRCA1 dysfunction, due to promoter methylation or deregulation of other genes involved in their transcriptional regulation (N. C. Turner et al., 2007), BER inactivation, MTMG promoter hypermethylation (Fumagalli et al., 2012) and lack of hOGG1 (Karihtala, Kauppila, Puistola, & Jukkola-Vuorinen, 2012). Given the importance of the cellular DNA repair in determining the cellular response to different anticancer agents, a priori knowledge of the repair status of a given tumor could play an important role in selection of the most appropriate therapy. Attempts to investigate the functionality of a given DNA repair pathway have been undertaken and shown to be feasible. Isolation of epithelial cells from breast tumor specimens and application of specific functional DNA repair assay systems led to the detection of specific defects in double strand breaks repair (Keimling et al., 2008), while homologous recombination status could be determined in ovarian cancer samples by RAD51 foci formation after in vitro treatment with a PARP inhibitor (Mukhopadhyay et al., 2010). However, all these assays still need to be technically

Genetic profile of DNA repair genes in TNBC Introduction

improved (i.e. requirement for an automated foci scoring and setting up of tumor specific primary cultures) rendering difficult their wide spread clinical use. An alternative approach is the study of biomarkers that, even if not completely validated (Lips et al., 2011 (Lips, Mulder, et al., 2011); (Konstantinopoulos et al., 2010), can be considered surrogate of DNA repair functionality, i.e. the mRNA and/or expression levels of key proteins involved in DNA repair pathways. Many recent study try to evaluate gene expression profile of DNA repair genes in TNBC with controversial results. Ribeiro et al. found that the levels of expression of all the genes involved in the NER and FA pathways were significantly lower in TNBC as compared to Luminal A samples (Ribeiro et al., 2013). The levels of the NER genes, ERCC1, XPA, XPF and XPD genes were significantly lower in TNBCs than in luminal BC. BRCA1, FANCD2, FANCF and PALB2 genes were significantly less expressed in TNBCs as well. On the contrary, PARP1 levels were higher in TNBCs. DNA repair gene expression in TNBC matched with normal tissues (Ossovskaya et al., 2011) revealed that several DNA repair pathways, such as homologous recombination (BRCA2, RAD51, RAD54B), mismatch repair (MLH1, PMS1, PMS2, MSH3) and DNA repair synthesis genes, were transcriptionally upregulated in TNBC. In contrast, most of the genes involved in excision repair pathways were transcriptionally repressed. Rodriguez et al. (Rodriguez et al., 2010) have derived a gene expression profile that is associated with DNA repair deficiency in sporadic TN breast cancers. Van’t Veer et al. published a gene expression signature that can potentially distinguish breast tumors from germline BRCA1 mutation carriers from sporadic tumors (van 't Veer et al., 2002). Using this gene signature and the genetic profiles of sporadic TN from three datasets, the authors obtained a signature of 334 with 136 genes overexpressed in and 198 underexpressed genes. PARP-1, RAD51, FANCA, and CHK1 were among the overexpressed genes in the group of BRCA1-like tumours. This signature was associated with sensitivity to DNA-damaging chemotherapy (anthracyclines) and relative taxane resistance, consistent with published preclinical data in BRCA1-deficient tumors (Wysocki, Korski, Lamperska, Zaluski, & Mackiewicz, 2008).

6. TNBC AND THERAPY

Management of TNBC is challenging because of a lack of targeted therapy, aggressive behavior and relatively poor prognosis. There are no specific treatment guidelines for TNBCs and they are managed with standard treatment. Treatment options are limited as most patients have been treated with adjuvant anthracyclines, taxanes and cyclophosphamide. Several limitations are recognized in the quest for determination of

Genetic profile of DNA repair genes in TNBC Introduction

optimal adjuvant chemotherapy for TNBC since the lack of robust prospective data in triple negative restricted trial populations. Due to the phenotypic similarities between TNBC and BRCA associated tumours, it is tempting to extend promising therapeutic strategies exploiting defective DNA repair in BRCA-associated tumors to the larger subset of sporadic TNBC. Importantly, TNBC, basal-like breast cancer and BRCA1 associated breast cancer are not synonymous so heterogeneity within the TNBC cohort have to be taken in account. Neoadjuvant treatment for this subtype of tumours, instead is very promising and interesting to investigate. It has been evidenced by various studies that these tumors are highly chemosensitive (Liedtke et al., 2008); (L. A. Carey et al., 2007) and in some cases are represented by complete pathological response (pCR), but the results remains unsatisfactory. Early TNBC is associated with a paradox: a minority of patients have highly chemosensitive disease but the subgroup as a whole has poor disease free and overall survival. This was highlighted by a neoadjuvant analysis in which TNBC patients obtaining pCR had an excellent prognosis (3 year overall survival 94% vs 98% for non TNBC) whereas TNBC patients not attaining a pCR had a high likelihood of systemic relapse (63% vs 76%, respectively) and death (74% vs 89%, respectively) within 3 years of primary diagnosis (L. A. Carey et al., 2007). Indeed pathological complete response is higher in the TNBC subset of patients but the disease free survival (DFS) and OS are still lower than non-TNBC patients (Liedtke et al., 2008); (L. A. Carey et al., 2007). Sporadic TNBCs show heterogeneity in response to chemotherapy, with pCR rates ranging from 12% for single-agent to 27%-65% in multi- agent neoadjuvant therapy trials (Rouzier et al., 2005). More recently pCR is considered a prognostic marker of survival in breast cancer patients that can be used as a surrogate outcome of survival. Neoadjuvant therapy has been evolving rapidly giving this benefit (von Minckwitz et al., 2012).

6.1. Standard therapies for TNBC

In the next paragraphs, the different therapeutical approach to TNBC in adiuvant and neoadiuvant settings are reported, divided for class of drugs: cytotoxic agents and targeted therapies.

Genetic profile of DNA repair genes in TNBC Introduction

6.1.1. Anthracyclines

Anthracyclines, specifically doxorubicin and epirubicin, are among the most active agents for breast cancer. In biologically unselected early breast cancer populations, anthracycline containing therapy imparts an overall survival benefit (Early Breast Cancer Trialists' Collaborative, 2005). These agents that act by destabilizing the DNA through intercalation also prove useful in TNBC due to a degraded DNA repair cascade. Many studies show that TNBC is sensitive to anthracyclines. The curative potential of anthracyclines must be balanced against the small but real risk of long term secondary haematological malignancy and congestive cardiac failure. An intergroup study (C9741) found differences in favor of dose density with adriamycin and paclitaxel I n patients with negative ERs, but not in ER-positive patients (32% vs 19%). This study highlights the importance of chemotherapy in hormone-independent tumors. Recently, (Skripnikova et al., 2011) in a prospective pilot trial evaluated the efficacy of doxorubicin, cyclophosphamide and capecitabine in locally advanced and metastatic TNBC. The overall response rate (RR) was 58% with 24% of CR and 34% of PR. Although the response was good, this combination is quite toxic.

6.1.2. Taxanes

The principal mechanism of action of the taxane class of drugs is the disruption of microtubule function, as they inhibit cell division. Thus, in essence, taxanes are mitotic inhibitors. In early breast cancer trials, in populations unselected for biology, the addition of taxanes to adjuvant anthracycline based therapy has been tested in in several clinical trials. Most trials and a recent meta-analysis reveal additional benefit from taxane for disease free and overall survival (De Laurentiis et al., 2008). Results specifically in TNBC are limited, however preferential benefit of microtubule stabilizing agents has not been demonstrated. Potential biological bases for taxane sensitivity in TNBC are the high tumor proliferative rate and the presence of aberrant TP53 in about 50% TNBC. In pre-clinical studies, BRCA1 deficient breast cancer cell lines which showed increased sensitivity to topo IIa inhibitors and cisplatin showed decreased sensitivity to paclitaxel and vinorelbine (Berry et al., 2009) The absolute benefit of taxanes when seen is modest and seems to be restricted to a minority of patients. Many studies reveal the benefits produced by paclitaxel when added to other chemotherapeutic agents.

Genetic profile of DNA repair genes in TNBC Introduction

In the neoadjuvant setting, (Rouzier et al., 2005) showed that TN and Her2-positive subtypes of breast cancer are more sensitive to paclitaxel and doxorubicin chemotherapy than the luminal and normal-like cancers. pCR was seen in 45% patients with basal like breast cancer (BBC) compared to 6% in luminal subtypes. In an adjuvant setting, two meta-analyses have shown benefit with taxanes (De Laurentiis et al., 2008). Many studies have demonstrated that taxanes are more effective in receptor negative patients. In a study by (Jacquemier et al., 2011), there was greater benefit with the addition of docetaxel to the conventional 6 cycles of FEC in BBC patients. A further study showed maximum benefit in TNBC patients when 4 cycles of FEC were followed by weekly paclitaxel for 8 wk compared to just 6 cycles of FEC (Martin et al., 2010). The role of anthracyclines alone in TNBC is still unclear but a definite benefit is seen when used in combination with taxanes. However, BRCA1/2 deficient cells have shown to be less sensitive to taxanes (Kriege et al., 2012). Since many TNBC harbor BRCA1 mutations or exhibit BRCAness phenotype is still very important to select patients more likely to respond. Enrichment of proliferation genes and increased Ki-67 expression in basal-like TNBC tumors suggest that this subtype would preferentially respond to antimitotic agents such as taxanes. This is indeed the case when comparing the percentage of patients achieving a pCR in 42 TNBC patients treated with neoadjuvant taxane in 2 studies (J. A. Bauer et al., 2010); (Juul et al., 2010). In these combined studies, TNBC patients whose tumors correlated to the basal-like (BL1 and BL2) subtype had a significantly higher pCR (63%; P = 0.042) when treated with taxane-based therapies as compared with mesenchymal-like (31%) or LAR (14%) subtypes.

6.1.3. Platinum compounds

TNBC has phenotypic and molecular similarity to BRCA1 related cancers that would confer sensitivity to cytotoxic agents like cisplatin. The platinum agents act by producing intra and inter strand cross links of double stranded DNA, prevent the replication fork formation and produce double strand breaks and replication lesions. In the BRCA1 mutants, the DNA repair cascade is nonfunctional and produces cell death (Hastak, Alli, & Ford, 2010). Clinical studies have also suggested that TNBC are more sensitive to DNA damaging agents like cisplatin. In a phase-Ⅱ study, it was observed a pCR of 21% with neoadjuvant cisplatin in patients with TNBC. Among 28 patients, two were BRCA1 carriers, both (100%) of whom achieved pCR; 4 (15%) of the 26 women with sporadic TNBC also achieved pCR to cisplatin. Overall, 50% of the patients had a good response to cisplatin.

Genetic profile of DNA repair genes in TNBC Introduction

In a similar study by Silver (Silver et al., 2010) with 4 cycles of single agent cisplatin, a pCR rate of 22% was seen. Two patients with BRCA1 mutation had pCR. They also found a significant association of tumor p53 protein-truncating mutations with cisplatin response. The largest series of BRCA1 mutation was reported by (Byrski et al., 2010); out of 6903 patients, 102 patients had BRCA1 mutation. Out of this, 12 patients were treated with neoadjuvant cisplatin and 10 (83%) had pCR. In another study which explored role of platins in TNBC with BRCA1 mutation, out of 25 patients, 72% had a pCR. Projected 5 year DFS and DDFS were 76% and 84% respectively (Byrski et al., 2009). At present, platinum agents in the neoadjuvant setting cannot be recommended over established regimens outside of a clinical trial. So, platins should always be used in combination with taxanes or anthracyclins to increase response and survival rates. However, patients with BRCA1 mutation tend to have maximum benefit in the neoadjuvant setting. In the metastatic setting, cisplatin or carboplatin have shown an overall response rate of 20%-40% and are often used in first and second lines. Cisplatin is very active in first line chemotherapy in MBC with a RR of 50%, whereas carboplatin is moderately active with an ORR of 30%. Carboplatin in combination with paclitaxel demonstrated an ORR of 41% in MBC. PFS was better in the paclitaxel and carboplatin arm compared to paclitaxel and epirubicin. However, there was no difference in ORR and OS between the two arms. Gemcitabine (GC) and platinum agents in combination have synergistic antitumor activity that results in inter strand DNA crosslinks and double strand DNA breaks, both of which are preferentially repaired by homologous recombination. Both agents have demonstrated activity in MBC (Heinemann, 2002). Lastly, in a retrospective study (Staudacher et al., 2011) was reported that median OS and median PFS were improved in patients responding to platinum based chemotherapy: 27 vs 8 mo (P < 0.001) and 10 vs 4 mo (P < 0.001), respectively. Therefore, combination of platins with taxanes or GC or vinorelbine are good alternatives for patients in whom anthracyclins may pose as toxic or who are already exposed to these in the adjuvant setting. The heterogeneous outcome with platins may be related to the heterogeneity of TNBC.

6.1.4. Alkylating agents

Cyclophosphamide is the most commonly used alkylating agent in breast cancer. Due to standard co-administration with an anthracycline and diversity in scheduling there is limited data to specifically ascertain the role of cyclophosphamide. Retrospective studies suggest that TNBC may have particular sensitivity to alkylating agents.

Genetic profile of DNA repair genes in TNBC Introduction

Cyclophosphamide in low continuous dosing has been shown to exert an anti-tumor effect in breast cancer, attributed to predominant anti-angiogenic effect. Specifically in TNBC, antiangiogenic therapy is promising due to high reported VEGF levels of 300 patients treated with neoadjuvant CMF, triple negative tumors presented the highest response rate as assessed by mammography. Response rate in TNBC was 64.9% compared with 51% for HER2 disease and 40% for luminal tumours. In node negative patients, classical CMF had differential effect across IHC subgroups, with the greatest benefit in TNBC (Colleoni et al., 2010).

6.2. Target therapy: PARP1 inhibitors

Currently, a lot of research is going on to further characterize TNBC with different molecular markers and find targets for therapy in order to improve its outcome. The inhibition of PARP1 potentiates the effects of ionizing radiation, DNA methylating agents, topoisomerase Ⅰ inhibitors and platinum compounds. Several PARP1 inhibitors are at different stages of clinical development. In a phase-Ⅱ study by Tutt et al. (Tutt et al., 2010) in 54 patients with known BRCA mutations, 27 received olaparib 400 mg twice a day, of which 11 (41%) experienced a response with a median PFS of 5.7 mo. A second cohort of 27 women received 100 mg of per day and 6 patients (22%) experienced a response with a median PFS of 3.8 mo. This agent was fairly well tolerated, with nausea and fatigue being the most common adverse events. In a phase-Ⅱ randomised study, O’Shaughnessy (O'Shaughnessy et al., 2011) found that the addition of iniparib to carboplatin and gemcitabine in metastatic TNBC resulted in significant improvements in RR, PFS and OS. The addition of iniparib was well tolerated. However, a randomized phase-Ⅲ study by the same investigators failed to prove significant benefit of iniparib in combination with GC in metastatic TNBC in terms of PFS (4.1 vs 5.1 mo) or OS (11.1 vs 11.8 mo). Another drug, veliparib, is a novel oral inhibitor of PARP1 and PARP2. It has shown a synergistic effect with temozolamide in TNBC (Isakoff, 2010). In BRCA1 and BRCA2 mutation carriers, ORR was 37.5% and CBR was 62.5% with a PFS of 5.5 mo. Since both the drugs are given orally, they can be good options for patients in whom there is difficulty in accessing a venous line from the above subgroup. PARP inhibitors have shown clinical activity in BRCA mutation carrier breast cancer and TNBC. These drugs are also being evaluated in the neoadjuvant setting but experience is limited.

Genetic profile of DNA repair genes in TNBC Introduction

One of the major challenges in PARP inhibitor therapies is how to identify biomarkers for the subset of the responder population with non-BRCA mutant, BRCAness and HR deficient cancers. At present it is generally assumed that same biomarkers of homologous recombination deficiency, will predict for the benefit of PARP inhibitors as single agents, as well as PARP inhibitors in combination with chemotherapy. However, it is probable that different molecular defects in HR may predict sensitivity to PARP inhibitors alone, whereas other defects for benefit to PARP inhibitors in combination with chemotherapy. For example, cancers with a ‘hard’ defect in HR, such as BRCA1/ 2 mutations, may be responsive to PARP inhibitors as single agents, whereas potentially cancers with a ‘soft’ or mild defect in HR may not show sensitivity to single agent PARP inhibition, but could show substantial benefit when the cell is stressed by the combination of PARP inhibition and more complex damage created by chemotherapy.

6.3. Neoadjuvant therapy as a tool to develop predictive biomarkers

Pathological complete response rate after neoadjuvant chemotherapy is a characteristic of TNBC, and pCR has been proved to be a typical marker predictive of clinical response and survival in TNBC patients (von Minckwitz et al., 2012); however, diverse pCR rates have been reported in various studies. A very recent meta-analysis by Wu and colleagues (Wu, Yang, Liu, Wu, & Yang, 2014) resuming 27 studies containing 9,460 cases: pCR rates were 28.9% (95% CI, 27.0 to 30.8%) for 2,952 cases of TNBC and 12.5% (95% CI, 11.7 to 13.4%) for 6,508 cases of non-TNBC. Patients with TNBC have a higher probability of achieving pCR than those with non-TNBC (OR, 3.02; 95% CI, 2.66 to 3.42); the TNBC pCR rate is about two times that of non-TNBC and TNBC exhibits a better response to NAC than non-TNBC. Of the 27 studies in this meta-analysis, 19 used anthracycline-based therapy and 11 used taxane containing regimens. The pCR rates for TNBC were 26.8% (95% CI, 24.1 to 29.6%) for the anthracycline-based group and 30.5% (95% CI, 25.9 to 35.5%) for the taxane containing group, a non-significant difference. Interestingly, the platinum- containing group had a higher pCR rate than either the anthracycline-based or taxane- containing groups. Some researchers have demonstrated that the addition of platinum agents to anthracycline and/or taxane regimens in neoadjuvant set gives good outcomes (Gelmon et al., 2012). Two others meta-analyses were published recently on breast cancer and pCR. (Von Minckwitz et al., 2012) presented a meta-analysis of 6,377 operable and non-metastatic breast cancer patients, who received neoadjuvant anthracyclines or taxanes. The authors

Genetic profile of DNA repair genes in TNBC Introduction

concluded that pCR should be conservatively defined as pT0 pN0 excluding ductal carcinoma in situ and that pCR is an effective mark of survival for TNBC, luminal B and nonluminal (HER2-positive). Kong et al. (Kong, Moran, Zhang, Haffty, & Yang, 2011) completed a meta-analysis that included 16 studies with 3,776 patients with breast cancer to determine whether pathologic response after neoadjuvant predicts outcomes. The authors concluded that the pathologic response is prognostic for relapse-free survival, disease-free survival and overall survival.

These findings highlight the importance of identifying biomarkers able to predict response to neoadjuvant chemotherapy. Only two perspective study have been performed giving indication about potential markers. In one study of 41 Chinese patients with locally advanced TNBC, tumor tissue was evaluated for Ki-67 proliferation index, CK5/6, EGFR, cyclin D1, and nm23-H1, a subunit of a gene encoding nucleoside diphosphate kinase, by IHC prior to neoadjuvant therapy with docetaxel and epirubicin. Tumors positive for CK5/6 and/or EGFR, as well as those with positive nm23-H1, as more likely to achieve pCR, with odds ratios of 3.15 and 1.93, respectively (X. R. Li et al., 2011). BRCA1 mRNA, BRCA methylation, p53 mutation, and ΔNp63/Tap73 ratio were evaluated as potential biomarkers in the seminal study of Silver et al. of cisplatin in the neoadjuvant setting for TNBC. None correlated with complete response, though lower BRCA1 mRNA expression and promoter methylation as well as p53 nonsense or frameshift mutations did correlate with good response (Silver et al., 2010). Nevertheless, BRCA1 germline mutations seems to predispose to a good or complete pathological response after cisplatinum+CMF neoadiuvant therapy (Byrski et al., 2010).

Many other potential biomarkers have been retrospectively evaluated through the use of tissue and outcome data from historical data sets to correlate potential biomarkers with the rate of pCR. Ki67: a retrospective study evaluating only Ki-67 as a marker of response to NAC on 105 TNBC patients showed 18.2% of had high Ki-67 achieved pCR, while no patients with low Ki-67 had a pCR. High Ki-67 expression was also associated with early recurrence and poorer OS after completion of adjuvant therapy. The authors suggest Ki-67 may thus be used to separate those TNBC tumors with a more aggressive phenotype in addition to predicting chemotherapy response (Keam et al., 2011). P53: The use of p53 status by IHC as a biomarker predictive of response to neoadjuvant chemotherapy has also been evaluated. In 120 patients with TNBC, there was a weak trend to association of p53status and pCR rate, with 22% of patients with positive p53 achieving pCR and 10% of patients with negative p53 with pCR (Bidard et al., 2008).

Genetic profile of DNA repair genes in TNBC Introduction

PARP1: A retrospective study evaluated tissue samples from participants of the GeparTrio trial for PARP expression by IHC. The breast cancer patients in this study were primarily treated with docetaxel, doxorubicin, and cyclophosphamide (TAC) chemotherapy in the neoadjuvant setting, with pCR rates evaluated at the time of surgery. Tissue samples from this patient population demonstrated increased cytoplasmic PARP expression in TNBC and HR–/HER2+ tumors, which correlated with higher pCR rates in these groups (41% and 42.9%, respectively). Methabolic pathways: Lactate dehydrogenase B (LDHB) was identified as highly expressed in basal-type and TNBC cell lines. Analysis of two combined patient datasets, demonstrated an association between high levels of LDHB expression and pCR following neoadjuvant therapy for TNBC with various standard regimens (odds ratio 3.18). Higher recurrence rates were also identified in TNBC patients with high levels of LDHB in the setting of residual disease following neoadjuvant therapy (Dennison et al., 2013). Pathologic features: tumor-infiltrating lymphocytes (TIL) and apoptosis scores have also been evaluated as predictive of response to neoadjuvant chemotherapy. In a retrospective study of 92 TNBC tumors from patients with stage II–III disease treated with neoadjuvant anthracycline- based or taxane-based chemotherapy alone or in combination, higher TIL scores of 3–5 were associated with higher rate of pCR (37%) than lower scores (16%) (p = 0.05). Increased apoptosis scores were also associated with higher rates of pCR in both the primary tumor and axillary nodes in the TNBC patients (p = 0.04) (Ono et al., 2012). As these studies demonstrate, neoadjuvant therapy is an attractive setting for evaluation of predictive biomarkers due to the ability to evaluate tissue pre- and post-therapy, and accessibility of an early readout of treatment response. Biomarkers predictive of response to adjuvant therapy have been more difficult to assess, largely due to limitations in outcome measures. The clinical trials evaluating such have largely used OS or disease free survival to evaluate response to chemotherapy, thus making associated biomarkers more prognostic than predictive (Jacquemier et al., 2011); (Kashiwagi et al., 2011). In one study, 138 of 190 TNBC patients were treated with adjuvant chemotherapy with either an anthracycline-based or 5FU-based regimen, while the remaining 52 patients did not receive adjuvant therapy. Tumor tissue expression of E-cadherin, Ki67 and p53 by IHC was correlated with OS. Interestingly, tumors positive for E-cadherin and negative for Ki67 were associated with improved OS in the group treated with adjuvant chemotherapy, but not in the group treated with surgery alone. The authors suggest this pattern may be useful to predict which patients have a greater chance of benefiting from adjuvant chemotherapy (Kashiwagi et al., 2011).

Genetic profile of DNA repair genes in TNBC Introduction

Clearly additional studies of predictive biomarkers for adjuvant and metastatic therapies for TNBC are required to further direct chemotherapy administration in these settings. Such investigation is limited, however, by the reliance on response or survival data to evaluate outcomes. This explains the preponderance of predictive biomarker studies performed in patients receiving neoadjuvant therapy for TNBC, where pathologic response can be used to document degree of response more accurately. However, given the poor prognosis of TNBC patients who do not respond to neoadjuvant chemotherapy, there is just as great a need for studies to identify predictive biomarkers in other phases of treatment.

Genetic profile of DNA repair genes in TNBC Introduction

7. AIM OF THE STUDY

TNBC accounts for 15%–20% of newly diagnosed breast cancer cases. In general, TNBC patients present larger, higher grade tumors with increased number of involved nodes, and poorer survival compared to other subtypes. Increasing evidence indicates that TNBC is a highly heterogeneous disease on a molecular and genetic level. Treatment of patients with TNBC has been challenging due to this heterogeneity, as well as the absence of well- defined molecular targets. Despite having higher rates of pathologic complete response (pCR) to neoadjuvant chemotherapy, TNBC patients have a higher rate of distant recurrence and worse prognosis. Among TNBC patients receiving neoadjuvant chemotherapy, only those with pCR have improved survival. In contrast, more than 70% of TNBC patients have residual invasive disease after neoadjuvant chemotherapy and are at high risk of disease relapse, with significantly worse survival, particularly in the first three years. These findings highlight the importance of identifying biomarkers able to predict response to neoadjuvant chemotherapy and adjuvant therapy as well. A substantial proportion (15-20%) of TNBCs arise as a result of inherited mutations in BRCA1 and BRCA2 genes; nevertheless 60% to 80% of breast tumors from BRCA1 mutation carriers display a TNBC phenotype. This similarity is defined BRCAness and suggests that TNBC and BRCA-related cancer may share also molecular and genetic features, maybe in the BRCA pathways and partners. Recently some studies based on panel-based testing has revealed that 10% of high-risk patients with no BRCA1 or BRCA2 mutation may carry inherited deleterious mutations in other breast cancer genes, even if further studies are needed to characterize the frequency and the impact of these “other genes” mutations in TNBC subgroup. The first aim of this study is to explore the genetic status of BRCA partners genes and many other DNA repair players in a set of patients with TNBC, with and without family history of breast cancer, who underwent neoadjuvant therapy. Breast susceptibility genes, DNA repair master genes, breast cancer related genes and novel target of therapy genes were included in a gene panel mutational screening with a Next Generation Sequencing platform. Therefore the entire coding region of BRCA1, BRCA2, CHEK2, BRIP1, PALB2, TP53, PTEN, STK11, CDH1, BARD1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C, RAD52, 53BP1, PARP1, ERCC1 were sequenced in 19 TNBC patients. This approach is motivated by two objectives: to evaluate the contribute of mutations and rare germline variants in DNA repair genes in the TNBC subgroups, and to get insights into the individual response to anthracyclines and taxanes neoadjuvant therapy. Indeed, the hypothesis of this study is that patients carriers of a mutation in DNA repair may have a similar behaviour to BRCA1-related cancer and a similar therapy

Genetic profile of DNA repair genes in TNBC Introduction

outcome. In other words, a carrier of pathogenetic or probable pathogenic variants in a BRCA related genes, may better respond to DNA alkylating drugs such as anthracyclines. On the other hand, is as much important to identify those patients who may not benefit from this neodiuvant regimen of therapy on the basis of a genetic profile. In this view, this little but homogeneous group of patients will be analyzed, and the genetic variations will be correlated to therapy response indicated as pathological complete response (pCR), partial complete response (pPR) and progression of disease (PROG). The second aim of this thesis is strictly linked to the first, as the same set of genes will be analyzed in 37 triple negative cancer tissues of patients in adjuvant setting of therapy and with a minimum of 5 years of follow-up. The primitive tumor of this patients is likely to harbour a number of mutations or genomic alterations, due to high genomic instability of TNBC. The intent is to find out if DNA repair pathways are involved even at a somatic level in progression and clinical characteristics of TNBC. For this purpose, the mutation landscape of these tumours will be correlated to DFS and other clinicopathological features. Moreover, this approach will allow to identify also germline variants. These data will be added to those of the germline screening in order to strengthen the hypothesis that many TNBC are altered in DNA repair pathways. Results from this study may help in understanding TNBC biology and give suggestion for TNBC genetic counseling and treatment. Indeed, mutational screening in TNBC patients, even without family history of breast cancer, may be relevant to identify those patient more likely to respond to classical therapy and to emerging target therapies such as PARP inhibitors.

Materials and Methods

Genetic profile of DNA repair genes in TNBC Materials and Methods

1. Germline screening: patients

For the germline screening of DNA repair genes, 19 patients afferred at Oncology Center of Santa Chiara Hospital in 2009-2013 and diagnosed with TNBC were selected. Among them, 10 had a familiar breast cancer history, and tested negative for mutation in BRCA1 and BRCA2 genes. Other 9 were unselected for family history and mutational status of BRCA genes was unknown. Pathological diagnosis was based on biopsy from the primary breast lesion even in the context of cases with metastasis. Triple negative” was defined as HER2 0/+1 on immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) less than 2, if IHC +2; estrogen and progesterone receptor negativity was considered with 5% or fewer cells positive on IHC. All patients were treated with neoadjuvant therapy including at least 3 cycles of FEC (fluorouracil, epirubicin, cyclophosphamide) every 4 days and 3 cycles of taxanes every 21 days. The residual tumor mass is evaluated in accord with neoadjuvant guide lines and RECIST guide lines. pCR was defined as no residual invasive tumor in the breast and lymph nodes. For each patient, a blood sample was collected and stored in EDTA at -80°C. Written informed consent was obtained from patients. All the clinical and hystopathological information are reported in Table 2 of the Results section. A collection of 50 healthyvolunteers blood samples was also available in the Laboratory of Genetical Oncology, AOUP Pisa.

2. Tumor Tissues Screening: patients

Since the need to have frozen tissues available and a minimum follow up of 5 years, we first check the data bank of tumor tissues stored in ultrafreezer at -80°C at Anatomia Patologica 1. First, hormone receptor negative tumours were selected; then, Her2 status was evaluated and clinical records were consulted. Patients enrolled includes 37 women with TNBC disease who underwent to surgery at Pisa Hospital from 1991 to 2006. Patients were unselected for family history, and included for TN phenotype. Hystopathological features of primary tumor, therapy regimens, distant metastasis sites, DFS and OS were collected. A complete clinical data summary is presented in Table 4 of the Results section.

Genetic profile of DNA repair genes in TNBC Materials and Methods

3. DNA extraction

For the neoadjuvant patient screening, genomic DNA was extracted from blood sample of all patients and volunteers according to a commercially DNA extraction kit (QIAamp DNA Blood Mini Kit, Qiagen). When available, tumoral DNA was extracted from FFPE samples, stored at Anatomia Patologica 1, AOUP, using a FFPE/tissues extraction kit (Genomic DNA from tissue, Macherey-Nagel). DNA quality and concentration was assessed by Nanodrop and, in some cases, Bioanalyzer (Agilent Technologies). For the analysis on tumor samples, cryostat sections were obtained from each tumor sample and, when available, each matched peritumor tissue. 2µm slices were stained with haematoxilyn/eosin and were evaluated from a pathologist to assess the percent of tumour cells. Samples with tumor cells present in <70% of total, were discarded. For the suitable samples, at least five 20µm slices were cut for DNA extraction (Genomic DNA from tissue, Macherey-Nagel). DNA quality and concentration was assessed by Nanodrop and Bioanalyzer (Agilent Technologies).

4. MLPA Analysis

In order to assess the Her2/neu state of each tumor, when the data was not available, we performed MLPA (Multiplex Ligation-dependent Probe Amplification). High level of concordance between MLPA and FISH results, supports the use of MLPA for assessment of HER2-amplification (Farshid, Cheetham, Davies, Moore, & Rudzki, 2011). MLPA is a variation of the multiplex polymerase chain reaction that permits multiple targets to be amplified with a single primer pair. Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA. Only when both probe are hybridised to their respective targets, can they be ligated into a complete probe. Each complete probe has a unique length, so that its resulting amplicons can be separated and identified by capillary electrophoresis. Because the forward primer used for probe amplification is fluorescently labeled, each amplicon generates a fluorescent peak which can be detected by a capillary sequencer. Comparing the peak pattern obtained on a given sample with that obtained on various reference samples, the relative quantity of each amplicon can be determined. This ratio is a measure for the ratio in which the target sequence is present in the sample DNA (Schouten et al., 2002). The P004-C1 ERBB2 kit (MRC-Holland) contains several probe for Her2/neu region, EGFR, BRCA1 and other genes on chr17 and related to Her2 overexpressing tumors.

Genetic profile of DNA repair genes in TNBC Materials and Methods

Along with Her2 status, MLPA has been exploited for the evaluation of other genomic events, such as deletion or duplication of other genes involved in carcinogenesis. We used the P105–Glioma 2 kit (MRC-Holland) to characterize a little the genomic profile for PTEN, P53, CDKN2A, CDK4, BRCA2 alterations. Area under the curve of each peak corresponding to a probe is compared with that of control samples. Tumor samples were compared with their matched healthy tissues, when available. Briefly, 70ng of genomic DNA are eluted in 5 µl of TE buffer and denatured for 15 minutes at 98°C. Then the annealing step takes place: a mix of 1,5 µl of the specific probemix and 1,5 µl of MLPA buffer are added to each samples, which are incubated 1’ at 95°C and then 16-20 hours of hybridization. After this step a ligation mix is prepared with two different buffers, dd H2O and a ligase enzyme. The reaction is lead to 56°C, and then added 32 µl of Ligase mix to each samples, maintaining them at 54°C in the thermocycler. The enzymatic reaction is performed at 54°C for 15 minutes and then deactivated at 98°C for 5 minutes. Finally, after the ligation of the contiguous probes, a PCR amplification is performed: 10µl consisting of H20, PCR Primer mix and SALSA polymerase are added for a 35cycles classic PCR reactions. PCR products are run by capillary electrophoresis with GeneScan 500 ROX dye Size Standard (Life Technologies) on the ABI PRISM 3130XL Genetic Analyzer. Differences between different DNA samples can be detected by comparing the resulting MLPA peak patterns. MLPA normalization consists of 2 steps: intrasample normalization (comparison of probe peaks WITHIN the sample) and intersample normalization (comparison or relative probe peaks BETWEEN samples.) Within each sample, each probe peak is compared to the peaks of the reference probes. Reference probes detect sequences that are expected to have a normal copy number in all samples. Almost all MLPA probemixes contain 8 or more reference probes located on various chromosomes. In the intersamples normalization, final probe ratios are determined by comparing the relative probe peak in the DNA sample of interest to all reference samples. Reference DNA samples are expected to have a normal copy number for both the reference and target probes.

5. Libraries preparation and sequencing : the ION PGM protocol

The Ion Torrent DNA sequencing technology is a scalable, low-cost semiconductor manufacturing technique for massive parallel sequencing. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides a semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells,

Genetic profile of DNA repair genes in TNBC Materials and Methods

which allow parallel, simultaneous detection of independent sequencing reactions (Rothberg et al., 2011). A discrete region of the genome can be easily amplified from the entire genome using PCR: this method for targeted sequencing it is often referred to as “amplicon sequencing”. Depending on the hypothesis, amplicon sequencing is typically used to investigate a few to several hundred genomic regions across multiple samples.

Fig 5. The Ion Torrent sequencing platform in brief. Target re-sequencing of known genes is performed by library construction by multiplex PCR and template amplification on sequencing spheres. Then the samples are loaded on sequencing chips and generated data are analyzed

For the present study a “targeted sequencing” approach was applied for studying the most interesting genes involved in DNA repair pathways and hereditary breast cancer. A custom panel of 783 primers pairs to amplify the entire coding sequence and the exon- intron junctions of 24 genes was designed using Ion AmpliSeq™ Designer software (https://www.ampliseq.com/), with a 94% of total coverage. The submitted region of interested was of 77kb; with the best design available all the genes were covered for >90% of the sequence with overlapping regions leading to 120kb of generated sequencing data. The genes amplified with this panel are: BRCA1, BRCA2, CHEK2, BRIP1, PALB2, TP53, PTEN, STK11, CDH1, BARD1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51c, RAD52, TP53BP1, PARP1, ERCC1 (Walsh, 2010).

Amplicon libraries preparation were performed according to the protocol of Ion AmpliSeq™ Library Kit 2.0 (Life Techonologies). Libraries quality and quantification were performed using both 2100 Bioanalyzer (Agilent Technologies) and Qubit® 2.0 Fluorometer (Life Technologies). Each library was linked to a unique molecular barcode (Ion Xpress™ Barcode Adapters 1- 16, Life Technologies), in order to sequence up to 8 patients/run. Each sample was clonally amplified on ION sphere particles (ISPs) by the One Touch instrument (Ion OneTouch™ 200 Template Kit v2 DL, Life Technologies). In this step each amplicon is

Genetic profile of DNA repair genes in TNBC Materials and Methods

linked to a single sequencing bead, and then amplified again by emulsion PCR. This sample preparation is essential for the correct sequencing, and is followed by a purification step with a biotin/streptavidin beads. Only the beads with a single amplicon linked, are selected for the next steps. ISPs were loaded and sequenced on a Ion 316™ Chip v2 or Ion Chip 318™. Both of this chips are semiconductor circuits able to sense the pH changes during the sequencing reaction, but differs in data output. The 316 v2 Chip can generated up 300 Mb of sequence, the 318 Chip up to 1Gb of sequence. These chips were used for different purposes since the need of different minimum coverage for a germline or a tumour analysis. As described, the size of the panel is approximately 120kb: in order to obtain a minimum coverage of 100X for the germline analysis, 8 patients for a 316 v2 chip were run. In order to obtain a 800-1000X coverage for the tumour tissue screening, to detected also somatic mutations at very low frequency, 4-7 patients were loaded on a 318 Chip. The sequencing was performed using Personal Genome Machine (PGM) instrument, using the kit “Ion PGM™ Sequencing 200 Kit v2” (Life Technologies). Each library can be discriminate from the others thanks to an indexing kit. The PGM sequencing run output was directly loaded on Torrent Server and stored as ‘.dat’ files. Torrent Server can serve as storage but also for basic data analysis: alignment of BAM files to hg19 genome, coverage analysis, run quality analysis, variant caller and others. A report after a PGM run is reported in Fig 6.

Genetic profile of DNA repair genes in TNBC Materials and Methods

Fig 6. ION PGM Sequencing Run Summary. The upper left panel describes how the chip have been loaded with the samples and the sequencing output; the central upper panel describes the quality of the libraries in term of clonality and purity; the right upper panel shows the amplicons read lenght distribution. The central panels give indications about the quantity and quality of the libraries sequenced, for each samples. Q20 represents the ideal quality.

6. Data processing and analysis

Data collection and processing were performed by Torrent Server and BAM files were uploaded on Ion Reporter 4.0 and Ion Reporter 4.2 (https://ionreporter.iontorrent.com/). The reference genome used is hg19, according to the GRCh37 human reference sequence assembling (February 2009). For the germline screening high quality parameters were set, including MAF (the frequency of the minor allele sequenced), threshold of 20%. This because germline variants are expected to have a frequency near the heterozygosity. For the mutational analysis of tumour tissues, high stringency quality somatic parameters were set, including detection of single nucleotide variations at MAF=2% and deletions/insertions at MAF=5%. This setting are in accord with somatic variants found in

Genetic profile of DNA repair genes in TNBC Materials and Methods

several studies and in cancer database as COSMIC (Catalogue Of Somatic Mutations In Cancer, www.cancer.sanger.ac.uk).

The annotation were also performed by wAnnovar (http://wannovar.usc.edu) and VeP! (http://www.ensembl.org/Homo_sapiens/Tools/VEP), in order to compare the efficiency of Ion-supported data analysis workflow. The functional effect of each variant was predict using bio-informatic prediction tools: SIFT (http://sift.jcvi.org), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) Grantham score, Mutation Taster (www.mutationtaster.org). SIFT (Sort Intolerant From Tolerant) predicts whether an amino acid substitution affects protein function. SIFT prediction is based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences, collected through PSI-BLAST (Kumar, Henikoff, & Ng, 2009). PolyPhen-2 (Polymorphism Phenotyping v2), available as software and via a Web server, predicts the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations (Adzhubei, Jordan, & Sunyaev, 2013). The Grantham score examines the difference in the physicochemical nature of the amino acid substitutions. The score ranges between 0 and 215. A higher Grantham score is indicative of a greater difference in chemical properties between two amino acids (ie, polarity and molecular volume) and can indicate a stronger (negative) effect on protein structure and function. MutationTaster is designed to predict the functional consequences of not only amino acid substitutions but also intronic and synonymous alterations, short insertion and/or deletion (indel) mutations and variants spanning intron-exon borders. MutationTaster2 includes all publicly available single-nucleotide polymorphisms and indels from the 1000 Genomes Project as well as known disease variants from ClinVar3 and HGMD Public4. Variants marked as pathogenic in ClinVar are automatically predicted to be disease causing, and the disease phenotype is displayed. MutationTaster is able to analyze sequence alterations spanning an intron-exon junction; these are likely to perturb normal splicing and hence have considerable pathogenic potential (Schwarz, Cooper, Schuelke, & Seelow, 2014). A further comprehensive literature review was carried on, to integrate in silico and functional reports for each variant. To the total variant number, a coverage filter was applied. Then, only the exonic variants and the canonical splicing site mutations were included. Homozygous frameshift in homopolimeric regions and variants with frequency >1% according to 1000 Genomes

Genetic profile of DNA repair genes in TNBC Materials and Methods

Project were removed from selection. Following this filter, only rare/new exonic and splicing variants were confirmed by direct Sanger sequencing.

7. Sanger sequencing

After filtering, Sanger sequencing was performed to confirm the identified variants. Primers were designed by Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and Beacon Designer; then primer pairs were checked by Primer-BLAST tool to ensure the amplification of target regions in a specific manner. PCR amplification was performed in a volume of 25µl according AmpTaq Gold protocol (Invitrogen, Life Technologies) and sequenced by BigDye Terminator v3.1 kit using an ABI PRISM 3130XL Genetic Analyzer. Sanger sequencing allowed to confirm variants with MAF >10%, i.e. germline variants and somatic variants in well established tumour clones. For the very low frequencies somatic variants, digital PCR and deep resequencing are in progress.

8. High Resolution Melting (HRM) in healthy controls

Genotyping analysis of 50 healthy controls by High Resolution Melting Analysis was performed to determine a frequency for the variants without population data in any database. Primer pairs for 3 SNPs (BRIP1 c.856C>T; RAD52 rs201623936, MSH2 rs34136999) were designed using Beacon Designer™ 8.13, and further verified and evaluated by Primer-Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and mfoldDNA Server (http://mfold.rna.albany.edu/?q=mfold/dna-folding-form). Primers sequence are reported in Table 1.

Genetic profile of DNA repair genes in TNBC Materials and Methods

Gene dbSNP Allele change Primer pair Sense: 5’-GGGTTCCAATGACTATTCTT-3’ Antisense: 5’-CTGTTCAAGTTACCGACTAC-3’ BRIP1 - c.856C>T Tm Product: 75,3°C TM Mutant Product:74,7 °C Sense: 5’-AGGTTGCAGTTTCATCAC-3’ Antisense: 5’-ATACTGGCTGAAGTCAAAAG-3’ MSH2 rs34136999 c.815C>T Tm Product: 74,3°C TM Mutant Product:73,9 °C Sense: 5’- CCTTGCCTTCTCCAAAGATAA-3’ Antisense: 5’-GTTCAAACTCCTTTTCTGCC-3’ RAD52 rs201623936 c.388G>A Tm Product: 77,1°C TM Mutant Product:77,4 °C

Table 1. SNPs and corresponding primer pairs for HRM-Analysis designed using Beacon Designer™ 8.13

HRM were performed using Type-it® HRM™ PCR kit (Qiagen, Crawley, UK) following manufacturer's instructions. The HRM-PCR reaction mixture contained 50 ng genomic DNA, 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 0.5X EvaGreen (Diatech), 0.7 mM forward and reverse primers, and 0.5 U AmpliTaq Gold® DNA Polymerase (Invitrogen, Life Technologies). Samples were run in duplicate, together with positive and negative controls, on a Rotor- Gene Q real-time PCR Thermocycler (Corbett Research, Sydney, Australia). Following a touchdown PCR programme, the products were denatured at 95°C for 5 s, and then annealed at 50°C for 30 s to randomly form DNA duplexes. Fluorescence data were acquired at the end of each annealing step during PCR cycles and each of the HRM steps with automatic gain optimisation. The melting data were normalized by adjusting start and end fluorescence signals, respectively, of all samples analysed to the same levels. Genotypes of the individuals were scored automatically by the software and verified manually.

9. Loss Of Heterozygosity (LOH) analysis in tumour tissues

LOH is frequent in cancer tissues, and suggest a pathogenetic role for the mutant allele. For germline variants without previous studies and no frequency data but with in silico suggestion for pathogenicity, LOH analysis was performed on the residual tumour mass. DNA was extracted from FFPE sections and amplified according PCR general conditions. Primers pairs were the same used for variant confirm by Sanger sequencing. PCR amplification was performed in a volume of 25µl according AmpTaq Gold protocol (Invitrogen, Life Technologies) and sequenced by BigDye Terminator v3.1 kit using an ABI PRISM 3100 Genetic Analyzer.

Genetic profile of DNA repair genes in TNBC Materials and Methods

10. Statistical Analysis

Well known pathogenetic mutations and in/del frameshift,” either in BRCA genes or other genes, were called “pathogenetic. Likely deleterious mutations were defined in the “probable-pathogenetic” category and considered as affecting protein function in the correlation analysis. Patients with TNBC without mutations were categorized as wild type. The Fisher’s exact test were used for evaluating associations with mutation status. P values<0.05 were considered statistically significant. For the second group of patients, the disease free survival (DFS) will be calculated from the date of diagnosis to the date of recurrence. Overall survival (OS) will be calculated from the date of diagnosis to the date of death or the last follow-up. DFS and OS will be calculated according to the method of Kaplan-Meier.

Results

Genetic profile of DNA repair genes in TNBC Results

1. GERMLINE MUTATIONAL ANALYSIS IN TNBC NEOADJUVANT PATIENTS

1.1. Neoadjuvant patients histopathological features

In the last decades neoadjuvant therapy has taken an emerging role among the cancer treatments, giving good results and also useful informations about how a tumor may respond even in adjuvant and metastatic settings. Indications for this kind of pre-operative therapy are usually massive lymph node involvement, extensive tumor mass, tumors invading the chest wall or the skin. However recently neoadjuvant therapy is also considered for stage II large tumors in patients who wants to have a breast-conserving surgery. This approach is relatively often used for TNBC patient, who are usually young. However, the high proliferation rate of this subtype sometimes made not possible to consider a breast conserving surgery. Despite having a poorer overall prognosis, patients with non-metastatic TNBC treated with neoadjuvant chemotherapy seem to have a better response than other breast cancer subtypes. In this study were included 19 patients with a diagnosis of TNBC, who underwent to surgery resection at U.O. Senologia of Pisa Hospital in the last five year. Among these, 10 had a family history of breast cancer, while 9 were unselected for family history. The median age of these two groups were 37.3 ± 4.3 and 57.7 ± 10,7 years, respectively. No substantial differences were reported in tumor grade (Bloom and Richardson scale), histotype and tumor size between familial and sporadic. However, sporadic samples tend to have lymph node involvement more often that the familial cancers (7/9 patients). As far the therapy response is concerned, the familial patients seem to have a more often a pathological complete response to this therapy design. This may be probably due to basal-like phenotype of the tumour, or mutations in BRCA1 gene, or the scarce lymph node involvement. Overall, the two groups are equivalent for the percentage of patients who, on the contrary, go to progression. A pathological complete response was achieved in 21% of patients, and a good pathological response in 78%; these data are in line with many studies on neoadjuvant treatment. It is known that TNBC are responsive to anthracyclines, but many BRCA related cancers are resistant to taxanes. The hypothesis of this study is that these tumours may be related to DNA repair defect and therefore resembles the BRCA-related ones: in this view, some tumors may be well responsive to anthracycline and not responsive to taxanes. A summary of the histopathological and clinical features of this set of patients is reported in Table 2.

Genetic profile of DNA repair genes in TNBC Results

Familial Sporadic Total

Age, median (range) 37.3 (31-47) 52.2 (38-77) 44,4 (31-77)

Grade G1/2 - 1 1 G3 7 8 14 NA 3 - 3 Histology IDC 10 8 17 Other - 1 1 T-stage T1/T2 4 4 7 T3/4 4 4 8 NA 3 1 3 Nodal Stage Negative 3 0 3 Positive 5 7 11 NA 2 2 4 Therapy Response pCR 4 1 5 pPR 4 6 10 PROG 2 2 4

Table 2. Histopathological features of neoadjuvant TNBC patients

1.2. Mutational screening in neadiuvant patients and selection of variants

Following the annotation by webAnnovar and Ion Reporter 4.0, 242 variants were detected and filtered by SIFT and POLYPHEN 2 prediction tools. For the first step of analysis, only the exonic variants and the canonical splicing site mutations with a minimum coverage of 60X were considered. Moreover, homozygous frameshift in homopolimeric regions and variants with frequency >1% according to 1000 Genomes Project were removed from selection. 31 exonic variants and 2 splice site variants were considered interesting because they were predicted by SIFT or Polyphen or due to their very low frequency in the population. This first phase of study was performed also to test the the specifity of the ION PGM sequencing, so all this variants were checked by direct Sanger sequencing. 3 false positive were found, in homopolimeric regions; two other variants were discarded since their localization in a PMS2 pseudogene. Among the 28 confirmed variants 25 were unique and 3 found in 2 patients. Here, the classes of selected variations: 28 single nucleotide variations (1 stopgain, 2 splicing variants, 1 synonymous, 24 missense), 2 indels frameshift , 2 indels in frame.

Genetic profile of DNA repair genes in TNBC Results

According to functional studies in literature, each reported variant was classified as “clearly pathogenic” or “neutral”. The variants of unknown clinical significance were classified as “probable pathogenic” or “probable benign” by bioinformatic prediction tools. In the Table 3 are reported all the variants; a clinical significance was derived by this integrative approach between previous studies and prediction scores. 20 variants with clearly and/or predicted pathogenic significance were found. 4 of them are alterations never described in literature. All the 20 variants are localized in 10 of the 24 genes analyzed. The clearly pathogenetic variations are located in BRCA1 (2 splicing variants and 1 frameshift deletion), PALB2 (1 frameshift insertion) and in RAD51c (1 SNV). The predicted pathogenetic variations are located in: BARD1 (1 SNV), MLH1 (1 SNVs, 1 in/del NFS), TP53BP1 (3 SNVs), RAD52 (2 SNVs), MSH2 (2 SNV), PARP1 (1 SNV), BRIP1 (1 SNV), PALB2 (2 SNV), RAD50 (1 SNV). The 4 novel variants are localized in RAD52, BRIP1, PALB2, TP53BP1.

Fig 7. Germline variants distribution. In the left pie chart the different classes of mutation are illustrated. In the right pie chart the distribution among genes is reported: HR pathway enrolls the majority of mutated genes (sectors in blue shades).

1.3. Variant distribution among genes and the most interested pathways

The screening evidenced that BRCA1 mutations are frequent , with 3 BRCA1 mutations in 20 patients (15%) in according with literature on TNBC. Moreover PALB2 and RAD51c, considered moderate risk genes of susceptibility, are mutated in 5% of cases (one patient for each gene). In PALB2 were found also two predicted pathogenetic missense, already reported in larger studies as possibly damaging. They are both placed in the WD40 domain of interaction with BRCA2, RAD51, RAD51c. Overall 3 patients have alterations in PALB2 gene that may distrupt the perfect machinery of DNA damage recognition and repair. Moreover one patient harbour a BRIP1 variant which fall in the Fe-S domain and

Genetic profile of DNA repair genes in TNBC Results

next to the M299I, which is involved in early onset breast cancer susceptibility. This missense has shown LOH in tumour tissue and its worth further investigation. Three other predicted mutations are found in MLH1 and one in MSH2: this last is already described in literature with controversial results, enforcing the possible contribute of MSH2 in the pathogenesis of breast cancer. A variant in the self-dimerization domain of RAD52 was also found in one patient in co-presence with a RAD51c pathogenetic mutation; the contribute of RAD52 variants may increase the deficiency driven by RAD51c mutated protein. Notably, few PARP1 variants are found in this set of patients. PARP1 is object of great interest for its multiple functions in DNA repair and as target of therapy. We found a very rare variant of PARP1 in a patient BRCA1-related. This substation is located near the BRCT domain and convert a Serine in a Tyrosine, maybe altering the pattern of phosphorylation. Interestingly, TP53BP1 is one of the genes with the major number of alterations. This gene have been recently investigated for its fundamental role in coordinating the choice between HR and NHEJ in response to DNA damage, and its strong connection with BRCA1 function (Zimmermann & de Lange, 2014). Mutations in its domains, may decrease affinity with a variety of interactors and lead to an abnormal signal cascade in response to stress. Recently also RAD50 have been proposed as a novel predisposition gene, since its involvement in DNA damage recognition. The two variants found are localized in the coil domain, essential for the correct structure and crucial for the interaction with partners. As described before, the well-known susceptibility genes of the HR patways are more altered than the other genes, mostly involved in other DNA repair pathways. Overall 5 out 20 patients have a germline mutation in HR genes (25%). Mutations in other genes may modulate the phenotype and may modify the risk in a synergistic way.

1.4. Neoadjuvant germine screening: mutation overview

A summary of the principal informations reported in literature for each variant, is reported in the next pages. Genetical and specific databases were consulted and the clinical informations integrated with the prediction scores of SIFT, PolyPhen, MutationTaster, PhyloP tools.

Genetic profile of DNA repair genes in TNBC Results

BARD1 c.1075_1095del p.Leu359_Pro365del This variant was found in one patient (#5). It is already described in the literature with controversial results. In dbSNP is reported but without clinical significance. It is an in frame deletion, located outside the putative functional domains even if is possible that it may result in a change in the secondary structure of the protein (Ghimenti, Sensi et al. 2002). In a recent study is considered not pathogenic (De Brakeleer, De Greve et al. 2010). In LOVD database is reported twice, as probably not pathogenic and as pathogenic. BARD1 c.33G>T p. Q11H This variant was found in one patient (#41). In dbSNP is registred but without any clinical significance. Is predicted “conserved” according to PhiloP but not predicted by other tools. There is no functional assays present in literature. In LOVD database is reported as pathogenic, but no studies that conclude its clinical significance. BRCA 1 IVS11 c.4096 + 1G> A: This variant was found in one patient (#690) This splicing variant has been reported as pathogenic on dbSNP and it was found in several HBOC families, as reported in the LOVD database. Neverthless, its effect on splicing process is still under investigation. It was shown that gives rise to transcripts missing exon 11 (Bonatti, Pepe et al. 2006). In the BIC database is indicated as important in the clinical setting. BRCA1 c.3481_3491del11 p.Glu1161Phefs This variant was found in one patient (#4). This deletion has already been observed: in most of the database is reported as pathogenetic, while the BIC ranks it as "important from the clinical point of view". Yet a recent study classified it as a disease-causing mutation (Tarabeux, Zeitouni et al. 2013). This frameshift deletion leads to a premature termination at codon 1161. BRCA1 IVS 8 c.547 + 2T>A This variant was found in one patient (#9). This splicing site variant is reported in dbSNP as pathogenetic. Two recent studies describe it as pathogenic (Colombo, De Vecchi et al. 2013; Vietri, Molinari et al. 2013). In particular, Colombo et al. have carried out functional studies and have shown that this mutation causes the loss of the entire exon 8. in silico predictions with ExPASy Proteomics (http://www.expasy.ch/) suggested the formation of a premature stop codon at residue 198: this prediction ranks the variant in Class 5. LOVD describes it as probably pathogenic (Steffensen et al. 2014). For the BIC database is of clinical interest.

Genetic profile of DNA repair genes in TNBC Results

BRCA2 c.6441 C>G p. H2147Q This variant was found in one patient (#1235). This is a Class 3 variants according to LOVD, otherwise a variant with discording reports about its pathogenicity. ClinVar and dbSNP report it as VUS. Prediction tools describe this BRCA2 variant as tolerated and probably benign. It falls out of the main BRCA2 domains of interaction. BRCA2 c.8187G>T p.K2729N This variant was found in one patient (#7). dbSNP clinical significance is "VUS”, but recent functional studies classify it as neutral variant (Farrugia, Agarwal et al. 2008; Biswas, Das et al. 2011). LOVD and BIC classify this variant as not pathogenic and VUS. BRCA2 c.A9976A>T K3326X This variant was found in one patient (#2) Various studies are reported on this variant but were inconclusive. Although the BIC database ranks it as "not important from a clinical point of view and dbSNP classified it as non-pathogenic, an interesting study notes an increase in the prevalence of this mutation in pancreatic cancer families, and suggest that this polymorphism may contribute to the risk of developing pancreatic cancer (Martin et al. 2005 ). A similar study suggested otherwise an association with breast cancer (Reid, Renwick et al. 2005). In a study in functional assays were used, to verify the effect of some VUS on the functionality of BRCA2. However a functional study on cell survival, homologous recombination capability and genomic instability suggested that this mutation is not pathogenic (Wu, Hinson et al. 2005). BRIP1 c.856C>T p.P286S This variant was found in one patient (#891). This variant was first observed in this study and no informations are available in dbSNP. Is predicted conserved and deleterious by all the algorithms used. BRIP1 c.2358T>C p.D846D This variant was found in one patient (#1235). It is not reported in dbSNP or ClinVar, no frequency data are available. It falls in the last residues of one helicase domain. According to MutationTaster may be affect splicing moderatly. It will be interesting to further investigate if the alternative transcript is stable and if BRCA1 interaction is lost. MLH1 c.1852_1853delinsGC p.K618A This variant was found in one patient (#4). In dbSNP is indicated as VUS and without frequency data. In literature there are contradictory studies: it has been described both as a neutral variant (Vogelsang and

Genetic profile of DNA repair genes in TNBC Results

Komel 2011) and as deleterious. In 2008, it was observed that this mutation reduces the amount of protein expressed, this probably for a deleterious effect on protein stability (Bapat and Perera, 2008). One study classify it neutral with respect to Lynch syndrome (Castillejo, Guarinos et al. 2011). Instead, more recent studies, focused on this syndrome, classify it as an incomplete penetrance mutation, suggesting that it may increase the risk of cancers related to Lynch syndrome approximately twice (Medeiros, Lindor et al. 2012). In the LOVD databases is registred as variant unknown meaning. MLH1 c.1939G>A p.V716M This variant was found in one patient (#740). This variant is predicted deleterious by MutationTaster and the nucleotide varied is considered "preserved" by PhyloP. A 2006 study, describes this rare variant in a Finnish family, finding it in a family with hereditary cases of prostate cancer, but not in controls (Fredriksson, Ikonen et al. 2006). Is located in exon 17, in the domain of interaction with PMS1, PMS2 and MLH3. There is no functional studies. LOVD and dbSNP classify it as VUS. MSH2 c.815C>T p.A272V This variant was found in one patient (#41). This variant, present in dbSNP without frequency, is reported in several works which defined it discordantly: pathogenic, probably neutral, unknown significance. A study, in an ex vivo assay observe that this variation may cause a partial exon skipping of exon 5 (Tournier et al. 2008). An in vitro study suggest that this variant can have a reduced efficiency of binding the mismatch than the wild-type protein (Ollila et al. 2008). In a more recent study, this mutation was found in association with deleteriuous mutations in Lynch syndrome patients, and notin the controls (Mueller, Gazzoli et al. 2009). Is considered deleterious by SIFT and MutationTaster. MSH2 c.965G>A p.G322D This variant was found in one patient (#10). This mutation is widely discussed in the literature. It is registered on dbSNP with a probably not pathogenic definition. Functional assays in yeast indicate it as non- pathogenic (Gammie, Erdeniz et al. 2007) while in a functional assay was observed a slightly decrease in the binding efficiency and release of DNA chains with mismatches compared to wild-type (Ollila et al. 2008). One study describes it as associated with breast cancer, and concluded that this polymorphism might be considered as a tumor marker (Poplawski, Zadrozny et al. 2005). This rare and low penetrant variant, togheter with other specific polymorphisms of MMR, may be associated with outcome after therapy, although further investigation is needed (Gargiulo, Torrini et al. 2009). In tuscany population the frequency is higher than in generale population (MAF=0,014 vs MAF=0,006).

Genetic profile of DNA repair genes in TNBC Results

MSH2 c.435T>G p.I145M This variant was found in one patient (#2). It is reported in LOVD database by different authors, with conflicting results: some report it as neutral, other as pathogenic, others with unknown meaning; there are no studies that conclude the nature. In one of the study was evaluated the interaction of the MSH2-MSH6 in the presence of this mutation and any deficiency in MMR was evidenced (Kariola, Otway et al. 2003). Recently has been described as pathogenic (Kurzawski, Suchy et al. 2006). PALB2 c.2993G>A p.G998E This variant was found in two patients (#5, #41). In dbSNP is reported as probably not pathogenic. Although it is predicted deleterious by the software analysis, has been observed that it is relatively common in patients with breast cancer, as in healthy controls (Bogdanova, Sokolenko et al. 2011). In the LOVD database is reported probably not pathogenic. PALB2 c.2816T>G p. L939W This variant was found in one patient (#946). This mutation according to dbSNP is probably not pathogenic. Is predicted deleterious by each algorithm. The LOVD database declares that is probably not pathogenic. Recently, a paper (Park et al., 2014) reported that L939W protein is stable but partially disrupt the PALB2–RAD51C–BRCA2 complex in cells. Functionally, the L939W mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. PALB2 c.38_39insG, K14Efs*29 This variant was found in one patient (#2). This frameshift variant has never been reported in any database. It is located in exon 1 and MutationTaster predicts a premature stop codon at residue 29. Always Mutation Taster predicts that the transcript originated from the mutated gene may be degraded due to the Nonsense Mediated Decay (NMD). Further investigations are needed to clarify the pathogenicity of this mutation. PARP1 c.1148C>A p.S383Y This variant was found in one patient (#690). This very rare variant is reported in dbSNP, but there are no functional studies. It is predicted deleterious by PhyloP and Mutation Taster. PARP1 c.2652G>A p.A884A This variant was found in one patient (#1114).

Genetic profile of DNA repair genes in TNBC Results

It is reported in dbSNP only, with frequency data but no clinical indication. This substitution takes place in the third-to-last residue of the exon. MutationTaster prediction suggest a weak effect on splicing. PMS1 c.605G>A p.R202K This variant was found in one patient (#690). This mutation have no information on the clinical significance in dbSNP. In the literature are not reported functional studies. The nucleotide is predicted conserved by PhyloP but this is not a deleterious substitution for the other softwares. RAD50 c.1544A>G p.D515G This variant was found in one patient (#891). It is registred in dbSNP, but are absent information on the clinical significance. There is no specific MAF and is predicted conserved and deleterious by each algorithm analysis. There are no functional studies already performed. RAD51C c.414G>C p. L138F This variant was found in one patient (#3). This rare variant is reported in dbSNP as probably pathogenetic. It is also described in the OMIM database. In 3 women from a German family with breast-ovarian cancer, Meindl et al. (2010) identified this germline heterozygous Leu138Phe substitution. Two individuals had ovarian cancer at age 53 years, and the third had breast cancer in her early fifties. Tumor tissue available from all tumors showed loss of heterozygosity at the RAD51C locus. The mutation was not found in 2,912 controls. In vitro studies showed that the L138F mutant was unable to restore normal RAD51C activity in RAD51C-deficient cells (Somyajit, Subramanya et al. 2010). Moreover,it would appear to have a role in the ATPase domain of RAD51c. This residue was found mutated in Fanconi anemia-related disorders, in complementation group O, ovarian cancer and breast cancer (Somyajit, Subramanya et al. 2012). RAD52 c.388G>A p.E130K This variant was found in one patient (#3) This variant has never been reported in either 1000genomes project nor in dbSNP. It’s predicted deleterious by all the algorithms used, and conserved even at the nucleotide level. In the literature there is no publication about it. This mutation is located in exon 6, in the self-dimerization domain. RAD52 c.598G>A p.V200M This variant was found in one patient (#7). This mutation has never been observed. It is predicted to be deleterious SIFT and Mutation Taster.

Genetic profile of DNA repair genes in TNBC Results

TP53BP1 c.895T>C p.S299P This variant was found in two patients (#6, #891). This variant is reported in dbSNP without any clinical suggestion. There is no functional assay for this variant. TP53BP1 c.3092T>C p. V1031A This variant was found in one patient (#9). This variant is inserted in dbSNP but there is no clinical significance. There are no functional assays in the literature and is predicted to be deleterious SIFT, PolyPhen-2 and Mutation Taster. TP53BP1 c.3440C>T p.P1147Q This variant was found in one patient (#1235). This missense in never reported in literature and no data of frequency are available. It is predicted deleterious by all bioinformatic tools.

Genetic profile of DNA repair genes in TNBC Results

2009

2005

, 2010

Wu, Wu,

Gargiulo,

;

Somyajit 2012 Somyajit

Brown, 2014 Brown,

2006

013

Wong

De Brakeller De

Park, 2 Park,

+ ALTRA REF REF ALTRA +

References

Bonatti

2002;

Tarabeux, 2013 Tarabeux,

Fredriksson, 2006 Fredriksson,

Perera, 2008; Medeiros, 2012 2008; Medeiros, Perera,

ajit, 2010; Vaz, 2010; 2010; Vaz, 2010; ajit,

Kariola, 2003; Kurzawsky, 2006 2003; Kurzawsky, Kariola,

Ghimenti,

Martin, 2005; Reid 2005 2005; Reid Martin,

Bogdanova, 2011; Bogdanova,

Gammie, Poplawsy,2007; 2005;

Chao, 2008; Tournir, 2008; Mueller, 2009 2008; Mueller, Tournir, 2008; Chao,

Somy

benign

pathogenic

pathogenetic

benign

pathogeni

pathogenic

probable

Clinical Significance Clinical

probable

MAF

T=0.000

T=0.041

A=0.000

A=0.031

A=0.0

A=0.005

G=0,000

G=0.000

G=0.005

T=0.0051

C=0.0051

G=0.0051

G=0.0000

1,0

0,01

0,74

0,976

0,973

0.233

0.928

0,976

0.928

0.993

0.999

score

PolyPhen

,78

0,4

0,0

0,8

0,0

0.0

0,0

0,00

0,78

0,06

0,04

0,15

0,01

0,04

SIFT SIFT

score

db SNP db

rs2066459

rs3219062

rs4987188

rs80358879

rs45478192

rs61751060

rs35831931

rs80358178

rs45551636

rs34136999

rs61751060

rs45551636

rs28997575

rs35502531

rs80357877

rs63750124

rs11571833

rs145843634

rs143914387

rs201623936

rs267606999

P1147

D846D

.K618A

p.Q11H

p.L138F

p.S383Y

p.S29

p.P286S

p.S299

p.E130K

p.K359P

p.R202K

p.I145M

p.G998E

p.A27

p.D515G

p.G322D

p.L939W

p.V647M

p.K3326*

p.H2147Q

pE13fs*42

p.E1161fs*

AA change AA

nsGC

c.G33T

c.T895C

c.C856T

c.C815T

c.T895C

c.T435G

c.G414C

c.G605A

c.G965A

c.G388A

c.39insG

c.A9976T

c.T2816G

c.C1148A

c.A1544G

c.G1939A

c.G2993A

c.2358T>C

c.3440C>T

c.6441C>G

NT change NT

c.4096+1G>A

c.1075_1095del

c.3481_3491del

c.1852_1853deli

NFS

ivs

synonim

Type of

In/del FS In/del

missense

nonsense

in/del

mutation

AD50

PMS1

BRIP1

MLH1

MSH2

Gene

PALB2

PARP1

BRCA2

BRCA1

BRCA2

RAD52

BARD1

no mut no

RAD51C

TP53BP1

#41

#946

#891

#833

#740

#690

#Pat

#1235

#1177

Positive Response (Responders)

Outcome

Genetic profile of DNA repair genes in TNBC Results

arrugia, 2008 arrugia,

References

Colombo, 2013 Colombo,

Gammie, Poplawsy,2007; 2005; Gargiulo, 2009

benign benign

pathogenic

benign

pathogenic

probable

Clinical Significance Clinical

MAF

A=0.000

A=0.015

G=0.015

T=0.0000

0.233

0,994

score

PolyPhen

0.15

0,02

0,01

SIFT SIFT

score

db SNP db

rs4987188

rs45482998

rs80358047

rs80359065

rs202143014

A884A

p.G322D

p.V200M

p.V1031A

p.K2729N

AA change AA

.G965A

c.G598A

c.T3092C

c.G8187T

c.2652G>A

c.547+2T>A

NT change NT

ivs

synonim

Type of

missense

mutation

MSH2

Gene

PARP1

BRCA1

BRCA2

RAD52

No mut No

TP53BP1

no mut no

#10

#478

#Pat #1114

Negative Response

utcome (Non Responders)

Table 3. Complete list of selected germline variants found in TNBC patients in neoadjuvant therapy. (A) Responders patients and (B) non Responders patients. SIFT and PolyPhen scores are reported, together with MAF according to 100genomes.

Genetic profile of DNA repair genes in TNBC Results

1.5. Characterization of novel variants

Since no frequency data are available and the prediction tools strongly suggested their pathogenity, we selected the 3 novel variants (RAD52 c.G388A , BRIP1 c.C856T , TP53BP1 c.3440C>T ) and the MSH2 c.C815T to further analysis. DNA from blood samples of 50 healthy controls was extracted and controls were screening by High Resolution Melting, in order to describe the presence of these variants in 100 chromosomes in the Tuscany population. HRM is able to discriminate two alleles that differs for a single specific nucleotide: this technique is really accurate but is not always easy to design the perfect primer pairs. It is the case of the TP53BP1 c.3440 variants: by now no reliable results are obtained for this variant. The other three variants, instead, were studied and the frequencies observed were MAF (A)=0,00 for RAD52 variant, MAF(T)=0,01 for MSH2 variant (one control was in heterozygosis) and MAF (T)=0,00 for BRIP1 variant. Even if the number of patients and control is really limited, this provide a further evidence of the rarity of this substitutions. LOH is often observed in breast cancer, especially for BRCA1 mutations. Loss of the WT allele in tumour tissue is usually associated with pathogenicity of variants, since the mutant allele is selected or preferred to WT allele. In order to evaluate if these novel mutations are able to drive tumor aggressiveness, LOH analysis in the residual tumor tissue was performed. The analysis for c.3440C>T of TP53BP1 is in progress, since no tumor tissue is yet available. No LOH events are associated with RAD52 and MSH2 variants, while complete LOH was observed for BRIP1 substitution (c.C856T, p.P286S) in patient #891, suggesting a probably role in tumorigenesis (Fig 8).

Fig 8. LOH in c.856C>T BRIP1 variants. The upper elettropherogram sequence is the tumor tissues in which the T allele is completely lost.

Genetic profile of DNA repair genes in TNBC Results

1.6. Pathogenetic variants carriers tend to have better clinical outcome

In order to understand if a particular gene signature in DNA repair genes is associated to a clinical outcome, the patients were divided in two groups of response. According to previous studies, patients were considered RESPONDERS when they achieved a reduction of tumour mass greater than 50% of the initial bulk. Patients were included in the NON RESPONDERS class when, instead, achieved a reduction of tumour mass after neoadjuvant therapy smaller than 50% or have a progression during therapy administration. With this selection, 6 patients were included in the NON RESPONDERS group, while the other 13 were REPONDERS patients. All the predicted pathogenetic variants were considered to affect protein function in a comparable way of known pathogenic ones. The hypothesis is that a dysfunctional DNA repair mechanism can have an effect in the response to anthracyclines and taxanes therapy. In the non-responders group, 2 patients are carriers of probable-pathogenetic mutation, and 4 of them have no predicted mutations. In the bigger group of responders women, 11 of them have at least one probable-pathogenetic variant, and only two are not mutated. A chi square test, corrected for small number of samples (Fisher Test) was applied. An altered DNA repair was found to be associated with the group of TNBC patients who well responded to anthracycline and taxane. Even adjusted for small groups, this test give back a p value=0,046 which is slightly significant and suggest a trend between pathogenetic mutations and a positive response to therapy. Indeed, the majority of mutations likely to affect protein function are carried by 11 of the 13 responsive patients, and 4 on 6 non responsive patients had any mutation (pathogenetic or predicted deleterious). Overall, the non responders patients are mutated in 33% of cases, in comparison with the 84% in the patients with a good response (Fig 9). This relationship is made stronger if only the known pathogenetic mutations are considered: indeed 4 of the deleterious variants are in the responsive patients, while the other one BRCA1 mutations in is one patient with progression. The frequency of mutated patients in the two groups of response is almost two-fold (31 vs 16%). This suggest that, in additionto BRCA1/2, maybe other DNA repair genes may be contribute to play a role in the response.

Genetic profile of DNA repair genes in TNBC Results

response/mutations 12

6 with probable/pathogenetic mut patients

° w/o probable/pathogenetic mut n 4

0 responders non responders

Fig 9. Correlation between mutational status and therapy outcome in TNBC patients. Patients are divided in class of response on the basis of % of reduction in tumor mass. In the class of responders patients, 11 are carrier of a probable pathogenetic mutation in comparison with 2 patients of the non responders class (p = 0,046).

Genetic profile of DNA repair genes in TNBC Results

2. MUTATIONAL SCREENING IN TNBC TISSUES

2.1. TNBC features

TNBCs are usually aggressive tumours of high grade and large in tumour size. The standard therapy in adjuvant setting is chemotherapy with FEC (5-fluorouracil, epidoxorubicin and cyclophosphamide) or CMF (cyclophosphamide, methotrexate, 5- fluorouracil), which have been almost equivalent employed over the years. In this second step of study, 37 patients with hormonal receptor negative status were evaluated. Their tumour tissues were stored in the tissue bank. For almost all the patients, data from the clinical record gave complete informations about histology and therapy. These patients were unselected for family history; age of breast cancer onset varied from 27 to 72 years old, with a median of 53,4 ±11 years. Almost all the patients developed a ductal infiltrating carcinoma (91%), with a prevalence for a high undifferentiated tumours G3 (87%). As far as TNM classification in concerned, the majority of patients had a large tumour at time of surgery with a T-stage>1, i.e a tumor mass larger than 2cm. 65% of patients had a nodal involvement, but only one patient presents distant metastasis at time of diagnosis. In patients in which it was possible to get the data of MIB1 (a Ki-67 antibody, used as a proliferation index), was observed that almost all had a very high MIB1 value indicating a high proliferating cancer. Regarding the therapy regimens of these patients, the majority of them was equally divided between FEC (34%) and CMF (31%); a small group have undergone to an experimental regimen with Epirubicin and Taxanes, followed by CMF, which was tested in the 90s. For 11 patients was not possible to define therapy regimens, but they have probably undergone to FEC or CMF. 12 patients had a relapse and/or a distant metastasis within 5 years from the primary tumor onset. The interested sites were skin, lung, breast chest, bone tissues, liver and brain. These represent the 37,5% of the 32 patients with a complete follow up; the other 62,5% had no recurrence of disease after 5 years. In many cases a follow up of 10 years is available. This clinical-pathological information are reported in Table 4.

Genetic profile of DNA repair genes in TNBC Results

Sporadic TNBC patients characteristics

Age range <40 3 40-49 10 50-59 11 60-72 13 Histology CDI 34 CDIS 2 Midoll Ca 1 Grade G2 4 G3 28 NA 5 T-stage T1 17 T+ 20 Nodal stage Positive 13 Negative 24 Mts at diag M0 27 M+ 1 NA 9 MIB-1 < 20% 1 >20% 12 NA 24 Therapy FEC 11 CMF 10 Epi+Tax/CMF 5 NA 11 Distant Mts (5yrs) yes 12 no 20 NA 5

Table 4. Histopathological features of TNBC tumours and patients.

Genetic profile of DNA repair genes in TNBC Results

2.2. Selection of interesting variants in tumor tissues

TNBC cancers are extremely heterogeneous; subpopulation of cell clones carrying different mutations may arise during tumour onset and progression due to the high genomic instability. Recent studies evidence that even low frequencies variants in a subset of genes may contribute to tumor behavior and clinical outcome. In order to characterize the main DNA repair pathways from a genetic point of view, 24 genes involved in DNA repair were sequenced in 37 TNBC frozen tumors. More than 2000 variants emerged from the raw analysis by high stringency settings from ION Reporter 4.2, including all the intronic variants and no minimum coverage and quality score. With the aim to find constitutional and somatic mutation the minimum coverage was set at 400X, according to other reports and other studies with gene panel sequencing. This threshold is low enough to include most of variants but high enough to find out very low frequencies variants, significantly and reliably. An additional selection was made: only the exonic variants and canonical splicing site variants were considered. This approach will allow to find those variant more likely to affect protein function. In this way, 1192 exonic variants were obtained: 93 were unique variants (find in one patient) and 76 were found in two or more patients. The variants were distributed in 23 of the 24 genes, as reported in Fig 10. The most variated genes are TP53, BRCA1, BRCA2, CDH1, PARP1. Many variants, especially those found in more patients are common polymorphism or validated variants with neutral impact on the protein function and not associated to cancer. To esclude all the common variants, all the variants with a MAF>1 % in the general population (according to 1000Genomes Project) were excluded by further investigation. From this selection 100 variants were submitted to bioinformatic tools to elucidate their possible pathogenetic role in TNBC progression and clinical response. Furthermore, many of these mutations have already reported as pathogenic or someway related to breast cancer. For each of them a revision of the literature and of the major clinical and genetical database was performed (dbSNP, ClinVar, LOVD, BIC, COSMIC) in order to establish an integrated “Clinical Significance”. In Table 5, all the interesting mutations are listed with SIFT, PolyPhen, PhiloP, Mutation taster scores. The global MAF and the identificative “rs” are reported, when available. After the revision, a putative clinical significance was proposed also on the basis of each variants germline/somatic origin.

Genetic profile of DNA repair genes in TNBC Results

variant number 5

0 TP53B MRE1 MUTY TP53 BRCA1 BRCA2 CDH1 PARP1 BARD1 PMS2 BRIP1 MSH6 NBN MLH1 MSH2 PALB2 RAD50 RAD52 ERCC1 PMS1 CHEK2 PTEN STK11 P1 1A H repetead 5 10 10 2 6 6 5 5 3 7 4 2 1 4 1 1 2 0 1 1 0 0 0 unique 23 9 4 9 4 3 4 3 4 0 3 4 5 2 4 3 1 3 1 1 1 1 1

Fig 10. Distribution of total variants in TNBC. The number of unique ad repeated variations for each gene is reported.

2.3. Overview of the rare and predicted pathogenetic variants in TNBC

tissues

BARD1 c.1670G>C Cys557Ser probable-pathogenetic This variant was found in 3 patients (EL260 50%, T348 50%, T108 20%). ClinVar reported this variants twice, as a risk factor or as benign variant. Karppinen et al. (2004) analyzed the index cases of 126 Finnish cancer families. A cys557-to-ser substitution was seen at elevated frequency in the cancer family patients compared to healthy controls (5.6% vs 1.4%, p = 0.005). The highest prevalence of C557S was found among a subgroup of 94 patients with breast cancer whose family history did not include ovarian cancer. Karppinen concluded that C557S may be a commonly occurring and mainly breast cancer-predisposing allele. Another study (Sauer et al., 2005), defined it as risk factor, since the aa substitution reduced capacity of growth suppression and apoptosis. Recently larger consortia studies have indicated BARD1 c.1670G>C as neutral and not associated at breast cancer risk (Spurdle et al., 2011), except for the Finnish and Iceland population (Stacey et al., 2006). BRCA1 c.5492_5492delC p.P1831Lfs*3 - pathogenetic This variant was found in one patient (EL261, 80%).

Genetic profile of DNA repair genes in TNBC Results

This is a pathogenetic germline variant associated with hereditary breast cancer and it is reported in ClinVar as well as in the BIC database. In the patient tumour LOH was observed. BRCA1 c.5035_5039delCTAAT p.L1679Yfs*2 pathogenetic This variant was found in two patients (T261, 80% - T487, 50%). This is pathogenetic germline variant associated with hereditary breast cancer and it is reported in ClinVar as well as in the BIC database. In the patient tumour LOH was observed. BRCA1 c.4956G>A Met1652Ile probable-benign This variant was found in one patient (T291, 40%). It is reported in ClinVar as benign; dbSNP refers a frequency of MAF= 0,011. Following several functional and association studies the M162I is now considered a Class 1 variant by LOVD database, suggesting its neutral role in breast cancer. BRCA1 c.4183C>T p.Gln1395* pathogenetic This variant was found in one patient (SC519, 82%). This is a validated pathogenetic substitution that leads to a stop codon and described several times in hereditary breast and ovarian cancer families. It’s reported in ClinVar and BIC as pathogenetic. In the patient tumour LOH was observed. BRCA1 c.3228_3229delAG G1077Afs*8 pathogenetic This variant was found in one patient (EL292, 70%). This is apathogenetic germline variant associated with hereditary breast cancer and it is reported in ClinVar as well as in the BIC database. In the patient tumour LOH was observed. BRCA1 c.2889_2890delTG p.G964Tfs*6 pathogenetic This variant was found in one patient (T351, 70%). This is pathogenetic germline variant associated with hereditary breast cancer and it is reported in ClinVar as well as in the BIC database. In the patient tumour LOH was observed. BRCA1 c.4071_4071delA p.E1358Sfs*8 pathogenetic This variant was found in one patient (T109, 50%). This is a rare pathogenetic germline variant associated with hereditary breast cancer and it is reported in ClinVar as well as in the BIC database. BRCA1 c.3024G>A Met1008Ile probable-benign This variant was found in one patient (G41, 50%) in association with other two rare BRCA1 synonymous variants, the c.2733A>G and the c.981A>G. All these three variants are reported as VUS in ClinVar and dbSNP, as their very low frequency in the general population (MAF= 0,001). They are considered Class 1 variants in LOVD database.

Genetic profile of DNA repair genes in TNBC Results

They form a rare aplotype on exon11, but no association with the disease has been clarified by now. BRCA2 c.1856A>T p.Gln619Leu probable-pathogenetic This variant was found in one patient (EL292 10%) and it’s probable a new somatic variation. No info are present in literature, in dbSNP in ClinVar or in LOVD database. It is predicted damaging by SIFT and Grantham score, but not from Polyphen2; MutationTaster suggests a weak role in affecting splicing. It is localized out of the main BRCA2 domains. BRCA2 c.6665A>G .Tyr2222Cys probable-pathogenetic This variant was found in one patient (EL261 40%). It is present in dbSNP with a MAF=0 and with “uncertain significance”. It is reported in ClinVar as a VUS. Previously BRCA2 T2222C has been observed in one case of male breast cancer (Edwards et al., 2003), as it’s reported in LOVD database. BRCA2 c.5718_5719delCT p.Leu1908Argfs pathogenetic This variant was found in one patient (T107, 80%), the only with a pathogenic mutation in BRCA2. This variant is reported by ClinVar and in the BIC database as germline pathogenic. In the patient tumour LOH was observed. BRIP1 c.3459T>C Asp1153Asp probable-benign This variant was found in one patient (EL311 75%) It’s described as benign in dbSNP with a MAF= 0, and never reported in other studies or databases. It’s localized in the C-terminal of BRIP1. BRIP c.2233G>A p.Ala745Thr probable-pathogenetic This variant was identified in one patient (G40, 20%) In ClinVar is reported by two company studies (GeneDx, Ambry genetics) and classified as VUS. In dbSNP is present but no frequency data are available. BRIP1 A745T is predicted pathogenic by SIFT, Poliphen and MutationTaster and is located in one of the helicase domains of BRIP1. This variant is likely to be a somatic variant since it is reported in COSMIC, in three tumour samples. BRIP c.577G>A p.Val193Ile probable-benign This variant was found in one patient (T107, 45%) ClinVar described it in a conflicting way since different studies have reported it as VUS/likely benign/benign. Wong et al. (Wong et al., 2011) found this variant in one family with strong family history but they classified this variant as probable neutral. dbSNP classified this variant as “other”. In the Tuscany population has a MAF=0,5%, a little higher than the general population. It is predicted neutral by SIFT and Polyphen.

Genetic profile of DNA repair genes in TNBC Results

BRIP1 c.139C>G p.Pro47Ala pathogenetic This variant was found in one patient (T261, 15%) ClinVar reported this variant as pathologic germline variant on the basis of the paper of Cantor. His work identified a heterozygous c.139C>G the BRIP1 gene, resulting in a P47A mutation, in an individual with early-onset breast cancer and a family history of breast and ovarian cancer. This mutation occurred in the helicase domain of the BRIP1 protein and was not found in 200 controls. It is not present in COSMIC database, and it seems never reported as somatic mutation. It is predicted pathogenic by all the bioinformatic tools. CDH1 c.268C>T p.Arg90Trp probable-pathogenetic This variant was found in one patient (T487, at 3,5%). This variant is not reported in ClinVar or dbSNp databases, but it was previously identified in one patient by one recent work of Fang et al. (Fang et al., 2013). It is predicted damaging by SIFT and Grantham score, but not from Polyphen2; MutationTaster suggest a weak role in affecting splicing. CDH1 c.1774G>A p.Ala592Thr probable-pathogenetic This variant was found in one patient (T104, 32%) In Clinvar is reported twice as VUS and likely benign; dbSNP assigned it a MAF=0,001. This variant has been previously identified as a somatic mutation in thyroid tumours and also at the germinal level in colon and lobular breast cancers (Boyault et al., 2012). One recent work performing structural analysis, suggests a non-pathogenic role of this variant as already confirmed by in vitro (Keller et al., 2004) and in silico studies (Suriano, Seixas, Rocha, & Seruca, 2006). In contrast to all these data, SIFT analysis suggest a damaging role for this variant. Also a paper described it as a known germline variant that is not associated with risk of familial breast cancer or HDGC. CDH1 c.1897G>T p.Gly633Trp probable-pathogenetic (new) This variant was found in one patient (T351, 3.5%). No informations are present in dbSNP or ClinVar. SIFT, Poliphen and MutationTaster scores defined it as damaging variant. This substitutions is located in the extracellular domain. CDH1 c.1906G>A p.Ala636Thr probable-benign This variant was found in one patient (T261, 30%). No informations are present in dbSNP or ClinVar. SIFT, Poliphen and MutationTaster scores defined it as neutral variant. This substitutions is located in the extracellular domain. CDH1 c.2004G>C p.Lys668Asn probable-pathogenetic (new) This variant was found in one patient (G26, 15%).

Genetic profile of DNA repair genes in TNBC Results

No informations are present in dbSNP or ClinVar. Polyphen score suggests for this variant a weak effect on the protein; also for MutationTaster this substitution may lead to defect in splicing. This substitutions is located in the extracellular domain. CDH1 c.2371C>T p.Leu791Phe probable-pathogenetic (new) This variant was found in one patient (T109, 50%). No informations are present in dbSNP or ClinVar. It is predicted probable-damaging by SIFT and MutationTaster, but not by Polyphen. This substitutions is located in the cytoplasmic domain. CDH1 c.594_595insA p.Thr199fs pathogenetic This variant was found in one patient (SC368, 20%). This insertion leads to a frameshift in the coding sequence and a premature stop after 10 aa. The mutated protein is 208aa in length, versus the 883aa of the full length. Most of the extracellular and citoplasmic domains are lost. However this altered transcript may be subjected to NMD. ERCC1 c.346G>A p.Val116Met probable-pathogenetic This variant was found in one patient (EL344, 50%). It is reported in dbSNP but with no frequency data, and is not in ClinVar database. This substitution is predicted pathogenic by SIFT and Polyphen, and is localized in the central domain. MLH1 c.1852_1853delinsGC p.K618A probable-pathogenetic This variant was found in one patient (G26, 80%) Clinvar shows many submission for this variant, concluding for a benign/likely benign significance. For dbSNP is classified as VUS. SIFT and Polyphen predicts it as deleterious. This variant in the MLH1 gene corresponds to an example of VUS in Lynch syndrome. When consulting the Leiden Open Variation Database (LOVD v.2.0), there are 120 entries for this variant. Available past studies reached contradictory conclusions about its pathogenicity reporting strong in silico predictions, absence of splicing or mRNA alteration (Tournier et al., 2008), presence in patients with a defective MMR tumor, co- occurrence with clearly pathogenic MMR mutations, apparent segregation with disease, and a majority of non-altered in vitro functional studies (Abuli et al., 2014). All previous data permitted to categorize it in LOVD as a class 1 variant. Therefore, it should be considered as a neutral variant in terms of its implication with Lynch syndrome. A recent work of screening of 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) described no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome. (Abuli et al., 2014).

Genetic profile of DNA repair genes in TNBC Results

MLH1 c.1360G>C p.Gly454Arg probable-pathogenetic This variant was found in one patient (G17, 50%). In ClinVar database is classified as VUS by recent submitters, while no info are present in dbSNP. LOVD database for MLH1 reports this variant two times and classified it as Class 3. Recently Loizidou et al. (Loizidou et al., 2014) have reported this mutation in a patient with two CRC tumours. MLH1 G454R is predicted deleterious by SIFT, MutationTaster and Grantham scores but not by Polyphen. MLH1 c.1959G>T p.693 probable-benign This variant was found in one patient (SC368, 70%). ClinVar reports this variant as benign; LOVD database described it as of Class 1. It has been reported as somatic variant. Mutation taster score it as slightly affecting splicing. MRE11 c.305G>T p.Gly102Val probable-pathogenetic This variant was found in one patient (EL311, 68%). It is reported once as VUS in ClinVar, no frequency data are present in dbSNP. It is predicted pathogenetic by Polyphen, MutationTaster and Grantham score. This mutation falls out of the main interaction domains. MSH2 c.1680T>C p. Asn560Asn probable-benign This variant was found in one patient (EL261, 50%). According to ClinVar this variant is benign/likely benign; in the LOVD database is a Class 2 variant. No frequency data are available. A minigene assay suggest a modest effect in splicing (Tournier et al., 2008). MSH2 c.199A>G p.Met67Val probable-pathogenetic (new) This variant was found in two patients (EL318 at 3%, EL346 at 3%) No info are reported in Clinvar, dbSNP and literature studies. It is predicted deleterious by SIFT and MutationTaster. This variant in the other reference gene (NM_001258281.1 ) for MSH2 is located in the first position of cds c.1A>G and result in skipping the first 16aa. MSH2 c.965G>A G322D probable-benign This variant was found in one patient (T107-T297-EL137 50%) In Clinvar is reported several times as benign; for the LOVD database is Class 1. For some authors, G322D is a common polymorphism of the MSH2 gene and not a disease-causing mutation. They found this exon 6 mutation in 9 of 170 colorectal cancer patients (5.3%) from high-risk families, and in 6 of those families this alteration was shown not to segregate with disease. They also found this alteration in 12 of 192 normal controls (6.3%) and in none of 104 sporadic colorectal cancer cases. Barnetson (Barnetson et al.,2008) screened for germline mutations in MLH1, MSH2, and MSH6 in 110 families finding a possible protective effect of three variants including the MSH2 c.965G>A.

Genetic profile of DNA repair genes in TNBC Results

MSH2 c.984C>T probable-benign This variant was found in one patient (T291, 50%) According to Clinvar is benign; a recent paper (Duraturo 2013) found that all subjects with the Lynch phenotype showed the c.984T allele of MSH2 and a germ-line variant in the MSH3 gene suggesting a cooperative role for variant in Msh3 in association with a weak mutation in the major MMR genes (MSH2 or MLH1) in tumorigenesis. MSH2 c.2264C>T p.Ser755Phe probable-pathogenetic This variant was found in one patient (T105 10%) No frequency data are available in dbSNP or ClinVar. This mutation is predicted pathogenetic by all the bioinformatic tools, since it is localized in the ATP-ase domain of MSH2 protein. MSH2 c.1666T>C p.Leu556Leu probable-benign This variant was found in one patient (SCxx, 45%) It is reported in Clinvar as benign and in dbSNP with a rare frequency. For the LOVD database is a Class 1 variant. This synonymous variant is located in the second residue of exon11 and it is predict to affect splicing by MutationTaster, even if two different studies showed no aberrant splicing detected in minigene assays (Auclaire 2006, Tournier 2008). MSH6 c.2511C>T synonimous This variant was found in one patient (G40 3,5%) No info are reported in Clinvar, dbSNP and literature studies. It seems a very low frequency somatic variant. MSH6 c.2633T>C p.Val878Ala probable-pathogenetic This variant was found in two patients (T107- T108, 50%) ClinVar reports this variant several times with contradictory assignment: benign, VUS, pathogenetic. Also in dbSNP is described as VUS/pathogenic. The LOVD database defines this mutation in Class 1. In the OMIM database is reported by Wu et al. (2001) in a HPNCC family. An in vitro study reported a reduced functionality of the V787A protein in comparison with the WT(Cyr and Heinen 2008) and an alteration in ATP-dependent conformation changes of hMSH2-hMSH6 complex. NBN c.628G>T p.Val210Phe probable-pathogenetic This variant was found in one patient (G43, 25%). It is described in dbSNP as a VUS and it has been observed as somatic also. ClinVar has few submission for this variant and classified it as VUS. It is predicted deleterious by SIFT and is localized in the Tandem BRCT linker region. NBN c.284A>G p.Asp95Gly probable-pathogenetic This variant was found in one patient (G44 3%).

Genetic profile of DNA repair genes in TNBC Results

It is reported in dbSNP with no frequency data and in ClinVar as a VUS. It is predicted deleterious by all the software and is localized in the FHA domain, responsible of focus formation and ATM activation. NBN c.1417C>A p.Gln473Lys probable-benign This variant was found in one patient (T105, 75%). No info are available for this missense, located in a structural domain of NBN. However none of the informatics tools predict it as deleterious. PALB2 c.2993G>A p.Gly998Glu probable-pathogenetic This variant was found in three patient (EL137 - EL344 - G26, 50%). This variant is classified as VUS by dbSNP and benign by ClinVar; it is predicted deleterious by all the bioinformatic tools (Teo et al., 2013; Blancoet al., 2013) but is not associated with breast cancer risk. Moreover has been observed that it is relatively common both in patients with breast cancer and in healthy controls (Bogdanova, Sokolenko et al. 2011). In the LOVD database is reported as probable not pathogenic. It is localized in the WD40 domains of interaction with BRCA2/RAD51. PALB2 c.1272C>T Ala424Ala probable-benign (new) This variant was found in one patient (EL102, 4%). No info are available for this silent mutation. PALB2 c.1010T>C p.Leu337Ser probable-benign This variant was found in one patient (EL346, 60%) Clinvar has several submission for this variant and classified it as VUS, or more often as benign variant. In dbSNP is reported as “other”. The frequency in 1000Genomes for the Tuscany population is slightly higher than in general population (MAF=0,014 vs MAF=0,006).Two recent studies (same references of G998E) evidenced no association with breast cancer risk. PARP1 c.942C>T Asp313Asp and c.339T>C p. F10107F probable-benign This two novel variants were found in the same patient (EL137, 50%). They are never reported in literature; are registred in dbSNP without any clinical informations. PARP1 c.115c>g p.Val39Met probable-pathogenetic This variant was found in two patients (T348, 50% - T106 70%). It is registered in dbSNP with no frequency data, and is not present in ClinVar. It is predicted deleterious by SIFT, Polyphen and MutationTaster. This missense substitution is located in the third-to-last residue of exon 1, suggesting a putative role in the splicing process. Moreover the residue 39 falls in the zinc finger domain, which participate to DNA binding.

Genetic profile of DNA repair genes in TNBC Results

PARP1 c.2919C>T p.Thr973Thr probable-benign This variant was found in one patient (T161, 40%). No frequency or functional information are present in ClinVar, dbSNP or literature. PARP1 c.2819A>G p.Lys940Arg probable-pathogenetic This variant was found in one patient (T348, 50%). This variant is reported in dbSNP with a MAF=0,027; in 1000Genome Project population frequencies shows that in Europe is less frequent (MAF=0) It is not present in ClinVar. SIFT and Polyphen prediction classified this missense as deleterious; the interested residue is located in the catalitic domain of PARP1 in the very last residues. One work (Cao et al., 2007) reported to have found 20 rare genetic variants of PARP1 in nine (10.8%) breast cancers of French patients. Among them the Lys940Arg was identified in the cases and not in the controls. PARP1 c.2124 p.Ala708Ala probable-benign Thi variant was found in one patient (EL329, 70%) It is not reported in ClinVar; in dbSNP is considered likely benign PARP1 c.2285T>C p.Val672Ala probable-pathogenetic This variant was found in 9 patient (EL102-EL163-EL253-EL261-EL292-T261-T346, 50% ; G72, 100%). Even if is not reported in ClinVar, this mutation has been object of many functional and association studies. A recent meta-analysis (Hua et al., 2014) on this variant and its cancer involvement report very discordant results. The Val762Ala polymorphism located within the COOH-terminal catalytic domain is associated with deficient poly (ADP-ribosyl)ation activity, which may impede DNA repair capacity of the BER, and thereby cause genome instability (Lockett 2004). Previously, some investigations demonstrated that genetic alteration of the PARP1 Val762Ala can modulate cancer susceptibility, and that the frequency of the Ala/Ala genotype was significantly higher in patients when compared with controls (Zhang 2008, Li 2013) Nevertheless, the association of Ala variants and cancer risk was not validated by others (Smith 2008, Zhang 2012, Landi 2006). PARP1 Val762Ala T>C polymorphism located in the catalytic domain has been shown to interact with XRCC1. In vitro, this polymorphism markedly reduces the enzymatic activity of PARP1, and has also been linked to cancer susceptibility (Wang 2007). Recently Alanazi et al. (2013) showed for the first time a significant association between the PARP- 1 Val762Ala genotype and increased risk of breast carcinoma in Saudi patients and suggested that PARP-1 Val762Ala may modulate the occurrence of other mutations and contribute to breast carcinogenesis.

Genetic profile of DNA repair genes in TNBC Results

PMS1 c.1609G>A p.Glu537Lys probable-pathogenetic This variant was found in one patient (EL329 50%). Clinvar reports only one submission and does not provide clinical significance; dbSNP indicates this variant as VUS, with a very rare frequency (MAF=0,05 in Tuscany population). It is predicted by Polyphen and MutationTaster PMS1 c.1706G>A p.Arg569Gln probable-pathogenetic This variant was found in one patient (T261, 20%). Neither dbSNP or ClinVar report this missense mutation. It is predicted deleterious by SIFT, Polyphen and MutationTaster. This residue is localized in the linker domain an may interfere with protein folding. PTEN c.855A>G p. Glu285Glu probable-benign (new) This variant was found in one patient (G41, 50%) No information about this synonimous variant are in ClinVar or dbSNP. RAD50 c.280A>C p.Ile94Leu probable-pathogenetic This variant was found in one patient (T348,50%). According to ClinVar is benign, while dbSNP describes it as “other”. It is reported in the LOVD database for RAD50, but always in combination with other variants. The first paper reporting this variation (Heikkinen et al., 2003) described it in 1.3% (2/151) affected individuals and in 0.3% (3/1000) controls.Isoleucin94 is a well conserved residue in the aminoterminal ATPase. Interestingly, one of the breast cancer families displaying the RAD50 Ile94Leu variant also showed partial co-segregation with the previously identified CHEK2 Ile157Thr unknown variant. RAD50 c.695C>A p.Ala232Asp probable-pathogenetic This variant was found in one patient (T487, 50%). This variant is not present in ClinVar and no frequency data are reported in dbSNP. The LOVD database reports this variant in association with others in a work of Moisor (Moisor et al, 2010), which found the A232D in 1/280 patients and in 0/328 controls. It is predicted deleterious by SIFT, Grantham score and MutationTaster. RAD50 c.1094G>A p.Arg365Gln probable-benign This variant was found in one patient (EL344, 50%). It is reported in ClinVar and dbSNP as a VUS; no frequency data are available. The prediction tools indicate that this substitution is tolerated; MutationTaster suggest a weak role in splicing. RAD50 c.3168A>G p. Glu1056EGlu probable-benign This variant was found in two patients (T105, 86% - G26 , 70%). No functional and clinical informations are reported in dbSNP or ClinVar. This residue is located in the Coiled Coil domain.

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Genetic profile of DNA repair genes in TNBC Results

RAD52 c.1186C>T p.Arg396Cys probable-pathogenetic This variant was found in one patient (G41, 50%). It is reported in dbSNP as “unknown” and is not described in ClinVar. In Tuscany population has been found with a MAF=0,009. All the prediction tools suggest a pathogenetic role for the R396C. TP53BP1 c.3077 p.Val1026Ala probable-pathogenetic This variant was found in one patient (EL 137, 45%). It is reported in dbSNP as “unknown” and it’s not present in ClinVar. In the Tuscany population is more frequent than in the general population (MAF=0,014 vs MAF=0,006). It is predicted deleterious by SIFT, Polyphen and MutationTaster. Val1026 is the third-to-last residue of the exon 15. TP53BP1 c.1546A>T p.Met516Leu probable-benign This variant was found in one patient (EL329, 70%) Although is present in dbSNP, no frequency data are available and no ClinVar definition is made. The main prediction tools define it as tolerated; only MutationTaster suggest a weak role in splicing process. TP53BP1 c.1347_1352delTATCCC p.Ile450Ser probable-benign This variant was found in one patient (G41,50%). This rare variant was first observed by (Frank et al., 2005); this inframe deletion causes the loss of an isoleucine and a proline residue at positions 450 and 451. In this study, comparing the occurrence of this rare 6 bp deletion between cases and controls an inverse association (not statistically significant) with breast cancer risk was suggested. TP53 variants For the mutations in TP53, the TP53 IARC database was consulted in order to investigate the origin (germline or somatic) of the variation and the functional impact. Among the total number of TP53 variations, 19 were found in the IARC database: some of these are validated as known pathogenetic variants. In Table 6 these variants are described reporting the following info: if the variants affects splicesite or CpG site; the function of the residue and the domain; the functional impact according to AGVGD and Transactivation assay; the Somatic, Germline and Cell line counts. Other 8 variants, which are small deletion, are not reported in IARC database and are submitted to MutationTaster prediction to evaluate the effect of the mutated protein. TP53 c.415_471del57 p.Lys139Arg This variant was found in one patient (EL253)

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Genetic profile of DNA repair genes in TNBC Results

This inframe deletion has never been described before; it leads to the loss of 19 aa from the Lys139 to the Valine in position 157. This loss in exon 5, strongly alters the DNA binding domain. TP53 c.322_325del4 p.G108Sfs*14 This variant was found in one patient (T107, 30%). It’s a frameshift deletion that leads to a truncated protein of 121 aa versus 394 of the TP53 full length. No other information are reported in IARC database. TP53 c.328_331del4 p.R110Wfs*12 This variant was found in one patient (T109, 15%). It’s a frameshift deletion that leads to a truncated protein of 121 aa versus 394 of the TP53 full length. No other information are reported in IARC database. TP53 c.454_466del13 p.Pro152fs This variant was found in one patient (G58, 60%). It’s a frameshift deletion that leads to a truncated protein. The IARC database report this variation 7 times, always somatic. TP53 c.608_609del2 p.V203Gfs*5 This mutation was found in one patient (EL163, 20%). It’s a frameshift deletion that leads to a truncated protein of 207 aa versus 394 of the TP53 full length. This variation is reported once in IARC database and no other info are available. TP53 c.617_617del1 p.L206Wfs*41 This mutation was found in one patient (G17, 43%). It’s a frameshift deletion that leads to a truncated protein of 246 aa versus 394 of the TP53 full length. The IARC database reports this variant 5 times, in somatic origin. TP53 c.652_652del1 p.V218Cfs*29 This mutation was found in one patient (EL311, 43%). It’s a frameshift deletion that leads to a truncated protein of 246 aa versus 394 of the TP53 full length. This variation is reported once in IARC database and no other info are available. TP53 c.365_366del2G p.V122Dfs*26 This mutation was found in one patient (EL102, 35%). It’s a frameshift deletion that leads to a truncated protein of 147 aa versus 394 of the TP53 full length. The IARC database reports this variant 4 times, as somatic mutation.

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Genetic profile of DNA repair genes in TNBC Results

pathogenic

pathogenetic

My significance My

probable-benign

probable-pathogenic

probable-pathogenetic

- -

dbSNP

rs4988346

rs4987050

rs1800740

rs1800704

rs1799967

rs80357582

rs35187787

rs28903098

rs80357890

rs80357635

rs80357260

rs28997576

rs587780235

rs397507875

MutationTaster

splicingaffected; affectedfeatures protein

protein features affectedfeatures protein

splicingaffected; affectedfeatures protein

protein features affectedfeatures protein

splicingaffected; affectedfeatures protein

protein features affectedfeatures protein

splicingaffected; affectedfeatures protein

- -

-0,5

-1,00

-1,66

-0,45

-4,05

-2,65

-1,94

-6,46

-0,26

-1,87

-1,48

-36,0

-2,59

PhyloP

- -

94,0

58,0

27,0

29,0

58,0

10,0

184,0

101,0

194,0

113,0

112,0

score

Grantham

- -

1,0

0,0

1,0

0,0

0,17

0,99

0,01

0,02

0,14

0,04

0,071

0,718

0,592

score

Plyphen2

- -

0,1

0,4

0,3

0,05

0,44

0,01

0,04

0,07

SIFT

0,571

0,055

0,911

0,178

0,004

score

- -

0,001

0,009

0,007

GMAF

0,0001

Protein

p.Thr199fs

p.Pro47Ala

p.Arg90Trp

p.Val193Ile

p.Gln1395*

p.Ala636Thr

p.Ala592Thr

p.Ala745Thr

p.Glu1358fs

p.Gly964Thr

p.Gly633Trp

p.Gln619Leu

p.Lys668Asn

p.Met1008Ile

p.Met1652Ile

p.Cys557Ser

p.Leu791Phe

p.Gly1077Ala

p.Leu1679Tyr

p.Tyr2222Cys

p.Leu1908Arg

p.Pro1831Leu

Coding

c.268C>T

c.577G>A

c.981A>G

c.139C>G

c.1856A>T

c.2371C>T

c.1897G>T

c.3459T>C

c.4183C>T

c.2004G>C

c.1906G>A

c.1774G>A

c.2233G>A

c.6665A>G

c.2733A>G

c.3024G>A

c.4956G>A

c.1670G>C

c.594_595insA

c.4071_4071delA

c.5492_5492delC

c.5718_5719delCT

c.2889_2890delTG

c.3228_3229delAG

c.5035_5039delCTAAT

Exon

missense

Function

nonsense

synonymous

frameshiftDeletion

frameshiftInsertion

G26

G40

G41

T109

T261

T351

T104

T487

T261

T107

T109

T351

T291

E261

sc368

EL311

EL261

EL292

SC519

#patient

T261-T487

EL260-348-T108

CDH1

BRIP1 BRIP1

BRCA2 BRCA2

BRCA1 BRCA1

BARD1 BARD1

GeneID

NM_004360.3

NM_032043.2

NM_000059.3

NM_007294.3 NM_000465.2

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Genetic profile of DNA repair genes in TNBC Results

My significance My

probable-benign

probable-pathogenic

probable-pathogenetic

- -

dbSNP

rs2020912

rs4987189

rs4987188

rs1800146

rs45494092

rs45551636

rs61754796

rs61756466

rs63750527

rs35502531

rs200056411

rs199736271

rs200459924

MutationTaster

splicingaffected; affectedfeatures protein

protein features affectedfeatures protein

affected,firstmissing 16 aa

splicingaffected; features protein

splicingaffected; affectedfeatures protein

- -

-1,60

-6,22

-0,34

-6,70

-1,90

-5,60

-1,97

-0,92

-1,49

-1,92

PhyloP

- -

98,0

2,21

94,0

50,0

64,0

6,45

94,0

21,0

145,0

109,0

125,0

score

Grantham

- -

1,0

0,0

1,0

0,29

0,017

0,403

0,034

0,433

0,788

0,975

score

Plyphen2

- -

0,5

0,3

0,0

0,12

0,03

0,01

0,12

0,18

0,14

0,02

0,01

SIFT

0,006

score

- -

0,006

0,008

0,001

0,004

0,002

0,008

0,006

0,002

0,004

GMAF

Protein

p.Asp95Gly

p.Val878Ala

p.Gly102Val

p.Gly998Glu

p.Gly454Arg

p.Val116Met

p.Gln473Lys

p.Leu337Ser

p.Val210Phe

p.Gly322Asp

p.Ser755Phe

p.Met67Val|p.Met1Val

p.Lys618Thr,p.Lys618Ala

Coding

c.984C>T

c.284A>G

c.628G>T

c.965G>A

c.305G>T

c.346G>A

c.1010T>C

c.1272C>T

c.1417C>A

c.2633T>C

c.2511C>T

c.1666T>C

c.2264C>T

c.1680T>C

c.1959G>T

c.2993G>A

c.1360G>C

c.199A>G|c.1A>G

c.1852_1853dellinsGC

1|2

Exon

missense

Function

synonymous

missense,missense

T43

T17

G44

G40

G26

T105

T291

scXX

sc368

EL346

EL102

EL261

EL311

EL344

#patient

T107-T108

EL318EL346

T107-T297-EL137

EL137EL344 - G26 -

NBN NBN

MSH6

MSH2

MLH1

PALB2

ERCC1

GeneID

MRE11A

NM_024675.3

NM_002485.4

NM_000179.2

NM_000251.2

NM_005590.3

NM_000249.3 NM_001983.3

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Genetic profile of DNA repair genes in TNBC Results

My significance My

probable-benign

probable-pathogenic

probable-pathogenetic

- -

dbSNP

rs1136410

rs3219145

rs56354945

rs45482998

rs28903089

rs28903085

rs61735401

rs61750986

rs61750984

rs145031495

rs112677599

rs146370443

rs151325573

rs142025196

MutationTaster

splicingaffected; affectedfeatures protein

splicingaffected

protein features affectedfeatures protein

splicingaffected; affectedfeatures protein

- -

-1,0

0,17

-7,53

-1,64

-1,84

-0,17

-2,40

-0,88

-1,50

-1,33

-2,86

-2,14

PhyloP

- -

5,0

15,0

64,0

43,0

56,0

64,0

26,0

21,0

180,0

126,0

score

Grantham

- -

0,0

0,01

0,99

0,013

0,018

0,997

0,893

0,728

0,884

score

Plyphen2

- -

0,1

0,24

0,03

0,28

0,03

0,06

0,16

0,17

0,02

SIFT

0,006

score

- -

0,02

0,19

0,006

0,001

0,004

0,001

0,004

0,027

0,001

0,004

GMAF

0,0003

Protein

p.Ile94Leu

p.Val39Met

p.Ile450Ser

p.Val762Ala

p.Arg365Gln

p.Arg569Gln

p.Glu537Lys

p.Lys940Arg

p.Ala232Asp

p.Met516Leu

p.Arg396Cys

p.Val1026Ala

Coding

c.339T>C

c.942C>T

c.264C>A

c.695C>A

c.280A>C

c.855A>G

c.115G>A

c.1546A>T

c.3077T>C

c.1186C>T

c.2124C>T

c.2285T>C

c.2919C>T

c.3168A>G

c.1094G>A

c.1706G>A

c.1609G>A

c.2819A>G

|c.1347_1352delTATCCC

Exon

missense

Function

synonymous

nonframeshiftDeletion

G41

T487

T348

T261

T348

T161

sc519

EL329

EL137

EL344

EL329

EL137

#patient

G26-T105

T261-T346

T348,T106

EL261-EL292-G72-

EL102-EL163-EL253-

PTEN

PMS1

STK11

RAD52 RAD52

RAD50 RAD50

PARP1 PARP1

GeneID

TP53BP1

NM_000455.4

NM_005657.2

NM_134424.2

NM_005732.3

NM_000314.4

NM_000534.4 NM_001618.3

105

Genetic profile of DNA repair genes in TNBC Results

My significance My

- -

dbSNP

rs1800372

rs28934576

rs11540654

rs11540652

rs28934574

rs11575997

rs28934578

rs142813240

rs121912666

rs121913343

MutationTaster

splicingaffected; affectedfeatures protein

protein features affectedfeatures protein

splicingaffected; affectedfeatures protein

- -

-4,78

-1,92

-8,48

-5,60

-3,89

-8,59

-2,00

-2,93

-3,92

-7,65

-4,82

-4,87

PhyloP

- -

1,21

2,33

5,04

56,0

45,0

58,0

43,0

29,0

194,0

180,0

101,0

180,0

score

Grantham

- -

1,0

0,65

0,803

0,999

0,697

score

Plyphen2

- -

0,0

0,04

0,01

SIFT

score

- -

0,002

0,007

GMAF

Protein

p.Trp146*

p.Gln192*

p.Arg110fs

p.Pro152fs

p.Val218Glu

p.Val203Gly

p.Arg273His

G108Sfs*14

p.Ser241Thr

p.Arg175His

p.Lys319Glu

p.Cys141Tyr

p.Lys132Glu

p.Tyr220Cys

p.Arg248Gln

p.Arg282Trp

p.Val218Cys

p.Lys139Arg

p.Val122Asp

p.Leu206Trp

p.Glu224Asp

p.Arg273Cys

p.Arg337Cys

Coding

c.653T>A

c.721T>A

c.817C>T

c.844C>T

c.574C>T

c.818G>A

c.955A>G

c.422G>A

c.394A>G

c.438G>A

c.639A>G

c.659A>G

c.743G>A

c.524G>A

c.672G>C

c.1009C>T

c.376-1G>A

c.993+1G>A

c.672+1G>A

c.617_617delT

c.652_652delG

c.454_466del13

c.415_471del57

c.365_366delTG

c.608_609delTG

c.328_332delCGTC

c.322_325delGGTT

Exon

spicesite

splicesite

missense

Function

nonsense

synonymous

frameshiftDeletion

nonframeshiftDeletion

T17

T43

G58

G40

G72

T108

T291

T105

T108

T106

T109

T107

T261

T351

T161

T291

sc368

sc519

EL102

EL344

EL318

EL163

EL346

EL261

EL311

EL260

EL253

#patient

EL260-EL346-T17-

EL329-T487 (male)

TP53

GeneID NM_000546.5

Table 5. Complete list of selected variants found in TNBC tissues. SIFT, PolyPhen, MutationTaster, PhiloP prediction score and 1000g MAF are reported. The variants are classified by the integration of prediction tools scores and literature data.

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Genetic profile of DNA repair genes in TNBC Results

114

CellLine

Germline

577

851

706

933

396

111

103

1210

Somatic

Class

functional

non-functional

Transactivation

partiallyfunctional

deleterious

AGVGDClass

NLS

Domain

DNA bindingDNA

Tetramerisation

Buried

Exposed

DNA bindingDNA

Tetramerisation

Partiallyexposed

Residue_function

yes

site

CpG CpG

Splice_site

consensusSD

consensusSA

p.S241T

p.R337C

p.K319E

p.R273C

p.E224D

p.Y220C

p.V218E

p.C141Y

p.K132E

p.R273H

p.R248Q

p.Q192*

p.R175H

p.R282W

p.W146*

c.844C>T

c.817C>T

c.721T>A

c.653T>A

c.574C>T

c.955A>G

c.818G>A

c.743G>A

c.672G>C

c.659A>G

c.524G>A

c.438G>A

c.422G>A

c.394A>G

c.1009C>T

c.782+1G>T

c.376-1G>A

c.993+1G>A

c.672+1G>A

C>T

G>T

T>A

G>C

T>A

G>A

A>G

G>A

A>G

G>A

A>G

Type

9-exon

8-exon

7-exon

6-exon

5-exon

Ex/ Ex/ Int

9-intron

7-intron

6-intron

4-intron

10-exon

T108, 4% T108,

G72, 30% G72,

G40, 27% G40,

EL344, 6% EL344,

G43, 65% 65% G43,

T291, 15% T291,

T105, 50% T105,

T161, 20% T161,

T351, 20% T351,

T261, 50% T261,

T487, 50% T487,

T106, 73% T106,

T108, 45% T108,

Pat/ Freq

EL261, 60% EL261,

EL346, 30% EL346,

EL260, 43% EL260,

EL329, 70% EL329,

EL318, 25% EL318,

SC368, 15% SC368, SC519, 50% SC519,

Table 6. TP53 variants and IARC database significance.

107

Genetic profile of DNA repair genes in TNBC Results

2.4. Pathways and genes mutated in TNBC

DNA repair pathways integrity is critical to prevent tumorigenesis and is well known that different pathways may contribute in resolving the same DNA damage in a redundant and cooperative mechanism. When for example the HR is impaired due to mutation in BRCA genes, DNA DSBs are fixed by NHEJ error-prone system. Moreover BRCA1-mutated cells, with an additional deregulation of TP53BP1, can restore HR ability and Rad52 foci formation. Therefore it is very important to understand which genes and which pathways are more deregulated, even at genomic level, in TNBC. A tumor with several mutations in different DNA repair genes may acquire an extra genomic instability and therefore grow in aggressiveness. From the analysis of this 37 TNBC emerged that rare interesting variation are distributed on 19 out 24 genes. No mutation were found in CHEK2, STK11, PMS2, MUTYH, RAD51c. The first two genes are highly penetrant susceptibility genes, responsible for a very small slice of the familial cancer. The tumors analyzed in this study are not selected for family history, and so maybe these two genes are not emerged as important. The other two genes are mainly involved in MMR and as DNA damage sensor and are frequently mutated in colorectal cancers. Surprisingly no alterations were found on RAD51c, one of the new susceptibility genes for breast cancer and major responsible of foci formation. In the next section the mutations will be described by genes, grouped by pathway as showed in Fig 11 and Table 7.

2.4.1. Homologous recombination genes

HR, which was found here very mutated, is the most important pathways in DSBs repair. Interestingly BRCA1 was the more mutated gene with 7 patient carrying a deleterious germline mutation. This data evidenced that 16% of this little cohort of TNBC unselected for family history are BRCA1-related. This data is in line with recent literature and huge TNBC analysis, suggesting a BRCA1-driven tumorigenesis for this subtype of cancer. Two other patients were carrier of a VUS, a variant of unknown clinical significance even if, in this case, more likely to be benign. BRCA2 deleterious mutation were found in one patient (3%) and two patient are carriers of predicted missense mutations. BRCA2 is relatively related to TNBC biology, and BRCA2 mutation and rearrangement are not frequent in TN. BRIP1, which directly interacts with BRCA1 in HR, is interested by 1 pathogenetic missense mutation, 2 predicted missense mutation, rare or never reported, and a new synonymous variant. This data suggest that BRIP1 may be involved in TN progression or

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Genetic profile of DNA repair genes in TNBC Results

may modulate the effect of BRCA1 mutation. In one patient a BRCA1 truncating mutation and a pathogenetic germline missense on BRIP1 are present. Interestingly three patients were found mutated in BARD1, with a known yet discussed missense mutation in the BRCT domain of BARD1. The C557S may be not predispose di per se to breast cancerogenesis, but may contribute to some tumor features or to a TN phenotype. This variant was found in 10% of this TNBC cohort. PALB2 harbour only three alterations in sequences, 2 predicted missense and 1 new synonymous substitution. One of the missense is a well-known variant largely discussed, which is found in three patients here. It is a germline variant probable non associated with breast cancer risk but in the domain of interaction with BRCA2. A single predicted missense variant was found in RAD52 also.

2.4.2. Mismatch Repair genes

The genes of MMR are frequently found mutated in several type of cancers. In this study all the MMR genes, except PMS2, present at least two predicted mutation. MLH1 are interested by two missense mutations and a rare synonymous variant. One of this variant was found also in the screening for germline mutations in the neoadjuvant treated patients. Three interesting (two predicted) and rare missense and two rare silent substitutions were found in MSH2. One of the missense was previously reported in germline screening: in the tumor screening was found other 3 times. The frequency in this group of TNBC patients is significantly higher than that of the general population as it is reported in 1000G. This variant is worth to be further investigated in a larger cohort of TN. MSH6 was found to carry one missense and one synonymous never described before. The missense variant is controversy described in literature and seems to have an effect on MSH6 activity; it is here reported in two patients both mutated in BRCA genes yet. Two patients are mutated in PMS1, one with a germline predicted missense mutation and the other with a novel somatic mutation predicted pathogenetic. Interestingly the first patient have only this PMS1 germline variants, while the somatic variant is harboured by one of the most mutated patient. This may explain a constitutional role for the first variant and an acquired occurrence in tumor progression for the other.

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Genetic profile of DNA repair genes in TNBC Results

2.4.3. Non Homologous End Joining genes

The MRN complex has recently been rediscovered since the evidence that mutations in RAD50 may predispose to breast cancer. Moreover the MRN complex is the alternative to HR in repairing DSBs in HR-deficient tumors. In this little group of TNBC MRE11 was found mutated only in one patient: this woman is carrier of a predicted germline missense. Three rare missense variants were found on NBN: two predicted somatic variants and one probable-benign germline variant. However is RAD50 the most interesting gene of this complex: three very rare germline missense mutations (two of them predicted) and a new synonymous variant were found. This last was found in two patient. Overall all these genes carry mutations to further investigate, although none of them is clearly suggestive of pathogeneticity.

2.4.4. Other pathways

Only one germline missense mutation was found in ERCC1, involved in the resolution of the nucleotide excision repair. PARP1, on the other hand, is often mutated in this group of TNBC: two predicted pathogenic missense and a new somatic silent variant were found. One of the missense was found in two patients, one of them with LOH in tumor tissue and it is worth to indagate soon. Interestingly the Val762Ala, one of the most studied PARP1 varants, was found in 8 patient with a frequency slightly higher than that of the general population (22% vs 17%) Loss of PTEN function was found to be one of the most important somatic event in breast tumor progression and development: in this study only one new germline silent mutation was found in PTEN. However this gene is more often interested with large rearrangement than point mutations, as it will be discussed in the next sections.

CDH1 in one of the most altered genes in this study with 7 missense variants, rare or predicted, and a frameshift insertion never described before. Most of these variants are somatic mutations, occurred in tumor progression. Loss of CDH1 expression or altered form of CDH1 may alter cell-to-cell adhesion and promote tumor invasiveness. TP53BP1, involved in the recruitment of protein on DNA damage site and in coordinating HR and NHEJ switch, have also shown to be mutated: two rare missense (one predicted) and one del inframe mutations were reported in three patients. This del inframe is poorly studied and it is interesting. Extensive analysis of TP53BP1 expression and regulation may be useful to better understand BRCA1-related tumorigenesis an PARPi resistance.

110

Genetic profile of DNA repair genes in TNBC Results

Not surprisingly, the major number of mutations were found in TP53 gene. All classes of variation were reported: missense mutations, stopgain, frameshift mutations, non frameshift indels, splicing variants. 25 out 37 patients had a TP53 mutation: this means that 68% of this group of TNBC have in TP53 somatic inactivation one of the fundamental hit of tumorigenesis. This data are in accord with literature. Almost all the single nucleotide substitution in TP53 were already reported in the IARC database, while 3 frameshift deletions were not registered but they strongly affect the DNA binding domain of TP53.

20 somatic mut germline mut

of mutationsof 10

° n

Fig 11. Schematic distribution of predicted pathogenetic/pathogenetic variants in TNBC patients. TP53 is the most mutated gene with 25 somatic variations, followed by CDH1. BRCA1 mutations are only germline. Only 8 genes had somatic mutations.

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Genetic profile of DNA repair genes in TNBC Results

2.5. TNBC are enriched in genomic rearrangement involving DNA repair

genes

Breast cancer, and in particular the TN subtype, is characterized by high genomic instability which often lead to accumulation of mutations or rearrangements of entire genes and part of chromosomes. Specifically, TNBC have a high rate of PTEN, P53, PI3K, RB1 genomic gain or loss. To further characterize this cohort of TNBC, these tissues were analyzed by MLPA for small rearrangements with two different commercial kits. One kit contains probes for some of the most altered genes in a large variety of tumours, and some genes specifically for the gliomas tumorigenesis. From this analysis, data for TP53, PTEN, CDKN2A, CDK4, MDM2, EGFR, platelet-derived growth factor receptor (PDGFRA) were obtained. CDKN2A and CDK4 are cell cycle regulators, often amplificated in tumor tissues. MDM2 is the principal inhibitor of TP53: it interacts with TP53 by inducing its degradation. Nevertheless TP53 increases the activity and the production of MDM2 both directly and indirectly, thus forming an autoregulatory negative feedback. EGFR, already discussed in this thesis, is one of the most important membrane receptor for signal transduction. It is considered on oncogene, as activating mutations rapidly promote cancer development. Its heterodimerization with Her2/neu leads to the activation of Ras and PI3K pathways. Mutations, amplification or dysregulation of EGFR or related proteins are implicated in about 30% of all epithelial cancers. PDGFRA, when activated by its ligand, plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue. Uncontrolled angiogenesis is a characteristic of cancer. Overexpression of PDGF and PDGF receptors has been reported in some human mesenchymal tumors. The second kit included probes for Her2/neu2 and other genes of its pathway, plus a set of genes on the chromosome 17 including BRCA1, mi21,GRB7. Moreover probes for BRCA2 and Estrogen Receptor are present. This other approach is useful to better understand the genomic status of BRCA genes in the selected tissues. Furthermore, may suggest new genes to investigate. GRB7 encodes a growth factor receptor-binding protein that interacts with EGFR and other tyrosine kinases. GRB7 is an SH2-domain adaptor protein that binds to receptor tyrosine kinases and provides the intra-cellular direct link to the Ras proto-oncogene. Human GRB7 is located next to the HER2/neu and are commonly co-amplified in breast cancers. GRB7 is thought to be involved in migration. A number of targets for microRNA-21 have been experimentally validated and most of them are tumor suppressors. Notable targets include PTEN and hMSH2.

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Genetic profile of DNA repair genes in TNBC Results

The pattern of genomic alterations in these genes for the 37 patients is reported in Table 8. The most altered genes are, as expected, TP53 and PTEN which are often loss in tumour tissues, with 11 (29%) and 10 (27%) cases respectively. 31 of 37 patients have at least one gene rearranged. One patient has 6 different genes, on chr17 and not, which are partially or completely deleted. Interestingly, two patients present a BRCA2 deletion: this two are not mutated in BRCA genes but harbor two different predicted missense. Another patient has a partial amplification of BRCA2 gene, while at a mutational level is carrier of a in/del frameshift in BRCA1. BRCA1 is amplificated in one patient and this is the only somatic rearrangement found in 37 patients. Notably, MLPA for TP53 has evidenced that many of those tumours which do not harbor a mutation in the gene sequence have often a rearrangement in the genomic TP53 locus. With this integration, 30 of 37 patients have a somatic hit in TP53 (81%)

BRCA1

GRB7

mir21

MDM2

cdk4

ESR

BRCA2

PDGFRA

p16

PTEN

EGFR

P53

0 2 4 6 8 10 12

Fig 12. Genomic rearrangements in TNBC.

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Genetic profile of DNA repair genes in TNBC Results

2.6. Correlation of genomic status of tumor tissue and disease free

survival

In order to define if a particular genetic condition may be associated with a better prognosis in this second group of patients in adiuvant therapy, DFS was correlated with the mutation status. The analysis was conducted gene by gene and for grouped mutations. Since the very small number of patients and the inhomogeneous stratification no results was statistically significant. However, some interesting trends have emerged in accord with recent huge studies and in line with the general state of art of genomic landscape of TNBC. Interestingly, 25 patients had a somatic TP53 mutation and many of them have a recurrence event. The Fig 13B clearly shows that the group of the mutated patients has a worse outcome. Moreover, considering all the known pathogenetic mutations in breast cancer susceptibility genes a (BRCA1, BRCA2, CDH1, PALB2, BRIP1, BARD1) genes in 37 patients, the Kaplan-Meyer analysis have evidenced a trend to a better outcome for the carrier patients (Fig 13A). This is similar to results obtained in the neoajduvant setting; the correlation is supported by some recent literature data from BRCA1 and BRCA2 related TNBC and serous ovarian cancers and good outcome (Gonzalez-Angulo, 2011; Yang 2011). Another interesting data is concerned with the polymorphism Val762Ala of PARP1: this largely discussed variant was found in 8 patients, most of which had a good prognosis. The Fig 13C shows that patients carrier of Val762Ala have generally a better response to therapy and longer DFS. This data must be confirmed in a large cohort of TNBC but suggests an interesting role for this polymorphisms, clinical outcome, and PARP1 involvement in TNBC phenotype.

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Genetic profile of DNA repair genes in TNBC Results

A B

C Fig A: with mutation

w/o mutation

Fig B: with TP53 mutation

w/o TP53 mutation

Fig C: with PARP1 snp

w/o PARP1 snp

Fig 13. Disease free survival and mutation status. (A) DFS in subjects with at least one germline mutation in one breast cancer susceptibility gene. (B) DFS in subjects with a somatic mutation in TP53 (C ) DFS in subjects with the polymorphism Val762Ala in PARP1.

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Genetic profile of DNA repair genes in TNBC Results

not recurred patients recurred patients EL253 EL329 EL260 EL346 EL292 EL261 T351 EL344 EL311 G26 EL102 T291 sc368 T487 G41 T161 T105 EL318 T104 T297 T261 T348 G72 EL137 T346 G17 G43 G58 T109 T108 G40 EL163 T107 sc519 G44 scxx T106

X X X BARD1 XXX X X X X X X X X BRCA1

X X X BRCA2

X X X X BRIP1

X X X X X X X CDH1

CHEK2

X ERCC1

X X X MLH1

X MRE11

X X X X X X X X X MSH2

X X X X MSH6

X X X NBN

X X X X X PALB2

X X X X X X X X X X X PARP1

X X PMS1

X PTEN

X X X X X RAD50

RAD51c

X RAD52

STK11

X X X TP53BP1

X X X X X X X X X X X X X X X X X X X X X X X X X TP53

Table 7. Variants distribution in TNBC patients. All the patients are reported in the rows: 12 recurred patients, with one or more metastasis; 20 not recurred patients, which are disease-free after 5 years; for the last 5 patients was not possible to obtain therapy data and outcome and are excluded by correlation analysis. In the columns are reported 19 genes with at least one probable-pathogenitic variant. The clearly pathogenetic variants are in red, the predicted variants are in orange, the new synonymous variants are in yellow. All the somatic variants in TP53 are in white.

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Genetic profile of DNA repair genes in TNBC Results

P53 p16 ESR

cdk4

PTEN EGFR

GRB7

mir21

BRCA2 BRCA1

MDM2

PDGFRA

T106 scxx G44 sc519 T107 EL163 G40 T108 T109 recurredpatients G58 G43 G17 T346 EL137 G72 T348 T261 T297 T104 EL318 T105 T161 G41 T487 sc368 T291

notrecurred patients EL102 G26 EL311 EL344 T351 EL261 EL292 EL346 EL260 EL329 EL253

Table 8. Genomic rearrangements profile in TNBC patients. All the patients are reported in the rows; in the columns are reported 12 genes which have been found to be deleted (in blue) or amplified (in green) by MLPA.

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Discussion

Genetic profile of DNA repair genes in TNBC Discussion

Triple Negative Breast Cancer constitutes 10–20% of all breast cancers and more frequently affect younger patients. TN tumors are generally large in size, higher grade cancers with lymph node involvement at diagnosis, and are biologically more aggressive than other subtypes. Sporadic 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. The strong phenotypic similarities between BRCA1-related breast cancers and TNBC suggest a common deficiency in DNA repair genes. Recent reviews reported that about 25% of TNBC carries mutations in BRCA, but a more complete molecular characterization of these tumors is still object of great interest.

Treatment of TNBC patients has been challenging due to the heterogeneity of the disease and the absence of well-defined molecular targets. Despite good rates of clinical response to neoadjuvant chemotherapy, TNBC patients have a higher rate of distant recurrence and a poorer prognosis than women with other breast cancer subtypes (Dent et al., 2007). For the majority of patients with TNBC who have residual disease after neoadjuvant treatment, there is a high risk of relapse with a peak of metastases occurring in the first 3 years (Liedtke et al., 2008) . These tumors are regarded as primary resistant and therefore, response to systemic therapy and long-term favorable outcomes are rare. The lack of drug-targetable receptors on TNBC tumours has led to develop novel therapeutic strategies for these patients. The routine use of neoadjuvant anthracycline/taxane combinations in TNBC is currently being supplemented with other types of agents. The substantial proportion of TNBC tumours associated with BRCA1 mutations is driving clinical research into the use of DNA-damaging agents such as platinum. A recent retrospective study which considered 144 women with locally advanced TBNC treated with neoadjuvant platinum therapy, showed a cisplatinum advantage in PFS (Progression Free Survival) and OS (Overall Survival) (Petrelli et al., 2014). Following promising results in the neoadjuvant setting, there has been renewed interest in platinum either as a single agent or in combination therapy for TNBC after prior exposure to classical agents such as anthracyclines, cyclophosphamide and taxanes. The use of platinum compounds is supported by the high frequency of germline mutations in the BRCA1 gene in TNBC tumours. Nevertheless, it is important to understand which other DNA repair pathway may be deficient, at germinal or tumor level, in this kind of cancers in order to select likely more responsive patients to this therapies. New potential approaches in TNBC disease have included targeting vascular endothelial growth factor (VEGF), EGFR tyrosine kinases and PARP1 inhibitors. In particular seems worth to investigate in those patients the efficacy of PARP1 inhibitors, a class of drugs that specifically targets cells defective in DNA repair. Several phase I and phase II trials of

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Genetic profile of DNA repair genes in TNBC Discussion

PARP inhibitors have been performed with BRCA1 mutation carriers. For instance, in a phase I trial, the PARP inhibitor olaparib showed selective activity against BRCA1/2- mutated breast cancer, whereas BRCA-unrelated tumors remained unaffected. Based on this finding, Tutt et al. demonstrated, in a phase II trial, that olaparib at a higher dose was also associated with an improved objective response rate in BRCA1/2 carriers (Tutt et al., 2010). Recently, PARP-1 has been shown also to regulate another DNA repair pathway, the Non Homologous End Joining, and to guide repair by forming PARP/DNA adducts (Murai et al., 2012). These findings provided rationale for investigation into the effect of PARPi in sporadic TNBC with acquired defect in DNA repair other than germline BRCA mutations. All these evidence have focused the attention on DNA repair pathways in TNBC. A DNA repair deficient condition is unfavorable and convenient at the same time: the most a tumor is genetically unstable and accumulate mutation in DNA repair genes, the most it can be sensitive to DNA targeted therapies. Based on the suggestion that DNA repair defects sensitize cells to chemotherapy with anthracyclines, platinum derived compounds and PARP-1 inhibitors, the hypothesis of this study is that germline and tumoral patient’s DNA repair genetic profiles may be predictive for efficacy of these therapeutic agents. Therefore, we aim to define a genetic signature that could potentially distinguish TNBCs into those that exhibit DNA repair defect, and for such reason will eventually respond better to these classes of drugs, from TNBCs that, not carrying a deficiency in DNA repair, might not respond.

The first objective of this study is to better understand the contribution of germline mutations in moderate- and high-risk genes to TNBC . For this purpose, a panel-based mutation screening has been carried out in a little cohort of TNBC in neoadjuvant therapy. While is well known the contribute of BRCA1 and BRCA2 mutations in TN patients, is still poorly investigated the importance of other genes. Recently, PALB2 status have been studied in TNBC larger cohorts (A. C. Antoniou et al., 2014) and the results indicate that the prevalence of PALB2 germline mutations in sporadic TNBC is 1%, similar to the frequency found in familial non-BRCA1/2 breast cancer cohorts.

In this study we found that BRCA1 mutations are frequent even in patients unselected for family history. BRCA1 mutations were found in 3 out 19 patients (15%) in according to literature. Moreover PALB2 and RAD51c, considered moderate penetrant genes, are mutated in 5% of cases (one patient for each gene). In PALB2 were found also two predicted pathogenetic missense, already reported in larger studies as possibly damaging. They are both located in the WD40 domain of interaction with BRCA2, RAD51, RAD51c. Overall 3 patients have alterations in PALB2 that may destroy the perfect machinery of DNA damage recognition and repair, accounting for another 15%. Moreover

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Genetic profile of DNA repair genes in TNBC Discussion

one patients harbour a BRIP1 variants which fall in the Fe-S domain and next to the M299I, involved in early onset breast cancer susceptibility. Therefore, around 26 %of patients have a pathogenetic mutation, and a further 15% is carrier of a very probable pathogenetic variant in other well-known breast predisposition gene. Moreover, patient cases of TNBC with mutations in the non-BRCA1/2 genes were not significantly associated with family history for either breast or ovarian cancer. In this view is reasonable to analyze these results together from those emerged from the other set of patients studied in this thesis. 37 TNBC tissues have been screened and germline mutations are confirmed in the non-tumoral tissue: this second set of patients evidenced the presence of 7 patients mutated in BRCA1, 1 patient in BRCA2 and 1 patient in CDH1. Overall, in the 56 TNBC patients BRCA1 mutations were found in the 18% of the cases (10/56). The other germline defects are due to mutations in BRCA2, RAD51c, PALB2, BRIP1 and CDH1 genes for another 9% of cases (5/56).

By now there are no guidelines or standard criteria for BRCA1/2 testing breast cancer patients on the basis on a particular phenotype. General criteria include age at diagnosis and presence of family history; only BRCA genes screening is suggested. In the next generation sequencing era, many genetic centers have started gene panel testing for breast cancer with family history, often including other few high and moderate risk genes. A very recent paper (Couch et al., 2015), published only few days ago, have described a very similar approach to that presented in this thesis. The authors have performed a panel-based mutation screening of breast cancer predisposition and DNA repair genes in a large cohort of patients with TNBC. 14.6% of 1,824 patients with TNBC unselected for family history of cancer carried germline deleterious mutations in 14 of 17 predisposition genes tested. BRCA1 and BRCA2 mutations were found in 11.2% of patients, whereas mutations in other genes were found in 3.7% of patients. In addition, 1% to 3% of patients carried missense mutations predicted by in silico methods to be deleterious. These huge study suggests a distinct enrichment for predisposition gene mutations in unselected TNBCs. Furthermore, genes involved in homologous recombination, including PALB2, BARD1, BRIP1, RAD51C, RAD51D, and XRCC2, accounted for 54 (81%) of the 67 mutations in non BRCA1/2 predisposition genes, suggesting that disruption of homologous recombination repair may be an important event in the development of triple- negative breast tumors.

This is in line with the data presented in our study: the majority of patients, indeed, harbour a defect in homologous recombination genes while the other pathways are less often deficient. Again, the BRCAness phenotype is more probably linked to defects in genes which interact with BRCA1/2 or participate in the same repair pathway.

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Genetic profile of DNA repair genes in TNBC Discussion

The paper of Couch and colleagues, support and give strength to our hypothesis that TNBC patients are very likely to be carriers of germline mutations in breast cancer predisposition genes, even in unselected cases. Therefore, it seems urgent to identify these patients in order to plan diagnostic surveillance and optimize therapy regimens. Gene panel testing for TNBC patients, with and without family history of breast and ovarian cancer, may be very useful in this purpose. TNBC tumours have an high cell proliferation rate leading to very large tumors in usually young patients: early detection of mutations in BRCA1/2 may be guide to surgical and therapy approach soon after the diagnosis. The contribute of other genes is far to be established but it is reasonable that some therapeutic options in patients mutated in other double strand breaks genes will be tested soon. For example, analogous to BRCA1/2-deficiency, PALB2-deficient cells are sensitive to PARP inhibitors (Buisson et al., 2010). Increased PARPi susceptibility was shown in a series of cell lines with PTEN mutation, confirmed in xenograft experiments using the PARPi, olaparib. There is also clinical evidence that olaparib may have a therapeutic utility in PTEN-deficient endometrioid endometrial cancer (Forster et al., 2011). Recent studies have demonstrated a distinct role for PTEN in the maintenance of chromosomal integrity and repair of DNA DSB: in cells with PTEN loss, RAD51 does not appear at sites of DNA DSB to mediate repair by HR. Deficiency of HR caused by dysfunctional MRN complex may sensitize cancer cells to PARP inhibitors. Colorectal cancer cell lines with biallelic mutations in MRE11 have been shown to be significantly more sensitive to olaparib and ABT-888 than MRE11 wild-type cells (Vilar et al., 2011); similarly, NBS1 deficient human fibroblasts are also sensitive (Horton, Stefanick, Zeng, Carrozza, & Wilson, 2011). Sequencing of panel of genes involved in HR DNA repair may help to define a repertoire of mutations that predict PARP inhibitor sensitivity. Moreover, EGFR, a proto-oncogene that belongs to a family of four transmembrane receptor tyrosine kinases that mediate the growth, differentiation, and survival of cells, is often overexpressed in TNBC and is associated with aggressive disease phenotype [Nakajima, 2012]. A recent study reports that a contextual synthetic lethality can be achieved both in vitro and in vivo with combined EGFR and PARP inhibition with lapatinib and ABT-888, respectively. The mechanism involves a transient DNA double strand break repair deficit induced by lapatinib and reveals that EGFR and BRCA1 can be found in the same protein complex (Nowsheen, Cooper, Stanley, & Yang, 2012)

A further objective of this study was to define a genetic signature of DNA repair that can be predictive for neoadjuvant therapy response. Evidence from accumulated neoadjuvant studies revealed that pCR provides a surrogate marker that is predictive for long-term clinical response and survival in TNBC patients. As previously discussed, BRCA related cancers have been shown to have good outcome to anthracyclines neoadjuvant

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Genetic profile of DNA repair genes in TNBC Discussion

regimens, as well as sporadic TNBC. Anthracycline/taxane based regimens are routinely used in the neoadjuvant setting for TNBC, but there is a clinical need to identify those patients more likely to respond. In this study 19 TNBC patients underwent to anthraciclynes/taxane neoadjuvant therapy have been tested: 5 of them have experienced a pathological complete response, 4 of them had no benefit from therapy and had tumor progression. The remaining 10 patients have good response, but only 8 of them had a reduction of tumor mass greater than 50% and were included in the responder group. This data are in accord with the response rates reported in literature. Interestingly, 5 patients have found to be mutated in BRCA1, PALB2 and RAD51c: 4 of these patients had a very good response to therapy while only one patient, carrier of splicing variant on BRCA1, had disease progression. Therefore, in this small cohort of patients, it seems that patients carrier of a deficient DNA repair, and in particular a defective homologous recombination pathway, had a better response to this kind of therapy. Moreover, the gene panel screening has detected a number of exonic variants very likely to affect protein expression and function in 10 genes. The clinical outcome was correlated to the condition of being carrier of a probable pathogenetic DNA repair variant. This analysis revealed an interesting trend for a better outcome for the patients with any DNA repair deficiency. These findings suggest the existence of a group of patients, among TNBC, with genetic features similar to BRCA1-related and a similar behavior in terms of response. The gene panel testing designed in this study, may be an efficient tool to discriminate these patients rapidly before start of treatment. Moreover, neoadjuvant treatment can be used as a research tool to assess the efficacy of new drugs: TNBC patients with a genetic signature of DNA repair deficiency may be selected and proposed for experimental or non-standard therapy as PARP inhibitor, platinum agents and others. Overall, this gene panel testing in neoadjuvant TNBC patients has evidenced that many TNBC have defects in DNA repair genes, other than BRCA1, and that this condition is associated with a good response to anthraciclyne/taxane therapy. Moreover it get insights into the contribute of the “other” breast cancer genes to TNBC phenotype.

A further aim of this thesis is to characterize the somatic changes in the tumor tissues also. DNA repair defects may lead to an accumulation of variants in several genomic regions and increase genomic instability, reflecting aggressive tumor properties. This may lead to an advantage to the tumor, which acquire a mutator phenotype where one or more of the mechanisms that preserve genomic integrity are loss such us DNA repair pathways. Among breast cancer types, basal-like TNBC group displays the most instable genome: mutations in BRCA1, PTEN and RB1 gene are associated with high chromosomal instability (Hu et al.2009). Besides mutations, other genetic changes such as copy number alterations involving EGFR, PTEN, P53, PIK3CA occur very often in TNBC. Rodriguez

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Genetic profile of DNA repair genes in TNBC Discussion

(Rodriguez et al., 2010) have derived a gene expression profile that is associated with DNA repair deficiency in sporadic TN breast cancer: PARP1, RAD51, FANCA, and CHK1 were some of the overexpressed genes in a group of BRCA1-like tumours. This signature was associated with sensitivity to DNA-damaging chemotherapy and relative taxane resistance. Given the importance of the cellular DNA repair in determining the cellular response to different anticancer agents, a priori knowledge of the repair status of a given tumor could play an important role in selection of the most appropriate therapy. For this approach, 37 TNBC patients have been selected. TNBC usually have an high risk of recurrence in the first 5 years, so the including criteria for this second set of patient was to have at least 5 years of follow up and all the clinicopathological data available. Comparing tumor tissue with matched healthy tissue, germline mutations have been evidenced in BRCA1 (18%) , BRCA2 (2,7%), BRIP1 (2,7%), CDH1 (2,7%) genes and many predicted deleterious missense variants in 14 of the selected genes. These data support the finding that TNBC patients are often mutated in BRCA1 and other breast susceptibility genes. Notably, the largely discussed Val762Ala polymorphism of PARP1 was found in 8 patients and survival analysis have evidenced that patients with this SNPs have often longer DFS. This data is not statistically significant due to the small number of patients, but give a suggestion for a positive effect, at least in this cohort of patients.

Somatic variants have been found in 8 genes: BRCA2, BRIP1, CDH1, MLH1, MSH2, NBN, PMS1, TP53. As reported in literature TP53 is very often mutated in TNBC; here is largely the most altered gene in 25/37 patients (68%). A somatic inactivation classically represents a key drive event of BRCA1-related cancers and TNBC. Mutations are distributed particularly in the central domains of protein, the DNA binding protein domain and the transactivation domain. The increase in TP53 mutations in hormone-receptor- negative tumors and their exceptionally frequent occurrence in TNBC suggests that in the absence of hormone-related stimulatory signaling characteristic to the mammary epithelium, mutant p53 may become critical for breast cancer progression. This is supported by the fact that TP53 mutations are more frequent in high-grade, large-size, node-positive cases, and thus possessing a prognosis-worsening driving role (Walerych, Napoli, Collavin, & Del Sal, 2012). Indeed, even in our little cohort of TNBC, patients with a mutated TP53 seem to have a worse disease progression. Comparing DFS from patients with a TP53 mutation with the WT ones, it is evident a trend to a shorter time of recurrence in the first group. This association is not evident when TP53 rearrangement are analyzed. In contrast, EGFR amplification is strongly associated with a worse outcome in TNBC and emerged also from our data. Recently Shapira and colleagues proposed a possible explanation for the outcome of EGFR positive TNBC and a novel TP53 mechanism (Shapira, Lee, Vora, & Budman, 2013). A TP53 mutated protein increased

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Genetic profile of DNA repair genes in TNBC Discussion

potency of the EGFR signaling due to increased endosomal recycling. Mutant TP53 acquires oncogenic functions and binds TP63 protein, a member of TP53 family with tumor suppressor activities. In the absence of functional TP63 there is an upregulation of EGFR and integrin with increased proinvasive abilities of cancer cells.

The mismatch repair pathway was also found mutated. Recently have been observed that a load of mutation in MMR pathway may be associated to poor clinical prognosis of patients with ER+ tumours (Haricharan, Bainbridge, Scheet, & Brown, 2014). In contrast to these results, a recent publication analyzing mutational signatures of various cancers was unable to identify any correlation between MMR deficiency and mutational signature in breast cancer (Alexandrov et al., 2013). Interestingly a novel MSH2 variants was identified in this thesis work: the Ser755Phe is localized in the ATP-ase domain of MSH2 protein and involves an important serine residue. This domain is of dramatic importance in recognizing and repairing mismatched bases and in controlling trinucleotide repeats expansions, involved in microsatellite instability. MMR have been studied also for its involvement in cisplatin response: although a complete knockout in MMR results increased cisplatin tolerance, it is not clear how single point mutations in MMR genes affect the response to a chemotherapeutic agent (Clodfelter, M, & Drotschmann, 2005). MMR-defective cells show a high spontaneous mutation rate after drug exposure (Karran, 2003). This acquired genome instability has been attributed to the growth advantage of MMR-deficient cells after chemotherapeutic treatment, which results in the expansion of cultures of repair-defective cells and the accumulation of mutations in downstream genes. This mechanism may interest other DNA repair pathways.

Moreover, the analysis of rearrangements by MLPA technique has evidenced that TNBC have a great number of gain and loss events in cancer genes and BRCA related genes. PTEN and TP53 are entirely or partially lost, while CDKN2A and EGFR are the most amplified genes. Interestingly, also BRCA1 and BRCA2 can undergo to somatic rearrangements and this simple analysis may be a useful tool to characterize TNBC and guide therapy choice. Indeed, three patients without germline BRCA mutations, presented in their tumours completely loss of BRCA2 and gain of exons 14-20 of BRCA1. These patients may be sensitive to PARP inhibitors or platinum compounds.

This gene panel approach has proved useful to identify a large number of germline mutations in TNBC patients. It will important to further investigate the functional impact of the never reported variants, in particular those in known breast cancer risk genes. Biochemical properties and subcellular localization of the mutated protein, are necessary to understand how the mutation can guide a dysfunctional DNA repair process. Moreover a critical issue to deal with, is the evaluation of the penetrance of a certain mutation to a

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Genetic profile of DNA repair genes in TNBC Discussion

specific phenotype; for this purpose case-control studies are needed. 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, this thesis have evidenced that altered DNA repair pathways may play a role in TNBC progression and that a tumor characterization can be useful to further improve the accuracy of treatments.

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Zhou, X. P., Waite, K. A., Pilarski, R., Hampel, H., Fernandez, M. J., Bos, C., . . . Eng, C. (2003). Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3- kinase/Akt pathway. Am J Hum Genet, 73(2), 404-411. doi: 10.1086/377109 Zimmermann, M., & de Lange, T. (2014). 53BP1: pro choice in DNA repair. Trends Cell Biol, 24(2), 108-117. doi: 10.1016/j.tcb.2013.09.003

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Acknowledgments

I’d like to thank my tutor Dr. Maria Adelaide Caligo for giving me the opportunity to be part of her laboratory. Thanks for let me approach to the world of genetics and oncology and for the chance to work with the latest technologies (even if in a “vintage” lab!).

Thanks to Prof. Giuseppe Naccarato and to all the technicians and pathologists of the Anatomia Patologica 1, for the support and expertise. Thanks to Dr. Katia Zavaglia for the friendly advices...thanks for giving me free space, computer and desk in your lab! Thanks to the doctors and interns of the Oncology Unit for the patience of listening to my doubts and to help me with the medical records.

A great thank to all members, past and present, of my lab. Elisabetta, Anita, Mariella thanks for your experience, presence, advices and laughter. Thanks a lot to my PhD friends, Chiara, Gaga, Luisa and Rosa, for sharing joys and especially pains, in the life of a PhD student. We sustained each other in following a passion...but never forgetting that real life is the most important thing!

Thanks to old and new friends, because time spent with you is worth more than any scientific success.

Most of all, I’d like to give a special thanks to my family, from my grandma to my lovely nieces&nephews, for their love and support and for respecting all my choices.

Gianluca, thank you with all my heart for choosing to stay with me and to love me in good times and bad times. Thanks for our love, laughter, understanding. And thanks for your technical support throughout the write up! Now I'm ready to finish decorating the house 