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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES Faculty of medicine
DEPARTEMENT OF GENETICS AND MOLECULAR BIOLOGY
THESIS TITLE:
BRCA1, BRCA2 AND TRIPLE NEGATIVE BREAST CANCER Systematic review
Student: Mohammad Mahamid Consultant: Dr. Marius Šukys Supervisor: Dr.Virginija Ašmonienė
Kaunas 2019-2020
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TABLE OF CONTENT
ABSTRACT...3
ABBREVIATIONS ...4
AKNOWLEDGEMENT ...5
INTRODUCTION ...6
AIM AND OBJECTIVES ...8
LITERATURE REVIEW ...9
1. BRCA1 and BRCA2 in Breast Cancer ...9
1.1 Epidemiology ...9
2. THE GENETICS BEHIND BREAST CANCER ... 11
2.1 BRCA 1 ... 11
2.2 BRCA2 ... 12
2.3 BRCA Genes as Caretakers of Genomic Stability ... 14
3. METHODS ... 20
4. RESULTS ... 22
5. DISCUSSION ... 27
6. CONCLUSION... 29
REFERENCES ... 30
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ABSTRACT
Triple-negative breast cancer (TNBC) is a type of aggressive breast cancer and it is characterized by a lack of the expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2). BRCA genes are tumor-suppressor genes that are involved in DNA damage repair and mutations of BRCA genes may increase the risk of developing breast cancer and/or ovarian cancer due to defective DNA repair mechanisms.
However, the relationship between BRCA status and TNBC needs to be further investigated and validated.
Aim and Objectives: The aim of this study was to systematically review and analyze the latest studies in order to determine the association between BRCA1/2 mutation and triple negative breast cancer. In addition, the frequency and the pathogenic variants of BRCA1/2 in TNBC had to be identified.
Methods: articles were systematically searched the electronic databases of MEDLINE (PubMed) and Google scholar to identify relevant publications from 2010 to 2020. The data from the
studies were collected and summarized in the Results section. 11 qualified studies from 634 publications have been identified.
Results: The results showed that breast cancer patients with BRCA1 mutation carriers were more likely to have TNBC than those of BRCA2 mutation carriers (or non-carriers. Furthermore, high expression of nuclear grade and large tumor burden (>2 cm) were significantly more
common in breast cancer patients with BRCA1 mutation carriers than those of BRCA2 Mutation carriers or non-carriers. The data suggest that breast cancer patients with BRCA1mutation are more likely to have TNBC, high nuclear grade, and larger tumor size. The most commonly identified pathogenic variants of BRCA1/2 genes in TNBC are c.5266dupC, c.2411_2412delAG, c.68_69delAG and c.3481_3491del for BRCA1 and c.7878G > C, c.5946delT, c.5744C>T. for BRCA2.
Conclusion: The results suggest that BRCA1/2 testing should be done in case of TNBC due to a strong association found. Also a wider based meta-analysis must be done to identify the most common BRCA1/2 pathogenic variants in TNBC and other pathologic types of BC.
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ABBREVIATIONS
BC: breast cancer
BRCA: breast cancer susceptibility gene TNBC: triple negative breast cancer ER: estrogen receptors
PR: progesterone receptors
HER2: human epidermal growth factor 2 P53: tumor protein 53 gene
DCIS: ductal carcinoma in situ
BARD1: BRCA1 associated RING domain protein 1 NLS: nuclear localization signals
DSB: double strand break RNF8: Ring Finger Protein 8
RAP80: receptor associated protein 80 HR: homologous recombination NHEJ: non-homologous end joining
XRCC: x-ray repair cross-complementing protein WT: Wild type
DNA-PK: DNA dependent protein kinase
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AKNOWLEDGEMENT
I would like to thank my supervisor Dr. Virginija Ašmonienė and Dr. Marius Šukys for their great support and guidance.
I would like to dedicate this thesis to all my collages who are fighting COVID-19 worldwide.
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INTRODUCTION
Breast Cancer (BC) is the most common type of cancer among women, with an estimated value of 1.67 million new cancer cases diagnosed in the year 2012[1] BC is the most common cause of death among other cancers in women after lung cancer.
Of all cases of breast cancer, about 5%-10% have a strong inherited component, of which 25% is explained by germline mutations in the high -penetrance breast cancer predisposition genes, BRCA1[2] and BRCA2[3]. The risk of breast cancer for BRCA1 mutation carriers at age 70, has been estimated to be within the range of 40%-87%, the corresponding risks for BRCA2 mutation carriers were estimated to be 40%-84% for breast cancer [4]. The risk not explained by the high penetrance genes is due to moderate or low penetrance genes, each having a small effect on breast cancer risk.
The prevalence and type of BRCA1 or BRCA2 germline mutations varies considerably, among diverse ethnic groups and geographical areas. Population specific and recurrent mutations have been described among the Ashkenazi Jews, Iceland, The Netherlands, Sweden, Norway, Germany, France, Spain, Canada, countries of eastern and southern Europe [5] and in Latin American populations, such as Chile, México, Brazil and Costa Rica [6]. Thousands of
mutations found in BRCA genes, in families with breast/ovarian cancer are now described in the recent literatures.
TNBC is characterized by a lack of the expression of estrogen receptor (ER),
progesterone receptors (PR) and human epidermal growth factor receptor two (HER2/neu), thus, we are left with no molecular targets for treatment (7). The BRCA1 and BRCA2 genes are tumor- suppressor genes and responsible for DNA damage repair and recombination, cell-cycle
checkpoint control, apoptosis and transcriptional regulation (8). Mutations in BRCA genes can lead to abnormal DNA repair mechanisms, which are associated with the increased risk of BC development (9). Some studies showed that BRCA1 mutation carriers were more likely to have ER-negative/PR-negative breast cancer (10). In contrast, BRCA2 mutation carriers seem to share the pathologic characteristics similar to those of patients with normal BRCA genes (non-carriers) (11). However, it has been found that the association between TNBC and BRCA mutations was not only limited to BRCA1, but also a significant proportion of women with TNBC had BRCA2
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mutation [12]. Currently, the relationship between the status of BRCA mutation and the statuses of ER, PR, HER2/neu and P53 have been inconsistent (13).
With the development of targeted therapies for breast carcinomas, designation of
treatment regimens has become more specific, and breast cancer patients with BRCA mutations should be treated differently from the patients without BRCA mutations. Therefore, the exact relationship between BRCA status and TNBC needs to be further investigated and validated.
Here in this review we will focus on the molecular mechanisms in which BRCA is involved and how a defect in these pathways may lead to BC, also the relationship between TNBC and BRCA mutation will be discussed in details.
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AIM AND OBJECTIVES
Study Aim: The aim of this study was to systematically review and analyze the latest studies in order to determine the association between BRCA1/2 mutation and triple negative breast cancer.
Objectives:
1. To analyze BRCA1 and BRCA2 pathogenic variants frequency in case of triple negative BC and the clinicopathologic differences between them.
2. To Analyze most common BRCA1 pathogenic variants in case of triple negative BC.
3. To Analyze most common BRCA2 pathogenic variants in case of triple negative BC.
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LITERATURE REVIEW
1. BRCA1 and BRCA2 in Breast Cancer 1.1 Epidemiology
Breast cancer is the most commonly diagnosed cancer among US women, with an estimated 268,600 newly diagnosed women with invasive disease in 2019, accounting for approximately 15.2%-30% of all new cancer cases among women, depending on the data sources. [14] Each year, nearly 40,000 women die of breast cancer in the US, making it the second-leading cause of cancer deaths among US women after lung cancer [15] worldwide around 500,000 people die of breast cancer. On average 1 in 8 women will receive the diagnosis of breast cancer in her lifetime. [2,3,4] The lifetime risk of dying of breast cancer is
approximately 2.6%.
The incidence rate of breast cancer has been increasing historically by 1% till 1980s when incidence increased abruptly likely due to the increasing use of mammography. In the 1990s, incidence remained fairly stable and in the 2000s it has decreased slightly, likely due to the decrease in the use of hormonal replacement therapy (16). Incidence varies greatly with race and ethnicity, in the white population, incidence (per 100.000) is 127.4. total mortality is 12.3 and 5-year survival is 90.4%. in the black population incidence is 121.4. total mortality is 18.2 and 5-year survival is 78.6%. BC incidence is also higher in developed countries thought to be linked to industrialization (high fat intake, high BMI, early menarche, lactation and reproductive pattern such as fewer pregnancies and late first pregnancy). However, the mortality rates of breast cancer are higher in less developed regions mostly due to less developed health care system. Differences in incidence have been observed with age (fig 1), the total risk of
developing BC increases with age, ER- positive BC incidence increases with age. In contrast, TNBC incidence increases with age until the age of 50, and the remains constant. ER-positive tumors therefore are more likely to occur in postmenopausal women.
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Figure 1: Age-specific rates of BC in the US
BRCA1 and BRCA2 mutations are a well-known genetic factors that contribute to the development of BC. These gene mutations are inherited in 50% of the cases and accounts for approximately 5% to 10% of all breast cancer diagnoses. A deleterious mutation in either gene corresponds to a 10-fold increase in the risk of BC development. The prevalence of both genes in the general population varies among geographic regions and ethnicities, but is generally low (0.4%-0.7% for BRCA1 ,1%-3% for BRCA2); however, penetrance of these genes is high.
TNBC accounts for approximately 15 percent of breast cancers diagnosed worldwide, which amounts to almost 200,000 cases each year (17). Compared with hormone receptor- positive breast cancer, TNBC is more commonly diagnosed in women younger than 40 years a feature that is also shared by BRCA+ BC.
11 2. THE GENETICS BEHIND BREAST CANCER
2.1 BRCA 1
Approximately 10% of all cases of breast cancer exhibit a familial pattern of incidence.
Efforts to identify the genetic basis of familial breast cancer reached fruition some five years ago, when the breast- cancer-susceptibility genes BRCA1 and BRCA2 were identified through positional cloning. (18)
The BRCA1 gene is responsible for making a BRCA protein which functions as a tumor suppressor (FIGURE 2). Thus it inhibits the rapid uncontrolled proliferation of the cells. The BRCA1 protein is involved in repairmen of the damaged DNA. These breaks of DNA can occur in different ways, such as radiation, chemotherapy and cellular division, saying that, BRCA protein plays a critical role in maintaining a stable genetic structure of the cells.
2.1.1 Location of BRCA1 GENE
Cytogenetic Location: 17q21.31, which is the long (q) arm of chromosome 17 at position 21.31
Molecular Location: base pairs 43,044,295 to 43,125,364 on chromosome 17 (Homo sapiens Updated Annotation Release 109.20200228, GRCh38.p13) (NCBI)
2.1.2 Structure
The BRCA1 gene consists of 24 exons and codes for a 220 kD protein consisting of 1,863 amino acids (aa) (19,20).
BRCA1 has several domains that are essential for its function. The RING finger domain of BRCA1, commonly found in many DNA repair proteins, consists of a conserved core of approximately 50 amino acids in a pattern of seven cysteine residues and one histidine residue to form a structure that can bind to two Zn++ ions. This motif aids in mediating protein-protein interaction, as exemplified by the interaction of BRCA1 with BARD1 (BRCA1 associated RING domain). This interaction is critical since mutations in the Zn++ binding regions, crucial for heterodimerization with BARD1, have been found in tumors.
12 2.1.3 Mutations in BRCA1
The first reported mutations in BRCA1 were truncations; small insertions or deletions (indels), or nonsense mutations leading to an early stop codon. In 1995, the Breast Cancer Information Core (BIC) was established, and subsequently all kinds of mutations have been reported for BRCA1 (24). BRCA1 has several different transcripts that result from alternative splicing of exons 1- 11 (21).
It also has two alternative start codons, one resulting in a protein lacking the first 17 aa.
In addition, there is a pseudogene consisting of only exon 1 and 2 of BRCA1, further complicating the investigation of mutations in BRCA (22). According to Ensembl genome browser, there are 32 different transcripts (splice variants) of BRCA1 (23). Each gene carries as many as 1000 different disease associated mutations, the mutations are at a high penetrance therefore, women who carry these mutations have a lifetime risk of 80-90% to develop breast cancer. Founder mutations such as the BRCA1-185delAG and 5382insC are found among Ashkenazi Jews. Larger and complex genomic rearrangements in the exons 21 and 22 of the BRCA1 gene, resulting in a lack of the BRCT motif have been reported.
2.2 BRCA2
The BRCA2 gene is composed of 27 exons and spans approximately 84.2 kb of genomic DNA located at 13q13.1. The BRCA2 gene encodes a 11386 bp mRNA transcript. Transcription site is located 227 bp upstream the first ATG of the BRCA2 ORF. The translation start site is located in exon 2. It is translated to the BRCA2 protein. BRCA2 protein is a nuclear protein that is composed of 3418 amino acids (384 kDa).
The N-terminal part of the BRCA2 protein contains a transcriptional activation domain (aa 18-105). BRCA2 exon 11 encodes eight conserved motifs termed BRC repeats. Each of these repeats is composed of about 30 residues.
A DNA-binding domain has been located in the C-terminal region of the BRCA2 protein (aa 2478-3185). It is composed of a conserved helical domain and three OB folds. Two nuclear localization signals (NLS) have been identified in the C-terminal region of BRCA2.
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BRCA2 expression is different in different tissues, the rapidly dividing tissues express high amounts of the proteins such as epithelial cells including breast epithelium during puberty and pregnancy. other non-dividing cells do not express BRCA2 protein such as neurons and muscles.
The BRCA2 expression is regulated during the cell cycle, with highest expression during the S phase of the cell cycle. Most of the BRCA2 proteins are associated to DSS1. The presence of DSS1 was demonstrated to stabilize the BRCA2 protein.
BRCA2 has been implicated in maintenance of genomic integrity and in the cellular response to DNA damage (FIGURE 2). The BRCA2 protein interacts with the RAD51 recombinase to regulate homologous recombination (HR). BRCA2 regulates the intracellular localization of RAD51. It also targets the RAD51 to ssDNA and inhibits dsDNA binding, thus regulating/enhancing DNA strand exchange activity of RAD51. CHEK1 and CHEK2 both phosphorylate the RAD51/BRCA2 complex and regulate the functional association of this complex in response to DNA damage.
BRCA2 is also implicated in cell cycle checkpoints. Following exposure to X-rays or UV light, cells expressing truncated BRCA2 protein exhibit arrest in the G1 and G2/M phases.
BRCA2 protein plays a role in mitotic spindle assembly checkpoints through modulation of the level of spindle assembly checkpoint proteins including Aurora A and Aurora B.
A role in regulation of transcription has been attributed to BRCA2. BRCA2 binding to the DSS1 protein appears to be required for proper completion of cell division in yeast.
The BRCA2 protein demonstrated the ability to stimulate transcription. For example, exogenous expression of BRCA2 can stimulate transcription of androgen receptor-regulated genes. This function of BRCA2 is regulated by the binding of the EMSY protein to the region of BRCA2 responsible for transcriptional activation. An excess of EMSY results in silencing of BRCA2-driven transcriptional activation.
14 2.2.1 Mutations in BRCA2
Germinal - High risk of breast and ovarian cancer is associated with germline BRCA2 mutations. Cumulative risk of breast cancer in BRCA2 mutation carriers was estimated to 45%
by the age of 70 years while ovarian cancer risk in carriers was estimated to 11%. Increased risk of several other cancers are associated with BRCA2 mutations, especially for prostate and pancreatic cancer.
Somatic mutations in BRCA2 are infrequent in sporadic breast cancer. Methylation of the BRCA2 promoter has not been detected in normal tissues nor in breast and ovarian cancers. Loss of heterozygosity at the BRCA2 locus has been frequently found in sporadic breast and ovarian tumors. As for BRCA1, the first reported mutations in BRCA2 were truncating mutations. These mutations were easier to classify, in contrast to some of the later detected missense and intronic variants (24, 25).
Figure 2: BRCA functions
The BRCA1 protein has multiple functions in different cellular processes, including DNA repair, transcriptional activation, cell cycle regulation and chromatin remodeling. BRCA2 plays a role in transcriptional and cell cycle regulation, DNA repair, mitophagy and stabilization of replication fork. BRCA proteins and interacting partners are shown.
Functions of proteins are shown in rectangles. Interacting partners are shown in blue ovals.
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2.3 BRCA Genes as Caretakers of Genomic Stability 2.3.1 BRCA Relocation
BRCA1 relocates to DNA damage sites and forms nuclear foci following DNA double- strand breaks (DSBs)(26).
Following DNA damage, chromatin-associated histone H2AX that locates close to DNA damage sites is phosphorylated by ATM and ATR (Burma et al., 2001), and subsequently recruits a phospho-module binding mediator MDC1 and an E3 ubiquitin ligase RNF8 to DNA damage sites (27); RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly (28; 29; 30).
RNF8 functions together with an E3 ubiquitin conjugase Ubc13 to ubiquitinate histone H2A and H2B at chromatin lesions, which regulates the translocation of BRCA1 to DNA damage sites (31-32).
Following DNA damage, RAP80 recognizes ubiqutinated histone at the site of DNA damage via its ubiquitin-interacting motif (UIM) and recruits the big complex, including BRCA1 to DNA damage sites (Wu et al., 2009).
BRCA1 was first implicated in DNA damage repair because it translocated to DNA damage sites and co-localized with RAD51, an essential protein in homologous recombination repair (Scully et al., 1996, 1997b). Later on, studies from different groups showed us that BRCA deficient cells are more susceptible to DNA damage from UV light, radiation alkylating agents and IMPAIRED DNA DAMAGE REPAIR. (Scully et al., 1999).
2.3.2 BRCA Genes and Proteins Role in DNA Repair
BRCA1 and BRCA2 proteins play a crucial role in the process of DNA double strand break (DSB) repair by regulation of homologous recombination (HR) (33). HR is a high fidelity mechanism of DNA repair with using of homologous template such as sister chromatids.
Therefore, this process can be active in S and G2 phase of cell cycle when sister chromatids are available for HR. The homology-directed DNA repair process includes a few steps such as pre-
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synapsis, synapsis and post-synapsis (34). In the first step the Mre11-RAD50-Nbs1 (MRN) complex and its interacting partner C-terminal binding protein interacting protein (CtIP) with nuclease activity performs resection of the DSB ends to generate a 3′ - single strand (ss) DNA tail protected from degradation by replication protein A (RPA). Next, dependent on BRCA1 and BRCA2 proteins, RAD51-ssDNA filament invades homologous duplex DNA which serves as a template. This generates a D-loop (displacement loop) structure, which is a DNA structure where the strands of a double strand (ds) DNA are separated for a stretch by a third strand of DNA. The resulted nucleoprotein-filament searches for homologous DNA sequence on the sister chromatid and invades the duplex to form a joint molecule. Finally, during post-synapsis RAD51
dissociates from dsDNA and the 3′ end of the damaged DNA is elongated by DNA polymerases and followed by DNA ligation (35). The resulted intermediate structures of DNA recombination called Holliday junctions are further resolved by the different mechanisms as discussed
elsewhere (36) resulting in an error-free repair.
In addition to its role in HR-dependent DNA repair, BRCA1 also regulates the non- homologous end-joining (NHEJ) repair pathway. NHEJ is one of the main DNA repair pathways when the broken DNA ends are directly ligated without the need for a homologous template. HR is an error-free repair with a high fidelity at the site of correction, but it needs more time to complete. In contrast, NHEJ is an error-prone DNA reparation that causes mutations at the site of damage with a high frequency. However, it is relatively fast and the most common repair
mechanism for DNA DSBs (37). Classical (C) NHEJ predominates in G0 and G1 but can operate in all phases of the cell cycle. It consists of a few steps: break recognition, end-processing and ligation. First, DSB ends are recognized by the Ku70/Ku80 heterodimer, which recruits DNA- dependent protein kinase (DNA-PK) and other NHEJ proteins such as DNA polymerase, helicase and ligase. Then DNA-PK phosphorylates and recruits endonuclease Artemis, which further processes DSB ends. End ligation is facilitated by the XRCC4 (X-ray repair cross- complementing protein 4)/Lig4 (38).
In contrast to C-NHEJ mechanism, alternative (A)-NHEJ depends on other factors such as MRN complex, CtIP, and poly (ADP-ribose) polymerase-1 (PARP-1) that plays a role in the DNA lesion detection and recruitment of the protein to the sites of DNA damage (39), (40). A-
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NHEJ functions as a backup repair pathway when C-NHEJ is compromised, and its mechanism is less defined than for C-NHEJ (41).
BRCA1 is involved in both, C-NHEJ and A-NHEJ pathways (FIGURE 4). BRCA1 interaction with the C-NHEJ factor Ku80 stabilizes the Ku heterodimer at DSB sites that is required for precise end-joining repair. In contrast, a growing body of evidence suggests that BRCA1 blocks A-NHEJ through phosphorylation of BRCA1 at S988 by checkpoint kinase 2 (Chk2) (42), (43), but the exact mechanism of this regulation is unknown.
Studies of BRCA genes revealed their interaction with HR factors such as RAD51 [44], (45) a central regulator of the strand exchange (46). Recent studies demonstrated that BRCA1 promotes HR-dependent DNA repair by dephosphorylation of 53BP1 (p53-binding protein 1) (47) that consequently results in the repair pathway switch from NHEJ to HR. Interaction between BRCA1, CtIP and MRN complex has been shown to be important for activation of HR by the mechanisms involving CDK (cyclin-dependent kinase)-mediated phosphorylation of CtIP at Ser327 (48). BRCA1 is important for BRCA2 recruitment to the sites of DNA DSBs during HR, and association between these two proteins is mediated through interaction PALB2/FANCN (Fanconi anemia, complementation group N) protein (49), (50).
The role of BRCA2 in repair of DSBs has also been extensively studied. It was demonstrated that BRCA2 plays an essential role in HR by the mechanisms involving recruitment of RAD51 to the sites of DSBs (FIGURE 5) (51). The loss of BRCA2 leads to genomic instability and tumorigenesis (52). This can be, in part, explained by the role of BRCA2 in regulation of the intracellular localization and DNA binding ability of RAD51 recombinase.
After detection of DSB by the MRN complex, the consequent protein phosphorylation and ubiquitination events recruit BRCA1 and CtIP proteins to the site of DNA DSB. Together with Exo1 and DNA2-BLM (Bloom syndrome protein) exonucleases, this complex triggers DNA end resection and mobilizes BRCA2 to the sites of DNA DSBs. BRCA2 promotes HR by the
displacement of RPA and recruitment of RAD51 recombinase on the sites of DNA damage.
BRCA2 directly binds to RAD51 through its BRC repeats and TR2 domain and therefore facilitates the loading of RAD51 on ssDNA and a search of homologous DNA template (53)-(
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54) ,( 55). BRCA2 may function as a complex with RAD51 paralogs such as XRCC2 and XRCC3 to facilitate an assembly of RAD51 with ssDNA (56).
BRCA2 plays a protective role for the maintenance of genomic stability upon replication stress. It has been characterized as a regulator of the stalled DNA replication fork by loading and stabilization of polymerized RAD51 onto DNA through binding to its BRC repeats (57). At the same time BRCA2 can prevent formation of chromosomal aberrations during replication stalling by inhibition of MRE11 nuclease (58) (Figure 3). BRCA2 is also recruited by 3′-repair
exonuclease 2 (TREX-2) complexes for processing of R-loops, the structures formed during transcription and composed of a DNA-RNA hybrid and associated ssDNA (59). BRCA2 can protect telomere integrity via loading of RAD51 on telomeres during S/G2 phase that is
evidenced by the accumulation of telomere dysfunction-induced foci and telomere shortening in Brca2- but not Brca1- deficient mice (60).
FIGURE 4. BRCA1 PATHWAY
BRCA1 works as a signal processor (1) during DNA damage responses in complex with proteins that bind to aberrant DNA structures (sensors), and the kinases that signal their
presence. Phosphorylation of BRCA1 may be essential for local functions [control of DSB resection (2), altering DNA topology (3)] near a DNA lesion, as well as for distant functions such as transcriptional control of checkpoint genes (4) (e.g., GADD45) or targets of estrogen receptor signaling (see Note Added in Proof), or transcription-coupled DNA repair (5). BRCA1 works with BARD1 (6) as an ubiquitin ligase
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FIGURE 5. PROTECTIVE ROLE OF BRCA2
DNA damage or replication arrest cause DSBs that activate signaling mechanisms (1) and are then resected (2) by exonuclease activity to generate ssDNA tracts. RAD51 is loaded onto the ssDNA (3) to form a nucleoprotein filament that mediates homologous pairing (4) followed by strand extension (5), exchange, and repair (6).
BRCA testing:
Genetic testing of BRCA1/2 done in many countries. The selection of candidates fitting for testing is according to the recommendations as listed (table 1). A peripheral blood sample is used for this test. Written informed consent should be signed by the individuals taking the test.
Genetic consulting is also necessary before performing the test and when giving the results.
Sequence variants in BRCA1 and BRCA2 can be subdivided into three broad classes: single- nucleotide changes, small insertion or deletion events (indels) and large genomic rearrangements (LGRs). BRCA testing is usually done by (Sanger) DNA sequencing. This method is considered the ‘gold standard' of DNA sequencing this because it is reliable, widely available and simple to use. The limitations of Sanger sequencing when compared to Next Generation Sequencing (NGS) are that it has low throughput and lower cost-effective testing. In addition, Sanger sequencing cannot detect LGRs, which require alternative polymerase chain reaction (PCR)- based techniques for analysis. NGS offers many advantages over Sanger sequencing, such as high throughput, lower cost Automated Analysis Uses less DNA can run in parallel with other genetic tests. A major disadvantage is that it needs Sanger sequencing for confirmation of the results (76).
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Table1: BRCA mutation testing recommendation:
3. METHODS
This literature review was conducted in accordance to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The search was performed in Medline (PubMed) and Google Scholar. The following search strategy was implemented:
(((BRCA1 AND BRCA2 MUTATION AND BREAST CANCER AND TRIPLE NEGATIVE) OR BRCA AND TRIPLE NEGATIVE BREAST CANCER) OR BRCA CARRIERS IN TRIPLE NEGATIVE BREAST CANCER))
Table2: selection of studies
INCLUSION CRITERIA EXCLUSION CRITERIA
BRCA mutation carriers Unknown BRCA status
TNBC (triple negative breast cancer) Men
Women (<60 y.o) Low statistical power / Biased article Early onset breast cancer Recurrent / post- treatment breast cancer Less than 10 years old (2010-2020) Other comorbid malignancies
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English language – full access Not meeting the inclusion criteria
The last PubMed search was on Feb 10th, 2020. This resulted in 3066 citations at the time. There were no duplicates removed as none could be found. All articles were conducted in English, that utilized humans as the focus of study, were published within the last 10 years (2010-2020), and centered around women between 20 – 50 years old were included. Any article that focused on men or old women as the sample to study, and did not meet all the inclusion criteria, was excluded. Of the 3066 citations, 634 articles were retrieved after the first
title/abstract screening. 552 more articles were excluded after a full text because of no proper relation to the main topic. Then other 70 articles were excluded due to low power and bias in the study, to end up with 12 articles (figure 6).
Figure 6. Inclusion and exclusion flow chart that describes the data collection process.
634 Titles and abstract were reviewed
82 full text reviews was done
12studies were included in this systematic review 3066 works found on initial
search
2432 works were excluded based on exclusion criteria
552 works were excluded based on title and abstract
review
70 studies were excluded based on bias inappropriate statistical
analysis and duplication of information
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STUDY LIMITATIONS
This study has several limitations. To begin with, only 2 sources, PubMed and Google Scholar were used in finding articles for the purpose of this review. Moreover, only one individual carried out the research behind this thesis. This could potentially cause errors in overlooking essential articles and important data that could have been implemented in this study.
In addition, there were not enough data found when searching the pathogenic variants of
BRCA1/2 for specific types of breast cancer, this mainly because of limited resources and limited access to some articles and large databases. Also this information wasn’t provided in some of the articles that were reviewed.
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4. RESULTS
In the study by Wong-Brown et al. The mutation prevalence was 9.3 % in Australia and was 9.9 % in Poland. the mean age of diagnoses of BRCA1 mutation carriers was significantly lower than that of non-carriers, while the age of onset of BRCA2 mutation carriers was similar to that of non-carriers. In the Australian cohort, 59 % of the
mutation-positive patients did not have a family history of breast or ovarian cancer. The pathogenic variants of BRCA1/2 in this study were not presented. In other study by Gonzalez-Angulo et al., the prevalence of BRCA1/2 in TNBC was 19.5%. 12 (15.6%) in BRCA1, and 3 (3.9%) in BRCA2. Patients with BRCA mutations tended to be younger than WT (wild type). Grade, histology and stage were not associated with mutation status. patients with BRCA mutations had a significantly better RFS (recurrence free survival). The most commonly seen mutations were deleterious type. The pathogenic variants which were found in the cohort are (BRCA1 187delAG, BRCA1 2795delAAAG, BRCA1 M1775R (5443T>G), BRCA1 3829delT, BRCA1 C61G (300T>G) BRCA1 E29X (204G>T), BRCA1 S451X (1471C>G), BRCA1 E1134X (3519G>T), BRCA1 Del Exon 17. And for it was BRCA2 5804del4, BRCA2 5578delAA, BRCA2 E3111X (9559G>T).
Hartman et al. prevalence was not similar, the prevalence of BRCA1/2 in TNBC is 10.6% the median age of the cohort was 54, and 51.5% were postmenopausal. 66% of our cohort was Caucasian, 15.7% Hispanic, and 13.6% African American. The majority (76.9%) did not have a significant family history of BC. 21 (10.6%) of the patients had a mutation, 13 mutations (8 BRCA1 and 5 BRCA2) were identified among the 86 patients diagnosed at <50 years, and 8 mutations (5 BRCA1 and 3 BRCA2) were identified among the remaining 113 patients diagnosed at ≥50 years.
Pogoda et al. BRCA1/2 pathogenic variants were detected in 30 patients, 29 of them had the BRCA1 mutation and only one case had BRCA2 mutation, their prevalence was 24%. The following BRCA1 pathogenic variants were found: c.5266dupC (5382insC) in 18 patients, c.181T>G (C61G, 300T>G) in 5 patients, c.3700_3704delGTAAA
(3819del5) in 2 patients, and c.5251C>T (5370C>T), c.5345G>A (p.Trp1782X), c.3756_3759delGTCT (3875del4), and c.68_69delAG (185delAG) in 1 patient each, respectively. One patient harbored BRCA2 gene mutation c.5744C>T (C5972T). When comparing BRCA1/2 pathogenic variant carriers to non-carriers we can notice that carriers were younger at the age of diagnosis, 41vs47 years old. Contralateral breast cancer developed in 26.5% of BRCA1/2 mutation carriers and in 14% of non-carriers.
Non-carriers had more often G3 tumors. while carriers had a smaller tumor size. While Couch et al., the BRCA1/2 mutations were found in 11.2% of TNBC patients (BRCA1 (8.5%) and BRCA2 (2.7%)). Deletion mutations were also identified in other 15 in 3.7%
of the patients. Patients with TNBC with mutations were diagnosed at an earlier age (P <
.001) and had higher-grade tumors (P = .01) than those without mutations. For BRCA1 145 truncated mutations were identified and 10 missense mutations:
185delAG (c.68_69delAG) detected in 18 patients, 5382insC (c.5266dupC) detected in 19 patients. As for BRCA2 the variant 6174delT (c.5946delT) was found in 6 patients.
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Large rearrangement mutation in BRCA1 was identified in two African–American patients. Patients with a BRCA1 mutation were younger at TNBC diagnosis compared to patients without a BRCA mutation (median age 40.2 vs. 55.7 years’ p <0.0001). No difference in age was identified between those with BRCA2 mutation carriers and the non-carriers. BRCA2 mutation prevalence was 4.3% (9/207) which were also deleterious.
The pathogenic variants of these genes were not listed in this study (Sharma et al.,2014).
Liu et al. study have had the highest BRCA1/2 prevalence among the TNBC patients (34%). Their median age at diagnosis was 47 years.16 (12.8%) were diagnosed when they were younger than 35 years; 11 (8.8%) had family histories of breast or ovarian cancer. Invasive ductal carcinoma was common (90.4%).
The study done on Sardinian population have demonstrated a prevalence of 14.3% for BRCA1/2 mutation carriers in TNBC patients. Overall, looking at the prevalence of BRCA1/2 mutation in TNBC and non-TNBC, deleterious BRCA1/2 mutations were detected in 21/726 (2.9%) breast cancer cases :7(BRCA1) and 14(BRCA2). (3.9%) patients with an age at diagnosis of ≤50 years were found to carry a BRCA1/2 mutation;
no statistical difference was found when looking at age otherwise. The rate of BRCA1/2 mutations was significantly higher in the group of patients with a triple-negative primary tumor compared with those without such a feature [7/49 (14.3%) vs. 14/677 (2.1%), respectively; P=0.012.The presence of a triple-negative phenotype was strongly
associated with the BRCA1 mutations at a highly significant level (P<0.001), whereas no association was found with the BRCA2 mutations (P=0.837). Other study von Minckwitz et al. have focused on the association between BRCA1/2 pathogenic variant frequency in TNBC and the presence of family history of BC, results show that 31 mutation carriers were identified in patients with Family history of BC, in compare to only 19 of 181 (10.5%) patients without a positive family history of cancer. While Yadav et al. have studied the survival and age at diagnosis of those who have BRCA positive TNBC to those who are BRCA negative, he found that BRCA mutation carrying patients were more likely to get diagnosed at younger age than non-carriers (46 VS 52 years old). Also there was no significant difference in 2 and 5 years’ survival between the BRCA
mutation carriers and non-carriers.
In study by Hoyer et al. the prevalence of BRCA1/2 mutation was 20.5% (61/299).
Deleterious BRCA1 mutations in 14.8% of TNBC patients. These were predominantly frameshift mutations (24/34, 70.6%). The most frequent mutations both among them and in total were the founder mutations: c.5266dupC and c.2411_2412delAG.
Other mutations were found: c.3481_3491del in 3 patients; c.181 T > G in 2 patients;
c.1504_1508delTTAAA in 2 patients; c.3700_3704delGTAAA in 2 patients. Deleterious BRCA2 mutations occurred in 5.7% of patients, all being unique, but one (c.1813dupA), that have been found in 2 patients. Domagala P et al. have showed similar results, the most common BRCA1 pathogenic variants that were detected in this cohort are including the 5 founder mutations c.5266dupC, c.4035delA, c.181T>G, c.3700_3704delGTAAA, c.5251C>T. and other rare mutations (c.5030_5033delCTAA, c.1687C>T, c.3936C>T).
The most frequent BRCA1 mutation (c.5266dupC) was detected in 0.17% of population
25
controls. But there were no BRCA2 pathogenic variants identified. While the prevalence of BRCA1/2 mutation in TNBC was 18.4%.
Table 3: BRCA1/2 mutation prevalence in triple-negative breast cancer (TNBC) patients
Author Study
type
Year Country TNBC
N=
TNBC (BRCA1/2 carriers n=) Wong-Brown et al., 2015
(61)
Cohort study
2015 Australia Poland
774 9.6 %
(74 /774) Gonzalez-Angulo et al.,
2011 (62)
Cohort study
2011 US- Texas M.D. Anderson
Cancer Center
77 19.5%
(15/77)
Hartman et al.,2011 (63) Cohort study
2011 US-TEXAS 199 10.6%
(21/199)
Pogoda et al.,2020 (73)
Cohort study
2020 Poland 124 24%
(30/124) Ouch et al.,2014
(64)
Cohort study
2014 United States 1,824 11.2%
Sharma et al.,2014 (65)
Cohort study
2014 US- KANSAS CITY
207 15.4 %
(32/207) Liu et al.,2017
(66)
Cohort study
2017 Z`China 26 34.6%
(9/26)
PALOMBA et al.,2014 (67)
Cohort study
2014 Sardinia 49/726 14.3%
(7/49) von Minckwitz et
al.,2014 (68)
Clinical trial
2014 Germany 110 28.2%
(31/110)
26 Yadav et al.,2018
(74)
Retrospe ctive cohort
2018 US-
MICHIGAN
266 27.0%
(72/266) Hoyer et al.,2018
(75)
Cohort study
2018 Germany 299 20.5%
(61/299)
27
5. DISCUSSION
In this review a strong association between BRCA1/2 genes and TNBC was reviewed.
The Cancer Genome Atlas found germline BRCA1/2 mutations in 5.3% of unselected breast cancers, while Couch et al. [64] showed that 11.2% of unselected TNBC cases had deleterious mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) gene, respectively. Hence we can conclude that the presence of BRCA1/2 pathogenic variants increases the risk of TNBC development.
TNBC patients with pathogenic variants were diagnosed at a younger age and had higher-grade tumors than those without pathogenic variants. A mean age at first diagnosis of 44 and 47 years was reported for TNBC patients with BRCA1 and BRCA2 mutations, respectively, while TNBC patients without pathogenic germline mutations show a mean age at first diagnosis of 51 years. Another study by Yadav et al. showed that BRCA mutation carriers TNBC patients were more likely to get diagnosed at younger age than non-carriers (46 VS 52 years old).
Another 5 articles have had similar results (74) (67) (66) (65) (73).
When stratified by family history, the study by Couch et al. (64) revealed that 66 of 539 (12.2%) TNBC patients with a family history of cancer carry pathogenic BRCA1/2 mutations, compared to 83 of 969 (8.6%) patients without a family history of cancer. In contrast, Wong- Brown et al., results showed that in the Australian cohort, 59% of the BRCA1/2 mutation carrying patients did not have a family history of breast or ovarian cancer. While Hartman et al concluded that the majority (76.9%) did not have a significant family history of BC. von Minckwitz et al. have also presented that only 19 of 181 (10.5%) patients without a positive family history of cancer compare to 31 mutation carriers that were identified in patients with Family history of BC. Therefore, most of the studies agreed that there are higher percentage of BRCA1/2 pathogenic variants in TNBC patients without a family history of BC.
Looking at the ratio of BRCA1/BRCA2 mutations the numbers have always been in favor for BRCA1 as was shown in the study by PALOMBA et al. (67). The results showed that 7/49 have BRCA1/2 mutations in which 6/7(85.7%) had the BRCA1 mutation. The presence of a triple-negative phenotype was strongly associated with the BRCA1 mutations at a highly
significant level (P<0.001).
28
The pathogenic variants of BRCA1 and BRCA2 were also reviewed and compared. The most commonly seen pathogenic variant in BRCA1 gene was the c.5266dupC (Pogoda et al.) (73). It has been found to be strongly associated with TNBC, especially when considering the results that were provided by Couch et.al (64), the c.5266dupC mutation was found in 19 patients followed by c.68_69delAG mutation in 18 patients. This finding is also pronounced in other studies such as Hoyer et al. (75), where c.5266dupC and c.2411_2412delAG were the two most common pathogenic variants observed. Domagala et al. (72) presented that the most commonly seen pathogenic variants in BRCA1 that result in abnormal protein product are frameshift (e.g., c.5266dupC, c.4035delA, c.3700_3704delGTAAA, c.68_69delAG, c.5030_5033delCTAA), nonsense (c.5251C>T, c.1687C>T, c.3936C>T), and some deleterious missense mutations (e.g.;
c.181T>G). In contrast, c.4035delA variant was not as commonly seen in other studies as described in the results table (table2). These conflicting findings may occur due to the different regions and populations where the studies were conducted. Functional studies of the c.181T>G mutation show that it results in inactivation of BRCA1 E3 ligase activity and is defective in homologous recombination (70,71). The BRCA1/2 germline mutations were more commonly seen in TNBC when compared to hereditary non-TNBC as the frequency was 18.4% and 2.3%
respectively (72).
The BRCA2 pathogenic variants varied when comparing the survival of those who tested positive for BRCA mutation and those who tested negative. It can be noticed that there was no significant statistical difference in 2 and 5 years of survival between mutation carrying patients and non-carriers(74).
29
6. CONCLUSION
1. BRCA1/2 mutation frequency in triple negative BC was 7.2% (691/9496).
Comparing the two pathogenic variant frequencies, BRCA1 is more strongly associated with TNBC than BRCA2 is. Furthermore, a high expression of nuclear grade and large tumor burden were significantly more common in patients with BRCA1 mutation than that of BRCA2 mutation.
2. The most common BRCA1 pathogenic variants in this study were: c.5266dupC, c.2411_2412delAG, c.68_69delAG and c.3481_3491del
The most common BRCA2 pathogenic variants found in this study were:
c.7878G > C, c.5946delT, c.5744C>T.
Theses pathogenic variants that were the most frequently seen in the 12 studies reviewed, however it is clear that a lot of contradictory findings exist. Therefore, a more thorough, wide- scale meta-analysis is required to properly assess the frequencies of the different pathogenic variants of BRCA1/2 in TNBC patients and to compare with other pathologic types of BC. Also a wider study should be performed in order to compare different pathogenic variants frequencies in different populations.
30
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