Somatic Mutations in BRCA2 BRC Repeat Associated with Outcome in Patients with High Grade Serous Ovarian Cancer

Background The interaction between BRCA2 BRC repeats and RAD51 is one of the great important factors affecting the homologous recombination in DNA damage repair of tumor cells. We investigated the effect of BRCA2 BRC repeat mutations on outcome in patients with high grade serous ovarian cancer (HGSOC) who received platinum-based chemotherapy. Methods We identied the type and location of BRCA2 BRC repeat mutations by PCR and DNA sequencing in tumor and peripheral blood leukocytes (PBL) samples of 113 patients with stage IIIC/IV high grade serous ovarian cancer (HGSOC), and assessed chemotherapy-free interval (CFI), progression-free survival (PFS) and overall survival (OS).


Abstract Background
The interaction between BRCA2 BRC repeats and RAD51 is one of the great important factors affecting the homologous recombination in DNA damage repair of tumor cells. We investigated the effect of BRCA2 BRC repeat mutations on outcome in patients with high grade serous ovarian cancer (HGSOC) who received platinum-based chemotherapy.

Methods
We identi ed the type and location of BRCA2 BRC repeat mutations by PCR and DNA sequencing in tumor and peripheral blood leukocytes (PBL) samples of 113 patients with stage IIIC/IV high grade serous ovarian cancer (HGSOC), and assessed chemotherapy-free interval (CFI), progression-free survival (PFS) and overall survival (OS).

Conclusions
Somatic mutations in BRCA2 BRC5-8 repeat motifs are associated with platinum-based chemotherapy sensitivity and a better outcome in patients with HGSOC.

Background
High-grade serous ovarian cancer (HGSOC) is the most common histological subtype (about 70%) of epithelial ovarian cancer (EOC) [1,2] and is a genetically heterogeneous disease that exhibit highly individual evolution and genomic diversity [3][4][5]. Approximately 50% of HGSOCs harbour genetic and epigenetic alterations in gene members of the homologous recombination (HR) DNA repair pathway, most commonly in BRCA1 and BRCA2 [6]. For a woman with a BRCA2 mutation, the risk of EOC is 12-20% [7]. Several studies of multidimensional genomics and clinical data have revealed that BRCA2 mutations are associated with bene cial survival and platinum-based chemotherapy sensitivity in patients with HGSOC [8][9][10]. However, analysis of two independent cohorts indicated that only HGSOC patients whose germline or somatic mutations of BRCA2 occur most commonly in exon 11 which codes functionally distinct BRC repeat motifs for RAD51 binding domain (RAD51-BD) prolong platinum-free interval and have better survival [11,12]. These ndings suggest that the location and type of BRCA2 BRC repeat mutations may be associated with highly sensitive to platinum-based therapy in EOC patients.
BRCA2 plays crucial role in regulating the actions of RAD51, a recombinase essential for homologydirected repair of DNA double strand breaks (DSBs) [13][14][15][16]. The direct interaction between BRCA2 and RAD51 and their colocalization in nuclear foci after DNA damage was the rst evidence for a role for BRCA2 within this DNA repair pathway. BRCA2 is directly involved in RAD51-mediated repair, affecting the choice between gene conversion (GC) and single-strand annealing (SSA). BRCA2 BRC repeats bind RAD51 and are essential for the function of both proteins. BRCA2 binds directly with RAD51 and delivers RAD51 to DNA DSBs through the eight conserved BRC repeats [13,[17][18][19] and promotes RAD51 speci c recruitment to DNA damage sites where homologous recombination (HR) process is initiated for cellular response to genotoxic agents by mediating DNA strand exchange during recombination [17,20]. BRCA2 BRC repeats are be provided with functionally distinct effects on RAD51 function. These repeats have been shown to bind distinct regions of RAD51, con rming nonequivalent interactions between the different BRC repeats and RAD51. BRCA2 BRC repeats and their intervening sequences mediate HR repair of DNA damage by two functionally different RAD51-BD binding modules, BRC1~4 and BRC5~8 repeat domains. Distinct binding of BRCA2 BRC repeat domains to RAD51 modulates DNA-binding selectivity and confers differential DNA-damage sensitivity [19,21,22]. It is proved that RAD51-mediated HR repair of DNA damage is dependent on the modular architecture BRC repeats of BRCA2 [23][24][25].
Almost all HGSOC patients in a clinical setting receive platinum-based chemotherapy, inducing interstrand adducts, and then resulting in DSBs in DNA. In response to DNA DSBs, the interaction between BRCA2 BRC repeat motifs and RAD51 is of great importance in creating a BRC-RAD51 complex for HR repair. Defective binding at a single BRC repeat or a single point mutation within an individual BRC repeat domain can be enough to impair this interaction [14,26]. Therefore, BRCA2 BRC repeat mutations may affect platinum sensitivity.
In the present study, we aimed to determine the effect of BRCA2 BRC repeat mutations on platinum-free interval (PFI), progression-free survival (PFS) and overall survival (OS) in patients with stage IIIc/IV HGSOC. Here we provide a clinical implication that the location and type of BRCA2 BRC repeat mutations may disrupt BRCA2 function and be associated with platinum-based chemotherapy sensitivity in HGSOC.

Ethics statement
The current study was approved by the Ethics Committee of the Institutional Review Board of Sichuan Cancer Hospital and Institute performed in strict accordance with the Declaration of Helsinki. All participants signed informed consent prior to enrollment.

Study subjects
Tumor tissue and peripheral blood leukocytes (PBL) were obtained from 113 HGSOC patients who received primary debulking surgery (PDS) followed by a 6 cycles of platinum-based chemotherapy between March 2015 and December 2017. The tumor specimens and PBL were immediately ash frozen in liquid nitrogen and stored at −80°C in the bio-bank of Oncology Research Laboratory at our institute until extraction of genomic DNA was performed. The amplicons from the obtained genomic DNA were used to identify if mutations in the type and location of BRCA2 BRC repeat by PCR sequencing. All patients enrolled in this study were histopathologically con rmed as HGSOC with FIGO stage III/IV and showed no family history of ovarian cancer. Clinicopathologic and follow-up data of patients were collected by our Hospital Patient Information Reporting System.

DNA Extraction
Genomic DNA with a size of greater than 5Kb was extracted from tumor samples and PBL using DNA sequencing and sequence alignment PCR ampli ed fragments (Fig 1 C) were bi-directionally sequenced using 5 primer pairs (Supplementary Table 1) with ABI-3730XL Sequencer. All the nucleotide changes identi ed were con rmed by repeating the PCR and sequencing reaction using the corresponding forward and reverse primers and by aligning with a homologous sequence of human BRCA2 exon 11 from NCBI sequence database using BLAST (www.ncbi.nlm.nih.gov/Blast.cgi).

Detection of the BRCA2 N-and C-terminus by immunohistochemistry
Immunostaining for BRCA2 N-terminus (aa.188-563) and C-terminus (aa.3245-3418) using Anti-BRCA2 antibody ([3E6], ab97, Abcam) and anti-BRCA2 Ab-2 (Clone: CA1033, Millipore) was performed as described in the manufacturer's protocol. Brie y, 4 μm thick sections were cut from the para n-embedded tumor biopsies. These sections were mounted on amino-propyl-ethoxy-silan (APES) coated glass slides. Sections were depara nized in xylene and rehydrated in ethanol. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxidase for 30 minutes. The area around the tissue sections was scored with a Pap pen to limit the amount of antibodies and reagents used. Staining was visualized by 3'3-diaminobenzidine tetrahydrochloride and counterstaining was performed with hematoxylin. PBS without the primary antibody served as negative control.

Outcome evaluation
The primary end-point was progression-free survival (PFS). Secondary endpoints were chemotherapy-free interval (CFI) and overall survival (OS). Date of rst relapse was de ned as the rst instance of disease progression based on computed tomography imaging by Response Evaluation Criteria In Solid Tumors (RECIST ,version 1.1) or clinical progression [27]. Chemotherapy-free interval (CFI) was de ned as the interval between the time of completion of platinum-based chemotherapy and the date of rst progression/relapse or death; PFS was de ned as the interval between histologic diagnosis and rst relapse, death or the last follow-up (censored); OS was de ned as the interval between histologic diagnosis and the date of death from any cause or last follow-up (censored). CFI of <6, 6-12 and ≥12 months was classi ed as platinum-resistant, partially platinum-sensitive and platinum-sensitive, respectively by Gynecologic Oncology Group (GOG) [28].

Statistical Analysis
Standard statistical tests were used to analyze the clinical and mutation data, including the chi-square test, Fisher's exact test, log-rank test, and Cox proportional hazard analysis. Signi cance was de ned as two-sided P value less than 0.05. All statistical analyses were performed using SPSS software (version 18.0).

Results
Genomic DNA and ampli ed fragments are quali ed for DNA sequencing The genomic DNA samples with a size of >5Kb ( Fig 1B)isolated from all HGSOC tissues and PBL were fractionally ampli ed by PCR. First-round PCR amplicons of BRCA2 exon 11 (c.2803-c.6728) ( Fig 1A) were ampli ed from genomic DNA using the 8 pairs of primers; the second-round PCR amplicons covering BRCA2 BRC1~8 repeats and their spacing sequences (c.2915-c.6534) were ampli ed from the rst-round PCR amplicons using the other 8 pairs of speci c primers and available for DNA sequencing (Fig 1B and C).

The mutation sites in BRCA2 BRC repeat
No mutation in BRCA2 BRC repeat was identi ed in PBL of all patients with HGSOC. 44 different nucleotide mutation sites in BRCA2 BRC repeat were identi ed in tumor tissues of 27 HGSOC patients, inclunding 10 of them were silent mutation, 26 missense mutation and 8 nonsense mutation. Most of them were situated in the spacing sequences between the evolutionary conserved BRC domains. Only 2 (4.5%) missense mutations (c.5076G>A and c.5587A>T) and 2 (4.5%) nonsense mutations (c.5038T>C and c.5608T>C) were located within BRC5 and BRC6 domain, respectively, others (91%) were occurred in the spacing sequences between the BRC4~8 repeat domains( Fig.2A and Table 1). These results showed that all the patients identi ed were somatic but not germline mutations.
Nonsense mutation leads to C-terminal truncation of BRCA2 protein Immunohistochemistry results showed that all patients were positive immunostaining in the N-terminus of BRCA2 protein, but 8 cases with nonsense/frameshift mutation were negative immunostaining in the C-terminus (Fig. 2C), proving that nonsense/frameshift mutation leads to C-terminal truncation of BRCA2 protein.

Discussion
Up to 50% of patients with HGSOC are described as having identi able defects in the HR pathway, with the archetypal defects being germline or somatic BRCA2 inactivation that results in a distinct clinical phenotype comprising hypersensitivity to platinum and prolonged survival [2,7,9,29]. DNA DSBs are more problematic than SSBs since the complementary strand is not available as a template for repair. DSBs may arise as a result of either exogenous insults, such as exposure to ionizing radiation (IR) and platinums. Three DSB repair pathways have been identi ed within eukaryotic cells: nonhomologous endjoining (NHEJ), GC and SSA. Both GC and SSA rely on sequence homology for repair while NHEJ utilizes no, or little, homology. BRCA2 is the major recombination mediator and regulator in mammalian cells and involved in HR repair of DNA DSBs and maintaining genome stability [30,31]. BRCA2 can interact with RAD51 through multiple sites of BRCA2 and control recombination and/or of genomic integrity through binding to RAD51. Cells de cient for functional BRCA2 show various cellular abnormalities including increased sensitivity to genotoxic agents, accumulation of DNA damage, changes in cell cycle checkpoint and apoptotic responses [32,33]. Surprisingly, not all BRCA2 mutations are highly sensitive to DNA damage agents. Location and type of mutation in BRCA2 has been shown to be associated with inactivate BRCA2 [11,12]. Mutations in BRCA2 at other locations (either exons 1~10 or exons 12~27) than the BRC repeats (exon 11) do not impact the outcome in patients with HGSOC compared to those with non-mutation, suggesting that only germline or somatic mutations in the BRC repeats or RAD51-BD but not other domains are highly sensitive to DNA damage agents such as platinums.
Interaction between BRCA2 and RAD51 mediated by BRC repeats is critical for the cellular response to DNA damage [34]. The BRCA2 encodes the eight conserved BRC repeats (residues 1009~2082) with approximately 35 amino acids that are located well spaced from one another amidst the long and divergent exon 11 region, and the spacing sequences between individual repeats varies from 60 to 300 amino acids [21,25]. The role of the BRC repeats in DNA repair has been well characterized in the induction of ionizing radiation-or platinum-induced assembly of RAD51 complex, which is independent of the BRCA2 C-terminal domain [20,35].
Although the BRC repeats are highly conserved between mammalian species, the individual repeats differ greatly from one another within a species [18],suggesting a speci c role for each BRC in RAD51 binding. In fact, the BRC repeats has been shown to work in two classes of regulatory elements (BRC1~4 and BRC5~8) that, via distinct mechanisms, display unique functional characteristics to ultimately facilitate loading of RAD51 onto sites of DNA damage. BRCA2 binds to monomeric RAD51 via its BRC repeats and the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding revealed that two distinct clusters of residues in the BRC repeats can differentially regulate DNA-binding selectivity and sensitivity of RAD51 in targeting active RAD51 to single-stranded DNA and prohibiting RAD51 nucleation onto double-stranded DNA (dsDNA) [19,24,25]. The BRC1~4 repeats share the common property of inhibiting the binding of RAD51 to dsDNA, the BRC5~8 repeat domain can e ciently repair nuclease induced DNA DSBs and accelerate the assembly of RAD51 repair complexes upon DNA damage [23][24][25].
While the BRC repeats are themselves well conserved, the intervening sequences between consecutive BRC repeats are remarkably poorly preserved, suggesting that the intervening sequences are indispensable for preservation of the functional structure of BCR repeats [25,36] and that disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeats of BRCA2, might modulate the ability of RAD51 to promote recombinational repair [37]. Therefore, mutation at sites crucial for the interaction between BRC repeats and RAD51 disrupts the BRCA2-RAD51 complex formation and impair the ability of BRCA2 to recruit RAD51 to DNA DSBs. It is striking that the principal role of BRCA2 in HR is dependent on its interaction with RAD51 through the BRC repeats and yet, no deleterious missense mutations have been located in that region. One reason for this might be that the other BRC repeats can compensate for the mutated one. However, this explanation is at odds with the prediction from the structural analysis of mutations affecting this region by which the BRC repeats would form a Velcro-strip like structure where the mutation of one BRC repeat would affect the interaction of the other BRC repeats with RAD51 [14]. Consistent with the role of the BRC repeats in contacting RAD51, several mutations affecting the structure of the BRC repeats have shown that the weakening of RAD51 a nity in the case of even one repeat is su cient to affect BRCA2-RAD51 complex-mediated HR repair of DNA damage [21]. This provides insight into why mutation in just one of the BRC repeats of BRCA2 affects the way that RAD51-mediated HR of DNA damage.
Nonsense or missense mutation in BRCA2 BRC repeats, which results in an impaired function of BRCA2 protein, disrupts a RAD51-binding domain-mediated HR of DNA damage, then increasing the sensitivity to DNA crosslinking agents such as platinums.
In this study, we showed a relatively high ratio (21.23%) of BRCA2BRC repeat mutations in patients with HGSOC, including 7.1% nonsense mutation and 14.13% missenes mutation (Fig 2B and Table 1), this may be involved in mutation predisposition to stage IIIc/IV HGSOC that exhibits highly individual evolutionary trajectories prior to therapy [38][39][40]. Interestingly, our results showed that missense mutation in BRCA2BRC repeats signi cantly prolonged CFI, PFS and OS in patients with HGSOC compared to those with non-mutation (Fig 3). Surprisingly, 91% of the mutation sites identi ed in the BCR repeat region occurred in the intervening sequences between consecutive BRC4~8 repeat, proving that the BRC repeats are highly conserved across while most of the intervening sequences are not, suggesting that the BRC repeats are important for BRCA2 function mutation in the poorlyconserved intervening sequences contributes to inactivation of BRCA2 and disrupts the interaction of the BRC repeat domain with RAD51 for repair of DNA DSBs [24,25,35,41]. This can be explained by the absence of analysis of BRCA2 based on the two modules of BRC repeats that display unique functional characteristics of BRCA2. The BRC5~8 repeat domain of BRCA2 is responsible for repair of DNA DSBs induced by inter-strand crosslinks. DNA DSBs caused by platinum can be deemed as acting as a "targeted chemotherapy" in BRCA2 mutated HGSOC. Therefore, BRCA2 BRC repeat mutations can be used to identify HGSOC cells with an impaired function of BRCA2 protein that confers higher sensitivity to platinum, thus extending platinum-free interval that is a strong predictor of survival and relates to the response to subsequent platinum treatment in HGSOC. Additionally, the hyperactivation of PARP-1 due to the functional defects of BRCA2 protein contributes to an effective maintenance therapy of PARP inhibitors for patients with HGSOC[42].

Conclusion
In summary, our study demonstrates that somatic mutations in BRCA2 BRC5-8 repeat motifs could impact on BRCA2 function for DNA damage repair and confer a higher sensitivity to platinum-based therapy and are associated with a favourable outcome in patients with HGSOC.
Our ndings highlight the importance of location and type of somatic mutations in BRCA2 BRC repeat in the context of HGSOC.      BRCA2 BRC repeat mutations correlates with chemotherapy-free interval, progression-free survival and overall survival in the study. A: Chemotherapy-free interval for HGSOC patients with nonsense and missense mutation in BRCA2 BRC repeat and with non-mutation. B: Progression-free survival for HGSOC patients with nonsense and missense mutation in BRCA2 BRC repeat and with non-mutation; C: Overall survival for HGSOC patients with nonsense and missense mutation in BRCA2 BRC4~8 repeats and with non-mutation.

Supplementary Files
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