Individualized Novel Therapies for Patients with Tumor Suppressor Genes BRCA1 and BRCA2 Mutated Epithelial Ovarian Cancer

Ovarian cancer is the leading cause of death in women with gynecological cancer, since a large proportion of patients are diagnosed at later stages of the disease. The incidence of ovarian cancer in the general population is 2%, but patients with germline mutations in the BRCA genes have a risk of developing ovarian cancer of up to 2050% with a cumula‐ tive risk of ovarian cancer at 70 years of age of 40% in BRCA1 and 18% in BRCA2 muta‐ tion carriers. Although it is a chemosensitive tumor, most of the patients after surgery and chemotherapy based on taxanes and platinum will relapse later in life. Due to the high risk of developing ovarian cancer in patients with BRCA germline mutations, new treatments rely increasingly on histological and molecular characteristics of the primary tumor, achieving greater selectivity and lower toxicity compared with standard cytotoxic agents. Poly (ADP-ribose) polymerase (PARPS) inhibitors are the first biologically active agents for patients with ovarian cancer with alterations in the DNA repair pathway, par‐ ticularly in the high-grade serous subtype of ovarian cancer. The results of clinical trials published so far mean that olaparib has been approved, pend‐ ing the results of the Phase III trials. The European Medicines Agency (EMA) adopted olaparib (lynparza ®) on the December 18, 2014, as a maintenance therapy after response to platinum-based chemotherapy in relapsed platinum-sensitive ovarian cancer patients with a BRCA mutation. By contrast, the Food and Drug Administration (FDA) approved olaparib on December 19, 2014, in patients with high-grade ovarian epithelial serous tu‐ mors and a BRCA mutation who have progressed during three or more lines of chemo‐ therapy. Olaparib is also used in primary fallopian tube and peritoneal cancers with BRCA mutations.


Introduction
The usual treatment of advanced disease of ovarian cancer is surgery [1] followed by taxane and platinum-based chemotherapy, although a large proportion of patients will relapse throughout their lives. Therefore, current clinical trials focus on the detection of molecular targets that can act more selectively and efficiently on ovarian cancer [2].
It is known that chemotherapy treatments damage the DNA and there are molecules that are responsible for repair and proper maintenance of the genome such as poly (adenosine diphosphate-ribose) polymerase (PARP), which plays a key role in the repair of DNA singlestrand breaks; so, researchers have focused on the mechanism for the development of new therapies, including the PARP inhibitors.
Olaparib is the first PARP-inhibitor class recently approved for the treatment of ovarian cancer with mutations in BRCA (breast cancer), delivered orally and with good tolerance, with myelosuppression and gastrointestinal toxicity as the most frequent adverse effects. Throughout the chapter, we describe the features of hereditary ovarian cancer with BRCA mutation, and the steps to follow once the mutation is detected in patients at risk of ovarian cancer. The characteristics of PARP inhibitors are discussed, focusing on olaparib, and their use and dosage recommendations after reviewing the main Phase II trials for which it has been approved; also, we comment on Phase III olaparib trials that are currently underway.

BRCA1 and BRCA2 genes
Most breast cancers and hereditary ovarian cancers are associated with mutations in two genes, breast cancer type 1 and 2 susceptibility genes (BRCA1 and BRCA2), whose prevalence varies among different geographical areas and ethnic groups, being known as the founder effect among Ashkenazi Jews, whose descendants have an increased risk with any of the three founder mutations (two BRCA1 mutations, 187delAG and 5385insC, and one BRCA2 mutation, 6174delT). Founder mutations come from a single carrier ancestor initially extending from a small town with some degree of inbreeding, highly recurrent alterations, or even characteristics of an ethnic group [3].
BRCA1 and BRCA2 are tumor suppressor genes and are involved in the repair of double-strand breaks of DNA, maintaining genome integrity. Germline mutations in the BRCA1 and BRCA2 genes are caused by the loss of one of their wild type alleles, and therefore have a single functioning allele, promoting genomic instability and tumorigenesis [4].
Studies suggest that mutation in p53 favor loss of functionality of the BRCA 1/2 genes inducing tumorigenesis [5,6]. The function of p53 is to detect DNA damage during the cell cycle, allowing repair; so if p53 is altered, DNA repair is incomplete or inadequate, causing cell death in normal cells [7]. The p53 mutation was detected in almost 90% of patients with high-grade serous carcinoma (HGSC) in patients with BRCA 1/2 mutation. The BRCA1 is located on the long arm of chromosome 17 (17q21) gene. It has a sequence of 5,592 nucleotides, divided into 24 exons. The BRCA1 protein is localized with BRCA2, PALPB2, and RAD51 (essential proteins in homologous recombination) in areas of repair of doublestrand breaks of DNA. BRCA1 is part of BASC (BRCA1 Associated genome surveillance) complex multiprotein complex responsible for the detection, removal, and repair of DNA breaks. In conclusion, BRCA1 interacts with other oncogenes, repressors, and activators of transcription, cell cycle regulators, etc., involved in genomic stability. It has also been linked to the development of other cancers, particularly pancreas, uterus, and prostate cancers [8].
The BRCA2 gene is located on chromosome 13q (13q12), and has a sequence of 11,385 nucleotides in 27 exones. BRCA2 plays a key role in the cell cycle, especially in cytokinesis and meiosis, as well as in homologous recombination DNA repair [9]. Mutations in this gene have been linked to other cancers such as cancer of the gallbladder, pancreas, stomach, and malignant melanoma.
Hereditary breast and ovarian cancer (HBOC) syndrome is characterized by an autosomal dominant inheritance with high penetrance, presenting increased susceptibility to breast and ovarian cancer, although it has been shown that BRCA1 and BRCA2 genes are expressed in most tissues and cells analyzed, suggesting that the pathological impact of a mutation is tissuespecific and that there must be alternative pathways that compensate for their loss of function in other tissue types [10].
Women with hereditary ovarian cancer may have higher rates of response to chemotherapy and improved survival rates in cases of sporadic cancer.
In 2012, the results of an analysis were published [11] in which data from 26 observational studies on the survival of women with ovarian cancer with germline mutations in BRCA1 and BRCA2 mutations.
Data from 1,213 cases with a germline mutation in BRCA1 (n = 909) and BRCA2 (n = 303) and 2,666 no mutation carriers were included. The observed overall survival (OS) for 5 years was 36% in non-carriers of mutation patients versus 44% for patients with a BRCA1 mutation and 52% for patients with a mutation in BRCA2. There was an increased survival in BRCA mutation carriers versus non-carriers. BRCA2 carriers had a better prognosis. There were several significant differences in the clinical characteristics of BRCA1 and BRCA2 compared with noncarriers. The BRCA1 and BRCA2 tumors were more likely to be serous histology and less likely to be mucinous histology. Patients with BRCA1 and BRCA2 mutation were more likely to have a tumor stage III/IV and present greater degree of differentiation compared to non-carriers. BRCA1 carriers were also younger at diagnosis. Detection of BRCA1 and BRCA2 genes is accomplished by DNA extraction from peripheral blood lymphocytes. Detection techniques must be able to identify everything from small changes to large duplications or deletions of exons.
There are over one thousand different mutations to BRCA1 and BRCA2, most of them being small insertions or deletions causing a change in the reading frame (frameshift) and producing a stop codon. However, amino acid substitutions producing a stop codon (nonsense) or mutations located at sites of exon splicing that alter the splicing of the genes and producing full or partial loss of exons [12] can also be found to occur.
It is known that the mutation in the BRCA1 gene presents a risk of ovarian cancer throughout life of up to 40%, while the BRCA2 gene has a 20% risk. Although penetrance may vary in the same family carrying a mutation, suggesting that the risk can be influenced by allelic heterozygosity, modifier genes, and environmental and hormonal cofactors [13,14].
After the diagnosis of breast cancer in a patient with a BRCA mutation, there is a subsequent risk of developing ovarian cancer of up to 12.7% in women for BRCA1 and 6.8% for BRCA2 [15].
Diagnosis in elderly or the absence of family history does not exclude the presence of a germline mutation as approximately 35% of the BRCA mutation carriers have no family history. Genetic tests are expensive, so you should select the most appropriate individuals for genetic testing, varying recommendations between populations and countries.
The presence of BRCA somatic and germline mutations are predictors of response to different chemotherapy treatments because they exhibit greater sensitivity and response to platinumtype drugs or PARP inhibitors, which are involved in the repair of DNA single-strand breaks [16].
It is important that families carrying this mutation are informed about the risks of developing various types of cancer, including education about prenatal diagnosis and assisted reproduction. Another option is IVF with previously selected embryos. Although, the decision should finally be made on an individual basis and will depend on the preference of each patient.

Patients with ovarian cancer BRCA genes mutations syndrome
Ovarian cancer is the principal cause of death in women with gynecological cancer, due to the late onset of symptoms and the absence of a method for early detection. Nulliparity, early menarche, and late menopause are associated with an increased risk of occurrence; however, the strongest risk factor is the history of ovarian cancer in a first-degree relative [17].
Malignant primary ovarian tumors fall into three main groups: epithelial, sex cord / stromal, and germ cell tumors. Epithelial tumors being ovarian carcinomas (CBs), which are the most common group, represent up to 90% of ovarian cancers. Low-grade and high-grade serous carcinoma (LGSC and HGSC), mucinous carcinoma (MC), endometrioid carcinoma (EC), and clear cell carcinoma (CCC) are the five histological subtypes of OCs that are known. It is important to make a proper histological typing to determine the prognosis and response to different treatments, including cisplatin [18].
A large proportion of ovarian tumors are sporadic, and only a minority is due to an inherited cause. BRCA1 and BRCA2 mutations have been identified in approximately 15% of all epithelial ovarian cancers and up to 22.6% in HGSC. Somatic mutations in BRCA1 and BRCA2 have also been identified in as much as 7% of all ovarian cancers [19,20]. Although up to 50% of patients with HGSC harbor homologous recombination defects including the homologous recombination pathway independent of BRCA 1/2, this is known as BRCA-like behavior.
BRCA-like behavior is similar to when there is a loss of function or mutation of the BRCA genes with the same clinical and molecular characteristics. Examples include promoter methylation BRCA1 (observed up to 35% of patients with epithelial ovarian cancer) and p53 mutation, c-myc amplification or other proteins are needed for proper homologous recombination [21][22][23][24]. The loss of function of suppressor gene PTEN has also been shown to produce BRCA-like behavior [25], more common in breast and prostate cancers [26,27].
The HGSC subtype has a greater sensitivity to PARP inhibitors without a BRCA mutation, probably due to changes in DNA repair that occur up to 50% of cases as we have mentioned previously [28,18,29].
Studies describe that BRCA mutation carriers diagnosed with ovarian cancer have higher survival rates compared with sporadic cases [30][31][32]. This could be due to increased sensitivity to cisplatin.
In HBOC syndrome, mutations in the BRCA-1 and BRCA-2 genes associated with the development of ovarian cancer [29] occur. Other inherited syndromes have also been associated with an increased occurrence of ovarian cancer such as Lynch syndrome (hereditary nonpolyposis colorectal cancer syndrome), characterized by mutations in the DNA repair genes MLH1, MSH2, MSH6, and PMS2 genes [33].

Prevention of ovarian cancer in women who have mutations in BRCA1 and BRCA2 genes
Primary prevention strategies consist of primarily risk-reducing surgeries, the procedure of choice being prophylactic salpingo-oophorectomy bilateral from 35 to 40 years, or after childbearing. Some experts also recommend prophylactic hysterectomy to dry the small portion of remaining fallopian knotweed, although 92% of fallopian tube neoplasms originate in the middle or distal portion of the tube [34]. Although the final decision will be made by the patient.
Patients with BRCA1 mutation are at increased risk of developing ovarian cancer from the age of 40; the recommendations as explained above are made from that age. This is not so with patients carrying the BRCA2 mutation; the increased risk of ovarian cancer starts after age 50, so surgery can be postponed for a few years and secondary effects of surgery reduced [35].
After prophylactic surgery, one of the most important side effects which can deteriorate the quality of life of patients is premature menopause, with increased risk of osteoporosis and cardiovascular diseases such as hypertension, diabetes, and hypercholesterolemia. So that closer monitoring is recommended for cardiovascular risk [36].
Secondary prevention is early detection strategies in women carrying mutations in the BRCA1 and BRCA2 genes. Current recommendations include performing transvaginal ultrasound twice a year (preferably day 1-10 of the menstrual cycle), together with detection of serum CA-125 levels (after day 5 of the menstrual cycle for), from age 30 or five to ten years earlier than the earliest age of first diagnosis of ovarian cancer in the family. But these methods have limited sensitivity and specificity, with no observed benefit in women carrying mutation since no mortality was reduced. All women who refuse to perform prophylactic surgeries should undergo screening every six months [37].
Despite the greatly reduced risk of developing ovarian cancer after prophylactic surgery, patients should know that a minority (from 3.9% to 4.3%) of them will develop primary peritoneal carcinoma 20 years after the last oophorectomy in patients with BRCA-1 mutations. So before you perform these procedures, patients should be informed of the risks and morbidities associated with these interventions [38,39].

Inhibitors of poly (ADP -ribose) polymerase
The preservation of the genetic code by DNA repair is essential for proper cell function. Currently, there is a better understanding of the DNA repair pathways, so it has been studied more carefully for potential drug targets [40].
There are at least five ways engaged in DNA repair, two of them involved in the repair of double-strand breaks (error-prone, non-homologous, end-joining, predominantly active in G1 cells, and error-free HRR, which predominates in dividing cells) [41].
There are certain external agents such as ionizing radiation producing damaging DNA strand breaks. Normal cells have the ability to repair this damage by a protection mechanism maintaining its normal function, but the tumor cells' ability to repair DNA is a radio-resistance mechanism. In recent years, studies have identified a number of agents in these pathways such as PARP inhibitors [43].
Poly (ADP-ribose) polymerase (PARP) inhibitors are a new class of targeted agents against ovarian cancer [44][45][46]. PARP is a nuclear enzyme whose function is to repair single-stranded DNA.
There are three generations of PARP inhibitor. The first generation of inhibitors included nicotinamide analogs. 3-Aminobenzamide was the first PARP inhibitor but was not considered powerful enough compared to the second generation [47]. Currently, clinical trials are aimed at third-generation inhibitors with greater potency and specificity, decreasing side effects, this includes olaparib.
DNA repair is essential for proper cell function. Each cell sustains many thousands of episodes of DNA damage every day, which will be repaired by a wide variety of repair mechanisms [48,49].
The BRCA 1/2 genes are responsible for DNA repair known as homologous recombination (HR) repair. HR is a form of double-strand break repair that occurs in the G2 phase of the cell cycle where the second double-stranded copy of the DNA is used as a template to form an error-free repair [49].
Other DNA repair pathway, such as the non-homologous, end-joining (NHEJ) pathway, also plays a role in the anti-cancer mechanism of action of PARP inhibitors [50].
PARP inhibitors act by trapping PARP-1 and PARP-2 on the double-strand break and blocks DNA replication, which is more toxic to cells than the accumulation of DNA breaks [51]. Overall, in tumors in where there is an apparent defect in homologous DNA repair (and thus a defect in the repair of double-stranded breaks), they seem to be susceptible to PARP-inhibitor therapy. These include tumors associated with germline or somatic mutations in BRCA 1/2 [52].
PARP-1 was the first to be reported in 1963 [57]. Durkacz [58] stated that modulating PARP1 could enhance the effect of chemotherapy.
PARP-1 contains three functional domains: the N-terminal DNA-binding domain (DBD), the center self-modification domain (DMA), and the C-terminal catalytic domain (CD). The DBD is involved in recognition of DNA-strand breakage and in the binding of PARP-1 to DNA. AMD can interact with many DNA damage response proteins and the CD includes a PARP signature motif and catalyzes the formation of PAR [59]. PARP-1 is essential for base excision repair (BER).
PARP-1 also contributes to other cellular processes such as gene transcription, and the regulation of the chromatin structure, to restart stalled replication forks due to nucleotide depletion or collisions with bulky lesions [52].
PARP-1 has been used in in vitro studies in combination with chemotherapy, to demonstrate its ability to inhibit the classical mechanisms of DNA repair, showing also increased distribution of cytotoxicity to the tumor, increasing their exposure by improving vascular perfusion. This resulted in further studies with PARP-1.
The DNA repair biology has allowed us to identify patients most likely to respond to treatment with PARP inhibitors [60].
PARP inhibitors act by synthetic lethality, which occurs when two independent conditions alone do not cause cell death but in combination are lethal. It occurs when a patient has an alteration in the homologous recombination (HR) such as in carriers of a mutation in BRCA1 and BRCA2 genes and the application of PARP inhibitors, causing cell death [61,62]. Up to 5% of cutaneous melanomas and gastric cancers, 1% of prostate cancers, and even 19% of familial pancreatic cancers will carry a germline mutation in BRCA 1/2, thus they have an altered HR and therefore they may also respond to PARP inhibitors [63].
PARP-2 cooperates with PARP-1 to synthesize poly (ADP-ribose) [pADPr] after damage in the DNA chain [41]. PARP-3 suppresses error-prone NHEJ [52,64,65] while associated with PARP-1 for DNA repair. The clinical development of PARP-inhibitors has lead to its use as monotherapy or in combination with chemotherapy agents. Olaparib has been recently approved for the treatment of hereditary breast ovarian cancer syndrome, and other PARPinhibitors such as veliparib, rucaparib, or niraparib are being studied [66].

Combination therapy of PARP inhibitors and radiotherapy
The efficacy of radiotherapy in the treatment of cancer have been known for several years either concomitantly with chemotherapy or as adjuvant use in therapy.
New clinical trials not only focus on researching new systemic treatments alone or in combination with other chemotherapy agents but also study their association with radiotherapy. These new therapies are the PARP inhibitors that have shown activity in conjunction with radiation therapy in several cancer cell lines. Data suggest that PARP inhibitors may enhance the effects of radiation in various types of tumors, such as lung cancer, colorectal, and cervical among others [67]. However, the mechanism of action is still unknown, one hypothesis is that it is due to mutual damage (of PARP-inhibitors and radiotherapy) of DNA or whether tumor re-oxygenation contributes to this radio sensitization via the vasoactive effects of the PARP inhibitors remains to be fully determined [43].
A recently published Phase I clinical trial [68] combined low-dose abdominal level fractionated radiotherapy with increasing doses of the PARP-inhibitor veliparib in patients with peritoneal carcinomatosis secondary to advanced malignant solid tumors. Patients were treated with veliparib (80-320 mg daily) for a total of 3 cycles.
The dose of radiotherapy consisted of 21.6 Gy in 36 fractions, 0.6 Gy twice daily on days 1 and 5 for weeks 1-3 of each cycle. Twenty-two patients were included. Disease stabilization (≥24 weeks) was observed in 7 patients (33%). Median progression-free survival (MPFS) was 4.47 months and median overall survival (MOS) was 13.04 months. In the trial, there were 8 patients with ovarian and fallopian cancers with an observed MPFS of 6.77 months and an MOS of 17.54 months, combined with a higher quality of life. The toxicity grade 3 and 4 lymphopenia were more frequent (68%), anemia (9%), and thrombocytopenia (14%). With these results, the authors concluded that the combination of radiotherapy and veliparib resulted in a stabilization of the response in patients with solid tumors and peritoneal carcinomatosis, especially in the subgroup of patients with ovarian cancer, besides being a well-tolerated regimen.

OLAPARIB
Because many cytotoxic agents work by damaging the DNA, there has been a great deal of interest in the use of inhibitors of DNA repair such as new treatments against cancer, especially in patients with mutations in the BRCA genes with altered function, and will be more likely to develop different types of neoplasms due to increased tumorigenesis [69].

Initial clinical trials with OLAPARIB
In 2008, Rottenberg et al. [70] postulated the hypothesis of the use of olaparib (then called AZD2281, KU-0059436) in cancer triple negative breast, because these tumors harbor defects in DNA repair and mutations in BRCA1. To do this, they used PARP inhibitors (AZD2281) in genetically engineered mouse models of breast cancer BRCA1, resulting in an inhibition of tumor growth and increased overall survival with no signs of toxicity. Drug resistance developed after long-term treatment due to upregulation of efflux pumps; however, this was overcome by co-administration of the P-glycoprotein inhibitor tariquidar. They observed that the combination of AZD2281 with cisplatin or carboplatin increased progression-free survival, suggesting the effectiveness of AZD2281 as DNA-damaging agents.
Evers et al. [71] studied sensitivity to conventional cytotoxic drugs AZD2281 in cell lines with BRCA2 mutations. AZD2281 was observed to be the drug that caused greater tumor reduction in the presence of BRCA2 mutations, alone or in combination with cisplatin.
Fong et al. [60] conducted a Phase I clinical trial with escalating doses of mg to 600mg olaparib, in a population of 60 patients, including 22 mutation carriers in the BRCA1 and BRCA2 gene. Dose-limiting toxicity was observed in one of eight patients receiving 400 mg twice daily (grade 3 fatigue and mood alteration) and two of five patients who received 600 mg twice daily (grade 4 thrombocytopenia and drowsiness grade 3). In 63% of patients with cancer and carriers of BRCA mutations, a clinical benefit for a period of 4 months or more was observed and 8 patients had ovarian cancer.
In this study, patients resistance to platinum response was observed.
A year later, the same team of Fong et al. confirmed previous results by expanding a cohort of patients with mutations in BRCA1 and BRCA2, including primary peritoneal cancer with ovary, and fallopian tube cancers (13 platinum-sensitive, 24 platinum-resistant, and 13 platinum-refractory) observing a clinical benefit response of up to 46%. The overall clinical benefit rate decreased due to insensitivity to platinum (platinum-sensitive patients: 69%, platinum-resistant: 46% refractory to platinum: 23%). The median response duration was 28 weeks, concluding that patients who were platinum-sensitive present a greater response to olaparib, in addition to showing a benefit in resistant and refractory patients [72].
Seventy five percent of BRCA1-mutated breast cancers are classified as triple-negative breast and BRCA1 or BRCA2 mutation carriers and have the tendency to develop ovarian cancer. In a Phase II clinical trial [63], the investigators administered 400 mg of olaparib twice daily in patients with ovarian cancer HGSC and triple-negative breast cancer. Patients were stratified according to whether they were carriers of BRCA1, BRCA2, and BRCA wild-type gene. The primary endpoint was objective response rate by Response Evaluation Criteria In Solid Tumors (RECIST). It was observed that 41% of patients with BRCA mutation carrier ovarian cancer and 24% of patients with wild-type BRCA showed no response to olaparib RECIST criteria. No confirmed objective responses were reported in patients with breast cancer and they concluded that olaparib is an efficient drug for treatment of BRCA mutant ovarian cancer.
Kaye et al. published the results of a Phase II trial in 2012. The study included 97 patients with ovarian cancer and BRCA1 or BRCA2 germline mutations that had recurred within 12 months of prior platinum therapy. They randomized in a 1:1:1 ratio to receive olaparib 200 mg or 400 mg bd or pegylated liposomal doxorubicin (PLD) 50 mg / m2 intravenously. The median PFS was 6.5 months, 8.8 months, and 7.1 months for the olaparib 200 mg, 400 mg olaparib, and PLD groups, respectively. Objective response rates were 25, 31, and 18% for olaparib 200 mg, 400 mg olaparib, and PLD, respectively. Proving that olaparib 400 mg twice daily is an appropriate dose. A surprising finding was the effectiveness of PLD [74]. These results affirm the data published by Adams et al. [75] which show increased PLD activity in patients carrying a BRCA mutation.
Lederman et al. [76] reported on a Phase II trial; they administered olaparib as a maintenance therapy in patients with recurrent ovarian cancer or fallopian tube or primary peritoneal cancer of high-grade, which was platinum-sensitive. Patients were randomized to receive olaparib 400 mg twice daily or placebo within 8 weeks after the last dose of platinum-based chemotherapy. The primary endpoint was progression-free survival (PFS). A first analysis performed after progression in 57.7% of patients showed that PFS was significantly longer in the olaparib group than in the placebo group. Median PFS was 8.4 months in the olaparib group versus 4.8 months in the placebo group (P < 0.001). Subgroup analysis of progression-free survival showed that, in the olaparib group, patients had a lower risk of progression than those in the placebo group. Having had a complete response to the treatment of platinum-based chemotherapy before entering the study was a significant prognostic factor for longer progression-free survival (hazard ratio, 0.46; P < 0.001). Time to progression according to the RECIST guidelines or CA-125 level was significantly longer in the olaparib group than in the placebo group (median, 8.3 months vs. 3.7 months (P < 0.001). The response rate was 12% (7 of 57 with measurable disease patients at study entry) in the olaparib group, as compared with 4% (P = 0.12).
In the interim analysis of overall survival (OS), 101 patients (38%) had died: 52 in the olaparib group and 49 in the placebo group. No significant difference in overall survival was observed (P = 0.75). The median overall survival was similar in the two study groups (29.7 months in the olaparib group and 29.9 months in the placebo group). Although BRCA mutation status was known for 37% of all patients who entered the study, a subgroup analysis suggested that olaparib could increase PFS in patients with a BRCA mutation.
The incidence of adverse events grade 3 or 4 was higher in the olaparib group (35.3%) compared to the placebo group (20.3%). The most common adverse events leading to discontinuation or dose reduction of olaparib were vomiting, nausea, and fatigue. There were no statistically significant differences in quality of life test performed patients.
In another study, Lederman et al. [77] presented test data 19, a second retrospective analysis of OS, and BRCA mutation status of the Phase II trial published by them in 2012. The primary endpoint was PFS, analyzed for the overall population and by BRCA status. BRCA mutation status was known in 96% of patients in the group of olaparib compared to 95% in the placebo group, of whom 56% versus 50% had a deleterious or suspected tumor or deleterious BRCA germline mutation. The median PFS was significantly longer in the olaparib group compared to the placebo group, 11.2 months vs. 4.3 months; in the wild-type BRCA patients the findings were similar, 7.4 months vs. 5.5 months. In a second interim analysis (58% maturity), the OS was similar in patients with mutated BRCA and wild-type BRCA, this may be secondary to the fact that 23% of patients receiving placebo could subsequently receive an inhibitor of PARP. In the results, they observed that some patients responded to olaparib in the absence of a BRCA mutation, this could be due to epigenetic silencing of BRCA or homologous recombination genes such as RAD51D.
There were more treatment interruptions and dose reductions in the olaparib group compared to placebo. Adverse effects grade 3 or 4 in the olaparib group were fatigue in 7% vs. 3% in the placebo group, and anemia 5% vs. <1%, respectively. Serious adverse effects were reported in 18% of patients receiving olaparib compared to 9% on placebo. Tolerability was similar regardless of the mutational status.
Although the number of patients treated with somatic BRCA mutations was lower, responses were also seen during maintenance therapy.
The test of olaparib superiority over the placebo in platinum-sensitive patients demonstrated that patients relapsed after treatment. In addition, it was shown that BRCA mutations, whether somatic or germ line, are the main determinants of response to olaparib.
After the results of test data19 were published, olaparib was approved by EMA as a maintenance therapy after response to platinum-based chemotherapy in relapsed platinum-sensitive ovarian cancer, fallopian tube, and primary peritoneal cancers patients with a BRCA mutation. In contrast, the FDA-approved olaparib in patients with high-grade serous ovarian epithelial tumors, primary fallopian tube and peritoneal cancers with a BRCA mutation who had progressed during three or more lines of chemotherapy.
Before starting treatment with olaparib, they confirmed the presence of a BRCA germline or somatic mutation, using a valid method of analysis in a specialized laboratory.
It also assessed the efficacy and tolerability of olaparib in combination with chemotherapy followed by maintenance olaparib versus chemotherapy alone in patients with high-grade serous ovarian cancer, including primary peritoneal and fallopian tubes, platinum-sensitive who had received three or more lines of platinum-based chemotherapy. AM Oza et al. [78] published data based on chemotherapy combination of carboplatin (area under the curve [AUC] 4 mg / mL per min) plus paclitaxel (175 mg / m2) every 21 days with olaparib 200 mg twice daily (during days 1--10 of each cycle of 21 days), 6 total cycles followed olaparib maintenance monotherapy (400 mg twice daily) until disease progression or unacceptable toxicity, compared to chemotherapy alone (carboplatin AUC 6 mg / mL paclitaxel 175 mg / m2) without maintenance. The primary endpoint was PFS, the secondary efficacy endpoints were overall survival; percentage change in tumor size; the proportion of patients with an objective response according to RECIST; cancer antigen 125 (CA-125) response.
The results concluded that the PFS was higher in the olaparib plus chemotherapy group (median 12.2 months) compared with chemotherapy alone (median of 9.6 months). BRCA mutation status was known in 107 patients. BRCA mutations were observed in 41 (38%) of 107 patients (20 in the chemotherapy group and 21 in more olaparib chemotherapy alone); of the 41 patients with BRCA mutation, the PFS at 12 months was 70% for patients in the combination arm vs. 12.5% in the chemotherapy alone arm. There were no statistically significant differences in OS or percentage change in tumor size. The proportion of patients with an objective response by the central review was similar between treatment groups. The two treatment groups had a similar proportion of patients with a CA-125 response and ovarian cancer response. The most frequent grade 3 or 4 adverse effects in the combination arm were neutropenia (43%) vs. (35%) in patients with chemotherapy alone and anemia ([9%] vs. [5%]). The most frequent adverse effects of mild or moderate intensity were alopecia, nausea, diarrhea, headache, and peripheral neuropathy among others, they were all more common in the combination group.
Not only olaparib has been studied in combination with chemotherapy, but their association was also analyzed with cediranib, an anti-angiogenic agent with activity against the VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. In Phase I clinical trials, combination responses and manageable toxicities were observed, so that a Phase II clinical trial was developed aimed at demonstrating whether their combination would result in increased PFS versus olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer. The trial was conducted by Liu [79].
They recruited women with ovarian (HGSC and EC), fallopian tube or primary peritoneal cancer, or patients with a BRCA germ line mutation. Patients were randomized to receive olaoparib in 400 mg capsules twice daily or the combination of cediranib 30 mg daily and olaparib 200 mg capsules twice daily. The primary endpoint was PFS. 46 women received 44 olaparib monotherapy and combination therapy women. Median PFS was 17.7 months for the women treated with cediranib plus olaparib compared with 9 months in patients receiving olaparib monotherapy. Grade 3 and 4 adverse events were more common with the combination therapy, including fatigue, diarrhea, and hypertension. Thus, the authors concluded that PFS increases considerably with the combination therapy, so Phase III clinical trials are necessary to confirm these results, including assessments of quality of life.
Because of the mechanism of action of olaparib, the use of PARP inhibitor in other tumors with mutations in the BRCA 1/2 gene could be effective, a Phase II clinical trial with different tumors was performed, all patients had BRCA 1/2 mutations and recurrent cancer. The study was conducted by Kaufman et al. [52]. It was a prospective, multi-center, randomized trial, and patients were recruited in several centers in Israel, Australia, Germany, Spain, Sweden, and the United States between February 21, 2010, and July 31, 2012. It included patients with ovarian cancer resistant to prior platinum; with three breast cancer chemotherapy regimens for metastatic disease; pancreatic cancer progression during treatment with gemcitabine; with prostate cancer progression or on hormonal and one systemic therapy. Olaparib was administered 400 mg twice per day. The primary efficacy end point was tumor response rate according to RECIST. Secondary end points included: objective response rate (in those with measurable disease at baseline), PFS, duration of response, safety, and tolerability. A similar response rate for patients with a BRCA1 mutation (26.3 % ; 95 % CI, 20.3 to 33.0) and those with a BRCA2 mutation was also noted.
The most common side effects were fatigue, nausea, and vomiting. About 54% of patients experienced grade 3 toxicity, most frequently fatigue and anemia, 40.3 % of patients had to modify (interruption and / or reduction) olparib dose due to adverse effects. Nine patients died as a result of severe adverse effects.
After these results, they concluded that the response to olaparib is independent of the anatomical organ of origin, provided that there is a mutation in BRCA 1/2.
Despite the demonstrated efficacy of PARP inhibitors in patients with BRCA 1/2 mutation, there has been less activity in patients with breast cancer, perhaps due to the biologic heterogeneity and low BRCA 1/2 mutation rate in somatic triple negative breast cancer [80][81][82][83].

Phase III clinical trials
Currently, there are two Phase III trials underway with olaparib, both sponsored by Astra Zeneca: SOLO 1 (NCT01844986) and SOLO2 (NCT01874353). Both are multi-center, doubleblind, in which randomly assigned patients (2: 1) receive 300 mg of olaparib daily maintenance in patients diagnosed with high-grade serous or endometrioid ovarian cancer, primary peritoneal including and / or fallopian tube cancer with a BRCA mutation, and having partial or complete response after completion of platinum-based chemotherapy. To be included in SOLO1 trial, patients must have been newly diagnosed, with advanced (FIGO stage III-IV) disease, and responding to first-line treatment with platinum. To be included in SOLO2, patients must have completed ≥2 lines of platinum therapy. The main objective for both clinical trials is progression-free survival [84].
Not only they are testing olaparib, but there are also other Phase III trials with PARP inhibitors, such as veliparib, rucaparib, and niraparib, trying to improve the identification of patients who might best respond to PARP inhibitors [80] and reducing associated toxicities.

Combination therapy of OLAPARIB with other therapeutic agents
Olaparib has been tested in several clinical trials in combination with other chemotherapy because it was thought that it could increase sensitivity to chemotherapy by inhibiting DNA repair which could be responsible for drug resistance. This has been associated with topotecan [85], dacarbazine [86], paclitaxel [87], and cisplatin and gemcitabine [88]. In several Phase I clinical trials, myelosuppression increased in the combination therapy versus monotherapy, especially with topotecan and cisplatin.
Olaparib has also been studied in combination with bevacizumab. Dean et al. [89] designed a Phase I clinical trial, as they hypothesized that bevacizumab antiangiogenic activity and hypoxia inducing DNA damage may enhance olaparib therapeutic activities. They showed that a dose of olaparib 400 mg twice daily in combination with Avastin 10mg / kg every two weeks was tolerated well. They are considering the combination for future clinical trials.

Dosage
The recommended lynparza® (olaparib) dose is 400 mg (eight capsules) twice a day, i.e., a total daily dosage of 800 mg. Treatment should begin before eight weeks of completion of the last cycle of chemotherapy with a regimen containing platinum. Continual treatment is recommended until disease progression. The recommended dose is reduced to 200 mg orally twice daily (total daily dose 400 mg). Elderly patients require a dose reduction initially, and it can be administered in patients with mild renal impairment (creatinine clearance> 50 mL / min).

Adverse reactions
Among the most frequent toxicities in clinical trials are hematologic toxicity with mild to moderate anemia, lymphopenia, neutropenia, and thrombocytopenia at manageable levels. Other frequently observed side effects are headache, fatigue, decreased appetite, abdominal discomfort, nausea, vomiting, diarrhea, and dyspepsia.
Another event that was observed was the development of myelodysplastic / AML syndrome, in only a small number of patients receiving olaparib alone or in combination with other antineoplastic during clinical trials. All had previously received platinum-based chemotherapy regimens, radiation, and other DNA-damaging agents.
There have been cases of pneumonitis, some of them being fatal. If patients are treated with Lynparza, respiratory symptoms such as cough, dyspnea, and fever should be closely monitored. If there is any alteration in the chest radiography, treatment must be stopped and the patient is treated appropriately. Paralyzer can cause birth defects if given to pregnant women. A reliable contraception should be recommended during treatment and one month after the last dose [90,63].

Mechanisms of resistance to PARP inhibitors
Targeted therapy based on the patient's mutation status is the future of the treatment of ovarian cancer. BRCA deficiency may be reversed by mutational changes in the reading frame, resulting in wild-type BRCA protein production. A second mutation (compensatory mutations or crossovers) can cause changes in the reading frame BRCA mutation, HR rebuilding, and restoring its functionality, explaining why not all tumors with BRCA mutation respond to PARP inhibitors [91]. Some BRCA1 mutant alleles encode functional proteins but are degraded, stabilizing the activity of the mutated protein and can reset the HR [92]. Another mechanism could be the upregulation of the pump glycoprotein efflux reducing concentrations of the intracellular PARP inhibitor [93,94] or loss of 53BP1, a key protein in the NHEJ pathway.

Conclusions
The context of the BRCA 1/2-mutant genotype has a significant impact on disease behavior and outcome. An encouraging data on responses to PARP inhibitors in BRCA 1/2-mutant carriers with prostate, pancreatic, and breast cancer have been reported and are likely to be associated with platinum sensitivity. It is apparent that establishing the germ-line and/or tumor BRCA 1/2 mutation status in patients with cancers known to be associated with BRCA 1/2 mutations is of notable importance due to the potential therapeutic options. Chemotherapy recommendation for patients with BRCA 1/2 mutant epithelial ovarian cancer ought to be based on rechallenging these patients with platinum-based treatment, and prolonging the platinum-free interval in the event of early relapse following platinum-based treatment. The timing and sequence of therapy, and the indications for rechallenging patients with platinum-based chemotherapy, including routes and schedules of administration (IV vs. IP, weekly vs. thrice weekly), olaparib plus paclitaxel and carboplatin followed by laparib maintenance monotherapy, significantly improved progression-free survival versus paclitaxel plus carboplatin alone, in patients with BRCA-mutated recurrent platinum-sensitive ovarian cancer, with acceptable tolerability profile. It differs as more data emerges from analyses of the mutation status of BRCA 1/2 genes and other HR-related genes in tumor samples from previously completed studies. In addition, stratification based on HR-deficiency phenotype/genotype may become the standard in future clinical trials involving patients with BRCA 1/2 mutant epithelial ovarian cancer. It has been established that olaparib plus paclitaxel and carboplatin followed by olaparib maintenance monotherapy significantly improved progression-free survival versus paclitaxel plus carboplatin alone, in patients with BRCA-mutated recurrent platinum-sensitive ovarian cancer, with acceptable tolerability profile.

Future directions
Tumors with alterations in DNA repair lead to a defective HR, based on synthetic lethality and being very sensitive to PARP inhibitors. This happens with HGSC BRCA mutation carriers demonstrating its response to olaparib. We still need to better identify patients who will respond to PARP inhibitors because a proportion of patients developed resistance to these treatments, so research is needed to understand the mechanisms of action of PARP inhibitors and mechanisms of resistance. The only biomarkers that have been shown to be predictive of response while using PARP inhibitors are the BRCA 1/2 mutations both somatic and germ line, in the absence of other biomarkers, we are limited to using the PARP inhibitors only in patients with BRCA mutation 1/2, although it may be effective in other tumors despite the absence of BRCA mutation.
We have studied the operation of Rad5 using antibody detection of Rad5 as new response PARP-inhibitors biomarkers, although initial results suggest that is not sufficient, specific and sensitive for use in clinical application, so we need to keep looking for new biomarkers or methods that help us to identify the appropriate patients for treatment with PARP inhibitors [52]. As not all genes responsible for DNA repair are known, another option that has not yet been implemented is to apply functional tests of DNA-repair capability, this would help us identify the abnormalities and tumors suitable for treatment with PARP inhibitors [95], the molecules involved in BRCA-like tumors whose information is essential to broaden the scope of action of PARP-inhibitors, without limiting its use for patients with BRCA 1/2 mutations. A current research topic is whether these new drugs work better alone or in combination with standard cytotoxic agents, avoiding toxicities and resistance mechanisms [96]. They have been shown to be well tolerated with manageable toxicity, but the long-term action is unknown. Some experts question whether inhibiting DNA repair can lead to deleterious effects such as an increased risk of developing other types of cancer in the future. Another topic of interest is its effect in combination with radiotherapy or as maintenance therapy. As of now, we do not know what effect PARP inhibitors may have in patients with low tumor burden, and what would be the benefit when these agents are used as maintenance therapy or chemoprevention.