Olaparib for the treatment of breast cancer

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1 Expert Review of Anticancer Therapy ISSN: (Print) (Online) Journal homepage: Olaparib for the treatment of breast cancer Gaia Griguolo, Maria Vittoria Dieci, Valentina Guarneri & PierFranco Conte To cite this article: Gaia Griguolo, Maria Vittoria Dieci, Valentina Guarneri & PierFranco Conte (2018): Olaparib for the treatment of breast cancer, Expert Review of Anticancer Therapy, DOI: / To link to this article: Accepted author version posted online: 27 Mar Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at

2 Drug Profile Olaparib for the treatment of breast cancer Gaia Griguolo 1,2, Maria Vittoria Dieci 1,2, Valentina Guarneri* 1,2, PierFranco Conte 1,2 1 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. 2 Division of Medical Oncology 2, Istituto Oncologico Veneto IRCCS, Via Gattamelata 64, 35128, Padova, Italy. *Corresponding author: Valentina Guarneri, M.D., PhD, Department of Surgery, Oncology and Gastroenterology, University of Padova Division of Medical Oncology 2 Istituto Oncologico Veneto IRCCS Via Gattamelata 64, Padova, Italy Phone: Fax: valentina.guarneri@unipd.it

3 ABSTRACT Introduction: Mutations in BRCA1 and BRCA2 genes account for around 2-3% of breast cancer events and more than 10% of triple negative breast cancers. Olaparib (Lynparza ), an orally administered PARP inhibitor, demonstrated clinical benefit in a phase III trial for mutated BRCApositive HER2 negative metastatic breast cancer. Areas covered: This review gives an overview of available preclinical and clinical data regarding olaparib, including its chemistry, mechanism of action, pharmacokinetics and pharmacodynamics, and evidence supporting antitumor efficacy and safety profile in breast cancer patients. Expert commentary: Olaparib improves progression-free survival in germline BRCA mutated HER2 negative metastatic breast cancer patients as compared to standard chemotherapy, with a manageable toxicity profile. Efficacy is of clinical relevance especially in the context of triple negative breast cancer. However, several aspects, such as sequencing or combination of these agents with other anticancer agents and identification of appropriate biomarkers, still need to be clearly defined. Keywords: olaparib, breast cancer, BRCA, synthetic lethality, OlympiAD trial, PARP inhibitors. -

4 1. Introduction Breast cancer (BC) is the most frequent female malignancy and one of the leading causes of death for women in western countries, accounting for more than deaths worldwide in 2012 [1]. Recent advances in the understanding of the cellular and molecular biology of BC have led to the development of a number of targeted therapeutic agents. Therefore, from a clinical and therapeutic point of view, BC can be sub-divided in several subgroups that can benefit from dedicated approaches. For patients with hormone-receptor positive BC several therapeutic options based on endocrine treatment, and more recently combinations of endocrine treatment and targeted agents such as mammalian Target of Rapamycin (mtor) or cyclin-dependent kinase (CDK) inhibitors, are available [2-7]. Patients with Human Epidermal growth factor Receptor 2 (HER2) overexpressing BC have been shown to benefit from anti-her2 targeted agents [8-10]. However, for triple negative (TN) BC, classically defined by the lack of estrogen receptor and/or progesterone receptor expression and the lack of HER2 overexpression, chemotherapy still remains the only available therapeutic option. This has fostered a major effort to better understand the molecular complexity of TNBC in order to identify actionable targets. Alterations in DNA repair pathways, such as mutations in breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2), have been identified as a potentially actionable target in a subset of TNBC. In fact, it has been well known for several decades that some families may show a strong predisposition for breast and ovarian cancer [11]. Two major genes associated with susceptibility to breast and ovarian cancer BRCA1 and BRCA2 have been identified in the last decades [12,13]. Even if heterozygous germline mutations in BRCA1 or BRCA2 confer a lifetime risk of BC between 60 and 80 percent, mutations in these genes account for only 5 to 10 percent of BC events in the overall population. [11, 14]. The presence of a germline mutation in BRCA1 is frequently associated with a TN basal-

5 like phenotype, characterized by hormone-receptor negativity, TP53 mutations, genomic instability and sensitivity to DNA-crosslinking agents [15,16]. This reflects in a higher frequency of BRCA alterations in patients diagnosed with TNBC (11-42% of cases varying with age and family history). On the contrary, the presence of a germline mutation in BRCA2 is more frequently associated with a luminal B phenotype, characterized by estrogen receptor positivity and high tumor grade, even if TN phenotype has been reported to modestly predictive of BRCA2 mutation status in patients diagnosed at 50 years or older [15]. 1.1 DNA repair defects in BC and rationale for targeting PARP. The tumor-suppressor proteins BRCA1 or BRCA2 are key components in the double-strand DNA repair by homologous recombination pathway [17]. Cells carrying heterozygous loss-of-function BRCA mutations, as those of patients carrying germline BRCA1 or BRCA2 mutations, can lose the remaining wild-type allele. This double-hit process results in homologous-recombination DNA repair deficiency, consequently leading to genetic aberrations driving carcinogenesis. In fact, the inactivation of the second wild-type allele is thought to be an obligate step in cancerogenesis in BRCA mutated patients. This process leads to the emergence of DNA-repair deficiency phenotype in the tumor that is not shared by the patient s normal tissues and that can be exploited to induce selective tumor cytotoxicity. In fact, in response to the continuous damaging of cellular DNA, several DNA repair pathways act in parallel in order to maintain genomic integrity and, in the absence of a functioning homologous recombination pathway, cells are more susceptible to the alteration of other DNA repair systems. One of these parallel systems is the repair of DNA single-strand breaks. The poly-adenosine diphosphate [ADP] ribose polymerases (PARPs) are a large family of multifunctional enzymes that play a key role in the repair of DNA single-strand breaks through base excision repair. PARP-1 is

6 the primary enzyme involved in DNA repair, while PARP-2 and PARP-3 are less involved. DNA damage stimulates the catalytic activity of PARP-1, which binds near the DNA single-strand break and activates the base excision repair system [17-19]. Through this mechanism, PARP inhibition induces the accumulation of DNA single-strand breaks. In addition, as PARP1 is involved in the control of transient fork reversal and replication fork restart following genotoxic stress, its inhibition might not only result in an accumulation of single-strand breaks, but also in a greater fraction of these lesions being processed into double-strand breaks [20]. This ultimately leads to an accumulation of DNA double-strand breaks at replication forks as a result of PARP inhibition. Homologous recombination deficient cells, such as tumor cells with inactivating mutations of the BRCA genes, cannot repair double-strand break DNA damage, therefore leading to cell death and/or cell cycle arrest. On the contrary, normal cells are heterozygous for BRCA mutations, therefore conserving an intact homologous-recombination function that allows to restore DNA damage through homologous recombination thus maintaining cell viability. On this basis, the concept of synthetic lethality was theorized [21-22]. Synthetic lethality occurs when there is a potent and lethal synergy between two otherwise nonlethal events, such as PARP inhibition and homologous recombination deficiency. They demonstrated that, in vitro, BRCA1- deficient and BRCA2-deficient cells were up to 1000-fold more sensitive to PARP inhibition than wild-type cells, and tumor growth inhibition was demonstrated in BRCA2-deficient xenografts [21-22]. Following these initial observations, other mechanisms of action of PARP inhibitors have been proposed, such as trapping of PARP-1 on damaged DNA, defective BRCA recruitment, and activation of non-homologous end joining [17-19].

7 1.2 PARP inhibitors in development in breast cancer Up to now, olaparib is one of the most investigated PARP inhibitors. It has been the first PARP inhibitor to receive FDA approval for the treatment of advanced ovarian cancer with germline BRCA-mutation and has been the first PARP inhibitor to report positive results from a phase III trial in metastatic HER2-negative BC with germline BRCA-mutation. However, several PARP inhibitors are currently under clinical development in BC patients (Table 1). In addition to olaparib, niraparib (MK4827), veliparib (ABT-888) and talazoparib (BMN673) have been tested in clinical phase III trials for metastatic BC patients. For olaparib and talazoparib, positive results from phase III trials have recently been presented. These agents differ slightly from each other in terms of activity, toxicity profile, pharmacodynamics, and capability of exerting secondary actions (such as PARP-1 trapping) [23, 24]. The first therapeutic agent to be thoroughly investigated as a PARP inhibitor, iniparib, was subsequently identified as having a different mechanism of action. After promising results of a phase II study [25], a randomized open-label phase III study of iniparib plus chemotherapy (carboplatin-gemcitabine) versus chemotherapy alone was conducted in metastatic TNBC patients, not selected for having BRCA mutations. This trial did not observe any statistically significant difference in term of overall survival or progression-free survival and clinical development of iniparib has stopped [26]. 1.3 Overview of the market Germline BRCA1-mutated patients more often present BC with TN phenotypes [15], while BRCA2 mutations are more frequently associated with a luminal B phenotype [15]. For patients with hormone-receptor positive BC, more effective endocrine treatments and the association with targeted agents, such as mtor inhibitors and CDK inhibitors, have improved

8 clinical benefit rates and progression free survival [27]. As a result, chemotherapy is often offered to these patients late in the clinical history of BC, when the disease has become resistant to endocrine-based treatment. On the contrary, chemotherapy still remains the cornerstone of systemic treatment in TNBC. Despite an initial chemosensitivity, TNBCs are associated with a high risk of relapse in the adjuvant setting and a worse prognosis in the metastatic setting [28]. Indeed, TN metastatic BC patients have a poor outcome, with a median overall survival that barely exceeds one year from diagnosis even for patients treated with chemotherapy in recent years [29]. Therefore, the treatment of metastatic TNBC still remains the most burning unmet need in the field of breast oncology and major efforts have been convened in understanding the molecular landscape of TNBC and uncovering actionable targets. Preclinical and clinical data have shown that patients with tumors harboring BRCA1/2 aberrations might selectively benefit from treatment with PARP inhibitors and DNA-damaging agents [30-31], and several of these agents are currently been investigated in phase III trials (NCT , NCT , NCT , NCT , NCT , NCT ). Patients with TNBC carrying defects in genes related to homologous recombination could also have a similar pattern of drug sensitivity [32]. 2.1 Introduction to the compound Olaparib (also known as AZD2281 or KU ) is a small molecule, orally active PARP-1, PARP-2 and PARP-3 inhibitor [33-34], capable of inducing synthetic lethality in BRCA-deficient cells. It has been developed in monotherapy as well as in association with radiation and chemotherapy in several malignancies.

9 In 2014, olaparib was licensed in ovarian cancer by both the European Medicines Agency and the U.S Food and Drug Administration. In addition, in January 2018 olaparib was licensed for the treatment of patients with germline BRCA-mutated, HER2-negative metastatic BC who have previously received chemotherapy by the U.S Food and Drug Administration. 2.2 Chemistry The chemical name for olaparib is 4-[3-(4-cyclopropylcarbonylpiperazin-1-carbonyl)- 4-fluorobenzyl]-2H-phthalazin-1-one [33]. (Figure 1) Olaparib is a phatalazinone [33]. Its core structure belongs to the planar bicyclic lactams with the carbamoyl moiety locked in an anti-conformation via a ring connection [35] Pharmacodynamics In vitro studies have demonstrated the efficacy of olaparib as PARP-1 inhibitor, highlighting the enhanced sensitivity of BRCA deficient tumor cell lines to this compound [21-22]. In fact, BRCA-1 and 2 deficient cells are 1000 times more sensitive to PARP inhibition than BRCA-viable cells, with a concentration for 50% inhibition (IC50) against PARP-1 of 5-6 nm, an IC50 against PARP-2 of 1nM, and an IC50 against PARP-3 of 4nM [33-34]. Olaparib also showed a synergistic activity with cisplatin in BRCA2 deficient mammary cell lines [36]. In vivo, olaparib has been shown to significantly reduce tumor growth in mouse xenograft models of human cancer, with increased antitumor activity observed in BRCA1 or BRCA2 deficient models [21,22,36,37]. Moreover, the combination of olaparib with a platinum drug increased overall survival of a genetically engineered mouse model of BRCA1-deficient BC as compared to monotherapy with olaparib or platinum drug alone [37].

10 In a phase I study, BRCA mutated patients receiving at least 60mg BID olaparib, showed a PARP inhibition of 90% or more (as compared with baseline value) [38] Pharmacokinetics and metabolism Two kinds of formulation exist for olaparib: capsules (50 mg) and tablets (100 and 150 mg). The tablet formulation has been developed in order to deliver the therapeutic dose of olaparib in fewer dose units. However, it should be highlighted that the capsule and tablet formulations are not bioequivalent, as demonstrated in a phase I bioavailability study of the two formulations [39]. In fact, the 200mg BID tablet formulation was observed to have similar Cmax as compared to 400 mg BID capsule formulation, but lower AUCss and Cmin [39]. Following multiple dosing, steadystate exposure with tablets 300 mg matched or exceeded that of 400 mg capsules. Based on this observation and on long-term toxicity from the randomized expansion phase, 300 mg twice daily was defined as recommended monotherapy dose of olaparib tablet formulation for phase III trials [40]. Olaparib is rapidly absorbed with peak plasma concentrations achieved within 1 to 3 hours after dosing for the 400 mg capsule dose [38], and within 1.5 to 4 hours of dosing for the 300 mg tablet dose depending on fasted/fed state [41]. Mean half-life is about 6 hours for the capsule formulation [38] and about 12 to 14 hours for the tablet formulation [41]. Based on trials evaluating olaparib tablet pharmacokinetics in patients with renal impairment, patients with mild renal impairment (creatinine clearance ml/min) do not require adjustments in olaparib dosing, while for patients with moderate renal impairment (creatinine clearance ml/min) the recommended starting dose is 200 mg twice daily. Olaparib has not been evaluated, and is not recommended, for patients with severe renal impairment or end-stage renal disease (creatinine clearance 30 ml/min). Patients with mild hepatic impairment (class A

11 according to Child-Pugh classification) do not require adjustments in the starting dose, as only a 15% increase in mean exposure was observed in patients with mild hepatic impairment as compared to patients with normal hepatic function. Currently, olaparib is not recommended in patients with moderate to severe hepatic impairment (serum bilirubin >1.5 times the upper limit of normal) [42]. Food intake has not been shown to significantly affect olaparib pharmacokinetics, as coadministration of a high-fat meal with olaparib slows the rate of absorption, but does not significantly alter the extent of olaparib absorption [41,43]. Olaparib is principally metabolized by CYP3A. Therefore, strong CYP3A inhibitors and inducers should be avoided (AstraZeneca prescribing information). 3. Clinical efficacy 3.1 Phase I Studies (Table 2 and Supplementary Table 1) Preclinical data on the efficacy of olaparib as PARP inhibitor and on the enhanced sensitivity of BRCA mutant cells led to the design of the first phase I trial of olaparib as a single agent. This phase I dose escalation study enrolled 60 patients with refractory solid tumors and was enriched for BRCA mutated patients [38]. The maximum tolerated dose of olaparib was 400mg twice daily in capsule formulation and olaparib was found safe with a majority of grade 1 or 2 adverse events. Dose-limiting toxicities included grade 4 thrombocytopenia, grade 3 mood alteration and fatigue, and grade 3 somnolence. This study was also the first to establish the proof-of-concept of olaparib activity as single agent in patients with BRCA deficient metastatic BC, as prolonged objective antitumor activity was only achieved in patients with confirmed BRCA-mutated tumors. This trial enrolled 9 patients with breast cancer; among them, 3 had a BRCA2 mutation. One of these three patients experienced complete remission, while another achieved a 7 months disease stability

12 [38]. This first pivotal phase I trial was followed by several early phase combination trials in BC patients (Supplementary Table 1). Lee et al. reported results from a phase I/Ib testing the association of olaparib (100 to 400mg BID in capsule formulation) and three-weekly carboplatin (AUC 3 to 5) in 45 BRCA mutated patients with BC or ovarian cancer. All patients were heavily pretreated. In this trial, dose-limiting toxicity was not reached so the single agent dose of olaparib 400 mg twice daily (day 1 to 7) with carboplatin AUC5 every 3 weeks has used for the expansion cohort. Toxicity was mostly hematologic: grade 3 and 4 neutropenia (42.2%), thrombocytopenia (20.0%), and anemia (15.6%) were reported. Among 8 BC patients enrolled, 1 experienced a complete response and 6 a partial response, with a total response rate of 87.5% [44]. A similar phase I/Ib trial was also conducted testing the association of olaparib in capsule formulation 400 mg twice daily, days 1-7, with three-weekly carboplatin (AUC 3 to 5) for 8 cycles followed by olaparib 400mg twice daily in 28 patients diagnosed with sporadic metastatic TNBC. The dose-limiting toxicities observed at the carboplatin AUC5 dose were grade 4 thrombocytopenia and grade 3 symptomatic hyponatremia. The association of olaparib capsule formulation 400mg twice daily (days 1-7) and three-weekly carboplatin AUC4 was defined as maximum tolerated dose. As expected, toxicity was mostly hematologic: grade 3 and 4 neutropenia (36%), thrombocytopenia (11%), and anemia (11%) were reported. However, the response rate was not striking: 1 complete response and 5 partial responses (22% response rate). Preplanned biomarker analysis subsequently identified a deletion of BRCA1 exons 1-2 in the only patient who achieved a long-term complete response [45]. The combination of olaparib in capsule formulation and cisplatin was also evaluated in a multipathology phase I trial enrolling patients with advanced BC, ovarian cancer, and other solid tumors. Among 54 patients included, 29 patients had BRCA1 or BRCA2 mutations and 42 BC

13 patients were enrolled. Dose-limiting toxicities occurred with cisplatin 75mg/m2 in combination with continuous olaparib 100mg or 200 mg twice daily (capsule formulation) and with cisplatin 75mg/m2 in combination with olaparib 100mg twice daily days 1-10 or 50mg twice daily days 1-5 (capsule formulation). A reduced dose of cisplatin 60mg/m2 in combination with olaparib 50mg twice daily days 1-5 was therefore tested achieving a manageable toxicity profile. The most frequently observed grade 3 adverse events were neutropenia (16.7%), anemia (9.3%) and leucopenia (9.3%). The objective response rate was 71% in BRCA mutated patients with BC [46]. Olaparib has also been tested in combination with non-platinum based chemotherapy. A combination phase I trial testing the association of olaparib with paclitaxel included 19 patients with TNBC. Patients were included with no regards to BRCA mutational status assuming that sporadic TNBC shared BRCA1-associated BC sensitivity to PARP inhibitors. Patients were treated with weekly paclitaxel 90 mg/m2 (day 1,8,15 in a four-week cycle) in combination with olaparib 200 mg twice daily continuously (capsule formulation). A high rate of neutropenia was observed, leading to the enrollment of a second cohort of patients (n=10) who were treated with granulocyte-colony stimulating factor if they experienced grade 2 neutropenia in cycle 1 and continued it prophylactically in subsequent cycles. The response rate was 33.3% in cohort 1 and 40% in cohort 2, with a median PFS of 6.3 and 5.2 months, respectively. However, relevant gastrointestinal and hematologic toxicity was mainly reported: adverse events were diarrhea (63%), nausea (58%) and neutropenia (58%), with grade 3 and 4 neutropenia observed in 44% of patients in cohort 1 and in 20% of patients in cohort 2 and the study was terminated [47]. On the contrary, a phase I trial testing olaparib (capsule formulation) in combination with pegylated liposomal doxorubicin reported a good safety profile [48]. Olaparib has also been tested in a phase I trial in association with paclitaxel or carboplatin or both in patients with advanced solid tumors refractory to standard therapies. 87 patients were

14 enrolled, 26% of which presented BC. Patients who completed at least 6 cycles of combination treatment and experienced substantial toxicity without any sign of disease progression were offered to continue olaparib monotherapy (400mg twice daily in capsule formulation) [49]. 21 patients (10 with BC) were included in this maintenance phase, 16 of them harboring BRCA mutation. During the maintenance phase, the most common adverse event was hematological toxicity, though the severity of hematological adverse events decreased over time. Nonhematological adverse events were grade 2 or less, the most frequent being fatigue, pain, nausea, cough, dyspnea, and diarrhea. BRCA1 or 2 mutated patients showed significant benefit; 9 patients presented complete response and 4 presented partial response, while patients with wild-type or unknown mutation status only presented stable or progressive disease [50]. The REVIVAL study, a phase I/II trial comparing the combination olaparib-carboplatin (2 cycles) followed by olaparib monotherapy to standard of care capecitabine, is currently recruiting (NCT ). Olaparib is also being tested in combination with several targeted agents, including immunotherapy (Supplementary Table 1). For some of these combinations, encouraging preliminary results have been presented [51-54]. Results are available for the phase I combination trial evaluating olaparib in association with cediranib in patients with recurrent ovarian cancer or recurrent TNBC. Only 8 BC patients were enrolled in the trial and none achieved clinical response [55]. Preclinical studies showed that PARP inhibitors can act as radio sensitizers, probably through the impairment cell radiation-induced single strand breaks repair [56]. This has led to the design of radiotherapy-based trials (Supplementary Table 1). At least two of these trials testing radiation in combination with olaparib are currently ongoing in BC (NCT , NCT ).

15 3.2 Phase II Studies (Table 3 and Supplementary Table 2) Based on the pivotal results by Fong et al, phase II studies of olaparib for BRCA1 or 2 mutated metastatic BC patients were developed. In a first trial, published by Tutt et al., olaparib was given in one cohort at the maximum tolerated dose (400mg twice daily in capsule formulation) and in a second cohort at 100mg twice daily. The objective response rate was 41% and 22% in the two cohorts respectively. Median progressionfree survival was 5.7 months (95%CI ) and 3.8 months (95%CI ), respectively. In this trial, olaparib showed significant activity even in heavily pretreated patients and toxicity was manageable. At the maximum tolerated dose of olaparib 400mg twice daily, the most frequent grade 3-4 adverse events were nausea (15%), fatigue (15%), vomiting (11%), and anemia (11%) [57]. Similar results were reported by a preliminary analysis of a phase II trial enrolling 298 germline BRCA1 or 2 mutated patients with ovarian, breast, prostate or pancreatic cancer. As expected, the most frequent adverse events were fatigue, nausea, and vomiting. 62 patients enrolled in this trial had heavily pretreated ( 3 chemotherapy regimens) metastatic BC. In this subgroup of patients, tumor response rate was lower, 12.9%, but almost half of the patients had received a prior platinum-based chemotherapy [58]. A third phase II trial evaluated the efficacy of olaparib (400mg twice daily capsule formulation) in recurrent ovarian cancer and TNBC, with no regards to BRCA mutational status. Among the 26 BC patients enrolled, 16 did not have a BRCA mutation. None of the BC patients had a confirmed objective response according to RECIST criteria [59]. Several ongoing phase II trials are currently testing the use of olaparib monotherapy in patients baring homologous repair alterations other than germline BRCA1 or BRCA2 mutations (NCT , NCT , NCT ; marked with * in Supplementary Table 2).

16 3.3 Phase III Studies (Supplementary Table 3) The efficacy of olaparib as a monotherapy agent has been evaluated in a randomized, open-label, phase III trial, the OlympiAD trial (NCT ). This study enrolled 302 patients with a germline BRCA mutation and HER2 negative metastatic BC who had received no more than two previous chemotherapy regimens for metastatic disease. Patients were randomized in a 2:1 ratio to receive olaparib tablets (300 mg twice daily) versus single-agent standard chemotherapy of physician s choice (capecitabine, eribulin, or vinorelbine in 21-day cycles). The primary end-point was progression-free survival, analyzed on an intention-to-treat basis and assessed by blinded independent central review. Patients were stratified according to having received previous chemotherapy in the metastatic setting, hormone-receptor expression, and previous platinumbased chemotherapy (patients could have received platinum for metastatic disease if they had not progressed during treatment). [60]. The study showed a significantly longer median progressionfree survival in the olaparib arm as compared to the chemotherapy arm (7.0 months vs. 4.2 months; HR 0.58; 95% CI 0.43 to 0.80; P<0.001). At subgroup analysis for progression-free survival, patients with TNBC seemed to benefit most from the experimental treatment with olaparib (HR 0.43, 95% CI ). In the overall population, the response rate was also significantly higher in the olaparib group (59.9%) than in the chemotherapy group (28.8%) and olaparib showed a better toxicity profile (grade 3 adverse event rate 36.6% and 50.5% in the olaparib arm and standardtreatment arm, respectively). The rate of treatment discontinuation due to toxicity was also lower in the olaparib arm (4.9% and 7.7%, respectively). Of interest, the trial included also patients pretreated with platinum and deemed to be platinum sensitive; also in these patients olaparib was more effective than chemotherapy (HR 0.67; 95% CI ). Anemia, nausea, vomiting, fatigue, headache, and cough occurred more frequently in the olaparib group, while neutropenia,

17 palmar plantar erythrodysesthesia, and an increase in liver-function enzymes were more common in the standard chemotherapy arm. No significant difference in overall survival was observed between the two treatment arms (median overall survival 19.3 months vs months, respectively; HR 0.90; 95% CI ; p=0.57), however the trial was not powered to assess differences in overall survival between treatment groups [60]. A confirmatory phase IIIb trial, the LUCY trial (NCT ), is currently ongoing in metastatic HER2 negative BC with germline BRCA1/2 mutations. Two other phase III trials are currently recruiting in the neo/adjuvant setting, enrolling patients with HER2 negative primary BC at high risk of recurrence. The OlympiA trial (NCT ) randomizes patients with high-risk HER2 negative BC and known germline BRCA1/2 mutations to one year of olaparib or placebo after completion of definitive local treatment and standard neoadjuvant or adjuvant chemotherapy. The SUBITO trial (NCT ) is a randomized, openlabel, (neo)adjuvant phase III study comparing optimized standard-dose chemotherapy followed by olaparib (dose-dense AC followed by carboplatin-paclitaxel followed by one year of olaparib) versus intensified, alkylating chemotherapy (cyclophosphamide, thiotepa and carboplatin) with stem cell rescue. This trial enrolls not only patients with germline BRCA1 or BRCA2 mutation, but also patients with BRCA1-like test positive BC (tumors with DNA copy number aberration profile similar to germline BRCA mutated tumors). 4. Safety and tolerability Olaparib monotherapy appears to have a manageable toxicity with doses up to 400mg twice daily. Several phase II trials confirmed the tolerability of olaparib 400mg twice daily (capsule formulation), while the phase III OlympiAD trial was conducted using the 300 mg twice-daily tablet formulation. Adverse events observed in these trials are mainly hematological toxicity (anemia,

18 leucopenia, neutropenia, and thrombocytopenia), gastrointestinal symptoms (nausea, vomiting, diarrhea, and stomatitis) and fatigue (Table 4 and 5). A meta-analysis of 731 patients with ovarian cancer and breast cancer treated with olaparib in 6 published trials reported nausea and vomiting as the most common toxicities [61]. Anemia is the most common hematological toxicity (40% of any grade anemia in the OlympiAD trial) and is generally manageable [60]. A small number of patients treated with olaparib have been reported to develop acute myeloid leukemia and myelodysplastic syndrome. Ledermann et al. originally reported three cases of patients who developed myelodysplasia and acute myeloid leukemia: two in the olaparib group (2.2%) and 1 in the placebo group (0.8%) [62]. All these patients had been previously treated with two lines of chemotherapy [62]. Three additional cases were reported by Kaufman et al. out of 298 patients treated with olaparib (1%) [58]. No cases of the myelodysplastic syndrome or acute myeloid leukemia were reported in either treatment group in the OlympiAD trial [60]. Overall, the incidence of myelodysplastic syndrome and acute myeloid leukemia in patients treated with olaparib monotherapy in clinical trials is <1.5% (21/1680), as reported by AstraZeneca prescribing information and additional cases have been documented in combination studies. All reported cases appear to have occurred in patients with other potential contributing factors, including extensive previous chemotherapy and/or radiotherapy. Therefore, a clear causal relationship between olaparib and the development of myelodysplastic syndrome and acute myeloid leukemia cannot be confirmed. However, special attention should be given to prolonged hematological toxicities in patients treated with olaparib. Data from phase I combination studies of olaparib with chemotherapy agents demonstrated more severe hematological toxicity (anemia, neutropenia, thrombocytopenia) than olaparib monotherapy [44-47,50].

19 5. Regulatory affairs Olaparib in capsule formulation was registered for use in Europe and in the United States in Based on the results of the phase II maintenance study [63], EMA approved olaparib as monotherapy for maintenance treatment of patients with platinum-sensitive relapsed BRCAmutated (germline and/or somatic) high-grade serous ovarian cancer who were responding to platinum-based chemotherapy. Some European countries, such as the United Kingdom, further restricted the indication: the National Institute for Health and Care Excellence only confirmed funding for patients who had received three or more lines of platinum-based chemotherapy. Only at the end of February 2018, olaparib received a positive opinion from the Committee for Medicinal Products for Human Use of the EMA recommending the use of the tablet formulation in Europe. Up to date, olaparib is not approved as treatment for BC patients in Europe. In the United States, the Food and Drug Administration approved the use of olaparib for the treatment of patients with deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy, based on the results from the ovarian cancer cohort of Study 42 [64]. The use of olaparib as maintenance therapy for patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer, who are in a complete or partial response to platinum-based chemotherapy, regardless of BRCA status, was only approved in the United States in August Concomitantly, the Food and Drug Administration granted approval to olaparib tablets for both indications. In addition, in January 2018 olaparib was licensed by the U.S Food and Drug Administration for the treatment of patients with germline BRCA-mutated, HER2-negative metastatic BC who have previously received chemotherapy.

20 6. Conclusion Olaparib is the first PARP inhibitor to demonstrate significant clinical benefit over standard therapy in metastatic BC patients [60]. In preclinical studies, treatment with PARP inhibitors has been shown to lead to highly selective toxicity in BRCA1 and BRCA2 mutated cancers cells. This observation has been further confirmed by clinical data from phase I and phase II studies, which demonstrated the activity of olaparib in BRCA-mutation associated BC patients both as monotherapy and in combination with chemotherapy or targeted agents. The phase III randomized OlympiAD trial, which enrolled patients with HER2-negative metastatic BC and a germline BRCA mutation, demonstrated that olaparib monotherapy improves progression-free survival as compared with standard chemotherapy and confirmed its favorable toxicity profile. Randomized phase III trials are now testing the use of olaparib as adjuvant treatment for high-risk HER2-negative BC patients with germline BRCA mutation. The combination with other targeted therapies or chemotherapies and improvement of patient selection through the identification of additional potential predictive biomarkers, such as homologous repair alterations different from germline BRCA mutations, are still under investigation. 7. Expert Commentary While multiple therapeutic options are currently available for hormone receptor and/or HER2 positive BC, the treatment of TNBC is still based on chemotherapy and lacking significant improvements in therapeutic efficacy. Molecular profiling of BC has led to the identification of possible molecular triggers such as germline BRCA1/2 mutations or BRCAness (homologous

21 repair defects in sporadically occurring tumors in the absence of germline BRCA1 or BRCA2 mutations). In this context, trials with PARP inhibitors have achieved promising results both as monotherapy and in combination in BRCA-mutated BC patients. Olaparib has been the first PARP inhibitor to show significant clinical benefit over standard therapy in terms of progression-free survival in a randomized phase III trial [60], shortly followed by talazoparib which, in the large phase III randomized EMBRACA trial, achieved very similar results [65]. Evidence from these two randomized phase III trials, the OlympiAD and the EMBRACA trial, will probably prompt the regulatory approval of PARP inhibitors in HER2-negative BRCA mutated metastatic BC (olaparib already received regulatory approval by the U.S. Food and Drug Administration in January 2018). However, despite the fact that both trials enrolled hormone receptor positive and triple-negative BC patients, the large unmet need in the treatment of metastatic BC is represented by TN disease. In fact, for patients with hormone-receptor positive BC several lines of no-chemotherapy containing treatment are available. In last decades, more effective endocrine agents, such as aromatase inhibitors and fulvestrant, have entered clinical practice; more recently, the association of targeted agents, such as mtor inhibitors and CDK inhibitors, have further boosted survival of patients diagnosed with metastatic hormone-receptor positive BC, which may now be estimated to exceed 3 years [27]. As a result, chemotherapy is often offered to these patients after several lines of therapy, when the disease has become resistant to endocrine-based treatment. It is thereby in metastatic TNBC that PARP inhibitors are more likely to be rapidly and widely used in clinical practice, due to the current lack of significant competitors and the good tolerability profile. Nevertheless, some open questions remain. Over the last decade, significant evidence has been building regarding the exquisite sensitivity of BRCA-associated BC to platinum salts [30, 66-67]. Since platinum agents were not included as

22 treatment options in the control group, neither olaparib in the OlympiAD nor talazoparib in the EMBRACA trial have been directly compared with a platinum-based chemotherapy. In the OlympiAD trial, patients could have received platinum for metastatic disease if they had not had progression during treatment, but only 29% of patients enrolled in the olaparib arm had actually received previous platinum-based therapy. Even if efficacy seemed to be maintained in the platinum pretreated subgroup, the trial does not allow for the assessment of olaparib in truly platinum-resistant BC and cannot address the relative benefits of olaparib and platinum-based chemotherapy in patients with BC and a germline BRCA mutation. Response rate and progressionfree survival observed with olaparib in the OlympiAD trial (59.9% response rate, 7.0 months median progression-free survival) are similar to those observed by first-line single-agent carboplatin in the TNT trial (68.0% response rate, 6.8 months median progression-free survival) in a similar population [30]. Therefore, indirect comparison does not strongly support the use of olaparib as an alternative to platinum salts. In addition, a randomized, phase II trial testing the addition of veliparib to the combination of carboplatin and paclitaxel, did not show any significant improvement in progression-free survival and overall survival from the use of the PARP inhibitor as compared to placebo in the context of platinum-based chemotherapy [68]. These results leave open questions regarding the real benefit of using platinum salts and PARP inhibitors in combination or in sequential lines of therapy. A head-to-head study, designed to determine the relative efficacy of olaparib and platinum-salts, and larger studies, investigating the differential treatment effects of olaparib among subgroups, would be helpful. In addition to platinum drugs, other DNA crosslinking agents, such as nimustine, have been reported to have a pronounced activity in cancers harboring BRCA alterations [69] and comparisons or combinations between these agents and PARP inhibitors should be considered as well. Another critical need is to identify biomarkers to guide the selection of patients most likely to

23 benefit from PARP inhibition. Even if preclinical studies have identified BRCA1/2 mutations as biomarkers of sensitivity to PARP inhibition [18-19,21-22,32,36-37], not all BRCA-mutated patients respond to PARP inhibitors, while some patients not harboring mutations respond. Homologous recombination repair is based on a complex system of multiple interacting proteins; therefore, defects in homologous recombination secondary to alterations other than BRCA mutation might be used as biomarkers of response to PARP inhibition. This situation has been defined as BRCAness [32]. In fact, in addition to mutations in BRCA1/2, abnormalities in other homologous repair deficiency genes have been identified, include mutations in ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D [17-18,70]. In addition to these, several BCs have been reported to harbor somatic BRCA1 or BRCA2 mutations which might also contribute to a homologous recombination deficit phenotype [71]. Due to the complex and variable nature of homologous recombination deficits in cancer cells, several diagnostic assays have been proposed in the attempt to recapitulate a BRCAness phenotype, more than a single molecular alteration. These include tests evaluating Loss of Heterozygosity in regions longer than 15 Mb, and more complex assays, such as the HRD score, a composite of Loss of Heterozygosity, telomeric allele imbalances, and large-scale state transitions, and the HRDetect, a score based on mutational signatures [72]. Several phase II trials are currently testing in BC the possibility of selecting patients for PARP inhibition by alterations other than germline BRCA1 or BRCA2 mutations (NCT , NCT , and NCT ). However, up to date, olaparib has only been approved in BC patients harboring germline BRCA mutations. As the use of PARP inhibitors in BC will expand in the next few years, the understanding of the mechanisms of resistance to these agents will be of extreme importance in order to accurately design therapeutic sequences in homologous recombination repair deficient BC. The acquisition of

24 secondary BRCA mutations that result in restoration of BRCA function has been described [73]; in these cases, the subsequent activity of alternative treatments based on the alteration of the homologous recombination repair, such as platinum salts or other DNA-crosslinking agents, might be limited. However, other mechanisms of resistance have also been described, such as drug efflux by P-glycoprotein [37] or BRCA-independent restoration of homologous recombination (e.g. somatic mutations of tumor protein p53 binding protein 1 or loss of MAD2 mitotic arrest deficientlike 2) [74-75]. In these cases, the patients might still benefit from treatment with DNA crosslinkers. In the next few years, data from several phase III trials with new therapeutic agents in BRCA associated BC should become available. In this scenario, several new therapeutic agents, ranging from different PARP inhibitors, DNA-damaging agents, targeted agents to checkpoint inhibitors, may be competing for the now empty niche of BRCA-associated TNBC treatment. The ongoing combination trials, which are testing the use of olaparib with other targeted therapies, immune therapy, and chemotherapy, should be considered with particular interest. 8. Five Year View It is likely that the future treatment of TNBC, in particular in presence of germline BRCA mutations, will continue to evolve in the next few years as other major clinical trials reach their completion dates. A series of phase III trials are currently investigating the use of several other PARP inhibitors (i.e. talazoparib, niraparib, veliparib) in metastatic HER2-negative BRCA-associated BC. For one of these trials, positive results, showing an improvement of progression-free survival with the use of talazoparib as compared to standard chemotherapy, have already been presented [65]. In the next few years, we might probably expect to see similar results for the use of niraparib from the phase III randomized BRAVO trial (NCT ).

25 In this scenario, several PARP inhibitors might reach regulatory approval in the next five years, and we are currently lacking solid clinical trials comparing head to head these new treatment options. In addition, the use of chemotherapy in BRCA-associated metastatic BC is currently changing with the increased use of platinum-based regimen [30]. In this context, the decision on how to sequence PARP inhibitors and platinum-based regimens in this setting will be a difficult and uncertain one, and results from the randomized phase III trial testing the addition of veliparib to a platinum-based regimen (NCT ) will be of particular interest. Moreover, if ongoing randomized phase III trials confirm the encouraging data from phase II trials, other new agents, such as immune therapy, might be expected to reach clinical practice in metastatic BC in the next five years. Understanding how best to choose, combine or sequence treatments for metastatic BRCA-associated BC is a key question that will need to be formally addressed. Combination trials of these agents with olaparib are already ongoing. Another key result for the use of olaparib in BC will come from the randomized phase III adjuvant OlympiA trial (NCT ). If this study shows positive results, olaparib might probably move at least partially from the increasingly crowed metastatic setting to a high-risk adjuvant setting. Clinical trials with appropriate endpoints will also be critical for addressing many of the questions that lie ahead in BC, one of the most relevant being the possibility to use defects in homologous recombination secondary to alterations other than BRCA mutation (BRCAness) as biomarkers of response to PARP inhibition. It is likely that the management of patients with homologous recombination deficient BC will change in the next few years, as multiple effective agents might be expected to enter clinical practice. Yet, large gaps in our understanding of how to sequence and combine these agents remain. A more comprehensive understanding of the molecular biology of BC, assessing

26 biomarkers of resistance and sensitivity to these new therapeutic targets, will allow for a more precise selection of therapy at an individual patient level. Key issues: Mutations in BRCA1 and BRCA2 genes account for around 5-10% of breast cancers Preclinical studies have shown that treatment with PARP inhibitors induces selective toxicity in BRCA1 and BRCA2 mutated cancers cells Olaparib is the best studied orally administered PARP inhibitor Phase I and phase II studies have confirmed the clinical activity of olaparib in BRCAmutation associated breast cancer patients both as monotherapy and in combination with chemotherapy or targeted agents The phase III randomized OlympiAD trial demonstrated that olaparib monotherapy improves progression-free survival as compared with standard chemotherapy in patients with HER2-negative metastatic breast cancer and a germline BRCA mutation with a favorable toxicity profile The combination with other targeted therapies or chemotherapies and improvement of patient selection through the identification of additional potential predictive biomarkers are under investigation Funding This paper was not funded. Declaration of interest V Guarneri discloses being a part of the speaker s bureau for Novartis, Pfizer, and AstraZeneca, as well as acting as a consultant for Eli Lilly. P Conte discloses work with Roche, Novartis,

27 AstraZeneca, Merck, and Celgene. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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