**4. PARP inhibitor therapy**

QOL at 1 year [34], filing for regulatory approval for cediranib had been previously withdrawn.

Pazopanib inhibits VEGR1,2 and 3, c-Kit and PDGFR. The AGO-OVAR 16 study [35] evaluated first-line maintenance pazopanib. PFS was 17.9 months for pazopanib compared to 12.3 months for control. Grade 3/4 adverse events were significantly higher for pazopanib including hypertension (30.8%), neutropenia (9.9%) and diarrhoea (8.2%). Discontinuation due to AEs occurred in 33% in the pazopanib arm compared to 5.6% in the placebo arm. Regulatory approval filing was withdrawn due to perceived imbalance in benefit–risk ratio. Other VEGFR TKIs have been studied in ovarian cancer [35]. Nintedanib was given in the first-line setting with chemotherapy and then maintenance. Again, a PFS benefit was seen but no significant OS advantage [36]. Other multitargeted VEGFR TKIs such as sunitinib and sorafenib have also been studied with similar outcomes. As a class the TKIs appear to have some effect however their multi-targeted nature and unpredictable bioavailability means that

Nonetheless cediranib maintenance is undergoing investigation in ICON9 (see below).

their perceived risk:benefit ratio has not led to any regulatory approvals as yet.

The Ang-Tie pathway is distinct from the VEGF axis, involved in vascular remodelling. Trebananib is peptide-Fc fusion protein that binds Angiopoietin 1 and 2 and prevents interaction with Tie on endothelium. Although promising results were seen in phase II [37], a phase III trial (TRINOVA-2) [38] failed to meet its PFS endpoint and a third terminated early for

Vascular disrupting agents (VDAs), in contrast to inhibiting formation of new vessels, target existing tumour vasculature. The VDA's combretastatin and fosbretabulin disrupt the endothelial cytoskeleton (by binding tubulin) aiming to cause endothelial detachment and eventual vessel obstruction. Tumour vasculature lacks pericytes and smooth muscle making them selectively susceptible. Fosbretabulin is being examined for synergy with bevacizumab and

There is pre-clinical rationale for the combination of VEGF-targeted therapy with poly (ADPribose) polymerase inhibitors (PARPi); anti-VEGF induced hypoxia can impair DNA repair and sensitize otherwise insensitive cells to PARPi. In a phase II trial of olaparib and cediranib [39] PFS with the combination was prolonged (17.7 vs. 9.0 months) and, consistent with preclinical rationale, the difference was most marked in BRCA wild-type patients. Grade 3/4 toxicity however was 70% with the combination vs. 7% for olaparib monotherapy. The combination is currently undergoing phase III testing (ICON 9). The combination of bevacizumab

Combining VEGF blockade and immunotherapy also has pre-clinical rationale (see below). Combinations of anti-angiogenesis and chemotherapy have been discussed in the paragraphs above. Of note, an early phase trial of pazopanib with carboplatin/paclitaxel was terminated

chemotherapy in platinum-resistant disease in a phase II/III trial (NCT02641639).

and olaparib in first-line maintenance is also being studied (NCT02477644).

early because of toxicity (GI perforations and myelotoxicity).

**3.3. Other antiangiogenic strategies**

336 Ovarian Cancer - From Pathogenesis to Treatment

futility (NCT01493505).

**3.4. Combination therapy**

DNA constantly undergoes single and double-strand breaks (SSBs/DSBs). SSBs are repaired predominantly by base excision repair (BER). PARPs are nuclear proteins with diverse functions including in BER and chromatin remodelling. PARP-1 is the most abundant member which upon binding to SSBs activates its ADP-ribosyltransferase catalytic domain allowing PARylation and recruitment of DNA repair effectors [41]. DSBs are mostly repaired by homologous recombination (HR) or non-homologous end joining (NHEJ), the latter being error-prone [42]. HR involves a number of key proteins including BRCA1, BRCA2, RAD51 and PALB2. A detailed discussion is beyond the scope of this chapter but the process of HR is reviewed here [43]

#### **4.1. Homologous recombination repair in ovarian cancer**

The Australian Ovarian Cancer Study Group screened 1001 patients with stage I-IV ovarian cancer for point mutations or large deletions in BRCA genes. 14.4% of patients overall had a germline mutation (including 17.1% with serous histology) [44]. A similar frequency was found in The Cancer Genome Atlas (TCGA) [45] although globally the prevalence varies between ethnic groups. In addition to germline mutations, BRCA genes can be somatically mutated, epigenetically silenced or the protein inactivated through post-translational mechanisms, e.g. EMSY amplification [46]. Various series have found somatic mutations of BRCA in 3–6% of EOC [47]. In contrast to somatic mutations, epigenetic silencing by promoter methylation is a dynamic process and may be harder to quantify. Studies report prevalence in the region of 5–30% of ovarian cancers.

However, BRCA1 and 2 are just two of many proteins involved in HR. TCGA undertook exomic analysis of 316 ovarian cancers as well as studies of promoter methylation, RNA expression and copy number changes [45]. Pathway analysis demonstrated that 51% of tumours had either mutations or silencing of components in the HR pathways. (**Figure 2**).

#### **4.2. PARP inhibitors in ovarian cancer**

HR deficiency (HRD) in EOC provides a target that can be exploited therapeutically. It was noted that cells with non-functioning PARP develop increased nuclear foci of Rad51 implying an increased burden of lesions being repaired by HR in these cells [48]. Farmer et al. [49] tested

trials at 400 mg BD. Objective responses were seen mainly in patients with germline BRCA mutations (gBRCAm) [52] Further support for the efficacy of olaparib in in the gBRCAm population came from a proof-of-concept phase II where the ORR in the 400 mg BD cohort was 33% including some complete responses (CRs) [53]. Of note, one heavily pre-treated patient

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A further phase II study gave 193 heavily pre-treated EOC platinum-resistant/unsuitable patients with gBRCA mutations olaparib at a dose of 400 mg BD [54]. The ORR was 31%. AEs were similar to those seen in earlier trials with a grade 3/4 rate of 54% including anaemia (17%) and fatigue (6%). Two patients developed leukaemia and one myelodysplastic syndrome, all were heavily pre-treated (25, 26 and 34 cycles each). These results (along with other applicant-submitted data) earnt olaparib FDA approval as monotherapy for patients with gBRCA mutations after three prior lines. The recent phase III SOLO3 study randomised patients with gBRCA mutations who have received at least 2 prior lines of platinum-based therapy and who are deemed at least partially platinum-sensitive to either Olaparib 300 mg BD or single agent chemotherapy of investigators choice [55]. Results are awaited. While the previous formulation of Olaparib required 16 capsules a day, the current tablet formulation requires only four raising hopes that some of the gastrointestinal toxicity

In the aforementioned studies olaparib was given as monotherapy for treatment of 'active' disease. In contrast, Study 19 randomised patients with recurrent platinum-sensitive cancer with at least 2 prior lines to *maintenance* olaparib or placebo post-chemotherapy [56]. In a predefined subset analysis of patients with known germline or somatic BRCA mutation (most retrospectively determined), median PFS in the gBRCAm group was 11.3 vs. 4.3 months with Olaparib and placebo respectively (HR 0.18). OS was not significantly different (23% crossover). The findings led to EMA approval. SOLO2 was a phase III double-blind placebocontrolled study in patients with recurrent platinum-sensitive EOC who had received at least 2 prior chemotherapy lines. Patients either got maintenance olaparib 300 mg BD or placebo. Investigator-assessed median PFS was 19.1 vs. 5.5 months (HR 0.30). Median PFS2 was also

developed acute myeloid leukaemia (AML) 9 months after cessation.

**Figure 3.** Schematic of synthetic lethality of PARP inhibition in HR deficient cells.

will be mitigated.

**Figure 2.** Distribution of HR gene mutations in EOC. Adapted from Ref. [47].

the hypothesis that BRCA 1/2 dysfunction would hypersensitize cells to PARP inhibition and were able to demonstrate this in BRCA deficient cell lines. This example of 'synthetic lethality' whereby either defect alone is tolerable but the combination is fatal has been exploited in the generation of a family of drugs, the PARP inhibitors. (**Figure 3**).

Following this, further work began on designing a PARP inhibitor (PARPi) suitable for clinical use. Early agents mimicked the substrate-enzyme interaction between NAD+ and the catalytic domain of PARP1/2 and further optimization led to the design of Compound 47, that would be developed as Olaparib [50]. Since Olaparib, several agents have been developed (discussed later) designed to inhibit PARP 1/2 catalytic activity.

In addition to catalytic inhibition, a distinct antitumour mechanism of PARPi, 'PARP-trapping' has been described. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs in PARP deficient cells and different PARP inhibitors had different PARP-trapping potency which was not correlated with their catalytic inhibitory properties [51].

#### **4.3. Olaparib**

Olaparib is an orally bioavailable small molecule with a nicotinamide moiety that competes with NAD+ for binding to PARP. The MTD for olaparib was established from early phase

**Figure 3.** Schematic of synthetic lethality of PARP inhibition in HR deficient cells.

the hypothesis that BRCA 1/2 dysfunction would hypersensitize cells to PARP inhibition and were able to demonstrate this in BRCA deficient cell lines. This example of 'synthetic lethality' whereby either defect alone is tolerable but the combination is fatal has been exploited in the

Following this, further work began on designing a PARP inhibitor (PARPi) suitable for clini-

lytic domain of PARP1/2 and further optimization led to the design of Compound 47, that would be developed as Olaparib [50]. Since Olaparib, several agents have been developed

In addition to catalytic inhibition, a distinct antitumour mechanism of PARPi, 'PARP-trapping' has been described. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs in PARP deficient cells and different PARP inhibitors had different PARP-trapping

Olaparib is an orally bioavailable small molecule with a nicotinamide moiety that competes

for binding to PARP. The MTD for olaparib was established from early phase

and the cata-

cal use. Early agents mimicked the substrate-enzyme interaction between NAD+

potency which was not correlated with their catalytic inhibitory properties [51].

generation of a family of drugs, the PARP inhibitors. (**Figure 3**).

**Figure 2.** Distribution of HR gene mutations in EOC. Adapted from Ref. [47].

338 Ovarian Cancer - From Pathogenesis to Treatment

(discussed later) designed to inhibit PARP 1/2 catalytic activity.

**4.3. Olaparib**

with NAD+

trials at 400 mg BD. Objective responses were seen mainly in patients with germline BRCA mutations (gBRCAm) [52] Further support for the efficacy of olaparib in in the gBRCAm population came from a proof-of-concept phase II where the ORR in the 400 mg BD cohort was 33% including some complete responses (CRs) [53]. Of note, one heavily pre-treated patient developed acute myeloid leukaemia (AML) 9 months after cessation.

A further phase II study gave 193 heavily pre-treated EOC platinum-resistant/unsuitable patients with gBRCA mutations olaparib at a dose of 400 mg BD [54]. The ORR was 31%. AEs were similar to those seen in earlier trials with a grade 3/4 rate of 54% including anaemia (17%) and fatigue (6%). Two patients developed leukaemia and one myelodysplastic syndrome, all were heavily pre-treated (25, 26 and 34 cycles each). These results (along with other applicant-submitted data) earnt olaparib FDA approval as monotherapy for patients with gBRCA mutations after three prior lines. The recent phase III SOLO3 study randomised patients with gBRCA mutations who have received at least 2 prior lines of platinum-based therapy and who are deemed at least partially platinum-sensitive to either Olaparib 300 mg BD or single agent chemotherapy of investigators choice [55]. Results are awaited. While the previous formulation of Olaparib required 16 capsules a day, the current tablet formulation requires only four raising hopes that some of the gastrointestinal toxicity will be mitigated.

In the aforementioned studies olaparib was given as monotherapy for treatment of 'active' disease. In contrast, Study 19 randomised patients with recurrent platinum-sensitive cancer with at least 2 prior lines to *maintenance* olaparib or placebo post-chemotherapy [56]. In a predefined subset analysis of patients with known germline or somatic BRCA mutation (most retrospectively determined), median PFS in the gBRCAm group was 11.3 vs. 4.3 months with Olaparib and placebo respectively (HR 0.18). OS was not significantly different (23% crossover). The findings led to EMA approval. SOLO2 was a phase III double-blind placebocontrolled study in patients with recurrent platinum-sensitive EOC who had received at least 2 prior chemotherapy lines. Patients either got maintenance olaparib 300 mg BD or placebo. Investigator-assessed median PFS was 19.1 vs. 5.5 months (HR 0.30). Median PFS2 was also improved from not reached vs. 18.4 months (HR 0.5) and OS data are immature. Although nausea (76% vs. 33%) and vomitting (37% vs. 19%) were higher in the olaparib arms, grade 3/4 events were infrequent (2.6% for both). Grade 3/4 anaemia occurred in 20%. Patient-reported outcomes showed no detriment for olaparib [57].

Part 1 of the ARIEL2 trial (from which the gBRCA mutation data was pooled in the above analysis) enrolled 206 patients who had been received at least 1 prior platinum containing chemotherapy regimen and who had progressed after at least 6 months after their most recent course [62]. Patients were prospectively divided into three subgroups based on their HRD status: 1) germline or somatic BRCA mutations 2) BRCA wild-type and LOH-high 3) BRCA wild-type and LOH-low. LOH was assessed using a next generation sequencing assay and a cut-off of 14% was assigned using microarray and survival data from TCGA. Based on this pre-specified score, PFS was 12.8 months, 5.7 months and 5.2 months in the BRCA mutated, BRCA wild-type/LOH-high and BRCA wild-type/LOH-low subgroups. Although median PFS was similar in the latter groups, the HR for PFS was significantly in favour of the LOHhigh subgroup (0.62 95% CI 0.42–0.90), and ORR by RECIST (29% vs. 10%) and 1 year survival (28% vs. 10%) were also better for the LOH-high subgroup. Of note, LOH exists on a continuum and exploratory post-hoc analysis revealed that a cut-off of 16% provided better discrimination between the two subgroups [63]. Also importantly, there were patients in the LOH-negative group with very good partial and even complete responses (by ca125). In this single arm phase II study, it is not possible to exclude the possibility that LOH-high tumours simply have a better prognosis and that LOH is a prognostic rather than predictive marker. In order to address this question (in a maintenance setting at least) the NGS assay is being prospectively applied in the phase III Ariel 3 study which is investigating maintenance rucaparib in platinum-sensitive ovarian cancer. The phase III Ariel 4 study is will compare rucaparib as an active treatment vs. standard of care chemotherapy in platinum-sensitive disease after at

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Another orally bioavailable PARP inhibitor, veliparib is far less potent at PARP-trapping than the previously mentioned agents although it is a more potent catalytic inhibitor than niraparib and has been shown to cross the blood–brain barrier [51]. In a phase I trial 40% of the 28 BRCAm positive evaluable patients had an ORR at the MTD (400 mg BD). Commonest toxicities were nausea, vomiting and lymphopenia and 2 patients had grade 2 seizures

In a phase II trial in patients with gBRCAm who had been treated with 3 or fewer prior regimens (median 2) and of whom 60% were platinum resistant, the ORR was 26% (35% in the platinum-sensitive cohort). Grade 3 fatigue, nausea and neutropenia occurred in 6%, 4% and 2% respectively with no other grade 3 toxicities. Veliparib is currently being explored in phase III trial concurrently with carboplatin/paclitaxel and then continued as maintenance

Talazoparib is a novel PARPi that traps PARP approximately 100-fold more efficiently than olaparib and rucaparib and exhibits cytotoxicity at nanomolar (compared to micromolar) concentrations) [60]. At an MTD of 1 mg/kg, 5/12 patients with BRCAm ovarian cancer achieved

least 2 prior lines.

**4.6. Veliparib**

[NCT01472783].

**4.7. Talazoparib**

(NCT02470585, see below).

The phase III SOLO1 has completed accrual and randomised patients with BRCAm following first-line platinum-based chemotherapy to either Olaparib 300 mg BD or placebo.
