A phase II study of single-agent RO4929097, a gamma-secretase inhibitor of Notch signaling, in patients with recurrent platinum-resistant epithelial ovarian cancer: A study of the Princess Margaret, Chicago and California phase II consortia
Ivan Diaz-Padilla a,1, Michelle K. Wilson a,1, Blaise A. Clarke b, Hal W. Hirte c, Stephen A. Welch d,
Helen J. Mackay a, Jim J. Biagi e, Michael Reedijk f, Johanne I. Weberpals g, Gini F. Fleming h, Lisa Wang a, Geoffrey Liu a, Chen Zhou a, Chantale Blattler a, S. Percy Ivy i, Amit M. Oza a,⁎
aDivision of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Ontario, Canada
bDepartment of Laboratory Medicine, University of Toronto, Toronto, Canada
cDivision of Medical Oncology, Juravinski Cancer Centre, Hamilton, Ontario, Canada
dDivision of Medical Oncology, London Regional Cancer Program, London, Ontario, Canada
eDepartment of Oncology, Cancer Centre of Southeastern Ontario, Kingston, Ontario, Canada
fCampbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, Toronto, Ontario, Canada
gDivision of Gynecologic Oncology, The Ottawa Hospital, Ottawa, Ontario, Canada
hThe University of Chicago Medical Center, Chicago, IL, USA
iNational Cancer Institute, Division of Cancer Treatment and Diagnosis, Cancer Therapy Evaluation Program, Investigational Drug Branch, Rockville, MD, USA

H I G H L I G H T S

•RO4929097 (a gamma-secretase inhibitor) has insufficient activity as monotherapy in platinum-resistant ovarian cancer.
•Agents targeting cancer stem cells should be studied earlier in the clinical course or potentially as an adjunct to chemotherapy.
•Future studies need to consider cohort enrichment with potential biomarkers such as intracellular Notch (NICD) protein expression.

a r t i c l e i n f o a b s t r a c t

Article history:
Received 30 December 2014 Accepted 4 March 2015 Available online 11 March 2015

Keywords: Ovarian cancer
Gamma-secretase inhibitor RO4929097
Notch
Phase II clinical trial
Purpose. A phase II study was performed to evaluate the effi cacy and safety of single-agent RO4929097 (a gamma-secretase inhibitor) in patients with recurrent platinum-resistant ovarian cancer.
Experimental design. Women with progressive platinum-resistant ovarian cancer treated with ≤ 2 chemo- therapy regimens for recurrent disease were enrolled in this trial. Patients received oral RO4929097 at 20 mg once daily, 3 days on/4 days off each week in a three week cycle. The primary endpoint was progression-free survival (PFS) rate at the end of 4 cycles. Secondary objectives included assessment of the safety of RO4929097 and exploration of molecular correlates of outcome in archival tumor tissue and serum.
Results. Of 45 patients enrolled, 40 were evaluable for response. Thirty-seven (82%) patients had high-grade ovarian cancer. No objective responses were observed. Fifteen patients (33%) had stable disease as their best re- sponse, with a median duration of 3.1 months. The median PFS for the whole group was 1.3 months (1.2–2.5). Treatment was generally well tolerated with 10% of patients discontinuing treatment due to an adverse event. In high grade serous ovarian cancer patients, the median PFS trended higher when the expression of intracellular Notch (NICD) protein by immunohistochemistry was high versus low (3.3 versus 1.3 months, p = 0.09). No clear relationship between circulating angiogenic factors and PFS was found despite a suggestion of an improved out- come with higher baseline VEGFA levels.
Conclusions. RO4929097 has insufficient activity as a single-agent in platinum-resistant ovarian cancer to warrant further study as monotherapy. Future studies are needed to explore the potential for cohort enrichment using NICD expression.
© 2015 Published by Elsevier Inc.

⁎ Corresponding author at: Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, 610 University Avenue, 5-700, Toronto M5G 2M9, Canada. Tel.: +1 416 946 2818; fax: +1 416 946 4467.
E-mail address: [email protected] (A.M. Oza).
1 Dr Ivan Diaz-Padilla and Dr Michelle K Wilson contributed equally as first authors.

http://dx.doi.org/10.1016/j.ygyno.2015.03.005 0090-8258/© 2015 Published by Elsevier Inc.

Introduction

Epithelial ovarian cancer (EOC) is diagnosed in more than 225,000 women worldwide each year and remains a significant cause of gyneco- logical cancer mortality [1]. Despite a high initial response rate of ovar- ian cancer to platinum-based chemotherapy, the majority of women with advanced disease ultimately recur [1]. Disease recurrence within six months of completing platinum-based therapy is defi ned as platinum-resistant disease and is associated with a poor prognosis and limited response to further chemotherapy [1].
Two models for tumor heterogeneity and initiation have been pro- posed: (i) the stochastic model where most tumor cells can reinitiate and cause tumor progression and (ii) the cancer stem cell model where- by only some cells can self-renew and sustain tumor growth [2,3]. Many advocate that EOC, particularly, high-grade serous carcinoma, adheres to the cancer stem cell (CSC) model [4,5] with tumor initiation, treat- ment failure and relapse reflecting intrinsically resistant CSCs and our relative inability to effectively target them. Many CSCs stay in the G0 phase of the cell cycle and as such are not susceptible to cell cycle specific chemotherapeutic agents [6].
Deregulation of key signaling pathways (e.g. Notch, WNT, Hedgehog and PTEN) involved in the functionality of such CSCs has been theorized to be associated with tumor recurrence and the development of platinum-resistance in ovarian cancer [7–9]. Furthermore, a compre- hensive genomic analysis of a prospectively annotated sample set of high-grade serous ovarian cancers identified that Notch signaling was altered in approximately 20% of cases [10], thus making Notch a rational therapeutic target (Fig. 1). The Notch signaling pathway is important in regulating cell differentiation, proliferation, apoptosis and cell–cell communication [11,12]. Notch signaling is primarily oncogenic but has been shown to have the potential to also act as a tumor suppressor [13,14]. In mammals, two structurally distinct families of Notch ligands (delta-like (DLL) 1, 3, and 4 and Jagged 1 and 2) have been described. These interact with four Notch transmembrane receptors (Notch1–4) causing a conformational change which allows proteolytic cleavage by metalloprotease and gamma-secretase [11]. This releases the Notch intracellular domain (NICD) that translocates to the nucleus, forming a transcription-activating complex. In EOC alterations in this pathway

have been frequently described and have been associated with poor outcomes [15,16]. The Notch3 receptor appears to be of particular interest with amplifi cation and upregulation associated with worse outcome [11,15,16]. Targeting Notch3 has been associated with growth inhibition and induction of apoptosis [15].
Formation of the Notch intracellular domain (NICD) is an important step in this pathway. Gamma-secretase inhibitors (GSIs) prevent the final cleavage step consequently decreasing the level of NICD. To date over 100 GSIs have been synthesized with more than 20 clinical trials investigating their role [17]. These novel therapeutic agents are being investigated as monotherapy or in combination with chemotherapy or targeted agents in a variety of solid tumors [17]. In EOC, data supporting [9,16] and refuting [18] the effect of GSIs on cellular proliferation and apoptosis exists. However, even in the absence of inhibition of cellular proliferation, gamma-secretase inhibition is proposed to be of therapeutic importance due to its effect on angiogenesis and microvessel density [18].
RO4929097 is a selective oral small molecule inhibitor of gamma- secretase that produces a less transformed, slower-growing phenotype in a variety of cancer cell lines [17,19]. In a phase I study, RO4929097 was well tolerated, with the most common reported toxicities being nausea, vomiting, diarrhea, fatigue, hypophosphatemia and skin rash [20]. Prelimi- nary evidence of activity was observed,withprolonged (≥3 months) stable disease (SD) seen in three of nine patients with recurrent ovarian cancer [20]. In this phase I study a dose-dependent decrease in drug exposure at doses above 24 mg occurred, consistent with auto-induction of CYP3A4 [20]. Furthermore, a weak-to-moderate pharmacokinetic– pharmacodynamic association was demonstrated between several putative markers of Notch inhibition including Aβ-40 protein and vascular endothelial growth factor receptor 2 protein in plasma, and Hes1 mRNA expression in hair follicles, but these did not appear predictive of response [20,21].
We conducted a phase II study to investigate the activity of RO4929097 in patients with advanced, platinum-resistant EOC, fallopian tube carcinoma, or primary peritoneal cancer. Secondary objectives were to describe the safety profile of RO4929097 and explore the expression of Notch biomarkers in EOC specimens and correlate these biomarkers with response.

Fig. 1. Schematic representation of the Notch pathway. The Notch pathway is a short-range communication system in which contact between a cell expressing a membrane-associated ligand and a cell expressing a transmembrane receptor sends the receptor expressing cell a cell-fate regulatory signal. Mammals have four Notch proteins (Notch1–4) that function as receptors for five Notch ligands (delta-like 1, 3, and 4, and Jagged 1 and 2). Notch ligand–receptor interaction on neighboring cells leads to two activating proteolytic cleavages of the re- ceptor. The first, mediated by the protease TACE (tumor necrosis factor alpha converting enzyme) occurs on the extracellular side of Notch. The second cleavage, mediated by a presenilin- protease (gamma-secretase) complex, releases the cytoplasmic domain fragment, intracellular Notch (NIC) from the plasma membrane. NIC translocates to the nucleus where it binds a transcriptional repressor known as C-promoter-binding factor (CBF-1) or CSL (CBF-1/Supressor of Hairless/Lag1), and activates Notch target genes, Myc, p21 and Hes (hairy/enhancer of split).

Patients and methods Study overview
The study reported herein is an open-label, single-arm, multicenter phase II clinical trial evaluating the efficacy and safety of RO4929097, a GSI in platinum-resistant recurrent EOC (NCT01175343). The transla- tional component included correlation of clinical response with blood and tumor-based markers of the Notch signaling pathway. This study was conducted by the Princess Margaret Hospital, Chicago and California phase II consortia. Patients provided written consent consis- tent with local institutional requirements. The protocol was approved by the institutional review board at each of the participating institutions.

Eligibility

Women (age ≥ 18) with histologically or cytologically confi rmed recurrent or metastatic, platinum-resistant EOC, fallopian tube carcino- ma, or primary peritoneal carcinoma were eligible for this trial. Central pathology review was undertaken by a pathologist (B.A.C.) blinded to outcome results. Cases were classified according to the two-tier grading system [22]. Platinum-resistant disease was defined as a treatment-free interval of less than six months since the completion of the last platinum-based chemotherapy. A maximum of two prior chemotherapy lines for recurrent disease was allowed. Prior hormonal treatments and/
or biological agents were allowed. Patients had to have measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 [23], or evidence of progression based on an elevated CA- 125 (defined as a value of N 2 times the upper limit of normal document- ed on two separate determinations made N 2 weeks apart, the most recent being completed within 7 days prior to study treatment) [24]. Other key eligibility criteria were an Eastern Cooperative Oncology Group Performance Status ≤ 2, and adequate hematologic, hepatic, and renal function. Key exclusion criteria included uncontrolled electrolyte abnormalities, and QTc on baseline electrocardiogram (ECG) N 470 ms.

Treatment plan and on-study evaluations

RO4929097 was administered as a 20 mg tablet orally daily on an empty stomach, on a 3 day-on and 4 day-off weekly schedule in a 21- day cycle (total of 9 doses per cycle). This modifi ed dosing schedule was based on concerns of autoinduction at higher doses [20]. Treatment was continued until disease progression or adverse events (AEs) prohibited further therapy. RO4929097 was supplied by the Cancer Treatment Evaluation Program of the National Cancer Institute. Toxicity was graded using National Cancer Institute Common Toxicity Criteria (NCI CTC) version 4.0.
Dose reductions to 10 mg and 5 mg were allowed if any patient developed grade 3–4 toxicity. If a treatment-related toxicity failed to recover to grade 0–1 or tolerable grade 2 within 14 days or if patients experienced treatment-related AEs requiring dose modification despite the number of permitted dose reductions they were removed from the study.

Response assessment

Patients were assessed clinically every three weeks and radiological- ly every 6 weeks. Further assessments were allowed at any time there was clinical suspicion of progressive disease (PD). Response criteria were defi ned by RECIST 1.1. Gynecologic Cancer Intergroup (GCIG) criteria were used to assess biochemical response by CA125 but were not used for treatment decisions [24].

Immunohistochemical analysis of archival specimens

Archival paraffin-embedded tumor specimens were requested on all subjects for immunohistochemical analysis of components of the Notch pathway: Jagged-1, NICD and Notch3. Immunohistochemistry was performed using standard techniques. Briefl y, 4 micron paraffi n- embedded tumor sections were preheated to 57 °C for 15 min, cooled to room temperature, deparaffinized in xylene and rehydrated in grad- ed alcohols. Endogenous peroxidase activity was blocked in 0.3% hydro- gen peroxide in PBS for 30 min. Heat induced epitope retrieval was accomplished using 10 mM citrate buffer (pH 6) in a Biocare Digital Decloaking Chamber. Anti-NICD (Cell Signaling #2421; 1/50) and anti- Notch3 (Santa Cruz Biotechnologies #sc-5593; 1/150) staining was per- formed using the VECTASTAIN ABC Kit (Rabbit IgG) (Vector Labs #PK- 4001) with the following modifications: permeabilization with 0.3% Tri- ton X100 in PBS for 2 × 10 min before block; block for 1 h at room tem- perature in PBS with 3% BSA, 20 mM MgCl2, 5% FBS, and 0.3% Tween20, plus Vector blocking sera; incubation with the primary antibody (diluted in blocking buffer plus vector blocking sera) overnight at 4 °C and with the secondary antibody diluted 1/100 in PBS with 5% BSA plus Vector blocking sera; and washing for 2 × 5 min in PBS, followed by 2 × 5 min in PBS with 0.3% Triton X100. Anti-Jagged-1 (R&D Systems #AF1277; 1/50) staining was performed using a Cell and Tissue Staining kit (Goat IgG) (R&D Systems #CTS008) with a 4 °C overnight incubation with the primary antibody. All immunohistochemistry experiments were performed using Shandon Sequenza immuno-staining cover plates (Fisher #7219950) and peroxidase substrate kits, DAB (Vector Labs #SK-4100). Slides were counterstained with hematoxylin, dehydrated in graded alcohols to xylene, and mounted using Permount.
The slides were scored for the presence of Jagged-1, Notch3, and NICD. The positive antibody reaction was scored into four grades, according to the staining intensity: 0, 1+, 2+ and 3+ [25]. The percentage of positive cells was also scored into four categories: 0 (0%), 1 (1–33%), 2 (34–66%) and 3 (67–100%). The product of the inten- sity and percentage scores was used as the final score and classified as negative (0–4) or positive (5–9) [26].

Circulating angiogenic factors

Soluble markers of angiogenesis were measured before treatment and post-treatment (on cycle 2, day 1): SDF-1alpha, basic fi broblast growth factor (bFGF), interleukin-6 (IL-6), IL-8 and vascular endothelial growth factors (VEGFs) A, C, and D. Venous blood samples were collect- ed, centrifuged at 3000 rpm for 10 min and stored at – 70 °C until analysis. Analytes were measured by an enzyme-linked immuno- absorbent (ELISA) assay (ELISA kit from Kamiya Biomedical Company, Thousand Oaks, CA). A standard concentration curve was produced for each ELISA plate with the manufacturer’s control solution and used to calculate plasma concentrations. Serial samples were assessed on the same ELISA plate to reduce inter-experimental variability. Baseline levels and changes at cycle 2 were correlated with PFS.

Statistical methods

The primary objective was to assess the effi cacy and toxicity of RO4929097. The primary endpoint for efficacy was the four-cycle PFS rate. Secondary endpoints included overall response rate (RR) and toxicity according to the NCI CTCAE version 4.0. Response was assessed by both RECIST 1.1 and CA-125 criteria (Gynecological Cancer Inter- group — GCIG) [24].
A Simon’s two-stage design with a target sample size of 37 patients was utilized. A clinically interesting 4-cycle PFS rate (p1) was defined as 40%, with the minimal 4-cycle PFS rate set at 20% (p0). The significance level of this design was α = 0.1 with a power of 1 – β = 0.9. A first- stage interim analysis was planned after enrollment of 17 evaluable

patients. If ≤ 3 instances of 4-cycle PFS were observed, the study was terminated (this resulted in a 55% probability of ending the study during

Table 1
Clinical and histological characteristics of enrolled patients.

stage I), whereas if ≥ 4 instances of 4-cycle PFS were observed, 20 more patients were to be enrolled in the second stage. If 11 or more instances of 4-cycle PFS were observed among the 37 evaluable patients, further investigation of RO4929097 in EOC was warranted. A 4-cycle PFS was chosen in preference to the standard 6 month PFS given the median PFS is often less than this in the platinum-resistant patient population.
Standard descriptive statistics were used to summarize the patient sample. Kaplan–Meier plots were used to estimate all time-to-event functions. PFS was defi ned as time from the start of treatment until disease progression or death as a result of any cause. The association between clinical characteristics, pathologic factors and immunohisto- chemical markers was evaluated using the chi-square test or Fisher’s exact test. Univariate cox proportional hazard analysis was performed to assess the association between protein expression and PFS. All tests were two-sided, with p b 0.05 considered significant.

Median age (range) ECOG performance status

Disease site

Histology

No. of prior chemotherapy regimens
(including first-line therapy)b Median number of cycles per patient
(range)
ECOG — Eastern Cooperative Oncology Group.

0
1
2 Ovarian
Peritoneal Fallopian tube
High grade serousa Low grade serousa Endometrioid Unknown
1
2
3
N = 45 (%) 58 (26–81)
13 (29) 30 (67)
2(4) 41 (91)
3(7)
1(2) 34 (76) 7 (16)
2(4) 2 (4)
17 (38) 16 (36) 12 (27) 2 (1–18)

Logarithmic transformation to a normal distribution was performed to analyze baseline levels of SDF-1alpha, bFGF, IL-6, IL-8 and VEGFs. The paired t-test was used to assess changes in circulating angiogenic factors (CAFs) from baseline to cycle 2. Univariate analysis using the Cox-ph model was carried out to identify the association between baseline expression of the biomarkers of interest (SDF-1alpha, bFGF IL-6, IL-8 and VEGFs A, C and D), changes in expression of these markers (%) and PFS. Statistical analysis was performed using the SAS 9.2 software.

Results

Patient characteristics and tumor sub-types

Forty-fi ve patients were enrolled from six participating centers between July 2010 and March 2012. After the fi rst 17 patients, the stage I bar was met and the study entered the second stage. Forty-four patients were eligible and evaluable for toxicity, with 40 patients evaluable for response. Table 1 summarizes the pretreatment patient and disease characteristics. The majority of patients had serous histolo- gy (n = 42, 93%). High grade histology by the two-tier system was seen in 82% of patients (n = 37). Four patients had progressive disease prior to completing the first cycle of therapy. At the time of data cutoff (June 17, 2014), the median follow-up duration was 2.6 months. All patients are off study.

Clinical outcomes

Among 40 evaluable patients, 34 underwent at least one follow-up scan. Nine patients did not complete the second cycle of treatment due to PD. No objective radiologic responses were observed. Fifteen patients (38%) had SD and 24 patients (60%) experienced PD as their best response. The median duration of SD was 3.1 months (2.1– 22.7 months). One patient with low-grade serous ovarian cancer had prolonged stabilization of her disease (12.2 months). The median PFS was 1.3 months (95% CI, 1.2–2.5; Fig. 2) but the median OS has not been reached.

Safety profile

In total, 164 cycles of therapy were administered to 44 patients. The median number of cycles was two (range: 1–33). RO4929097 was generally well tolerated with the most common toxicities (at least possibly related) described being nausea (34%) and fatigue (27%)
aCentral pathology review was undertaken. Tumor grading was performed according to the two-tier grading system.
bNo more than 2 prior chemotherapy regimens for recurrent disease were allowed. Prior hormonal treatment and/or biological agents were permitted. Note: chemotherapy for initial disease did not count towards the total of 2 lines for recurrent disease. No pa- tients had more than the allowed 2 lines of chemotherapy for recurrent disease.

not necessitate a dose modifi cation. Anemia was the most common hematological toxicity (n = 5, 11%), but no grade ≥ 3 episodes were observed. One patient experienced grade 4 liver transaminitis requiring a treatment interruption during cycle 2. Liver enzymes returned to normal within 2 weeks after treatment cessation. Four patients stopped therapy due to AEs.

Notch pathway biomarkers

An exploratory analysis for potential predictive biomarkers was performed on archival, paraffin-embedded tumor tissue. Twenty-five patients were assessed for Jagged-1 expression. Twenty-three were positive (92%), with a median intensity score of 7 (range: 3–8). Twenty-five patients were assessed for NICD expression, of which 17 had high-grade serous histology. Of these 17, six were positive (35%) for NICD (Fig. 3). A trend towards longer PFS was observed for patients with positive NICD expression in contrast to patients with negative expression of NICD (3.3 vs 1.3 months, p = 0.09) (Fig. 4).

PFS Estimate 95% CI

(Table 2). Six patients experienced grade 1–2 transient QT interval pro- longation with no associated electrolyte imbalances.
Three grade 3 toxicities were described: diarrhea, headache and
0
2
4
6
months
8
10
12

hypophosphatemia. The episode of grade 3 hypophosphatemia required oral and intravenous phosphate supplementation, but did
Fig. 2. Kaplan–Meier estimate of progression-free survival (patients evaluable for re- sponse; n = 40).

Table 2
Treatment-related toxicity at least possibly related to therapy (n = 44).
RO4929097-related toxicitya Any grade (%) Grade 3/4 (%)
Nausea 15 (34) 0
Fatigue 12 (27) 0
Hypophosphatemia 7 (16) 1 (2)
Anorexia 6 (14) 0
Anemia 5 (11) 0
Headache 5 (11) 1 (2)
Vomiting 5 (11) 0
ALT increase 5 (11) 1 (2)
AST increase 4 (9) 1 (2)
Diarrhea 4 (9) 1 (2) a Common toxicity criteria for adverse events (v. 4.0).

3.5. Circulating angiogenic factors

An exploratory analysis was performed investigating potential circulating angiogenic biomarkers on peripheral blood samples prior to therapy and post cycle 2. Forty-four patients had at least one sample available for analysis, 38 of whom had paired samples collected. No as- sociations between baseline levels of IL6 (p = 0.94), IL8 (p = 0.66), SDF (p = 0.58), bFGF (p = 0.52) and PFS were detected. With respect to VEGFs A, C and D, only higher baseline levels of VEGFA appeared to cor- relate with a better PFS (HR: 0.996, p = 0.04) but the clinical

A

B

Fig. 3. Immunohistochemical expression of NICD in two samples of high-grade serous ovarian carcinoma. A. Absence of NICD protein expression. B. Strong NICD protein expres- sion (+3).

Fig. 4. Kaplan–Meier estimate of progression-free survival comparing high intracellular Notch (NICD) protein expression to low NICD (3.3 versus 1.3 months, p = 0.09). Seven- teen patients were evaluable for response. The score was based on the percentage of cells and the intensity of staining. The final scores were classified as negative (0–4) or positive (5–9).

significance of this was uncertain. No statistically significant correlation was found between change in expression with treatment and clinical outcome.

4. Discussion

Resistance to platinum therapy is a major obstacle to treatment for EOC patients and conveys a poor prognosis [9,27]. Novel therapeutic options are needed. The high rates and patterns of treatment failure have been proposed to be consistent with an accumulation of drug- resistant CSCs [8,9]. Evidence has shown that recurrent EOC is enriched with CSCs and stem cell pathway mediators [8]. Targeting pathways integral to CSC development such as Notch, appears to be a rational and innovative approach for the treatment of EOC.
CSC marker expression is not static but constantly evolving as a result of differentiation and environmental stimuli. The phenotypical properties of CSCs parallel those seen in chemoresistant cell populations with high expression of multi-drug resistant transporters, enhanced DNA repair ability and the propensity to proliferate slowly [9,28]. Repeated courses of chemotherapy have been shown to generate chemoresistant cell-lines consistent with CSCs [29]. Importantly, these cells appear to emerge even after a single dose of a chemotherapeutic agent [30].
The Notch pathway is deregulated in approximately 20% of high- grade serous ovarian carcinomas and appears to be associated with a worse clinical outcome [10]. As proteolytic cleavage by the gamma- secretase complex represents an important step in the downstream cascade of the Notch pathway (Fig. 1), targeting this step either alone or in combination may have important ramifi cations in recurrent disease. The cleavage releases the intracellular domain of Notch from the membrane, allowing it to move to the nucleus and form a short- lived transcriptional activation complex with the DNA-binding factor RBPJ (also known as CSL) and co-activators of the mastermind-like family. Despite the apparent straightforward nature of this signaling pathway, the outcome of Notch activation varies widely with disease context, from differentiation to maintenance of stemness, apoptosis to cell survival and uncontrolled growth to growth arrest [31]. This incom- pletely understood pleiotropy emphasizes the complexities inherent to attempts to use Notch inhibition as a therapy [31].

Gamma-secretase inhibition with drugs including RO4929097 has demonstrated signifi cant anti-tumor activity in preclinical and early clinical studies [19,20,32]. Despite this, in the present phase II study in an unselected population of platinum-resistant EOC patients, RO4929097 did not demonstrate meaningful activity to warrant further study. A post-hoc analysis in a limited subgroup of 17 patients revealed a non-signifi cant trend towards better PFS (3.3 vs 1.3 months) in women with positive NICD expression. Positive expression was present in 6 of the 17 patients (35%).
In this study, there were no objective responses to RO4929097 and median PFS was 1.3 months (1.2–2.5 months). This does not compare favorably with historical data in platinum-resistant ovarian cancer [27]. Several reasons may explain these negative results. Firstly, while the 3-day on, 4-day off dosing schedule of RO4929097 was used, it is unclear whether this is an optimal dose to cause Notch inhibition [21]. Integration of biopsies pre- and post-treatment would have allowed better assessment of this. The absence of pharmacodynamic and pharmacokinetic evaluation is a weakness of this study. Prior studies have shown that plasma concentrations with human doses greater than 6 mg exceed efficacious exposure levels established in xenograft models. Secretory diarrhea has been proposed as a clinical pharmacody- namic marker of Notch inhibition due to the critical role of Notch in differentiation of crypt cells of the small intestine [19]. Inhibiting gamma-secretase shifts the balance of cell types from nutrient- absorbing cells to mucus-secreting cells leading to secretory diarrhea. Diarrhea was infrequently observed in our study (4 patients, 9%), suggesting that the doses and schedule may not have been adequate to inhibit Notch. Furthermore, the development of this drug has been discontinued due to an unfavorable pharmacology profile and concerns over efficacy [33].
Secondly, similar to differentiated cancer cells, CSCs are likely to be a heterogeneous population. In multi-passaged cancer cell-lines, key CSC populations appear to be composed of small overlapping CSC groups defi ned by various arbitrary markers [7]. Given the multitude of pathways and mechanisms involved in drug resistance in CSCs blockade of a single pathway i.e. the Notch pathway may be insufficient. Success- ful tumor elimination may require a combination of therapies in order to target both the differentiated cancer cells and CSCs [9,34]. CSCs are likely to represent a minority component of a patient’s disease [5], with the rest of the tumor bulk not necessarily sensitive to Notch inhibition alone. Based on this premise, earlier introduction of agents targeting pathways intrinsic to CSC development may be more effective.
Given the limited effi cacy seen with monotherapy with GSIs, a combination approach of a Notch inhibitor with either chemotherapy or another targeted agent (or agents) may yield higher response rates. Trials are investigating Notch inhibition in combination with several other inhibitor families including: tyrosine kinases, mammalian target of rapamycin, and conventional chemotherapeutics. Pharmacodynamic studies from MK-0752 (another GSI) have shown compensatory upreg- ulation of Wnt and other tyrosine kinases at 48 h, further supporting the hypothesis that combination therapy is necessary [21,32]. Given that Notch signaling interacts with many other pathways including PI3K/
Akt, NF-KB and STAT3, combinational therapy may prove essential for efficacy [35]. In pancreatic cancer cell lines, synergistic anti-tumor effect has been shown with the combination of rapamycin and a GSI [36]. Synergistic activity between platinum therapy and a GSI has also been shown in vivo and in vitro [9]. Additionally, the feasibility of the combi- nation of MK-0752 with docetaxel has been demonstrated in breast cancer [37]. In vivo models have shown the combination of a GSI with paclitaxel was more effective than single agent paclitaxel or single agent GSI at reducing tumor growth [38]. Interestingly, no additional benefit was seen when added to carboplatin and paclitaxel in platinum- sensitive disease. Further exploration of combination therapy may be warranted in platinum-resistant EOC. Other GSI agents are undergoing ongoing investigations as monotherapy (including NCT01292655 and

NCT01986218) [39]. Alternate ways to target the Notch pathway are also being investigated, with Jag1 inhibition of particular interest [39,40].
Thirdly, an additional limitation of this study is the lack of drug specificity to the Notch receptors. Given the described pleiotropy, the four Notch receptors may have distinct and even opposite roles depend- ing on cell context and tumor type. Illustrating this, Notch2 is oncogenic in embryonal brain tumor growth whereas Notch1 inhibits it [14]. Notch3 seems to be of particular interest in EOC. Higher expression of Notch3 is seen in recurrent disease than in primary tumors, suggesting that Notch3 signaling may be important in chemoresistance and relapse [41]. Targeting the gamma-secretase component of the Notch pathway may not be specific enough in these circumstances. A further criticism of this study is the inclusion of both low grade histology and high grade histology in the same cohort, as one would expect very different Notch signaling and stem-like behaviors refl ecting the underlying tumor biology of these two distinct diseases.
Finally, while Notch signaling seems to be deregulated in approxi- mately 20% of high-grade serous ovarian cancer patients, this study did not pre-screen for the presence of Notch overexpression or gene amplification [10]. It is conceivable that Notch inhibition may only be effective in patients whose tumors are primarily driven by altered Notch signaling. Sensitivity to GSI has been shown in earlier studies to correlate with Notch3 gene expression [9]. We performed an explorato- ry analysis of selected components of the Notch signaling pathway (NICD) which to our knowledge, has not been previously reported. The differential response seen in patients with positive expression of NICD versus negative expression warrants highlighting in this study but it is unclear whether the relatively improved PFS demonstrated is due to the predictive or perhaps a prognostic role of NICD. This is some- thing that should be explored further. Recently, contrasting data has demonstrated a worse OS with high Notch1 intracellular domain expression [42]. A PFS of 3.3 months in NICD positive patients appears to be in keeping with expected outcomes of patients with platinum- resistant disease treated with standard therapy. NICD expression was evaluable in only 25 patients (56%) in our study. The investigation of Notch expression in tumor tissue, CSCs and endothelial cells at baseline and after Notch inhibition should be included in future trials.
A biomarker signature that correlates with response to Notch inhib- itors has not yet been identifi ed. In a phase I study by Tolcher et al. several PD markers were evaluated including Aβ-40 protein and VEGFR2 in plasma and HES1 mRNA expression in hair follicles with only a weak–moderate relationship with pharmacokinetics demon- strated [20]. A further phase I study by Krop et al. investigating another GSI (MK-0752) developed a nine-gene signature consisting of ADAM19, CCND1, DVL1, HES4, HES5, HEY1, HEYL, NOTCH1 and NRARP, which corre- lated with PK studies but not clearly with response [32]. To our knowl- edge, no studies to date have published data utilizing paired biopsies to evaluate the effect of Notch inhibitors. Notch signaling has been implicated in angiogenesis and has been shown to play an important role in the proliferation and migration of endothelial tip and stalk cells [43]. In vivo data has shown that inhibition of DLL4-Notch signaling in endothelial cells led to increased sprouting angiogenesis but reduced tumor growth — so called non-functional angiogenesis [44,45]. In ovar- ian cancer, objective response to aflibercept/bevacizumab therapy was observed in patients whose tumors had low expression of DLL4 in contrast to those with high expression [46]. In breast cancer, high expression of DLL4 in the tumor-associated endothelium was thought to evoke resistance and raised the potential need to simultaneously target VEGFs and DLL4 [44,47]. In our study, we found that there was a suggestion of better outcome and higher baseline expression of VEGFA but the clinical significance of this was unclear (HR: 0.996). No correlation between alterations in circulating angiogenic factors post- therapy and PFS was found.
In summary, this negative study demonstrated no evidence of objec- tive response to treatment with single-agent RO4929097, a gamma- secretase inhibitor of Notch signaling, in an unselected population of

patients with platinum-resistant EOC. The median PFS was less than 1.3 months, suggesting a lack of signifi cant clinical activity of RO4929097 at the study dose and schedule. Further studies with agents targeting the Notch pathway in EOC should investigate com- bination therapy. Subsequent trials should include biomarker assess- ment, particularly NICD and/or Notch3 expression with exploration of potential enrichment designs if its predictive role is confi rmed.

Funding

This study has been supported by the NCI Contract No. N01-CM- 2011-00032. ID-P was partially funded by the Jan Vermorken grant supported by the Grupo Español de Investigación en Cáncer de Ovario (GEICO).

Disclosures

None of the authors declare any conflict of interest.

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