A phase I study of an oral selective gamma secretase (GS) inhibitor RO4929097 in combination with neoadjuvant paclitaxel and carboplatin in triple negative breast cancer

Summary

Upregulation of Notch pathway is associated with poor prognosis in breast cancer. We present the results of a phase I study of an oral selective gamma secretase (GS) inhibitor (critical to Notch signaling), RO4929097 in combination with neoadjuvant chemotherapy for operable triple negative breast cancer. The primary objective was to determine the maximum tolerated dose (MTD) of RO4929097. Secondary objectives were to determine real-time pharmacokinetics of RO4929097 and paclitaxel, safety and pathologic (pCR) complete response to study treatment. Eligible patients, initiated carboplatin at AUC 6 administered intravenously (IV) on day 1, weekly paclitaxel at 80 mg/m2 IVand RO4929097 10 mg daily given orally (PO) on days 1–3, 8–10 and 15–17 for six 21-day cycles. RO4929097 was escalated in 10 mg increments using the 3 + 3 dose escalation design. Two DLTs were observed in 14 patients - Grade (G) 4 thrombocytopenia in dose level 1 (10 mg) and G3 hypertension in dose level 2 (20 mg). Protocol-defined MTD was not determined due to discontinuation of RO4929097 development. However, 4 of 5 patients enrolled to 20 mg dose of RO4929097 required dose reduction to 10 mg due to toxicities (including neutropenia, thrombocytopenia and hypertension) occurring during and beyond the DLT observation period. Thus, 10 mg would have been the likely dose level for further development. G3 or higher hematologic toxicities included neutropenia (N = 8, 57%) and thrombocytopenia (N = 5, 36%) patients. Six (43%) patients had G2–3 neuropathy requiring paclitaxel dose reduction. No signs of drug-drug interaction between paclitaxel and RO4929097 were evident. Five patients (36%) had pCR.

Introduction

Triple negative breast cancer (TNBC) is defined by the lack of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 (HER-2) expression [1, 2]. TNBC is associated with an increased likelihood of relapse and death within 5 years of diagnosis as compared to other breast cancer subtypes [3, 4]. While recent results from large phase III randomized trials have confirmed that a subset of patients with metastatic TNBC derive benefit from chemo-immunotherapy combination or a PARP inhibitor [5–7], there is a lack of available targeted treatment options for patients with early stage disease [8].

Avast majority of patients with early TNBC receive standard anthracycline – taxane (A-T) based neoadjuvant chemotherapy. Evidence from neoadjuvant studies highlight that pathologic complete response (pCR), defined as no residual invasive disease in breast or axilla, serves as a valid surrogate endpoint for long term survival, given that patients with pCR have favorable disease free survival (DFS) and overall survival (OS) [9–11]. This makes the neoadjuvant setting an excellent platform to study novel agents. For instance, the use of platinum-based chemotherapy drugs to treat TNBCs has been studied in multiple neoadjuvant clinical trials, based on findings showing that a DNA-repair defect sensitizes TNBC cells to these agents in animal models [12]. Results from CALGB 40603 and GeparSixto confirm significant improvement in pCR rates with addition of carboplatin to standard neoadjuvant A-T therapy [13, 14]. Larger adjuvant trials with platinum-based drugs in early TNBC are ongoing to assess impact on long-term survival outcomes (NCT02445391, ECOG ACRIN 1131).

Notch inhibitors have been investigated in treatment of TNBC as a potential therapy that targets the developmental Notch pathway [15, 16]. The Notch pathway has been shown to be central for the maintenance of breast cancer stem cells (BCSCs) [17, 18], with preclinical studies showing that specific Notch ligand or receptor inhibitors prevented the formation of secondary mammospheres from cell lines and primary patient samples. Notably, these inhibitors also led to depletion of BCSCs [19, 20]. High levels of Notch1 and Notch3 receptors and JAG1 ligand have been detected in breast tumors with poor prognostic features and with shorter overall survival compared to breast cancer patients with low tumor levels [15, 21, 22]. Notch pathway activation involves three proteolytic cleavages [23], the last of which is carried out by the gamma secretase (GS) complex that releases the intracellular domain of Notch (NICD) critical for the activation of Notch signaling [24]. Pharmacological agents identified as GS inhibitors demonstrate preclinical evidence of antitumor activity in animal models and show favorable safety profile in phase I studies [25–28]. Notably, RO4929097, a potent, selective small molecule inhibitor of GS was shown to be well-tolerated when given in intermittent and continuous schedules [29–31].

Based on high clinical activity of pre-operative platinum-taxane regimens in TNBC, feasibility of administering RO4929097 with weekly paclitaxel and the synergy observed with these agents [32], we propose a first-in-human phase I trial of neoadjuvant intermittent RO4929097 in combination with paclitaxel and carboplatin in women diagnosed with stage II-III TNBC.

Methods

Objectives

The primary objective of the study was to determine the maximum-tolerated dose (MTD) and dose limiting toxicities (DLT) of RO4929097 administered on a weekly (3 days on, 4 days off) schedule, in combination with weekly paclitaxel, and carboplatin given on day 1 of six every 3 week cycles to patients with stage II-III TNBC. Secondary objectives of the study were to measure real-time pharmacokinetics (PK) of paclitaxel and RO4929097 to enable dose-adjustment of RO4929097 if paclitaxel PK were altered by direct drug-drug interaction or by auto-induction. RO4929097 has previously been shown to be an auto-inducer of CYP enzyme activity which could increase metabolism of paclitaxel by activation of CYP3A4/5 enzyme [33]. The rate of pCR and clinical response was also evaluated.

Patient population

Eligible patients were those with histologically proven TNM AJCC 7th edition stage II-III invasive, HER-2 and hormone receptor negative breast cancer who were candidates for neoadjuvant chemotherapy Hormone receptor negative breast cancer was defined as the presence of estrogen receptor and progesterone receptor in less than 10% of invasive cancer cells based on immunohistochemistry (IHC) staining. Negative HER2 status was defined as 0 or 1+ IHC staining or 2+ IHC staining and negative fluorescence in-situ hybridization (FISH) for amplification of the HER2 gene (based on HER2/CEP17 ratio of <2.0). The participants were required to have clinically or radiographically measurable primary breast cancer, with tumor size ≥2.0 cm. Other eligibility criteria included: ECOG Performance Status ≤1, age ≥ 18 years, absolute neutrophil count (ANC) >1500/dL, hemoglobin >9 g/dL, platelet count >100,000/ dL, creatinine: < 1.5 × upper limit of normal (ULN), an adequate liver function: AST and ALT <2.5 × ULN, alkaline phosphatase <2.5 × ULN, and bilirubin <ULN. Women of childbearing potential and men had to use two forms of contraception at least 4 weeks prior to study entry, for the duration of study participation, and for at least 12 months post-treatment. The Human Institutional Review Board at The Ohio State University Comprehensive Cancer Center approved the study (OSU IRB 2010C0043), and all patients provided written informed consent prior to initiation of study procedures and treatment. This investigator initiated study was supported by the National Cancer Institute Experimental Therapeutics Clinical Trials Network (5UM1CA186712–03).

Treatment design

This was a single institution phase I trial. Enrolled participants were to receive carboplatin AUC = 6 IV on day 1 and weekly paclitaxel 80 mg/m2 IV in combination with escalating doses of RO4929097 on days 1–3, 8–10 and 15–17 for six 21-day cycles. During cycle 1, paclitaxel was given on day −1 to allow PK assessment. The starting dose of RO4929097 was 10 mg and this dose was escalated by 10 mg increments according to the standard 3 + 3 design. Initially, 3 patients were treated at each dose level and observed for 1 cycle. Dose escalation proceeded if no patients experienced a DLT. If a DLT was observed in one of the first three patients at a dose level, then three more patients were enrolled at the same dose level. If 2 or more of the 6 patients developed a DLT, the dose was considered to be too high and the dose immediately below was expanded to 6 patients if not done previously. Adverse events were graded according to the NCI Common Terminology Criteria for Adverse Events, version 4.0 (http://ctep.cancer.gov/protocol).

Definition of DLT and MTD

DLT was assessed during the first 21-day treatment cycle and defined as a treatment-related event meeting the following pre-specified criteria: (1) any Grade 3 or 4 non-hematologic toxicity (except for untreated nausea), (2) Grade 3 electrolyte abnormalities that do not resolve within 72 h after appropriate electrolyte replacement therapy or are associated with EKG changes; (3) Grade 4 thrombocytopenia, (4) Grade 4 neutropenia complicated by fever, or (5) Grade 4 neutropenia without fever but lasting more than 7 days. MTD was defined as the dose at which no more than 1 of 6 patients developed DLT and which did not result in a 30% or greater reduction in paclitaxel exposure.

Pharmacokinetic studies

Since both RO4929097 and paclitaxel are substrates of CYP3A4/5 and RO4929097 has the potential to induce CYP3A4, real-time PK analysis was conducted to determine the effects of RO4929097 on paclitaxel PK in order to ensure that exposure to standard cytotoxic drugs was not impaired by co-administration of RO4929097. To accomplish this, the first dose of paclitaxel was given on day −1 (in cycle 1 only). RO4929097 PK was assessed on days 1, 15 and 17, while paclitaxel PK was evaluated on days −1 and 15 of the first cycle. Modifications of RO4929097 dosing were planned if paclitaxel PK were affected by direct interaction or by auto-induction. Dosing and sampling schema for PK studies for both drugs are shown in Fig. 1. PK would also be re-assessed on day 15 of cycle 2 for patients whose paclitaxel AUC decreased by more than 30% between days −1 and 15 of cycle 1.

Fig. 1.

Dosing and sampling schema for PK studies for paclitaxel, carboplatin, and RO4929097. Pharmacokinetic (PK) assessments were performed for paclitaxel alone on day −1 and RO4929097 alone on cycle 1, day 1. The combination of paclitaxel and RO4929097 was assessed on day 15, cycle 1; RO4929097 alone, accumulation, was measured on day 17, cycle 1. The combination of paclitaxel and RO4929097 was measured on cycle 2, day 15 only if paclitaxel exposure decreased by more than 30% on days 15 and 17 of cycle 1

Sample analysis

Plasma (100 μL) was mixed with 1 ml of ethyl acetate, and the mixture was agitated for 30 min then centrifuged at 14,000 rpm for 2 min. The bottom plasma layer was frozen on dry ice, and the supernatant was transferred to glass tubes. Next, the organic solvent was dried under nitrogen, and the dry residue was reconstituted in 50% acetonitrile with 0.1% formic acid. The reconstitute was analyzed for RO4929097 and paclitaxel using a quantitative liquid chromatography/tandem mass spectrometry LC-MS/MS method. Briefly, extracted plasma samples (25 μL) were run on a Betabasic C8 column using isocratic elution with 50% acetonitrile and 0.1% formic acid. The LC-MS/MS assays were developed and validated in the Comprehensive Cancer Center Pharmacoanalytical Shared Resource (PhASR) at The Ohio State University prior to study accrual and sample analysis.

Follow-up assessments

Safety was reported and graded based on Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 on day 1 of each cycle. ECG monitoring was performed for the first 10 patients before and after RO4929097 dosing on days 1 and 17 during cycle 1 only. Clinical tumor measurements were obtained at baseline (prior to starting protocol treatment), after cycle 2, cycle 4, and prior to surgery. Baseline tumor measurements on ultrasound and/or mammogram imaging were also recorded in the study database. Patients with a radiographically measurable primary tumor were assessed with a breast ultrasound and/or mammogram at baseline, after cycle 2 and cycle 4. The determination of pCR was performed by the local pathologist following examination of breast tissue and lymph nodes removed at the time of surgery. PCR was defined as no histologic evidence of residual invasive cancer within the breast and regional lymph nodes. Patients with only residual ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS) in the breast after preoperative therapy were also considered to have pCR.

Statistical methods

Summary statistics were calculated for patient and clinical characteristics. Toxicities were also summarized by grade per the NCI CTCAE v4.0 criteria using frequency and percentage. Patients who received at least one dose of RO4929097 were evaluable for toxicity. Clinico-pathological response was also summarized using frequency table for all patients and the change of tumor measures from baseline for each patient was explored longitudinally using spider plots. These analyses were conducted in SAS version 9.04 (SAS Institute, Cary, North Carolina). Non-compartmental analyses of the PK parameters for both RO9429097 and paclitaxel were carried out using Phoenix® 7.0 (Certara, New Jersey, USA). Differences in plasma exposure were evaluated using area under the curve (AUC) and compared using student t-tests. Statistical significance was concluded at p value ≤0.05.

Results

Patients

Fourteen women with TNBC were enrolled on the study between August 2011 and October 2012. The majority of women (64%) were premenopausal, and the median age of patients was 57 years (range 36–70 years; see Table 1 for patient characteristics). The majority of patients (71%) had stage II disease. All but 2 patients had axillary lymph node evaluation by core biopsy (9 patients) or by sentinel lymph node biopsy (3 patients) before the neoadjuvant chemotherapy. Nine (64%) patients had evidence of lymph node involvement and 11 (79%) had grade 3 tumors.

Table 1.

Patient Characteristics (N = 14)

Characteristic	N (%)
Median age (range)	57 (36–70)
Race
White	13 (93)
Black	1 (7)
Stage a
I	1 (7)
II	10 (71)
III	3 (21)
Node
Negative	5 (36)
Positive	9 (64)
Grade
2	3 (21)
3	11 (79)
Menopausal status
Premenopausal	9 (64)
Postmenopausal	5 (36)

^aOne patient received RO4929097 with stage I (cT1c pN0(sn)) disease

Dose escalation

The first cohort of three patients was entered on dose level 1 testing 10 mg of RO4929097 in combination with carboplatin at AUC 6 and paclitaxel at 80 mg/m². One patient developed a DLT (Grade 4 thrombocytopenia) on day 14 of cycle 1, prohibiting administration of paclitaxel on that day and collection of blood samples for planned PK studies. It was determined that carboplatin, not RO4929097, was predominantly contributing to this toxicity. Therefore, the protocol was amended to lower the dose of carboplatin to AUC 5 moving forward. At dose level 1A, the dose of RO4929097 was kept at 10 mg, the new dose of carboplatin was AUC 5 and the dose of paclitaxel remained at 80 mg/m². At this level, three patients were enrolled, and no DLTs were observed. At dose level 2A (20 mg dose of RO4929097), only one DLT (Grade 3 hypertension) was observed. However, 4 out of 5 patients enrolled on this expanded cohort required dose reductions of RO4929097 during and beyond cycle 1 due to clinically significant toxicities (Grade 3 hypertension in cycle 1, Grade 4 neutropenia in cycle 3, Grade 2 thrombocytopenia in cycle 5 and Grade 3 neutropenia and thrombocytopenia in cycle 1 each). As a result, the dose of RO4929097 was de-escalated to 10 mg. An additional 3 women were treated with 10 mg RO4929097 and carboplatin AUC 5 (level 1 A). At this point, the study was prematurely terminated as the drug sponsor discontinued further clinical development of RO4929097 due to lack of clinical efficacy in other studies. Therefore, the MTD could not be determined based on DLT criteria. However, it was determined that 10 mg of RO4292097 should be considered as the MTD, given that 4 of 5 patients enrolled to 20 mg of RO4929097 required dose reduction to 10 mg following significant treatment related toxicities occurring during and beyond the DLT observation period (cycles 1—5). Dose levels and associated DLTs are summarized in Table 2.

Table 2.

Summary of dose levels and dose limiting toxicities (N = 14, 1 patient did not receive study drug)

Dose Levela	N	Paclitaxel	Carboplatinb	RO4929097	DLT (N)	Type of DLT
1	3	80 mg/m2	AUC = 6	10 mg	1	Grade 4 thrombocytopenia
1A	6	80 mg/m2	AUC = 5	10 mg	0	–
2A	5	80 mg/m2	AUC = 5	20 mg	1	Grade 3 hypertension
3A	0	80 mg/m2	AUC = 5	30 mg	–	–
4A	0	80 mg/m2	AUC = 5	40 mg	–	–

AUC area under curve, DLT dose limiting toxicity

^aOnly one patient completed dose level 2A without dose reduction; remaining patients treated with dose level 1A following study amendment

^bCarboplatin reduced to AUC = 5 following incidence of Grade 3 and Grade 4 thrombocytopenia in two patients

Safety

The most common toxicities observed in the study patients are listed in Table 3. Six (43%) patients had Grade 2/Grade 3 neuropathy that required dose reduction of paclitaxel. ECG data were collected for 10 patients during cycle 1. No QTc prolongation or other clinically significant ECG changes were observed. Grade 3 or higher hematologic toxicities included Grade 3 neutropenia in 6 (43%) patients, Grade 4 neutropenia in 2 (14%) patients, Grade 3 thrombocytopenia in 4 (29%) patients, Grade 4 thrombocytopenia in 1 (7%) patient, and Grade 3 anemia in 4 (29%) patients. Grade 3 or higher non-hematologic toxicities included Grade 3 sensory neuropathy in 2 (14%) patients, Grade 3 hypertension in 1 (7%), and Grade 3 fatigue in 1 (7%) patient. Eleven serious adverse events (SAEs) were reported during the duration of this study including hypertension (N = 3, 21%), depression (N = 1,7%), glucose intolerance (N = 1, 7%), hyperglycemia (N = 1, 7%), leukopenia (N = 1, 7%), neutropenia (N = 1, 7%), thrombocytopenia (N = 1, 7%), wound dehiscence (N = 1, 7%), and wound infection (N = 1, 7%). Aside from glucose intolerance, hyperglycemia and Grade 3 hypertension, all were thought to be at least possibly related to study therapy. There were no hospitalizations for neutropenic fevers or other treatment-related toxicities.

Table 3.

Treatment-related adverse events (N = 14, 1 patient did not receive study drug)^a,b

Adverse Event	Grade 1 N (%)	Grade 2 N (%)	Grade 3 N (%)	Grade 4 N (%)	All Grades N (%)
Hematological
Neutropenia	0 (0)	3 (21)	6 (43)	2 (14)	11 (79)
Leukopenia	1 (7)	7 (50)	5 (36)	0 (0)	13 (93)
Lymphocytopenia	5 (36)	5 (36)	2 (14)	0 (0)	12 (86)
Anemia	0 (0)	8 (57)	4 (29)	0 (0)	12 (86)
Thrombocytopenia	4 (29)	2 (14)	4 (29)	1 (7)	11 (79)
GI
Constipation	10 (71)	0 (0)	0 (0)	0 (0)	10 (71)
Nausea	9 (64)	3 (21)	0 (0)	0 (0)	12 (86)
Anorexia	8 (57)	1 (7)	0 (0)	0 (0)	9 (64)
Elevated aspartate aminotransferase	6 (43)	0 (0)	0 (0)	0 (0)	6 (43)
Neurological
Peripheral sensory neuropathy	6 (43)	4 (29)	2 (14)	0 (0)	12 (86)
Headache	8 (57)	1 (7)	0 (0)	0 (0)	9 (64)
Skin
Rash maculo-papular	7 (50)	3 (21)	0 (0)	0 (0)	10 (71)
Alopecia	3 (21)	6 (43)	0 (0)	0 (0)	9 (64)
Miscellaneous
Hypertension	4 (29)	3 (21)	1 (7)	0 (0)	8 (57)
Hyperglycemia	7 (50)	0 (0)	0 (0)	0 (0)	7 (50)
Dyspnea	7 (50)	3 (21)	0 (0)	0 (0)	10 (71)
Fatigue	5 (36)	5 (36)	1 (7)	0 (0)	11 (79)
Epistaxis	7 (50)	1 (7)	0 (0)	0 (0)	8 (57)

^aAdverse events related to RO4929097, paclitaxel, and/or carboplatin treatment

^bThirteen patients included because one patient did not receive study treatment and was not considered evaluable per protocol

Real-time pharmacokinetics

Plasma samples were collected from 12 patients on day 1 for initial assessment of RO4929097 PK. RO4929097 PK was also evaluated on day 15 and 17 to estimate accumulation and auto induction. Individual and mean concentration-time profiles are shown in Fig. 2, and calculated pharmacokinetic parameters for the different dose levels are presented in Table 4. There were no significant differences in RO4929097 PK comparing day 1 and day 15 at 10 and 20 mg dose levels. Elevated exposure of RO4929097 was observed on day 17 at both dose levels. Maximum concentration (C_max) and AUC values were 158% and 220% respectively compared to day 1.

Fig. 2.

Concentration time profiles of a) RO4929097 (10 mg) b) RO4929097 (20 mg) and c) Paclitaxel (80 mg/m²). PK analysis of RO4929097 was performed on samples from 12 patients on day 1, cycle 1 for initial assessment and again on day 15 and 17 to estimate accumulation and auto induction. There were no significant differences in RO4929097 PK comparing Day 1 and Day 15 at 10 (panel a) and 20 mg dose levels (panel b). Elevated exposure of RO4929097 was observed on day 17 at both dose levels. No significant differences in paclitaxel PK were observed (panel c)

Table 4.

RO4929097 pharmacokinetic parameters by day and dose level (mean ± SD)

Parameter	10 mg Dose			20 mg Dose
	Cycle 1 Day 1 N = 8	Cycle 1 Day 15 N = 7	Cycle 1 Day 17 N = 6	Cycle 1 Day 1 N = 4	Cycle 1 Day 15 N = 3	Cycle 1 Day 17 N = 2
Cmax (ng/mL)	203 ± 92.7	177 ± 93.6	259 ± 113	324± 116	234 ± 25.0	422 ± 42.6
Vz/F (L)	68.2 ± 20.2	76.8 ± 46.8	55.6 ± 25.4	115 ± 38.6	139 ± 37.5	66.3 ± 2.4
CL/F (L/h)	3.21 ± 0.84	2.70 ± 1.07	1.97 ± 1.12	3.87 ± 1.09	3.90 ± 2.20	2.47 ± 2.55
Half life (h)	14.7 ± 2.2	20.1 ± 11.2	20.6 ± 7.0	22.8 ± 12.4	36.0 ± 31.7	39.3 ± 39.9
AUC INF (h*ng/mL)	3285 ± 749.0	4118 ± 2135	6212 ± 2607	5525 ± 1683	6727 ± 4228	17,415 ± 18,001

Non-compartmental pharmacokinetic parameters determined for RO4929097 include observed maximum concentration (Cmax) and calculated volume of distribution (Vz), clearance (CL), terminal phase half life, and area under the concentration vs. time curve estimated to infinity (AUC INF). Note that Vz and CL are represented as ratios relative to oral bioavailability (F) which was not determined in these patients

Paclitaxel PK was assessed when given alone (day −1) and in combination with RO4929097 (day 15) to determine if paclitaxel exposure is affected by direct drug-drag interaction or by auto-induction. The concentration time profiles and estimated pharmacokinetic parameters for paclitaxel are shown in Fig. 2c and Table 5, respectively. No significant differences in paclitaxel PK were observed, and mean clearance and AUC values on day 15 showed non-significant deviation from baseline. Given these findings, no additional PK assessment was conducted on cycle 2, day 15.

Table 5.

Paclitaxel mean parameters = (mean ± SD)

Parameter	Week 1 (Day – 1) N = 14	Week 3 (Day 15) N = 12
Cmax (ng/mL)	2932 ± 757	3287 ± 970
CL (ml/min/m2)	295 ± 78.6	286 ± 77.0
Half life (h)	10.3 ± 2.5	11.3 ± 2.5
AUC INF (h*ng/mL)	4797 ± 1174	4911 ± 1078

Non-compartmental pharmacokinetic parameters determined for paclitaxel include observed maximum concentration (Cmax) and calculated clearance (CL), terminal phase half life, and area under the concentration vs. time curve estimated to infinity (AUC INF)

Clinico-pathological response

Five of the fourteen patients (36%) enrolled in the study showed a pCR. In addition, there were 4 patients (29%) with <ypT1b ypN0 at the time of surgery, and 5 patients (36%) had residual disease in the breast and lymph nodes (1 patient with residual disease did not receive the full study treatment due to adverse reaction to chemotherapy; see Table 6). Figure 3 provides the change of tumor measurements on imaging evaluation between baseline and for each measurement time point for each patient.

Table 6.

Pathologic response to neoadjuvant treatment (N = 14)

Response assessment	N (%)
Complete pathologic response	5 (36)
Residual disease	9 (64)a,b

^aFour patients had < ypT1b ypN0 at surgery

^bOne patient did not receive RO4929097.

Fig. 3.

Spider plot of response as measured by ultrasound (N = 13). Each line provides the change of tumor measures from baseline for each patient longitudinally (at 3 months, 5 months, and post-treatment)

Discussion

This phase I study of a potent and selective GS inhibitor RO4929097, given on intermittent dosing schedule, in combination with neoadjuvant carboplatin and paclitaxel to women with early stage TNBC prior to definitive surgery is one of the first few phase I trials with notch inhibitors to be conducted in patients with operable breast cancer. Based on poor tolerance of the 20 mg dose leading to dose reductions to 10 mg in 4 of 5 patients treated at this dose (occurring in cycles 1—5), it was determined that the MTD of RO4929097 in this clinical setting (3 days on followed by 4 day break) should be considered 10 mg in combination with carboplatin at AUC 5 on day 1 and paclitaxel at 80 mg/m2 weekly per 21 day cycles. There were 2 DLTs (Grade 4 thrombocytopenia and Grade 3 hypertension) observed in thirteen evaluable patients. Major AEs were consistent with previously reported studies with RO4929097 in advanced solid tumors [29, 34] or with the combination of carboplatin- paclitaxel in early TNBC [13, 14, 35]. Importantly, there were no hospitalizations for neutropenic fever or other treatment related toxicities. These results are consistent with the data reported from other studies in advanced solid tumor patients showing that GS inhibitors are well tolerated in combination with cytotoxic chemotherapy [26, 31, 36].

No effect on the PKs of paclitaxel were observed when administered in combination with RO4929097 at the two dose levels evaluated. The AUC exposures for paclitaxel ranged from 80% in week 3 compared to 134% in week 1. In addition, RO4929097 exposure at pre-specified time points during neoadjuvant therapy was evaluated. Previous studies have reported reduced exposure of RO4929097 with 20 mg doses or higher [33] secondary to auto-induction as RO4929097 is both a known substrate and inducer of CYP3A4 activity. This was not the case in the present study, and neither increased clearance nor decreased exposure (AUC) were observed. In fact, RO4929097 exposure on days 15 and 17 were 125% and 220%, respectively, compared to day 1. Higher concentrations observed on day 17 suggested accumulation of the drug upon daily dosing for three consecutive days.

In the 14 patients who completed chemotherapy (including 1 patient who never received RO4929097), the overall response rate was 64% (9 patients), with 36% (5 patients) experiencing pCR and 29% (4 patients) having residual tumor measuring <1 cm in the breast with absence of lymph node metastases). Previous studies have reported higher pCR rates (40–55%) with platinum-taxane based neoadjuvant therapy [37, 38]. However, the number of patients in this Phase I study is fairly limited to make any definitive conclusions on efficacy of RO4929097 in combination with carboplatin and paclitaxel. In the metastatic setting, phase I studies with RO4929097 in combination with gemcitabine or capecitabine showed limited antitumor activity with partial response rates ranging from 10 to 16%. However, a large number of patients in these trials received more than 3 prior lines of treatment, which could explain this difference [26, 29]. Another study with the novel selective GS inhibitor PF-03084014 showed a partial response of 16% and stable disease of 36% in combination with first line docetaxel in metastatic TNBC patients [25, 26].

To our knowledge, this is the first phase I study of a GS inhibitor conducted in the neoadjuvant setting in breast cancer. The neoadjuvant trial design allows for early assessment of toxicity as well as preliminary response to study intervention in treatment naive population. Most dose escalation phase I studies are conducted in the metastatic setting, yet in early stage disease, the biology and treatment response to experimental agents can be distinct from metastatic disease. Therefore, lack of efficacy in heavily pre-treated metastatic disease may not translate into low activity in early stage or treatment naïve disease. Unfortunately, most agents that are found to lack efficacy in early phase clinical trials of patients with treatment refractory metastatic malignancy are abandoned from further clinical development [39]. Treatment refractory, advanced disease can consist of clonally evolved pan-resistant tumor as the result of exposure to variety of different anti-neoplastic therapeutic interventions. Agents found to be ineffective in such patients could have much more promising activity if given in early, treatment naïve stage. This issue prompted the design of this study in patients with untreated, operable breast cancer. The main disadvantage of this approach is the risk of exposing patients with curable disease to agents with an unestablished toxicity and efficacy profile. In the case of RO4929097, prior phase 1 studies in treatment refractory metastatic malignancies (including breast cancer) have shown that this agent can be safely administered in combination with chemotherapy. More recent trials (e.g. I-SPY-2, NCT01042379) are taking advantage of the neoadjuvant platform to design biomarker driven studies and augment the process of drug discovery in early breast cancer, allowing for promising targeted therapies to move faster from early clinical testing to definitive randomized trials.

Limitations

The findings of this study, while promising, have limitations due to the small number of patients enrolled. This was a result of early termination of the study by the sponsor allowing only half of the intended number of patients to be enrolled on study. Another limitation includes the single institution experience and lack of paired biopsies to study effects of GS inhibition on Notch signaling pathway in the tumor. However, the strengths of this study are the prospective design, selection of patients with TNBC phenotype, who are suitable for neoadjuvant chemotherapy and a correlative study design that allowed reliable analysis of effect of RO4929097 on metabolism of paclitaxel and auto-induction. This phase I trial in patients with operable TNBC demonstrates that the drug combination can be safely administered without significant drug interactions between the cytotoxic agents and RO4929097.

Conclusion

RO4929097 administered on a weekly oral intermittent dosing schedule at 10 mg, 3 days on 4 days off in combination with neoadjuvant carboplatin (AUC 5) and weekly paclitaxel (80 mg/m2) in patients with operable TNBC showed an acceptable safety profile with no unexpected toxicities. All patients who received the study drag completed planned chemotherapy, and the administration of RO4929097 did not affect paclitaxel exposure during neoadjuvant therapy. The combination of RO4929097 with carboplatin and weekly paclitaxel showed antitumor activity in the neoadjuvant setting. Further assessment of GS inhibitors in this patient population is warranted.