Pyrotinib alone or in combination with docetaxel in refractory HER2‐positive gastric cancer: A dose‐escalation phase I study

Pyrotinib (an irreversible pan‐ErbB small‐molecular tyrosine kinase inhibitor) was approved in human epidermal growth factor receptor 2 (HER2)‐positive breast cancer and showed great antitumor activity in preclinical studies of gastric cancer (GC). This study was first designed to prospectively assess pyrotinib in pretreated HER2‐positive GC.

Pyrotinib was a novel oral, irreversible pan-ErbB smallmolecular TKI blocking HER1, HER2, and HER4, which was approved in HER2-positive breast cancer (BC). And it has been reported that pyrotinib could inhibit the proliferation of gastric cells overexpressing HER2 (NCI-N87 and BT474 cell line) in vitro. 8 Although a phase I trial demonstrated that pyrotinib at 400 mg had well-tolerated toxicity and promising efficacy in patients with HER2-positive metastatic BC, 9 rare safety and efficacy data of pyrotinib in HER2-positive GC was ever reported. Considering the high heterogenicity of GC, pyrotinib monotherapy might contribute to limited efficacy. To further improve the efficacy, pyrotinib in combination with chemotherapy would also be evaluated.
Docetaxel, a broad-spectrum phase M cycle-specific cytotoxic drug which promotes the polymerization of tubules into stable microtubules, inhibits their polymerization and significantly reduces the tumor proliferation. 10 It was generally used in refractory GC alone and in combination with target drugs or other cytotoxic drugs as salvage therapies. 11,12 Thus, docetaxel was chosen as a combined medication with pyrotinib in this study.
Hereby, we first performed a multicenter, phase I study to prospectively assess the safety and pharmacokinetic (PK) characteristics of pyrotinib alone or plus docetaxel in refractory HER2-positive GC patients, and explore the preliminary antitumor activity as well.

| Patient eligibility
Patients with pathologically confirmed advanced HER2positive (3+ or 2+ staining intensity by immunohistochemistry or gene amplification by fluorescence in situ hybridization amplification [HER2:CEP17 ratio ≥2]) GC who suffered previous standard treatments (without docetaxel or other anti-HER2 TKIs) failure or intolerance were considered. Other key inclusion criteria were age 18-70 years, measurable lesions per RECIST V1.1, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, adequate organ and bone marrow function, and a life expectancy of at least 3 months.
Patients were excluded if they had a history of autoimmune diseases or requirement of long-term use of steroids (≥50 days); brain metastases; symptomatic or uncontrolled third space effusion; unable to take the drugs orally; uncontrolled hypokalemia and hypomagnesemia; a history of other antitumor therapy within 4 weeks before or during study; active hemorrhage within 2 months before study; chronic disease or gastrointestinal obstruction effecting absorption; other malignancies (except for cured locally cancers) within 5 years before study entry; a history of active hepatitis B virus, hepatitis C virus, syphilis or human immunodeficiency virus infection; uncontrolled heart disease. Each patient provided written informed consent.

| Study design and treatment
This multicenter, open-label, phase I dose escalation study was conducted in China from September 2014 (ClinicalTrials. gov, NCT02378389). The study was approved by the ethics committee of each study center and performed in accordance with the International Conference on Harmonization Guidelines for Good Clinical Practice and Declaration of Helsinki.
The phase I study was composed of two parts: dose escalation of pyrotinib monotherapy and in combination with docetaxel. This study followed a standard "3+3" design ( Figure 1). In the pyrotinib monotherapy part, four dose levels (240, 320, 400, and 480 mg qd, d1-21; q3W) were designed for evaluation. Based on the findings of the pyrotinib monotherapy part, one-three dose levels would be selected for combination with docetaxel (60 mg/m 2 d1; q3W). All patients were orally administered once-daily continuous doses of pyrotinib within 30 min after breakfast in 21-day cycles. In pyrotinib combined with docetaxel part, patients also received docetaxel injection via a 60 min infusion. Patients could receive study drugs until confirmed disease progression, death, intolerable toxicity, loss to follow-up or withdrawal of informed consent. In all, 21-42 patients would be enrolled.

| Endpoints
The primary endpoints were to assess the maximum tolerated dose (MTD), safety and the recommended phase II dose (RP2D) of pyrotinib monotherapy and pyrotinib in combination with docetaxel. The secondary endpoints included PK evaluation and preliminary efficacy of pyrotinib monotherapy and pyrotinib combined with docetaxel.

| Safety assessment
Safety was evaluated throughout the study. All adverse events (AEs) were recorded, the severity was assessed according to NCI-CTCAE (V.4.0), and the relationship with pyrotinib or docetaxel was judged.
DLT criteria included Grade 4 neutropenia with a duration of ≥7 days, Grade 3 or 4 neutropenia with fever, Grade 3 thrombocytopenia with bleeding tendency or Grade 4 thrombocytopenia, Grade 3 or 4 anemia; ≥ Grade 2 heart and renal insufficiency, Grade 3 or 4 non-hematologic AE (except for Grade 3 fatigue lasting ≤3 days, recoverable diarrhea, nausea, and vomiting after supportive care). DLTs were assessed in 21 days following the first dose.

| PK evaluation
The main PK parameters were estimated by a standard noncompartmental method using Phoenix WinNonLin (Pharsight, version 6.3, Pharsight Corp.), including elimination half-life (t 1/2 ) and area under the concentrationtime curve from time zero to the last measured time point (AUC 0-t ). Validated liquid chromatography-tandem mass spectrometry method was used to measure plasma concentrations (linearity range, 1-200 ng/mL). The peak plasma concentration (C max ) and time to C max (t max ) were directly obtained from the measured concentration.

| Efficacy assessment
Tumor responses were evaluated based on RECIST 1.1 at screening and every two cycles until disease progression or the start of new antitumor treatment. The complete and partial response would be confirmed after 4 weeks. All the progression-free survival (PFS) was defined as the time from the start of the first dose to disease progression or death of any cause. The overall survival was defined as the time from the start of the first dose to death. Survival status was obtained from medical records and followed up by telephone.

| Statistical analysis
Statistical analysis was performed by SPSS Version 22.0 (SPSS Inc.). The safety was assessed in patients who received at least one dose of study drugs. Patient characteristics and efficacy were assessed in the full analysis set, including patients who received at least one dose of study drugs. Descriptive statistics were used to assess demographic characteristics, safety, and tumor response outcomes. The Kaplan-Meier method was used for timeto-event endpoints.

| Patient characteristics
Between September 2014 and February 2017, 25 patients with HER2-positive GC were finally enrolled in this study. The median age was 58 years (range 39-65). And the majority of patients (n = 21, 84%) were male. The baseline characteristics of enrolled patients are detailed in Table 1.
All the patients (n = 25) received prior chemotherapies, and 10 patients (40.0%) had ≥3rdline treatment. More than half of the patients had surgery of gastrectomy.

| Safety
A total of 25 patients were included in safety analysis (Table 2a,b). All patients experienced treatment-related adverse events (TRAEs) of any grade. In the pyrotinib monotherapy part, MTD was not reached. Diarrhea (n = 12, 80%), anemia (n = 5, 33%), leukopenia (n = 4, 27%), and neutropenia (n = 4, 27%) were most commonly observed. Diarrhea was the most common grade ≥3 TRAE (n = 2). One of them (Grade 3) discontinued pyrotinib treatment for uncontrolled diarrhea after supportive care, which was defined as DLT at a dose level of 400 mg. Grade 3 neutropenia, transaminase, and bilirubin were also observed at a dose level of 400 and 320 mg.
In the combination with docetaxel part, for no DLT observation of pyrotinib alone at dose level of 240 mg and occurrence of effective case (n = 1), we set the initial dose of pyrotinib at 240 mg for combination with docetaxel. One DLT (Grade 4 neutropenia and leukopenia) was observed in patients received pyrotinib at 400 mg plus docetaxel. The majority of patients suffered hematologic toxicities including leukopenia (n = 9, 90%), neutropenia (n = 9, 90%), and anemia (n = 8, 80%). The most common non-hematologic toxicity was diarrhea (6/10, 60%), which is similar to the results in the monotherapy part. Neutropenia and leukopenia were the most common ≥3 TRAEs.

| PK
Totally, 22 patients provided plasma samples for PK analysis (monotherapy, n = 15; combination therapy, n = 7). The serum concentrations and PK data are shown in Tables S1 and S2 and in Figure 2. After single dose of pyrotinib monotherapy, the median t 1/2 was approximately 20 h (14-34 h). A linear regression model was adopted to assess the relationship between dose and exposure at steady state. The result indicated that C max and AUC 0-24 did not increase in proportion to pyrotinib doses, which suggested a nonlinear relationship for C max and AUC 0-24 versus dose. Besides, there were large variations in the ratio of combination therapy to monotherapy for C max and AUC 0-24 of pyrotinib.

| Antitumor activity
In all, from September 2014 to February 2017, 24 patients finished efficacy assessment (monotherapy, n = 14; combination therapy, n = 10). One patient at 400 mg dose level of pyrotinib monotherapy part withdrew from the study because of DLT (Grade 3 diarrhea) before efficacy assessment. So, the patient was not evaluable. A summary of the antitumor activity per investigator is shown in Table 3 and Figure 3.
In the pyrotinib monotherapy part, the overall objective response rate (ORR) was 21% and the disease control rate (DCR) was 43%, with three patients achieving PR, three patients achieving stable disease, eight patients suffering progressive disease. The patients achieving PR were distributed in dose levels of 240, 400, and 480 mg. None of the patients achieving PR received prior anti-HER2 regimens. The PFS ranged from 2.67 to 20.47 months. Of note, at the time of data cut-off (February 28, 2017), one patient with PFS of 20.47 months was at the 400 mg dose level and was still ongoing with pyrotinib. Based on the safety data, PK analysis in this study and the results of phase I study in BC, 9 the dose of 400 mg was considered as RP2D.
In the pyrotinib combined with docetaxel part, there were two patients achieving PR, five patients experiencing SD, with the ORR and DCR of 20% and 70%, respectively. At the dose level of 400 mg, although only one patient suffered DLT, three of four patients experienced quick disease progression. Hence, the enrollment of this dose level was stopped early for poor efficacy. Based on the data of safety, PK, and efficacy, the RP2D of pyrotinib was 400 mg.

| DISCUSSION
Pyrotinib is an irreversible pan-ErbB small-molecular TKI, which has been approved in HER2-positive BC. While the safety and efficacy data were limited in HER2-positive GC, only several small-sample retrospective studies and case reports involved it. 13,14 And the effect of chemotherapy combined with pyrotinib in GC was unknown. A study was necessary to prospectively explore the safety and antitumor activity in HER2-positive GC. This multicenter, phase I, open-label study first demonstrated that pyrotinib, both alone and combined with docetaxel, was generally well tolerated and showed comparable antitumor activity in patients with HER2-positive advanced GC. In the monotherapy part, diarrhea was the most common TRAE. And two patients (13%) experienced Grade 3 diarrhea in the 400 mg dose cohort, one of which was defined as DLT. In the phase I study in HER2-positive BC, pyrotinib monotherapy was generally well tolerated at doses up to 480 mg. 9 DLT was Grade 3 diarrhea in the 480 mg dose cohort, and MTD was 400 mg. This study showed that dose-producing DLT was lower in GC patients compared with BC patients (400 mg vs. 480 mg). Generally, the performance status of the GC patients was poorer than that of BC patients, making it difficult to ascend to high dose level in GC patients. So, the RP2D of the pyrotinib was determined as 400 mg. Moreover, the overall spectrum of AEs observed in BC was similar to that in GC. Besides, the AEs of pyrotinib were similar to those reported for other anti-HER2 TKIs (such as lapatinib and neratinib), with the most common AEs of diarrhea. 7,15 After combination with docetaxel, the most common TRAEs were hematologic toxicities (n = 9, 90%). The most common nonhematologic toxicity was still diarrhea (n = 6, 60%), with one Grade 3 diarrhea (10%). The combination therapy showed a higher incidence of Grade ≥3 TRAEs compared with monotherapy (80% vs. 27%). However, we speculated that these hematologic and Grade 3 toxicities were more likely caused by docetaxel.
Pyrotinib exposure was dose-dependent with a nonlinear relationship versus dose, which was different from the PK profile data reported in the metastatic BC population. Besides, limited data suggested coadministration with docetaxel had no apparent effect on the PK of pyrotinib.
The ORR of pyrotinib alone and pyrotinib combined with docetaxel was 21% and 20%, which was relatively unsatisfied in patients with pretreated HER2-positive GC. Unlike BC, pyrotinib alone could not show encouraged efficacy in HER2-positive GC. But there were indeed some patients who achieved long-lasting disease control time for more than 12 months. So, pyrotinib might be effective in some selected patients. This similar result was also shown in studies of other anti-HER2 TKIs, including lapatinib, afatinib, and neranib. 6,16 The underlying biological differences between HER2-positive GC and BC might be one of the reasons, while the differences have influence on the responses to HER2-directed therapies. GC showed more heterogeneity of HER2 expressions and incomplete membrane staining as compared with BC. Discordance in HER2 status was reported between primary and metastatic lesions, which could affect the efficacy in patients. 17,18 Besides, some HER2-positive patients converted to HER2negative after failure of chemotherapy. 19,20 It reminded us of the reassessment of HER2 status after disease progression. Unfortunately, our study did not re-evaluate HER2 status before initiating anti-HER2 therapy. Besides, only one of five PR patients had prior anti-HER2 regimes. For the small sample size, whether pervious exposure of trastuzumab affects the efficacy of pyrotinib warrants further evaluation. Hence, future studies should highlight the importance of confirmation of HER2 status before giving drugs targeting HER2, as well as exploring the effective biomarkers of pyrotinib and better identifying HER2driven GC patients who might benefit more from anti-HER2-targeted therapies.
The main limitations of this study are typical of earlyphase clinical studies. Most clinical cohorts had a small size and required further investigations. The lack of a control arm makes it difficult to accurately confirm the effect of combination with docetaxel. But based on the early signs demonstrated in this study, the combination with docetaxel increased the toxicity, rather than the efficacy, which might suggest us to try other combination strategy. Furthermore, the resistance mechanism of pyrotinib had been explored in cell lines and patient-derived xenograft models. Dysregulation of the CCND1-CDK4/6-Rb pathway was found to be to the main cause of pyrotinib resistance in preclinical AVATAR mouse. 21 Hence, combination pyrotinib with CDK4/6 inhibitor might improve

| CONCLUSIONS
In brief, pyrotinib alone and combined with docetaxel had shown acceptable safety profile in HER2 positive gastric cancer. Pyrotinib plus docetaxel might not further improve the efficacy.