2.1 Research subjects and experimental design

This was a prospective, multicenter, randomized, controlled clinical study. The purpose of this study was to compare the efficacy between inetetamab and sirlimus group (trial group) and pyrotinib group (control group) in HER2-positive metastatic breast cancer patients with abnormal activation of the PAM pathway and disease progression after trastuzumab treatment. The study was approved by the Institutional Review Board of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital (approval number: NCC2445).

Fifty-nine patients from a total of 21 centers, including the Cancer Hospital of the Chinese Academy of Medical Sciences, were enrolled in this study between July 2021 and September 2022. The inclusion criteria were as follows: women >18 years of age; HER2 overexpression and +++ detected by immunohistochemistry (IHC) or positive fluorescence in situ hybridization (FISH) test; histologically or cytologically confirmed invasive breast cancer, with local recurrence or radiological evidence of metastatic lesions; HER2-positive breast cancer patients with disease progression after treatment with trastuzumab (including trastuzumab, trastuzumab combined with pertuzumab, and T-DM1); patients with PAM pathway-related gene mutations, as determined by genetic testing; Eastern Cooperative Oncology Group Performance Status (ECOG PS) score ≤2, expected survival ≥6 months, and able to be followed up; clear measurable and/or evaluable lesions according to RECIST 1.1 criteria; normal cardiopulmonary function, with left ventricular ejection fraction (LVEF) ≥ 50% within 4 weeks before starting treatment; sufficient liver function and bone marrow function, generally normal coagulation function, and no history of serious heart, kidney, and other important organ or endocrine system diseases; negative pregnancy test for women of childbearing age and voluntarily use of effective and reliable contraception; and patients who voluntarily signed an informed consent form. The exclusion criteria were as follows: previous use of mTOR inhibitors; previous use of pyrotinib in the advanced first-line treatment stage (previous use of lapatinib was allowed); chronic corticosteroid use for >3 months or within 4 weeks plus immunosuppressant use or radiation therapy for bone marrow replacement of 25% or more; evidence of symptomatic central nervous system metastasis or leptomeningeal disease; LVEF < 50% of cardiac function; clinical manifestations of significant arrhythmias, myocardial ischemia, severe atrioventricular block, cardiac dysfunction, or severe valvular heart disease; gastrointestinal dysfunction or gastrointestinal diseases (including active ulcers); chronic liver disease; and other factors considered by the investigators to be inappropriate for participation in this trial, such as any other medicines, social factors, or psychological factors that may affect their safety or compliance with study procedures.

PAM pathway mutations were detected as follows: Blood was collected in a cell-free DNA (cfDNA)-stabilizing collection tube, and then plasma was separated within 5 days of blood draw. Plasma cfDNA was extracted with the QIAamp Circulating Nucleic Acid Kit (Qiagen) following the manufacturer's instructions. PIK3CA mutation detection was performed using digital PCR with a custom panel (Mission Medical Technologies [Ningbo] Co. Ltd.) covering the six most frequently observed mutations in Chinese patients (see Table S1) on a Bio Digital Qing digital PCR system (Shanghai Turtle Technologies).

2.2 Specific medication regimens

In the inetetamab and sirolimus group, the patients were given the first dose of inetetamab at 8 mg/kg by intravenous infusion over 90 min, followed by a maintenance dose (6 mg/kg) every 3 weeks by intravenous infusion over 30–90 min until disease progression or other termination criteria were met; patients were also administered sirolimus (2 mg orally) once a day. In the pyrotinib group, the patients were given pyrotinib (400 mg orally) once a day. The chemotherapeutic drugs for the patients in the two groups were not limited, with the physicians selecting such drugs based on the conditions of the patients. All patients included in this study voluntarily signed an informed consent form. The enrolled patients received the corresponding treatments until tumor progression, intolerable adverse reactions, or requested to withdraw from the study for personal reasons.

2.3 Evaluation of efficacy

Efficacy was evaluated according to the RECIST v1.1 standard. At baseline, patients underwent computed tomography (CT) or magnetic resonance imaging (MRI) scans, and patients with bone metastasis received whole-body bone emission computed tomography (ECT) scans to evaluate tumor status. Efficacy assessments were performed every two treatment cycles (21 days as one treatment cycle), and safety assessments were performed once every treatment cycle (21 days) until progressive disease (PD).

2.4 Safety assessment

All adverse events were rated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 5.0 [11].

2.5 Statistical analysis

All statistical analyses were performed using SPSS 22.0 statistical analysis software (IBM Corp.) or GraphPad Prism 8.0 (GraphPad Software). Descriptive statistics were used to summarize the clinicopathological features, such as the median and percentage of patients. Categorical variables were compared using the chi-square test or Fisher's exact test. PFS was defined as the duration from when patients first received a study drug to the day of disease progression or death. The objective response rate (ORR) was defined as the ratio of patients with complete response (CR) and partial response (PR) among the total population. The disease control rate (DCR) was defined as the ratio of patients with CR, PR, and stable disease (SD) among the total population. Survival curves were created using the Kaplan–Meier (KM) method, and the log-rank test was used to compare PFS between the inetetamab and pyrotinib groups. Cox regression models were used to compare the relationships between the clinical and pathological factors and PFS between the inetetamab and pyrotinib groups. The median follow-up time was calculated by the reverse KM method. In this study, p < 0.05 was considered statistically significant.

3.1 Clinicopathological characteristics of patients and specimens

A total of 59 HER2-positive metastatic breast cancer patients with abnormal activation of the PAM pathway confirmed by gene detection were included. Figure 1 summarizes the study flow. The most frequent mutations were p.H1047R (37.9%), followed by p.E545K (15.6%), p.E542K (8.6%), p.H1047L (5.5%), p.N345K (5.5%), and p.E726K (3.0%). Among them, 37 patients received inetetamab and 22 patients received pyrotinib. The patients were between 31 and 74 years of age, with a median age of 53 years. Fifty-one patients were younger than 65 years (86.44%), and eight patients were older than 65 years (13.56%). Thirty patients (50.8%) were HR-positive/HER2-positive, and 29 patients (49.2%) were HR-negative/HER2-positive. After breast cancer recurrence and metastasis, 46 patients (78.0%) developed visceral metastasis [26 patients (70.3%) in the trial group and 20 patients (90.9%) in the control group], 31 (52.5%) of whom developed lung metastasis and 30 (50.8%) of whom developed liver metastasis. The average number of previous treatment lines was 2.6 in the inetetamab group and 1.4 in the pyrotinib group (p = 0.016). Six patients (16.2%) in the inetetamab group and seven patients (31.8%) in the pyrotinib group had never received chemotherapy after recurrence and metastasis. Four patients (10.8%) in the inetetamab group and eight patients (36.4%) in the pyrotinib group had received first-line treatment. For chemotherapy, 12 patients (32.4%) in the inetetamab group and three patients (13.6%) in the pyrotinib group had received second-line chemotherapy, while 10 patients (27.0%) in the inetetamab group and two patients (9.1%) in the pyrotinib group had received more lines of chemotherapy. The differences between groups were statistically significant. The baseline characteristics of all the included patients are shown in Table 1.

There was no significant difference observed in age, HR status, or the presence of visceral metastases between the inetetamab and pyrotinib groups (p > 0.05).

The median follow-up duration was 5.33 months; the median follow-up duration was 5.53 months in the inetetamab group and 4.53 months in the pyrotinib group.

3.2 Efficacy

The best efficacy achieved by each patient during the study was recorded. The best efficacy could not be evaluated for ten patients, six because of withdrawal of informed consent and four because of intolerable toxicity. For the efficacy analysis, 33 patients were included in the inetetamab group, and 17 patients were included in the pyrotinib group. The median PFS of inetetamab group patients was 4.64 (2.96–7.83) months, which was similar to that of pyrotinib group patients (5.69 [3.09–14.24] months). The difference between the two groups was not significant (p = 0.507; hazard ratio [HR] = 0.41; 95% confidence interval [CI] = 0.12–1.45) (Figure 2). The Cox multivariate analysis results showed that among the subjects with visceral metastasis (liver metastasis and lung metastasis), the median PFS and 95% CI were 4.34 (2.80–5.63) months for the inetetamab group and 4.47 (3.09–14.24) months for the pyrotinib group. This difference was also not significant (p = 0.327; HR = 0.40; 95% CI = 0.08–2.00). Among the subjects with fewer than two previous treatment lines, the median PFS and 95% CI were 4.54 (1.48–8.88) months in the inetetamab group and 4.14 (2.83–NA) months in the pyrotinib group, with no significant difference (p = 0.686; HR = 0.23; 95% CI = 0.01–3.66). Among the subjects with two or more previous treatment lines, the median PFS and 95% CI were 4.34 (1.41–7.73) months in the inetetamab group and 2.96 (2.83–NA) months in the pyrotinib group, with no significant difference (p = 0.352; HR = 0.63; 95% CI = 0.05–8.43). Among the subjects who were previously treated with trastuzumab, the median PFS and 95% CI were 4.61 (2.93–7.83) months in the inetetamab group and 5.69 (3.09–14.24) months in the pyrotinib group, with no significant difference (p = 0.596; HR = 0.41; 95% CI = 0.12–1.45).

The overall evaluation of best efficacy was performed based on RECIST 1.1. In the inetetamab group, nine patients achieved PR, 16 patients achieved SD, and seven patients had PD. In the pyrotinib group, five patients achieved PR, and 12 patients achieved SD. Among the patients who received inetetamab combined with sirolimus, nine (27.3%) patients experienced disease remission, while the disease was controlled in 25 patients (75.8%). Among the patients in the pyrotinib group, five patients (29.4%) achieved disease remission, while the disease was controlled in all 17 patients (100%). The ORR and 95% CIs were 27.3% (13.75–46.75) for the inetetamab group and 29.4% (10.31–55.96) for the pyrotinib group, with no significant difference (p = 1.000). The clinical benefit rate (CBR) and 95% CIs were 33.3% (17.96–51.83) for the inetetamab group and 41.2% (18.44–67.08) for the pyrotinib group, with no significant difference (p = 0.584). The median duration of remission (DOR) and 95% CI were 7.57 (1.48–NA) months for the inetetamab group and 3.03 (1.55–NA) months for the pyrotinib group, with no significant difference (p = 0.726) (Table 2).

3.3 Safety

The overall incidence of treatment-related adverse events was 86.1 (31/36) in the inetetamab group and 78.9% (15/19) in the pyrotinib group. The incidence of grade 3 and above adverse events was 25.0% (9/36) in the inetetamab group and 21.1% (4/19) in the pyrotinib group. Among them, increased aminotransferase (41.7% vs. 21.1%), reduced neutrophil count (33.3% vs. 31.6%), anemia (33.3% vs. 26.3%), and reduced white blood cell count (27.8% vs. 26.3%) were the most commonly reported adverse events. A total of nine patients (25.0%) in the inetetamab group and four patients (21.1%) in the pyrotinib group had treatment-related grade 3 and above adverse events. The adverse events with a frequency of ≥10% were diarrhea (13.9% vs. 42.1%), nausea/vomiting (13.9% vs. 26.3%), oral mucositis (22.8% vs. 15.8%), increased bilirubin (2.8% vs. 10.5%), increased blood glucose (13.9% vs. 10.5%), hyperlipidemia (11.1% vs. 5.2%), hypertriglyceridemia (19.4% vs. 10.5%), and hand-foot syndrome (0% vs. 15.8%) (Table 3).

In the inetetamab + sirolimus group, three patients (8.1%) were treated with a reduced sirolimus dose because of grade 3 and above adverse events, two patients (5.4%) were permanently discontinued because of intolerance, and two patients (5.4%) were treated with a reduced dose of chemotherapy drugs. In the pyrotinib group, seven patients (33.3%) received a reduction of pyrotinib because of grade 3 and above adverse events, while three patients (14.3%) received a reduced dose of chemotherapy drugs.