The ALTER-C-001 study was a multicenter, single-arm, phase II, exploratory study (Study registration ID: ChiCTR1900028417) and was conducted at 4 centers in Sichuan and Chongqing, China. Each center had independent ethics committee that approved the research protocol. The primary goal of this study was to evaluate the efficacy and safety of anlotinib plus XELOX in patients with mCRC. The study was performed in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki, as well as all relevant regulations and laws in the applicable countries. All patients gave written informed consent before enrollment.

Eligible patients were aged between 18 and 75 with histologically or cytologically confirmed metastatic colon and rectum adenocarcinoma (TNM stage IV). Patients had at least one untreated and measurable lesion within 3 months per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (21). Patients who received study treatment at least 2 weeks after palliative treatment in non-target lesions, or who received neoadjuvant/adjuvant chemotherapy, and targeted radio-/chemo-therapy for locally advanced treatment and relapsed more than 6 months from the last administration of peri-operation chemotherapy were included. Other inclusion criteria were Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1, adequate organ function, a minimum of six-month predicted survival duration, and left ventricular ejection fraction (LVEF) of ≥50%.

Patients who were diagnosed with mucinous adenocarcinoma or ovarian implantation metastasis and received prior antiangiogenic agents were ineligible. Patients who developed previous unrelieved treatment-related toxicity (≥grade 1) per the National Cancer Institute Evaluation Criteria for Common Adverse Events (NCI CTCAE), version 4.03 (22) were excluded from the study. Other main exclusion criteria included uncontrolled severe diseases, a history of psychotropic substance abuse with the inability to quit, and arterial and venous thrombosis within 6 months prior to the study. Pregnant or lactating patients, as well as patients with childbearing potential who did not use contraception if sexually active, were also excluded. Detailed exclusion criteria were listed in Additional File 1: Table S1 .

Eligible patients received 10 mg oral anlotinib once daily on days 1 to 14, and the XELOX regimen (130 mg/m² intravenous [>2 h] oxaliplatin on day 1, followed by 1000 mg/m² oral capecitabine twice daily on days 1 to 14) as initial therapy on a 21-day cycle for a minimum of 6 cycles. Patients who had a complete response (CR)/partial response (PR), or stable disease (SD) in cycle 4 and cycle 6, or subsequent adjacent cycles during the initial therapy were then administered 12 mg oral anlotinib monotherapy as maintenance therapy once daily on days 1 to 14 every 3 weeks. In parallel, following the completion of 6 cycles of initial therapy, maintenance therapy in patients with achievements of CR/PR/SD while intolerance, or of clinical benefits with anlotinib alone per investigators was also recommended. The treatment was continued until progressive disease (PD), or intolerable toxicity. An overview of the therapeutic procedures was shown in Figure 1 .

Dose modifications of any drug in this combination regimen were allowed depending on the potential therapeutic benefits for patients or treatment-emergent adverse events (TEAEs), which were determined by investigators. Dose reduction and interruption should give priority to anlotinib, then to chemotherapy. A maximum of two dose reductions was permitted (anlotinib: to 10 mg or even 8 mg; oxaliplatin: to 85 mg/m² and then 65 mg/m²; capecitabine: to 75% or even 50% of the initial dose and then discontinuation). Patients could resume the initial dose or a reduced dose of anlotinib due to the recovery from toxicity. If the dose of capecitabine was reduced, it could not be increased in subsequent cycles. To allow a patient to recover from any toxicities, the drug could be interrupted. However, the cumulative duration of dose interruption should be limited to 2 weeks; otherwise, study treatment should be discontinued. Detailed criteria for dose modifications were listed in Additional File 1: Table S2 .

Tumor response was assessed by investigators using magnetic resonance imaging (MRI) or computed tomography (CT) according to RECIST version 1.1 (21) at baseline and every 2 cycles until objective disease progression and intolerance. During the treatment, safety, and tolerability were monitored by physical examination, ECOG PS, vital signs, 12-lead electrocardiogram, and laboratory analyses (hematology, biochemistry, urinalysis, and coagulation). The TEAEs were evaluated by investigators and graded using the NCI-CTCAE, version 4.03 (22).

The primary endpoint was PFS, which was defined as the time from the start of the treatment to disease progression (radiological or clinical progression) or death from any cause, whichever came first. The secondary endpoints were ORR (calculated as the proportion of patients achieving CR and PR), DCR (referred to the proportion of patients with CR, PR, and SD), duration of response (DoR, defined as the duration from the day when patients firstly had response [CR or PR] to the day that they had PD firstly or death from any cause), safety and tolerability. Safety and tolerability were assessed by TEAEs. A serious TEAE (SAE) was defined as any AE that was fatal, life-threatening, required prolonged hospitalization, and resulted in persistent or significant disability/incapacity.

Sample size estimation was based on the primary endpoint of PFS. Approximately 13 PFS events were expected if 23 patients were enrolled within a 12-month accrual period with a 12-month follow-up. This number of events would provide 80% power at a two-sided 5% significance level, corresponding to the improvement in PFS from 6.6 months to 14 months based on the OPTIMOX-2 study (23). A total of 29 patients were required for this study, considering an approximate dropout incidence of 20%.

The efficacy analysis was performed in the full analysis set (FAS), which included all patients who received at least one dose of the study drug. Moreover, the analysis of ORR and DCR was additionally conducted in the per-protocol set (PPS; all FAS patients who had no protocol violations that directly impinged on or affected the efficacy endpoint and had evaluable imaging data). The safety analysis was performed in the safety analysis set (SAS), which was defined as all patients who received at least one dose of the study drug and had safety records.

Patient characteristics, safety outcomes, and tumor response were summarized descriptively. Categorical variables were summarized as frequencies (percentage [%]) and continuous variables were presented as medians with interquartile range (IQR) or range. The PFS and DOR were calculated by the Kaplan-Meier method with a 95% confidence interval (CI). The 95% CI of the ORR and DCR was calculated by the Clopper-Pearson method. Statistical comparisons of ORR according to RAS/BRAF status were performed using the Chi-square test or Fisher’s exact test, while those of PFS was performed using a two-sided exact log-rank test. All statistical tests were two-sided, with significance set at p<0.05. All analyses were conducted with obtained data, using SAS software version 9.4 (SAS Institute, Cary, NC, USA).

Between December 2019 and March 2022, 50 mCRC patients from 4 centers across China were screened and 19 were excluded from enrollment due to failure to meet inclusion criteria (n=14) and failure of consent (n=5). A total of 31 patients were enrolled and received initial therapy and were included in the FAS and SAS ( Figure 2 ). Moreover, 7 patients did not have available imaging data because of loss to follow-up (n=4), TEAEs (n=2), and other therapies (n=1); thus, 24 patients were included in the PPS.

The baseline characteristics of 31 patients were presented in Table 1 . The median age of patients was 57 (range, 51-66) years. The majority (23/31, 74.2%) were male. The primary tumor sites were commonly found in the colon (13/31, 41.9%) and rectum (18/31, 58.1%). Most patients (27/31, 87.1%) had left colonic carcinoma. Nine (29.0%) mCRC patients were identified as RAS/BRAF wild-type; totally 10 (32.3%) cases did not detect mutation status due to financial burden. The majority (28/31, 90.3%) had ≥2 metastasis, most commonly involving the liver (20/31, 64.5%), lung (15/31, 48.4%), peritoneum (5/31, 16.1%), and bone (4/31, 12.9%). Most patients (23/31, 74.2%) experienced prior surgery. Over half (18/31, 58.1%) of the patients had an ECOG PS of 0.

Sixteen (51.6%) patients subsequently received maintenance therapy following initial therapy. At the data cut-off (June 30, 2023), all 31 patients have discontinued treatment. Common reasons for discontinuation were disease progression (16/31, 51.6%), loss to follow-up (6/31, 19.4%), TEAEs (5/31, 16.1%), and other therapies (4/31, 12.9%). The regimens with bevacizumab plus chemotherapy (22.6%), capecitabine (16.1%), or cetuximab in combination with chemotherapy (12.9%) were commonly used in out-group patients ( Additional File 1: Table S3 ).

The median duration of treatment for patients was 6.3 (range, 0.0-17.2) months, with a median treatment cycle of 8.0 (range, 0.0-24.0). A total of 13 (42%) patients had TEAEs that resulted in dose reduction, interruptions, or delays, in which three (10%) patients due to TEAEs releated with anlotinib, and the others (90%) due to TEAEs releated with chemotherapy.

The median follow-up time was 17.5 (95% CI, 3.0-17.5) months. Seventeen (54.8%) PFS events were observed in the FAS population. The median PFS was 8.3 (95% CI, 6.6-10.0; Figure 3A ) months, with 6-, 9-, and 12-month PFS rates of 82.3% (95% CI, 59.2%-93.0%), 44.2% (95% CI, 21.6%-64.7%), and 18.9% (95% CI, 4.8%-40.1%), respectively. The PFS of maintenance therapy was also analyzed, which was named PFS2 and was defined as the time from maintenance therapy to disease progression or death from any cause. The median PFS2 was 4.1 (95% CI, 3.0-5.1) months in a total of 16 patients who received maintenance therapy. The median PFS was 11.0 (95% CI, 4.1-NE) months for mCRC with RAS/BRAF wild-type, and 6.9 (95% CI, 1.4-9.3) months for mCRC with mutation ( Figure 3B ).

No CR was observed from the FAS population and 15 (48.4%) patients achieved PR. Thus, the ORR was 48.4% (95% CI, 30.2%-66.9%) per RECIST version 1.1 ( Table 2 ). Additional 7 (22.6%) patients had an SD, for a DCR of 71.0% (95% CI, 52.0%-85.8%). The ORR for patients with RAS/BRAF wild-type and any mutation was repectively 66.7% (95% CI, 29.9%-92.5%) and 60.0% (95% CI, 26.2%-87.8%). Of 15 responders, the median DoR was 6.0 (95% CI, 3.6-8.0; Figure 4A ) months.

Moreover, of the 24 patients with evaluable imaging in the PPS, the ORR, and DCR were 62.5% (95% CI, 40.6%-81.2%) and 91.7% (95% CI, 73.0%-99.0%), respectively. Twenty-three (74.2%) obtained tumor shrinkage and 16 (51.6%) achieved decreased target lesion size (≤30%) from baseline ( Figures 4B, C ). Notably, despite decreased target lesion size of <30%, 1 patient was considered PD due to new lesions. One patient had the longest ongoing response of 17.3 months and the treatment duration for all participants was shown in Figure 4D .

All TEAEs of any grade with an incidence of >3% in the safety population were presented in Table 3 . All 31 patients in the SAS experienced at least one TEAE, mainly including vomiting (58.1%), nausea (51.6%), decreased white blood cell count (51.6%), decreased neutrophil count (35.5%), hypertension (35.5%), diarrhea (25.8%), and peripheral neurotoxicity (22.6%). The majority were grade 1-2 TEAEs. Grade 3 or higher TEAEs occurred in 14 (45.2%) patients and primarily included decreased neutrophil count (12.9%), hypertension (12.9%), increased lipase (9.7%), hypertriglyceridemia (6.5%), and decreased lymphocytes count (6.5%). Besides, grade 3 or more oxaliplatin-related allergic reaction was reported in 1 (3.2%) of patients. Only 1 (3.2%) patient experienced grade 5 TEAEs (respiratory failure and heart failure). Four (12.9%) patients developed SAEs including abdominal pain, incomplete intestinal obstruction, anaphylaxis, bone marrow suppression (grade 4), and stroke. SAEs were considered irrelevant to the study drug. Dose reduction of anlotinib occurred in 5 (16.1%) patients due to TEAEs. Besides, 6 (19.4%) patients required chemotherapeutic dose reduction.