Phase II study of apatinib in combination with oral vinorelbine in heavily pretreated HER2-negative metastatic breast cancer and clinical implications of monitoring ctDNA ========================================================================================================================================================================== * Anjie Zhu * Peng Yuan * Nanlin Hu * Mingzhou Li * Wenmiao Wang * Xue Wang * Jian Yue * Jiayu Wang * Yang Luo * Fei Ma * Pin Zhang * Qing Li * Binghe Xu * Shanbo Cao * Giuseppe Lippi * Yoichi Naito * Mohammed A. Osman * Gustavo N. Marta * Gianluca Franceschini * Armando Orlandi ## Abstract **Objective:** Apatinib is an oral TKI targeting VEGFR-2. Single-agent apatinib treatment has been shown to produce an objective response in patients with pretreated mBC. Oral vinorelbine also holds promise as a treatment of choice in patients with mBC. This study aimed to investigate the efficacy and safety of the oral vinorelbine-apatinib combination in patients with pretreated mBC. In addition, we detected gene variants in ctDNA to explore the therapeutic implications. **Methods:** This study enrolled patients with HER2-negative mBC who were pretreated with anthracycline/taxanes. Patients were treated with apatinib at 500 mg/425 mg daily plus oral vinorelbine 60 mg/m2 on days 1, 8, and 15 of every cycle (3 weeks). The primary endpoint was PFS. The secondary endpoints were ORR, CBR, OS, and safety. Patients eligible for ctDNA detection were evaluated before and during treatment. **Results:** Forty patients were enrolled. The median PFS was 5.2 months (95% CI, 3.4–7.0 months), and the median OS was 17.4 months (95% CI, 8.0–27.0 months). The ORR was 17.1% (6/35), and the CBR was 45.7% (16/35). The most common AEs included gastrointestinal reaction, myelosuppression, and hypertension. In 20 patients, ctDNA was detected at baseline and during treatment. A significant difference was found in PFS for undetected *vs.* detected baseline ctDNA (13.9 months *vs.* 3.6 months, *P* = 0.018). **Conclusions:** All-oral therapy with apatinib plus vinorelbine displayed objective efficacy in patients with heavily pretreated HER2-negative mBC, with acceptable and manageable toxicity profiles. Patients with no gene variant detected and lower variant allele frequencies in ctDNA at baseline showed longer PFS. * Metastatic breast cancer * apatinib * oral vinorelbine * ctDNA ## Introduction Despite a recent decline in breast cancer (BC) mortality, metastatic BC (mBC) remains an incurable disease1. Angiogenesis is an important factor in tumor growth, invasion, and metastasis2. Vascular endothelial growth factor (VEGF) and its receptor, vascular endothelial growth factor receptor (VEGFR), are key factors regulating neovascularization3. Studies on antiangiogenic agents have continued to develop applications in the treatment of mBC. Although prolonged progression-free survival (PFS) has been observed with chemotherapy, the lack of improvement in overall survival (OS) along with the presence of severe adverse events (AEs) has limited the application of antiangiogenic agents4–11. Apatinib is a novel small-molecule oral tyrosine kinase inhibitor that selectively binds VEGFR-2, thus decreasing VEGF-mediated endothelial cell migration, proliferation, and tumor microvessel density12. Apatinib, an inhibitor of VEGFR-2 through selective competition for ATP binding sites, is more specific to VEGFR-213. Preclinical studies suggest that apatinib can reverse P-glycoprotein (P-gp/ABCB1)- and BC resistance protein (BCRP/ABCG2)-mediated multidrug resistance, thus amplifying the cytotoxicity of chemotherapeutic drugs such as anthracyclines, taxanes, and vinca alkaloids14,15. Two multicenter phase II studies have reported an objective response rate (ORR) of apatinib in mBC treatment of 0.7%–16.7%, a median PFS of 3.3–4.0 months, and a median OS of 10.3–10.6 months16,17. On the basis of preclinical and clinical data, researchers have become increasingly interested in evaluating the efficacy of apatinib combined with chemotherapy in patients with mBC18–20. Vinorelbine is a semisynthetic vinca alkaloid antitumor agent. Single-agent or combination chemotherapy with vinorelbine has shown efficacy in patients with mBC. The ORR of oral single-agent regimens in the first-line treatment of metastatic HER2-negative BC has been reported to be 11%–31%21–24 and to reach 50%–60% when these regimens are combined with agents such as capecitabine25–29. In addition, vinorelbine exhibits anti-angiogenic properties30. For low-dose chemotherapy combined with anti-angiogenesis, Klement et al.31 have validated the rationale in which any anti-vascular effects of the low-dose chemotherapy would be selectively enhanced when survival signals mediated by VEGF were inhibited. Preclinical and clinical studies have revealed that anti-angiogenetic agents and vinorelbine have synergistic anti-tumor functions in NSCLC and BC32–35. On the basis of this evidence, and given the relatively benign AE profile and convenience of administration, we selected oral vinorelbine as a combination agent to be used with the antiangiogenic tyrosine kinase inhibitor (TKI) apatinib. Analysis of circulating tumor DNA (ctDNA) is a widespread method of liquid biopsy, which enables disease diagnosis36,37, prognostication38,39, detection of recurrence40,41, monitoring of tumor burden, and identification of therapeutic responses42 and resistance43,44 in patients with BC. This phase II study aimed to prospectively explore the efficacy and safety of apatinib combined with oral vinorelbine in patients with metastatic HER2-negative BC. This study further detected somatic mutations in plasma ctDNA at baseline and during treatment in patients treated with apatinib plus oral vinorelbine, to explore the potential association between ctDNA and clinical outcomes. ## Materials and methods ### Patients and methods #### Ethical approval The present study was approved by the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Approval No. CH-BC-046) and registered at clinicaltrials.gov (No. [NCT02768415](http://www.cancerbiomed.org/lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02768415&atom=%2Fcbm%2F18%2F3%2F875.atom)). Written informed consent to participate in the study was obtained from all patients or their legal guardians. The study was performed in accordance with the relevant guidelines and regulations. #### Included patients The study population included patients with HER2-negative mBC for whom previous treatment failed. The specific inclusion criteria and exclusion criteria are described in **Supplementary material 1**. #### Therapeutic protocol The initial oral dose of apatinib was 500 mg/day with 21 days/cycle. Investigators could adjust the initial dose down to 425 mg/day according to age, ECOG score, and body surface area. If patients had grade 3/4 hematologic AEs, hypertension, proteinuria, hand-foot skin reaction, mucositis, or grade 3/4 nonhematologic AEs requiring intervention, delayed administration and dose adjustment were considered: if the initial dose was 500 mg/day, then it was reduced to 425 mg/day for the first dose and 250 mg/day for the second dose; if the initial dose was 425 mg/day, it was reduced to 250 mg/day. Apatinib was taken continuously until disease progression, intolerance of AEs after dose modification, withdrawal of informed consent, or administration delays of > 14 days due to toxicity. Oral vinorelbine 60 mg/m2 (body surface area) was administered on days 1, 8, and 15 of the 21-day cycle. If patients had grade 3/4 hematologic AEs or grade 3/4 nonhematologic AEs that required intervention, delayed administration and dose adjustment were considered. Vinorelbine was taken continuously until disease progression, intolerance to AEs after dose adjustment, withdrawal of informed consent, or administration delays of > 21 days due to toxicity. #### Study design This was a single-arm, open-label phase II study. The primary endpoint was PFS. The secondary endpoints were ORR, clinical benefit rate (CBR), OS, and safety. PFS was defined as the time from registration to the date of disease progression or death from any cause. OS was defined as the time from registration to the date of death from any cause or the last follow-up visit. Efficacy was evaluated every 2 cycles until disease progression, intolerable AEs, withdrawal of informed consent, or delayed dosing beyond the prescribed period. Delayed administration was defined as failure to take the drug on time for ≥ 3 days during treatment. According to the response evaluation criteria in solid tumors (RECIST) 1.1, efficacy was categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). ORR was defined as the proportion of eligible patients who achieved confirmed CR or PR. CBR was defined as the proportion of patients who achieved CR, PR, or SD for at least 24 weeks. AEs were assessed and graded in accordance with the common terminology criteria for adverse events (CTCAE) 4.0. ### ctDNA detection Patients who consented to at least one blood draw were eligible for further ctDNA analysis and constituted the study population. Library preparation, NGS sequencing, and bioinformatics analysis were performed as described in **Supplementary material 2**. ### Statistical analysis Previously reported data have indicated that the median PFS with oral vinorelbine combination with capecitabine as a second-line treatment for metastatic HER2-negative BC is 3.8 months45. The study was designed to be two-sided, with an α-error of 5% and a power of 80%. We expected that the median PFS for patients receiving apatinib combined with oral vinorelbine would be 6 months. Assuming a 15% dropout rate and 6-month follow-up period, the final accrual number was 40. Data were summarized as frequency and percentage for qualitative variables, and as medians and ranges for quantitative variables. The PFS and OS were calculated from the date of registration to the first documented date of disease progression and date of death, respectively, with the Kaplan-Meier method. Associated 95% CIs were calculated with the Brookmeyer-Crowley method. The log-rank test was used for comparison of PFS and OS between groups. The multiple Cox model was used to evaluate significant differences in PFS and OS between groups. Statistical analyses were conducted with SPSS 21.0 statistical software (SPSS, Chicago, IL, USA). ## Results ### Patient information The baseline patient characteristics are shown in **Table 1**. Forty patients with HER2-negative mBC were enrolled at our institution between May 2016 and January 2018 (median age, 55 years; range 30–70 years). Twenty-one patients (52.5%) received apatinib combined with oral vinorelbine as a second-line treatment, and 19 patients (47.5%) received it as a third-line treatment or beyond. All 40 patients were included in the survival and safety analyses. Five patients were discharged before the first efficacy evaluation. A total of 35 patients were included in the efficacy evaluation analysis. At the time of the final follow-up (November 30, 2019), 33 (82.5%) patients had disease progression, and 28 (70.0%) patients had died. Twenty-seven (67.5%) patients had delayed administration of apatinib or oral vinorelbine during treatment. Twenty-one (52.5%) patients experienced apatinib or oral vinorelbine dose modification. View this table: [Table 1](http://www.cancerbiomed.org/content/18/3/875/T1) Table 1 Patient characteristics at baseline ### Safety No treatment-related deaths occurred, but 28 patients died because of disease progression. The initial dose of apatinib was 500 mg/day for the first 17 patients. During the first cycle of treatment, 6 patients (35.3%) developed grade 3 hypertension with poor control from the combined antihypertensive therapy, 1 patient (5.9%) had grade 3 proteinuria, 1 patient (5.9%) developed a grade 3 hand-foot skin reaction, and 1 patient (5.9%) was hospitalized for grade 3 symptomatic thrombocytopenia. Because of tolerance concerns, all patients enrolled after the 17th patient were given a lower initial dose of 425 mg/day. Among patients who underwent dose modification with apatinib (*n* = 8), 5 had a final dose of 425 mg/day, and 3 had a final dose of 250 mg/day. Twenty-three patients had an initial apatinib dose of 425 mg/day. No significant correlation was found between the initial apatinib dose and the onset of delayed administration or grade 3/4 AEs. The specifics of the initial dose, delayed administration, and dose modification of apatinib are shown in **Supplementary Table S2**. Most AEs were grade 1–2 and were well tolerated (**Table 2**). The most common AEs included gastrointestinal reaction, myelosuppression, and hypertension. One patient was hospitalized for grade 3 symptomatic thrombocytopenia. Among the patients with an initial apatinib dose of 500 mg/day, the incidence of grade 3/4 AEs was 58.8%, which was higher than that in the patients with an initial dose of 425 mg/day (43.5%) (**Supplementary Table S3**). View this table: [Table 2](http://www.cancerbiomed.org/content/18/3/875/T2) Table 2 Summary of adverse events The main drug-related specific AEs, the median time of first delayed administration, and the median time of first dose modification were analyzed from the initiation of combined treatment (**Supplementary Table S4**). ### Efficacy Thirty-three patients had disease progression, and 28 patients died. The median PFS was 5.2 months (95% CI, 3.4–7.0 months, **Figure 1A**), and the median OS was 17.4 months (95% CI, 8.0–27.0 months, **Figure 1B**). Among the 35 patients with evaluable efficacy, 6 (17.1%) achieved a better response to PR, 23 (65.7%) achieved SD, and the ORR was 17.1% (6/35). Sixteen patients achieved PR or remained in SD for > 24 weeks, and the CBR was 45.7% (16/35). The efficacy by number of treatment lines and HR expression is shown in **Supplementary Table S5**. ![Figure 1](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/18/3/875/F1.medium.gif) [Figure 1](http://www.cancerbiomed.org/content/18/3/875/F1) Figure 1 Kaplan-Meier curve of progression-free survival (PFS) and overall survival (OS) in patients with pretreated advanced breast cancer who received apatinib combined therapy. (A) Kaplan-Meier curve of PFS, indicating a median PFS of 5.2 months (95% CI: 3.4–7.0); (B) Kaplan-Meier curve of OS, indicating a median OS of 17.4 months (95% CI: 8.0–27.0). The median PFS was significantly longer for patients who showed clinical benefit (i.e., CR, PR, or SD for at least 24 weeks) (*n* = 16) than for those who did not (*n* = 19) [11.2 months (95% CI, 3.7–18.7 months) *vs.* 3.3 months (95% CI, 1.7–4.9 months), respectively, *P* < 0.001, **Figure 2**]. Nevertheless, no significant OS difference was found between these patient groups (*P* = 0.271). ![Figure 2](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/18/3/875/F2.medium.gif) [Figure 2](http://www.cancerbiomed.org/content/18/3/875/F2) Figure 2 Kaplan-Meier curve of PFS comparing patients who achieved a clinical benefit after apatinib combined therapy, with a median PFS of 11.2 months, and those who did not, with a median PFS of 3.3 months. A statistically significant difference was found between these patient groups (*P* < 0.001). In the survival analysis, univariate analysis of the correlation between the characteristics and treatment-related conditions and PFS/OS was performed (**Table 3**). The results showed significant prolongation of median PFS in patients with an ECOG PS score of 0 (*P* = 0.022) and delayed administration during treatment (*P* = 0.008) (**Figure 3A and 3B**). View this table: [Table 3](http://www.cancerbiomed.org/content/18/3/875/T3) Table 3 Subgroup analysis comparing median PFS and OS among patients with different characteristics ![Figure 3](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/18/3/875/F3.medium.gif) [Figure 3](http://www.cancerbiomed.org/content/18/3/875/F3) Figure 3 Kaplan-Meier curve of progression-free survival (PFS) with statistical significance in subgroups. (A) Kaplan-Meier curve of PFS comparing patients whose ECOG PS score was 0, with a median PFS of 6.1 months (95% CI: 4.1–8.0), and those whose ECOG PS score was 1, with a median PFS of 3.6 months (95% CI: 1.6–5.7). A statistically significant difference was found between these patient groups (*P* = 0.022); (B) Kaplan-Meier curve of PFS comparing patients who experienced administration delay, with a median PFS of 7.0 months (95% CI: 3.3–10.6), and those who did not, with a median PFS of 3.6 months (95% CI: 1.4–5.8). A statistically significant difference was found between these patient groups (*P* = 0.008). Variables including age, hormone receptor, ECOG PS, apatinib initial dose, proteinuria, and delayed administration were included in multivariate Cox proportional hazard models predicting PFS. An ECOG PS score of 0 and delayed administration remained independent predictive factors of PFS (**Figure 3A, 3B, Table 4**). In addition, chest wall recurrence was an independent predictor of OS (*P* = 0.025) (**Supplementary Table S6**). View this table: [Table 4](http://www.cancerbiomed.org/content/18/3/875/T4) Table 4 Multivariate Cox proportional hazard models predicting PFS for patients receiving combined therapy ### Patient characteristics for ctDNA analysis and detection of mutation profiling in ctDNA Twenty patients had their first blood drawn at baseline (**Supplementary Table S7**). A total of 52 blood samples were collected, and 57 variant alterations were detected. The most frequently altered genes at baseline were *TP53* (35%), *PIK3CA* (25%), *PTEN* (15%), *ERBB2* (10%), and *FGFR1* (10%). The distribution of the main genomic alterations is shown in **Figure 4**. Specific genomic alterations at baseline and serial monitoring of alterations in ctDNA are shown in **Supplementary Table S8**. ![Figure 4](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/18/3/875/F4.medium.gif) [Figure 4](http://www.cancerbiomed.org/content/18/3/875/F4) Figure 4 Distribution of the main genomic alterations in the entire population at baseline. ### The associations between baseline gene variants and PFS/OS In analysis of genomic alterations in ctDNA at baseline, the median PFS was significantly longer for patients without detectable gene variants in ctDNA [13.9 months (95% CI, 0.0–31.8 months) *vs.* 3.6 months (95% CI, 1.6–5.7 months), *P* = 0.018, **Figure 5A**]. The median value of the maximum variant allele frequency (maxVAF) was used as the cutoff value (0.985%). Patients whose maxVAF was less than the median value achieved longer PFS than those with higher maxVAF [7.0 months (95% CI, 3.1–10.8 months) *vs.* 3.3 months (95% CI, 0.8–5.8 months), *P* = 0.037, **Figure 5B**]. No statistically significant difference was found in the analysis of OS. ![Figure 5](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/18/3/875/F5.medium.gif) [Figure 5](http://www.cancerbiomed.org/content/18/3/875/F5) Figure 5 Kaplan-Meier curve of progression-free survival (PFS) in subgroups with ctDNA at baseline. (A) Kaplan-Meier curve of PFS comparing patients with detected gene variants and those without gene variants in ctDNA at baseline. The median PFS was significantly longer for patients who had no gene variants detected in ctDNA compared with patients who had detectable gene variants [13.9 months (95% CI, 0.0–31.8 months) *vs.* 3.6 months (95% CI, 1.6–5.7 months), *P* = 0.018]. (B) Kaplan-Meier curve of PFS comparing patients with different values of maxVAF in ctDNA. Patients whose maxVAF was less than the median value (0.985%) achieved longer PFS than those with higher maxVAF [7.0 months (95% CI, 3.1–10.8 months) *vs.* 3.3 months (95% CI, 0.8–5.8 months), *P* = 0.037]. ### Dynamic changes in ctDNA gene alterations during follow-up A total of 12 patients had blood drawn at baseline, and at least one blood sample was collected at the subsequent time points. Four patients were free from genomic alterations at baseline and during treatment, whereas 8 patients displayed genomic alterations in ctDNA. The entire follow-up of these patients and the patterns of changes in ctDNA are shown in **Supplementary Figure S1**. ## Discussion This is the first study exploring the efficacy and safety of apatinib combined with oral vinorelbine for the treatment of mBC. In this study, nearly half the patients (19/40, 47.5%) had already received at least 2-line metastatic treatment. The patients enrolled in our study were all HER2-negative. Notwithstanding the limits imposed by comparing different studies, our subgroup analysis of patients with TNBC showed better efficacy than that of the single-agent treatment in Hu’s study17. The median PFS and OS of HR-positive patients were also longer in our study than in a non-TNBC apatinib single-agent study16, although a discrepancy in the molecular subtypes of these 2 studies makes the studies poorly comparable. Most previous studies of vinorelbine combined with chemotherapy (mainly capecitabine) have focused on first-line therapy for advanced BC. A recent pooled analysis for stage II–III clinical trials of oral or intravenous vinorelbine plus capecitabine45 has suggested that combined therapy for second-line treatment has an ORR of 41.0%, a median PFS of 3.8 months, and a median OS of 11.3 months. All our patients received apatinib plus oral vinorelbine as a second-line treatment or beyond, and our data showed that the median PFS and OS for second-line treatment were 4.8 months and 14.7 months, respectively, with an ORR of 20.0%. Compared with the efficacy of previous vinorelbine combined chemotherapy, the PFS and OS for our combination therapy as a second-line treatment were both longer, although the ORR did not improve. A recent Chinese randomized clinical trial comparing vinorelbine and eribulin mesylate has reported a median PFS and median OS for vinorelbine of 2.8 months and 12.5 months in patients with locally recurrent BC or mBC who had at least 2 prior regimens46, whereas our results in patients who had received at least 2 regimens showed longer PFS and OS (5.2 months and 27.0 months, respectively). Because PFS and OS are the main objectives in clinical practice, apatinib plus oral vinorelbine as a second-line treatment for mBC may offer better disease control. Simultaneously, our combined therapy may be considered in patients with at least 2 prior regimens. Beyond its anticancer activity against angiogenesis, apatinib was found to reverse multidrug resistance by decreasing expression of P-glycoprotein (ABCB1) and BC resistance protein (ABCG2) *in vitro*. It also effectively enhanced the drug susceptibility of drug-resistant cell lines. Further studies have demonstrated that a certain concentration of apatinib significantly increases the toxicity of chemotherapy agents such as taxane and vinca alkaloids14,15. On the basis of the knowledge regarding the antitumor mechanism and the potential efficacy of apatinib combined with chemotherapy gained from this study, we conclude that synergistic or additive effects may exist between apatinib and chemotherapy. The combination of apatinib and oral vinorelbine may be a promising treatment after failure of other therapies for advanced BC. Therefore, the development of therapies in subsequent research is highly important. The major AEs in our study were gastrointestinal reaction, myelosuppression, hypertension, pain, malaise, anorexia, elevated transaminase, and hand-foot skin reaction (incidence > 40%). The more frequently observed severe AEs (hypertension, diarrhea, and neutropenia) were reversed after symptomatic treatment, delayed administration, or dose modification, thus suggesting that collecting patient information before treatment may be advisable. The incidence of grade 3–4 AEs was decreased by downregulation of the initial dosage of apatinib in late-enrolled patients, but no significant difference in efficacy between these dose groups was noted. Therefore, we recommend an initial dose of apatinib of 425 mg/day in combination therapy. Our study also showed that patients who gained clinical benefit after combination therapy achieved longer PFS, thus implying that an ideal short-term outcome predicts good disease control. Huang et al.47 have found that the response to apatinib is significantly associated with clinical outcomes in advanced gastric cancer, a finding consistent with our data. Our study found that patients with an ECOG PS score of 0 had longer PFS, thereby suggesting that patients in good condition are more likely to benefit. Previous studies on the treatment of solid tumors have shown that patients with ECOG PS scores of ≥ 2 often have poorer outcomes48,49. Therefore, our results confirm that patients with better health assessment scores would benefit more, thus indicating that patient health condition is essential in evaluating treatment choices50. Our multivariate analysis additionally showed that the treatment schedule may affect PFS. For example, patients with at least one delayed administration had significantly longer PFS than those who strictly followed the schedule. The leading cause of delayed administration was the onset of AEs, and hence the duration of treatment was longer for these patients. The efficacy and AEs may simultaneously increase in patients with higher blood concentrations, whereas drug susceptibility differs because of different targets among individuals. Given the potential therapeutic value of apatinib combined with oral vinorelbine, the optimal administration pattern for improving compliance and efficacy should be further investigated. Although our study did not find a significant difference in PFS or OS between HR-positive and HR-negative patients, the efficacy results suggested a significantly improved ORR in patients with TNBC compared with HR-positive individuals. Microvessel density is an important biomarker of tumor angiogenesis51,52, and high expression of VEGF/VEGFR is associated with greater vascular density53. Previous studies have found increased tumor VEGF levels in TNBC54. However, VEGFR, particularly VEGFR2, is simultaneously expressed with VEGF on the tumor endothelium53. Therefore, tumor microvessel density might increase in TNBC with high expression of VEGF/VEGFR, thus leading to a greater benefit from antivascular therapy. However, larger samples of clinical studies are needed to validate this analysis. In the current study, we also explored the roles of ctDNA variants during antiangiogenic combination therapy. PFS was relatively shorter in patients with more mutations/variants or with higher frequencies of ctDNA alterations. Recently, Rossi et al.55 have longitudinally detected ctDNA in patients with mBC, in whom the number of mutations in ctDNA along with the maximum mutant allele fraction at baseline were both predictive of progression and death. Dawson et al.42 have confirmed that increasing levels of ctDNA are significantly associated with poorer outcomes. Regarding the factors predicting the efficacy of antiangiogenic-based therapy, no efficient biomarkers have been identified to date. Most studies have focused on the plasma or tissue levels of specific protein expression and single nucleotide polymorphisms in the VEGF signaling pathway. Nevertheless, all these biomarkers lack validation in further clinical studies. For the exploration of gene mutations, a single-arm, phase II study of apatinib in refractory metastatic colorectal cancer has analyzed a panel of 1,021 cancer-related genes by ctDNA, but has not found any positive results associated with PFS or OS56. One possible explanation is that by blocking VEGF signal transduction, antiangiogenic therapies act not only on tumor cells but also on the microenvironment57,58, thus making identification of a biomarker in ctDNA difficult. Our investigation revealed an association between gene alterations in ctDNA at baseline and outcomes during antiangiogenic-based therapy, thus demonstrating that clinical outcomes depend on somatic variants, in terms of both mutation burden and frequency. However, the association between tumor mutation burden and therapeutic effects is more frequently discussed in immune therapies. Our exploration of ctDNA was based on a single arm study with a small sample size. The nature of the study (e.g., the absence of a control arm) did not enable clarification of the predictive or prognostic role of ctDNA. Larger randomized controlled double-blind clinical trials should be conducted to identify possible biomarkers for effective prediction. Despite the limited data for the patients with ctDNA examined and the impossibility of distinguishing the predictive or prognostic role, some results of the exploratory analysis deserve further discussion. One patient originally treated with letrozole had an *ESR1* mutation at baseline before combined treatment, thus demonstrating that endocrine therapy resistance might occur59–61. Two additional patients with HER2-negative primary tumors had acquired *ERBB2* amplification in ctDNA at baseline sampling before combined treatment, thus suggesting clonal evolution toward a more aggressive subtype62. This finding may indicate that patients could benefit from anti-HER2 therapy. Another patient with triple-negative BC had *EGFR* amplification in ctDNA at baseline, and the copy number was highest when the disease progressed. According to ctDNA detection, patients with *EGFR* amplification may be considered for targeted therapy with lapatinib and may benefit from this approach63. Overall, these findings suggest that ctDNA detection might provide effective information for treatment guidance64. Serial monitoring could be performed in only 8 patients. Dynamic changes in mutations and copy number variants in ctDNA can provide useful data in terms of response to treatment, particularly the association between *PIK3CA/TP53* mutation and tumor burden. This finding supports the results of previous studies42,63. Some variants not originally detected appeared during treatment, thus reflecting treatment resistance and/or clonal evolution65. A patient was identified to bear *PDGFRA* and *KIT* amplification when the disease progressed. Alterations in these genes are involved in *PDGF* signaling pathway activation, which is associated with tumor angiogenesis66–68. A previous study has shown that PDGFR expression may reflect *VEGF* signaling pathway resistance, and consequently that inhibition of *VEGFR-2* plus *PDGFR* induces tumor vessel regression69,70. In our study, no genetic variations were detected in 4 patients from baseline to progression until the last follow-up. A possible explanation is that alterations were not present within the detectable variants in our panel. Hence, we believe that whole-exome analysis of plasma ctDNA should be performed to explore heterogeneity and evolutionary cloning in BC to clarify the mechanism of drug resistance. ## Conclusions In conclusion, this prospective study shows that apatinib combined with oral vinorelbine may enable good disease control in patients with HER2-negative mBC who previously received treatments that failed. Patients in better condition and those with delayed administration achieved longer PFS. The main AEs were similar to those of apatinib or vinorelbine alone, and were limited through use of appropriate care. Full oral administration was more acceptable, particularly in patients who had received intravenous therapy for a long time. To our knowledge, this is the first study demonstrating a potential clinical role of liquid biopsy applied to antiangiogenic combination therapy. The results of ctDNA target sequencing indicated that the mutation burden and VAF of ctDNA may be informative in antiangiogenic-based therapies. Patients with gene variants or higher VAF displayed poorer outcomes. A dynamic change in ctDNA might thus mirror gene clonal shifts, disease progression or resistance. However, the results of our ctDNA research remain to be further explored, owing to the small sample size in this study. Additional prospective studies with large randomized controlled trials may be needed to assess the long-term clinical benefits. Our results also implied that the resistance mechanism of angiogenic therapy, particularly in combination therapy, may be highly complicated. Further validation of our findings in a larger number of patients, with adequate control arms and comprehensive analysis of multiomics data, is needed. ## Supporting Information [[cbm-18-875-s001.pdf]](pending:yes) ## Grant support The current study was funded by the National Natural Science Foundation of China (Grant No. 81472753 and 81672634). ## Acknowledgements We thank Giuseppe Lippi, Yoichi Naito, Mohammed A. Osman, Gustavo N. Marta, Gianluca Franceschini, Danilo Di Giorgio (Gynecology and Breast Care Center, Mater Olbia Hospital, Olbia, Italy), and Armando Orlandi for suggestions in the writing of the manuscript. ## Footnotes * **Conflict of interest statement** No potential conflicts of interest are disclosed. * Received July 21, 2020. * Accepted November 11, 2020. * Copyright: © 2021, Cancer Biology & Medicine [https://creativecommons.org/licenses/by/4.0/](https://creativecommons.org/licenses/by/4.0/) This is an open access article distributed under the terms of the [Creative Commons Attribution License (CC BY) 4.0](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. ## References 1. 1. Harbeck N, Gnant M. Breast cancer. Lancet. 2017; 389: 1134–50. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/S0140-6736(16)31891-8&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=27865536&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 2. 2. Schneider BP, Miller KD. Angiogenesis of breast cancer. J Clin Oncol. 2005; 23: 1782–90. [FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiRlVMTCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjk6IjIzLzgvMTc4MiI7czo0OiJhdG9tIjtzOjE4OiIvY2JtLzE4LzMvODc1LmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 3. 3. Banerjee S, Dowsett M, Ashworth A, Martin L-A. Mechanisms of disease: angiogenesis and the management of breast cancer. Nat Clin Pract Oncol. 2007; 4: 536–50. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/ncponc0905&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=17728712&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 4. 4. Brufsky AM, Hurvitz S, Perez E, Swamy R, Valero V, O’Neill V, et al. RIBBON-2: a randomized, double-blind, placebo-controlled, phase III trial evaluating the efficacy and safety of bevacizumab in combination with chemotherapy for second-line treatment of human epidermal growth factor receptor 2–negative metastatic breast cancer. J Clin Oncol. 2011; 29: 4286–93. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjEwOiIyOS8zMi80Mjg2IjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 5. 5. Miles DW, Chan A, Dirix LY, Cortés J, Pivot X, Tomczak P, et al. Phase III study of bevacizumab plus docetaxel compared with placebo plus docetaxel for the first-line treatment of human epidermal growth factor receptor 2–negative metastatic breast cancer. J Clin Oncol. 2010; 28: 3239–47. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjEwOiIyOC8yMC8zMjM5IjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 6. 6. Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA, et al. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007; 357: 2666–76. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1056/NEJMoa072113&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18160686&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000251964500005&link_type=ISI) 7. 7. Bergh J, Bondarenko IM, Lichinitser MR, Liljegren A, Greil R, Voytko NL, et al. First-line treatment of advanced breast cancer with sunitinib in combination with docetaxel versus docetaxel alone: results of a prospective, randomized phase III study. J Clin Oncol. 2012; 30: 921–9. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjg6IjMwLzkvOTIxIjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 8. 8. Crown JP, Diéras V, Staroslawska E, Yardley DA, Bachelot T, Davidson N, et al. Phase III trial of sunitinib in combination with capecitabine versus capecitabine monotherapy for the treatment of patients with pretreated metastatic breast cancer. J Clin Oncol. 2013; 31: 2870–8. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjEwOiIzMS8yMy8yODcwIjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 9. 9. Gradishar WJ, Kaklamani V, Sahoo TP, Lokanatha D, Raina V, Bondarde S, et al. A double-blind, randomised, placebo-controlled, phase 2b study evaluating sorafenib in combination with paclitaxel as a first-line therapy in patients with HER2-negative advanced breast cancer. Eur J Cancer. 2013; 49: 312–22. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.ejca.2012.08.005&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=22954665&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000313193000005&link_type=ISI) 10. 10. Baselga J, Segalla JGM, Roché H, del Giglio A, Pinczowski H, Ciruelos EM, et al. Sorafenib in combination with capecitabine: an oral regimen for patients with HER2-negative locally advanced or metastatic breast cancer. J Clin Oncol. 2012; 30: 1484–91. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjEwOiIzMC8xMy8xNDg0IjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 11. 11. Baselga J, Zamagni C, Gómez P, Bermejo B, Nagai SE, Melichar B, et al. Resilience: phase III randomized, double-blind trial comparing sorafenib with capecitabine versus placebo with capecitabine in locally advanced or metastatic HER2-negative breast cancer. Clin Breast Cancer. 2017; 17: 585–94.e4. 12. 12. Li J, Zhao X, Chen L, Guo H, Lv F, Jia K, et al. Safety and pharmacokinetics of novel selective vascular endothelial growth factor receptor-2 inhibitor YN968D1 in patients with advanced malignancies. BMC Cancer. 2010; 10: 529. 13. 13. Tian S, Quan H, Xie C, Guo H, Lü F, Xu Y, et al. YN968d1 is a novel and selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase with potent activity in vitro and in vivo. Cancer Sci. 2011; 102: 1374–80. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1111/j.1349-7006.2011.01939.x&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21443688&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 14. 14. Mi Y-J, Liang Y-J, Huang H-B, Zhao H-Y, Wu C-P, Wang F, et al. Apatinib (YN968d1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res. 2010; 70: 7981–91. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NjoiY2FucmVzIjtzOjU6InJlc2lkIjtzOjEwOiI3MC8yMC83OTgxIjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 15. 15. Tong X-Z, Wang F, Liang S, Zhang X, He J-H, Chen X-G, et al. Apatinib (YN968d1) enhances the efficacy of conventional chemotherapeutical drugs in side population cells and ABCB1-overexpressing leukemia cells. Biochem Pharmacol. 2012; 83: 586–97. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.bcp.2011.12.007&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=22212563&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 16. 16. Hu X, Cao J, Hu W, Wu C, Pan Y, Cai L, et al. Multicenter phase II study of apatinib in non-triple-negative metastatic breast cancer. BMC Cancer. 2014; 14: 820. 17. 17. Hu X, Zhang J, Xu B, Jiang Z, Ragaz J, Tong Z, et al. Multicenter phase II study of apatinib, a novel VEGFR inhibitor in heavily pretreated patients with metastatic triple-negative breast cancer. Int J Cancer. 2014; 135: 1961–9. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1002/ijc.28829&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=24604288&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 18. 18. Li Y-H, Zhou Y, Wang Y-W, Tong L, Jiang R-X, Xiao L, et al. Comparison of apatinib and capecitabine (Xeloda) with capecitabine (Xeloda) in advanced triple-negative breast cancer as third-line therapy: a retrospective study. Medicine. 2018; 97: e12222. 19. 19. Hu T, Liu C, Li Q, Xiong J, Ma Y, Wu G, et al. Apatinib+ CPT-11+ S-1 for treatment of refractory brain metastases in patient with triple-negative breast cancer: case report and literature review. Medicine. 2018; 97: e0349. 20. 20. Lin Y, Wu Z, Zhang J, Hu X, Wang Z, Wang B, et al. Apatinib for metastatic breast cancer in non-clinical trial setting: satisfying efficacy regardless of previous anti-angiogenic treatment. Tumour Biol. 2017; 39: 1–9. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1177/1010428317698338&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=28443474&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 21. 21. Blancas I, Morales S, Diaz N, Barnadas A, Gonzalvez M, del Barco S, et al. Efficacy and safety of oral vinorelbine (NVBO) in first-or second-line metastatic breast cancer (MBC). J Clin Oncol. 2010; 28: 1090. 22. 22. Freyer G, Delozier T, Lichinister M, Gedouin D, Bougnoux P, His P, et al. Phase II study of oral vinorelbine in first-line advanced breast cancer chemotherapy. J Clin Oncol. 2003; 21: 35–40. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjc6IjIxLzEvMzUiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 23. 23. Winer EP, Chu L, Spicer DV. Oral vinorelbine (navelbine) in the treatment of advanced breast cancer. Semin Oncol. 1995; 22: 72–8; discussion 78–9. [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=7740337&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=A1995RA78400011&link_type=ISI) 24. 24. Mansour M, Mourad C. Phase II study of single agent oral vinorelbine as first-line treatment in patients with HER-2 negative metastatic breast cancer. Cancer Chemother Pharmacol. 2013; 72: 429–35. 25. 25. Delcambre C, Veyret C, Levy C, Switsers O, Allouache D, Raban N, et al. A phase I/II study of capecitabine (X) combined with oral vinorelbine (X) as first-or second-line chemotherapy in patients (pts) with locally advanced breast cancer (MBC). Breast Cancer Res Treat. 2005; 94: S67. 26. 26. Finek J, Holubec L, Svoboda T, Sefrhansova L, Pavlikova I, Votavova M, et al. A phase II trial of oral vinorelbine and capecitabine in anthracycline pretreated patients with metastatic breast cancer. Anticancer Res. 2009; 29: 667–70. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTA6ImFudGljYW5yZXMiO3M6NToicmVzaWQiO3M6ODoiMjkvMi82NjciO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 27. 27. Gil-Delgado M, Rocher M, Boostandoost E, Khayat D. First step of oral vinorelbine and capecitabine combination in advanced breast cancer patients: feasability study. J Clin Oncol. 2008; 26: 1135. 28. 28. Nolè F, Crivellari D, Mattioli R, Pinotti G, Foa P, Verri E, et al. Phase II study of an all-oral combination of vinorelbine with capecitabine in patients with metastatic breast cancer. Cancer Chemother Pharmacol. 2009; 64: 673–80. [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19184601&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 29. 29. Tubiana-Mathieu N, Bougnoux P, Becquart D, Chan A, Conte P, Majois F, et al. All-oral combination of oral vinorelbine and capecitabine as first-line chemotherapy in HER2-negative metastatic breast cancer: an international phase II trial. Br J Cancer. 2009; 101: 232–7. [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19584872&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 30. 30. Mavroeidis L, Sheldon H, Briasoulis E, Marselos M, Pappas P, Harris AL. Metronomic vinorelbine: anti-angiogenic activity in vitro in normoxic and severe hypoxic conditions, and severe hypoxia-induced resistance to its anti-proliferative effect with reversal by Akt inhibition. Int J Oncol. 2015; 47: 455–64. 31. 31. Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, et al. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest. 2000; 105: R15–24. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1172/JCI8829&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=10772661&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000086538400009&link_type=ISI) 32. 32. Li H, Sun L, Li H, Lv X, Semukunzi H, Li R, et al. DT-13 synergistically enhanced vinorelbine-mediated mitotic arrest through inhibition of FOXM1-BICD2 axis in non-small-cell lung cancer cells. Cell Death Dis. 2017; 8: e2810. 33. 33. Qin RS, Zhang ZH, Zhu NP, Chen F, Guo Q, Hu HW, et al. Enhanced antitumor and anti-angiogenic effects of metronomic Vinorelbine combined with endostar on lewis lung carcinoma. BMC Cancer. 2018; 18: 967. 34. 34. Tan EH, Tan DS, Li WY, Haaland B, Ang MK, Chau NM, et al. Metronomic vinorelbine (oral) in combination with sorafenib in advanced non-small cell lung cancer. Lung Cancer. 2015; 88: 289–96. 35. 35. Tsui KH, Wu MY, Lin LT, Wen ZH, Li YH, Chu PY, et al. Disruption of mitochondrial homeostasis with artemisinin unravels anti-angiogenesis effects via auto-paracrine mechanisms. Theranostics. 2019; 9: 6631–45. 36. 36. Gong B, Xue J, Yu J, Li H, Hu H, Yen H, et al. Cell-free DNA in blood is a potential diagnostic biomarker of breast cancer. Oncol Lett. 2012; 3: 897–900. [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=22741014&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 37. 37. Huang ZH, Li LH, Hua D. Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett. 2006; 243: 64–70. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.canlet.2005.11.027&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=16412565&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 38. 38. Madic J, Kiialainen A, Bidard FC, Birzele F, Ramey G, Leroy Q, et al. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients. Int J Cancer. 2015; 136: 2158–65. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1002/ijc.29265&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=25307450&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 39. 39. Fujita N, Nakayama T, Yamamoto N, Kim SJ, Shimazu K, Shimomura A, et al. Methylated DNA and total DNA in serum detected by one-step methylation-specific PCR is predictive of poor prognosis for breast cancer patients. Oncology. 2012; 83: 273–82. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1159/000342083&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=22964822&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 40. 40. Olsson E, Winter C, George A, Chen Y, Howlin J, Tang MHE, et al. Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO Mol Med. 2015; 7: 1034–47. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NjoiZW1ib21tIjtzOjU6InJlc2lkIjtzOjg6IjcvOC8xMDM0IjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 41. 41. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med. 2015; 7: 302ra133. 42. 42. Dawson S-J, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin S-F, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013; 368: 1199–209. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1056/NEJMoa1213261&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=23484797&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000316869000007&link_type=ISI) 43. 43. Murtaza M, Dawson S-J, Tsui DW, Gale D, Forshew T, Piskorz AM, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013; 497: 108–12. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nature12065&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=23563269&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000318221500043&link_type=ISI) 44. 44. Murtaza M, Dawson S-J, Pogrebniak K, Rueda OM, Provenzano E, Grant J, et al. Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer. Nat Commun. 2015; 6: 8760. 45. 45. Petrelli F, Di Cosimo S, Lonati V, Barni S. Vinorelbine with capecitabine, an evergreen doublet for advanced breast cancer: a systematic literature review and pooled-analysis of phase II-III studies. Clin Breast Cancer. 2016; 16: 327–34. 46. 46. Yuan P, Hu X, Sun T, Li W, Zhang Q, Cui S, et al. Eribulin mesilate versus vinorelbine in women with locally recurrent or metastatic breast cancer: a randomised clinical trial. Eur J Cancer. 2019; 112: 57–65. 47. 47. Huang L, Wei Y, Shen S, Shi Q, Bai J, Li J, et al. Therapeutic effect of apatinib on overall survival is mediated by prolonged progression-free survival in advanced gastric cancer patients. Oncotarget. 2017; 8: 29346–54. 48. 48. Laird BJ, Kaasa S, McMillan DC, Fallon MT, Hjermstad MJ, Fayers P, et al. Prognostic factors in patients with advanced cancer: a comparison of clinicopathological factors and the development of an inflammation-based prognostic system. Clin Cancer Res. 2013; 19: 5456–64. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTA6ImNsaW5jYW5yZXMiO3M6NToicmVzaWQiO3M6MTA6IjE5LzE5LzU0NTYiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 49. 49. Bourgeois H, Grudé F, Solal-Céligny P, Dupuis O, Voog E, Ganem G, et al. Clinical validation of a prognostic tool in a population of outpatients treated for incurable cancer undergoing anticancer therapy: PRONOPALL study. Ann Oncol. 2017; 28: 1612–7. 50. 50. Zimmermann C, Swami N, Krzyzanowska M, Hannon B, Leighl N, Oza A, et al. Early palliative care for patients with advanced cancer: a cluster-randomised controlled trial. Lancet. 2014; 383: 1721–30. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/S0140-6736(13)62416-2&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=24559581&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000336267300025&link_type=ISI) 51. 51. Uzzan B, Nicolas P, Cucherat M, Perret G-Y. Microvessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta-analysis. Cancer Res. 2004; 64: 2941–55. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NjoiY2FucmVzIjtzOjU6InJlc2lkIjtzOjk6IjY0LzkvMjk0MSI7czo0OiJhdG9tIjtzOjE4OiIvY2JtLzE4LzMvODc1LmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 52. 52. Gasparini G, Weidner N, Bevilacqua P, Maluta S, Dalla Palma P, Caffo O, et al. Tumor microvessel density, P53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinoma. J Clin Oncol. 1994; 12: 454–66. [Abstract](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MzoiamNvIjtzOjU6InJlc2lkIjtzOjg6IjEyLzMvNDU0IjtzOjQ6ImF0b20iO3M6MTg6Ii9jYm0vMTgvMy84NzUuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 53. 53. Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications. Lancet Oncol. 2001; 2: 667–73. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/S1470-2045(01)00556-3&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=11902537&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000174092400019&link_type=ISI) 54. 54. Linderholm B, Hellborg H, Johansson U, Elmberger G, Skoog L, Lehtiö J, et al. Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer. Ann Oncol. 2009; 20: 1639–46. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1093/annonc/mdp062&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19549711&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000270217700004&link_type=ISI) 55. 55. Rossi G, Mu Z, Rademaker AW, Austin LK, Strickland KS, Costa RLB, et al. Cell-free DNA and circulating tumor cells: comprehensive liquid biopsy analysis in advanced breast cancer. Clin Cancer Res. 2018; 24: 560–8. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTA6ImNsaW5jYW5yZXMiO3M6NToicmVzaWQiO3M6ODoiMjQvMy81NjAiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 56. 56. Chen X, Qiu T, Zhu Y, Sun J, Li P, Wang B, et al. A single-arm, phase II study of apatinib in refractory metastatic colorectal cancer. The Oncologist. 2019; 24: 883–e407. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTM6InRoZW9uY29sb2dpc3QiO3M6NToicmVzaWQiO3M6ODoiMjQvNy84ODMiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 57. 57. Lacal PM, Graziani G. Therapeutic implication of vascular endothelial growth factor receptor-1 (VEGFR-1) targeting in cancer cells and tumor microenvironment by competitive and non-competitive inhibitors. Pharmacol Res. 2018; 136: 97–107. 58. 58. Romero-Laorden N, Doger B, Hernandez M, Hernandez C, Rodriguez-Moreno J, Garcia-Donas J. Predictive biomarker candidates to delineate efficacy of antiangiogenic treatment in renal cell carcinoma. Clin Transl Oncol. 2016; 18: 1–8. 59. 59. Schiavon G, Hrebien S, Garcia-Murillas I, Cutts RJ, Pearson A, Tarazona N, et al. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer. Sci Transl Med. 2015; 7: 313ra182. 60. 60. Jeselsohn R, Buchwalter G, De Angelis C, Brown M, Schiff R. ESR1 mutations—a mechanism for acquired endocrine resistance in breast cancer. Nat Rev Clin Oncol. 2015; 12: 573–83. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nrclinonc.2015.117&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=26122181&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 61. 61. Chu D, Paoletti C, Gersch C, VanDenBerg DA, Zabransky DJ, Cochran RL, et al. ESR1 mutations in circulating plasma tumor DNA from metastatic breast cancer patients. Clin Cancer Res. 2016; 22: 993–9. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTA6ImNsaW5jYW5yZXMiO3M6NToicmVzaWQiO3M6ODoiMjIvNC85OTMiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 62. 62. Page K, Guttery DS, Fernandez-Garcia D, Hills A, Hastings RK, Luo J, et al. Next generation sequencing of circulating cell-free DNA for evaluating mutations and gene amplification in metastatic breast cancer. Clin Chem. 2017; 63: 532–41. [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6ODoiY2xpbmNoZW0iO3M6NToicmVzaWQiO3M6ODoiNjMvMi81MzIiO3M6NDoiYXRvbSI7czoxODoiL2NibS8xOC8zLzg3NS5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 63. 63. Liang DH, Ensor JE, Liu Z-B, Patel A, Patel TA, Chang JC, et al. Cell-free DNA as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients. Breast Cancer Res Treat. 2016; 155: 139–49. 64. 64. Wan JC, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017; 17: 223–38. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nrc.2017.7&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=http://www.n&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 65. 65. Yates LR, Gerstung M, Knappskog S, Desmedt C, Gundem G, Van Loo P, et al. Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nat Med. 2015; 21: 751–9. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nm.3886&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=26099045&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) 66. 66. Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine Growth Factor Rev. 2004; 15: 229–35. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.cytogfr.2004.03.010&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=15207814&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000222747400005&link_type=ISI) 67. 67. Fletcher JA. Role of KIT and platelet-derived growth factor receptors as oncoproteins. Semin Oncol. 2004; 31: 4–11. [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=14970932&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000189005000002&link_type=ISI) 68. 68. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature. 2001; 411: 355–65. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/35077225&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=11357143&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000168710000058&link_type=ISI) 69. 69. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest. 2003; 111: 1287–95. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1172/JCI200317929&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=12727920&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom) [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000182605400007&link_type=ISI) 70. 70. Erber R, Thurnher A, Katsen AD, Groth G, Kerger H, Hammes HP, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J. 2004; 18: 338–40. [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1096/fj.03-0271fje&link_type=DOI) [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=14657001&link_type=MED&atom=%2Fcbm%2F18%2F3%2F875.atom)