Apatinib as targeted therapy for sarcoma

Sarcomas are a group of malignant tumors originating from mesenchymal tissue with a variety of cell subtypes. Despite several major treatment breakthroughs, standard treatment using surgery, radiation, and chemotherapy has failed to improve overall survival. Therefore, there is an urgent need to explore new strategies and innovative therapies to further improve the survival rates of patients with sarcomas. Pathological angiogenesis has an important role in the growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) play a central role in tumor angiogenesis and represent potential targets for anticancer therapy. As a novel targeted therapy, especially with regard to angiogenesis, apatinib is a new type of small molecule tyrosine kinase inhibitor that selectively targets VEGFR-2 and has shown encouraging anticancer activity in a wide range of malignancies, including gastric cancer, non-small cell lung cancer, breast cancer, hepatocellular carcinoma, and sarcomas. In this review, we summarize the preclinical and clinical data for apatinib, focusing primarily on its use in the treatment of sarcomas.


INTRODUCTION
Sarcomas are a group of heterogeneous malignant tumors derived from mesenchymal tissue [1]. These malignancies, including more than 50 subtypes, have unique clinical and histologic features [1]. Although sarcomas are less common than other tumors, they account for almost 21% of all solid tumors in children and are the third leading cause of cancer-related death among people under 20 years [1,2]. In recent years, despite significant developments in new multidisciplinary therapies, such as surgery, chemotherapy and radiation therapy, the 5-year survival rate has remained relatively unchanged [3]. This phenomenon is particularly evident in metastatic or recurrent advanced disease, with a median overall survival (mOS) of about 15 months and with only 10% of patients still alive at 5 years [4]. Therefore, there is an urgent need to develop new strategies and explore innovative therapies to further improve the survival rate of patients with sarcomas.
Apatinib, also known as YN968D1, is a novel, oral, small molecule TK inhibitor of VEGFR-2 [24]. Preclinical and clinical studies have shown that apatinib has promising efficacy and manageable side effects in the treatment of a variety of solid tumors [24][25][26][27][28][29][30]. Especially in advanced gastric cancer, apatinib has been shown to significantly prolong the survival time of patients after standard chemotherapy has failed, and can effectively improve treatment compliance [27,31]. Based on these findings, apatinib was approved by the China Food and Drug Administration (CFDA) in 2014 as a subsequentline treatment for patients with advanced gastric cancer. To date, several phase II/III clinical trials of apatinib for the treatment of various cancers, such as non-small cell lung cancer (NSCLC), breast cancer, hepatocellular carcinoma, and sarcomas, have been completed or are ongoing [28][29][30]32].

Preclinical findings for apatinib
In vitro, apatinib has demonstrated anti-angiogenic properties including the inhibition of budding rat aortic rings, the proliferation and migration of human umbilical vein endothelial cells, and tube formation induced by VEGF. The inhibition of cell migration and vessel formation was similar to that observed for sunitinib. In addition, apatinib suppressed the phosphorylation of VEGFR-2/KDR and ERK1/2, a downstream of VEGF signaling, in a concentrationdependent manner at the cellular level [25,33].
In vivo, once-daily oral administration of apatinib produced a statistically significant, dose-dependent inhibition of tumor growth in established NCI-H460 human lung tumors, HCT-116 human colon tumors, and SGC-7901 human gastric tumors compared with control nude mice [25]. In addition, apatinib appeared to exert synergistic effects when assessed in combination with docetaxel or doxorubicin in lung cancer models, and with oxaliplatin or 5-FU in colon cancer models [25]. Apatinib also appeared to reverse multidrug resistance by inhibiting the transport function of multidrug resistance protein 1 (ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) [34,35]. This suggests that apatinib may act synergistically with standard chemotherapy agents in the treatment of certain malignancies. Tong et al. showed that the addition of apatinib to standard chemotherapy improved tumor inhibition of ABCB1-overexpressing leukemia cell lines [34].
As for the preclinical data of apatinib in sarcoma field, there are two important studies reported by Prof. Wei Guo group. The first study reported by Liu K et al. shows that apatinib can inhibit the growth of osteosarcoma in vivo and in vitro [23]. In vitro, apatinib can inhibit osteosarcoma growth by inducing autophagy and apoptosis of osteosarcoma cells. The mechanism is that apatinib can directly inhibit the expression of Bcl-2, an anti-apoptotic gene, and inactivate signal transducer and activator of transcription 3 (STAT3) mediated by VEGFR2 ( Figure 1). Interestingly, the effect of apatinib on apoptosis of osteosarcoma cells was enhanced by inhibition of autophagy. This suggests that autophagy is a protective factor in the treatment of osteosarcoma with apatinib. In vivo, apatinib also reduced the volume of osteosarcoma compared with controls. This study suggests that the combined use of autophagy inhibitors may enhance the antitumor activity of apatinib. The second study reported by Zheng B, et al. that apatinib attenuates migration and invasion by suppressing epithelial-mesenchymal transition (EMT) and inactivating STAT3. Furthermore, apatinib reduces PD-L1 expression in osteosarcoma cells ( Figure 1). [36] So these data suggest that apatinib might effect as not only a target therapeutic drug, but also an immunotherapy modulator for sarcoma patients.

Clinical trials of apatinib for epithelial tumors
The efficacy of apatinib for the treatment of a variety of solid tumors has been investigated in several phase I-III clinical trials in gastric cancer, [27,31] NSCLC, [28] triple-negative breast cancer, [29] non-triple-negative breast cancer, [32] advanced solid tumors, [24] and advanced hepatocellular carcinoma [30] (Summarized in Table 1).
A phase I clinical trial was conducted by Li et al. to determine the maximum tolerated dose (MTD), www.oncotarget.com  Apatinib has been well tolerable in patients; most of the adverse event could be managed by dose interruptions or reductions.
Abbreviations: mPFS, median progression-free survival; mOS, median overall survival; ORR, objective response rate; RR, response rate; DCR, disease control rate; CBR, clinical benefit rate; MTD, maximum tolerated dose; mTTP, median time to progression; BID, twice a day; QD, once a day. www.oncotarget.com safety profile, pharmacokinetic variables, and antitumor activity of apatinib in advanced solid malignancies (NCT00633490) [24]. The MTD was determined to be 850 mg once daily and a dose of 750 mg once daily was recommended. With respect to efficacy, partial remission (PR) was observed in 7% of patients and stable disease (SD) in 24%, and the disease control rate (DCR) was 83.8% (7+24/37). The safety profile was acceptable and the regimen was found to be well tolerated.
Phase II clinical trials of apatinib have been performed in patients with gastric cancer, breast cancer, NSCLC, and hepatocellular carcinoma to evaluate the efficacy and safety of apatinib in heavily pretreated patients with malignant tumors. In the trial in gastric cancer, [27] 144 patients were enrolled and divided into three groups: group A, placebo; group B, 850 mg of apatinib once daily; and group C, apatinib 425 mg twice daily. Median progression-free survival (mPFS) for groups A, B, and C was 1.40 months, 3.67 months, and 3.20 months, respectively; and mOS was 2.50 months, 4.83 months, and 4.27 months, respectively. A statistically significant difference in PFS and OS was observed between the apatinib and placebo groups (P <0.001), but no difference was found between the two apatinib groups (Figure 2). In the study of nontriple-negative metastatic breast cancer, mPFS for the 38 enrolled patients was 4.0 months, [32] the objective response rate (ORR) was 16.7% (6/36), DCR was 66.7% (24/36), and mOS were 10.3 months (Figure 3). In the study of triple-negative metastatic breast cancer in 59 patients, [29] mPFS and mOS were 3.3 months and 10.6 months, respectively ( Figure 4). In the 56 evaluable patients, ORR and clinical benefit rate (CBR) were 10.7% and 25.0%, respectively. In the study of NSCLC, 135 patients (90 in the apatinib arm, 45 in the placebo arm) were included, [28] and mPFS, response rate (RR), and DCR were significantly better in the study arm (4.7 months, 12.2%, and 68.9%, respectively) than in the placebo arm (1.9 months, 0%, and 24.4%, respectively). In the study of hepatocellular carcinoma, 144 patients were enrolled and assigned to stage 1 (36 patients, 850 mg once daily) and stage 2 (85 patients, 750 mg once daily) [30]. Median time to progression (mTTP), mOS, and DCR in the stage 1 group were 4.2 months, 9.7 months, and 48.57% respectively; in the stage 2 group were 3.3 months, 9.8 months, and 37.25%, respectively.
A phase III trial was conducted by Li et al. to evaluate the efficacy and safety of apatinib in 267 patients with advanced gastric cancer or gastroesophageal junction adenocarcinoma who had received prior chemotherapy [31]. The outcomes demonstrated that mOS and mPFS of the apatinib arm were significantly prolonged compared with placebo (6.5 months vs. 4.7 months, and 2.6 months vs. 1.8 months, respectively) ( Figure 5).
These clinical trial results indicate the therapeutic efficacy and manageable adverse effects (AEs) of apatinib in epithelial tumors.

Effectiveness of apatinib in case reports of sarcomas
To the best of our knowledge, a total of six case reports regarding the treatment of sarcoma with apatinib have been published, and the details of these reports are summarized in Table 2.
Of the six case reports, five patients were given apatinib daily 500mg, and one patient was 425mg per day. The final efficacy of all these six patients was evaluated as PR. The main AEs were grade 1-4 nonhematologic toxicities, including skin rash, mild hand-foot syndrome (HFS), hypertension, and transient elevation of aminotransferase and aspartate aminotransferase, which were adequately controlled after symptomatic treatment or reduce the dose. No serious drug-related side effects were observed. Ji et al. reported a 78-year-old male patient with malignant fibrous histiocytoma (MFH) who had multiple lung metastases a year after surgery and symptoms of sputum and hemoptysis [37]. After two cycles of targeted treatment, expectoration was markedly reduced, without hemoptysis. The size of the lung metastasis was significantly reduced and was considered PR. At the time of the report, 6-month PFS had been achieved. Dong et al. described a 68-year-old Chinese female patient initially diagnosed with advanced multiple abdominal and  pelvic cavity round cell liposarcomas, accompanied with liver metastasis [38]. After nearly 1 month of treatment with apatinib, the middle and right abdominal mass had diminished and the metastatic lesion in the liver remained stable, and the patient was considered to be in PR. At the time of the report, 6-month PFS had been achieved. Ji et al. reported a 74-year-old male patient with angiosarcomas of the scalp, and 2 months after surgery, whole-body positron emission tomography/computerized tomography confirmed local tumor recurrence with multiple metastases in the right temporal lobe, subcutaneous adipose tissue, and bilateral lung lobar [39]. After 1 cycle of treatment with apatinib, the volume of multiple pulmonary metastases had significantly decreased or even disappeared, and the recurrent tumor of the scalp had completely disappeared and the patient was considered to be in PR. The PFS was 12 months. A 50-year-old male patient diagnosed with osteoblastic osteosarcomas was reported by Zhou et al. [40]. Although the patient had undergone multiple resections, he developed multiple pulmonary metastases. After 11 months of targeted therapy with apatinib, a total of nine lesions disappeared and no new lesions were observed, so he was evaluated as in PR. Zhou et al. also described a case of an 18-yearold male patient with alveolar soft part sarcomas (ASPS) and multiple pulmonary metastases at the initial visit [41]. After 1 month of treatment, the size and number of pulmonary metastases decreased, and almost all metastatic lesions had disappeared by 3 months of treatment. The patient was evaluated as in PR, and 12-month PFS had been achieved at the time of the case report. Zhou et al.
presented an 81-year-old Chinese woman with advanced pleomorphic liposarcomas (PLS) who received apatinib 425mg per day after failure chemotherapy [42]. One week later, symptoms such as abdominal distension, nausea and vomiting gradually eased. CT examination showed that the tumor was slightly reduced and the internal necrosis area increased. The case was eventually evaluated as PR and received a 3-month PFS.
The results of these studies show that apatinib is efficacious and its adverse effects are manageable in the treatment of malignant sarcoma and thus represent a promising treatment option for patients with metastatic or recurrent sarcomas.

Safety and efficacy of apatinib in sarcoma treatment: two retrospective studies
Two retrospective studies of apatinib for the treatment of sarcomas have been conducted to date, to the best of our knowledge.
The medical records of 31 patients who received apatinib between September 2015 and August 2016 were retrospectively reviewed by Yang et al. to evaluate short-term efficacy, mTTP, and safety [43]. Nineteen (61.3%) patients received apatinib after failing second-line or further cytotoxic chemotherapy, 8 (25.8%) patients received apatinib as second-line therapy, and 4 (12.9%) patients who refused chemotherapy were given apatinib as first-line therapy. In the study cohort, one patient was treated with apatinib as adjunctive therapy after ablation, while the remaining 30 received apatinib as salvage therapy. AEs led to treatment discontinuation in 3 patients, and 3 more discontinued for personal reasons. Among the 24 remaining patients eligible for evaluation of tumor response to apatinib, 25 patients were available for survival analysis. The ORR was 33.3% and the CBR was as high as 75.0%. The mTTP was 4.3 months (range, 1.8-11.6 months). The median PFS of all patients was 4.25 (95% CI, 2.22-5.11) months, while the median OS was 9.43 (95% CI, 6.64-18.72) months ( Figure 6A-6B) [44]. Most AEs were of grade 1 or 2, and grade 3 AEs included hypertension (n = 2, 6.5%), HFS (n = 2, 6.5%), and diarrhea (n = 1, 3.2%). No grade 4 AEs or drug-related mortality occurred.
The other retrospective analysis was conducted by Li et al., [45] with the aim of evaluating the efficacy and safety of apatinib in patients with stage IV sarcomas who had failed to respond to previous standard chemotherapy. Data were examined from 16 patients treated with apatinib as salvage treatment at a dose of 500 mg/d; 28 days per cycle. Patients underwent an average duration of 3.2 cycles (range 0-9 cycles). Median follow-up time was 8.4 months (1-12 months). Of the 16 patients evaluated, 10 received apatinib treatment more than one cycle, so these 10 patients were evaluated for efficacy. The mPFS was 8.84 months, and in the final efficacy evaluation 2 patients were evaluated as having a PR, 6 patients had SD and the remaining 2 patients had progressive disease (PD). ( Figure 6C) The ORR was 20.0% (2/10) and the DCR was 80.0% (8/10). The most common grade 3-4 treatmentrelated AEs were hypertension (n = 3, 18.7%), HFS (n = 2, 12.5%), and proteinuria (n = 1, 6.3%). No drug-related severe AEs were reported.
In terms of efficacy, the study by Yang et al. had a better ORR than the study by Li et al. (33.3% vs. 20%); however, mPFS and DCR were worse (4.3 months vs. 8.84 months; 75.0% vs. 80.0%, respectively). These studies show subtle differences in efficacy, possibly arising from differences in the cohort sizes. Toxicities were similar between the studies, and were mainly mild to moderate non-hematologic toxicities including grade 1-2 skin rash, mild HFS, hypertension, and transient elevation of aminotransferase and aspartate aminotransferase, which were well controlled after symptomatic treatment. No serious drug-related side effects were observed. In short, as these retrospective studies included small study populations, they support only a preliminary role of apatinib for the treatment of sarcoma. To further confirm the effect of apatinib in this type of cancer, phase II/III clinical trials are required. www.oncotarget.com

Safety and efficacy of apatinib compared with other anti-angiogenic drugs for the treatment of sarcoma
Despite a lack of phase II/III clinical trials of apatinib for sarcoma, the preliminary studies described above, with mPFS of 4.3 and 8.84 months, support the potential superiority of apatinib over other targeted antiangiogenesis drugs for sarcoma, including pazopanib, sunitinib, and sorafenib. Sleijfer et al. conducted a phase II trial of pazopanib in 142 patients with relapsed or refractory advanced soft tissue sarcomas (STS). The study population was divided into four groups according to disease category: adipose cell STS, leiomyosarcomas, synovial sarcomas, and other STS types. PFS in the groups was 2.9 months, 3.3 months, 5.8 months, and 3.3 months, respectively [46]. A phase II, multicenter clinical trial of sunitinib in 53 patients with advanced non-gastrointestinal stromal tumor soft tissue sarcomas was reported by George et al. One patient achieved confirmed PR and 10 patients (20%) achieved SD for at least 16 weeks [47]. In a phase II clinical study of sorafenib for metastatic or recurrent sarcomas, 147 patients were enrolled. The median follow-up time was 6 months and PFS was 3.2 months [48]. The most common side effects of these reported targeted anti-angiogenesis agents include hand-foot skin reaction, hypertension, proteinuria, rash, diarrhea, hyperbilirubinemia, rash/desquamation, fatigue, thrombocytopenia, leukopenia, diarrhea, nausea, vomiting and so on [46][47][48]. The toxicity of apatinib appears to be similar to that of other targeted anti-angiogenesis agents, with no serious adverse events reported.

Ongoing trials of apatinib for sarcomas
Based on clinical trials for apatinib in epithelial tumors and retrospective analysis and case reports for sarcomas, a number of phase II/III clinical trials of apatinib for sarcoma have been initiated in China. Tianjin Medical University Cancer Institute and Hospital has registered a prospective, open-label, single-arm, multicenter phase II trial to evaluate the efficacy and safety of apatinib for chemotherapy failure IV stage STS, with an estimated enrollment of 80 patients. The clinical trial registered by Shanghai Jiao Tong University Affiliated Sixth People's Hospital is also expected to enroll 80 patients, with the purpose of exploring the safety and effectiveness of apatinib for advanced STS. Peking University People's Hospital has registered two clinical trials, a one-armed, phase II, open-label, multicenter prospective trial of apatinib for advanced STS patients after failure of traditional therapy, and a single arm, phase II/III, singlecenter trial of apatinib for advanced osteosarcoma after failure of standard multimodal therapy. Each trial is expected to enroll 37 patients. Finally, Henan Cancer Hospital has registered a single-center, one-armed clinical study to evaluate the efficacy and safety of apatinib as second-line treatment for advanced osteosarcoma and STS. Four out of five of these clinical trials have begun recruiting patients, and the efficacy and safety outcomes may contribute to the eventual approval of apatinib for sarcoma by the U.S. Food and Drug Administration as the second anti-angiogenesis targeted treatment for this disease. The details of these phase I/III trials are summarized in Table 3.

Future perspectives
Apatinib is a novel, orally bioavailable smallmolecule TK inhibitor of VEGFR-2. Previous studies have shown that apatinib is a promising agent for the treatment of a variety of tumor types, including gastric cancer, NSCLC, triple-negative breast cancer, non-triple-negative breast cancer, and advanced hepatocellular carcinoma, exhibiting improved outcomes and a tolerable safety profile as subsequent-line therapy. Retrospective analyses and case reports have shown that apatinib also exerts a marked effect on sarcomas, with acceptable toxicity, and that it may be superior to other anti-angiogenesis targeted drugs such as pazopanib, sunitinib, and sorafenib. Recently, an interesting study found that the presence of hypertension, proteinuria, or HFS during the first cycle of apatinib treatment correlated with clinical outcomes in gastric cancer patients and was a viable biomarker of antitumor efficacy in metastatic gastric cancer patients [49]. However, no such phenomenon was observed in sarcoma patients, possibly due to fewer cases. Finally, although several phase II/III clinical trials to confirm the efficacy and safety of apatinib for sarcoma are ongoing in China, it may be necessary to establish specific biomarkers to identify appropriate patients for apatinib and to assess the disease prognosis.