Antitumour effects of apatinib in progressive, metastatic differentiated thyroid cancer (DTC)

Purpose Management of progressive, metastatic radioactive iodine refractory differentiated thyroid cancer (RAIR-DTC) has been a great challenge due to its poor prognosis and limited treatment options. Recently, apatinib, an orally anti-angiogenic tyrosine kinase inhibitor (TKI) is reported to be useful for treatment of progressive RAIR-DIC. The aim of this study was to evaluate the antitumour effect of apatinib and the combination therapy with radioactive iodine (RAI) in patients with progressive metastatic DTC. Methods Five patients (all female, mean age 62 ± 8 years, ranged from 51 to 69 years) with distant metastatic DTC (dmDTC) after total thyroidectomy (TTE) and neck lymph node dissection were treated with apatinib at a dose 500 mg per day after 18F-Fluorodeoxyglucose (18F-FDG) PET/CT. The effects of apatinib on DTC were evaluated at 4 ± 1 months after treatment with apatinib. RAI therapy was then initiated. The response to apatinib and the combination therapy with RAI treatment was evaluated by Response Evaluation Criteria in Solid Tumours (RECIST, version 1.1) and metabolic activity using serum thyroglobulin (Tg) and 18F-FDG PET/CT. Results Positive 18F-FDG PET/CT results were found in all patients before apatinib therapy. The immunohistochemical analysis of primary tumour tissues showed high expression of vascular endothelial growth factor receptor-2 (VEGFR-2). Four patients with follicular thyroid carcinoma (FTC) showed partial response (PR) with significant decrease in tumour size and maximum standardized uptake value (SUVmax) after 4 ± 1 month’s treatment with apatinib. Further significant reduction of tumour size and SUVmax were observed in three patients after combination therapy with apatinib and RAI. Only one patient with both FTC and papillary thyroid cancer (PTC) demonstrated progressive disease (PD) after treatment with apatinib alone, however, a decrease in tumour size and SUVmax as well as serum Tg levels was achieved after the combination with RAI therapy and apatinib. Conclusions Apatinib had significant antitumour effects on progressive distant metastatic DTC. Moreover, beneficial synergistic and complementary effects were shown when apatinib combined with RAI therapy. Clinical Trial Registration NCT 04180007, Registered November 26, 2019.


Introduction
Approximately 10-20% of differentiated thyroid cancer (DTC) patients have progress distant metastatic DTC (dmDTC) [1,2]. A part of them belongs to radioactive iodine refractory DTC (RAIR-DTC) [1,2]. The RAIR-DTC is leading cause of thyroid cancer related death [3]. The dmDTC presents a major management challenge. FDA has approved two agents targeting vascular endothelial growth factor receptors (VEGFRs), lenvatinib and sorafenib for treatment of RAIR-DTC [4,5]. Although sorafenib and lenvatinib were found to prolong progression-free survival (PFS) in patients with RAIR-DTC, however, no significant benefit on overall survival (OS) was observed, except for a subgroup of patients older than 65 years in the SELECT trial [4,5]. Therefore, alternative treatment options are needed for patients with RAIR-DTC. Recently, our study showed that apatinib, a small-molecule tyrosine kinase inhibitor (TKI) targeting vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor (PDGFR) β [6], demonstrates significant clinical benefits in both prolonged PFS and OS in patients with progressive locally advanced or metastatic RAIR-DTC [7,8]. In this preliminary study, we want to evaluate the role of apatinib on dmDTC before RAI and the effects of combination therapy with RAI and apatinib.

Patients
This study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice guidelines and approved by the Ethics Committee of Nanjing First Hospital. Written informed consent was obtained from all patients.
Five patients, who had total thyroidectomy (TTE) and neck lymph node dissection before the study and at least one 18 F-Fluorodeoxyglucose ( 18 F-FDG)-avid distant metastatic lesion as well as no previous RAI or TKI therapy, were included in the study (Table 1). Treatment with apatinib and RAI with radioactive iodine-131 ( 131 I) 500 mg apatinib (Hengrui Medicine, Jiangsu, China) was administrated orally once daily until intolerable toxic effects occurred. Adverse events (AEs) were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (AEs) version 4.0 (CTCAE 4.0). All AEs were assessed every 4 weeks after apatinib administration. According to institutional guidelines at the time of treatment, median administered RAI activity was 9.25 GBq [200 mCi] (minimum-maximum: 7.40-8.33 GBq (200-250 mCi)) per each 131 I treatment. The Patients were treated with 131 I every 6-12 months according to decision of the Multidisciplinary Team (Table 2). 18

F-FDG PET/CT imaging and response evaluation
PET imaging from the top skull to mid-thigh was performed 60 ± 5 min post intravenous injection of 3.7 MBq 18 F-FDG per kg bodyweight. Patients fasted at least six hours before 18 F-FDG injection. Before the 18 F-FDG injection, the fasting blood glucose level was less than 9 mmol/L. All scans were performed on United Imaging, U780 PET/CT (Shanghai, China). Follow-up 18 F-FDG PET/CT was performed at 4 ± 1 months after apatinib initiation and 3 months after combination therapy of apatinib and RAI treatment. Tumour response was evaluated according to RECIST (version 1.1) [9]. Serum thyroglobulin (Tg) and anti-thyroglobulin antibody (Anti-Tg Ab) level were measured at the baseline, at 4 ± 1 months after apatinib therapy and at 3 months after RAI treatment, respectively. Figure 1 showed the flow diagram of this study. Followup was performed at the beginning of apatinib therapy.

Statistical analysis
All continuous variables are presented as mean ± standard deviation and were analyzed statistically using SPSS 23 software (SPSS, Dallas, TX, USA). Two-tailed paired sample t-tests were used to compare the tumour diameters and metabolic parameters before and after apatinib or RAI treatment. p < 0.05 were considered as statistically significant.

Case 1
A 64-year-old woman (No 1 in the Table 1) presented with pain in her left hip and right ribs with an increased serum Tg level of 8644 ng/mL. 18 F-FDG PET/CT showed high uptake in the thyroid masses and multiple lytic bone lesions on the right humerus, 10 th right rib, and the left acetabulum accompanied by a large tumour infiltration ( Fig. 2A, Aa, Ab, Ca and Cb). Follicular thyroid carcinoma (FTC) was diagnosed after a total thyroidectomy. High expression of VEGFR-2 was confirmed in immunohistochemical (IHC) analysis (Fig. 2F). Four months after apatinib treatment, 18 F -FDG PET/CT showed remarkable decrease in tumour size and 18 F-FDG uptake in metastases (Fig. 2B, Ba, Bb, Da, Db). Serum Tg level decreased to 887 ng/mL. Four cycles of RAI therapy were then performed with a total dose of 33,3 GBq (900 mCi) 131 I. Post therapeutic 131 I whole-body image showed radioactive iodine uptake in the bone  2G). Follow-up 18 FDG-PET/CT showed clearly further decrease in tumour size and reduced FDG uptake in bone lesions ( Fig. 2Ea and Eb). The serum Tg level reduced to 6 ng/mL (Table 1).

Case 2
A 69-year-old woman (No 2 in Table 1) had severe pain in her left hip. 18

Case 3
After TTE and neck lymph node dissection, a 68-year-old woman (No 3 in Table 1) with histologically verified FTC underwent 18 F-FDG PET/CT, which showed higher uptake in multiple bone metastases (9 th left posterior rib, Th11, Th12, L1 vertebras with spinal cord compression, and left humerus) and multiple pulmonary metastases. The serum Tg level was 4581 ng/mL. Rapid regression of lung metastases and bone metastases with decreased Tg level to 4341 ng/mL were observed only three months after apatinib treatment. After one cycle of RAI therapy with 9.25 GBq (250 mCi) 131 I. Tg level decreased further to 2482 ng/mL. The post-therapeutic 131 I whole-body image showed positive bone metastases (Fig. 3D).

Case 4
11 years after TTE and neck lymph node dissection, a 51-year-old woman (No 4 in Table 1) with FTC had an increasing serum Tg level of 74465 ng/mL. Positive 18 F-FDG PET/CT results were revealed in multiple mediastinal lymph nodes, bone (sternum, the 3rd and the 7th thoracic vertebra, the left 5 th to 9 th rib, bilateral iliac crest, and sacrum), pulmonary, liver and thoracic wall metastases (Fig. 4A, Aa and Ab). After 4 months treatment with Apatinib, the serum Tg decreased to 4439 ng/mL. 18 F-FDG PET/CT showed notable reduction in both 18 F-FDG uptake and tumour size of all metastatic lesions (Fig. 4B, Ba and Bb). However, three months after one cycle of RAI therapy with 9.25 GBq (250 mCi) 131 I, no further regression of metastases and reduction of metabolic activities were observed (Fig. 4C, Ca and Cb). Tg level was constant at 4700 ng/mL. All metastases were negative in the posttherapeutic 131 I whole-body image (Fig. 4D).

Case 5
A 58-year-old woman (No 5 in the Table) suffered a lumbar pathological fracture with a cold nodule in the thyroid and    (Fig. 5D). 18 FDG-PET/CT revealed a reduction in tumour size and SUVmax (Fig. 5C, Ca to Cd) as well as a decrease in Tg level to 9123 ng/mL in 3 months after the combination therapy with RAI and apatinib. As shown in Table 1, statistically significant decreases in tumour size and SUVmax (both p < 0.01) were found in cases 1 to 4 after apatinib treatment compared with those before the apatinib therapy. Further significant reductions in tumour size and SUVmax (both p < 0.01) were demonstrated in cases 1, 2 and 3 after the combination therapy with RAI and apatinib compared with those after the apatinib therapy. The serum Tg levels showed remarkably reduced after apatinib therapy or after the combination with RAI, however, no statistical significance were observed due to the small numbers of patients. The anti-Tg-antibody values in all patients before and after therapy were under the reference range (≤4.11 IU/ml).
Positive post-therapeutic 131 I whole-body images were shown in cases 1, 2, 3 and 5. All metastases were negative in the post-therapeutic 131 I whole-body image in case 4.
All patients suffered from apatinib treatment-related AEs. The most common AEs were hand-foot skin reaction (which worsened after thyroxine withdrawal), hypertension, fatigue and proteinuria. Cases of 1, 3 and 4 had only grade 1 to 2 AEs. Only two patients (No. 2 and No. 5) had grade 3 AEs (hand-foot reaction and hypertension) for a short time (2 weeks). There were no severe AEs during and after treatment with apatinib. Apatinib combined with iodine therapy did not cause more ≥ grade 3 side effects than apatinib alone ( Table 2).
The median follow-up time were 24 months (ranged from 10 to 52 months, Table 2). All patents were still alive after a year treatment with apatinib.

Discussion
Apatinib is an orally anti-angiogenic TKI targeting VEGFR2 and PDGFR β [6] and has been approved by the  [10]. Recently, apatinib has been shown to be a promising treatment option for progressive locally advanced or metastatic RAIR-DTC [7,8]. Here, we reported the beneficial antitumour effects of apatinib alone or in combination with RAI therapy on progressive dmDTC. To the best of our knowledge, this is the first investigation into the neoadjuvant therapy effects of apatinib and the combination therapy with RAI on progressive dmDTC. Our results suggested that apatinib induced significant decrease in tumour size in patients with aggressive dmDTC. Apatinib alone has antitumour effect, and beneficial synergistic and complementary effects were shown when apatinib combined with RAI therapy in the treatment of dmDTC.
Patients with dmDTC usually have an unfavorable prognosis, because some of dmDTC are RAIR-DTC [2,3]. Furthermore, in our present study, all patients had FTC. Previous studies [11,12] demonstrate that patients with aggressive variants of FTC have an unfavorable prognosis and exhibit lymph node or distant metastases. In many cases, these aggressive variants of thyroid carcinoma require more Increased tumour sizes and a new metastasis formation in the right ilium were revealed after 6-month therapy with apatinib (B, Ba to Bd). After combined therapy with RAI, decreases in tumour sizes and SUVmax were shown (C, Ca to Cd). The post-therapeutic 131 I whole body scans in anterior and posterior views (D) and CT (Da) as well as SPECT/CT (Db) of 5 th rib metastases were shown. Red arrows indicated metastasis in the right 5 th rib and orange arrows metastases in the right acetabulum aggressive or innovative forms of clinical management. TKIs such as lenvatinib and sorafenib have been approved by FDA and European Medicine Agency (EMA) for progressive, metastatic RAIR-DTC [4,5]. However, the therapy options for RAIR-DTC patients in China are still very limited due to limited availability of lenvatinib and sorafenib. TKI has been reported as a successful neoadjuvant for total thyroidectomy to reduce tumor burden [13] and enhance RAI sensitivity of thyroid cancer including an increased sodium/iodide symporter expression [14]. 18 F-FDG PET/CT has been widely used in the diagnosis of dmDTC and 18 F-FDG-avid lesions are usually more aggressive with poor prognosis [15,16]. Previous studies have shown less effective of high-dose RAI therapy in patients with FDG-avid DTC than in patients with non-FDG-avid DTC [16] and there is an inverse relationship between RAI and 18 F-FDG accumulation in DTC cells [17]. In our present study, all patients have 18 F-FDG-avid dmDTC lesions suggesting aggressive diseases and less effective RAI therapy. We therefore explore the use of neoadjuvant treatment with apatinib prior to RAI therapy for evaluation of effect of apatinib on tumour progression and possible improvement of following RAI treatment results. Our present study demonstrated interestingly the effective treatment of apatinib alone on progressive dmDTC as shown in cases 1, 2, 3 and 4. Further decreases in tumour size and SUVmax, as well as serum Tg level, were found in combination with apatinib and RAI therapy in cases 1, 2 and 3. This might be explained by the synergistic antitumour effects of combination therapy with apatinib and RAI, since TKI has been reported to be able to enhance the RAI sensitivity of thyroid cancer due to the enhancement of sodium iodide symporter (NIS) function [14]. However, it could be also possible that this effect was only due to the longer duration of apatinib therapy. In case 4 the patient was effectively treated by apatinib, however, RAI therapy showed no effect on the dmDTC, since all metastatic lesions, in this case, were negative in post-therapeutic 131 I scan, these metastatic lesions may be defined as RAIR-DTC. The reasons why the patient in case 5 showed no response to apatinib remain unclear. In contrast to other four patients with FTC in cases 1 to 4, this patient had both FTC and PTC. It is well known that DTC may have heterogeneous response to TKIs [18]. Apatinib suppresses tumour progression via blocking the VEGFR2 cascade in malignant cells [6]. Unfortunately, the tumour tissue VEGFR2 status, in this case, was unknown. We can only speculate that the tumour progression in this case might be through other molecular pathways rather than VEGFR2 pathway. Fortunately, this patient had response to RAI therapy leading to stable disease (SD) after RAI therapy. It might be possible that the positive effect of RAI on the metastatic lesions in case 5 might be partly due to the apatinib therapy since TKI may enhance the RAI sensitivity of thyroid cancer [14]. Our results may have implication for the complementary effect of combination therapy with apatinib and RAI as shown in cases 4 and 5.
Despite the small number of patients, our results demonstrated statistically significant reduction of tumour size and SUVmax under apatinib therapy indicating the clinical potential of apatinib therapy. Further significant decrease in tumour size and SUVmax demonstrated the synergistic and complementary effects of the combination of RAI with apatinib. The serum Tg levels which may indicate the tumour activity showed remarkably reduced after apatinib therapy and after the combination therapy with RAI, however, no statistical significance were observed due to the small numbers of patients.
There were some limitations in this study. Firstly, the number of investigated cases was limited. Secondly, there was no control group. The present study was a pilot study to evaluate the effects of apatinib on progressive dmDTC. More patients should be included in a randomized study to verify the results. Meanwhile, all patients are still alive.
In conclusion, apatinib is effective to inhibit the tumour progression of dmDTC. Furthermore, apatinib combined with RAI therapy might have beneficial synergistic or complementary antitumour effect on progressive dmDTC.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request and Development Project (201911042), the Second Round Fund of Nanjing Clinical Medical Center "Nanjing Nuclear Medicine Centre", Open access funding provided by Medical University of Vienna.

Compliance with ethical standards
Conflict of interest The author declares no competing interests.
Consent to participate and for publication Informed consent was obtained from all patients.
Ethical approval All procedures performed in studies involving human participants were approved by the Institutional Review Board of Nanjing First Hospital and with the principles of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
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