Novel recombinant human thyroid-stimulating hormone in aiding postoperative assessment of patients with differentiated thyroid cancer—phase I/II study

Thyroid hormone withdrawal (THW) inevitably induced hypothyroidism in patients with differentiated thyroid cancer (DTC), and we aimed to evaluate the safety and efficacy of a novel recombinant human thyroid-stimulating hormone (rhTSH, ZGrhTSH) as an alternative of THW in China. Totally, 64 DTC patients were enrolled with 24 in the dose-escalation cohort equally grouped into 0.9 mg × 1 day, 0.9 mg × 2 day, 1.8 mg × 1 day, and 1.8 mg × 2 day dosage, and 40 further enrolled into 0.9 mg × 2 day dose-expansion cohort. All patients underwent both ZGrhTSH phase and levothyroxine (L-T4) withdrawal phase for self-comparison in terms of TSH levels, the radioactive iodine (RAI) uptake, stimulated thyroglobulin level, and the quality of life (QoL). In ZGrhTSH phase, no major serious adverse events were observed, and mild symptoms of headache were observed in 6.3%, lethargy in 4.7%, and asthenia in 3.1% of the patients, and mostly resolved spontaneously within 2 days. Concordant RAI uptake was noticed in 89.1% (57/64) of the patients between ZGrhTSH and L-T4 withdrawal phases. The concordant thyroglobulin level with a cut-off of 1 μg/L was noticed in 84.7% (50/59) of the patients without the interference of anti-thyroglobulin antibody. The QoL was far better during ZGrhTSH phase than L-T4 withdrawal phase, with lower Billewicz (− 51.30 ± 4.70 vs. − 39.10 ± 16.61, P < 0.001) and POMS (91.70 ± 16.70 vs. 100.40 ± 22.11, P = 0.011) scores which indicate the lower the better. Serum TSH level rose from basal 0.11 ± 0.12 mU/L to a peak of 122.11 ± 42.44 mU/L 24 h after the last dose of ZGrhTSH. In L-T4 withdrawal phase, a median of 23 days after L-T4 withdrawal was needed, with the mean TSH level of 82.20 ± 31.37 mU/L. The half-life for ZGrhTSH clearance was about 20 h. The ZGrhTSH held the promise to be a safe and effective modality in facilitating RAI uptake and serum thyroglobulin stimulation, with better QoL of patients with DTC compared with L-T4 withdrawal.


Introduction
According to the data of the International Agency for Research on Cancer (IARC) in 2020, the new cases of thyroid cancer in China (221,093) accounted for more than one-third of the global number, indicating the relatively high thyroid cancer burden in terms of incidence in China [1,2]. Differentiated thyroid cancer (DTC) accounts for over 94% of thyroid cancer [3]. Successful management of DTC is based on total thyroidectomy followed by selective radioactive iodine (RAI) therapy and thyroxine therapy for thyroid-stimulating hormone (TSH) suppression [4,5]. RAI therapy of DTC is based on the sodium iodide symporter (NIS) expressing in DTC cells to some extent, allowing its ability of trapping circulating RAI. Notably, though a more than 98% of 5-year survival rate revealed by Surveillance, Epidemiology and End Results (SEER) database [6], it was only 84.3% for Chinese patients, indicating the less favorable survival status in China [7], and urging the need This article is part of the Topical Collection on Oncology -Head and Neck of strengthening thyroid cancer management, particularly a more comprehensive assessment in active surveillance of recurrence and more accurate subsequent therapeutic tailoring such as RAI therapy. During the pre-RAI assessment and active surveillance, diagnostic 131 I-whole body scan (Dx-WBS) and serum stimulated thyroglobulin (s-Tg) commonly require TSH stimulation by means such as temporary thyroid hormone withdrawal (THW) for better detection of residual or functional DTC lesions [8][9][10]. However, during THW period, patients would inevitably experience symptomatic hypothyroidism; of note, in those with the high tumor burden, an adequate TSH stimulation might not be reached due to the extrathyroidal tumorigenic thyroid hormone excretion, or even risk to stimulate the tumor growth and cause disease progression, which all may prevent the patients from optimal assessment.
Thyrogen®, a kind of recombinant human TSH (rhTSH), has been approved by Food and Drug Administration (FDA) in 1998, is an exogenous TSH, which can stimulate thyroid cells without THW and has been recommended as an alternative for the preparation of RAI ablation for over 20 years. As a replacement of THW prior to 131 I-WBS or s-Tg examination, rhTSH utilization greatly maintains quality of life during RAI ablation or assessment [11][12][13]. However, so far rhTSH remains unavailable in China where THW is the only modality for TSH stimulation. The preclinical studies showed that ZGrhTSH, a Chinese rhTSH analog, could bind to TSHR on the cell surface and result in rising of intracellular second messenger cyclic adenosine monophosphate (cAMP), which indicated its biological activity was similar to Thyrogen®. The current phase I and II self-control study was designed to assess the safety, tolerance, and efficacy of ZGrhTSH in diagnostic evaluation and its impact on patient's quality of life (QoL) in comparison with levothyroxine (L-T 4 ) withdrawal.

Materials and methods
The study was registered at ClinicalTrials.gov (NCT04137185) and approved by the ethical board of each participating clinical institution. All patients were fully informed and provided written informed consent before enrollment.

Study participants
From May, 2019 to April, 2021, patients who underwent total thyroidectomy and pathologically diagnosed as DTC were enrolled. All the following inclusion criteria should be met: (i) aged 18 ~ 75 years; (ii) Dx-WBS planned for the disease status evaluation postoperatively or after initial therapy; (iii) the serum TSH level was controlled below 0.5 mU/L before enrollment; (iv) low-iodine preparation for more than 4 weeks before enrollment. Patients who were pregnant, lactating, or not suitable for L-T 4 withdrawal were excluded. rhTSH ZGrhTSH was produced by Suzhou Zelgen Biopharmaceutical Co., Ltd., with a specification of 1.1 mg per piece. The storage condition should be 2 ~ 8 ℃. ZGrhTSH is a hTSH produced by recombinant DNA technology. rhTSH freezedried is made from Chinese hamster ovary (CHO) cells that efficiently express hTSH α and β subunit genes, after cell culture, isolation, and high purification.

Study design
In this phase I/II, open-label, multicenter self-control study, we evaluated the safety and efficacy of ZGrhTSH in patients with DTC who were referred for postoperative assessment including primarily Dx-WBS and s-Tg. The study consisted of the following I and II parts: dose-escalation (phase I) and dose-expansion (phase II). A total of 64 eligible patients were enrolled with 24 for phase I and followed by 40 for phase II study (Fig. 1). Patients in phase I (n = 24) were equally grouped into 0.9 mg × 1 day, 0.9 mg × 2 day, 1.8 mg × 1 day, and 1.8 mg × 2 day dose regimen sequentially. During phase I study, the dose escalation allowed to initiate only after the safety was confirmed in the prior lower dose regimen. Similar as Thyrogen®, based upon the results of phase I study, 0.9 mg × 2 day was selected for further phase II study. Pharmacokinetic analysis was performed in all patients of phase I and first 10 patients of phase II.
Each enrolled patient received ZGrhTSH injection firstly, then followed by L-T 4 withdrawal. The sequence throughout the trial was always the same, which was consistent with the design of Thyrogen® [14]. The reason for this sequential design is to avoid the possibly delayed therapy for those a therapeutic dose of RAI was indicated, who would experience L-T 4 withdrawal again for another 4-6 weeks if the patient underwent a L-T 4 withdrawal scan firstly, followed by ZGrhTSH scan. Two phases of ZGrhTSH and L-T 4 withdrawal were successively underwent to compare the influence on elevated TSH level, RAI uptake, Tg secretion, and QoL. In ZGrhTSH phase, ZGrhTSH was injected intramuscularly with dosage of the corresponding dose group. In L-T 4 withdrawal phase, the L-T 4 withdrawal was conducted to raise TSH above 30 mU/L.

Anti-TSH antibody testing
The presence of antibodies against rhTSH in human serum samples is detected by the typical bridging assay on the MESO Scale Discovery (MSD) platform. Briefly speaking, biotinylated-rhTSH (Bio-rhTSH) and ruthenylated-rhTSH (Ru-rhTSH) are added to human serum samples to form an antibody-drug complex with ADA in the samples during incubation. The complex is then captured and detected on the MSD plate with Streptavidin pre-coated.

Safety assessment
Adverse events (AEs) were observed and documented for all patients during the study. Common terminology criteria for adverse events (CTCAE) version 4.03 were used to evaluate the severity of AEs.

Dx-WBS imaging and interpretation
Each patient was instructed to oral 131 I (74-148 MBq) for Dx-WBS after 24 h of the last ZGrhTSH injection and after at least 2 weeks of discontinuation of L-T 4 for ZGrhTSH and L-T 4 withdrawal phase respectively. The interval between the two scans was at least 4 weeks. TSH level ≥ 30 mU/L and low-iodine diet for more than 4 weeks were required in all patients before oral 131 I administration. Dx-WBS was performed after 48 h of 131 I administration.
Each Dx-WBS was interpreted independently by two nuclear medicine physicians who were unaware of the patient's informations such as the center, sequence of the scans, the Tg levels, or the surgical management. The scans were defined as either positive when showing the 131 I concentration in the thyroid bed or abnormal uptake in the neck, the lungs, or other extrathyroidal sites, or negative when no 131 I uptake in the thyroid bed or extrathyroidal sites. The locations of the 131 I uptake were compared within each pair of scans to assess whether the two scans were concordant.

Serum measurements
Serum TSH, Tg, and anti-Tg antibody (TgAb) levels were measured upon baseline (TSH ≤ 0.5 mU/L) and prior to each Dx-WBS (TSH ≥ 30 mU/L). For pharmacokinetic analysis, serum samples were obtained at 30 min before and 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h, 120 h after ZGrhTSH administration. TSH was tested by laboratories of local sites using standard method. Tg and TgAb were tested by central laboratory using electrochemiluminescence method. The measurable range of Tg and TgAb was 0.1-5000.00 μg/L and 10.00-4000 kU/L, respectively, and a reference range of TgAb for normal value was < 115 kU/L.

Hypothyroid symptoms and QoL measurements
Hypothyroid symptoms were assessed with the Billewicz Scale (score range: − 53 ~ 72), which is an international Fig. 1 Flow chart of the trial. rhTSH, recombinant human thyroid-stimulating hormone; Dx-WBS, diagnostic. 131 I-whole body scan; THW, thyroid hormone withdrawal standard for hypothyroidism evaluation, with higher score indicates more obvious hypothyroid symptoms. The QoL was evaluated by the short-form Profile of Mood States (POMS) (score range: 56 ~ 216). Similarly, the higher POMS score suggests the more negative emotional state. The Billewicz Scale and POMS were tested in all patients at the baseline, 1 day before 131 I administration, and 3 days after 131 I administration for both ZGrhTSH and L-T 4 withdrawal phases.

Statistical analysis
All efficacy outcomes were assessed in the intention-totreat population. Proportions were presented with a twosided 95% CI using Clopper-Pearson method. Comparison of the concordant rate of RAI uptake between ZGrhTSH and L-T 4 withdrawal phases was performed using the McNemar chi-square test. The differences of serum TSH, Tg levels, and hypothyroid symptoms on the Billewicz Scale and QoL on the short-form POMS were analyzed by the Wilcoxon signed-rank test. All statistical analysis was performed in two-side model using SAS 9.4. All safety analyses were made in the population received any dose of ZGrhTSH.

Immunogenicity
With available samples in 39 patients, none of them had detectable serum anti-ZGrhTSH antibodies (ADA).

Adverse events
Of the 64 patients enrolled in the study, AEs were observed in 36 (56.3%) patients during ZGrhTSH phase, of which 13 were identified to be related to ZGrhTSH, but usually mild and resolved within 2 days. The most common AE related to ZGrhTSH was headache, which occurred in 4 patients (6.3%). Other AEs included lethargy (4.7%) and asthenia (3.1%). ZGrhTSH-related AEs are listed in Table 2. No grade 2 and above AEs were observed in ZGrhTSH phase. uptake during L-T 4 withdrawal phase, but negative during ZGrhTSH phase. The same consistency rate of 89.1% was achieved among the 46 patients in 0.9 mg × 2 day group, and the details are presented in Fig. 2. The images of concordant and discordant Dx-WBS findings are provided in Fig. 3.

Serum Tg response during ZGrhTSH and L-T 4 withdrawal phases
The mean Tg level was 0.40 ± 0.68 μg/L, and the median was 0.10 (0. 10

Quality of life during ZGrhTSH and L-T 4 withdrawal phases
No patient experienced hypothyroidism during ZGrhTSH phase while 3 of 49 available cases showed suspicious hypothyroidism during L-T 4 withdrawal phase. The Billewicz Scales score after ZGrhTSH administration was significantly lower than that after L-T 4 withdrawal (− 51.30 ± 4.70 vs − 39.10 ± 16.61, P < 0.001). The specific incidence of hypothyroidism symptoms and signs is shown in Fig. 6. The top three symptoms are periorbital puffiness (30/64), weight increase (28/64), and chills (16/64) during L-T 4 withdrawal. For all patients, the POMS score was 90.10 ± 14.95 at baseline and 91.70 ± 16.70 in ZGrhTSH phase, with the change value of 1.50 ± 12.58. In L-T 4 withdrawal phase, the POMS score rose to 100.40 ± 22.11, with the change value of 10.30 ± 21.77 compared with baseline. The POMS score significantly differed between ZGrhTSH and L-T 4 withdrawal phases (P = 0.002), indicating the mood state of patients after ZGrhTSH administration was significantly better than after L-T 4 withdrawal.

Serum TSH level change during ZGrhTSH and L-T 4 withdrawal phases
The changes of serum TSH had similar trends in all dose groups over time. For all 64 patients, the average level was 0.11 ± 0.12 mU/L at baseline, then reached to the maximum level of 122.11 ± 42.44 mU/L based on 24-h interval with feasible clinical monitoring sampling time and dropped to near baseline level (0.25 ± 0.28 mU/L) on 14th day after the last dose of ZGrhTSH. During L-T 4 withdrawal phase, serum TSH level reached to the peak (82.20 ± 31.37 mU/L) with a median of 23 days, and maintain stable thereafter, which was significantly lower than that on 24 h after last dose of ZGrhTSH (P < 0.001) (Fig. 7).

Pharmacokinetics of ZGrhTSH
The mean peak TSH concentrations for 0.9 mg × 1 day, 0.9 mg × 2 day, 1.8 mg × 1 day, and 1.8 mg × 2 day groups were 7.49 ± 1.64, 11.94 ± 5.37, 24.09 ± 9.14, and 26.42 ± 9.54 ng/mL, respectively, with approximately 12 h for single dose groups and approximately 8-9 h for double doses groups after the last dose of ZGrhTSH (Fig. 8). The half-life of ZGrhTSH clearance from circulation was similar among four regimens with about 20 h after last dose of ZGrhTSH.

Discussion
In this self-control phase I/II clinical study, a rapid rise of serum TSH is observed after ZGrhTSH administration, with a mean peak concentration much higher than that after an average of 23 days of L-T 4 withdrawal, indicating ZGrhTSH could effectively increase serum TSH level instead of the modality of THW. No one developed ZGrhTSH antibodies, suggesting the applicability of multiple ZGrhTSH administration.
The pharmacokinetics of ZGrhTSH were characterized by the durable elevation of TSH levels (> 30 mU/L) for at least 72 h after the last dose administration, indicated a relatively slow clearance which is quite similar as it reported in studies of Thyrogen® when comparing with the endogenous TSH [13]. Theoretically, the slow Fig. 2 Concordance of Dx-WBS in ZGrhTSH and THW phases. Dx-WBS, diagnostic. 131 I-whole body scan; ZGrhTSH, Zelgen recombinant human thyroidstimulating hormone; THW, thyroid hormone withdrawal clearance pharmacokinetics feature of rhTSH might be more favorable in aiding the adequate stimulation for RAI uptake and Tg secretion. Of note, serum TSH level could still maintain at about 80% of the peak concentration 24 h after the last dose with an average of 109.1 mU/L and 186.7 mU/L for 0.9 mg and 1.8 mg 2-day regimen, which allow an adequate TSH stimulation in terms of both duration and high concentration, and also well met clinical routine practice of TSH examination. Though we noticed that the blood concentration of ZGrhTSH increased in a dose-dependent manner, with TSH levels in 1.8 mg groups almost onefold higher than that in 0.9 mg groups, the current clinical need for TSH level is indicated as only 30 mU/L and above [15]. One of our prior studies showed that TSH level of 90-120 mU/L was enough in aiding RAI remnant ablation [16]. Hence, an average of 100 mU/L for 0.9 mg × 2 day regimen would be appropriate for both elevation of TSH, and also with a consideration of reducing the potential risk induced by higher TSH level. Thus, the 0.9 mg × 2 day regimen was selected for phase II and subsequent phase III study.
All 64 patients experienced both ZGrhTSH administration and L-T 4 withdrawal for TSH stimulation. Only 13 of the 64 patients suffered ZGrhTSH-related AEs, which were generally mild and 11/13 recovered within 1-2 days. The most common AE was headache, with an incidence of 6.3%, and the incidence of other AEs was less than 2%. There was no grade 2 and above ZGrhTSH-related AEs, demonstrating the great safety and tolerance of ZGrhTSH. Meanwhile, the Billewicz Scales and POMS score of patients was remarkably higher during L-T 4 withdrawal phase than ZGrhTSH phase, indicating the more hypothyroidism symptoms and dysphoric mood states after THW than ZGrhTSH administration and ZGrhTSH greatly improved the quality of life.
The efficacy of ZGrhTSH has been well demonstrated in terms of promoting RAI uptake and Tg stimulation in this study. A high consistency rate (89.1%) of Dx-WBS findings between ZGrhTSH or L-T 4 withdrawal phases was observed, suggesting ZGrhTSH could effectively enhance NIS expression and corresponding function, thereby improve the iodine uptake. Furthermore, ZGrhTSH seems to be more advantageous in the detection of residual thyroid tissue, with the fact that 5 patients showed positive thyroidal 131 I uptake in ZGrhTSH phase while negative in L-T 4 withdrawal phase. Similar phenomenon was also found in Thyrogen® [13]. With regard to stimulate Tg secretion, the modality of L-T 4 withdrawal appears to be more efficient than ZGrhTSH. Serum Tg levels after L-T 4 withdrawal increased almost twofold to that after last dose of ZGrhTSH, which is similar to the findings of Thyrogen®. One reason is that the extended period of TSH stimulation following L-T 4 withdrawal lead to more adequate Tg secretion from thyroid follicular cells or DTC cells than an acute rise of TSH following rhTSH injection [17]. Another speculated reason is that the radiation effect of 131 I administration at the activity of 3 mCi during ZGrhTSH phase may exert partial ablation effect and cause the damage of follicular cells, which leads to the destructive release of Tg and manifested as higher level in L-T 4 withdrawal phase. The latter could be further evidenced by the mismatch of Dx-WBS finding among patients showed the positive thyroidal 131 I uptake during ZGrhTSH phase  while negative during L-T 4 withdrawal phase, while the diverse interpretation of such clinical manifestation indicates the need for further exploration and evidence [12,18].
In conclusion, ZGrhTSH was well tolerated and showed the comparable potential to increase TSH level, improve iodine uptake, and stimulate Tg secretion in DTC patients comparing with the L-T 4 withdrawal. Together with the demonstrated better quality of life, ZGrhTSH could be a safe and effective alternative in aiding postoperative evaluation and active surveillance in DTC patients.