1 Introduction
The pulmonary metastasis treatment from differentiated thyroid carcinoma (DTC) is an increasingly challenging issue in clinic oncology. Over the past years, the large dose 131I internal irradiation was used for such metastases in clinical practice, but many pulmonary metastatic foci failed to uptake 131I due to leading to DTC relapse[1]. Thus, these patients need effective adjuvant therapeutic modalities.
In the past decade, the radioimmunotherapy (RIT) utilized the radiolabeled monoclonal antibodies (MABs) to target specific tumor-associated antigens, and was superior to the traditional chemotherapies due to its intrinsic low-toxicity and high-efficiency[2,3]. For immunotherapy against solid tumors, the iodine-131- labeled recombinant chimeric therapeutic monoclonal antibody (131I-chTNT) is a new targeted antibody agent against tumor cell nucleus protein has been developed[4,5]. Also, the 131I-chTNT was potential against various tumors in vitro and in vivo, including lung cancer, lymphoma, glioblastoma and colorectal carcinoma[6,7,8,9]. In this paper, the effects of 131I-chTNT against DTC is further studied, this agent is apply to the pulmonary metastases patients from DTC for the first time, The chest computer tomography (CT) was performed before and after treatment. Responses and safety were assessed during the treatment. The results show that the 131I-chTNT infusion was well tolerated with the 31.3% overall response, indicating that the most treatment-related adverse effects are mild transient and reversible.
2 Materials and methods
The protocol was approved by the institutional review board and independent ethic committees of China-Japan Union Hospital, Changchun, China. The written informed consent was obtained from all participants prior to the study commencement.
2.1 Patient eligibility
The 16 patients (10 males and 6 females) in the study have a mean age of 63.3±7.9 years and body weight of 52±10.8 kg, including 12 papillary carcinomas and 4 follicular carcinomas. All patients were non-uptaking 131I pulmonary metastases from DTC, and had no residual thyroid confirmed by the prior detections. No patient underwent radiotherapy or chemotherapy at least 30 days prior to the study entrance, and had abnormal findings in regular biological and biochemical assays for hemogram, hepatic and renal functions. The human anti-mouse antibody (HAMA) and the iodine allergy tests were performed before agent administrations.
2.2 Major apparatuses and drugs
The apparatuses in this study included SkyLight SPECT (single photon emission computerized tomography, Philips), RM-905a radioactivity counter (China Metrology Development Corp. Group), GC-2016 radioimmunoassay (RIA) γ counter (Xi'an Zhongjia Co), and TDL-5Z centrifuge (Toshiba). The 131I-chTNT in a clear primrose liquid state, which was purchased from Shanghai Meien Biotechnology Corp., Ltd., had a radioactivity of 370 MBq·mL–1, radiochemical purity of ≥ 95%, specific antibody binding activity of ≥ 50%, bacterial endotoxin of <10 EU·mL–1, and pH of 6.5‒7.5.
2.3 Drug administration
The 131I-chTNT (29.6±3.7 MBq·kg‒1 and 5 mL) was intravenously infused, and the RM-905a counter was applied to determine its radioactivity.
2.4 Treatment evaluation
CT was performed at 1 day before treatment as well as the 28 and 70 days after treatment. These treatments were evaluated by their response criteria in solid tumors (RECIST, version 1.1). The complete response (CR) shows that all target lesions disappeared for 28 days, the partial response (PR) meets that at least 30% diameters of target lesions decreased for 28 days, the overall response (OR) means the sum of both, progressive disease (PD) show that at least 20% diameters of target lesions increased, and the stable disease (SD) is no sufficient shrinkage or increase to qualify for PR or PD[10].
2.5 Safety evaluation
2.5.1 Conventional examination
To evaluate the status of haptic and renal functions, each patient underwent a physical examination and a batch of laboratory tests at 1 day before treatment as well as the 14, 42, and 70 days after treatment, including radiologic studies, electrocardiogram (ECG), and complete blood cell count (CBC), and biochemistry panels.
2.5.2 Micronucleus assay and karyotyping
Peripheral blood samples were collected at 1 day before treatment as well as the 7, 90, and 180 days after treatment. Peripheral blood lymphocytes were isolated as previously described[11].
Human lymphocytes are seeded in RPMI1640 medium (Sigma) containing the 5% heat-inactivated fetal bovine serum (FBS, Whittaker Bioproducts, Walkersville, MA), the 100 U/mL penicillin (Sigma), and the 100 g/mL streptomycin (Sigma). The cells were cultured in a 5% CO2 incubator until adding Cytochalasin-B 4 h after the first cell division. After the second division, the cultured cells (1 mL) were collected and centrifuged at 1000 rpm for 1 min, thus discarding the supernatant. Washed 3 times by culture medium supplemented with 2% FBS, the cells were swollen for 15 min in an hypotonic solution (wash medium : distilled water=1:4). The slides were prepared by using a cytospin, and the cells were located in holder equipped with a filter and a chamber and cytospinned for 7 min. After the slides were recovered, air-dried, fixed and stained in a Giemsa solution, the stained interphase cells were microscopically used for analyzing their micronuclei and chromosome mutation[12].
The scored micronuclei in those cells were complete nuclear division, and exposure to the agent. The 2000 metaphases cells were analyzed, and micronucleus rate of less than 4‰ was set as the normal cutoff.
The metaphases were karyotyped according to ISCN 1995 classification[13]. After analyzing 1000 cells, the chromosome changes and aberrations were determined, calculating the chromosome mutation rates. A total mutation rate of lower than 2.5% and a dicentromere plus centric ring rate of lower than 0.05% were considered as the normal cutoffs.
2.6 Statistical analysis
Data were expressed as mean ± SD, and analyzed by student’s t test using SPSS Package 10.0.
3 Results
3.1 Clinical efficacy
Among 16 patients, 2 (12.5%) was CR (Fig.1); and 3 (18.8%), PR; and 4 (25.0%), PD; and 7 (43.8%), SD. The OR was 31.3%.
3.2 Safety
All 16 patients well tolerated the 131I-chTNT infusion. During the study course, 7 patients (43.8%) reported treatment-related adverse events (AEs), including 1 nausea, 1 erythra, 2 leucopenias and 3 thrombocytopenias. Most treatment-related AEs were mild and transient. The hemogram status, haptic and renal functions were shown in Tables 1 and 2.
| Day 1 | Day 14 | Day 42 | Day 70 | |
|---|---|---|---|---|
| RBC (×1010/L) | 405.0±27.2 | 396.7±28.2 | 390.0±33.3 | 393.3±35.6 |
| Platelet (×109/L) | 201.3±42.6 | 203.1±42.6 | 187.3±30.2 | 192.5±34.5 |
| WBC (×109/L) | 7.9±1.6 | 7.4±3.0 | 7.8±1.4 | 7.4±1.6 |
| Neutrophils (×109/L) | 5.7±1.4 | 5.1±2.4 | 5.5±1.1 | 5.6±1.9 |
| Day - 1 | Day 14 | Day 42 | Day 70 | |
|---|---|---|---|---|
| ALT (U/L) | 17.0±10.0 | 17.9±11.9 | 16.1±9.5 | 17.1±9.9 |
| AST (U/L) | 22.5±11.1 | 22.7±15.1 | 21.5±6.4 | 20.9±10.9 |
| BUN (mmol/L) | 4.8±1.4 | 4.3±0.8 | 4.4±1.2 | 4.5±0.8 |
| Day 1 | Day 7 | Day 90 | Day 180 | |
|---|---|---|---|---|
| Lymphocyte micronucleus rate (‰) | 1.00±0.22 | 4.40±0.73 | 1.35±0.75 | 1.00±0.75 |
| Chromosome aberration rate (%) | 1.10±0.40 | 3.00±0.44 | 1.30±0.55 | 1.10±0.43 |
| DCB-CR rate (%) | 0.03±0.01 | 0.90±0.30 | 0.02±0.01 | 0.02±0.01 |
The lymphocyte micronucleus rate, the chromosome aberration rate and the double centromere body plus centromere ring (DCB-CR) rate of all patients experienced a significant rise 7 days post-treatment, returning to normal levels the 90 days apogee (Table 3 and Fig. 2).
4 Discussion
As one of the most common malignancies worldwide, the pulmonary metastases by DTC increase with mortality. To date, the mega-dose 131I therapy is considered as a golden modality in clinical treatment. However, due to its little or non-intake by pulmonary metastasis foci, about 20% patients still had the intractable residual foci after treating several rounds[14], and its high-dose and frequent administration would also induce radiation associated pathological damages to normal tissues and organs, thus increasing micronucleus scores and chromosome mutation rates[15]. Thus, it is urgent to induce the improved therapeutic regimens, such as tumor specific molecular targeted radioimmunotherapy for treating the pulmonary metastases.
Monoclonal antibodies, which represent an emerging strategy in clinical oncology, can mostly kill target tumor cells directly, and deliver cytotoxic toxins, drugs and radionuclides to tumor microenvironments [16,17,18,19]. In recent years, several antibodies in US and Europe have been approved for solid tumor treatment. Exposed by dead and dying cells at the center of solid tumors, the 131I-chTNT as target monoclonal antibody can design to bind the DNA histone complex. Its targeting mechanism is to bind the dying tumor cells and deliver the radioactive payload to the adjacent living tumor cells, then the tumor from the inside out are essentially destroyed by minimal radiation exposure to healthy tissue[20,21,22]. The 131I-chTNT has been approved in China for radioimmunotherapy of advanced lung cancer or glioblastoma[8,20]. These findings prompted us to investigate the safety and efficacy of the 131I-chTNT in the patients by DTC using the non-uptaking 131I pulmonary metastases.
This study shows that the 31.3%OR was consistent with previously published works, revealing the characteristic of the TNT approach. The adverse event of 131I-chTNT was considered as the bone marrow-related toxicity. Also, this study detected the 2 transient leucopenias and 3 transient thrombocytopenias, which is lower than patients receiving systemic chemotherapy. Another 2 patients, which experienced non-serious nausea and erythra, required no special treatment. These results show that 131I-chTNT was safe for clinic application.
The 131I-mediated radiotherapy for DTC with pulmonary metastases is currently the first therapeutic regimen, but it can damage normal tissues adjacent to tumor cells. Analyzing the radioactive risk of the treated patients was based on chromosomal mutation and micronucleus aberrations rates in peripheral blood lymphocytes, and associated with 131I administration in the range of 0.25 to 5 Gy. For chromosomal mutation, the double centromere body and centromere ring was a sensitive criterion.
Seven days after the 131I radiotherapy, the aberration rates of the lymphocyte micronucleus, chromosome and DCB-CR experienced a significant rise at the 7 days post-treatment, and recovered after 90 days, indicating that the radiopathological effects were reversible and associated with the 131I dose but not disease category. Our results were in line with existing reports, confirming that the micronucleus assay and karyotyping analysis as biological radiation dosimeters can be used to evaluate the clinical outcomes of DTC patients by 131I treatment. As reported[23,24], the transient chromosomal mutation of germ cells would be produced by damaging the peripheral blood lymphocyte chromosome. Therefore, a long-term follow-up is necessary for the young patients and the fertility should be considered only after recovering chromosome.
5 Conclusion
The non-uptaking 131I pulmonary metastases from DTC are difficult to treat in clinic oncology, but the 131I-chTNT appears to cure them. In this pilot clinical trial, the OR by 131I-chTNT mediated radioimmunotherapy was 31.3%. Most treatment- related AEs were mild, transient and reversible. The 131I-chTNT mediated radioimmunotherapy is expected as a new approach for the non-uptaking 131I pulmonary metastases. Further, the better clinical outcomes should obtained by studying more patients and long-term follow-ups.
