Introduction
Hepatocellular carcinoma (HCC) is the 7th most common malignancy and the fourth leading cause of cancer-related death worldwide.1,2 Approximately 10–40% of HCC patients suffer portal vein tumor thrombus (PVTT) complication at the time of diagnosis.3 Based on Barcelona Clinic Liver Cancer staging, sorafenib is the only evidence-based and recommended treatment option for patients with vascular invasion (stage C). However, several studies have shown only modest clinical efficiency when sorafenib monotherapy has been provided for HCC patients with PVTT.4–6 In recent studies, transarterial chemoembolization (TACE) combined with sorafenib or radiotherapy has shown survival benefits for HCC patients with PVTT.7–9 Although survival benefits have been provided through the adoption of these combinations, the survival duration of HCC patients with PVTT remains poor, which may be attributed to the obstructed blood flow in the PV. Owning to compromised PV blood supply, the interruption of hepatic arterial flow may result in severe ischemic liver damage.10 Therefore, HCC patients with PVTT are generally considered as contraindicated for TACE.
In China, HCC patients with PVTT often receive treatment through multiple methods, such as surgery, external beam radiotherapy (EBRT), chemotherapy, and iodine-125 (125I) seeds strand and stent implantation.11 A recent multicenter study proved that patients with limited PVTT could benefit from liver resection.12 However, patients with main PV tumor thrombus (MPVTT), can barely meet the requirements for liver resection. A systematic review and meta-analysis compared TACE plus EBRT with TACE alone for unresectable HCC.13 The results of the study indicated that the pooled median survival for TACE plus EBRT-treated patients was significantly better than of TACE alone-treated patients. However, the pooled analysis also showed that compared with TACE alone, TACE plus EBRT significantly increased the occurrence of gastroduodenal ulcers, as well as elevated levels of alanine aminotransferase and total bilirubin. Since normal hepatic tissues show poor tolerance to radiation, it is also impossible to increase the external irradiation dose provided for the treatment of PVTT. Endovascular brachytherapy (EVB) using 125I seeds strand implantation could offer an adequate dose of radiation to exert a tumor killing effect with subtle damage during irradiation, leading to the relative protection of healthy tissues.14 Animal models with vascular tumor thrombus have been established and employed to demonstrate the safety and efficacy of 125I seeds strand in exerting an antitumor effect.15,16 For patients with MPVTT, estimated overall survival was only 2 to 4 months.17 Our previous studies have also established that TACE combined with 125I seeds strand and portal vein stent implantation (PVSI) can prolong the overall survival of HCC patients with MPVTT.18–21 Moreover, this method has been widely used for HCC patients with MPVTT in many tertiary hospitals in China.
Due to the eccentric location of 125I seed strands in MPV, the uneven distribution of radiation in the PV was a potential deficiency that could compromise the efficacy of endovascular radiotherapy. Moreover, the usage of a higher dosage of 125I seeds strand was restricted due to the close association to the vascular wall. Hence, even after undergoing 125I seeds strand combined with stent MPV implantation, some patients may still suffer from re-occlusion of the PV owing to tumor thrombus progression. To further improve the antitumor effect of EVB using PV stent and 125I seeds strand insertion, we introduced an innovative EVB-Stent, which is a biconical stent with an 125I seed strand attached coaxially at the center of the stent. The aim of this study was to develop and evaluate the technical feasibility and safety of the EVB-Stent.
Methods
Assembling of the EVB-Stent
A self-expanding Nitinol stent (Hongpu Medical Device Corporation, Shanghai, China) with a mesh-like structure, was tapered into two rings at the proximal and distal ends. The stent used in this study was 18 mm × 60 mm in size. A 4-Fr sterile plastic tube (Boston Scientific Co., Marlborough, MA, USA) containing 125I seeds was inserted through the rings at both ends and the seeds strand was fixed to the stent using sterile sutures (Fig. 1). The stent, once equipped with the 125I seeds stand, was known as an EVB-Stent.
As described in our previous study,19 Model 6711 125I seeds (XinKe, Shanghai, China) were encapsulated in a 4-Fr sterile plastic tube (Boston Scientific Co.) to assemble the seeds strand. The radioactivity of each 125I seed was 25.9 MBq, with a half-life of 59.4 days. The principal photon emissions were 27.4–31.4 keV X-ray and 35.5 keV γ-ray. The half-value thickness of tissue for 125I seeds was 17 mm, and the initial dose rate was 7 cGy/h. The effective irradiating range was 20 mm. The number of 125I seeds was determined by the length of the plastic tube (L mm; N = L/4.5). In this study, the number of 125I seeds used in each strand was 14.
Animals
The animals selected for the experiment were six ordinary white pigs (weight 35–40 kg) provided by the Experimental Animal Center of our hospital. This study was approved by the institutional Animal Ethics Committee of our hospital. The animals were fasted for 12 h before operation. Intramuscular injection of xylazine hydrochloride (2–4 mg/kg) and diazepam (2 mg/kg) were used for sedation and anesthesia, respectively. Blood pressure, heart rate, and respiration rate were monitored during the procedure.
Interventional procedures
Under fluoroscopic and ultrasound guidance, the right intrahepatic PV was punctured using a 21G Chiba needle (Cook Medical Inc., Bloomington, IN, USA) and a 0.018-inch wire (Cook Medical Inc.) was inserted into the PV. A 6-F Neff Percutaneous Access set (Cook Medica Inc.) was introduced into the PV over the wire. Through the outer cannula of the Neff Percutaneous Access set, a 0.035-inch, 150 cm-long wire (Terumo, Tokyo, Japan) was manipulated across the MPV into the superior mesenteric vein, followed by the insertion of a 4-F pigtail catheter (Cook Medical Inc.). PV venography was performed via the pigtail catheter. After venography, the catheter was removed, and the outer cannula of the Neff Percutaneous Access set was replaced by a 7-Fr, 23 cm-long sheath (Cordis, Hialeah, FL, USA) over the wire. After 100 U/kg heparin (XinYi, Shanghai, China) was administered through the sheath, and the novel EVB-Stent was loaded into the sheath and pushed into the target main PV. The stent was deployed from the distal MPV into the proximal patent intrahepatic PV under fluoroscopic guidance. PV venography was repeated through the pigtail catheter to confirm the appropriate location and patency of the EVB-Stent. Finally, the transhepatic puncture track was occluded using n-butyl-2-cyanoacrylate (NBCA) (Compont, Beijing, China).
Radiation safety protection measures were followed by physicians during the 125I seed preparation and PV implantation. Lead protective gloves were provided to animal house workers during the follow-up period. At the end of the study, the 125I seeds were restored and processed properly.
Post-procedure management
After implantation, 100 U/kg heparin was subcutaneously administered once a day for 2 months. The animals were raised by conventional methods and monitored for the loss of appetite, vomiting, diarrhea, and weight loss after implantation. Blood samples were collected before implantation and at 1 week and 2 months after implantation, to determine liver function and blood toxicity. Single photon emission computed tomography (SPECT) combined with CT (SPECT/CT) scan was performed to evaluate the radiation distribution emitted by the 125I seeds strand immediately after implantation.21 Tissue accumulated absorbed doses 10 mm from the midpoint of the 125I seeds strand were theoretically calculated using 125I Radiation Field Distribution Calculation Software.16 CT scan was performed at 2 months post operation, and three-dimensional (3D) reconstructions were made to evaluate the patency of the stented PV. The contrast agent used was Ultravist Injection (300 mgI/mL). Intravenous access was through the femoral vein, and the dose of the contrast agent was 80 mL (2.0 mL/kg) on average, at an injection rate of 4 mL/s, delay time of arterial phase enhancement of 15 s, and a portal venous phase enhancement delay time of 50 s. After CT scans were performed, the animals were sacrificed through intravenous injection of potassium chloride and necropsied, immediately. The stented MPV and adjacent organ tissue were harvested and fixed in 10% formalin for pathological examination. Hematoxylin and eosin staining were performed, to evaluate adjunct liver tissue change and to evaluate thrombus organization within the stented PV and neointima coverage of the PV.
Statistical analysis
Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA) software. One-way analysis of variance was used to compare differences in hematological indices between the different time points. Measurement data are presented as mean±standard deviation. A two-sided p-value of <0.05 was considered statistically significant.
Results
The EVB-Stent was successfully implanted into the MPV of all six pigs without major complications. After stent placement, venography of the PV confirmed the proper location of the EVB-Stent in the MPV. All animals were in good condition without a loss of appetite, bleeding, weight loss, or death. Hematological indices taken throughout the examination period were summarized, and showed that no significant liver function deterioration or blood toxicity were detected (Table 1).
Table 1Comparison of laboratory test results pre-procedure and post-procedure
| Preoperative | One-week post-intervention | Two-month post-intervention | p |
|---|
| RBC as ×1012/L | 7.2±0.3 | 6.7±0.6 | 7.0±0.5 | 0.261 |
| HB as ×109/L | 116.8±9.4 | 107.8±13.9 | 111.3±10.3 | 0.352 |
| WBC as ×109/L | 11.7±2.2 | 13.9±2.2 | 11.0±1.6 | 0.074 |
| PLT as ×109/L | 514.2±82.8 | 507.1±77.4 | 513.8±67.3 | 0.984 |
| TB in μmol/L | 9.7±0.4 | 10.4±0.6 | 10.2±0.5 | 0.079 |
| DB in μmol/L | 6.5±0.4 | 7.2±0.5 | 7.0±0.5 | 0.053 |
| AST in U/L | 37.2±1.9 | 38.0±1.9 | 38.2±2.3 | 0.655 |
| ALT in U/L | 44.0±2.4 | 45.2±2.1 | 46.5±2.8 | 0.240 |
| ALB in g/L | 23.6±1.6 | 22.9±1.4 | 22.8±0.8 | 0.508 |
The SPECT/CT scan showed that the EVB-Stent had been correctly placed in the MPV without collapse, occlusion, or over-expansion. Radiation emitted by the 125I seeds strand was distributed symmetrically in the MPV, and presented as a cylindrical shape that completely covered the targeted MPV (Fig. 2). Based on the 125I Radiation Field Distribution Calculation software, the tissue accumulated absorbed dose was 31,822.11 mGy 10 mm transverse from the midpoint of the 125I seeds strand, with a half-life of 59.4 days (Fig. 3). The three-dimensional reconstruction CT image showed that the stented vessel remained in position and that the 125I seeds strand was tightly fixed to the center of the stent (Fig. 4). Additionally, no malposition, collapse, thrombosis, or stenosis occurred in the stented vessels, when observed using CT scans.
Necropsy and pathological examination results showed that none of the stents were covered or occluded at the two tapered ends by neointimal overgrowth in all six pigs (Fig. 5A). However, slight intimal hyperplasia and incomplete neointimal growth covered the tapered end near the hilar side of the stent in one pig (Fig. 5B). Hematoxylin-eosin staining revealed that the central grid of the EVB-Stent and even the densest part of the stent mesh were almost completely covered by neointimal growth, which resulted in the proper incorporation of the EVB-Stent, and its surface was smooth and free from tears, peelings, or injuries (Fig. 5C–D). No obvious abnormalities were found in nearby organs, including the liver parenchyma, duodenal wall, and pancreas, as examined through gross observations and pathological analysis (Supplementary Fig. 1).
Discussion
In this study, we demonstrated the technical feasibility of the percutaneous transhepatic placement of a novel radioactive stent, the EVB-Stent, in the MPV of a live porcine model. Furthermore, during the 2-month follow-up period, the stented PV remained in position in all cases, with minimal neointimal growth covering it. All these results present evidence that the EVB-Stent has the potential to be used for further clinical exploration.
PVTT is a prognostic factor for poorer overall survival among patients with HCC. Stent implantation promptly restores blood flow in the obstructed MPV and provides an opportunity for TACE to be applied for tumor lesions. However, in-stent stenosis can occur due to tumor growth and/or tumor thrombosis.22 EVB along with 125I seeds implantation, which can inhibit and prevent the progression of tumor thrombosis, has been provided an option to prolong stent patency.21,23 In a previous study, better overall response rate as well as a significantly favorable level of survival were observed in patients who received TACE combined with EVB with 125I seeds strand and stent placement.24–26 Numerous studies have demonstrated that stent devices that are combined with EVB exert therapeutic efficacy in PVTT, unresectable malignant esophageal and biliary obstruction.21,27,28 The eccentric distribution of the 125I seeds strand may cause the delivery of an insufficient radiation dose to the contralateral blood vessel wall, thus compromising the therapeutic efficacy. Moreover, the eccentric distribution of the 125I seeds in the MPV may limit the maximum dose of radiation delivered. Hence, we constructed a novel EVB-Stent to further improve the therapeutic efficacy of brachytherapy using 125I seeds for MPVTT by improving the radiation dosage delivered. The 125I seeds were presented as a sequential string coaxially at the center of the EVB-Stent, and the radiation emitted by the 125I seeds was homogeneously distributed and covered the entire stent canal. To examine the safety and feasibility of the novel radioactive stent, we conducted an animal study through the percutaneous transhepatic PV deployment of the EVB-Stent in a live porcine model.
Previous experimental studies have proven that transhepatic puncture and catheterization in a porcine model can be technically feasible without bleeding complications.29 In our study, ultrasound was used to assess intrahepatic vascular dissection and provided clear guidance for PV puncture. Moreover, owning to fine needle puncture, no bleeding or hematoma was observed after operation. A 7-Fr long sheath was used to create a transhepatic track and the EVB-Stent was released through the long sheath. The EVB-Stent could be easily introduced into the PV without any technical difficulties or challenges. Our previous experience showed us that the procedure used for the implantation of the 125I seeds strand combined stent was complicated to some extent.21 The outer cannula of the Neff set can be difficult to be delivered to the obstructed MPV and the release of the 125I seeds strand can be complicated if the stent has already been inserted. In this study, the 125I seeds were arranged linearly and continuously sealed into a 4-Fr sterile catheter to construct an 125I seeds strand. Then, the strand was fixed at the center of the stent. Compared with previously used techniques, the transhepatic MPV deployment of the EVB-Stent performed in this study was relatively simple. Hence, we demonstrated the technical feasibility of the transhepatic PV placement of an EVB-Stent into a pig model. However, its manipulative feasibility for HCC patients with MPVTT remains to be elucidated through clinical trials.
In this study, 14 125I seeds were encapsulated in a 4-Fr sterile plastic tube to create a seeds strand and then fixed at the center of the stent. SPECT/CT showed that the radiation emitted by the 125I seeds was uniformly distributed in the PV. The overall isodose of radiation can exert an elongating tumor killing effect and minimize the irradiation of the surrounding normal tissue. Moreover, the potentially irregular and asymmetric radiation emitted by the 125I seeds strand for the eccentric location in the PV can be avoided and the potential heterotopia of implanted 125I seeds strand can also be avoided. None of the animals were found to show signs of PV thrombosis after EVB-Stent placement during the 2-month follow-up period. On one hand, proper anticoagulant therapy with heparin is of great importance in preventing thrombosis. On the other hand, the anti-neointimal hyperplasia effect provided by the implantation of the 125I seeds strand30 allowed for a longer patency period to be achieved by the MPV stent. Moreover, the central location of the 125I seeds strand in the stented PV contributed to full conformal radiotherapy implementation on the MPV. Hence, incomplete neointimal hyperplasia coverage of the stent was observed but without thickening or resulting in PV stenosis after 2 months of deployment of the EVB-Stent.
Studies have established that persistent low-energy 125I irradiation therapy may keep tumor cells in the sensitive resting period, resulting in tumor cell apoptosis, which can induce epigenetic changes that reactivate silenced tumor suppressor genes, and damage to the DNA to kill cancer cells.14,31 For end-stage patients, the combination of EVB and PV stent implantation may not only provide a long period in which symptoms of portal hypertension are relieved, but may also suppress the progression of tumor thrombus. Furthermore, compared with external radiotherapy, brachytherapy using 125I seeds provides a high local dose close to the seeds and a steep fall in the dose provided to the surrounding tissues, which exerts an adequate tumor suppression effect with limited damage exerted onto the surrounding normal tissue.15 Since 125I seeds have a long half-life (59.4 days), a sustained level of radiation can be exerted to inhibit the replication of tumor cells and induce tumor cell apoptosis.32 In this study, the EVB-Stent, which was used as a novel endovascular brachytherapy stent kit, provided three obvious advantages compared with its current usage. First, it may induce tumor cell apoptosis more effectively and inhibit the progression of tumor thrombus, since the gamma rays are evenly distributed at the center of the obstructed PV. Second, the support provided by the stent resulted in the 125I seeds strand being firmly fixed at the center of the stent without displacement. Third, the central deployment of the radiative strand provided an opportunity to further improve the antitumor effect by increasing the dosage provided by the 125I seeds without obvious damage to the PV wall.
There are several limitations in our study. First and foremost, only six pigs were used, which may introduce a case-by-case bias. Second, the use of healthy animals without tumor thrombus or portal hypertension can barely reproduce the complex environment in a real human diseased vessel in which a stent must be implanted. Finally, long-term results of the efficacy and safety of the EVB-Stent transhepatic PV implantation, as well as its impact on the vessel wall over longer periods of time, are still pending. The creation of an ideal animal model with PVTT remains a daunting challenge but is urgently required to demonstrate the antitumor efficacy and safety of the transhepatic PV placement of the EVB-Stent.
Conclusions
Mid-term preclinical results demonstrated the feasibility and safety of the percutaneous transhepatic MPV implantation of a novel EVB-Stent into a live porcine model. The implantation of the stent did not produce thrombosis or stenosis. Further studies using large samples of animals with or without PVTT are needed to further assess the efficacy and safety of this innovative stent before it can be considered suitable for clinical application.
Supporting information
Supplementary Fig. 1
Histomorphology observations of adjacent organs and tissues.
(A) No swelling, necrosis, edema, atrophy, or inflammatory changes were observed in liver cells in the hilar area. (B) Pathological manifestations of the duodenal section showed that no duodenal papilla atrophy was observed. (C) No inflammation, swelling or necrosis of the pancreas was observed. (D) Lymphocyte infiltration was observed in certain enlarged lymph nodes around the portal vein.
(TIF)
Abbreviations
- 125I:
iodine-125
- EBRT:
external beam radiotherapy
- EVB:
endovascular brachytherapy
- HCC:
hepatocellular carcinoma
- MPV:
main portal vein
- MPVTT:
main portal vein tumor thrombus
- NBCA:
n-butyl-2-cyanoacrylate
- PVSI:
portal vein stent implantation
- PVTT:
portal vein tumor thrombus
- SPECT:
single-photon emission computed tomography
- TACE:
transarterial chemoembolization
Declarations
Acknowledgement
We give our thanks to Hongpu Medical Device Corporation for their help in building the self-expanding Nitinol stent. The authors are very grateful to Dr. Xianglin Hu of Fudan University Shanghai Cancer Center for his professional suggestions for the English writing.
Data sharing statement
All data are available upon reasonable request.
Funding
This study has received funding by the Shanghai Science Committee (16411968600), Clinical Research Special Fund from Zhongshan Hospital, Fudan University (2013SY060) and National Clinical Research Center for International Medicine.
Conflict of interest
The authors have no conflict of interests related to this publication.
Authors’ contributions
Study concept and design (JL, ZY), acquisition of data (ND, JM, YZ), analysis and interpretation of data (MY, WZ, ZZ), obtained funding (ZY), drafting of the manuscript (ND, JM), critical revision of the manuscript for important intellectual content (MY, WZ), administrative, technical, or material support, study supervision (JL).