Based on an increasing course of alpha‐fetoprotein (AFP), as well as findings obtained in dynamic CT,24 magnetic resonance imaging,25 (link), 26 (link) contrast enhanced ultrasonography with perflubutane (Sonazoid®, Daiichi Sankyo Co., Ltd., Tokyo, Japan) examinations,27 (link), 28 (link) and/or pathological findings, HCC was diagnosed. To evaluate tumor progression, we used Barcelona Clinic Liver Cancer (BCLC) stage29 and tumor node metastasis (TNM) stage, determined as previously reported in a study for TNM staging of HCC conducted by the Liver Cancer Study Group of Japan (LCSGJ) 6th edition30 (TNM‐LCSGJ).
Sonazoid
Manufactured by Daiichi Sankyo
Sourced in Japan
Sonazoid is an ultrasound contrast agent. It is a suspension of perfluorobutane-filled microbubbles.
Automatically generated - may contain errors
Lab products found in correlation
Gf uct260, by Olympus (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Aplio xg, by Toshiba (2 mentions)
Aplio 400, by Canon (2 mentions)
Prosound ssd α 10, by Aloka (2 mentions)
Prosound f75, by Hitachi (1 mentions)
Prosound α10, by Hitachi (1 mentions)
Eu me2, by Olympus (1 mentions)
Sodium citrate tribasic, by Merck Group (1 mentions)
Penicillin streptomycin amphotericin b, by Fujifilm (1 mentions)
Hydroquinone, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Gold 3 chloride haucl4, by Merck Group (1 mentions)
Bis p sulfonatophenyl phenylphosphine dihydrate dipotassium salt bspp, by Merck Group (1 mentions)
Tween 20, by Tokyo Chemical Industry (1 mentions)
Potassium carbonate, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Tannic acid, by Merck Group (1 mentions)
Prosound f75, by Fujifilm (1 mentions)
Cisplatin, by Fujifilm (1 mentions)
30 protocols using sonazoid
1
Comprehensive Hepatocellular Carcinoma Evaluation
2
Contrast-Enhanced Endoscopic Ultrasound Imaging of Pancreatic Tumors
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CE-EUS was performed using a convex-type echoendoscope (GF-UCT260; Olympus, Tokyo, Japan) and ultrasound processors (ProSound F75 or ProSound α10; Hitachi-Aloka Medical, Tokyo, Japan, or EU-ME2; Olympus, Tokyo, Japan). The setting of the ultrasound processor was changed to dual imaging with the extended pure harmonic detection mode on the right side and standard B-mode on the left side (Figure 1 ). The gain and range levels were adjusted so that the entire tumor could be visualized with appropriate contrast. Microbubbles were composed of perfluorobutane, created to a median diameter of 2–3 μm (Sonazoid: Daiichi-Sankyo, Tokyo, Japan; GE Healthcare, Milwaukee, WI, USA), and were used as the ultrasound contrast agent. A video image of the screen was recorded while observing the pancreatic tumor for 90 seconds after intravenous administration of the contrast agent.
3
Hepatocellular Carcinoma Prognosis Analysis
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HCC was diagnosed based on findings showing an increasing course of alpha‐fetoprotein (AFP), as well as results obtained with dynamic CT17 or MRI,18 , 19 contrast enhanced ultrasonography with perflubutane (Sonazoid®, Daiichi Sankyo Co., Ltd.),20 , 21 and/or pathological results. For evaluation of tumor progression, tumor node metastasis (TNM) staging, determined based on a previous study of TNM staging for HCC conducted by the Liver Cancer Study Group of Japan (LCSGJ), 6th edition22 (TNM‐LCSGJ), was utilized. Surgical resection and radiofrequency ablation (RFA) were each defined as curative treatment.
To analyze the prognostic impact of BTR for overall survival (OS) in Analysis 2, eight clinical factors [gender, age, AFP, Child‐Pugh class, basal hepatic disease etiology, curative treatment method, Eastern Cooperative Oncology Group performance status (ECOG PS), up to 7 criteria score (sum of maximum tumor size and tumor number)] were used for calculating inverse probability weighting (IPW) and propensity score.23 This study was based on the Guidelines for Clinical Research issued by the Ministry of Health and Welfare of Japan and all procedures were performed in accordance with the declaration of Helsinki. Informed consent was obtained in the form of opt‐out. Those who rejected were excluded from this study.
To analyze the prognostic impact of BTR for overall survival (OS) in Analysis 2, eight clinical factors [gender, age, AFP, Child‐Pugh class, basal hepatic disease etiology, curative treatment method, Eastern Cooperative Oncology Group performance status (ECOG PS), up to 7 criteria score (sum of maximum tumor size and tumor number)] were used for calculating inverse probability weighting (IPW) and propensity score.
4
Synthesis and Characterization of Gold Nanoparticles
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Gold (III) chloride (HAuCl4), sodium citrate tribasic, tannic acid, potassium carbonate, Bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP), hydroquinone, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 20 was purchased from Tokyo Chemical Industry (Tokyo, Japan). Thiol-terminated metoxypolyethylene glycol (PEG-SH) (Sunbright ME-050SH, MW = 5 kDa) was purchased from NOF Corp. (Tokyo, Japan). Sonazoid was purchased from Daiichi Sankyo Co. Ltd. (Tokyo, Japan). bEND.3 mouse brain endothelial cell was provided by the RIKEN cell bank. Penicillin–streptomycin-amphotericin B and 20% formalin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Fetal bovine serum (FBS) and ZO-1 rabbit polyclonal antibodies were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). TRITC-conjugated anti-rabbit IgG was purchased from Agilent Technologies Inc. (Santa Clara, CA, USA). DAPI was purchased from Dojindo (Kumamoto, Japan). A silver enhancement kit was purchased from BBI Solutions (Crumlin, UK).
5
Radiological Diagnosis and Staging of HCC
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HCC was diagnosed based on an increasing trend of alpha-fetoprotein (AFP), as well as typical findings obtained in dynamic CT10 (link), MRI11 (link),12 (link), and contrast enhanced ultrasonography (CEUS) with perflubutane (Sonazoid®, Daiichi Sankyo Co., Ltd., Tokyo, Japan) examinations13 (link),14 (link), and/or pathological findings. To evaluate tumor progression, Barcelona Clinic Liver Cancer (BCLC) stage15 (link) and tumor node metastasis (TNM) stage were used, and determined as previously reported in a study for TNM staging of HCC conducted by the Liver Cancer Study Group of Japan (LCSGJ) 6th edition (TNM-LCSGJ)16 .
6
Contrast-Enhanced EUS for Lymph Node Evaluation
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All patients were moderately sedated with midazolam and pentazocine during the EUS procedure. EUS was performed with a convex-type echoendoscope (GF-UCT260; Olympus, Tokyo, Japan) connected with US systems (ProSound F75 or Pro-Sound alpha 10; FUJIFILM, Tokyo, Japan). Before CE-EUS, the size, shape (round or oval), border (sharp or fuzzy), echogenicity (hyperechoic or hypoechoic), and echo texture (heterogeneous or homogeneous) of each lymph node was evaluated. With regard to CE-EUS, the mechanical index was set at 0.2, and the transmitting frequency was set at 5 MHz, respectively. The extended pure harmonic detection mode was used for CE-EUS. Sonazoid® (Daiichi-Sankyo, Tokyo, Japan, or GE Healthcare, Milwaukee, WI, USA) was injected as the contrast agent for CE-EUS. A measure of 0.015 mL/kg body weight of the contrast agent was injected through a peripheral vein by bolus injection. After injection of the contrast enhancer, the images acquired by CE-EUS were recorded for 90 s. Digital Imaging and Communications in Medicine (DICOM) format and Audio Video Interleaved (AVI) format were used for recording all the examinations. EUS-FNA was performed for all lymph nodes, followed by CE-EUS. Patients were observed in the endoscopy unit for at least 2 h after the procedure.
7
Contrast-Enhanced HFUS Imaging of Tumor-Bearing SiLNs
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Tumor-bearing SiLNs were visualized using a contrast-enhanced HFUS (CE-HFUS) imaging system with a 25 MHz center-frequency transducer (RMV-710B), on day 0 (the day before inoculation), day 6 and day 9. Sonazoid (Daiichi Sankyo, Tokyo, Japan), used as the microbubbles for imaging, was prepared according to the manufacturer’s instructions. Two-dimensional consecutive images of the tumor-bearing SiLN (slice thickness, 100 μm) were collected before and after injection of 100 μL Sonazoid into the tail vein. The differences in the video intensities between pre-injection and post-injection image frames were measured and areas exceeding a set threshold value were considered to be vessel extraction images. The images were reconstructed three-dimensionally to obtain the blood vessel density of each tumor-bearing SiLN as previously described [5 (link)]. The vessel densities of the tumor-bearing SiLNs were normalized to values obtained on day 0.
8
Evaluating Lymph Node Vascularity and Pressure
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After the measurement of LN volume, 100 μL microbubbles (Sonazoid; Daiichi Sankyo) was injected through the tail vein to detect the intranodal blood vessels. One minute after injection, 200 images at the maximal cross‐sectional area were acquired using HF‐US in M‐mode and VEVO 770 software. The data were analyzed according to our newly developed method,23 and an image of the intranodal blood vessels was constructed. Blood vessel density was defined as the ratio of the blood vessel area to the LN area expressed as a percentage. After the intranodal blood vessel density measurements had been made, the INP of the SiLN was recorded using the method described in our previous study.18 A schematic diagram of the experimental procedure is shown in Figure S1 A. The INP was measured using a 31‐G side‐hole needle (Lot No. 15113; Ito) (Figure S1 B). Data were recorded for 5 minutes.
9
Cisplatin-Sonazoid Microbubble Preparation
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Cisplatin (Wako Pure Chemical Industries Ltd., Osaka, Japan) was dissolved in saline at a concentration of 1 mg/mL (stock solution). An US contrast agent (Sonazoid™; DaiichiSankyo, Tokyo, Japan) was used as microbubbles. The contrast agent is comprised of a phospholipid shell encapsulating perfluorobutane gas. Sonazoid™ was reconstituted in 2 mL of sterilized distilled water according to the manufacturer’s instructions, which contains 1.2 x 109 microbubbles/mL with an average diameter of 3.2 μm [19 (link),20 (link)].
10
Hepatocellular Carcinoma Diagnosis Protocol
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HCC was diagnosed based on an increasing course of alpha‐fetoprotein (AFP), as well as dynamic CT,15 magnetic resonance imaging (MRI),16 , 17 contrast enhanced ultrasonography with perflubutane (Sonazoid®, Daiichi Sankyo Co., Ltd. Tokyo, Japan),18 , 19 and/or pathological findings. Tumor node metastasis (TNM) stage proposed by the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) was used for evaluation of tumor progression, as well as that previously reported in studies for staging of HCC conducted by the Liver Cancer Study Group of Japan, 6th edition (LCSGJ 6th).20
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