The value of dynamic contrast enhanced ultrasound (DCE-US) in monitoring treatment effect of high-intensity focused ultrasound (HIFU) in locally advanced pancreatic cancer (LAPC)

Abstract

PURPOSE:

To evaluate the feasibility of dynamic contrast enhanced ultrasound (DCE-US) in predicting treatment response of high-intensity focused ultrasound (HIFU) in patients with locally advanced pancreatic cancer (LAPC) lesions.

PATIENTS AND METHODS:

In this prospective study, 10 patients with pathologically confirmed LAPC lesions (7 men, 3 women; average age, 61.13±5.80 years) were prospectively enrolled. All patients received HIFU treatment with peak intensity at 12000 W/cm². Contrast enhanced ultrasound (CEUS) was performed with an ACUSON Oxana 2 ultrasound equipment and a 6 C-1 transducer (1–6 Hz). A dose of 2.4 ml SonoVue was injected for each examination. Time intensity curves (TICs) were generated and quantitative analyses were performed by SonoLiver software. B mode ultrasound (BMUS) features, CEUS enhancement patterns, TICs, CEUS quantitative parameters and serum carcinoma antigen 19-9 (CA19-9) levels were compared before and 4 weeks after HIFU treatment. Statistical analyses were performed with SPSS Version 20.0 and GraphPad Prism 5.

RESULTS:

While comparing before and after HIFU, no significant difference was obtained on mean size of lesion, BMUS or CEUS features. After HIFU treatment, TICs showed decreased and delayed enhancement. Among all CEUS quantitative parameters, significant decrease could be found in maximum intensity (MI) (60.66±23.95% vs 41.31±26.74%) and mean transit time (mTT) (76.66±47.61 s vs 38.42±28.35 s). CA19-9 level decreased significantly after HIFU (2747.92±4237.41 U/ml vs 715.08±1773.90 U/ml) (P = 0.05).

CONCLUSION:

DCE-US combining with quantitative analysis might be a useful imaging method for early treatment response evaluation of HIFU in LAPC lesions.

Keywords

Dynamic contrast enhanced ultrasound (DCE-US)locally advanced pancreatic cancer (LAPC)high-intensity focused ultrasound (HIFU)therapeutic response blood perfusion

1 Introduction

Pancreatic cancer is one of the leading courses of cancer-related death, with an overall 5-year survival rate as 9% according to annual reports of American Cancer Society in 2020 [1]. Surgical resection represents the only curative treatment for pancreatic cancers [2]. Although diagnostic accuracy for pancreatic cancer is up to 92.4%, more than 80% patients are not amenable for surgical resection due to metastases or locally advanced lesions when first diagnosed [1 –3]. Locally advanced pancreatic cancer (LAPC) is defined as lesions not suitable for surgical resection due to cancer cells’ invasion to nearby major vessels, such as the superior mesenteric artery, celiac axis and abdominal aorta, with or without the existence of distant metastasis [2]. A large proportion of patients who are not candidates for surgical operation may accept palliative therapies, including chemotherapy, radiotherapy, targeted therapy or ablation therapy [2, 4].

High intensity focused ultrasound (HIFU) is a non-thermal ablative modality with promising therapeutic effect and low mortality [5]. It has been widely used for local tumor control in various solid tumors [6 –9]. Comparing with other local palliative treatment, HIFU has unique advantages including convenient, noninvasive and less pain. It was reported to be an optimal alternative for unresectable pancreatic cancer and end stage cancer patients [10, 11].

Early and accurate evaluation of post-treatment response plays an important role in clinical decision-making process. Microvascular perfusion of the tumor is reported to be exactly correlated with prognosis of LAPC [12 –16]. Currently, computed tomography (CT) was recommended as the most widely available and reproducible imaging method to evaluate treatment response of tumors, however, it has potential risk of exposure to radiation [12]. Magnetic resonance image (MRI) is also widely accepted in post treatment evaluation, but its high-cost and long examination time are limitations for clinical application [16]. Currently the modified response evaluation criteria in solid tumors (m-RECIST) based on CT/MRI mainly focuses on change of tumor numbers, size of lesions and number of pathological lymph nodes [12]. Neither CT nor MRI is sensitive to demonstrate early microvascular perfusion changes of tumor after HIFU treatment.

The sulphur hexafluride microbubbles of contrast enhanced ultrasound (CEUS) is pure blood pool agent which stays strictly intravascular [17]. CEUS offers a real-time and noninvasive evaluation of microvascular perfusion of tumors [12]. Besides diagnostic purpose, intraoperative CEUS is helpful to optimize therapeutic procedures of tumors as well [18]. Dynamic contrast enhanced ultrasound (DCE-US) could provide further quantitative analysis results based on wash-in and wash-out dynamic process of CEUS. Previously, it was reported to be effectively used in diagnosis and follow up of radiotherapy or chemotherapy in liver [19, 20], pancreas [21] and renal tumors [22]. Time intensity curve (TIC) and relative quantitative parameters were proved to reflect the microvascular perfusion of lesions [23].

However, up till now, little is known about the value of DCE-US in monitoring the treatment response of HIFU in pancreatic tumors. The purpose of this prospective study is to investigate the capability of DCE-US with quantitative analysis in monitoring the early treatment response of HIFU in pancreatic malignant tumors.

2 Patients & methods

This prospective study was approved by the ethical board of our institution. Written informed consents for DCE-US examination were signed by all the patients.

2.1 Patients

From September, 2018 to July, 2020, LAPC patients received HIFU treatment and followed up by CEUS examinations were prospectively included. The inclusion criteria were: (1) Pancreatic cancers were confirmed by biopsy and histopathological results; (2) Patients were confirmed with LAPC by CT or MRI imaging results; (3) Patients were scheduled to underwent HIFU treatment by a multi-disciplinary team of pancreatic tumors; (4) Patients planned to accept HIFU treatment; (5) CEUS examinations were performed before and 4 weeks after HIFU treatment.

The exclusion criteria included: (1) Patients with poor cardio-pulmonary function, severe tendency of haemorrhage or severe jaundice; (2) Patients could not cooperate well during CEUS examinations; (3) Patients without complete DICOM cines for 2 minutes of CEUS; (4) Pancreatic lesion was invisible on BMUS.

2.2 Research protocol

First, patient’s clinical data were recorded, including age, sex and serum carcinoma antigen-19-9 (CA19-9) level. All ultrasound data were recorded in JPEG and DICOM format. TICs were generated and quantitative analysis were created with SonoLiver 1.0 software (TomTec Imaging Systems, Munich, Germany). BMUS features, CEUS features, TICs, CEUS quantitative parameters and serum CA19-9 levels were compared before and 4 weeks after the HIFU treatment Fig. 1.

Fig. 1

Research protocol. LAPC: locally advanced pancreatic cancer; HIFU: high-intensity focused ultrasound; BMUS: B-mode ultrasound; CEUS: contrast enhanced ultrasound; CT: computed tomography; MRI: magnetic resonance image; TIC: time intensity curve

2.3 Ultrasound examinations

All ultrasound examinations were performed by an ACUSON Oxana 2 equipment (Siemens Medical Solutions, Munich, Germany) with a 6 C-1 transducer (1–6 MHz). BMUS features including lesions’ location, size, echogenicity, shape, dilation of main pancreatic duct and color flow signals of LAPC lesions were observed and recorded.

After the injection of 2.4 ml SonVeu (SonoVue, Bracco Group, Milan, Italy), a 2 minutes DICOM clip were recorded and stored for further dynamic analyses. The CEUS enhancement patterns and enhancement degrees were recorded and analyzed according to current WFUMB guideline [17]. All the examinations were performed by 2 radiologists with more than 10-year experience in CEUS.

2.4 Creation of time intensity curves (TICs)

Two regions of interest (ROIs) were placed inside tumor and in the surrounding pancreatic parenchyma accordingly. ROIs were set with the same size, at the same depth, and avoiding major vessels and necrosis area. TICs of CEUS were then automatically generated. The shape and peak enhancement of TICs were observed and recorded.

2.5 Curve fitting and quantitative analysis

When quality of fit (QOF)>75%, the quantitative values were considered to be valid. CEUS quantitative perfusion parameters were then generated, including maximum intensity (MI) - the difference between the maximum and minimum enhancement of TIC, rise time (RT) - the time from the injection of sulphur hexafluride microbubbles to the start of enhancement, time to peak (TTP) - the time from zero intensity to maximum intensity, mean transit time (mTT) - the mean time that bubbles pass through the ROI.

2.6 Algorithm of HIFU treatment

Extracorporeal HIFU ablation was performed with the HIFUNIT9000 (Aishen Technology Development Limited Corporation, Shanghai, China). Patients lied on supine position. The local treatment temperature was monitored under real time ultrasound guidance. Therapeutic ultrasonic beam was generated by HIFU transducer, with a focal area of 3×3×8 mm. The HIFU treatment parameters included: acoustic intensity 12000 W/cm²; electrical power 60% – 100% which was adjusted depending on patients’ tolerance to thermo; ultrasonic transmission time at 200 ms and interval time at 400 ms. The tumor ablation procedure was repeated until the whole targeted regions were completely ablated. Each procedure lasted approximately 30 minutes. The standard algorithm of HIFU for each patient contained 5 sessions in 4 weeks.

2.7 Statistical analysis

Descriptive statistics were calculated for patients’ age, sex, tumor location, diagnostic method and existence of main pancreatic ductal dilation. A Kolmogorov-Smirnov test was performed on continuous data to determine the normal distributions. For normally distributed data, a paired-sample two-sided t test was used to determine whether significant differences existed in CEUS perfusion parameters between pre- and post- HIFU treatment. For non-normally distributed data, a Wilconox test was performed. All statistical tests were analyzed with SPSS version 20 (IBM, USA, New York) and GraphPad Prism 5 (GraphPad Software, California, USA). A P value < 0.05 was considered to be statistically significant.

3 Results

3.1 Clinical features

From September, 2018 to July, 2020, a total of 10 patients with 10 single LAPC lesions were included. The baseline features of the 10 patients were showed in Table 1. Four weeks after HIFU treatment, serum CA19-9 level decreased significantly (2747.92±4237.41 U/ml vs 715.08±1773.90 U/ml, P = 0.05).

Table 1
Baseline characteristics of patients

Characteristic LAPC (n = 10)

Age (years)

Mean±SD 61.2±5.3

Range 55 –70

Female/male 3/7

Size (mm) 53.4±17.7

Location of lesion

Head 4

Body 5

Tail 1

Main pancreatic duct dilation 3

Mean diameter of pancreatic duct (mm) 4

Histological confirmation

Biopsy during surgery 2

Percutaneous biopsy 8

Characteristic	LAPC (n = 10)
Age (years)
Mean±SD	61.2±5.3
Range	55 –70
Female/male	3/7
Size (mm)	53.4±17.7
Location of lesion
Head	4
Body	5
Tail	1
Main pancreatic duct dilation	3
Mean diameter of pancreatic duct (mm)	4
Histological confirmation
Biopsy during surgery	2
Percutaneous biopsy	8

LAPC: locally advanced pancreatic cancer

All 10 patients accepted HIFU treatment, no complications such as infection, thrombosis or necrosis of pancreas was observed.

3.2 B-mode ultrasound features

Before and after HIFU treatment, all LAPC lesions were heterogeneous hypoechoic solid mass with irregular shape and unclear margin (P > 0.05) Fig. 2. Color blood flow signals could be detected in 2 lesions (P > 0.05). No significant change could be found in size of lesions before and after HIFU treatment (53.4±17.7 mm vs 48.8±16.4 mm, P > 0.05).

Fig. 2

A locally advanced pancreatic cancer (LAPC) lesion located on pancreatic head in a man aged 55 years old. Before high-intensity focused ultrasound (HIFU) treatment, the LAPC lesion was a heterogeneous hypoechoic solid lesion with irregular shape and unclear margin (a). After injection of SonVeu, the lesion showed heterogeneously hypoenhancement during arterial phase (b), venous (c) and late phase (d). After HIFU treatment, the LAPC lesion showed similar features on BMUS (e) and CEUS examination (f-h).

3.3 Contrast enhanced ultrasound features

Comparing to the surrounding pancreatic parenchyma, LAPC lesions showed heterogeneously hypoenhancement (n = 9) or isoenhancement (n = 1) during arterial phase of CEUS. During venous and late phases, all lesions showed hypoenhancement Fig. 2. After HIFU treatment, no changes could be found on CEUS enhancement features.

3.4 Dynamic contrast enhanced ultrasound TICs

CEUS perfusion images were then converted into TICs with SonoLiver software. Four weeks after HIFU treatment, TICs of LAPC showed decreased and delayed enhancement Fig. 3.

Fig. 3

Comparing between contrast enhanced ultrasound (CEUS) features before and after high-intensity focused ultrasound (HIFU) treatment, no significant changes could be detected (a, c). While comparing of time intensity curve (TIC) before and after HIFU treatment, the curve showed decreased and delayed enhancement in ablative area after HIFU treatment (b, d).

3.5 Curve fitting and CEUS quantitative indexes

When QOF > 75%, the quantitative values were considered to be valid. Taking the surrounding pancreatic parenchyma as the reference area with maximum intensity at 100%. Four quantitative indexes including MI, RT, TTP and mTT were created. While comparing quantitative parameters before and after HIFU treatment, significant decrease could be found in MI (60.66±23.95% vs 41.31±26.74%, P < 0.05) and mTT (76.66±47.61 s vs 38.42±28.35 s, P < 0.05). No significant changes could be found in RT (16.93±8.11 s vs 10.58±6.23 s, P > 0.05) or TTP (20.21±10.04 s vs 15.37±10.21 s, P > 0.05) (Table 2) Fig 4.

Table 2
Comparison of quantitative indexes before and after HIFU treatment

CEUS quantitative indexes Value P value

Before HIFU After HIFU

MI (%) 60.66±23.95 41.31±26.74 0.009^*

RT (sec) 16.93±8.11 10.58±6.23 0.067

TTP (sec) 20.21±10.04 15.37±10.21 0.299

mTT (sec) 76.66±47.61 38.42±28.35 0.014^*

CEUS quantitative indexes	Value	P value
MI (%)	60.66±23.95	41.31±26.74	0.009^*
RT (sec)	16.93±8.11	10.58±6.23	0.067
TTP (sec)	20.21±10.04	15.37±10.21	0.299
mTT (sec)	76.66±47.61	38.42±28.35	0.014^*

MI: maximum intensity; TTP: time to peak; RT: rise time; mTT: mean transit time; HIFU: high-intensity focused ultrasound; CEUS: contrast enhanced ultrasound. ^*: Significantly decreased

Fig. 4

Comparison of dynamic contrast enhanced ultrasound (DCE-US) quantitative indexes before and after high-intensity focused ultrasound (HIFU) treatment. Significant changes could be found in maximum intensity (MI) and mean transit time (mTT). DCE-US: dynamic contrast enhanced ultrasound; HIFU: high-intensity focused ultrasound; MI: maximum intensity; TTP: time to peak; RT, rise time: mTT, mean transit time; ^***: P < 0.05

4 Discussion

Traditionally, BMUS and CDFI are noninvasive imaging methods for follow up of treatment response during minimal invasive treatments. It has been reported that significant tumor volume reductions measured by BMUS was about 60% 3 months after HIFU treatment [6, 24]. However, neither BMUS nor CDFI is specific for early evaluation of treatment response. In our study, no difference was observed on BMUS or CDFI before and 4 weeks after HIFU treatment (P > 0.05).

Traditional ablative treatments for tumors were divided into thermal and non-thermal options. Thermal ablative treatment such as radiofrequency ablation is widely used to treat different solid tumors, but its application in pancreas is still limited [25]. Typical non-thermal ablative treatment included irreversible electroporation (IRE) and HIFU. IRE is now mainly used for the ablation of tumors near bile ducts and blood vessels [26]. However, complications after pancreatic IRE remain severe, including pancreatitis, abdominal pain, spontaneous pneumothorax and portal vein thrombosis [26]. IRE causes cell death by electric energy, without any damage to nearby vessels thus, it is different to evaluate microvascular perfusion changes after IRE treatment [27]. Different from IRE, during HIFU treatment, rapid temperature rise causes coagulative necrosis in targeted tumor area and thrombus in capillary vessels (<2 mm) [5 , 29]. Microvascular perfusion changes might be assessed in early tumor treatment response evaluation [21, 29]. CEUS is a widely available image method to make real time evaluation of microvascular perfusion. CEUS has unique advantages including easy to perform, noninvasiveness, real-time and high sensitivity [6]. In our study, all LAPC lesions showed typical hypoenhancement on CEUS. However, no significant changes could be observed visually on CEUS features before and 4 weeks after HIFU treatment.

TICs is a fitted curve and the shape of the curve including the ascending and descending slope, peak intensity and the time of peak intensity may reflect the real time microvascular perfusion of tumors [30]. It has been reported that DCE-US is efficient and sensitive in assessing treatment response to antiangiogenic therapy, targeted therapy and transhepatic arterial chemotherapy and embolization (TACE) for hepatocellular carcinoma [31 –34]. The recently updated WFUMB liver CEUS guidelines recommended the application of DCE-US for treatment response assessment in patients with liver malignant tumors [35]. Pathologically, typical changes could be observed in treated area after HIFU treatment, including damaged small blood vessels, abnormal blood spaces and scattered intravascular thrombi, which might leading to changes of microvascular perfusion of tumor area [29]. In our study, decrease of peak intensity of TICs could be found 4 weeks after HIFU treatment, which might relate to the changes of microvascular perfusion [21]. DCE-US and TICs are more sensitive than CEUS examination features observed by naked eyes.

By curve fitting, multiple CEUS quantitative parameters were created, including MI, RT, TTP and mTT. By analyzing the microvascular perfusion, DCE-US offers possibility to make quantitative evaluation of early response to HIFU therapy. Among all quantitative indexes, decrease of MI and mTT could be observed 4 weeks after HIFU treatment in our study. Previously, MI was proved to positively correlate with the intralesional microvascular density which is associated with tumor prognosis [13]. Similar changes of MI have been reported during follow up of treatment response after chemoradiotherapy [21] and TACE [19] in malignant liver tumors. The peak intensity in the center of liver tumor was observed significantly lower than the margins of liver tumor lesions after intervention treatment by Wiesinger I et al using VeuBox [19]. CEUS combining with VeuBox provides new quantitative parameters that might facilitate the discrimination of malignancy and benignity and the evaluation of treatment response [19, 36]. Time associated parameter mTT was correlated with the wash out time of sulphur hexafluride microbubbles and might be influenced by the procedure of its injection and the malformation of vascular [29, 37]. The decrease of mTT have been observed in our study, which was similar to previous study [19]. Thus, MI and mTT may be used as possible biomarkers for the early evaluation of treatment response in LAPC. All the CEUS examinations were performed by 2 experienced examiners. Our results were independent from the Siemens ACUSON Oxana 2 ultrasound equipment.

5 Limitations

The main limitation of our study was limited number of patients. Further studies with larger samples size should be performed to further verify the value of those CEUS quantitative analysis. In our future studies, we will use new software such as VueBox to make further analysis of perfusion in pancreatic tumors.

6 Conclusion

In conclusion, DCE-US combined with TICs and quantitative analysis provide real-time and sensitive assessment of the microvascular perfusion changes in LAPC tumors after HIFU treatment. MI and mTT may be useful biomarkers to predict treatment effect of HIFU in patient with LAPC.

Funding

Supported by Shanghai Municipal Science and Technology Medical Guidance Project (Grant No. 18411967200). Supported by Shanghai Municipal Health and Family Planning Commission Research Project (Grant No. 201840215).

References

Siegel

, Miller

, Jemal

. Cancer statistics, 2020. CA-Cancer J Clin. 2020;70(1) :7–30.

Kamisawa

, Wood

, Itoi

, Takaori

. Pancreatic cancer. Lancet. 2016;388(10039):73–85.

Xie

, Liu

, Li

. Quantitative shear wave elastography for noninvasive assessment of solid pancreatic masses. Clin Hemorheol Microcirc. 2020;74(2):179–87.

Pollom

, Koong

, Ko

. Treatment Approaches to Locally Advanced Pancreatic Adenocarcinoma. Hematol Oncol Clin North Am. 2015;29(4):741–59.

Izadifar

, Izadifar

, Chapman

, Babyn

. An Introduction to High Intensity Focused Ultrasound Systematic Review on Principles, Devices and Clinical Applications. 2020.

Marinova

, Huxold

, Henseler

, Mücke

, Conrad

, Rolke

, et al. Clinical Effectiveness and Potential Survival Benefit of US-Guided High-Intensity Focused Ultrasound Therapy in Patients with Advanced-Stage Pancreatic Cancer. Ultraschall Med. 2019;40(5):625–37.

Anzidei

, Marincola

, Bezzi

, Brachetti

, Nudo

, Cortesi

, et al. Magnetic resonance-guided high-intensity focused ultrasound treatment of locally advanced pancreatic adenocarcinoma: preliminary experience for pain palliation and local tumor control. Invest Radiol. 2014;49(12):759–65.

, Wang

, Zhu

, Chen

, Zou

, Bai

, et al. Feasibility of US-guided high-intensity focused ultrasound treatment in patients with advanced pancreatic cancer: initial experience. Radiology. 2005;236(3):1034–40.

, Liu

, Yu

. The effect of high intensity focused ultrasound on the treatment of liver cancer and patients’ immunity. Cancer Biomark. 2019;24(1):85–90.

10.

Wang

, Zhou

. Preoperative ultrasound ablation for borderline resectable pancreatic cancer: A report of 30 cases. Ultrason Sonochem. 2015;27:694–702.

11.

Vidal-Jove

, Perich

, Del Castillo

. Ultrasound Guided High Intensity Focused Ultrasound for malignant tumors: The Spanish experience of survival advantage in stage III and IV pancreatic cancer. Ultrason Sonochem. 2015;27:703–6.

12.

Eisenhauer

, Therasse

, Bogaerts

, Schwartz

, Sargent

, Ford

, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47.

13.

Dalah

, Tai

, Oshima

, Hall

, Erickson

, Li

. PET-based Treatment Response Assessment for Neoadjuvant Chemoradiation in Pancreatic Adenocarcinoma: An Exploratory Study. Transl Oncol. 2018;11(5):1104–9.

14.

Tanaka

, Kamata

, Takenaka

, Yoshikawa

, Ishikawa

, Okamoto

, et al. Contrast-enhanced harmonic endoscopic ultrasonography for evaluating the response to chemotherapy in pancreatic cancer. Dig Liver Dis. 2019;51(8):1130–4.

15.

Kim

, Shin

, Park

, Jeong

, Jeon

, Choi

, et al. Vascular enhancement pattern of mass in computed tomography may predict chemo-responsiveness in advanced pancreatic cancer. Pancreatology. 2017;17(1):103–8.

16.

Kim

, Morgan

, Schexnailder

, Navari

, Williams

, Bart Rose

, et al. Accurate Therapeutic Response Assessment of Pancreatic Ductal Adenocarcinoma Using Quantitative Dynamic Contrast-Enhanced Magnetic Resonance Imaging With a Point-of-Care Perfusion Phantom: A Pilot Study. Invest Radiol. 2019;54(1):16–22.

17.

Sidhu

, Cantisani

, Dietrich

, Gilja

, Saftoiu

, Bartels

, et al. The EFSUMB Guidelines and Recommendations for the Clinical Practice of Contrast-Enhanced Ultrasound (CEUS) in Non-Hepatic Applications: Update (Long Version). Ultraschall Med. 2018;39(2):e2–e44.

18.

da Silva

NPB

, Beyer

, Hottenrott

, Hackl

, Schlitt

, Stroszczynski

, et al. Efficiency of contrast enhanced ultrasound for immediate assessment of ablation status after intraoperative radiofrequency ablation of hepatic malignancies. Clinical Hemorheology and Microcirculation. 2017;66(4):357–68.

19.

Wiesinger

, Wiggermann

, Zausig

, Beyer

, Salzberger

, Stroszczynski

, et al. Percutaneous Treatment of Malignant Liver Lesions: Evaluation of Success Using Contrast- Enhanced Ultrasound (CEUS) and Perfusion Software. Ultraschall Med. 2018;39(4):440–7.

20.

Wildner

, Schellhaas

, Strack

, Goertz

, Pfeifer

, Fiessler

, et al. Differentiation of malignant liver tumors by software-based perfusion quantification with dynamic contrast-enhanced ultrasound (DCEUS). Clin Hemorheol Microcirc. 2019;71(1):39–51.

21.

Zhang

, Wu

, Yang

, Qiu

, Yu

, Dong

, et al. Clinical application of dynamic contrast enhanced ultrasound in monitoring the treatment response of chemoradiotherapy of pancreatic ductal adenocarcinoma. Clin Hemorheol Microcirc. 2020.

22.

Dong

, Wang

, Lin

, Fan

, Mao

. Assessment of renal perfusion with contrast-enhanced ultrasound: Preliminary results in early diabetic nephropathies. Clin Hemorheol Microcirc. 2016;62(3):229–38.

23.

Dietrich

, Averkiou

, Correas

, Lassau

, Leen

, Piscaglia

. An EFSUMB introduction into Dynamic Contrast-Enhanced Ultrasound (DCE-US) for quantification of tumour perfusion. Ultraschall Med. 2012;33(4):344–51.

24.

Strunk

, Henseler

, Rauch

, Mücke

, Kukuk

, Cuhls

, et al. Clinical Use of High-Intensity Focused Ultrasound (HIFU) for Tumor and Pain Reduction in Advanced Pancreatic Cancer. Rofo. 2016;188(7):662–70.

25.

Scopelliti

, Pea

, Conigliaro

, Butturini

, Frigerio

, Regi

, et al. Technique, safety, and feasibility of EUS-guided radiofrequency ablation in unresectable pancreatic cancer. Surg Endosc. 2018;32(9):4022–8.

26.

Scheffer

, Nielsen

, de Jong

, van Tilborg

, Vieveen

, Bouwman

, et al. Irreversible electroporation for nonthermal tumor ablation in the clinical setting: a systematic review of safety and efficacy. J Vasc Interv Radiol. 2014;25(7):997–1011; quiz

27.

Dollinger

, Muller-Wille

, Zeman

, Haimerl

, Niessen

, Beyer

, et al. Irreversible Electroporation of Malignant Hepatic Tumors–Alterations in Venous Structures at Subacute Follow-Up and Evolution at Mid-Term Follow-Up. PLoS One. 2015;10(8):e0135773.

28.

She

, Cheung

, Jenkins

, Irwin

. Clinical applications of high-intensity focused ultrasound. Hong Kong Med J. 2016;22(4):382–92.

29.

, Chen

, Bai

, Zou

, Wang

, Zhu

, et al. Pathological changes in human malignant carcinoma treated with high-intensity focused ultrasound. Ultrasound Med Biol. 2001;27(8):1099–106.

30.

Piscaglia

, Nolsoe

, Dietrich

, Cosgrove

, Gilja

, Bachmann Nielsen

, et al. The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall Med. 2012;33(1):33–59.

31.

Maconi

, Nylund

, Ripolles

, Calabrese

, Dirks

, Dietrich

, et al. EFSUMB Recommendations and Clinical Guidelines for Intestinal Ultrasound (GIUS) in Inflammatory Bowel Diseases. Ultraschall Med. 2018;39(3):304–17.

32.

Frampas

, Lassau

, Zappa

, Vullierme

, Koscielny

, Vilgrain

. Advanced Hepatocellular Carcinoma: early evaluation of response to targeted therapy and prognostic value of Perfusion CT and Dynamic Contrast Enhanced-Ultrasound. Preliminary results. Eur J Radiol. 2013;82(5):e205–11.

33.

Williams

, Hudson

, Lloyd

, Sureshkumar

, Lueck

, Milot

, et al. Dynamic microbubble contrast-enhanced US to measure tumor response to targeted therapy: a proposed clinical protocol with results from renal cell carcinoma patients receiving antiangiogenic therapy. Radiology. 2011;260(2):581–90.

34.

Tian

, Wang

. Quantitative analysis of microcirculation blood perfusion in patients with hepatocellular carcinoma before and after transcatheter arterial chemoembolisation using contrast-enhanced ultrasound. Eur J Cancer. 2016;68:82–9.

35.

Dietrich

, Nolsoe

, Barr

, Berzigotti

, Burns

, Cantisani

, et al. Guidelines and Good Clinical Practice Recommendations for Contrast-Enhanced Ultrasound (CEUS) in the Liver-Update 2020 WFUMB in Cooperation with EFSUMB, AFSUMB, AIUM, and FLAUS. Ultrasound Med Biol. 2020;46(10):2579–604.

36.

Wiesinger

, Kroiss

, Zausig

, Hornung

, Zeman

, Stroszczynski

, et al. Analysis of arterial dynamic micro-vascularization with contrast-enhanced ultrasound (CEUS) in thyroid lesions using external perfusion software: First results. Clin Hemorheol Microcirc. 2016;64(4):747–55.

37.

Wiesinger

, Beyer

, Zausig

, Verloh

, Wiggermann

, Stroszczynski

, et al. Evaluation of integrated color-coded perfusion analysis for contrast-enhanced ultrasound (CEUS) after percutaneous interventions for malignant liver lesions: First results. Clinical Hemorheology and Microcirculation. 2018;69(1-2):59–67.