The effect of high intensity focused ultrasound on the treatment of liver cancer and patients’ immunity

Abstract

OBJECTIVE:

To investigate the effects of high intensity focused ultrasound on liver function, tumor markers and survival rate of hepatocellular carcinoma patients.

METHODS

: Ninety six cases with primary liver cancer patients, consisting of 66 males and 30 females, were enrolled in this study and treated with high intensity focused ultrasound combined with stereotactic segmentation dose radiation, low frequency for 10 times, followed by analysis of KPS score of liver cancer, Child-Pugh, grading and staging of liver cancer, 3 months, 6 months, 1 year of clinical symptom remission rate, tumor markers, liver function, survival rate, as well as the change of immune related cytokines.

RESULTS:

Three months after high intensity focused ultrasound treatment, abdominal distension abdominal pain, jaundice symptoms, anorexia and ascites were significantly relieved compared with before treatment ( $P<$ 0.05). At 3 months after treatment, levels of AFP and CA199 were significantly reduced than before treatment ( $P<$ 0.05). Meanwhile, Child-Pugh classification score was significantly decreased at 3 months after treatment compared with before treatment, which was further decreased at 6 months after treatment than 3 months after treatment ( $P<$ 0.05). In addition, ALT, AST, AKP, propagated and TBIL level at 3 months after treatment displayed no differences to those before treatment but was significantly decreased at 6 months treatment ( $P<$ 0.05). Moreover, the late stages of liver cancer, the lower survival rate after treatment. Furthermore, the levels of NK, CD3, CD4, CD8 and CD4/CD8 cytokines were significantly increased at 3 months after treatment ( $P<$ 0.05), together with significantly increased levels of IFN-r and IL-2 and decreased levels of IL-4 and IL-10 ( $P<$ 0.05).

CONCLUSION:

High intensity focused ultrasound can effectively improve liver function, increase the survival rate and enhance immune function of patients with liver cancer.

Keywords

Ultrasonic treatment liver cancer curative effect immunizing

1. Introduction

If the primary liver cancer is diagnosed in early stage, especially in China, the best treatment is surgical excision of the cancer lesions. However, only 22% patients can be diagnosed early, and most of patients have already been in middle or late stage at the time when they are detected [1], so chemotherapy, radiotherapy or interventional therapy are usually applied for the treatment of these cases. However, the effects of such treatments are not satisfactory [2]. More and more studies have been performed to discover a better treatment approach that is most suitable for patients who have lost the surgical opportunities or who have refused to receive operations. High-intensity focused ultrasound (HIFU) has been gradually initiated and emphasized with several advantages [3]. HIFU can effectively enhance the radiation intensity of local cancer tissues and protect normal tissues during the process of treating liver cancer. In addition, the fraction dose is relatively high and the treatment course is short, so it is not only noninvasive compared with the surgical treatment, but also not affected by the influences of the size and shape of cancer during treatment [4, 5]. The primary principle of HIFU in treating liver cancer is a series of effects caused by the focused ultrasound in the bodies of patients including high heat effect and mechanical effect. The cancer tissues, especially the cancer vessels, are damaged by such effects [6]. Several studies have shown that HIFU can precisely damage the malignant cancer tissues during treating primary liver cancer, which is irreversible to some extent [7]. Moreover, it can not only improve the symptoms of patients, liver functions, increase post-treatment survival rate, but also enhance immune function [8].

Table 1
General clinical characteristics of liver cancer patients (n)

Item	Cases
Age (years)
$\geqslant$ 60	35
$<$ 60	61
Gender
Male	66
Female	30
KPS score (score)
60	20
70	25
80	31
90	20
Child-Pugh staging
A stage	33
B stage	53
C stage	10
Liver cancer grading
IIa	10
IIb	36
IIIa	32
IIIb	18

2. Patients and methods

2.1 Patients

A total of 96 patients definitely diagnosed as primary cancer from November 2016 to September 2017 were selected in this study, consisting of 66 men and 30 women. They were aged 30–78 years old with an average of (48.32 $\pm$ 5.41) years old. The Karnofsky performance status (KPS) of all included patients was over 60. The Child-Pugh staging was A, B and C stage, while liver cancer grading was IIa, IIb, IIIa, IIIb grade (Table 1). All included patients were diagnosed with liver cancer by the elevation of peripheral serum alpha-fetoprotein (AFP) or biopsy pathological morphology as well as meeting the diagnosis criteria which is proposed by the Research and Cooperation Meeting on the Prevention and Treatment of Liver Cancer in 1997. Exclusion criteria: patients with diffuse liver cancer; prothrombin time lower than 50%; severe heart or renal disease; platelet count lower than 5000/L; intolerant of anesthesia. This study was approved by ethics committee in The Sixth People’s Hospital of Qingdao and all the enrolled objects had signed informed consent.

2.2 Treatment methods

Before treatment, all patients received tracheal intubation and general intravenous anesthesia with skin being degreased and degassed and the posture for treatment was selected according to the specific location of liver cancer. At first, the location, size, shape of tumor and the relation with adjacent tissues were detected by B-ultrasonography, and the sections were prepared via X, Y and Z axes covering all predetermined target section. When the echo of tumor was enhanced with the treatment time of about 4200 s, treatment was stopped. The HIFU tumor treating system was provided by Beijing HIFU Company.

Table 2
Comparisons of symptom remission rates before and after HIFU in treating liver cancer

Time	Abdominal distension and abdominal pain	Poor appetite	Jaundice	Ascites
Before treatment	52	70	41	23
3 months after treatment
Disappear	16	19	16	0
Relieve	25	31	18	9
Remission rate	78.9%*	71.4%*	82.9%*	39.1%*

Note: Compared with before treatment, * $p<$ 0.05.

2.3 Treatment effect and immunologic detection

Patients were fasting for solids and liquids for 10 h overnight on 1 day before treatment and 3, 6 months after treatment, respectively, and 15 mL peripheral blood was extracted followed by isolation of serum for detecting the levels of AFP, CA199, NK cell, and T cell subsets, IFN-r, IL-2, IL-4 and IL-10 via enzyme-linked immunosorbent assay before treatment and 3 months after treatment. The reagents and instruments were provided by Shandong Bio-Instruments Ltd. Changes in liver function, including alanine transaminase (ALT), aspartate aminotransferase (AST), total albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (AKP) and prothrombin time, before treatment and 3, 6 months after treatment were detected by an automatic biochemical analyzer. Changes in survival rates of patients in different stages at 3, 6, 9 months and 1 year after treatment were also recorded.

2.4 Statistical analysis

Statistical Product and Service Solutions (SPSS) 19.0 software was used for data processing. The collected data were presented as mean $\pm$ standard deviation (SD). Student $t$ -test was used for the comparison of measurement data, and chi-square test was used for the comparison of enumeration data. $p<$ 0.05 suggested that the difference was statistically significant.

3. Results

3.1 Comparisons of symptom remission rates before and after HIFU

At 3 months after HIFU, the remission of abdominal distension and abdominal pain as well as jaundice was the most significant with a remission rate of 78.9% and 82.9%, respectively, followed by poor appetite and ascites. The above symptoms were significantly relieved after treatment compared with those before treatment, and the differences were statistically significant ( $p<$ 0.05) (Table 2).

3.2 Comparisons of tumor markers before and after HIFU

The serum tumor markers, such as AFP and CA199, were measured at 3 months after treatment and showed that they were significantly lowered compared with those before treatment ( $p<$ 0.05) (Table 3).

3.3 Comparisons of Child-Pugh staging of liver function before and after HIFU

The Child-Pugh staging scores at 3 months after HIFU were significantly reduced compared with those before treatment ( $p<$ 0.05). The scores at 6 months after treatment were also significantly reduced compared with those at 3 months after treatment and before treatment ( $p<$ 0.05). The number of patients in stage A in terms of Child-Push staging was significantly increased, while the number of patients in stage B and C was significantly decreased. The differences were statistically significant ( $p<$ 0.05) (Table 4).

Table 3
Comparisons of tumor markers before and after HIFU

Time	n	AFP (ng/ml)	CA199 (U/L)
Before treatment	96	204.5 $\pm$ 30.7	79.8 $\pm$ 20.3
3 months after treatment	96	36.3 $\pm$ 7.6	22.8 $\pm$ 5.7
P		0.001	0.001

Table 4

Comparisons of Child-Pugh staging of liver function before and after HIFU in treating liver cancer

Time	Child-Pugh	A	B	C
	staging (score)	stage	stage	stage
Before treatment	8.32 $\pm$ 1.31	33	53	10
3 months after treatment	7.29 $\pm$ 1.27*	44*	47*	5*
6 months after treatment	5.08 $\pm$ 1.06#	64#	31#	1#

Note: Compared with that before treatment, * $p<$ 0.05. Compared with 3 months after treatment, # $p<$ 0.05.

3.4 Comparisons of liver functions before and after HIFU

Liver functions at 3 months after treatment were detected and indicated that there were no significant differences in the levels of ALT, AST, AKP, ALB and TBIL before and after treatment, but the levels of ALT, AST, AKP, ALB and TBIL in serum at 6 months after treatment were significantly decreased compared with those before treatment, but ALB level was significantly elevated ( $p<$ 0.05) (Table 5).

Table 5
Comparisons of liver functions before and after HIFU

Time	ALT (IU/L)	AST (IU/L)	AKP (IU/L)	ALB (g/L)	TBIL (uumol/l)
Before treatment	60.6 $\pm$ 13.8	61.2 $\pm$ 17.4	139.3 $\pm$ 12.9	29.3 $\pm$ 2.8	29.3 $\pm$ 5.8
3 months After treatment	70.5 $\pm$ 12.3	72.3 $\pm$ 16.5	152.3 $\pm$ 20.6	30.7 $\pm$ 2.4	26.2 $\pm$ 7.4
6 months After treatment	29.3 $\pm$ 10.8*	38.1 $\pm$ 12.6*	30.8 $\pm$ 7.9*	35.1 $\pm$ 2.5*	16.3 $\pm$ 2.3*

Note: Compared with before treatment, * $p<$ 0.05.

Table 6

Comparisons of survival rates in different stages after HIFU

Grading (grade)	n	3 months after treatment	6 months after treatment	9 months after treatment	1 year after treatment
IIa	10	80.0%	90.0%	67.2%	38.9%
IIb	36	75.0%	58.3%	23.1%	13.6%
IIIa	32	71.9%	8.1%	1.1%	0
IIIb	18	33.3%	0	0	0
Total	96	66.7%	35.3%	18.9%	10.9%

Table 7

Comparisons of NK cell and T cell subset after HIFU (%)

Time	n	NK cell	CD3	CD4	CD8	CD4/CD8
Before treatment	96	26.5 $\pm$ 5.9	39.6 $\pm$ 5.3	29.8 $\pm$ 5.5	30.6 $\pm$ 5.4	1.1 $\pm$ 0.1
3 months after treatment	96	33.7 $\pm$ 5.6	46.8 $\pm$ 5.1	37.2 $\pm$ 4.8	36.3 $\pm$ 6.7	1.7 $\pm$ 0.2
P		0.001	0.001	0.001	0.001	0.001

Table 8

Comparisons of cytokines after HIFU (pg/mL)

Time	IFN-r	IL-2	IL-4	IL-10
Before treatment	92.3 $\pm$ 27.6	72.3 $\pm$ 16.7	80.3 $\pm$ 13.8	75.6 $\pm$ 20.2
3 months after treatment	114.3 $\pm$ 23.2	96.3 $\pm$ 20.2	59.2 $\pm$ 10.3	54.5 $\pm$ 18.1
P	0.001	0.001	0.016	0.022

3.5 Comparisons of survival rates in different stages after HIFU

Patients in different liver cancer stages had different survival rates in different time periods after treatment. The higher the cancer stage, the lower the survival rate after treatment. There were significant differences in survival rates after treatment among different stages ( $p<$ 0.05). The average survival rate of stage III patients after treatment was 3.6 months, which was significantly longer than that of patients with natural course (2 months on average) (Table 6).

3.6 Comparisons of NK cell and T cell subset after HIFU

The cytokines levels (NK, CD3, CD4, CD8 and CD4/CD8) at 3 months after treatment were significantly elevated compared with those before treatment ( $p<$ 0.05) (Table 7).

3.7 Comparisons of cytokines after HIFU

IFN-r and IL-2 levels at 3 months after treatment were significantly increased, but IL-4 and IL-10 levels were significantly decreased compared with those before treatment ( $p<$ 0.05) (Table 8).

4. Discussion

The primary liver cancer seriously threatens human life. The most common cause of liver cancer in China is hepatitis B virus (HBV) infection [9], so most liver cancer patients are often accompanied with chronic liver hepatitis or liver cirrhosis. If regular follow-up and monitoring of disease changes haven’t been performed, patients are often in intermediate and advanced stage at the time of diagnosis [10]. At present, radiotherapy has become the main treatment approach in clinical practice for patients who have lost operation opportunity. But radiotherapy is difficult to achieve an ideal treatment effect [11] due to high radiation dose which may cause much more damages to liver tissue cells and often result in severe hepatic insufficiency after treatment. HIFU is a treatment way which can achieve the purpose of real-time monitoring and strong control [12] with the use of imageology. It has fewer lesions than operation as it can flexibly select ablation zone in accordance with the tumor size, which can not only avoid excess energy release but also protect the normal tissue cells which are adjacent to tumor and reduce the occurrence rate of complications after treatment [13]. The primary principle of HIFU in treating liver cancer is a series of effects caused by the focused ultrasound in patients’ bodies including high heat effect and mechanical effect. The cancer tissues, especially cancer vessels, are destroyed by such effects [14]. A lot of research have shown that HIFU in treating primary liver cancer can precisely destroy the malignant cancer tissues, which is irreversible to some extent [15], and can effectively improve the symptoms of the patients with primary liver cancer, especially abdominal distension abdominal pain [16]. In the present study, we found that the remission rates of jaundice and abdominal distension and abdominal pain after HIFU were increased, and symptoms were significantly ameliorated after treatment compared with those before treatment.

In terms of diagnosis of primary liver cancer, AFP is more sensitive and accurate than other diagnostic markers, except for histopathological diagnosis. Thus, CA199 combined with AFP is often used in clinical practice to help the diagnosis of primary liver cancer [17]. In addition to diagnosis, the related tumor markers can also be useful for the evaluation of treatment efficacy. In this study, it was found that AFP and CA199 levels in peripheral serum were significantly decreased after HIFU treatment. On the other hand, among the 96 patients included in this study, the Child-Pugh staging of liver function at 3 months after treatment was not obviously decreased, but it was significantly improved at 6 months after treatment and liver function recovered to normal. The average survival rate of stage III patients after treatment was 3.6 months, which was significantly longer than that in natural course. In addition, we also found that the survival rates after treatment in different time varies in patients with different disease stages, suggesting that different stages of liver cancer might also be the important factor affecting the prognostic survival rate, in addition to the treatment approaches [18].

Cellular immunity, especially that induced by T cell, plays an important role in the regulation of the immunologic process associated with tumor. During immunosuppression, CD4 cells are involved in the synthesis and release of cytokines and CD8 cells play a critical role in the synthesis and release of inhibiting cytokines. Thus, CD4 and CD8 cell can reflect the status of cellular immunity function [19]. It is concluded in this study that CD4 and CD8 levels were significantly increased and immunity function was improved after receiving HIFU treatment. IL-2 and IFN-r can be expressed by Th1 subset which is also the main subset of CD4 cells. IL-4 and IL-10 are mainly synthesized by Th2 subset. Th1 subset will be transformed into Th2 subset in the immunologic process of tumor [20]. Consistent with the role of Th1 and Th2 in the pathogenesis of tumor, after the treatment with HIFU, we found IL-2 and IFN-r levels were increased while IL-4 and IL-10 levels were significantly decreased.

In conclusion, HIFU treatment can effectively improve symptoms and enhance immune function of patients with liver cancer, indicating it might be widely applied in the treatment of liver cancer in clinic.

Footnotes

Conflict of interest

All author declare that they have no conflict of interest.

References

Rowland

I.J.

Rivens

and Chen

, MRI study of hepatic tumours following high intensity focused ultrasound surgery, Br J Radiol 70 (1997), 144–153.

Wang

and Chen

, Pathological study of extracorporeally ablated hepatocollular carcinoma with high intensity focused ultrasound, Chinese Journal of Oncology 23(3) (2001), 237–239.

Pemick

D.G.

Strieter

R.M.

and Eskandari

M.K.

, Role of tumor necrsis factor alpha in lipopolysaccharide induced pathologic alterations, AM J Pathol (136) (1990), 49.

Mengozzi

and Chezzi

, Cytokine down regulation in endotoxin tolerance, Eur Gytokine Netw (4) (1993), 89.

Watsuki

L.H.

and Starzl

T.E.

, Personal experience with 411 hepatic resection, Ann Surg 208 (1998), 421–431.

Padhya

K.T.

Marrero

J.A.

and Singal

A.G.

, Recent advances in the treatment of hepatocellular carcinoma, Curr Opin Gastroenterol 29(3) (2013), 285–292.

Zhang

and Wang

Z.B.

, High-intensity focused ultrasound tumor ablation: Review of ten years of clinical experience, Front Med China 4(3) (2010), 294–302.

Harvey

C.J.

Pilcher

J.M.

and Eckersley

R.J.

, Advances in ultrasound, Clinical Radiology 57(3) (2002), 157.

Chen

W.Z.

and Bai

, Tumor vessel destruction resulting from high-intensity focused ultrasound in patients with solid malignancies, Ultrasound Med Biol 28(4) (2002), 535.

10.

Madersbacher

Kratzik

and Susani

, Transcutaneous high intensity focused ultrasound and irradiation: An organ-preserving treatment of cancer in a solitary testis, European Urology 33(2) (1998), 195.

11.

Parkin

D.M.

Bray

and Ferlay

, Global cancer statistics, 2002, CA Cancer J Clin 55(2) (2005), 74–108.

12.

EI-Serag

H.B.

and Rudolph

K.L.

, Hepatocellular carcinoma: Epidemiology and molecular carcinogenesis, Gastroenterology 132(7) (2007), 2557–2576.

13.

Park

H.C.

Seong

and Han

K.H.

, Do se-response relationship in local radiotherapy for hepatocellular carcinoma, Int J Radiat Oncol Biol Phys 54(1) (2002), 150–155ï¼Ž

14.

Kampinga

H.H.

Dynlacht

J.R.

and Dikomey

, Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines, Int J Hyperthermia 20(2) (2004), 131–139.

15.

Takahashi

Matsumoto

and Nagayama

, Evidence for the involvement of double-strand breaks in heat-induced cell killing, Cancer Res 64(24) (2004), 8839–8845.

16.

Myerson

R.L.

and Hunt

, Transfection of human tumour cells with Mrell siRNA and the increase in radiation sensitivity and the reduction in heat-induced radiosensitization, Int J Hyperthermia 20(2) (2004), 157–162.

17.

Y.Y.

Sha

W.H.

and Zhou

Y.J.

, Short and long term efficacy of high intensity focused ultrasound therapy for advanced hepatocellular carcinoma, J Gastroenterol Hepatol 22(12) (2007), 2148–2154.

18.

Livraghi

Goldberg

S.N.

and Lazzaroni

, Hepatocellular carcinoma: Radiofrequency ablation of medium and large lesions, Radiology 214(3) (2000), 761–768.

19.

Inokuchi

Seki

and Ikeda

, Percutaneous microwave coagulation therapy for hepatocellular carcinoma: Increased coagulation diameter using a new electrode and microwave generator, Oncology Reports 24(3) (2010), 621–627.

20.

Jung

S.E.

Cho

S.H.

and Jang

J.H.

, Highintensity focused ultrasound ablation in hepatic and pancreatic cancer: Complications, Abdom Imaging 36(2) (2011), 185–195.