Liver stiffness assessed by shear-wave elastography declines in parallel with immunoregulatory proteins in patients with chronic HCV infection during DAA therapy

Abstract

BACKGROUND:

A rapid decline of liver stiffness (LS) was detected by non-invasive methods in patients with chronic hepatitis C (HCV) infection during treatment with direct-acting antivirals (DAA).

OBJECTIVE:

To investigate the influence of inflammation on LS.

METHODS:

We prospectively examined LS by sonographic shear-wave elastography in 217 patients during DAA therapy from treatment initiation (BL) to 12 weeks after end of therapy (SVR12). Demographic data, laboratory findings and serum levels of cytokines were determined.

RESULTS:

Values of LS decreased from 1.86 m/s to 1.68 m/s (p = 0.01) which was most pronounced in patients who had F4 fibrosis at BL (3.27 m/s to 2.37 m/s; p < 0.001). Initially elevated values of aminotransferases, ferritin, IgG (p < 0.001 each) and international normalized ratio (p < 0.003) declined, thrombocyte count (p = 0.007) increased. Correlations of these laboratory parameters with BL levels of LS measurement (LSM) were most apparent in patients with F1-F3 fibrosis. Tumor necrosis factor (TNF)-α (p = 0.031), interleukin (IL)-10 (p = 0.005) and interferon y inducible protein (IP)-10 (p < 0.001) decreased in parallel with LSM under DAA therapy and corelated with BL values.

CONCLUSION:

Decrease of systemic inflammatory parameters correlated with LSM under DAA therapy. We conclude that regression of LSM is attributable to the decline of inflammation rather than reflecting fibrosis.

Keywords

HCV shear-wave elastography inflammation fibrosis

1 Introduction

Chronic hepatitis C virus (HCV) infection is of global concern, with 130 to 150 million people infected worldwide. HCV infection can lead to liver cirrhosis and hepatocellular carcinoma, causing about 112,500 deaths a year [1]. Complications of chronic HCV infections are driven by inflammation, triggering liver fibrosis, which can be assessed by ultrasound based non-invasive liver-stiffness measurement (LSM) like fibroscan or shear-wave elastography (SWE) [2]. With implementation of direct acting antivirals, cure rates of HCV have improved up to 95%within treatment regimens of 8 to 12 weeks. Cure of HCV, defined as sustained virological response (SVR12), is reached, if there is no more proof of virus RNA in polymerase chain reaction 12 weeks post-treatment (EOT) [3]. Quick and effective elimination of HCV raises the question which short-term and long-term effects can be achieved. A fast decline of transaminases and a decrease of LSM have been reported within the treatment period [4 –6], along with improvement of liver function parameters [7, 8] and scores of fibrosis [9]. Additionally, to the decline of transaminases, interpreted as improvement of liver inflammation, soluble inflammatory markers improve after reaching SVR12 [10].

The reciprocal influence between liver stiffness and inflammation remains unclear [11]. So far, no study analysed markers of fibrosis and inflammation in correlation to SWE. We hypothesised that a fast decline of non-invasive measurement of liver stiffness by SWE is due to a decrease of inflammation rather than fibrosis. The aim of the present study was to prospectively evaluate the impact of HCV clearance following direct-acting antiviral agent (DAA) treatment on biomarkers of fibrosis and inflammation and to the change of LSM.

2 Material and methods

This study was performed at the Department of Internal Medicine I at the University Hospital Regensburg (Regensburg, Germany). The study was approved by the local ethical committee (Nr. 14-101-0049) and was performed according to the updated guidelines of good clinical practice and the updated Declaration of Helsinki. All patients gave informed consent prior to inclusion in the study. 217 patients, who have been treated with DAAs from October 2014 to September 2017, were included in this study. All patients suffered from chronic hepatitis C and were treated with one of the following regimens: sofosbuvir/daclatasvir (DCV/SOF), sofosbuvir/ledipasvir (LDV/SOF), sofosbuvir/velpatasvir (VEL/SOF), sofosbuvir/ribavirin (SOF/RBV), dasabuvir/ombitasvir/paritaprevir/ritonavir (DSV/OBV/PTV/RTV)±ribavirin (RBV). Treatment was performed according to international guidelines [12]. Serum was collected at initiation of DAA therapy (Base line/BL), at the end of therapy (EOT) and 12 weeks after EOT (SVR12). Tissue elastography was quantified by dynamic SWE, using acoustic radiation force imaging (ARFI), measuring speed of shear waves (m/s). At BL and SVR12 SWE was performed by a trained physician with respect to the quality standards defined by the EFSUB consensus guidelines 2013 and 2017 [13, 14]. Operators had appropriate knowledge and training in ultrasound elastography (technical procedure by operator: Measurement through a right intercostal space in supine position, right arm in extension, breath hold in neutral position, adequate B-mode liver image, measurement 40 to 50 mm below the liver capsule) (Fig. 1). Devices used in this study were GE LOGIQ E9 and GE LOGIQ S8 (1–6 MHz probe), Siemens Acuson S2000 (1–5 MHz probe) and Siemens Acuson S3000 (1–6 MHz probe). ACUSON S3000 and S2000 (Siemens®) use point shear-wave speed elastography (pSWE), providing a single value expressed in m/s in a region of interest (ROI) selected by means of B-mode imaging. GE® LOGIQ S8 and E9 use two-dimensional shear-wave elastography (2D-SWE), which provides a high imaging frequency down multiple simultaneous lines, displaying an image of stiffness within a ROI [14]. The median value of 8–12 valid measurements was obtained and grading of fibrosis was done using cut of values defined by Colombo et al. [15]. (F0 = 0.00–1.39 m/s, F1 = 1.40–1.79 m/s, F2 = 1.80–1.99 m/s, F3 = 2.00–2.59 m/s, F4≥2.60 m/s). Median values were documented in the electronic patient record. The cut-off values for LSM were defined as proposed by Colombo et al., however intermodality agreement may differ in dependence of the system used [16, 17]. Clinical data, ultrasound findings documenting steatosis of the liver or sonographic signs of liver cirrhosis [18] as well as results of routine laboratory tests were obtained from the electronic patient record. APRI score (aspartate amino-transferase to platelet ratio index) and FIB4 score (fibrosis 4: including GOT, GPT, platelets, age] are established non-invasive fibrosis scores. Cut-offs used to determine fibrosis were the following for the FIB4-score: fibrosis > 3.25, no fibrosis: < 1.3 for patients < 65 years, < 2 for patients > 65 years; and for the APRI-score: cirrhosis > 2, fibrosis 1,5 to 1,9, no fibrosis < 0,5 [19, 20]. Serum used for cytokine/chemokine analysis was stored at 4°C for a maximum of 12 hours, centrifuged and stored at –80° C. Cytokine levels of interferon (IFN)-γ, interleukin (IL)-1α, IL-4, IL-10, tumor necrosis factor (TNF) and interferon y inducible protein (IP)-10 were analysed and quantified using Milliplex® multiplex-ELISA-assays for Luminex (Merck, KGaA, Germany), following the manufacturer’s instructions. Patient characteristics are shown in Table 1.

Fig. 1

Liver stiffness measured by ARFI using Siemens S3000 and Shear-point elastography using GE LOGIQ E9.

Table 1

Patients characteristics

Parameter	n	%
Sex
Male	125	57.6%
Female	92	42.4%
Genotype
1a	68	31.3%
1b	96	44.2%
2a/c	5	2.3%
2b	4	1.8%
3a	38	17.5%
4	1	0.5%
5a	1	0.5%
Time of DAA therapy
8 weeks	20	9.2%
12 weeks	170	78.3%
24 weeks	27	12.4%
Fibrosis
By LSM
F0	65	30.0%
F1	49	22.6%
F2	11	5.1%
F3	25	11.5%
F4	46	21.2%
Missing data	21	9.7%
By FIB4 score
Fibrosis	54	24.9%
Possible fibrosis	66	30.4%
By APRI-score
Cirrhosis	29	13.4%
Fibrosis	16	7.4%
Possible fibrosis	20	9.2%
Sonographic findings
Diagnosis of liver cirrhosis	46	21.2%

2.1 Statistical analysis

Statistical differences were analysed by Student’s t-test (Ms Excel) or two-sided Pearson correlation (SPSS Statistics 25.0 program, IBM, Leibniz Rechenzentrum, München. Germany). A value of p < 0.05 was regarded significant.

3 Results

3.1 Patient characteristics

217 patients were included in this prospective single centre study from 2014 to 2017. Mean age of the patients was 53 years. Main genotypes of HCV were 1a (n = 68, 31.3%) 1b (n = 96, 44.2%) and 3 (n = 38, 17.55%). 78.3%(n = 170) of the patients were treated for 12 weeks.

21.1%(n = 46) of the study cohort had liver cirrhosis, and 41.0%(n = 89) had steatosis hepatis as assessed by ultrasound examination. 24.9%(n = 54) had fibrosis due to FIB4 score, 30.4%(n = 66) possible fibrosis and 13.4%(n = 29) had cirrhosis. 7.4%(n = 16) of the patients had significant fibrosis according to APRI score, 9.2%(n = 20) had possible fibrosis. By SWE 21.2%(n = 46) of the patients had F4 fibrosis, 11.5%(n = 25) F3 fibrosis, 5.1%(n = 11) F2 fibrosis and 22.6%(n = 49) F1 fibrosis. 30.0%(n = 65) of the patients did not show signs of fibrosis. In n = 21 patients (predominantly patients with sonographic characteristics of cirrhosis) LSM was not performed at BL and in n = 43 patients LSM was not carried out at SVR12 (n = 46 patients with cirrhosis: ARFI at BL n = 28, ARFI at BL and SVR12 n = 16).

Patients’ characteristics are shown in Table 1. Scoring systems for fibrosis (APRI and FIB4), diagnosis of cirrhosis by clinical and sonographic criteria and F4 fibrosis by LSM did only overlap in parts. So, patients with sonographic signs of cirrhosis and patients with cirrhosis defined by APRI score had both F4 and F3 fibrosis defined by LSM. Furthermore, patients with fibrosis defined by APRI and FIB4 score had both F3 and F4 fibrosis defined by LSM. (Table 2)

Table 2
Distribution of the grade of liver stiffness measurement in scores of fibrosis / cirrhosis

n F0–2 F3 F4

Cirrhosis by ultrasound 28 1 (4%) 8 (28%) 19 (68%)

Cirrhosis by APRI score 18 1 (5%) 5 (28%) 12 (67%)

Fibrosis by APRI score 14 1 (7%) 5 (36%) 8 (57%)

Fibrosis by FIB4 score 37 2 (5%) 11 (31%) 24 (64 %)

	n	F0–2	F3	F4
Cirrhosis by ultrasound	28	1 (4%)	8 (28%)	19 (68%)
Cirrhosis by APRI score	18	1 (5%)	5 (28%)	12 (67%)
Fibrosis by APRI score	14	1 (7%)	5 (36%)	8 (57%)
Fibrosis by FIB4 score	37	2 (5%)	11 (31%)	24 (64 %)

Table 2 shows the distribution of grades of fibrosis defined by LSM in patients with cirrhosis, APRI score and FIB4 score and value of LSM at BL.

SVR12 was achieved in 97.2%of patients (n = 211). In 2.8%(n = 6) of patients a relapse occurred after DAA therapy; in one patient this was possibly due to incompliance. 50%of the patients with relapse had clinical signs of liver cirrhosis (n = 3). However, LSM in these patients was F0 in n = 1, F1 n = 1, F2 in n = 1, F3 in n = 0 and F4 in n = 2, one patient had missing LSM.

3.2 Change of LSM under DAA therapy

Values of LSM declined significantly during DAA therapy. The mean value declined by 9.6%during the observation period (BL 1.86 m/s, SD 0.84; SVR12 1.68 m/s, SD 0.78; p = 0.01). This was most apparent in patients with F4 fibrosis in LSM (27.5%, BL 3.27 m/s, SD 0.54; SVR12 2.37 m/s, SD 1,04; p < 0.001) (Fig. 2). In patients with sonographic signs of cirrhosis, LSM was performed in 16 out of 46 patients at both timepoints, BL and SVR12. In these patients LSM decreased, without significance, possibly because of the small sample size (12.2%, BL 2.79 m/s, SD 0.74; SVR12 2.45 m/s, SD 0.72; p = 0.103). Patients without fibrosis (F0) showed an increase of LSM to SVR12, most likely because measurement of values < 1.4 m/s are imprecise [13, 14]. Patients with relapse did not show any significant change between BL and SVR12 (BL 2.26 m/s, SD 1.03; SVR122.27 m/s, SD 1,3) (Table 3).

Fig. 2

Median values of liver stiffness measurement (LSM) by shear-wave elastography at BL and SVR12. ^*p < 0.05, ^**p < 0.001.

Table 3

Decline of liver stiffness measurement in subgroups of patients under DAA therapy

LSM by shear-wave elastography	[m/s] (SD)
	BL	SVR12	n	p
All	1.86 (0.84)	1.68 (0.78)	154	0.010
Relapse	2.26 (0.78)	2.27 (0.75)	5	0.970
Cirrhosis	2.79 (0.74)	2.45 (0.72)	16	0.103
No cirrhosis	1.75 (0.79)	1.60 (0.75)	138	0.032
F0	1.14 (0.17)	1.44 (0.64)	56	0.002
F1–3	1.80 (0.35)	1.57 (0.56)	67	< 0.001
F4	3.27 (0.54)	2.37 (1.04)	31	< 0.001

Table 3 shows the mean LSM values assessed by shear-wave elastography at baseline (BL) and SVR12 are listed. N indicates the number of patients enrolled with values for LSM at both timepoints BL and SVR12. SD shows the standard deviation. Paired Student’s t-test was used for statistical analysis and the respective p-values are given in the last column.

3.3 Correlation of LSM with biomarkers of liver damage and function

For the correlation of biomarkers with fibrosis measured by LSM, we defined the following groups: all patients (all), patients with diagnosis of cirrhosis of the liver by clinical and sonographic criteria (cirrhosis), patients without cirrhosis by clinical and sonographic criteria (no cirrhosis), patients with F0 fibrosis in LSM (F0), patients with F1 to F3 fibrosis in LSM (F1-3) and patients with F4 fibrosis LSM. Transaminases (GOT/AST and GPT/ALT) showed a fast, significant decline during DAA therapy as has been described before [15]. This was true for all defined groups and for all grades of fibrosis defined by LSM, except for patients with relapse (GOT: all patients; BL 60 U/l, SVR12 22 U/l, p = < 0.001; GPT: all patients; BL 86 U/l, SVR12 30 U/l, p < 0.001, GOT: relapse; BL 307 U/l, SVR12 342 U/l; GPT relapse: BL 84 U/l, SVR12 161 U/l). Notably, there were no further significant changes from EOT to SVR12. BL levels of GOT and GPT showed significant correlations with BL levels of LSM in the group ‘all patients’ and in patients without cirrhosis. GOT levels additionally were correlated with LSM in patients with F1-F3 fibrosis. There was no correlation between the decline of transaminases and the change in LSM (Table 4).

Table 4
Change of Glutamat-Oxalacetat-Transaminases (GOT) and Glutamat-Pyruvat-Transaminases (GPT) in subgroups of patients under DAA therapy and correlation to liver-stiffness measurement

Group GOT [U/l] GPT [U/l]

n⁰ BL¹ EOT² SVR12³ r^a4 r²⁵ n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5

All 188 60^** 25^* 22^ 0.01 0.37^ 191 86^** 32^* 30^** 0.06 0.15^*

Cirrhosis 36 86^ 33 30^ 0.20 –0.09 38 89^ 29 38^ 0.02 –0.48^*

No cirrhosis 150 53^ 24 20^ 0.04 0.32^ 153 86^ 32 28^** 0.08 0.22^*

F0 58 43^** 20^* 18^ 0.19 –0.02 60 72^ 29^* 26^ 0.14 –0.19

F1–F3 73 59^ 27 21^** 0.13 0.35^* 73 96^ 36 28^ 0.01 0.08

F4 41 79^ 27 28^ 0.23 –0.16 41 98^ 33 42^ 0.13 –0.08

Relapse 5 307 191 342 0.02 –0.56 5 84 31 161 0.07 –0.49

Group	GOT [U/l]	GPT [U/l]
All	188	60^**	25^*	22^**	0.01	0.37^**	191	86^**	32^*	30^**	0.06	0.15^*
Cirrhosis	36	86^**	33	30^**	0.20	–0.09	38	89^**	29	38^**	0.02	–0.48^*
No cirrhosis	150	53^**	24	20^**	0.04	0.32^**	153	86^**	32	28^**	0.08	0.22^*
F0	58	43^**	20^*	18^**	0.19	–0.02	60	72^**	29^*	26^**	0.14	–0.19
F1–F3	73	59^**	27	21^**	0.13	0.35^*	73	96^**	36	28^**	0.01	0.08
F4	41	79^**	27	28^**	0.23	–0.16	41	98^**	33	42^**	0.13	–0.08
Relapse	5	307	191	342	0.02	–0.56	5	84	31	161	0.07	–0.49

n⁰ = number of patients in T-Test; number of participants in Pearson correlation differs from that. BL: baseline, EOT: end of therapy, SVR12: sustained virological response 12 weeks after EOT. ¹compares BL to EOT, ²compares EOT to SVR12, ³compares BL to SVR12. r^a 4 is the Pearson correlation coefficient for the change of GOT or GPT to the change of LSM values from BL to SVR12. r^2 5 is the Pearson correlation coefficient of GOT or GPT to LSM at BL. ^*p < 0.05; ^**p < 0.001.

Bilirubin, INR and albumin levels, reflecting liver function and synthesis, significantly improved in all groups. Taken together, there was no significant correlation of these parameters with a change in LSM. If, correlation was only apparent in patients without cirrhosis.

Thrombocyte levels did significantly improve solely in the group of all patients (BL 190/nl, SVR12 195/nl; p = 0.007) and in the group of patients without cirrhosis (BL 210/nl, SVR12 214/nl; p = 0.011). In patients with cirrhosis and F4 fibrosis there was an improvement from BL to EOT (F4 p = 0.007, cirrhosis = 0.006) which did not persist to SVR12. BL levels of thrombocytes were below the lower normal value (182/nl) in the group of patients with diagnosis of cirrhosis of the liver (108/nl), in patients with F4 fibrosis (153/nl) and in patients with relapse (165/nl). Interestingly, from BL to EOT, thrombocytes increased significantly in all patients but not in patients with relapse. Of note, thrombocyte count declined from EOT to SVR12, and this was significant in the group of all patients (EOT 210/nl, SVR12 195/nl, p = 0.004) and in the group of patients with F4 fibrosis. Thrombocyte count at BL showed a correlation with LSM in the group of all patients (r² = –0.461, p < 0.001), in patients without cirrhosis (r² = –0.29, p < 0.001), in patients with F1-F3 fibrosis (r² = 0.49, p < 0.001) and in patients with relapse (r² = –0.88, p < 0.05). However, there was no significant corelation between changes of thrombocytes and LSM (Table 5).

Table 5

Change of bilirubin, INR, albumin and thrombocytes in subgroups of patients under DAA therapy and correlation to liver-stiffness measurement

	Bilirubin [mg/dl]						INR
	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5
All	189	0.71	0.69	0.65	0.13	0.31^*	189	1.088^*	1.072	1.065^*	0.04	0.29^**
Cirrhosis	37	1.17	1.22	1.06	0.28	0.05	37	1.290^*	1.249	1.283	0.21	0.08
No cirrhosis	152	0.60	0.56	0.55^*	0.12	0.10	152	1.038^*	1.029^*	1.012^*	0.03	0.07
F0	59	0.58	0.56	0.52^*	0.13	0.00	60	1.026^*	1.011	0.989^*	0.16	0.11
F1–F3	73	0.67	0.64	0.61	0.25^*	0.44^**	73	1.067	1.061	1.048^*	0.02	0.36^**
F4	40	0.85	0.80	0.87	0.22	–0.11	40	1.172	1.144	1.169	0.13	–0.14
Relapse	5	0.68	0.53	0.71	0.97^*	0.84	5	1.076	1.072	1.058	0.50	0.97^*
	Albumin [g/l]						Thrombocytes [/nl]
	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5
All	181	37.6^*	38.9	39.2^**	0.00	–0.31^**	189	190^**	201^*	195^*	0.08	–0.44^**
Cirrhosis	38	33.8^*	35.9	36.2^*	0.08	–0.19	36	108^*	117	114	0.04	–0.15
No cirrhosis	143	38.6^*	39.7	40.0^**	0.01	–0.10	153	210^**	220	214^*	0.09	–0.29^**
F0	59	38.8	39.4^*	40.1^**	0.12	0.18	61	224^**	237	230	0.01	–0.19
F1–F3	69	38.3	40.0	39.6^**	0.01	–0.42^**	72	196^*	206	203^*	0.03	–0.49^**
F4	36	36.0^*	37.5	37.7^*	0.07	0.11	41	153^*	163	155^*	0.23	–0.07
Relapse	5	38.3	37.7	38.5	0.20	–0.56	5	165	175	175	0.10	–0.88^*

n⁰ = number of participants in T-Test; number of participants in Pearson correlation differs from that. BL: baseline, EOT: end of therapy, SVR12: sustained virological response 12 weeks after EOT. ¹compares BL to EOT, ²compares EOT to SVR12, ³compares BL to SVR12. r^a 4 is the Pearson correlation coefficient for the change of variables to the change of LSM values from BL to SVR12. r^2 5 is the Pearson correlation coefficient of variables to LSM at BL. ^*p < 0.05; ^**p < 0.001.

3.4 Correlation of LSM measurement with scores of fibrosis

APRI score (all patients: BL 0.95, SVR12 0.32, p < 0.001) and FIB4 score (all patients: BL 2.92, SVR12 1.77, p < 0.001) declined throughout all groups of patients, except in patients with relapse. Improvement of these scores was significant from BL to EOT but did not further change from EOT to SVR12: An exception was the APRI score in patients with F0 fibrosis which also decreased during this time period (EOT 0.19, SVR12 0.16, p = 0.025). Cirrhosis estimated by APRI and FIB4 score at BL correlated with the grade of fibrosis by LSM. However, mean values of the APRI score in patients with F4 fibrosis at BL (APRI at BL 1.61) was recognized as fibrosis (cut off 1.5–1.9) but not as cirrhosis (cut off > 2). This supports the findings shown in Table 2, suggesting that diagnosis of F4 fibrosis by LSM does often not match with clinical diagnosis of cirrhosis. BL levels of fibrosis scores showed a correlation with LSM levels in the group of all patients (APRI: r² = 0.375, p < 0.001, FIB4: r² = 0.493, p < 0.001), in patients without cirrhosis (APRI: r² = 0.41, p < 0.001, FIB4: r² = 0.39, p < 0.001) and in patients with F1 to F3 fibrosis (APRI: r² = 0.55, p < 0.001, FIB4: r² = 0.62, p < 0.001). Changes of these scores during DAA therapy did not correlate with changes in LSM. (Table 6) Noteworthy, both scoring systems are strongly influenced by transaminase levels and thrombocyte count, which are both affected by HCV-induced inflammation.

Table 6
Change of APRI-score and FIB4-score in subgroups of patients under DAA therapy and correlation to liver-stiffness measurement

APRI-score FIB4-score

n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5 n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5

All 189 0.95^ 0.35 0.32^ 0.06 0.48^ 188 2.92^ 1.80 1.77^ 0.03 0.50^

Cirrhosis 36 2.24^ 0.77 0.74^ 0.11 0.07 36 7.65^ 4.55 4.52^ 0.04 0.15

No cirrhosis 153 0.65^ 0.26 0.23^ 0.04 0.41^ 152 1.79^ 1.15 1.11^ 0.02 0.39^

F0 61 0.43^ 0.19 0.16^ 0.13 0.07 60 1.28^ 0.90 0.83^ 0.13 0.16

F1–F3 72 0.77^ 0.34 0.26^ 0.12 0.55^ 72 2.17^ 1.45 1.37^ 0.11 0.62^

F4 41 1.61^ 0.52 0.56^ 0.23 –0.06 41 4.63^ 2.60 2.64^ 0.25 –0.05

Relapse 5 3.01 1.77 2.83 0.08 0.48 5 6.47 6.49 5.37 0.23 0.21

	APRI-score	FIB4-score
All	189	0.95^**	0.35	0.32^**	0.06	0.48^**	188	2.92^**	1.80	1.77^**	0.03	0.50^**
Cirrhosis	36	2.24^**	0.77	0.74^**	0.11	0.07	36	7.65^**	4.55	4.52^**	0.04	0.15
No cirrhosis	153	0.65^**	0.26	0.23^**	0.04	0.41^**	152	1.79^**	1.15	1.11^**	0.02	0.39^**
F0	61	0.43^**	0.19	0.16^**	0.13	0.07	60	1.28^**	0.90	0.83^**	0.13	0.16
F1–F3	72	0.77^**	0.34	0.26^**	0.12	0.55^**	72	2.17^**	1.45	1.37^**	0.11	0.62^**
F4	41	1.61^**	0.52	0.56^**	0.23	–0.06	41	4.63^**	2.60	2.64^**	0.25	–0.05
Relapse	5	3.01	1.77	2.83	0.08	0.48	5	6.47	6.49	5.37	0.23	0.21

3.5 Correlation of LSM with systemic markers of inflammation

Commonly used systemic inflammatory markers, which are associated with chronic infections, are the acute phase protein ferritin and the immunoglobulin (Ig)G. Median ferritin levels (upper limit 400 ng/ml) and IgG levels (upper limit 1600 mg/dl) were inside the normal range at BL, except IgG values in patients with cirrhosis (BL 1744 mg/dl) and patients with relapse (BL 1685 mg/dl). IgG and ferritin levels declined significantly in all groups of patients under DAA therapy (e.g. ferritin all patients: BL 220.6 ng/ml, SVR12 107.7 ng/ml, p < 0.001; IgG all patients: BL 1417 mg/dl, SVR12 1248 mg/dl, p < 0.001), except in the group of patients with relapse. Ferritin and IgG levels declined from BL to EOT. A further decrease from EOT to SVR12 was observed in all groups except in patients with relapse and expect IgG levels in patients with cirrhosis. BL levels of IgG and ferritin showed a correlation to LSM in the group of all patients (ferritin r² = 0.15, p = 0.045, IgG r² = 0.35, p < 0.001) and in patients with F1-F3 fibrosis (ferritin r² = 0.42, p < 0.001, IgG r² = 0.27, p = 0.014). In patients with F0 fibrosis (ferritin: r^a = 0.29, p = 0.029; IgG r^a = 0.28, p = 0.038) there was a correlation between change of ferritin and IgG values and LSM (Table 7). The decline of IgG and ferritin levels suggests that virus elimination rapidly improves systemic inflammation. Leucocyte counts, PCT and CRP values were not significantly increased in our patients during the study period. Thus, non-viral infections can be ruled out as reason for inflammation.

Table 7
Change of IgG and ferritin in subgroups of patients under DAA therapy and correlation to liver-stiffness measurement

IgG [mg/dl] Ferritin [ng/ml]

n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5 n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5

All 185 1417^** 1291^* 1248^ 0.10 0.35^ 179 220.6^ 135.8^ 107.7^** 0.12 0.15^*

Cirrhosis 35 1744^** 1509^* 1433^** 0.02 0.21 4 150.9 109. 137.7^* 0.25 –0.08

No cirrhosis 150 1341^** 1240^* 1204^** 0.14 0.21^* 34 238.7^ 175.4^ 138.1^** 0.20 0.16^*

F0 61 1259^* 1177 1164^* 0.28^* 0.02 145 216.4^** 126.5^* 100.6^ 0.12 –0.11

F1–F3 71 1430^ 1294^ 1238^ 0.19 0.27^* 61 188.3^** 124.9^* 101.0^ 0.29 0.42^

F4 36 1576^ 1418^ 1343^** 0.27 0.04 68 233.7^* 143.3 104.8^* 0.17 –0.22

Relapse 5 1685 1477 1578 0.51 0.95^* 34 317.7 166.5 154.0 0.38 –0.12

	IgG [mg/dl]	Ferritin [ng/ml]
All	185	1417^**	1291^*	1248^**	0.10	0.35^**	179	220.6^**	135.8^**	107.7^**	0.12	0.15^*
Cirrhosis	35	1744^**	1509^*	1433^**	0.02	0.21	4	150.9	109.	137.7^*	0.25	–0.08
No cirrhosis	150	1341^**	1240^*	1204^**	0.14	0.21^*	34	238.7^**	175.4^**	138.1^**	0.20	0.16^*
F0	61	1259^*	1177	1164^*	0.28^*	0.02	145	216.4^**	126.5^*	100.6^**	0.12	–0.11
F1–F3	71	1430^**	1294^**	1238^**	0.19	0.27^*	61	188.3^**	124.9^*	101.0^**	0.29	0.42^**
F4	36	1576^**	1418^**	1343^**	0.27	0.04	68	233.7^*	143.3	104.8^*	0.17	–0.22
Relapse	5	1685	1477	1578	0.51	0.95^*	34	317.7	166.5	154.0	0.38	–0.12

3.6 Correlation of LSM with cytokines and chemokines

Chronic HCV infection causes a systemic inflammation [21]. Serum cytokines/chemokines showed a differential response to DAA therapy and were elevated in subgroups of our patient cohort (IL-4 in 29%, IFN-α in 39%, IL1-α in 40%, IFN-γ in 70%, IL-10 in 74%, TNF-α and IP10 in 100%). Proinflammatory cytokines showed a decrease from BL to EOT in some groups (IFN-γ cirrhosis p = 0.04, F4 p = 0.043; IL1-α all patients p = 0.026, no cirrhosis p = 0.026). Cytokine / chemokine concentrations reached the BL level in the timespan from EOT to SVR12. TNFα levels declined from BL to SVR12.

In patients with cirrhosis BL of TNF-α levels (r² = 0.57, p = 0.003), in patients with F1-F3 fibrosis BL levels of TNFα (r^a = 0.33, p = 0.003) and IFNy (r² = 0.28, p = 0.014), showed a correlation with LSM. In patients with cirrhosis, F1-F3 fibrosis and patients with relapse, there was a correlation between the change of TNF-α levels and LSM. (cirrhosis: r^a = 0.54, p = 0.037; F1-3: r^a = 0.26, p = 0.036; relapse r^a = –0.97, p = 0.006).

The anti-inflammatory cytokine IL-10 showed a significant decline in all patients (p = 0.048), in patients with no cirrhosis (p < 0.001) and in patients with F1-F3 fibrosis (p = 0.012). Furthermore, IL-4 increased under DAA therapy but without significance. BL levels of IL-10 showed no correlation to LSM, change of IL-10 levels from BL to SVR12 correlated with change of LSM in patients with F1-3 fibrosis (r^a = 0.32, p = 0.009). Of note, IP10 showed the highest reduction of all biomarkers during DAA therapy, and this was apparent in all groups (p < 0.001), except in patients with relapse. BL levels showed correlations to LSM in all patients (r² = 0.244, p < 0.001), in patients without cirrhosis (r² = 0.3, p < 0.001) and in patients with F1-F3 cirrhosis (r² = 0.25, p = 0.024). However, this change showed no correlation with change of LSM (Table 8).

Table 8
Change of TNF-α, IFN-γ, IL-10 and IP10 in subgroups of patients under DAA therapy and correlation to liver-stiffness measurement

TNF-α [pg/ml] IFN-γ [pg/ml]

n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5 n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5

All 174 17.0^* 13.0 14.1 0.01 0.14 174 8.9 12.1 7.5 0.02 –0.13

Cirrhosis 33 20.0 16.6 18.5 0.54^* 0.57^* 33 2.4^* 25.5 11.4 0.15 0.07

No cirrhosis 141 16.3^* 12.1 13.0 0.08 0.08 141 10.4 9.0 6.6 0.02 –0.10

F0 56 17.3 12.8 12.7 0.21 –0.16 56 15.0 7.3 7.4 0.07 –0.13

F1–F3 69 14.5 11.7 11.9 0.26^* 0.33^* 69 8.7 10.1 6.3 0.11 0.28^*

F4 36 20.4 14.5 15.7 0.06 0.28 36 1.7^* 25.1 7.3 0.12 –0.17

Relapse 5 15.7 13.3 12.1 0.97^* 0.13 5 0.2 1.1 1.2 0.34 –0.43

IL-10 [pg/ml] IP-10 [pg/ml]

n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5 n⁰ BL¹ EOT² SVR12³ r^a 4 r^2 5

All 174 5.8^* 3.4 2.3^* 0.09 0.08 174 911^** 355^* 293^** 0.03 0.24^*

Cirrhosis 33 5.8 3.1 4.6 0.04 0.19 33 941^ 482 426^ 0.17 0.12

No cirrhosis 141 5.8 3.5^* 1.8^* 0.10 0.05 141 904^ 325^ 262^ 0.02 0.30^

F0 56 6.8 4.3 1.7 0.14 0.23 56 758^ 272 228^ 0.05 0.13

F1–F3 69 6.2 3.5 2.2^* 0.32^* 0.18 69 848^** 336^* 283^** 0.06 0.25^*

F4 36 2.3 2.5 1.4 0.01 0.03 36 1170^** 498^* 385^** 0.11 0.16

Relapse 5 0.0 0.5 1.0 0.0 - 5 921 282 560 0.55 0.32

	TNF-α [pg/ml]	IFN-γ [pg/ml]
All	174	17.0^*	13.0	14.1	0.01	0.14	174	8.9	12.1	7.5	0.02	–0.13
Cirrhosis	33	20.0	16.6	18.5	0.54^*	0.57^*	33	2.4^*	25.5	11.4	0.15	0.07
No cirrhosis	141	16.3^*	12.1	13.0	0.08	0.08	141	10.4	9.0	6.6	0.02	–0.10
F0	56	17.3	12.8	12.7	0.21	–0.16	56	15.0	7.3	7.4	0.07	–0.13
F1–F3	69	14.5	11.7	11.9	0.26^*	0.33^*	69	8.7	10.1	6.3	0.11	0.28^*
F4	36	20.4	14.5	15.7	0.06	0.28	36	1.7^*	25.1	7.3	0.12	–0.17
Relapse	5	15.7	13.3	12.1	0.97^*	0.13	5	0.2	1.1	1.2	0.34	–0.43
	IL-10 [pg/ml]	IP-10 [pg/ml]
	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5	n⁰	BL¹	EOT²	SVR12³	r^a 4	r^2 5
All	174	5.8^*	3.4	2.3^*	0.09	0.08	174	911^**	355^*	293^**	0.03	0.24^*
Cirrhosis	33	5.8	3.1	4.6	0.04	0.19	33	941^**	482	426^**	0.17	0.12
No cirrhosis	141	5.8	3.5^*	1.8^*	0.10	0.05	141	904^**	325^**	262^**	0.02	0.30^**
F0	56	6.8	4.3	1.7	0.14	0.23	56	758^**	272	228^**	0.05	0.13
F1–F3	69	6.2	3.5	2.2^*	0.32^*	0.18	69	848^**	336^*	283^**	0.06	0.25^*
F4	36	2.3	2.5	1.4	0.01	0.03	36	1170^**	498^*	385^**	0.11	0.16
Relapse	5	0.0	0.5	1.0	0.0	-	5	921	282	560	0.55	0.32

n⁰ = number of participants in T-Test; number of participants in Pearson correlation differs from that. BL (baseline), EOT (end of therapy), SVR12 (sustained virological response 12 weeks after EOT). ¹compares BL to EOT, ²compares EOT to SVR12, ³compares BL to SVR12. r^a 4 is the Pearson correlation coefficient for the change of variables to the change of LSM values from BL to SVR12. r^2 5 is the Pearson correlation coefficient of variables to LSM at BL. ^*p < 0.05; ^**p < 0.001.

4 Discussion

This is to our knowledge the first study assessing the change of clinical biomarkers of liver disease and cytokine profiles in comparison with non-invasive liver stiffness measurement (LSM) in patients with HCV during DAA therapy.

Several studies observed a rapid decline of liver stiffness by non-invasive LSM since admission of DAA therapies [4 , 22–25]. A meta-analysis described a rapid decline of LSM measured by vibrant controlled transient elastography from BL to EOT, which continued less pronounced after EOT [26]. However, some studies found a pronounced decrease of transient elastography during EOT and SVR12 especially after DAA regimes with shorter treatment periods [24]. We observed a decline of ultrasound mediated LSM from BL to SVR12 values under DAA therapy. In accordance with the data published by Tag Adeen, Singh and Knop et al., the most prominent decline of values occurred in patients with F4 fibrosis [4, 26].

Later findings suggest that LSM measured by shear wave elastography in patients without fibrosis is not reliable, and indeed, LSM values even increased in patients without liver fibrosis under DAA therapy.

Interestingly, patients with LSM-defined F4 fibrosis at BL were only in part diagnosed with liver cirrhosis by ultrasound imaging. On the other hand, a subgroup of patients with diagnosis of cirrhosis of the liver, defined by APRI score, FIB 4 score or sonographic signs, displayed values of LSM < 2.4 m/s (F4). Therefore, elevated values of LMS may reflect not only fibrosis but other signs of liver disease.

We confirmed a correlation of BL values of LSM and transaminases in the group of all patients, which was recently described by Knop et al. [24], however we could specify this effect to affect predominantly patients without cirrhosis and with F1-F3 fibrosis. This was also true for the correlation of baseline values of LSM with albumin, thrombocytes, bilirubin, INR, APRI score and FIB4 score. Change of these parameters did not significantly correlate with change of LSM. Notably, Knop et al used FibroScan and not shear-wave elastography to evaluate LSM. Values of SWE measurement and FibroScan may differ in early stages of fibrosis [2, 27].

We confirmed an increase of albumin and INR levels during DAA therapy [7 , 28–30]. Interestingly, INR levels did not improve in the subgroup of patients with cirrhosis. Bilirubin levels did neither significantly improve in patients with fibrosis nor cirrhosis. There are, as far as we know, no data available on changes of LSM of patients with liver cirrhosis during DAA therapy. A recent study showed that in patients with liver cirrhosis a significant change of MELD score was only achieved in 24%of patients in short term and 29%in long term follow up [8]. This implicates that changes of liver function in advanced liver disease are marginal. Overall BL parameters of liver function show that liver disease was not severe in our cohort. The fact, that LSM correlates with parameters of liver function only in patients with F1-F3 fibrosis implicates, that the dynamic of liver stiffness by SWE is mostly relevant in patients with fibrosis but not cirrhosis.

There are only limited data available about histologically proven decline of fibrosis in patients with HCV under DAA therapy. However, in a cohort analysing patients after liver transplantation an ongoing inflammation even after reaching SVR12 has been reported [31] and another study described that inflammation did improve at SVR12, but not so did fibrosis [32]. This implies that the decline of values of SWE reflects regression of necroinflammation rather than improvement of fibrosis. Thus, LSM measured by SWE and transient elastography correlated in both cases not only with fibrosis but also with necroinflammation [2].

The grade of fibrosis can be defined either by LSM or by scoring systems, most commonly FIB4 and APRI scores. Both scores are predominantly influenced by transaminases and thrombocytes [19]. There is a consensus that rapid decline of transaminases in the first weeks of DAA therapy is due to direct effects of the clearance of the virus. We could confirm findings of Hsu et al. [5], that the course of thrombocyte count under DAA increases initially, but declines again after EOT. This suggests again a direct effect of virus eradication on thrombocyte counts. Decline of thrombocytes in patients with F4 fibrosis from EOT to SVR12 argues against an effect of portal hypertension herein. Portal hypertension may affect SWE of liver and spleen [33], however if portal hypertension would decline due to improvement of fibrosis, this would cause an increase of thrombocyte levels. The direct influence of the clearance of the virus on thrombocytes and transaminases has great impact on APRI and FIB4 score. We confirmed a fast decline of these scores. Of clinical relevance, the course of thrombocytes and transaminases support the hypothesis of Hsu et al. that non-invasive fibrosis scores in HCV patients may reflect rather improvement of necroinflammation than improvement of fibrosis [5].

In addition to the decline of transaminases, we observed a decline of IgG and ferritin levels in all patients, again suggesting an interconnection of the virus with inflammation. IgG is known to be an important marker for disease activity in patients with autoimmune hepatitis [34] and may be associated with autoimmune phenomena in patients with HCV [35]. Ferritin is an acute phase protein which is associated with inflammation in many diseases [36]. Both have not been analysed yet in patients with DAA therapy. These common systemic markers of inflammation showed correlation with BL levels of LSM in all patients and in the group of patients without cirrhosis and F1-F3 fibrosis. This implicates a major role of inflammation on the dynamics of liver stiffness measurement.

Regarding specific markers of inflammation, we observed a dominant effect of DAAs on the chemokine IP10, which showed a significant decline in all groups of patients but not in those with relapse. This effect has only been described in much smaller groups of patients yet [37, 38]. IP10 is a chemokine which is associated with fibrosis and inflammation in HCV [39] and has been associated with SVR in patients on therapy with interferon-based regimes infected with genotype 1 and 4 [40]. There was a correlation of IP10 with BL values of LSM in the group of all patients, and more specifically in the subgroups of patients without cirrhosis and F1-3 fibrosis. In accordance with El-Nahaas et al. [41] we could not see any correlation of change of LSM and change of IP10. The proinflammatory cytokine TNFα may contribute to inflammation and fibrosis of the liver and may be influenced by DAA therapy according to animal models [42]. We observed a decline of TNFα to EOT, which was significant in all patients and in patients without cirrhosis, followed by an increase from EOT to SVR12. The correlation of TNFα with subgroups of fibrosis defined by LSM indicates a central role of the eradication of the virus and inflammation on LSM. The anti-inflammatory cytokine IL-10 showed the same pattern as described for other variables. In patients with F1-F3 fibrosis and patients without cirrhosis, there was a correlation with the change of values of LSM from BL to SVR12. The pattern of change of cytokines and chemokines under DAA therapy and their correlations to LSM strongly supports the hypothesis that LSM is influenced mainly by necroinflammation. Findings of this study reflect changes in SWE in patients with hepatitis C, which is one of the leading cause for liver cancer worldwide [43]. The interaction in between necroinflammation and fibrosis and its influence on oncogenesis plays an important role in genesis of hepatocellular carcinoma [44]. SWE is commonly used for diagnosis of liver fibrosis, however this technique is more and more transferred to the characterisation of HCC [45, 46]. Furthermore SWE is now used to define alterations in other organs than the liver, like the thyroid, the pancreas and the breast, especially for evaluation of malignancies [47 –58]. Therefore, the hypothesis that SWE reflects rather necroinflammation than fibrosis could have impact on the diagnosis of malignancies and should be content of further research.

Limitations of the study are the single center setting. Furthermore, due to ethical standards there was no liver biopsy taken to histologically correlate fibrosis and inflammation with LSM. LSM was only analysed at BL and SVR12 but not at EOT, therefore we could not correlate variables with LSM at EOT. Furthermore, LSM measurement was not performed at BL and SVR12 in all patients, especially in those with cirrhosis. In this subgroup sample size was limited and may affect statistical analyses. Last, we have been using two different systems for measurement of SWE, both working with dynamic, quantitative acoustic radiation force impulse (ARFI): ACUSON S3000 and S2000 (Siemens®) use point shear-wave speed elastography (pSWE) and GE® Logiq S8 and E9 use two-dimensional shear-wave elastography (2D-SWE). Both techniques show comparable results [59].

5 Conclusion

This study for the first time provides comprehensive data including LSM, fibrosis scores, laboratory markers of liver disease and inflammatory markers in a large study group. Our results support the hypothesis that LSM by shear-wave elastography does not only reflect the grade of fibrosis but is substantially affected by inflammation. Histological correlation with LSM and new ultrasound-based techniques like measurement of dispersion are needed to define limitations of shear-wave elastography for the detection of liver-fibrosis and to assess the potentials of sonographic techniques to measure inflammation of the liver.

Funding

The immune monitoring was funded by Novartis®.

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