The goal of this study was to evaluate differences in liver biopsies collected from patients pre- and post-treatment with DAAs for HCV infection. All 10 patients achieved SVR (i.e., undetectable viral RNA for 12 weeks or more post-treatment) independent of DAA regimen (Table 1) and had paired liver biopsies (pre-and post-DAA treatment) with adequate clinical follow-up. Patient demographics, viral status, and treatment regimens are shown in Table 1. The study group consisted of both male (6/10) and female (4/10) patients with a mean age of 51.8 ± SD 7.09 years when the first biopsy was collected and a mean age of 54.7 ± SD 7.5 years when the post-treatment biopsy was collected. Sixty percent of the patients were non-Hispanic whites (6/10), 20% were Hispanic whites (2/10), and 20% were African American (2/10). Patients had a mean body mass index (BMI) of 28.2 ± SD 7.27 kg/m2 and 28.9 ± SD 7.49 kg/m2 at the time of the first and second biopsies, respectively (P < 0.05). The HCV viral load ranged from 6.64 x 105 to 1.72 x 107 IU/ml before DAA treatment. The most prevalent genotype was 1a (in 5/10 patients), which was commonly treated with the sofosbuvir/simeprevir DAA combination (in 4 of 10 patients) (Table 1).
Table 1
Patient demographics, viral status and treatment regimens.
HCV Pt # a
|
Biopsyb
|
Age
|
Sex
|
Ethnicity/
Race
|
BMI
|
Genotype
|
Viral Load
(IU/mL)
|
DAA
Treatment
|
Treatment
Duration
|
1
|
Pre-Tx
|
58
|
M
|
Non-Hispanic/
White
|
25.0
|
1a
|
4,641,110
|
Sofosbuvir/
Simeprevir
|
12 weeks
|
Post-Tx
|
62
|
|
25.4
|
Not detected
|
|
2
|
Pre-Tx
|
45
|
F
|
Non-Hispanic/
White
|
45.0
|
1a
|
5,396,975
|
Sofosbuvir/
Ledipasvir
|
12 weeks
|
Post-Tx
|
47
|
|
46.3
|
Not detected
|
|
3
|
Pre-Tx
|
48
|
M
|
Hispanic/
White
|
26.8
|
1a
|
958,240
|
Sofosbuvir/
Simeprevir
|
12 weeks
|
Post-Tx
|
54
|
|
28.7
|
Not detected
|
|
4
|
Pre-Tx
|
56
|
F
|
Non-Hispanic/
White
|
32.1
|
2
|
664,007
|
Sofosbuvir/
Ribavirin
|
12 weeks
|
Post-Tx
|
63
|
|
31.3
|
Not detected
|
|
5
|
Pre-Tx
|
38
|
M
|
Non-Hispanic/
African American
|
21.0
|
1a
|
2,610,582
|
Sofosbuvir/
Simeprevir
|
12 weeks
|
Post-Tx
|
40
|
|
23.2
|
Not detected
|
|
6
|
Pre-Tx
|
61
|
M
|
Hispanic/
White
|
20.7
|
1a
|
3,083,562
|
Sofosbuvir/
Ledipasvir
|
12 weeks
|
Post-Tx
|
62
|
|
20.6
|
Not detected
|
|
7
|
Pre-Tx
|
55
|
F
|
Non-Hispanic/
White
|
33.6
|
1b
|
3,327,711
|
Sofosbuvir/
Simeprevir
|
12 weeks
|
Post-Tx
|
58
|
|
35.2
|
Not detected
|
|
8
|
Pre-Tx
|
57
|
M
|
Non-Hispanic/
White
|
23.4
|
2b
|
17,229,088
|
Sofosbuvir/
Ribavirin
|
12 weeks
|
Post-Tx
|
59
|
|
25.5
|
Not detected
|
|
9
|
Pre-Tx
|
53
|
M
|
Non-Hispanic/
White
|
25.9
|
2b
|
3,964,339
|
Sofosbuvir/
Simeprevir/
Ribavirin
|
12 weeks
|
Post-Tx
|
53
|
|
23.4
|
Not detected
|
|
10
|
Pre-Tx
|
47
|
F
|
Non-Hispanic/
African American
|
28.2
|
1b
|
2,940,158
|
Viekira Pak
|
12 weeks
|
Post-Tx
|
49
|
|
29.0
|
Not detected
|
|
Histological changes in liver biopsies pre-and post-treatment with DAA
Paired liver biopsies were collected from 10 study patients pre- and post-DAA therapy and were blindly evaluated for features of inflammation, fibrosis and steatosis as described in the methods Sect. 2. All of the patient’s pre- and post-treatment liver biopsies are shown in Fig. 1. Of these 10 samples, nine biopsies (patients 1–8 and 10) were collected pre-treatment and 10 were collected post-treatment. The duration between the patients’ first (i.e., pre-treatment) and second (i.e., post-treatment) liver biopsies ranged from 15.3 to 85.8 months (Table 2). The duration between DAA treatment completion and collection of their follow-up biopsies ranged from 6.0 to 28.4 months (Table 2). These were collected as standard of care to determine the modified hepatitis activity index (MHAI), the presence or absence of steatosis, and to stage the amount of hepatic fibrosis (Table 2). The fibrosis stage was determined two ways. It was first estimated using the Ishak criteria and then more objectively measured using the CPA “equation (1)” [22] (Fig. 2). The Ishak fibrosis stages ranged from 1 to 6 (out of 6) prior to treatment (Table 2). The calculated CPA in the study patients prior to treatment showed percentages of fibrosis from 1.6 to 15.7% and 1.3 to 32.0%, pre- and post-treatment, respectively (Table 2). Some of the patients showed decreases in the percentages of fibrosis (patients 1, 5, 6, 8, and 10), while others showed increases (patients 2, 3, 4, and 7), post-DAA treatment (Table 2). Persistent lymphocytic inflammation (i.e., those with a post-treatment MHAI > 1) was primarily observed within the portal tracts, in 60% of the patients (patients 1–4, 7, 10) (Table 2 and Fig. 1).
Table 2
Histopathological findings in liver biopsies pre- and post-DAA therapy.
HCV Pt #
|
Biopsy a
|
MHAI b
(n/18)
|
Fibrosis c
stage (n/6)
|
CPA c
%
|
Steatosis % d
(Pathologist)
|
Steatosis % d
(Visiopharm)
|
Duration between pre- and post-Tx Bx (mos)
|
Duration between SVR and post-Tx Bx (mos)
|
1
|
Pre-Tx
|
7
|
3
|
14.911
|
1
|
0
|
54.8
|
14.2
|
|
Post-Tx
|
5
|
2.5
|
13.461
|
1
|
0
|
|
|
2
|
Pre-Tx
|
9
|
2
|
5.299
|
40
|
6.994
|
16.6
|
9.4
|
|
Post-Tx
|
4
|
2
|
11.596
|
20
|
1.555
|
|
|
3
|
Pre-Tx
|
3
|
1
|
1.613
|
0
|
0
|
70.2
|
17.5
|
|
Post-Tx
|
2
|
0.5
|
3.009
|
0
|
0
|
|
|
4
|
Pre-Tx
|
8
|
6
|
6.744
|
5
|
0.861
|
85.8
|
28.4
|
|
Post-Tx
|
4
|
6
|
32.024
|
60
|
6.321
|
|
|
5
|
Pre-Tx
|
4
|
1
|
6.661
|
0
|
0
|
20.8
|
19.2
|
|
Post-Tx
|
1
|
1
|
3.496
|
0
|
0
|
|
|
6
|
Pre-Tx
|
2
|
1
|
3.677
|
0
|
0
|
15.3
|
6.0
|
|
Post-Tx
|
0
|
1
|
2.905
|
0
|
0
|
|
|
7
|
Pre-Tx
|
5
|
3
|
5.781
|
10
|
1.231
|
26.8
|
24.1
|
|
Post-Tx
|
2
|
1
|
6.767
|
10
|
2.735
|
|
|
8
|
Pre-Tx
|
3
|
2
|
8.021
|
15
|
0.183
|
24.6
|
20.1
|
|
Post-Tx
|
1
|
1
|
1.265
|
< 5
|
0.597
|
|
|
9
|
Pre-Tx
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
26.0
|
|
Post-Tx
|
1
|
1.5
|
7.461
|
10
|
nd
|
|
|
10
|
Pre-Tx
|
8
|
4.5
|
15.664
|
5
|
1.248
|
21.1
|
15.3
|
|
Post-Tx
|
3
|
4
|
14.362
|
2
|
0.201
|
|
|
*P value
|
|
0.001
|
0.063
|
1.0
|
0.875
|
0.459
|
|
|
Since hepatic steatosis may be caused by HCV infection itself [23] or may be caused by other risk factors, such as obesity and metabolic syndrome [24], we compared the amount of steatosis present in each patient’s liver biopsy before and after DAA treatment using traditional microscopy (Fig. 3, A-D). Five study patients had macrovesicular steatosis that was estimated to involve greater than or equal to 5.0% of hepatocytes prior to DAA treatment (Table 2 and Figs. 1 and 3) (patients 2, 4, 7–8, and 10). The estimated percentage of hepatocytes involved by macrovesicular steatosis decreased in three patients (patients 2, 8, and 10), remained stable in one patient (patient 7), and increased in one patient (patient 4), post-treatment (Table 2). The percentage of hepatic steatosis “equation (2)” was more objectively calculated in these five study patients using a quantitative imaging software algorithm (Visiopharm), as described in the methods Sect. 2.4 (Fig. 3, E-H). Using this method, two of the patients (patients 2 and 10) had decreased steatosis (4.49- and 6.20-fold, respectively) and three patients (patients 4, 7, and 8) had increased steatosis (7.34-, 2.22-, and 3.26-fold, respectively) post-DAA treatment (Table 2, Fig. 1). Representative examples that showed an increase (patient 2) and a decrease (patient 4) in the percentage of hepatic steatosis after treatment, using the Visiopharm steatosis algorithms, are shown in Fig. 3. Although the steatosis assessments made by the pathologist were mostly in agreement with the Visiopharm software regarding the relative increases and decreases in each patient’s pre- and post-treatment liver biopsies (Table 2, see patients 2, 4, and 10), the imaging analysis algorithm showed much smaller percentages of hepatic steatosis overall and was better at detecting subtle variations (Table 2, see patients 7 and 8).
Next, we compared all the patients’ MHAI scores, Ishak fibrosis stages, CPA, and hepatic steatosis percentages before and after treatment with DAAs (Fig. 4). Figure 4 also shows the changes for each of these parameters in each individual patient’s pre- and post-treatment liver biopsy. The MHAI scores significantly decreased after treatment, with a mean of 5.4 and 2.3, in the pre- and post-treatment biopsies, respectively (P < 0.001) (Fig. 4A, Table 2). Comparison of Ishak fibrosis stages pre- and post-treatment did not show significant differences, with a mean of 2.6 and 2.1, respectively (P = 0.063) (Fig. 4C). Although the combined CPA calculations were not significantly different between the pre- and post-treatment groups (P = 1.0) (Fig. 4E), they showed more striking differences when the biopsies were compared on an individual basis for each patient before and after treatment (Fig. 4F). The majority of patients had decreased steatosis in their post-treatment liver biopsies when compared to the one collected prior to treatment (Fig. 4G-4J). However, since patient 4 had such a marked increase in hepatic steatosis in her post-treatment biopsy, no significant difference was observed (P > 0.05).
Clinical outcome
All patients achieved SVR in the study, regardless of regimen, and transaminases returned to baseline (Tables 1 and 3). Serum ALT levels decreased from a mean of 105.1 U/L (median, 65 U/L; SD, ± 108.7) to a mean of 29 U/L (median, 28.5 U/L; SD, ± 10.3; P = 0.015). Aspartate aminotransferase (AST) decreased from a mean of 96.5 U/L (median, 59.5 U/L; SD, ± 104.6) to a mean of 27 U/L (median, 25 U/L; SD, ± 7.4; P = 0.013). Alkaline phosphatase (ALP) levels decreased from a mean of 117.3 U/L (median, 115.5 U/L;SD, ± 34) to a mean of 104.2 U/L (median, 111 U/L; SD, ± 26.3; P = 0.033). Two patients (patients 2 and 10) had persistently elevated ALP after DAA treatment (Table 3). Two patients (patients 2 and 8) were co-infected with HIV (> 75 copies/ml) at the time of pre-DAA treatment. However, after clinical follow-up (1–3 years) these patients had undetectable HIV copies (Table 4).
Table 3
Evaluation of liver injury and function pre- and post-DAA therapy.
HCV
Pt #
|
Blood a
|
ALT
(9–51 U/L)
|
AST
(13–40 U/L)
|
ALP
(34–122 U/L)
|
Albumin
(3.5-5.0 g/dL)
|
Total Bili
(0.1–1.1 mg/dL)
|
Platelets
(150-328x103/µL)
|
PT
(12.0-14.7 s)
|
INR
(< 1.1)
|
1
|
Pre-Tx
|
90**
|
66**
|
93**
|
4.6
|
0.3
|
392**
|
14.3
|
1.1
|
|
Post-Tx
|
27
|
22
|
77
|
4.0
|
0.3
|
358**
|
12.4
|
0.9
|
2
|
Pre-Tx
|
352**
|
361**
|
140**
|
4.1
|
0.8
|
157
|
13.4
|
1.0
|
|
Post-Tx
|
22
|
16
|
136**
|
4.0
|
0.5
|
223
|
12.6
|
0.9
|
3
|
Pre-Tx
|
82**
|
70**
|
128**
|
4.2
|
0.3
|
276
|
12.7
|
1.0
|
|
Post-Tx
|
39
|
29
|
108
|
4.7
|
0.7
|
259
|
12.9
|
1.0
|
4
|
Pre-Tx
|
239**
|
168**
|
94
|
4.3
|
1.0
|
101**
|
15.4**
|
1.2**
|
|
Post-Tx
|
33
|
38
|
78
|
4.0
|
0.9
|
121**
|
14.4
|
1.1
|
5
|
Pre-Tx
|
40
|
29
|
108
|
4.4
|
0.5
|
322
|
12.7
|
0.9
|
|
Post-Tx
|
25
|
25
|
114
|
4.9
|
0.1
|
344**
|
12.7
|
1.0
|
6
|
Pre-Tx
|
28
|
30
|
123**
|
4.1
|
0.7
|
197
|
13.3
|
1.0
|
|
Post-Tx
|
23
|
33
|
114
|
4.3
|
0.5
|
198
|
13.7
|
1.1
|
7
|
Pre-Tx
|
48
|
53**
|
138**
|
4.4
|
0.8
|
194
|
12.2
|
0.9
|
|
Post-Tx
|
30
|
25
|
120
|
4.0
|
0.3
|
210
|
13.8
|
1.1
|
8
|
Pre-Tx
|
15
|
23
|
99
|
4.0
|
0.5
|
159
|
12.6
|
0.9
|
|
Post-Tx
|
33
|
24
|
105
|
4.5
|
0.6
|
200
|
12.3
|
0.9
|
9
|
Pre-Tx
|
35
|
32
|
63
|
4.4
|
0.6
|
145**
|
13.0
|
1.0
|
|
Post-Tx
|
48
|
38
|
55
|
4.3
|
0.9
|
240
|
12.7
|
1.0
|
10
|
Pre-Tx
|
122**
|
133**
|
187**
|
4.3
|
0.8
|
179
|
13.2
|
1.0
|
|
Post-Tx
|
10
|
20
|
135**
|
4.0
|
0.5
|
247
|
13.8
|
1.1
|
*P value
|
|
0.015
|
0.013
|
0.033
|
0.940
|
0.316
|
0.057
|
0.634
|
0.798
|
Table 4
Clinical outcome of study patients.
Pt #
|
Data
Collection
|
HIV viral load (copies/mL)
|
Cancer
|
Ascites
|
SBP
|
Varices
|
Spleno-
megaly
|
Jaundice
|
Icterus
|
Encepha-lopathy
|
Coagulo-pathy
|
HRS
|
HPS
|
1
|
Pre-Tx
|
nd
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
nd
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
2
|
Pre-Tx
|
2471.0
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
nd
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
nd
|
nd
|
nd
|
nd
|
nd
|
Yes***
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
3
|
Pre-Tx
|
< 75
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
4*
|
Pre-Tx
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
Yes***
|
nd
|
nd
|
Post-Tx
|
nd
|
LI-RADs 2
|
No
|
No
|
Yes***
|
Yes***
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
HCC**
|
Yes***
|
No
|
Yes***
|
Yes***
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
5
|
Pre-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
6
|
Pre-Tx
|
< 75
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
7
|
Pre-Tx
|
< 75
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
No
|
No
|
No
|
8
|
Pre-Tx
|
27008
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
9
|
Pre-Tx
|
< 40
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
66
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
No
|
No
|
No
|
10*
|
Pre-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Post-Tx
|
Not detected
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
1–3 dfu
|
Not detected
|
cholangio-
carcinoma**
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes***
|
No
|
No
|
No
|
To assess for markers of liver injury and function, we documented levels of hepatic enzymes, albumin, prothrombin time (PT)/international normalized ratio (INR), and total bilirubin (Table 3). To determine if portal hypertension was likely, we documented platelet numbers and the presence or absence of ascites, esophageal varices, splenomegaly, and other manifestations of end-stage liver disease (Tables 3 and 4). Patient 4 had mild thrombocytopenia and an elevated PT (15.4 s) that improved after DAA treatment. Even though most of the patients had platelets in the normal range prior to treatment, the levels slightly increased after treatment, with means of (212.2 and 240 x 103/µL), respectively (P = 0.057). Patient 1 also had mild, persistent thrombocytosis pre- (392 x 103/µL) and post- (358 x 103/µL) DAA treatment (Table 3). Despite achieving SVR, several patients had persistent or new development of splenomegaly (patients 2, 4, 7 and 9). We also evaluated the patients for evidence of hepatic decompensation and other complications of chronic liver disease, including the presence or absence of ascites, spontaneous bacterial peritonitis, esophageal varices, jaundice, scleral icterus, hepatic encephalopathy, hepatorenal syndrome, and hepatopulmonary syndrome (Table 4). Three patients developed hepatic encephalopathy at follow-up (patients 4, 5, and 10). All patients had normal albumin and total bilirubin levels pre- and post-treatment.
Patient 4, a non-Hispanic Caucasian female that had an increased BMI, was the only patient who had well-developed cirrhosis at the time of her first biopsy. This patient started alpha-interferon/Ribavirin approximately three years after her first biopsy was obtained and failed treatment. Two years later, she was approved for DAA treatment and was given Sofosbuvir/Ribavirin for a duration of 12 weeks and successfully achieved SVR. Even though her her liver enzymes initially normalized (Table 3), her post-treatment liver biopsy showed a 4.7-fold higher percentage of fibrosis using the CPA calculation and a 7.34-fold increase in macrovesicular steatosis (Table 2 and Fig. 1–4). Her post-treatment liver biopsy also showed large areas of parenchymal extinction that had substantially increased from her initial, pre-treatment biopsy (Figs. 1 and 2). Two years after achieving SVR, she developed several clinical manifestations of portal hypertension and decompensated liver disease, including ascites, esophageal varices, splenomegaly, and hepatic encephalopathy (Table 4). Initial screening for HCC by CT scan post-treatment showed a liver lesion that was “probably benign” (i.e., a Liver imaging reporting and data system (LI-RADS) score of 2), however, HCC (LI-RADS score of 5) was diagnosed during clinical follow up at 3.5 years post-treatment and the patient died approximately 6 years after achieving SVR. A single patient (patient 10) developed cholangiocarcinoma with metastasis, two years after finishing her DAA treatment regimen, and died 4 years after achieving SVR.