# Persistence of Hepatitis C RNA in Liver Allografts Is  Associated with Histologic Progression Independent of  Serologic  Viral Clearance

Authors: M. Ghabril; R. C. Dickson; M. Krishna; R. Lloyd; J. Aranda-Michel; A. Keaveny; R. Satyanarayana; H. Bonatti
Journal: Journal of Transplantation
 (2009)

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## Abstract

Background. Hepatitis C virus (HCV) nondetectability in the liver may predict a sustained viral response (SVR) in patients with an end of treatment response. HCV RNA can be detected in liver tissue by in situ hybridization (ISH). Aim. To determine if HCV nondetectability in liver allografts by ISH can predict SVR in patients who cleared virus serologically on treatment. Methods. Twenty five patients with undetectable serum HCV on Interferon/Ribavirin therapy for HCV recurrence post liver transplant (LT) were studied. All had biopsies at 4 months post LT (baseline) and follow up with HCV ISH analysis performed. Results. 10 were ISH positive (group 1); 15 were ISH negative (group 2). Groups 1 and 2 had similar patient, donor, and viral characteristics at LT, as well as treatment duration at the time of the ISH assayed liver biopsy (13±16 versus 10±4 months P = .24). However, group 1 had longer total treatment duration (24±10 versus 14±5 months, P = .001). Eight (80%) group 1 and 9 (60%) group 2 patients achieved SVR. Mean grade and stage (modified Ishak score) were similar at 4 months, however, group 1 had higher grade (3±1.7 versus 1.6±1.3, P = .039) and stage (1.4±1.4 versus 0.5±0.6, P =  .084) on the ISH assayed biopsy, after similar post LT intervals (23±10 versus 24±12 months, P = .91). Conclusion. Allograft HCV ISH nondetectability does not predict SVR in treatment responsive HCV recurrence, but is associated with less severe histologic disease.


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## Body

## 1. Introduction

Hepatitis C virus (HCV) is the most common indication for liver transplantation (LT) in the US
with almost
universal recurrence following LT. The need for  antiviral therapy is common with
up to 30% of patients progressing to cirrhosis by
5 years [1].   Though the indication for antiviral therapy is
based on histologic findings, the primary goal of treatment goal is
serologically defined by a sustained viral response (SVR).The absence of HCV RNA in liver
tissue at the end of treatment has been associated with SVR in HCV patients
with chronic liver disease treated with interferon-based antiviral
therapy [2]. A
recently reported series in HCV LT patients suggested that the presence
or absence of allograft HCV RNA following treatment predicted a relapse or SVR
in patients with a loss of viremia at the end of treatment. All 7 patients with
a negative hepatic HCV RT-PCR at the end of treatment had an SVR while 3
patients with HCV RT-PCR present had
relapse [3].However, SVR status has not
uniformly resulted in histologic stabilization or fibrosis regression. It has
been observed in a previous report that twenty percent of post LT HCV patients
experienced fibrosis progression 3–5 years following SVR
[4, 5],  while fibrosis regression has been described
in treated patients without SVR [6]. Additionally, hepatic HCV RNA persistence has
been described in patients with SVR [7]. The correlation of hepatic allograft HCV RNA
detectability post LT with serum virologic endpoints and histologic outcomes
remains uncertain.HCV RNA can be detected in liver
tissue by polymerase chain reaction (PCR), immunohistochemical, and in situ
hybridization (ISH) assays. HCV ISH utilizes digoxigenin labeled riboprobes
with sense (genomic) and antisense (replicative intermediate) transcripts
corresponding to the5′  noncoding region of the virus. It can be performed on
paraffin-embedded sections, with positive and  negative controls, and yields a
qualitative result. It has been shown to correlate well with tissue HCV RNA
detection by PCR [8].The primary aim of this study was to
determine if HCV nondetectability in liver allografts by ISH can
predict SVR in patients who cleared virus serologically on antiviral treatment.
A secondary aim was to determine the correlation of allograft HCV-ISH status
with histologic disease in this patient subset.


## 2. Patients and Methods

We retrospectively reviewed the records of HCV patients undergoing LT
at our center between February 1998 and December 2005. The HCV ISH assay became
available at our institution in January 2003. It was performed in selected
patients with undetectable serum HCV by PCR while receiving
antiviral therapy. These patients were further analyzed.
Data was retrospectively collected through April 2007. The study was
approved by the study centers Institutional Review Board.The ISH assay was performed as previously
described [8]. Patients
were grouped by HCV ISH status (positive or negative), and compared for  (1) patient and donor characteristics.  (2) Antiviral therapy and virologic outcomes
of early viral response (EVR), end of treatment response (ETR) and SVR. SVR was
defined as undetectable HCV RNA in serum by qualitative assay ≥6 months after completion
of antiviral therapy.  (3) Histologic
findings of grade and stage at baseline 4 months post LT (protocol biopsy) and
at the ISH assayed biopsy. Grading and staging of all biopsies were performed
using the modified Ishak score by a single pathologist (MK) who was blinded to
the ISH result, serum viral status, and clinical
findings [9].All LT procedures were performed using piggy back technique. Initial
immunosuppression included a 3 drug regimen of tacrolimus, mycophenolate
mofetil and prednisone, and tacrolimus monotherapy after 4 months post LT. Protocol
liver biopsies were performed at 7 days, 4 months, and annually post LT, and as
clinically indicated.  Antiviral therapy
using Interferon (interferon alfa 2b prior to 2002 and peginterferon alfa 2a
since 2002) and ribavirin was initiated for significant HCV recurrence. HCV
recurrence was defined as liver enzyme elevation ≥2× the upper limits of
normal, detectable serum HCV RNA, and histologic features consistent with
hepatitis C with Batts Ludwig activity score of ≥2 and/or progressive fibrosis.
Minimum planned duration of therapy was 48 weeks for genotypes 1 and 4, and 24
weeks for genotypes 2 and 3. Growth factors were used as clinically indicated.
Prior to 2005 serum HCV detectability was determined by the Roche COBAS
Amplicor HCV Monitor 2.0 assay (Roche Diagnostics, Indianapolis, IN) with a
lower limit of detection of 50 IU/mL.
Since 2005 serum HCV was assayed with the COBAS TaqMan HCV Test
(TaqMan HCV; Roche Molecular SystemsInc., Branchburg, N.J.)
with a dynamic range of 10 IU/mL to 50,000,000 IU/mL.


## 3. Statistical Analysis

Patient groups were compared using the Mann-Whitney and chi-square
tests. SPSS 14.0 (SPSS Inc., Chicago,
IL) was used for the analysis.
AllP  values were 2 tailed, and P<.05  was considered statistically
significant. All values are shown as mean ± 1 SD, or percentage unless
otherwise specified.


## 4. Results

LT for HCV were performed in 460 patients
between 1998 and 2005 at our center and 231 (50%) underwent antiviral
treatment. Of the treated patients 73 (31.6%) had an on treatment response, 44
had an SVR, 16 relapsed and 13 remained on treatment.  HCV ISH assays of liver biopsies were
performed prospectively in 26/73 (36%) of the patients with undetectable serum
HCV by PCR while on treatment between July 2004 and June 2006. Ten patients
were ISH positive (group 1), 15 were ISH negative (group 2), and 1 was excluded
due to indeterminate ISH results. Serum HCV was not detectable at the time of
ISH assayed liver biopsies based on the highly sensitive COBAS Taqman serum HCV
RNA assay in 22 (88%) of the 25 patients since 2005, and based on the COBAS
Amplicor HCV Monitor assay in 3 (12%) patients before 2005.Groups 1 and 2 were similar for patient,
donor, and viral characteristics, with the exception of a trend toward more
female patients in group 1 (Table1). Antiviral therapy timing, duration at the
time of the ISH assayed biopsy, treatment tolerance, and virologic outcomes were
similar for groups 1 and 2, with the exception of longer total treatment
duration in group 1 (Table 2). All patients received at least the minimum
planned duration of treatment per genotype. Eight (80%) group 1 patients
achieved SVR, 1 (10%) relapsed and 1 (10%) remained on therapy with
undetectable serum HCV at last follow up. Nine (60%) group 2 patients achieved
SVR, and 6 (40%) relapsed.Table 1
Patient and donor characteristics, and
antiviral therapy at the time the ISH biopsy in HCV ISH positive (group 1) and
negative (group 2) patients.
VariablesGroup 1  ISH positiveN=10Group 2  ISH negativeN=15PPatient age at LT49 ±
1152 ±
11.66Female gender (patient)40%13%.13Caucasian (patient)90%87%.69Recipient body mass index31 ±
729 ±
6.51MELD at LT14 ±
518 ±
9.42Genotype 1 (unknown in 1 patient per group)56%86%.11Donor age47 ±
1343 ±
14.82Female gender (donor)40%53%.51Caucasian (Donor)90%67%.34Cold ischemia time, hours7 ±
27 ±
2.74Warm ischemia time, minutes39 ±
934 ±
10.28Tacrolimus as primary immune suppressant100%83%.23Steroid bolus treated ACR30%40%.61LT to ISH biopsy interval, months23 ±
1024 ±
12.91Table 2
Antiviral therapy timing, duration, dose
reductions, growth factor use, and virologic outcomes in treated group 1 and 2
patients.
VariableGroup 1  ISH positiveN=10Group 2  ISH negativeN=15PPegylated IFN100%86%.23Dose reduction40%33%.73Growth factors used80%73%.70LT-Treatment interval, months6 ±
39 ±
7.35Treatment duration at ISH biopsy months13 ±
610 ±
4.24Total treatment duration24 ± 1014 ±
5.001EVR90%86%.75ETR90%*100%NASVR80%*60%.291* patient was
still on treatment at last follow up.Antiviral treatment outcomes collated by
patient group, HCV genotype, duration of treatment at the time of ISH assayed
biopsy, total treatment duration, and timing of virologic response are described
for each case in Table3. After 12 weeks of therapy, HCV RNA was undetectable
by qualitative assay in 11 patients, and detectable in 8 (5 of whom had EVR). A
qualitative assay at 12 weeks was not performed in 6 patients (5 of whom had
EVR). Eight of  the 11 patients with
undetectable serum HCV by 12 weeks of therapy achieved SVR with treatment of at
least the minimum planned duration per genotype, while 2 relapsed, and 1 remained
on therapy for low levels of detectable HCV (<10 IU/mL) after >6 months
of therapy. Seven of the 8 patients with detectable HCV at 12 weeks had
undetectable HCV by week 24.  Four of
these 7 patients received ≥24 weeks of additional therapy beyond their minimum
planned treatment duration per genotype, 3 achieved SVR, and 1 relapsed. The
other 3 patients received <24 weeks of additional therapy, and all 3
relapsed. One patient with detectable serum HCV at week 24 became undetectable
at 19 months, and achieved SVR after a total of 33 months of therapy.Table 3
Antiviral treatment duration and outcomes
collated by patient group, HCV genotype, timing of ISH assayed biopsy and
virologic response.
Group, genotype and case numberISH StatusTreatment duration at ISH assay (months)Total treatment duration (months)Viral load responseHCV undetectable by 12 weeksHCV undetectable by 24 weeksOutcomeGroup 1,genotype 2 or 31+1212EVRNoYesSVR2+1218EVRNAYesSVR3+2126RVRYesSVR4+1437EVRYesOn therapyGroup 1,genotype 1 or unknown*5+418EVRNoYesRelapse6+519EVRYesSVR7*+918EVRYesSVR8+1421No EVRNoYesSVR9+2146EVRNAYesSVR10+1322EVRYesSVRGroup 2,genotype 2 or 311−711RVRYesSVR12−99No EVRNoYesRelapseGroup 2,genotype 1 or unknown*13−1011EVRNAYesSVR14−812EVRNAYesRelapse15−611RVRYesSVR16*−812EVRYesSVR17−1013EVRYesRelapse18−1414EVRNAYesSVR19−1112EVRNoYesRelapse20−2033No EVRNoNoSVR21−312EVRYesSVR22−1414NANAYesSVR23−712EVRNoYesRelapse24−1317EVRNoYesSVR25−1213EVRYesRelapseNA: Not available; EVR: Early virologic response; RVR: Rapid virologic response; SVR: Sustained viral response.*The patients that did not have a known genotype.ALT, histologic grade, and stage were similar
for the 2 groups at 4 months post LT; however, at the time of the follow up ISH
biopsy they were higher in group 1 patients (Table4). There were no deaths
during the study period.Table 4
HCV viral loads, ALT, and modified Ishak
score grade and stage for groups 1 and 2, at 4 months post LT and at the ISH
biopsy.
VariablesGroup 1  ISH positiveN=10Group 2  ISH negativeN=15P4 month HCV titer x1,000,000 IU/ml1.2 ±  152.4 ± 4.4.324 month ALT IU126 ±
92168 ±
205.684 month grade3.6  ±  2.53.6 ± 2.7.94 month stage1  ±
1.20.5 ± 0.5.394 month to ISH biopsy interval, months19  ±
1019 ±
12.93ISH biopsy ALT IU69  ±
4532 ±
27.016ISH biopsy grade3  ±
1.71.6 ± 1.3.039ISH biopsy stage1.4  ±  1.40.5 ± 0.6.084


## 5. Discussion

The data from this study supports two
primary findings. First the absence of allograft HCV RNA by ISH did not predict SVR in patients with an ETR. Second, there was a
correlation of liver allograft HCV RNA detectability by ISH with
increased disease activity and fibrosis in the liver allograft. This finding
was independent of serologic viral clearance.Viral relapse in 7 of 14 patients with
undetectable hepatic HCV RNA by
ISH on or at the end of completed treatment was unexpected. A lack of
sensitivity of the ISH assay is
one possibility. However, while there is no gold standard to define HCV
detectability in liver tissue, the ISH assay has been shown to
correlate well with tissue PCR techniques and immunohistochemical
assays [8]. Sampling
variation has been described in the grading and staging of liver biopsies
specimens in patients with chronic
HCV [10]. Thus,
this could also account for these findings as the ISH assay was taken from only a single liver biopsy specimen. Extrahepatic
compartmentalization of HCV has been well described and may theoretically
explain these contradictory
findings [7, 11]. Extrahepatic
sources may provide alternative viral reservoirs and account for reinfection of
the liver following HCV RNA clearance from the liver and serum while on
treatment.  The concept of extrahepatic
reservoirs of hepatotropic viruses has been well described with Hepatitis B
virus (HBV). HBV DNA has been found in serum and lymphocytes many years
following successful LT despite no clinical evidence of HBV
recurrence [12, 13].Virologic relapse in those with an ETR and
undetectable hepatic HCV by ISH post LT in our series is at odds with the
finding of SVR in all 7 patients with an ETR and undetectable hepatic HCV by
PCR post LT in a prior study [3]. Both
series suffer from small sample size, but a difference in the assay of hepatic
HCV may account for the difference.  ISH
involves in situ detection of viral RNA in hepatocytes, whereas PCR analysis
of homogenized liver tissue would include some circulating lymphocytes
harboring virus.Our series comprised a select group
including only patients with loss of HCV serum RNA on treatment, thus the
higher than expected SVR rates were anticipated. There was a statistically
insignificant but numerically higher rate of SVR in the ISH positive versus
negative group, 80%  versus 60%, respectively. However, the differences were most
likely due to the longer duration of antiviral treatment given and a higher
percentage of genotypes 2 or 3 in patients with positive ISH (44% versus 14%). Longer duration
of therapy in group 1 patients was attributed to a treatment bias based on the
finding of positive HCV ISH, and likely reduced relapse rates in the subset of
patients with EVR, detectable HCV at 12 weeks, and undetectable HCV at 24
weeks [14, 15]. Interestingly,
the patient with detectable serum HCV at 24 weeks cleared virus late into
treatment and achieved SVR.The second major finding of the study was
the correlation of hepatic HCV RNA detectability by ISH with increased
histologic activity despite similar demographic factors in the ISH positive and
negative groups. The presence of HCV RNA in native liver tissue has been
associated with increased histologic necroinflammatory activity and fibrosis in
patients with chronic liver disease secondary to
HCV [16–19]. Post LT, Neff et al. noted that 6 of 7
patients with detectable hepatic HCV by RT-PCR had grade 1-2 inflammation at
the end of treatment while in 3 of 4 patients with no inflammation the HCV RT-PCR
in the liver was undetectable [3]. Our data may suggest an important role of
hepatic HCV RNA in eliciting or maintaining an immune response regardless of a
loss of HCV RNA in the serum. Allograft histologic progression has been
described in 20% of patients three to five years following serologic SVR [4, 5], perhaps this could be
accounted for by remnant hepatic HCV RNA.In summary, this is a retrospective study
and is limited by sample size, but it suggests that hepatic HCV detectability
has limited value for predicting sustained virologic response in post LT HCV
patients achieving loss of HCV RNA on current antiviral therapy. The
correlation of hepatic HCV RNA with histologic activity represents a
preliminary finding which merits further investigation.



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