Download Iron Deposition and Progression of Disease in Chronic Hepatitis C

Anatomic Pathology / IRON AND HFE MUTATIONS IN CHRONIC HEPATITIS C
Iron Deposition and Progression of Disease
in Chronic Hepatitis C
Role of Interface Hepatitis, Portal Inflammation, and HFE Missense
Mutations
Mario Pirisi, MD,1 Cathryn A. Scott, MD,2 Claudio Avellini, MD,2 Pierluigi Toniutto, MD,1
Carlo Fabris, MD,1 Giorgio Soardo, MD,1 Carlo A. Beltrami, MD,2 Ettore Bartoli, MD1
Key Words: Hereditary hemochromatosis; Chronic hepatitis C; Iron; Histologic activity index; Stage; Grade; HFE mutations
Abstract
Histologically detectable iron (HDI) and HFE
mutations were searched for in liver biopsy specimens
from 58 Italian patients with chronic hepatitis C, and
morphologic features were compared to examine their
reciprocal relation and their contribution to disease
progression. HDI was evident in 48% of cases with
features of nonhemochromatosis iron overload. Total,
sinusoidal, and portal HDI increased with stage; grade
was related to all iron scores because of the contribution of portal inflammation and interface hepatitis. HFE
mutations were seen in 47% of patients with chronic
hepatitis C and in 28% of control subjects; they were
related to stage and the His63Asp mutation to portal
HDI. On multivariate analysis, grade but not stage or
HFE mutations was associated with HDI in all sites.
Interface hepatitis with its sequelae (sinusoidal
capillarization and microshunting) represents a major
factor in iron deposition in chronic hepatitis C and
justifies the features of HDI. HFE mutations are not
responsible for HDI deposition but could favor the
progression of virus-induced damage independently
from interference with iron metabolism.
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Excessive liver iron deposition in overload conditions,
genetic and iatrogenic, can lead to progressive architectural
damage, including cirrhosis, through the induction of oxidative stress.1 The pathogenetic role of iron, however, goes
beyond that seen in classic overload conditions.2 In fact,
excess iron is present in up to 67% of nonbiliary cirrhoses3;
furthermore, histologically detectable iron (HDI), using the
Perl Prussian blue method, frequently is found in biopsy
specimens of patients with chronic viral hepatitis C.4-13
In chronic hepatitis C, HDI usually is sparse and patchy,8
and hepatic iron concentration lies between the middle and
upper limit of the reference range.5,10,14 A slightly increased
liver hepatic index is present in up to 10% of cases.14 Nevertheless, the presence and distribution of iron in the liver of
these patients is claimed to predict a negative response to
interferon therapy.5,6,8,10-12
In normal livers, hepatocytes are the deposition site for
iron originating from gastrointestinal absorption, while
Kupffer cells are the storing location for iron bound to ferritin
and hemosiderin and released because of turnover or damage
to tissues, including the liver.15 In hereditary hemochromatosis (HH), an autosomal-recessive iron overload disease,
iron is accumulated in hepatocytes and, subsequently, in
Kupffer cells,15 both because of increased gastrointestinal
absorption and of greater cellular uptake.16 Two types of
altered molecules are thought to be responsible for the phenotypic expression of HH: 1 is the product of the divalent metal
transporter (DMT-1) gene,16 which transports iron from the
intestinal lumen into enterocytes and favors the passage of
transferrin receptor–cycled iron from the endosome into the
cell cytoplasm; the other is the product of the HFE gene,
which represses transferrin uptake and, therefore, intracellular
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
iron accumulation, by forming a stable complex with the
transferrin receptor.16,17 In humans, 2 missense mutations for
the HFE gene, Cys282Tyr and His63Asp, have been
identified18; the former is present in a minimum of 83%
patients with HH.18
In chronic viral hepatitis, iron is thought to accumulate
mainly because of a damage-release process, 8-10,19,20
whereby infected hepatocytes release hemosiderin that is
taken up by Kupffer cells, although other mechanisms, such
as hepatocyte regeneration, cytokine release, alterations in
iron uptake owing to chronic necroinflammation, and intrahepatic shunting, also have been implicated.21
Because of the high estimated carrier frequency of
mutations for HH22 and the high prevalence of hepatitis B
virus and hepatitis C virus (HCV) markers in patients with
clinically overt HH,23-25 the association between iron deposition and HFE mutations has been examined in patients with
chronic viral hepatitis C of different ethnic groups26-30 based
on the assumption that iron accumulation could be accounted
for by coincident carriage of mutations for HH. The influence of HFE mutations on HDI in chronic hepatitis C has
been variable26-30; 1 of the confounding factors is that the 2
HFE gene mutations show a different ethnic distribution in
HH and in control subjects.16,31 In fact, patients with HH
who are of Celtic and Nordic descent show the highest
prevalence of homozygosity for the Cys282Tyr mutation,16,18
which is seen in only 64% of Italian patients with HH,31
while the His63Asp mutation is present only in a minority of
cases.16,18 Last, there are HH cases lacking the mutations and
any other cause of secondary iron overload.32
The aim of the present study was to verify the existence
of an association between carriage of HFE mutations and
iron deposition in a series of 58 consecutive Italian patients
with chronic viral hepatitis C and to examine how the mutations and the morphologic features indicative of disease
progression according to Ishak et al33 influence cellular site
and zonal distribution of HDI deposition within the liver.8
Materials and Methods
Patients
Fifty-eight consecutive nonselected patients, aged 14 to
67 years, referred to our institution for complete diagnostic
workup because of suspected liver disease, were studied. All
were anti–HCV antibody–positive and underwent needle
biopsy of the liver. The demographic and clinical characteristics are given in ❚Table 1❚. None had evidence of other causes
of liver disease (viruses other than HCV, autoimmunity,
toxins and drugs, genetic other than HH), and none had clinical or laboratory evidence consistent with iron overload or
© American Society of Clinical Pathologists
❚Table 1❚
Demographic and Clinical Characteristics for 58 Patients*
Variable
Value
Age (y)
Male/female ratio
Alcohol consumption (any degree)
No
Yes
Albumin (g/L)
Bilirubin (µmol/L)
Iron (µmol/L)
Transferrin saturation (%)
Ferritin (µg/L)
Serum HCV RNA positive
Genotype 1a
Genotype 1b
Genotype 2a
Genotype 2b
Genotype 3a
47.8 ± 12.2
36:22
19
39
46.7 ± 2.8
14 (5-31)
25 (11-61)
29.3 (11.4-77.1)
160 (1-954)
51
9
25
7
8
2
HCV, hepatitis C virus.
* Categoric variables are given as frequencies; continuous variables with normal
distribution as mean ± SD; and continuous variables with nonnormal distribution
as median (range). Laboratory data are given in Système International units;
conversions to conventional units are as follows: albumin (g/dL34), divide by 10;
bilirubin (mg/dL), divide by 17.1; iron (µg/dL), divide by 0.179; and ferritin
(ng/mL), divide by 1.0.
had received interferon therapy before biopsy. A history of
alcohol consumption did not exclude enrollment in the study;
in particular, 18 patients reported a daily intake of more than
40 g. However, all patients had to observe a 6-month abstinence period before biopsy.
Biohumoral Determinations
Serum anti-HCV antibodies were tested by using a thirdgeneration enzyme immunoassay (Ortho Diagnostics, Raritan,
NJ); positive test results were confirmed by immunoblotting
(HCV Matrix, Abbott, Abbott Park, IL). Circulating HCV
RNA was detected by an in-house reverse transcriptase–polymerase chain reaction (PCR) assay, with nucleotide primers
derived from the 5´ noncoding region of HCV. HCV genotyping was performed by hybridization of PCR products of the
5´ noncoding region of the HCV genome with type-specific
probes.34 Ferritin was determined by an immunoenzymatic
method (Ferritin, Axsym System, Abbott).
Histologic Examination
All liver biopsies were obtained by using the Menghini
technique; they were at least 15-mm long and included at
least 4 portal tracts. Specimens were fixed in formalin and
embedded in paraffin. Slides were stained with H&E, periodic acid–Schiff with and without diastase predigestion,
Gomori reticulin stain, and Perl Prussian blue method and
evaluated by 2 experienced pathologists (C.A.S. and C.A.)
without knowledge of clinical and laboratory data.
All biopsy specimens were staged and graded
according to the criteria of Ishak et al.33 Grading was given
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by the sum of scores obtained evaluating the following 4
features: periportal or periseptal interface hepatitis;
confluent necrosis; focal lytic necrosis, apoptosis, and focal
inflammation (from here on referred to as acinar necrosis);
and portal inflammation.
Presence of HDI deposits was estimated according to
the system of Barton et al,8 which evaluates iron separately
in hepatocytes, sinusoids, and portal tracts. Hepatocyte and
sinusoidal scoring is based on the separate evaluation of the
presence of HDI deposits in each liver zone and on the
extent (none, less than one third, between one third and two
thirds, more than two thirds, all) of total zones involved.
Portal tract scoring is estimated separately in connective
tissue, vascular walls, and bile duct cells on the basis of the
relative proportion of portal areas involved (none, less than
one third, between one third and two thirds, more than two
thirds, all). The total iron score is given by the sum of each
score area.
women, aged 50.0 ± 12.3 years), living in the same geographic
area as the anti–HCV-positive patients, were tested.
Molecular Analysis of HFE Mutations
The presence of missense mutations in the HFE gene
(Cys282Tyr and His63Asp) was verified by means of
restriction fragment length polymorphism of PCR
products 35 on liver fragments obtained from paraffin
blocks. When adequate liver biopsy specimens were not
available, the presence of the missense mutations was evaluated on the patient’s mononuclear cells.
Oligonucleotide primers for amplification of the
Cys282Tyr and His63Asp loci were synthesized according
to previously reported sequences.18 Amplification, digestion, and visualization of the PCR products were
performed as previously reported.35
To determine the rates of the Cys282Tyr and
His63Asp mutations in a reference population, 138
anti–HCV-negative, healthy blood donors (84 men, 54
Results
Statistical Analysis
Statistical analysis of the data was performed by using
the BMDP/Dynamic statistical software package, release 7.0
(Statistical Software, Cork, Ireland). The Shapiro and Wilk
W test was applied to test normality of continuous data. The
associations between variables were analyzed by performing
Pearson chi-square tests. Differences among continuous variables were evaluated by using the Student t test and KruskalWallis test, as appropriate. Logistic regression analysis with
a stepwise forward approach was applied to determine the
variables independently associated with presence of HDI.
Values of P were considered significant when equal to or
below .05.
Morphologic Findings
❚Table 2❚ shows the distribution of cases by stage and,
within each stage, the percentage distribution by score for
each grading parameter; the median and range values of
grade also are given. Forty-five percent of cases were stage
1. Grade was significantly and positively related to stage.
Interface hepatitis and portal inflammation were distributed
mostly in low scores in the first 2 stages and progressively
shifted to higher ones with increasing stage; acinar necrosis,
regardless of stage, was distributed mostly in scores 1 and 2;
confluent necrosis was seen in 5 of 58 biopsy specimens and
increased significantly with stage.
HDI deposits were observed in liver biopsy specimens
from 28 (48%) of 58 patients; they were found in portal triads
❚Table 2❚
Distribution of Cases*
Distribution by Score
Interface Hepatitis
Stage
1
2
3
4
5
6
*
No. (%)
ofCases
26 (45)
11 (19)
7 (12)
4 (7)
6 (10)
4 (7)
Grade,
Median
(Range)
3 (1-7)
5 (1-8)
6 (3-9)
6 (4-8)
8.5 (5-10)
9.5 (9-11)
Confluent
Necrosis
Acinar Necrosis
0
1
2
3
4
0
1
2
3
0
69
18
0
0
0
0
27
55
43
50
17
0
0
18
43
25
17
50
4
9
14
25
33
0
0
0
0
0
33
50
11
9
0
0
0
0
54
18
14
50
33
25
31
73
86
50
50
50
4
0
0
0
17
25
100
100
100
50
83
50
Portal Inflammation
1
5
1
2
3
4
0
0
0
50
17
25
0
0
0
0
0
25
92
37
15
0
0
0
4
36
57
75
17
25
4
9
14
25
33
25
0
18
14
0
50
50
By stage according to Ishak et al33 and, within each stage, by score percentage distribution within each grading parameter. Median and range values for grade also are given.
Comparison of stage by grade and grading parameters: grade, Kruskal-Wallis = 37.6 (P = .0001); interface hepatitis, chi-square = 57.8 (P = .0001); acinar necrosis, chi-square
17.2 (P = .305); confluent necrosis, chi-square = 31.9 (P = .0004); and portal inflammation, chi-square, 49.9 (P = .0001).
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© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
in 22 cases, in hepatocytes in 19, and in sinusoids in 19. HDI
deposits appeared as fine, blue, intracytoplasmic granules
within hepatocytes, Kupffer cells, endothelial cells lining
venules, and macrophages in portal tracts; connective tissue
interstitial deposition also was seen in portal triads. HDI
deposits were sparse and patchy in distribution in all iron
score sites, even when deposition involved all zones; portal
tract HDI also was evident in cases lacking extensive acinar
deposition. No HDI was found in bile duct cells.
As stage increased, the total iron score rose progressively (Kruskal-Wallis, P = .004). In the first 2 stages,
median values were extremely low (0 and 1), but they
increased steadily in stages higher than 2, reaching a
maximum of 5.5.
Stage, grade, and the grading parameters were divided
using their median value as the cutoff (2 for staging, 4 for
grading, 1 for interface hepatitis, 0 for confluent necrosis, 2
for both acinar necrosis and portal inflammation) and
compared with the presence and absence of total, hepatocytic, sinusoidal, and portal HDI by means of chi-square
tests. Stage was related positively to sinusoidal, portal, and
total iron scores; grade was associated positively with all 4
iron scores ❚Table 3❚. Interface hepatitis and portal inflammation were related positively to the total iron score (chisquare, P = .007 and .024, respectively) and portal iron score
(chi-square, P = .006 and .008, respectively); both were
unrelated to hepatocytic and sinusoidal iron scores. Acinar
and confluent necrosis showed no association with any of the
iron score areas or with total iron score.
❚Table 4❚ shows the percentage distribution of negative
and positive cases for hepatocytic and sinusoidal iron in the
3 acinar zones in stages from 1 to 4, regardless of relative
proportion of biopsy specimen involvement. Hepatocytic
and sinusoidal iron increased greatly in zones 1 and 2 in
stages from 2 to 4; in zone 3, hepatocytic iron increased
only in stage 4.
HDI in Relation to Age and Sex
Patients with evidence of HDI had a significantly higher
mean age than those without (51.4 ± 9.3 vs 44.5 ± 13.7;
Student t test, P = .028). Significantly fewer women showed
HDI than men (5/22 vs 23/36; chi-square, P = .002).
Alcohol Consumption in Relation to Stage, Grade, and
Iron Scores
Thirty-nine patients consumed alcohol (drinkers), and
19 did not (nondrinkers). HDI deposits were documented in
23 of 39 drinkers vs 5 of 19 nondrinkers (chi-square, P =
.020). Sinusoidal HDI was found in 18 of 39 drinkers vs 1 of
19 nondrinkers (chi-square, P = .002). The association of
drinking status with stage (chi-square for trend, P = .106)
and with grade (Mann-Whitney U, P = .070) did not reach
statistical significance.
HFE Mutations
In patients with hepatitis C, homozygosity for the
Cys282Tyr mutation was seen in 1 patient (2%) and
heterozygosity in 5 (9%) of 58 patients; homozygosity for
the His63Asp mutation was present in 1 patient (2%), and
heterozygosity was found in 16 (28%) of 58 patients. Four
patients were compound heterozygotes (7%). No mutations
were found in 31 patients (53%). In the control population,
no Cys282Tyr homozygotes were present. The prevalence of
heterozygosity for the Cys282Tyr mutation was 2 of 138
(1.4%), 2 subjects were homozygous for His63Asp (1.4%),
and the prevalence of heterozygosity was 34 of 138 (24.6%).
No double heterozygotes were found, and no mutations were
seen in 100 subjects (72.5%).
❚Table 3❚
Histologically Detectable Iron (HDI) in Liver Biopsy Specimens of Patients With Chronic Hepatitis C in Relation to Stage and
Grade*
Stage
HDI
Portal tracts
Absent
Present
Hepatocytes
Absent
Present
Sinusoids
Absent
Present
Total
Absent
Present
<2 (n = 37)
>2 (n = 21)
29
8
7
14
28
9
11
10
29
8
10
11
25
12
5
16
Grade
P
<4 (n = 31)
>4 (n = 27)
<.001
P
<.001
26
5
10
17
25
6
14
13
26
5
13
14
23
8
7
20
.069
.020
.016
.004
.001
<.001
* Stage and grade were evaluated according to Ishak et al.33 The median values for stage (2) and grade (4) were chosen as cutoffs. P values by chi-square.
© American Society of Clinical Pathologists
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❚Table 4❚
Percentage of Negative and Positive Cases for Hepatocytic and Sinusoidal Iron in Each Acinar Zone in Biopsy Specimens From 48
Patients With Chronic Hepatitis C*
Zone
1
Stage
No. (%) of Cases
Hepatocytic iron
1
2
3
4
P
Sinusoidal iron
1
2
3
4
P
*
2
Negative
Positive
Negative
Positive
26 (54)
11 (23)
7 (14)
4 (8)
92
55
43
50
.003
8
45
57
50
.003
88
55
57
50
.021
12
45
43
50
.021
96
82
86
50
.017
4
18
14
50
.017
26 (54)
11 (23)
7 (15)
4 (8)
92
45
57
25
.001
8
55
43
75
.001
92
73
43
25
<.001
8
27
57
75
<.001
92
91
86
75
.303
8
9
14
25
.303
Negative
Positive
Stages 1 to 4 according to Ishak et al.33 P values by chi-square for trend.
❚Table 5❚
Number (Percentage) of HFE Missense Mutations in 138
Control Subjects and 58 Patients With Hepatitis C in Relation
to Stage*
Control subjects
Patients/stage
1 (n = 26)
2 (n = 11)
3 (n = 7)
4 (n = 4)
5 (n = 6)
6 (n = 4)
*
3
Wild Type
Mutated
100 (72.5)
31 (53)
17 (65)
6 (55)
3 (43)
2 (50)
3 (50)
0 (0)
38 (27.5)
27 (46)
9 (35)
5 (45)
4 (57)
2 (50)
3 (50)
4 (100)
Stage was evaluated according to Ishak et al.33 Control subjects vs patients, P =
.010 by chi-square. Comparison of stage, P = .034 by chi-square for trend.
❚Table 5❚ shows that carriers of either of the HFE mutations were significantly overrepresented among patients with
chronic viral hepatitis C and underrepresented in the control
population. When evidence of either mutation was related to
stage within the chronic viral hepatitis C group, its frequency
increased significantly above stage 1.
When grade and grading parameters were compared by
means of a chi-square test with the presence of either HFE
missense mutation using their respective median as cutoff,
no significant relations were found.
Presence of either HFE gene missense mutation was not
associated with a higher degree of HDI in the liver, with the
single exception of iron in portal triads, which was detected
more frequently when at least 1 allele carried the His63Asp
mutation ❚Table 6❚ . The patient homozygous for the
Cys282Tyr mutation, a 53-year-old woman, did not show
liver iron deposits and did not have increased transferrin
saturation or ferritin levels.
Fifty-three patients underwent interferon treatment,
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whereas 5 were not treated (because of the presence of
contraindications or because of refusal of treatment). The
minimum follow-up period after discontinuation of interferon treatment was 6 months. There was no association
between carriage of either of the 2 mutations and response to
treatment. In particular, carriage of the Cys282Tyr mutation
was observed in 2 of 15 patients with no response to interferon-alfa, in 3 of 26 patients with end-of-treatment response
(circulating HCV RNA negative initially, followed by relapse
on discontinuation of treatment), and in 3 of 12 patients with
sustained response (circulating HCV RNA negative 6
months or more after interferon treatment was stopped) (chisquare, P = .546). Nevertheless, nonresponders had significantly higher total HDI scores (median, 5; range, 0-19) than
did patients with end-of-treatment response (median, 0;
range, 0-12) and patients with sustained response (median, 1;
range, 0-9) (Kruskal-Wallis, P = .040).
Multivariate Analysis of Factors Associated With HDI
The factors independently associated with hepatocytic,
sinusoidal, portal, and total HDI were studied by means of
logistic regression analysis with a stepwise forward
approach. The set of explanatory variables used included
grade, stage, HFE mutations, alcohol consumption, age, and
sex. Grade was the only variable that contributed to the
model in all iron score areas ❚Table 7❚.
Discussion
This series of consecutive cases of chronic hepatitis C
represents a typical example of the disease with regard to
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
❚Table 6❚
Histologically Detectable Iron (HDI) in Liver Biopsy Specimens of Patients With Chronic Hepatitis C in Relation to HFE
Missense Mutations
Cys282Tyr
HDI
His63Asp
–/– (n = 48)
–/+ (n = 9)
+/+ (n = 1)
28
20
7
2
1
0
31
17
7
2
1
0
32
16
6
3
1
0
23
25
6
3
1
0
Portal tracts
Absent
Present
Hepatocytes
Absent
Present
Sinusoids
Absent
Present
Total
Absent
Present
P*
–/– (n = 37)
–/+ (n = 20)
+/+ (n = 1)
.175
P*
.015
27
10
9
11
0
1
24
13
15
5
0
1
26
11
13
7
0
1
21
16
9
11
0
1
.301
.911
.697
.334
.162
.229
–/–, wild type; –/+, mutated heterozygous; +/+, mutated homozygous.
* P values by chi-square for trend.
❚Table 7❚
Explanatory Variables Independently Associated With Histologically Detectable Iron (HDI) in the Various Deposition Sites*
HDI
Explanatory Variable
Age
Sex
Grade
Alcohol use
Stage
HFE mutations
*
Hepatocytic
NE
.001
.071
NE
NE
NE
Sinusoidal
NE
NE
.013
.001
NE
NE
Portal
.033
.059
< .001
NE
NE
NE
Total
.044
.010
< .001
NE
NE
NE
P values indicate improvement of chi-square by logistic regression analysis with a stepwise forward approach. Variables that yielded P values > .10 were not entered (NE).
epidemiologic features (age and sex distribution and HCV
genotype frequency characteristic of Western Europe)36,37
and to morphologic features on liver biopsy examination,
including evidence of steatosis, portal lymphoid aggregates,
and presence of HDI deposits. Most cases showed a mild
degree of architectural damage and inflammatory
activity.10,38 Stage was associated with grade, interface
hepatitis, and portal inflammation. Acinar necrosis was unrelated to stage, and confluent necrosis was present mostly
focally and in few cases.
The incidence of HDI in biopsy specimens of patients
with chronic hepatitis C reported in the literature5-9,11,29,30
varies from 36% to 73%; it is interesting to note that, with
few exceptions,5,6 the series with the higher incidences of
HDI also are those with a greater percentage of cirrhotic
cases. In the present study, 48% of biopsy specimens had
HDI deposits, and, accordingly, relatively few (17%) showed
advanced disease.
From a purely qualitative point of view, and in accordance with previous studies,6-9 the model of iron deposition
observed in the present series was similar to that described
for nonhemochromatosis iron overload. 15 In fact, HDI
© American Society of Clinical Pathologists
deposits were seen in hepatocytes and Kupffer cells, even
when iron was present only in zone 1. They were evident in
portal tracts even when not extensive at acinar level, they were
constantly absent in bile duct cells, and their distribution was
patchy and sparse even when zone 3 was involved. On the
other hand, as in most iron-overload conditions, a zone 1 to 3
decreasing frequency in iron deposition was seen, and portal
iron scores increased with progression of architectural
damage.15 HH iron overload, on the other hand, is characterized morphologically by massive iron accumulation in hepatocytes, starting in zone 1 and progressing to zone 3 with duration of disease; most hepatocytes belonging to the same zone
show HDI, while Kupffer cells start accumulating iron only
when extensive deposition is present within hepatocytes.15
Notwithstanding the qualitative similarity described in
the literature for HDI deposition in chronic hepatitis C, the
reported semiquantitative findings on HDI are difficult to
compare because of the use of different systems for evaluating staging, grading, and HDI scoring in liver biopsy specimens, most of which use HH as a model of reference for iron
deposition.5,6,9-12,28,29 We chose the scoring system of Barton
et al8 because it evaluates the extent of HDI in the whole
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biopsy sample, analogous to the system of Searle et al,15
with the added advantage of examining the features of
zoning; furthermore, at variance with the semiquantitative
method of Brissot et al,39 it does not introduce a bias in favor
of hepatocytic iron deposition, even though it gives no indication of the actual percentage of positive cells.
In the present study, at variance with findings of some
others,9,12 morphologic features were related strongly to the
semiquantitative features of iron deposition. In fact, on
univariate analysis, stage was associated with sinusoidal,
portal,6,7,10 and total iron scores.13,30 Grade was related to all
iron scores13; this association was due mostly to the contribution of interface hepatitis and portal inflammation,6,7
rather than to that of acinar and confluent necrosis.10
A higher overall incidence of missense mutations for the
HFE gene (47%) was seen in the present series of patients
with chronic hepatitis C compared with other studies,26-30 all
of which state that the mutation rate was similar to that for
control subjects. It must be said that some did not examine
the His63Asp mutation.26-28 Furthermore, attention was
given to the patterns of association of the mutations rather
than to overall mutation rate; therefore, the differences
seemed to be nonsignificant because numbers were low. As a
matter of fact, Piperno et al30 found a 10% increase in the
overall mutation rate in Italian patients with chronic hepatitis
C in comparison with control subjects and, more importantly, the appearance of what is considered an abnormal
pattern of mutation, ie, double heterozygosity.16 Our findings
are in every way similar, with a 20% overall increase in
mutation rate, mostly due to an increase in Cys282Tyr
heterozygotes and to presence of double heterozygotes. This
alteration in mutation frequencies in patients with chronic
hepatitis C is difficult to explain because it certainly differs
from Italian control populations,30,31 including our own; it
could be a chance finding, owing to low numbers, but
certainly not to inclusion of patients with HH. In fact, none
of the subjects in the present series showed clinical or laboratory findings consistent with HH, and the pattern of HFE
mutations was different from that reported for northern
Italian patients with this disease.31 On the other hand, if HFE
mutations were a cofactor capable of aggravating the liver
damage induced by HCV, there might be a “referral bias.” In
other words, patients with subclinical or inapparent infections would more often lack the HFE mutations and would
be underrepresented in the present retrospective study. In
fact, they may stay asymptomatic more often and for longer
periods, thus not seeking medical attention.40
HFE mutations were associated significantly with
disease progression.28 In fact, while in stage 1 the overall
mutation rate was similar to that of controls, from stage 2
upward it steadily increased; on the other hand, HFE mutations were unrelated to grade and the grading parameters.26,28-30
552
Am J Clin Pathol 2000;113:546-554
HFE mutations did not clearly discriminate cases with from
those without HDI liver deposits, with the exception of the
association of the His63Asp mutation with portal HDI, also
observed by Piperno et al.30 Furthermore, the presence of
HDI liver deposits was associated with poor response to
antiviral treatment, whereas carriage of HFE mutations was
not. Only in British patients with chronic hepatitis C,28 but
not in French and Austrian patients,27,29 was a relation
observed between the Cys282Tyr mutation and HDI. This
variability in results certainly is influenced by differences
in ethnic distribution of the mutations for HFE and possibly
by the different role of the 2 mutations in iron overload. In
particular, the His63Asp mutation is observed rarely in
overt HH, possibly because of lower penetrance.16 Furthermore, in relatives of patients with HH and in patients with
iron overloaded but without heterozygous Cys282Tyr mutation, it is insufficient on its own to cause significant iron
overload.41
Multivariate analysis confirmed the lack of an independent role of HFE mutations on iron deposition and demonstrated that grade, rather than stage, influenced HDI in all
deposition sites with a role also for sex, age, and alcohol
consumption.
Undoubtedly, a greater availability of iron in the subset
of patients with hepatitis C with a history of alcohol
consumption15 may represent the determining factor for the
higher total and sinusoidal iron scores observed,30 given that
alcohol intake was unrelated to stage and grade. In the
nondrinkers in the present series, the presence of increased
iron in the liver may be due to accumulation with age and
duration of disease and the influence of sex.
On the other hand, our results implicate the tissue alterations induced by the virus as responsible for a greater
hepatic availability of iron and its deposition by means of
mechanisms that do not entail the damage-release process. In
fact, in the present series, lytic acinar and confluent necrosis
did not contribute directly to increased HDI. A possible
explanation for this finding, which is in contrast with
previous reports,8-10,19,20 is that few cirrhotic cases were
included; therefore, lytic necrosis, which usually is mild in
chronic hepatitis C,8,38 was possibly not seen as exerting its
full effect over time.10
Interface hepatitis and portal inflammation, however,
were related directly to HDI. Interface hepatitis is morphologically identified by necrosis, mainly apoptotic, at a
parenchymal-stromal interface (ie, the external hepatic
limiting plate and hepatic septa) and is accompanied by
capillarization of sinusoids at the sites of necrosis with
preservation of adjacent sinusoids in uninvolved areas. Since
sinusoids, capillarized or not, provide a widely anastomosing
vascular bed to the liver, 1 of the consequences of capillarization is substantial shunting, leading to further and progressive
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
architectural damage. 42 It is possible that the iron that
reaches the liver from the gastrointestinal absorption sites, in
presence of sinusoidal capillarization, is unable to pass into
the space of Disse and, therefore, cannot be cleared by
hepatocytes and, given its relatively high concentration in
portal blood, is cleared by the only cells available, ie, the
Kupffer cells lining capillarized sinusoids. Kupffer cells are
possibly not as active as hepatocytes would be in clearing
iron from sinusoidal blood; therefore, more iron is available
to hepatocytes in zones 1 and 2, lining adjacent but
preserved sinusoids, thus determining also hepatocytic
accumulation of hemosiderin.
This hypothesis justifies the fact that iron in chronic
hepatitis C shows a patchy distribution dependent on the
well-known patchy distribution of interface hepatitis.
Furthermore, it explains the higher incidence of iron deposits
in zone 1 where the process of interface hepatitis initially
takes place and, last, the fact that iron is present in relevant
quantities in zone 1 and 2 from stage 2 onward. In fact, the
prevalence of interface hepatitis rises from 31% in stage 1 to
82% in stage 2. The tight relation between HDI and interface
hepatitis explains why stage is not an independent factor for
determining iron deposits. Interface hepatitis appears well
before histologically evident architectural damage in the
evolution of chronic viral hepatitis.42
The relation between portal inflammation and HDI
also could be due to architectural features. For interface
hepatitis to occur, lymphocytes must be present near the
hepatic external limiting plate; hence, the lymphoid infiltrate must be considerable.
The surprisingly high frequency of carriers of HFE
mutations in the present series of patients with chronic
hepatitis C deserves a final comment. Although one may
assume that HFE mutations in livers affected by chronic
hepatitis C may influence negatively the ability of hepatocytes and Kupffer cells to clear iron at the sites of interface
hepatitis, thereby facilitating iron deposition, the fact
remains that this feature was unrelated to evidence of mutations in the present and all other previous studies.26-30 On
the other hand, HFE mutations are clustered effectively
among patients with more advanced disease28; therefore, 1
suggestion could be that, if present, HFE mutations may
favor the progression of HCV-induced damage independently from their interference with iron metabolism as
evidenced by iron deposition.
From the 1Department of Clinical and Experimental Pathology and
Medicine and the 2Institute of Pathology, Medical School,
University of Udine, Italy.
Address reprint requests to Dr Pirisi: Clinica di Medicina
Interna, DPMSC, Università degli Studi, Piazzale Santa Maria
della Misericordia 1, 33100 Udine, Italy.
© American Society of Clinical Pathologists
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