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Received: 6 May 2021    Revised: 1 October 2021    Accepted: 14 October 2021

DOI: 10.1111/vcp.13085

ORIGINAL ARTICLE

Iron, hepcidin, and microcytosis in canine hepatocellular

carcinoma

Klaudia Z. Polak | Paula Schaffer | Dillon Donaghy | Madison C. Zenk |

Christine S. Olver

College of Veterinary Medicine and

Biomedical Sciences, Clinical Pathology
Section, Colorado State University, Fort
Collins, Colorado, USA
Correspondence
Christine S. Olver, Colorado State
University, 1644 Campus Delivery, Fort
Collins, CO 80523, USA.
Email: colver@colostate.edu
Funding information
Colorado State University Clinical
Pathology Research and Development
Fund; Clinical Pathology Section; Colorado
State University
Abstract
Background: Erythrocyte microcytosis in some dogs with hepatocellular carcinoma
(HCC) suggests a derangement in systemic iron. Hepcidin, the master regulator of
iron, is secreted by the liver in response to interleukin 6 (IL-­6) and/or bone morphoge-
netic protein 6 (BMP6) and can cause microcytosis.
Objectives: Pilot study to compare the quantities of hepcidin, IL-­6, and BMP6 RNA
molecules in archival tumoral (HCC) and adjacent peritumoral (non-­HCC) hepatic
tissue to determine if they are different between tissue types or associated with
microcytosis.
Methods: RNA was isolated from formalin-­fixed, paraffin-­embedded HCC and non-­
HCC tissue from seven microcytic dogs and four normocytic dogs. Digital RNA counts
of hepcidin, IL-­6, or BMP6, and six other iron-­regulatory genes were determined using
the Nanostring nCounter system. The area of blue on each section was digitally evalu-
ated to measure the extent of Prussian blue staining objectively. Parameters were
compared between HCC and non-­HCC tissue and between microcytic and normo-
cytic groups.
Results: Hepcidin was decreased, and transferrin receptor 1 (TfR1) was increased in
HCC tissue compared with non-­HCC tissue. Non-­HCC hepcidin RNA counts corre-
lated negatively with MCV and positively with the extent of iron staining. Hepcidin
expression was higher in non-­HCC tissue of microcytic cases than in normocytic
cases.
Conclusions: Canine HCC cases showed relatively increased iron staining in non-­HCC
tissue and decreased hepcidin RNA in HCC tissue. Microcytic cases had higher hepci-
din RNA in non-­HCC tissue than normocytic cases. Future studies may extend these
findings to protein quantification, cellular localization of RNA changes, and determin-
ing if iron loading in canine liver is a predisposing factor for HCC.

KEYWORDS
anemia, gene, liver, RNA

© 2022 American Society for Veterinary Clinical Pathology

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Polak et al.
1  |  I NTRO D U C TI O N
Hepatocellular carcinoma (HCC) is the most common primary liver
tumor of both dogs and people.1,2 Previous studies have reported ab-
normalities of the erythron associated with various histologic types
of canine HCC, including anemia (50% of cases) and microcytosis
(30% of cases).1-­4 In one study, humans with HCC had a lower mean
cell volume (MCV) and higher red cell distribution width (RDW) than
healthy controls, although the percentage of people with microcyto-
5
sis was not reported. Interestingly, excess hepatic iron is known to
be a risk factor for HCC in human beings.6
Microcytosis can indicate altered systemic iron metabolism that
results in iron-­restricted erythropoiesis (IRE). In general, IRE may
result from (1) absolute iron deficiency due to decreased absorp-
tion or loss of iron or (2) functional iron deficiency caused by a de-
creased availability of iron in the face of normal or increased iron
stores.7 Functional iron deficiency is seen in inflammatory and neo-
plastic conditions and is due in part to increased secretion of hep-
cidin, considered the master regulator of iron.8 Systemic hepcidin
secreted by the liver can be induced by interleukin 6 (IL-­6) and initi-
ates the downregulation of ferroportin (Fpn), the only known cellu-
9,10
lar exporter of iron. Ferroportin is responsible for the export of
iron from macrophages and enterocytes for delivery into the blood
stream.11 The degradation of Fpn due to the binding of hepcidin re-
sults in decreased serum iron, the main source of iron available for
erythropoiesis. The systemic effects of hepcidin were shown in an
early study in which mice that overexpressed the peptide became
hypoferremic and microcytic.12 Hepcidin expression can also be
induced by bone morphogenetic protein 6 (BMP6), which is locally
produced in liver tissue by sinusoidal endothelial cells (SECs) through
the sensing of high iron loads.13 We hypothesized that HCC tissue
or adjacent, peritumoral non-­HCC tissue would show overexpres-
sion of certain iron regulatory molecules, specifically hepcidin and/
or its upstream regulators IL-­6 and/or BMP6, which would support
systemic iron dysregulation and potentially IRE. To test this hypoth-
esis and further explore iron regulation in HCC, we compared the
expression of these three molecules and six other iron regulatory
genes in archival HCC and non-­HCC using a direct digital counting
method of RNA isolated from formalin-­fixed paraffin-­embedded tis-
sues. This method of counting does not require high-­quality RNA
and therefore is used commonly for RNA harvested from formalin-­
14,15
fixed tissues. In addition, we report the areas of Perl’s Prussian
blue staining in HCC and non-­HCC tissues to measure the extent of
iron storage as measured by digital imaging software.
2  |  M ATE R I A L S A N D M E TH O DS
2.1  |  Case selection
Cases with a diagnosis of “hepatocellular carcinoma” were found by
searching the electronic medical records of canine patients seen be-
tween 2004 and 2014 at the Colorado State University Veterinary
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Teaching Hospital. Permission for the use of collected tissues was
given at the time of patient admission. Cases were included in the
study if they had a complete blood count (CBC) and serum chem-
istry (including serum iron) performed at the time of diagnosis, and
adequate available tissue for RNA isolation in paraffin blocks of both
HCC and adjacent non-­HCC liver tissue. The age at surgery was
rounded to the nearest 0.5 years. CBCs were performed using EDTA
anticoagulated blood (BD Vacutainer, Fisher Biosciences) on an
ADVIA 120 automated hematology analyzer (Siemens Healthcare).
Serum chemistries were performed on the Roche Hitachi 911 (Roche
Diagnostics, Indianapolis, IN). Dogs were classified as “microcytic”
if the MCV was less than or equal to 62 femtoliters (fL), and “nor-
mocytic” if the MCV was greater than or equal to 65 fL; the lower
limit of the laboratory’s reference interval is 62 fL. Microcytic cases
were selected first, and normocytic case controls (in approximately
equal numbers to the microcytic cases) were chosen as the canine
HCC cases with the closest date to the last microcytic case (that also
had a CBC). In one case, a normocytic dog (MCV 62) was reclassified
as a microcytic dog to take into account the dispersion around the
lower limit of the reference interval. Cases were excluded if they
had co-­morbidities that would affect iron metabolism, such as renal
disease, gastrointestinal bleeding, and evidence of inflammation or
concurrent neoplasia other than HCC. This included clinical signs or
history suggesting an intestinal tumor or foreign body, evaluation
of the CBC and serum biochemistry data (for azotemia), history of
previous intestinal surgery, and any abdominal ultrasound findings
that would indicate a gastrointestinal mass. Histologic sections were
reviewed by a board-­certified anatomic veterinary pathologist, and
cases with concurrent hepatitis, hepatocellular necrosis, or other
primary hepatocellular injury were excluded.
2.2  |  Histologic evaluation of liver sections
Formalin-­fixed paraffin-­embedded (FFPE) blocks and correspond-
ing hematoxylin and eosin-­stained slides were collected from the
Colorado State University Diagnostic Laboratory and Veterinary
Teaching Hospital archives. For each case, a board-­certified anatom-
ical veterinary pathologist (PAS) confirmed the diagnosis of hepa-
tocellular carcinoma and annotated the location of HCC tissue and
non-­HCC tissue. Additionally, the degree of differentiation of the
tumor was scored using a subjective system, based on the degree of
anisocytosis and anisokaryosis (mild, moderate, or severe) and archi-
tectural organization, as well-­differentiated (score = 1), moderately
differentiated (score = 2), or poorly differentiated (score = 3).
2.3  |  Perl’s Prussian blue staining and tissue
iron estimation
Perl’s Prussian blue staining was performed on sections of paraffin-­
embedded samples as previously described,16 because there
was insufficient tissue for both atomic absorption spectroscopy
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210      Polak et al.
measurement of iron and RNA harvest. Four images (which included
both hepatocytes and macrophages) of randomly chosen sections
were captured at 400x magnification for each case and combined
into one composite tiff file on Adobe Photoshop CC 2015 (Adobe
Creative Cloud, https://www.adobe.com/creat​ivecl​oud.html). The
four images were chosen randomly by visualizing the HCC or non-­
HCC area at 40x magnification and then moving the section around
several fields at a time blindly so that each 400× magnification field
was chosen without subjective input. NIS Elements Software (Nikon)
was used to estimate iron quantity based on the total area of blue
staining in pixels. Iron quantity in this manuscript refers to the per-
cent area (in pixels) of the composite images that were measured as
positive (blue) for iron by the program.
2.4  |  RNA isolation and copy counting
RNA was isolated from the archived FFPE blocks using a commercial
kit (ReliaPrep FFPE Total RNA Miniprep System, Promega, Madison,
WI). Using the corresponding and annotated histology slides as ref-
erences, approximately 50 mg of tissue was excised from the HCC
and non-­HCC sections of each paraffin block for separate isola-
tion of RNA. The age of each block for each dog has been added to
Table S2. RNA from FFPE blocks has been shown to be stable for
up to 10  years17 (also, Dr. Enni Markannen, personal communica-
tion). The samples were deparaffinized with mineral oil followed by
sample lysis, protein digestion, DNase treatment to remove DNA,
and column isolation and elution of RNA. In cases where insufficient
RNA was harvested (usually in non-­HCC samples), the protocol was
repeated but was altered by reducing the deparaffinization volume
of mineral oil, and the final elution volume. RNA in each sample was
quantified using spectrophotometry (Synergy H1, Biotek). The RNA
in each sample was diluted to a standard concentration of 40 μg/μL
using RNase-­free water. RNA quality was evaluated with the High
Sensitivity RNA assay on the Agilent 5200 Fragment Analyzer
System with 33  cm capillary array (Agilent) at the University of
TA B L E 1  RNA levels of nine iron-­regulatory genes between HCC and non-­HCC tissue (n = 11 of each)
Gene Function
Hepcidin System iron regulator
TfR1 (CD71) Cellular iron acquisition
BMP6 Hepcidin expression via smad pathway
Ferritin Intracellular iron storage
Fpn Cellular iron exporter
HFE Iron sensor
Interleukin 6 Hepcidin expression via stat pathway
SFXN5 Mitochondrial iron storage
SLC25a37 Divalent metal transport
Abbreviations: BMP6, bone morphogenetic protein 6; Fpn, Ferroportin; HFE, Hemochromatosis gene; SFXN5, sideroflexin 5; SLC25a37, Solute
Carrier Family 25 Member 37; TfR1; transferrin receptor 1.
*Values are presented as a ratio of medians of HCC and non-­HCC when all HCC or all non-­HCC from microcytic and normocytic cases were
combined. HCC and non-­HCC values were compared using a Wilcoxon matched-­pairs signed-­rank test.
Arizona Genetics Core (University of Arizona), where the digital
counting was performed. RNA copy counting was performed using
the NanoString nCounter gene expression system.18,19 Briefly, the
optimal amount of total RNA (150-­4 00 ng) was hybridized with the
custom codeset in an overnight incubation at 65°C, followed by pro-
cessing on the NanoString nCounter FLEX Analysis System. The qual-
ity of the RNA is judged based on the concentration of RNA and the
percentage of RNA fragments that are greater than 300 nucleotides
in length as determined by the Bioanalyzer instrument (Agilent). The
recommended input of RNA is determined by the percentage of RNA
fragments that are greater than 300 nucleotides in length. The com-
mon names for the genes probed for in these experiments and their
functions are listed in Table 1, and the target sequences are listed in
Table S1. GAPDH and beta-­actin were used as housekeeping genes.
GADPH and beta-­actin were considered appropriate housekeeping
genes as the raw values of these genes were not statistically dif-
ferent between non-­HCC and HCC tissue, nor between the same
tissues of the microcytic and normocytic cases. GADPH and beta-­
actin were used to normalize the test counts. CD25 was chosen as a
negative control gene that was expected to be low and equal in both
HCC and non-­HCC tissues. The RCC files provided by NanoString
were analyzed using the nSolver software version 4.0. This software
allows for raw count normalization to the expression of housekeep-
ing genes and background correction. For background correction,
the means ±2 SD of the negative controls were subtracted from the
counts obtained for each gene.
2.5  |  Study design and statistical analyses
Data were assessed for normality using the Anderson-­Darling
normality test. Non-­p arametric tests were used since most of the
datasets were not normally distributed. Several analyses were
performed with data from the dogs with HCC. The first analysis
compared the non-­H CC and HCC tissue values for RNA and iron
from all dogs using a Wilcoxon matched-­p airs signed-­r ank test.
Fold difference* P-­value
0.03 0.001
3.04 0.019
0.51 0.4143
1.29 0.1831
0.80 0.1716
1.29 0.2146
0.98 0.2439
1.69 0.9097
1.04 0.7354
Polak et al.
The second analysis used a Spearman correlation to determine
correlations between (1) gene copy number and MCV of all dogs;
(2) the extent of iron staining and MCV from all dogs; and (3) tissue
iron quantity in non-­H CC tissue and non-­H CC gene copy number.
The third analysis compared parameters between normocytic and
microcytic groups using Mann-­W hitney U tests (non-­p aired). This
analysis included clinical parameters such as tumor differentiation
score and selected hematologic and clinical chemistry values. It
also included comparisons between HCC and adjacent non-­H CC
tissue for RNA copy counts for each gene and estimated iron. All
analyses were performed with commercial software (GraphPad
Prism 8.2.1).
3  |  R E S U LT S
3.1  |  Case selection results
Out of 224 cases with HCC, 83 had a CBC at presentation (34%). Of
these, 14 cases were microcytic. One Akita and one Shiba Inu mix
were removed due to breed-­associated microcytosis. One dog with
a gastric foreign body (possible blood loss) was removed. Of the re-
maining 10 cases (12%), only seven had paraffin blocks with enough
tissue for RNA isolation from both HCC and non-­HCC tissues. Seven
normocytic HCC cases were identified, but sufficient RNA was iso-
lated from only four. The detailed clinical data for each individual
dog are represented in Table S2.
3.2  |  RNA counts and iron staining in HCC
compared with non-­HCC tissue
Of the nine iron regulatory genes chosen for this analysis, only
hepcidin and TfR1 counts were different between HCC and non-­
HCC tissue according to Wilcoxon rank-­sum comparisons (Table 1).
When individual fold differences were calculated, hepcidin data
F I G U R E 1  Hepcidin RNA counts (left panel), TfR RNA counts (middle panel), and iron area (right panel) in non-­HCC tissue or HCC tissue.
Iron and hepcidin were significantly higher and TfR1 RNA significantly lower in non-­HCC tissue compared with HCC liver tissue. HCC,
hepatocellular carcinoma
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was most compatible with lower hepcidin counts in the HCC tis-
sue than the non-­H CC tissue by an observed median of a 63.1-­
fold decrease (95% confidence interval compatible with an actual
change of between 11.38-­and 845.8-­fold decrease). On the other
hand, median RNA counts of TfR1 were increased 1.8-­fold in the
HCC tissue compared with non-­H CC tissue, with a 95% confi-
dence interval of between 0.81-­and 9.95-­fold increase. There was
no difference in the RNA counts of the BMP6, ferritin, Fpn, HFE,
IL-­6 , SFXN5, and SLC25a37 genes between HCC and non-­H CC
samples. Values for four dogs with livers without HCC are shown
in Table S3. The extent of Perl’s Prussian blue staining was most
compatible with the increased extent of staining in non-­H CC tis-
sue, in which there was a median of 4.69-­fold higher in non-­H CC
tissue than HCC tissue, with a 95% confidence interval of 0.987 to
34.5). Figure  1 shows graphic comparisons of hepcidin and TfR1
counts and the extent of iron staining between HCC and non-­H CC
tissue.
3.3  |  Correlations of gene expression with
MCV and non-­HCC iron staining
Hepcidin RNA counts in non-­HCC tissue showed statistically sig-
nificant negative correlation with MCV (r = −0.76, CI = −.94 to −.27,
P  =  0.009) (Figure  2). The extent of iron staining in non-­HCC tis-
sue was weakly positively correlated with both hepcidin (r  =  0.65,
CI = 0.07 to 0.9, P = 0.034) and TfR1 RNA counts (r = 0.64, CI = 0.03
to 0.90, P = 0.04) in non-­HCC tissue (Figure 3).
3.4  |  Microcytic and normocytic groups
Summary clinical data, including tumor differentiation scores, are
presented in Table 2. HCC from microcytic cases were more poorly
differentiated based on our subjective histologic scoring. Of the
nine iron regulatory genes evaluated, only tissue hepcidin RNA
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212      Polak et al.
counts showed any significant expression differences between
microcytic and normocytic dogs; hepcidin counts were signifi-
cantly higher in the non-­H CC tissue of microcytic versus normo-
cytic dogs (P  =  0.0424). The median hepcidin count in non-­H CC
tissue of microcytic dogs was 52649 (CI = 2397-­22266), whereas
the median hepcidin count in the non-­H CC tissue of normocytic
dogs was 14421 (CI  =  6355-­103288). The extent of iron stain-
ing was higher in the non-­H CC tissue of dogs with microcytosis
compared with the non-­H CC tissue of normocytic dogs (1204 vs
8123 median pixels) but this did not reach statistical significance.
Three of the microcytic dogs had iron staining compared with nor-
mocytic dogs, while four microcytic dogs had much higher iron
staining. These parameters, with each group divided into HCC and
non-­H CC tissue, are shown in Figure 4. There was no difference
in the extent of iron staining or counts of any regulatory genes
F I G U R E 2  Scatterplot of MCV and non-­HCC hepcidin RNA
counts. There was a negative correlation between non-­HCC
hepcidin RNA counts and MCV (r = −0.76, P = 0.009). HCC,
hepatocellular carcinoma, MCV, mean cell volume
F I G U R E 3  Scatterplots of hepcidin RNA counts (left panel) or transferrin receptor 1 (TfR1) RNA counts (right panel) and non-­HCC iron
quantities. There was a positive correlation between non-­HCC iron load and both hepcidin (r = 0.65, P = 0.034) and TfR1 (r = 0.64, P = 0.04).
HCC, hepatocellular carcinoma
between HCC tissue from the microcytic or normocytic dogs.
Composite images used for digital assessments of the extent of
iron staining are available in Figure S1 (each case shows composite
images for non-­H CC and HCC).
4  |  D I S C U S S I O N
We performed a pilot study on archived canine HCC and non-­HCC
tissues to determine if the RNA of iron regulatory molecules is dif-
ferentially expressed and/or are quantitatively different between
microcytic and normocytic cases. Additionally, we investigated
whether differences in RNA expression and estimated iron quantity
between canine HCC and non-­HCC tissue are associated with micro-
cytosis vs normocytosis. One of the most interesting findings was
that hepcidin expression was markedly lower in all HCC tissue vs the
non-­HCC tissue. Although we did not find differences in hepcidin
RNA levels between the HCC tissues of microcytic and normocytic
cases, we did find higher hepcidin RNA counts in the adjacent non-­
HCC hepatic tissue of microcytic cases compared with normocytic
cases. Furthermore, the microcytic HCC cases were associated with
clinical findings such as increased ALT and lower HCT and more
poorly differentiated tumors.
Serum and cellular iron influence the expression of hepcidin in
the liver. Serum and cellular iron overload stimulate hepcidin gene
expression, whereas iron deficiency and erythropoiesis repress it. 20
In normal hepatic tissue, transferrin, and non-­transferrin bound iron
is taken up by sinusoidal endothelial cells (SEC), which then express
BMP6. BMP6 is secreted in a paracrine fashion to act on hepato-
cytes via BMP receptors, along with the co-­receptor hemojuvelin,
and initiates hepcidin transcription through the SMAD pathway.13
This regulatory mechanism may explain why higher non-­HCC liver
iron stores in our study correlated with higher hepcidin expression.
The low hepcidin expression in HCC compared with non-­HCC in
our study was surprising, but this phenomenon has been shown in a
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gene expression study in human HCC. 21 The increased TfR1 in our
HCC versus non-­HCC is an expected finding, as this is common in
neoplastic cells compared with their non-­neoplastic counterparts. 22
We speculate that the accelerated iron utilization in neoplastic he-
patocytes depletes their cellular iron stores and results in increased
TA B L E 2  Summary data for clinicopathologic variables for
microcytic or normocytic dogs
TfR1 expression. The increased iron utilization by neoplastic hepato-
cytes may also explain the decreased local hepcidin production. The
local iron depletion might be sensed by SEC, resulting in dampened
BMP6 secretion within the tumor. Inhibition of BMP6 release would
then disrupt the BMP-­SMAD pathway, possibly causing decreased
intratumoral hepcidin production.13 Although we did not see signif-
icant differences in BMP6 RNA expression between HCC and non-­
HCC tissue, differential expression of BMP6 may still be occurring

Variable Mean Minimum Median Maximum but below the lower limit of detection with our methods. Overall,
the amount of BMP6 RNA measured was very low throughout all
HCT (%)
samples. Since SEC represent a small proportion of the total hepatic
Micro 36 27 36* 50
cellular population and consequently contributes to a small percent-
Normo 47 43 45 54
age of the total RNA harvested, it may be that our analysis was not
MCV (fL) sensitive enough to demonstrate a difference in BMP6 RNA counts
Micro 59 56 59* 60 between HCC and non-­HCC tissues.
Normo 69 62 70 75 We also speculate that the neoplastic hepatocytes may actu-
SeFe (μg/dL) ally benefit from the effects of reduced tumor hepcidin levels on
Micro 114 64 114* 180 nearby iron-­containing Kupffer cells. Hepcidin binds to Fpn, the only
Normo 132 41 134 220 known cellular iron exporter, and results in its internalization and
ALT (IU/dL) degradation. Reduced local hepcidin expression would presumably

Micro 681 340 557 *

1296
result in increased Kupffer cell Fpn, which could allow enhanced iron
excretion and availability to adjacent tumor cells. Considering the
Normo 165 43 120 297
increased iron load in non-­HCC tissue and the absence of iron in
Tumor differentiation score
HCC tissue, it is surprising that Fpn RNA counts were not different
Micro 2.3 2 2* 3
between the two tissue types. Since Fpn expression is controlled
Normo 1.2 1 1 2
post-­transcriptionally, 23 we suspect that our RNA counts for Fpn
Age
may not reflect the actual protein content, especially since hepcidin
Normo 10 6.5 11 13.5 RNA expression is so dramatically different between HCC and non-­
Micro 11 10 11 13.5 HCC hepatic tissue.
Abbreviations: ALT, serum alanine aminotransferase; HCT, hematocrit; We found evidence of increased iron in the non-­HCC liver tissue
MCV, mean cell volume; SeFe, serum iron. of dogs with HCC. We measured the extent of iron staining with the
*Significantly different between microcytic and normocytic groups. rationale that measuring the area of the staining would be more likely

F I G U R E 4  RNA counts for hepcidin (left panel) and iron quantity (right panel) as determined by the digital area measurements of
blue-­staining (Perl’s Prussian blue) shown for non-­HCC or HCC liver tissue in the normocytic and microcytic groups. HCC = hepatocellular
carcinoma. Statistically significant differences are shown with the * (P < 0.05), P = 0.0424 for hepcidin and 0.016 for iron
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214      Polak et al.
to gather information on the extent of iron accumulation in hepato-
cytes. We felt that this was an important parameter, as the biochemi-
cal measurement of iron would not distinguish between iron in Kupffer
cells versus iron in hepatocytes. A review of the literature reveals a
comparable pattern of iron staining in human HCC, which similarly
shows a lack of iron accumulation in tumor tissue and excessive iron
in non-­HCC tissue, along with a similar differential expression of iron
regulatory genes between these tissue types.21,24 Iron overloading in
hepatocytes is associated with an increased risk of HCC in humans.
For example, there is a 200-­fold increased incidence of HCC in people
with hereditary hemochromatosis (HH), a genetic iron loading disor-
der6 and dietary iron overload in humans also comes with a 24-­fold
increased risk of HCC.25 When examining the relationship between
26
iron overload and liver disease, it is unclear which insult occurs first
or the specifics of subsequent HCC development. Iron-­induced car-
cinogenesis is thought to result from the DNA-­damaging effects of
reactive oxygen intermediates that result from the Fenton reaction
involving free iron,27 although iron also directly causes hepatocyte
proliferation.28 Our data introduce the possibility that iron loading in
the hepatocytes may be a risk factor for the development of HCC.
Increased liver iron concentration has been reported before in dogs
with chronic inflammatory liver disease, similar to what we saw in
the dogs of this study.29 Additionally, Harro et al. found that hepatic
iron concentrations, measured with inductively coupled plasma mass
spectrometry, were increased in non-­HCC tissue compared with HCC
30
tissue which parallels our findings, although with a different method.
The development of HCC as a sequela to hepatocyte iron loading has
not been reported in dogs, and thus this possible association requires
further investigation.
Given that it is a marker of hepatocellular damage, increased
ALT in the microcytic HCC group compared with the normocytic
group may be due to underlying liver disease or to oxidative injury
secondary to hepatic iron loading. Among the microcytic cases, the
increased hepcidin RNA in the non-­HCC liver tissue paired with the
anemia and low serum iron all point to the occurrence of IRE second-
ary to functional iron deficiency. Moreover, we found that hepcidin
RNA counts in adjacent non-­HCC tissue were negatively correlated
to MCV and serum iron. Taken together, these data suggest that the
presence of microcytosis in dogs diagnosed with HCC may be at-
tributed to elevated hepcidin levels in the non-­HCC liver tissue. This
increase in hepcidin RNA is most likely due to increased hepatic iron
stores in the non-­HCC tissue, as these were positively correlated
with non-­HCC hepcidin RNA counts. Histopathologic review of all
cases by a board-­certified anatomic pathologist (PAS) ruled out in-
flammation prior to conducting the study.
Several limitations are identified in our study. Our sample size
was small after excluding many HCC cases due to a lack of a pre-­
operative CBC and our strict exclusion criteria and is therefore sub-
ject to type II error. Thus, we might have seen differences in other
iron regulatory proteins if a larger sample size had been used. We
were also unable to measure certain serum iron values typically used
to describe iron status, such as total iron-­binding capacity, percent
saturation of transferrin, and serum ferritin. These values are most
commonly used to non-­invasively evaluate iron status in serum, al-
though they are not always sensitive in detecting functional vs ab-
solute iron deficiency.7 Additionally, we could not measure serum
or urine hepcidin31; therefore, we could not correlate the increased
hepcidin mRNA in liver with serum levels. We plan to investigate
further with immunohistochemistry and laser capture microdissec-
tion studies on a larger sample set of archived canine HCC tissue and
controls, as the samples in this study were consumed by harvesting
RNA. We will use antibodies against Fpn, ferritin, and BMP6 to as-
sess the location and staining intensity differences between the tis-
sue types. These studies are ongoing. Additionally, with LCM, we can
measure regulatory genes in subsets of hepatocytes, Kupffer cells,
and sinusoidal endothelial cells. This will serve not only to quantify
proteins and verify our RNA results but also to locate any changes
in protein expression to cell types located within the heterogeneous
sections of HCC (eg, SEC, Kupffer cells, and hepatic stellate cells, as
well as hepatocytes).32,33 A final limitation is that we were unable
to quantify iron using flame atomic absorption spectroscopy (spec-
trophotometry) because of limited tissue, so we used Prussian blue
staining and digital analysis as a proxy. Prussian blue staining does
have the advantage of localizing iron accumulation (hepatocytes
and/or Kupffer cells); however, an important consideration when
trying to understand the effect of hepatocyte iron accumulation as
an inducer of proliferation.
In conclusion, our iron staining and gene expression results sug-
gest that there is a local and systemic iron disturbance associated
with canine HCC that has not been previously described in the dog,
and which parallels findings observed in human HCC. We found that
microcytosis was related to iron storage and hepcidin expression in
hepatic tissue adjacent to the HCC. Additional work is needed to
determine whether microcytosis is a useful prognostic factor and to
strengthen the link between iron and oncogenesis in HCC.
ORCID
Christine S. Olver  https://orcid.org/0000-0002-9937-2706
REFERENCES
1. Patnaik AK, Hurvitz AI, Lieberman PH, Johnson GF. Canine
Hepatocellular Carcinoma. Vet Pathol Published Online. 1981;18:427-­
438. doi:10.1177/03009​85881​01800402
2. Patnaik AK, Hurvitz AI, Lieberman PH. Canine hepatic neo-
plasms: a clinicopathologic study. Vet Pathol. 1980;17(5):553-­564.
doi:10.1177/03009​85880​01700504
3. Liptak JM, Dernell WS, Monnet E, et al. Massive hepatocellu-
lar carcinoma in dogs: 48 cases (1992-­2002). J Am Vet Med Assoc.
2004;225(8):1225-­1230. doi:10.2460/javma.2004.225.1225
4. Matsuyama A, Takagi S, Hosoya K, Kagawa Y., Nakamura K.,
Deguchi T., Takiguchi M. Impact of surgical margins on survival of
37 dogs with massive hepatocellular carcinoma. N Z Vet J 2017. 65,
227, 231d oi:10.1080/00480​169.2017.1319304
5. Wei TT, Tang QQ, Qin BD, et al. Elevated red blood cell distribution
width is associated with liver function tests in patients with primary
hepatocellular carcinoma. Clin Hemorheol Microcirc. 2016;64(2):149-­
155. doi:10.3233/CH-­162053
6. Niederau C, Fischer R, Sonnenberg A, Stremmel W, Trampisch
HJ, Strohmeyer G. Survival and causes of death in cirrhotic and in
Polak et al.
noncirrhotic patients with primary hemochromatosis. N Engl J Med.
1985;313:1256-­1262. doi:10.1056/NEJM1​98511​14313​2004
7. Radakovich LB, Santangelo KS, Olver CS. Reticulocyte hemoglo-
bin content does not differentiate true from functional iron defi-
ciency in dogs. Vet Clin Pathol. 2015;44(4):511-­518. doi:10.1111/
vcp.12294
8. Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys
Acta -­Mol Cell Res. 2012;1823(9):1434-­1443. doi:10.1016/j.
bbamcr.2012.01.014
9. Ward DM, Kaplan J. Ferroportin-­mediated iron transport: expres-
sion and regulation. Biochim Biophys Acta. 2012;1823(9):1426-­
1433. doi:10.1016/j.bbamcr.2012.03.004
10. Steinbicker AU, Muckenthaler MU. Out of balance-­-­systemic iron
homeostasis in iron-­related disorders. Nutrients. 2013;5(8):3034-­
3061. doi:10.3390/nu508​3 034
11. Drakesmith H, Nemeth E, Ganz T. Ironing out Ferroportin. Cell
Metab. 2015;22(5):777-­787. doi:10.1016/j.cmet.2015.09.006
12. Nicolas G, Bennoun M, Porteu A, et al. Severe iron deficiency ane-
mia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci
U S A. 2002;99:4596-­4601. doi:10.1073/pnas.07263​2499
13. Canali S, Zumbrennen-­Bullough KB, Core AB, et al. Endothelial
cells produce bone morphogenetic protein 6 required for iron ho-
meostasis in mice. Blood. 2017;129(4):405-­414. doi:10.1182/blood​
-­2016-­06-­721571
14. Walsh HL, Sperry AJ, Blazer VS. The effects of tissue fixation on
sequencing and transcript abundance of nucleic acids from mi-
crodissected liver samples of smallmouth bass (Micropterus do-
lomieu). PLoS ONE 2020;15(8 August):1–­20. doi:10.1371/journ​
al.pone.0236104, e0236104
15. Hahn CM, Iwanowicz LR, Cornman RS, Mazik PM, Blazer VS.
Transcriptome discovery in non-­model wild fish species for the
development of quantitative transcript abundance assays. Comp
Biochem Physiol -­Part D Genomics Proteomics. 2016;20:27-­4 0.
doi:10.1016/j.cbd.2016.07.001
16. Sprague WS, Hackett TB, Johnson JS, Swardson-­Olver CJ.
Hemochromatosis secondary to repeated blood transfusions in a
dog. Vet Pathol. 2003;40(3):334-­337.
17. Guscetti F, Nassiri S, Beebe E, Rito Brandao I, Graf R, Markkanen
E. Molecular homology between canine spontaneous oral squa-
mous cell carcinomas and human head-­and-­neck squamous cell
carcinomas reveals disease drivers and therapeutic vulnerabilities.
Neoplasia. 2020;22(12):778-­788. doi:10.1016/j.neo.2020.10.003
18. Tsang H-­F, Xue VW, Koh S-­P, Chiu Y-­M, Ng LP-­W, Wong S-­CC.
NanoString, a novel digital color-­coded barcode technology: cur-
rent and future applications in molecular diagnostics. Expert Rev Mol
Diagn. 2017;17(1):95-­103. doi:10.1080/14737​159.2017.1268533
19. Geiss GK, Bumgarner RE, Birditt B, et al. Direct multiplexed mea-
surement of gene expression with color-­coded probe pairs. Nat
Biotechnol. 2008;26:317-­325. doi:10.1038/nbt1385
20. Nemeth E. Iron regulation and erythropoiesis. Curr Opin Hematol.
2008;15:169-­175. doi:10.1097/MOH.0b013​e3282​f 73335
21. Tseng H-­H, Chang J-­G , Hwang Y-­H, Yeh K-­T, Chen Y-­L , Yu H-­S.
Expression of hepcidin and other iron-­regulatory genes in human
hepatocellular carcinoma and its clinical implications. J Cancer
Res Clin Oncol. 2009;135(10):1413-­1420. doi:10.1007/s0043​
2-­0 09-­0585-­5
|
      215
22. Torti SV, Torti FM. Iron and cancer: more ore to be mined. Nat Rev
Cancer. 2013;13:342-­355. doi:10.1038/nrc3495
23. Muckenthaler MU, Rivella S, Hentze MW, Galy B. Review a red
carpet for iron metabolism. Cell. 2017;3(3):1-­18. doi:10.1016/j.
cell.2016.12.034
24. Tan MG, Kumarasinghe MP, Wang SM, Ooi LL, Aw SE, Hui KM.
Modulation of iron-­regulatory genes in human hepatocellu-
lar carcinoma and its physiological consequences. ExpBiolMed
2009;234(1535–­3702 [Print]):693–­702. doi:10.3181/0807-­RM-­227
25. Gordeuk VR, McLaren CE, MacPhail AP, Deichsel G, Bothwell TH.
Associations of iron overload in Africa with hepatocellular carci-
noma and tuberculosis: Strachan’s 1929 thesis revisited. Blood.
1996;87(8):3470-­3 476. doi:10.1182/blood.v87.8.3470.blood​journ​
al878​3 470
26. Kowdley KV. Iron overload in patients with chronic liver disease.
Gastroenterol Hepatol. 2016;12(11):695-­698.
27. Torti SV, Manz DH, Paul BT, Blanchette-­Farra N, Torti FM. Iron and
cancer. Annu Rev Nutr. 2018;38(1):97-­125. doi:10.1146/annur​ev-­
nutr-­0 8211​7-­051732
28. Ma S, Yang J, Li J, Song J. The clinical utility of the proliferating
cell nuclear antigen expression in patients with hepatocellular
carcinoma. Tumor Biol. 2016;37(6):7405-­7412. doi:10.1007/s1327​
7-­015-­4582-­9
29. Schultheiss PC, Bedwell CL, Hamar DW, Fettman MJ. Canine
liver iron, copper, and zinc concentrations and association with
histologic lesions. J Vet Diagn Invest. 2002;2002(14):396-­4 02.
doi:10.1177/10406​38702​01400506
3 0. Harro CC, Smedley RC, Buchweitz JP, Langlois DK. Hepatic copper
and other trace mineral concentrations in dogs with hepatocellular
carcinoma. J Vet Intern Med. 2019;33(5):2193-­2199. doi:10.1111/
jvim.15619
31. Vizi Z, Lányi K, Bagi M, Laczay P, Balogh N, Sterczer Á. Serum hep-
cidin measurements in healthy dogs using liquid chromatography/
tandem mass spectrometry. Vet Clin Pathol. 2020;49(2):292-­298.
doi:10.1111/vcp.12872
32. Liu J, Dang H, Wang XW. The significance of intertumor and intra-
tumor heterogeneity in liver cancer. Exp Mol Med. 2018;50:e416.
doi:10.1038/emm.2017.165
33. Zhang S, Chang W, Wu H, et al. Pan-­c ancer analysis of iron met-
abolic landscape across the cancer genome atlas. J Cell Physiol.
2020;235(2):1013-­1024. doi:10.1002/jcp.29017
S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found in the online
version of the article at the publisher’s website.
How to cite this article: Polak KZ, Schaffer P, Donaghy D,
Zenk MC, Olver CS. Iron, hepcidin, and microcytosis in
canine hepatocellular carcinoma. Vet Clin Pathol. 2022;51: 
208–215. doi: 10.1111/vcp.13085