Immunohistochemical differentiation of reactive from malignant mesothelium as a
diagnostic aid in canine pericardial disease
*1Elspeth Milne, BVM&S PhD DipECVCP FRCPath FRCVS, [email protected], Tel. +44
(0)131 650 6221
*Yolanda Martinez Pereira, LdaVet CertVC DipECVIM-CA (Cardiology) MRCVS,
[email protected]
ⱡClare Muir BSc BVM&S MRCVS, [email protected]
ⱡTim Scase BSc BVM&S PhD MRCVS DipACVP, [email protected]
*Darren J. Shaw, BSc PhD, [email protected]
*Gillian McGregor, BSc, [email protected]
†Lucy Oldroyd, BSc BVMS MACVS DipACVP MRCVS, [email protected]
¥Emma Scurrell, BVSc DipACVP MRCVS RCVS Recognised Specialist in Veterinary Pathology,
[email protected]
#Mike Martin, MVB DVC MRCVS, [email protected]
$Craig Devine, BVSc DVC MRCVS, [email protected]
€Hannah Hodgkiss-Geere, BVM&S MSc PhD DipECVIM-CA (Cardiology) MRCVS, H.Hodgkiss-
[email protected]
1Corresponding author
*Royal (Dick) School of Veterinary Studies and The Roslin Institute, The University of
Edinburgh, Easter Bush Campus, Roslin, Midlothian, EH25 9RG, United Kingdom
ⱡBridge Pathology Ltd., 637 Gloucester Rd, Bristol BS7 0BJ United Kingdom
†Abbey Vet Services, 89 Queen Street, Newton Abbot, Devon, TQ12 2BG, United Kingdom
¥Cytopath Ltd., Ledbury, Herefordshire HR8 2YD, United Kingdom
#Willows Referral Centre, Highlands Road, Shirley, West Midlands, B90 4NH United Kingdom
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$Borders Veterinary Cardiology Services, Smithy Cottage, Skirling, Peeblesshire, ML12 6HD
United Kingdom
€Small Animal Teaching Hospital, Institute of Veterinary Science, University of Liverpool,
Leahurst Campus, Chester High Road, Neston, Wirral, CH64 7TE, United Kingdom
Acknowledgements
The authors are very grateful to BSAVA Petsavers for funding the study, the original
pathologists and technical staff of The Royal (Dick) School of Veterinary Studies, Bridge
Pathology Ltd., Abbey Veterinary Services and Cytopath Ltd. for their expertise, and the dog
owners and clinicians who allowed storage of archive material and provided follow up
information.
Data accessibility
The authors comply with the journal’s data sharing accessibility requirements where this
does not breach client confidentiality.
Structured summary
Objectives: To develop a provisional immunohistochemistry panel for distinguishing reactive
pericardium, atypical mesothelial proliferation and mesothelioma in dogs.
Methods: Haematoxylin and eosin staining, immunohistochemistry for cytokeratin,
vimentin, insulin-like growth factor II mRNA binding protein 3, glucose transporter 1, and
desmin were carried out on archived pericardial biopsies, and scored for intensity and
number of cells stained.
Results: From haematoxylin and eosin staining, 10 reactive mesothelium (group R), 17 of
atypical mesothelial proliferation (group A), 26 of mesothelioma (group M) and 5 of normal
pericardium (group C) were obtained. Cytokeratin and vimentin were expressed in all,
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confirming mesothelial origin. Group C had the lowest scores for insulin-like growth factor II
mRNA binding protein 3, glucose transporter 1 and desmin. Of the disease groups, M and A
were similar to each other with higher scores for insulin-like growth factor II mRNA binding
protein 3 and glucose transporter 1 than group R. Desmin staining was variable. Insulin-like
growth factor II mRNA binding protein 3 was best to distinguish between disease groups,
with increasing scores from R to A to M, with group R indistinguishable from group C. In a few
mesotheliomas, glucose transporter 1 conferred an advantage over insulin-like growth
factor II mRNA binding protein 3.
Clinical significance: An immunohistochemistry panel of cytokeratin, vimentin, insulin-like
growth factor II mRNA binding protein 3 and glucose transporter 1 could provide useful
additional information over haematoxylin and eosin staining alone in the diagnosis of cases
of mesothelial proliferations in canine pericardium although further validation is warranted.
Keywords
Mesothelioma, idiopathic pericarditis, GLUT1, IMP3, canine
Introduction
Pericardial effusion is common in dogs and can lead to cardiac tamponade, collapse and
death. The most common causes in dogs in the UK are idiopathic pericarditis (IP) and
neoplastic effusion (Stafford Johnson et al. 2004, MacDonald et al. 2009). Malignant
mesothelioma of the pericardial lining is the third most common neoplastic cause of
pericardial effusion, after haemangiosarcoma and chemodectoma. Surgical intervention with
pericardiectomy in patients with recurrent IP is often successful in ameliorating clinical signs,
whereas in dogs with mesothelioma, there is typically a persistent and recurrent pleural
effusion and the prognosis is poorer (Stepien et al 2000).
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Canine mesothelioma is a neoplasm of mesodermal origin affecting the mesothelial lining of
the serosal surface of the coelomic cavities (WHO 2007), usually considered to be malignant
(Uzal et al. 2016). There is a clear association with aerosol exposure to asbestos in people,
but causal factors of naturally-occurring mesothelioma have not been well established in
dogs, although an association with chronic IP (Machida et al. 2004) and asbestos exposure
have been proposed in a few studies (Glickman et al. 1983, Harbison et al. 1983). In dogs, the
tumour most commonly affects the pleura followed by the peritoneum and pericardium, and
is characterised by pale, pedunculated or sessile nodules, plaques or villous projections (Uzal
et al. 2016). Reactive mesothelial cells can closely mimic both mesothelioma and carcinoma
on histopathological and cytological examination of tissues and effusions respectively, and it
is widely recognised in human and veterinary medicine that distinguishing reactive from
neoplastic mesothelium can be very challenging. This is particularly problematic in the
pericardium as mesothelial cell reactivity is more marked in this site, presumably due to the
increased local friction caused by the beating heart (Baker and Lumsden 2000). In dogs, an
intermediate category of “atypical mesothelial proliferation” has also been suggested (Cagle
and Churg 2005), as described in people (Churg et al. 2012).
Numerous studies have investigated the value of immunohistochemical (IHC) panels to aid in
the distinction of reactive from neoplastic mesothelium in human patients. Guidelines for the
pathological diagnosis of malignant mesothelioma in people recommend a combination of
histopathological features and specific IHC panels (Husain et al. 2013) e.g. desmin, p53,
epithelial membrane antigen (EMA), glucose transporter 1 (GLUT1) and insulin-like growth
factor II mRNA binding protein 3 (IMP3) (Galateau-Salle et al. 2015). A recent study on
human tissue reported a sensitivity of 100% and specificity of 95% for the diagnosis of
mesothelioma versus reactive mesothelial cells using IHC methods for GLUT1 and IMP3 in
formalin-fixed, paraffin wax-embedded tissues, with haematoxylin and eosin (HE) staining as
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the gold standard for comparison (Minato et al. 2014). Strong expression of both antibodies
was consistent with mesothelioma and negative reactions with benign proliferations in their
study. However, it is well recognised that while IHC may provide supporting evidence, this is
based on statistical probability and may not always be useful in an individual case (Husain
2014). Others have found the relationship to be less definitive (Husain et al. 2013, Lee et al.
2013).
Currently, the diagnosis of canine pericardial mesothelioma is based on histopathological
examination of HE-stained sections of tissue obtained at surgery. However, more reliable
differentiation of mesothelioma from atypical mesothelial proliferation and reactive
mesothelium is required. This would enable pathologists to provide a more accurate
definitive diagnosis and consequently help clinicians to make better decisions with respect to
treatment and prognostic advice.
It was hypothesised in this preliminary study that an IHC panel of cytokeratin, vimentin,
desmin, GLUT1 and IMP3 might aid in the differentiation of reactive mesothelium, atypical
mesothelial proliferation and mesothelioma warranting further prospective studies to assess
its potential as a new diagnostic tool for these conditions in dogs. The pericardium was
chosen for the study because IP presents a common diagnostic conundrum, and because the
marked reactivity shown by pericardial mesothelium makes distinction from mesothelioma
particularly challenging.
Materials and methods
Cases
This was a multicentre, retrospective study, carried out with approval of the local Veterinary
Ethical Review Committee (approval no. 06 14). The authors’ clinical and pathological records
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were searched for cases of IP, atypical mesothelial proliferation and mesothelioma, then the
archives of their four histopathology laboratories were searched for paraffin wax-embedded
tissue blocks from biopsies and post mortem examinations. Based on the diagnosis from
routine HE-stained sections they were classified as having simple hyperplasia (reactive
pericarditis [IP], group R), atypical mesothelial proliferation (group A) and mesothelioma
(group M). Those from the university laboratory were from cases referred to the university’s
Hospital for Small Animals. In addition, clinically normal dogs, euthanised for behavioural
reasons, were collected by the university laboratory for use as controls. HE sections were
reviewed and any cases in which the tissue was of poor quality were excluded. In all cases,
attempts were made to determine the clinical history, clinical findings and the outcome from
patient records where available, and by contacting the owners via the primary clinicians. The
information available was variable but included the main presenting signs and the presence,
site and volume of cavitary effusions, and survival times.
Tissue processing and staining
Routine histopathology
All tissues had been fixed in 10% phosphate-buffered formalin and embedded in paraffin wax.
4µm sections were cut and stained with HE. In cases where more than one tissue block was
available, the highest quality block based on the HE sections was chosen for IHC.
Immunohistochemistry
All IHC was carried out in the university laboratory. Sections (4µm) of formalin-fixed, paraffin
wax-embedded tissue were placed on SuperFrost® Plus coated slides (Thermo Electron Ltd.,
Altrincham, Cheshire, UK), dewaxed, hydrated, and rinsed in distilled water. To block non-
specific endogenous peroxidase, sections were treated with blocking agent (REAL blocking
agent S202386, Dako Ltd., Ely, Cambridgeshire, UK). All antibodies were diluted in antibody
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diluent (S0809, Dako). For cytokeratin, mouse monoclonal anti-cytokeratin antibody clone
MNF116 (M0821, Dako) was diluted 1/50 and incubated 30 minutes at room temperature
(RT) following antigen retrieval using proteinase K (S3020, Dako) for 30 minutes at RT. For
vimentin, mouse monoclonal anti-vimentin antibody clone V9 (NCL-L-VIM-V9, Novocastra
Laboratories, Newcastle upon Tyne, UK) was diluted 1/400 and incubated for 30 minutes at
RT following antigen retrieval using high pH antigen unmasking buffer (H-3300, Vector
Laboratories Ltd., Peterborough, UK) at 110oC for 5 minutes. For desmin, mouse monoclonal
anti-desmin antibody clone D33 (M0760, Dako) was diluted 1/50, incubated for 30 minutes at
RT following antigen retrieval using high pH antigen unmasking buffer (H-3300, Vector) at
110oC for 5 minutes. For GLUT1, rabbit polyclonal anti-GLUT1 antibody (15309, Abcam Ltd.,
Cambridge, UK) was diluted 1/500 and incubated overnight at 4oC following antigen retrieval
using 0.01M citrate buffer pH 6.0 at 110oC for 5 minutes. For IMP3, mouse monoclonal anti-
IMP3 antibody clone 69.1 (M3626, Dako) was diluted 1/50 and incubated for 30 minutes at
RT followed by antigen retrieval using high pH antigen unmasking buffer (H-3300, Vector) at
97oC for 50 minutes. Following incubation with primary antibody, the sections were
incubated with secondary antibody (Envision anti mouse HRP [K4007] or Envision anti rabbit
HRP [K4011], Dako, as appropriate) and visualised with DAB+ chromogen (K3468, Dako).
Sections were counterstained with Harris haematoxylin. All heat-induced epitope retrievals
were carried out using the Histos 5 microwave histoprocessor (Milestone SRL, Bergamo, Italy)
and all washings between steps were carried out using Tris buffered saline plus Tween (TA-
999-TT, Thermo Fisher Scientific Ltd., Warrington, Cheshire, UK). Positive controls were
canine tissues known to express the relevant antibody (GLUT1: oesophagus, IMP3: stomach,
CK: skin, liver, intestine and kidney, vimentin and desmin: intestine) and negative controls
were processed without the primary antibody. CK, vimentin and desmin IHC are well-
established diagnostic techniques in veterinary laboratories and preliminary studies in one of
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the authors’ laboratory showed cross-reaction of the GLUT1 and IMP3 antibodies used with
canine brain and squamous cell carcinoma respectively (data not shown).
Assessment of sections
The original histopathology reports were used for preliminary categorisation of the diagnoses
that were then refined by two pathologists who reached a consensus diagnosis. The
pathologists were blinded to the original diagnosis. The sections were categorised as either
normal, simple hyperplasia (reactive), atypical mesothelial proliferation, or mesothelioma,
based on the descriptions of Cagle and Churg (2005). Briefly, they state that normal
mesothelium is composed of flat, inconspicuous cells and simple hyperplasia results in a more
conspicuous but bland layer of cuboidal cells, sometimes with distinct nucleoli. Atypical
mesothelial hyperplasia is described as a more florid mesothelial proliferation that varies
from a single layer of cells with more prominent atypia to focal, raised accumulations of cells
with more marked anisocytosis and anisokaryosis and prominent nucleoli. Small tubule-like
structures may sometimes be present. In mesothelioma, there is expected to be invasion of
underlying tissues, cellular nodules with expansion of the stroma, lack of the normal layers of
the pleura and presence of atypical cells within the full thickness, severe cellular atypia and
areas necrosis (Cagle and Churg 2005).
The immunohistochemistry slides were assessed by two pathologists who were blinded to
the histopathological diagnosis. The sections were scored semi-quantitatively for each
antibody for each section using shared example images as a guide:
Percentage of mesothelium positive: 0 = completely negative, 1 = 1-25% of cells positive, 2 =
26-50% positive, 3 = 51-75% positive and 4 = >75% positive.
Intensity of staining: negative (0), weakly positive (1), moderately positive (2), strongly
positive (3)
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The pattern of staining within the cells was also recorded. All antibodies stained the
cytoplasm and/or cell membrane and the pattern was recorded as membranous
(predominantly on the cell membrane) and/or diffuse (all round the nucleus), focal (random),
focal (basal) or focal (apical). Focal (basal) and focal (apical) categories could only be
determined where the polarity of the cells was evident.
Statistical analysis
For each pathologist’s scores, the statistical analysis of the comparison between groups for
number of cells stained and intensity of staining was carried out separately i.e. the scores for
the two pathologists were not averaged. A two pronged approach to the analyses was
adopted. Firstly, whether combinations of scores from the different stains were able to
distinguish between groups in an expected way, i.e. a gradation from group C to R to A to M,
was evaluated by the production of heatmaps using the package gplots (v 3.0.1) in R (v 3.4.0
© 2017 The R Foundation for Statistical Computing), allowing the creation of hierarchical
dendrograms to determine closeness or distance of groups and stains from each other as
represented by a false image matrix. Only the average number of cells and intensity scores
for IMP3, GLUT1 and desmin were included. CK and vimentin were not included in this
comparison as they were only used to identify the cells as being mesothelial in origin (positive
for CK and vimentin) and to aid the subtyping of the mesothelioma cases.
Secondly, use was made of classification tree-based analysis involving partitioning of the
groups via binary recursive partitioning using maximum likelihood (Clark and Pregibon, 1997).
This technique is of particular use with multivariable analyses where the degree of imbalance
in group sizes makes more standard linear analysis approaches difficult (McCann et al. 2007).
Its use here was as an attempt to create an algorithm for identification of cases of R, A and M
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using intensity of staining and number of cells stained for IMP3, GLUT1 and desmin and was
carried out using the package tree (v 1.03-37).
Results
Cases
The tissues dated from 2000-2016 and comprised mainly pericardium with or without
myocardium and attached epicardium. Based on histopathological examination, there were
10 cases of simple mesothelial hyperplasia (group R), 17 cases with atypical mesothelial
proliferation (group A) and 26 cases considered to have mesothelioma (group M). There were
5 controls that consisted of young, pure or crossbred, adult Staffordshire bull terriers from a
pet shelter, euthanised for behavioural reasons. Exact ages were not available for the control
dogs.
The signalment and selected clinical details of the cases in groups R, A and M are shown in
Table 1. A range of breeds were represented with a marked predominance of large dogs in all
three groups. Golden retrievers were the commonest breed in all disease groups. In all three
groups, the age at onset and survival time from first presentation were similar, with mainly
middle-aged and elderly dogs affected. However, the amount of survival data was small. The
dogs in each group showed a range of similar clinical signs. The volume of pericardial fluid
obtained on pericardiocentesis was large in all three groups and the time to relapse of signs
post-pericardiocentesis was very variable and overlapped between groups.
HE staining
Examples of the histopathological patterns of staining, classified according to the method of
Cagle and Churg (2005) are shown in Figure 1. Normal mesothelial cells formed a single flat
cell layer lining the pericardium. These cells contained a small nucleus with an indistinct
nucleolus. The deeper adipose and fibrovascular connective tissues were unremarkable.
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Reactive mesothelial cells were cuboidal and formed a variably intact, single cell layer lining
to the pericardial surface. Nuclei were oval, contained a small prominent nucleolus and
dispersed chromatin. Atypical mesothelial cells were polygonal and formed short to
elongated, exophytic proliferations from the pericardial surface. The proliferations were
supported by variable amounts of fibrovascular connective tissue. Nuclei were oval,
contained multiple, variably sized nucleoli and dispersed chromatin and occasional mitotic
figures were identified. The supporting stroma was infiltrated by variable numbers of
lymphocytes, plasma cells and neutrophils. Atypical mesothelial cells were occasionally
identified within subserosal pericardial lymphatic vessels and there was occasional
entrapment of the mesothelial cells by layers of fibrous connective tissue within the
superficial subserosal stroma. In cases classified as mesothelioma, the cells had a similar
appearance to atypical mesothelial cells; however, there was extensive invasion of the
underlying subserosal adipose tissue. If invasion could not be identified clearly then the case
was assigned to group A. Lymphatic invasion was occasionally identified but this was not used
as a criterion of malignancy given that reactive mesothelial cells can also be present within
subserosal pericardial lymphatics. Additional findings included occasional marked
pleomorphism, atypical mitoses and areas of necrosis.
Mesotheliomas were either polygonal (epithelial pattern) or spindle shaped (sarcomatous
pattern) and both morphologies were sometimes identified within the same mesothelium
(biphasic pattern).
Of the mesothelioma cases, 15 had a tubular or tubulopapillary epithelial pattern, 3 were
sarcomatous and 8 were biphasic (Supplementary Table 1). Intravascular mesothelial cells
were evident in the subserosal layers in one case from group R (10%), 3 from group A (17.6%)
and 9 from group M (34.6%).
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The comparison between the clinical diagnosis, original histological diagnosis at the time the
sample was submitted to the respective laboratory as a diagnostic sample, and the consensus
diagnosis as part of the study is shown in Table 2. For group R, the original pathologist’s
diagnosis was 9/10 classed as reactive and 1/10 as mesothelioma. In group A, the original
diagnosis was 11/17 reactive, 5/17 mesothelioma and 1/17 atypical proliferation. For group
M, the original diagnosis was 19/26 mesothelioma, 5/26 reactive and 2/26 atypical
proliferation. In a majority of cases in groups R and M, there was good agreement between
the clinical and original histological diagnoses but not for group A; this was considered to be
due to lack of use of the group A category by clinicians, and the pathologists who originally
examined the samples. Similarly, the clinical diagnosis and the consensus histological
diagnosis for this study were in agreement for 7/9 cases in group R for which a clinical
diagnosis was available and 11/13 for group M, but only 4/11 for group A.
IHC staining
The IHC slides were scored independently by two pathologists, both of whom were
considered to be able to score consistently; one tended to score slightly higher than the other
for the intensity of staining and the number of cells stained although the results were
qualitatively similar. To avoid excessive complexity, the results for only one pathologist, who
had the most experience of scoring are presented, although the individual scores for each
dog by both pathologists are available in Supplementary Table 1. The sections in the controls
and all three disease groups showed strong staining for CK and vimentin in most of the
mesothelial cells confirming mesothelial origin (Figure 2).
Intracellular staining pattern
CK and vimentin were present intracytoplasmically in the mesothelial cells. Desmin staining
was more variable and was predominantly cytoplasmic in a diffuse location around the
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nucleus although occasionally more focal (3 (30%) group R, 1 (5.9%) group A, 5 (19.2%) group
M). In addition, mesenchymal cells (fibroblasts and myocytes) were positive for vimentin, and
myocytes were immunopositive for desmin but staining patterns were not further assessed in
these cell types.
GLUT1 staining was both cytoplasmic and membranous. IMP3 was diffusely cytoplasmic and
less often focal (4 [23.5%] of group A and 7 [26.9%] of group M [2 randomly focal, 3 apical
and 2 basal]). Only 3 cases in group R stained positively for IMP3 and in each case the staining
was focal, predominantly at the basal pole of the cells. Examples of sections stained for
GLUT1, IMP3 and desmin are shown in Figure 2.
IHC scoring for IMP3 and GLUT1 and desmin
Examples of staining for IMP3, GLUT1 and desmin are shown in Figure 2. The results of the
IHC scoring for intensity of staining and number of cells stained for IMP3, GLUT1 and desmin
are shown in the heatmap in Figure 3. Using a panel of all three antibodies, group C was
distinguishable from the disease groups in having lowest staining for all three and lowest
numbers of cells stained. Of the disease groups, M and A were similar to each other, while
group R differed from groups M and A.
IMP3 showed the greatest ability to distinguish between the disease groups, with a gradation
of increasing intensity and number of cells stained from R to A to M, with group R
indistinguishable from group C (Figure 3). For GLUT1, group R had less staining than groups M
and A which were similar to each other. In a small number of cases of mesothelioma, GLUT1
conferred an advantage over IMP3 alone e.g. of 4 cases in group M with low IMP3 staining, 3
were high for GLUT1.
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Group C had the lowest intensity and number of cells stained for desmin. Intensity of staining
for desmin was not useful in distinguishing groups A and M which had the greatest intensity;
group R was intermediate between control and A/M. For the number of cells staining for
desmin, groups A and R had the highest number with group M intermediate between groups
A/R and C (Figure 3).
An algorithm using the above variables with the aim of creating a decision tree for individual
cases confirmed the trends shown by the heatmaps but was not of additional value in making
a definitive diagnosis (data not shown).
There were no consistent differences between mesothelioma subtypes in intensity of staining
or numbers of cells stained for any of the five antibodies, although the numbers of
sarcomatous and biphasic cases were small (data not shown).
Discussion
The difficulty in establishing a definitive diagnosis in cases of pericardial effusion is well
recognised, even with diagnostic imaging, cytological examination of effusions and pericardial
biopsy. In addition, clinical signs, history and diagnostic imaging including radiography and
echocardiography are also similar in IP and mesothelioma (Smith and Hill, 1989, McDonough
et al., 1992, Stepien et al., 2000). Indeed in one study, masses were suspected on diagnostic
imaging in some cases of IP and were not evident in some dogs with mesothelioma (Stepien
et al., 2000). In the present study, similar difficulties were encountered. Survival time has
been reported to be longer in IP than mesothelioma (Stepien et al., 2000) but others report
similar survival times (Dunning et al., 1998). In the present study, the survival times were
similar between all three groups, although survival data was not available for all patients. This
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aspect would be better addressed in a prospective study, but the similar survival times were
not considered not to preclude a correct histological diagnosis (Dunning et al., 1998).
In group A, there were many discrepancies between the clinical diagnosis, the original
diagnosis by the laboratory at the time of submission of the material, and the consensus
diagnosis used in this study. This is likely to be mainly due to the atypical category not being
adopted by all clinicians and pathologists and some samples pre-dated its description (Cagle
and Churg 2005). However, for groups R and M, the agreement between the clinical, original
histological and consensus histological diagnoses was much closer, supporting the accuracy of
the consensus histological diagnosis used in this study. However, the possibility that some
histological diagnoses were incorrect is not ruled out, a common limitation of studies in
which the gold standard diagnostic method may be suboptimal.
The distinction between reactive and neoplastic mesothelial cells, and in some cases other
types of neoplasia, in cytological and histopathological samples is a common and challenging
dilemma in veterinary pathology (MacDonald et al. 2009, Cagle et al. 2014). This has
significant implications for patient management and prognosis. Simple methods such as
effusion pH (Fine et al. 2003) or biochemical analysis (Laforcade et al. 2005) have not proved
to be of value in this respect. However, there is currently an interest in both veterinary
(Höinghaus et al. 2008, Przezdziecki et al. 2014, D’Angelo 2014,) and human pathology
(reviewed by Churg et al., 2016) in the use of immunological techniques on cytological and
biopsy specimens to distinguish reactive and neoplastic processes affecting serosal surfaces.
The aim of the present study was to develop a preliminary IHC panel, based on that
recommended for use in human pathology, as a potential aid to definitive diagnosis on
pericardial biopsies.
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The age at presentation was similar to that reported previously for IP and mesothelioma
(Stepien et al. 2000, Stafford Johnson et al. 2004) although in our case series, males
predominated, especially in groups R and A. The reason for the apparent sex difference is
unclear but has been reported previously for IP (Gibbs et a. 1982). IP is known to occur
predominantly in large breed dogs with a predominance of Golden retrievers (Aronsohn et al.
1999, Stepien et al. 2000, Stafford Johnson et al. 2004, Cagle et al. 2014) although
mesothelioma is reported to be more common in small and medium-sized breeds (Stepien et
al. 2000). In contrast to these reports, large breeds also predominated in group M in our
study and Golden retrievers were the commonest breed in groups R, A and M suggesting that
the three histological categories could represent different stages of the same pathological
process, at least in this breed. Progression from reactive to neoplastic mesothelium was also
suggested by Machida et al. (2004) who described development of lesions they considered to
be consistent with mesothelioma in five Golden retrievers with a long-term history of IP.
However, the breed incidence in our study could not be compared with those of the
geographically dispersed study populations.
The histopathological lesions in routine HE-stained sections in canine IP and mesothelioma
have been previously described. In IP, there is reported to be diffuse and sometimes nodular
fibrosis, neovascularisation, congestion, variable haemorrhage and inflammation, and areas
of mesothelial hyperplasia and loss (Aronsohn et al. 1999, Day et al. 2002, Peters et al. 2003).
Expansion of the subserosal stroma by loose connective and granulation tissue with denser
expansion of connective tissue more deeply may be present, together with neutrophilic to
mixed inflammatory infiltrates and haemosiderin-laden macrophages. (Day et al., 2002).
More proliferative lesions in the present study were designated as atypical mesothelial
proliferations (group A) as they had features intermediate between simple hyperplasia and
mesothelioma, in particular the exophytic appearance and entrapment of mesothelial cells in
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the subserosal stroma (Cagle and Churg 2005). Previous studies of canine mesothelioma have
described multiple nodules of large cells in nests, cords and acini surrounded by fibrovascular
stroma (McDonough et al., 1992). In addition, epithelioid, fibrous (sarcomatous) and biphasic
types of canine mesothelioma have been described (WHO, 2007). This description formed the
basis for our subcategorization in this study.
Interestingly, the presence of embolizing non-neoplastic mesothelial cells in the lumen of
submesothelial lymphatics in the pericardium and mediastinal lymph nodes is reported in
dogs with reactivity of the pericardial mesothelium (Peters et al. 2003, Goupil et al. 2012).
This feature was also observed in several of our cases in all three disease groups; the highest
frequency was seen in group M but clearly this is not a reliable feature in distinguishing
mesothelioma from benign processes. The cause of embolization is unclear but it is also
recognised in people, and it has been postulated that desquamated mesothelial cells can pass
into lymphatics through stomata that are enlarged by serosal injury (Suárez Vilela et al.
1998). Recognition of this phenomenon is important to avoid an erroneous diagnosis of
metastatic malignancy.
In human pathology, numerous studies utilising IHC have been undertaken to improve
diagnostic confidence in cases of mesothelial proliferation. This is used in particular to
distinguish reactive mesothelium, metastatic disease affecting the pleura (e.g. carcinoma)
and mesothelioma because of the therapeutic, prognostic and legal implications, but their
value and interpretation remains controversial (Travis et al. 2015). In the absence of single
specific markers, panels are recommended. GLUT1, IMP3, p53, desmin and epithelial
membrane antigen (EMA) are the main markers that have been proposed for distinguishing
reactive mesothelium from mesothelioma in people, with desmin expected to be positive in
reactive mesothelium and the others more likely to be positive in mesothelioma. GLUT1 is a
member of the family of glucose transporters which is upregulated in some malignancies.
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IMP3 is a member of the insulin-like growth factor II mRNA binding protein family and is
almost absent from post-embryonic tissues, but is upregulated in some malignancies (King et
al. 2009). Several authors recommend a combination of GLUT1 and IMP3 (Minato et al. 2014,
Galateau-Salle et al. 2015) but others suggest caution as the differences may be statistically
significant between groups but difficult to interpret in an individual clinical case (Husain et al.
2013, Lee et al. 2013), or they distinguish reactive from neoplastic proliferations, but not the
type of neoplasia (Ikeda et al. 2010). The use of more specialised methods as an adjunct to
standard IHC, such as detection of loss of expression of the tumour-suppressor gene p16
have been advocated (Hiroshima et al. 2016) but such genetic markers have not been
validated for this purpose in dogs.
Previous studies of IHC in canine mesothelioma are limited. McDonough et al. (1992) found
CK and vimentin to be strongly expressed in mesothelioma while Machida et al. (2004) used
CK, vimentin and HBME-1 in five mesothelioma cases with strong positive staining for
cytokeratin and weak but positive staining for vimentin and HBME-1. However, at least in
people, mesothelial cells from the subserosal multipotential cells express only vimentin
whereas they co-express CK when they proliferate. Immunocytochemistry has been applied
more recently to cytological preparations in dogs (Przeździecki et al. 2014) and a combination
of CK, vimentin and desmin was found to be useful: all three markers were strongly
expressed in normal and reactive mesothelial cells and mesothelioma, there was weak
expression of vimentin in carcinomas, weak expression of CK in sarcomas and desmin
expression was negative in both carcinomas and sarcomas. However, this would not
distinguish reactive from neoplastic mesothelial cells. Using cytological preparations,
Höinghaus et al. (2008) also noted CK and vimentin positivity in canine mesothelioma but
found desmin staining to be variable. For these reasons, our panel was extended to include
GLUT1 and IMP3 in addition to CK and vimentin.
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In the present study, IMP3 was found to be the most valuable in distinguishing control, R, A
and M groups (Figure 3). Similar results were obtained with GLUT1. On a group basis,
inclusion of GLUT1 did not confer any additional advantage, but in a small number of
mesothelioma cases, IMP3 staining was low but GLUT1 was high, therefore the inclusion of
both antibodies may have advantages over IMP3 alone; this has also been reported in human
pathology (Minato et al., 2014). However, the less than perfect sensitivity and specificity of
IMP3 and GLUT1 for distinguishing benign from malignant disease of the mesothelium is also
recognised in human pathology by some authors (Shi et al. 2011; Chang et al. 2014) and our
findings in the dog agree with this reservation. Desmin was variable between groups and not
considered to be a useful addition to a mesothelioma panel, as previously reported in dogs
(Höinghaus et al. 2008). It was of interest that, at least for IMP3, there was an increasing
gradation in staining in reactive, atypical and mesothelioma cases suggesting that atypical
mesothelial proliferation may be an intermediate stage between reactive mesothelium and
mesothelioma, or a pre-cancerous stage of mesothelioma. Our conclusions are similar to
those in human pathology in that a combination of GLUT1 and IMP3 may be of value in
distinguishing between groups of mesothelial disease categories (Minato et al. 2014,
Galateau-Salle et al. 2015), but can still be difficult to interpret in an individual clinical case
(Husain et al. 2013, Husain 2014, Lee et al. 2013However, the results may still provide
additional supporting information when working up clinical cases.
The pattern of staining within cells has been examined in human tissues; strong membranous
staining of GLUT1 is said to favour mesothelioma (especially the sarcomatous form) over
reactive mesothelium, although no such difference in pattern was shown for IMP3 or desmin
which was mainly cytoplasmic (Minato et al. 2014). No consistent differences between the
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groups or between subtypes of mesothelioma in staining pattern within cells were observed
in our study.
The present study has a number of limitations. In common with many such studies, it was
difficult to be certain that all cases were accurately assigned to the disease groups using the
suboptimal existing gold standard method of HE-stained sections. However, there was good
agreement between the clinical diagnosis, original histological diagnosis and consensus
diagnosis by the study pathologists for groups R and M, suggesting that in most cases the
diagnosis was likely to be correct. Even allowing for the fact that some cases might have been
assigned to the wrong group, some differences in IHC were observed. We were unable to
obtain follow-up information from every case, and post mortem examinations were not
performed on all dogs. In addition, it became evident that the two pathologists scoring the
IHC scored them quantitatively slightly differently although the trends were similar. However,
determining a consensus or an average score between the two was not undertaken as this
was considered likely to complicate the interpretation and would not be representative of
standard practice in a diagnostic laboratory. The inherent subjectivity of IHC scoring is a
limitation of our study, as is the case in all pathological studies based on this method. In some
instances, for example, a low positive score was allocated by one pathologist and a negative
score by the other for the same section, and such discrepancies are difficult to avoid with this
commonly-used scoring method. In the future, image analysis that is rapid and easy to use in
diagnostic pathology laboratories may increase objectivity of scoring. A further limitation
was the relatively small number of cases and care must be taken not to over-interpret the
findings, but since pericardial mesothelioma is a relatively uncommon diagnosis, obtaining a
large case series is challenging.
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In conclusion, the use of an IHC panel including CK, vimentin, IMP3 and GLUT1 may be of
some value in the diagnosis of cases of mesothelial proliferation in canine pericardium.
Future prospective studies using a better characterised canine population and investigation
of novel methods such as genetic markers are warranted. As pericardial biopsy is highly
invasive, the successful application of similar techniques to cells in pericardial fluid could have
clinical and welfare advantages for canine patients presenting with pericardial effusions.
Conflicts of interest
No conflicts of interest have been declared.
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Figure and table legends
Figure 1. Examples of histological findings in different groups: A: Low-grade reactive
mesothelium with oedema and mild inflammation, B: atypical mesothelial proliferation with
severe inflammation and haemosiderin-laden macrophages, C: mesothelioma, epithelial type,
D: mesothelioma, sarcomatous type. HE x10, scale bars 200µm.
Figure 2. A-E: Atypical mesothelium with the expected staining pattern: A: Cytokeratin
positive x40, B: Vimentin positive x40, C: Desmin positive x40, D: GLUT1 low staining x40, E:
IMP3 negative x40. F-J: Mesothelioma with the expected staining pattern: F: Cytokeratin
positive x40, G: Vimentin positive x40, H: Desmin negative x40, I: GLUT1 positive x40, J: IMP3
positive x40. Scale bars 50µm.
Figure 3. Heatmap of a row and column dendrogram of the average number of cells and
intensity of the 3 stains (desmin, GLUT1 and IMP3) as observed in the 4 groups.
Table 1 Signalment and selected history and clinical features of cases of idiopathic pericarditis
(reactive), atypical mesothelial proliferation and mesothelioma included in the study
Table 2. Clinical diagnosis, original histological diagnosis and consensus diagnosis by the study
pathologists in cases of reactive mesothelium (group R, n=10), atypical mesothelial
proliferation (group A, n=17) and mesothelioma (group M, n=26). The original histological
diagnosis is the diagnosis made by a variety of pathologists at the time of submission of the
biopsy to the respective laboratory. Both histological diagnosis categories were made on
haematoxylin and eosin (HE) stained sections.
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Supplementary Table 1. Immunohistochemistry scoring results for cytokeratin, vimentin,
desmin, GLUT1 and IMP3 in groups R, A and M for each pathologist in all dogs, and the
pattern and subtype of mesothelioma diagnosed. The percentage of mesothelium positive
was scored as 0 = completely negative, 1 = 1-25% of cells positive, 2 = 26-50% positive, 3 = 51-
75% positive and 4 = >75% positive. The intensity of staining was scored as negative (0),
weakly positive (1), moderately positive (2) and strongly positive (3). The scores for
pathologist 1 are those discussed in the paper.
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Table 1 Signalment and selected history and clinical features of cases of idiopathic pericarditis
(reactive), atypical mesothelial proliferation and mesothelioma included in the study
Reactive (10 cases) Atypical mesothelial proliferation (17 cases)
Mesothelioma (26 cases)
Breed Golden retriever (3),
Labrador, Rhodesian
Ridgeback, St
Bernard, Grand
Basset Griffon
Vandéen, SBT,
GSD, GSHP (1
each)
Large (9), medium
and small (1)
Golden retriever (5),
Springer spaniel (2),
Dogue de Bordeaux,
Greyhound, Boxer,
Bull Mastiff, GSD,
Pomeranian,
Rottweiler, JRT,
Border collie, GSHP
(1 each)
Large (12), medium
and small (5)
Golden retriever (7),
GSD (3), Labrador
(2), Rhodesian
ridgeback (2),
GSHP, Soft-coated
wheaten terrier,
WHWT, Greyhound,
whippet, Springer
spaniel, Collie,
Crossbred (1 each),
unknown (4)
Large (16), medium
and small (6),
unknown (4)
Sex Male (9), female (1) Male (15), female (2) Male (12), female
(9), unknown (5)
Age when effusion
first reported
(median and range
in years)
8 (3.4-12.3)
(n=7)
8.4 (2.7-11.2)
(n=13)
8.9 (2-14)
(n=9)
Age at time of
biopsy/PME
(median and range
in years)
9.1 (4.1-13)
(n=10)
8.7 (2.7-11.7)
(n=17)
9.2 (2.1-14)
(n=20)
Dyspnoea 4/6 (66.7%) 3/7 (42.9%) 2/7 (28.6%)
Weakness/lethargy 3/6 (50%) 5/9 (55.6%) 7/7 (100%)
Presence of
pericardial effusion
7/8 (87.5%) 13/14 (92.9%) 10/14 (71.4%)
Volume of
pericardial fluid
removed (median
and range in ml)
200 (70-880)
(n=5)
500 (60-900)
(n=6)
400 (240-586)
n=5)
669
670
Presence of pleural
effusion
3/8 (37.5%) 8/13 (61.5%) 7/10 (70%)
Presence of
peritoneal effusion
7/8 (87.5%) 6/10 (60%) 7/8 (87.5%)
Time to clinical
relapse post
pericardiocentesis
(median and range
in days)
13 (5-180) 10 (1-240) 17.5 (1-210)
Survival from first
presentation
(median and range
in months)
7.5 (1-22)
(n=7)
10 (0-100)
(n=9)
4.5 (1-52)
(n=10)
Note that clinical information was unavailable for some cases.
Abbreviations: SBT = Staffordshire bull terrier, GSD = German shepherd dog, GSHP =
German short-haired pointer, JRT = Jack Russell terrier, WHWT = West Highland white terrier,
PME = post mortem examination
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674
675
676
Table 2. Clinical diagnosis, original histological diagnosis and consensus diagnosis by the study
pathologists in cases of reactive mesothelium (group R, n=10), atypical mesothelial
proliferation (group A, n=17) and mesothelioma (group M, n=26). The original histological
diagnosis is the diagnosis made by a variety of pathologists at the time of submission of the
biopsy to the respective laboratory. Both histological diagnosis categories were undertaken
on haematoxylin and eosin (HE) stained sections.
Case identific
ation
Clinical diagnosis
Original histologic
al diagnosis
Consensus
histological
diagnosis for this study
Group RR1 Mesothel
iomaReactive Reactive
R2 Reactive Mesothelioma
Reactive
R3 Reactive Reactive ReactiveR4 Reactive Reactive ReactiveR5 Reactive Reactive ReactiveR6 Reactive Reactive ReactiveR7 Atypical Reactive ReactiveR8 No
information
Reactive Reactive
R9 Reactive Reactive ReactiveR10 Reactive Reactive Reactive
Group AA1 Reactive Reactive AtypicalA2 Reactive Reactive AtypicalA3 Reactive Reactive AtypicalA4 Atypical Reactive AtypicalA5 Atypical Atypical AtypicalA6 Reactive Reactive AtypicalA7 Mesothel
iomaMesothel
iomaAtypical
A8 Atypical Mesothelioma
Atypical
A9 Mesothelioma
Mesothelioma
Atypical
A10 No informati
Reactive Atypical
677
678
679
680
681
682
onA11 No
information
Mesothelioma
Atypical
A12 No informati
on
Reactive Atypical
A13 No informati
on
Mesothelioma
Atypical
A14 Reactive Reactive AtypicalA15 No
information
Reactive Atypical
A16 No informati
on
Reactive Atypical
A17 Atypical Reactive AtypicalGroup M
M1 No informati
on
Reactive Mesothelioma
M2 No informati
on
Reactive Mesothelioma
M3 Reactive Reactive Mesothelioma
M4 Mesothelioma
Atypical Mesothelioma
M5 No informati
on
Reactive Mesothelioma
M6 Reactive Reactive Mesothelioma
M7 Mesothelioma
Mesothelioma
Mesothelioma
M8 Mesothelioma
Mesothelioma
Mesothelioma
M9 No informati
on
Mesothelioma
Mesothelioma
M10 No informati
on
Mesothelioma
Mesothelioma
M11 Mesothelioma
Mesothelioma
Mesothelioma
M12 Mesothelioma
Mesothelioma
Mesothelioma
M13 Mesothelioma
Mesothelioma
Mesothelioma
M14 Mesothelioma
Mesothelioma
Mesothelioma
M15 Mesothelioma
Mesothelioma
Mesothelioma
M16 No informati
on
Mesothelioma
Mesothelioma
M17 No informati
on
Mesothelioma
Mesothelioma
M18 No informati
on
Mesothelioma
Mesothelioma
M19 No informati
on
Atypical Mesothelioma
M20 No informati
on
Mesothelioma
Mesothelioma
M21 Mesothelioma
Mesothelioma
Mesothelioma
M22 No informati
on
Mesothelioma
Mesothelioma
M23 No informati
on
Mesothelioma
Mesothelioma
M24 Mesothelioma
Mesothelioma
Mesothelioma
M25 Mesothelioma
Mesothelioma
Mesothelioma
M26 No informati
on
Mesothelioma
Mesothelioma
683
684
