Oncology

Veterinary Pathology
49(3) 532-537
Comparison of CD34, CD31, and Factor ª The Author(s) 2012
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VIII–Related Antigen Immunohistochemical DOI: 10.1177/0300985811429312
http://vet.sagepub.com
Expression in Feline Vascular Neoplasms
and CD34 Expression in Feline Nonvascular
Neoplasms

R. N. Jennings1,2, M. A. Miller1, and J. A. Ramos-Vara1

Abstract
The diagnosis of vascular neoplasms is often facilitated by the use of immunohistochemical markers such as factor VIII–related
antigen, CD31, and CD34. However, the relative sensitivity and specificity of these markers have not been compared in cat
vascular neoplasms. In this study, these 3 immunohistochemical markers were evaluated in 61 endothelial neoplasms
(50 hemangiosarcomas and 11 hemangiomas) in 59 cats. All neoplasms were labeled by all 3 markers. CD34 had the highest
average immunolabeling intensity in neoplastic endothelial cells. CD31 had the lowest average background labeling, followed by
CD34 and factor VIII–related antigen, respectively. CD34 expression was also examined in 130 nonvascular neoplasms of cats;
14 of 62 epithelial neoplasms, 39 of 43 mesenchymal neoplasms, 8 of 23 leukocytic neoplasms, and 2 of 2 melanomas were
positive. Given the broad expression of CD34 in mesenchymal neoplasms, this marker has limited diagnostic relevance for
vascular neoplasms of cats.

Keywords
CD34, factor VIII–related antigen, CD31, neoplasia, hemangioma, hemangiosarcoma, immunohistochemistry

Vascular neoplasms, including hemangioma and hemangiosar-
coma, are uncommon in cats.5,12,15,24 Vascular neoplasms are
identified by the formation of blood-filled vascular spaces lined
by neoplastic endothelial cells.11 In well-differentiated vascu-
lar neoplasms, diagnosis is straightforward and seldom requires
immunohistochemistry. However, hemangiosarcomas may
regionally form solid sheets of spindloid neoplastic cells
without formation of vascular spaces; poorly differentiated
hemangiosarcomas may lack vascular channel formation alto-
gether and morphologically mimic nonvascular spindle cell
sarcomas. Telangiectatic osteosarcoma may grossly and histo-
logically resemble osseous hemangiosarcoma and is included
in the differential diagnosis for a lytic neoplasm of bone with
blood-filled spaces.10,11 Finally, an epithelioid phenotype of
hemangioma and hemangiosarcoma, described in several
domestic species,30 further complicates morphological diagno-
sis. For these reasons, immunohistochemistry is commonly
used to diagnose vascular neoplasms in domestic species.
Three well-established endothelial cell immunomarkers in
human tissues are factor VIII–related antigen (FVIII-ra),
CD31, and CD34. Factor VIII-ra is a protein packaged in
endothelial cell Weibel-Palade bodies and is also expressed in
megakaryocytes.31 CD31, also known as platelet-endothelial cell
adhesion molecule-1 (PECAM-1), is a transmembrane glycopro-
tein adhesion molecule expressed by platelets, megakaryocytes,
and endothelial cells.31 CD34 is a cell-surface marker expressed
in endothelial cells and hematopoietic stem cells, as well as
nerves, hair follicles, muscle bundles, and sweat glands.14,31
Although the function of CD34 has not been fully elucidated,
L-selectin has been identified as its ligand. CD34 may function
in part as a cell trafficking molecule for leukocytes and hemato-
poietic stem cells.18
The relative utility of FVIII-ra, CD31, and CD34 antibodies
in the diagnosis of cat vascular neoplasms has not been evalu-
ated. The aims of this retrospective study were (1) to evaluate
and compare specific and background immunohistochemical
labeling of vascular neoplasms of cats with anti-FVIII-ra, anti-
CD31, and anti-CD34 antibodies and (2) to evaluate the labeling
of anti-CD34 antibodies in nonvascular neoplasms as part of the
validation of CD34 in the diagnosis of vascular neoplasms.
1
Animal Disease Diagnostic Laboratory and Department of Comparative
Pathobiology, Purdue University, West Lafayette, Indiana
2
Current address for Dr. Jennings: Department of Comparative Medicine,
Wake Forest University School of Medicine, Winston-Salem, NC 27157
Corresponding Author:
José Ramos-Vara, Animal Disease Diagnostic Laboratory and Department of
Comparative Pathobiology, 406 South University, West Lafayette, IN 47907
Email: ramosja@purdue.edu
Jennings et al
Table 1. Immunohistochemical Techniques for Feline Vascular and Nonvascular Tumors
Animal
Antibody Source Antibody Source/Code
Factor VIII- Rabbit Dako, Carpinteria, CA, A0082
ra
CD34 Goat Santa Cruz, Santa Cruz, CA,
SC7045
CD31 Mouse Dako, Carpinteria, CA, M0823
a
HIER, heat-induced epitope retrieval with decloaker, 20-24 psi, 125 C for 30 seconds.
Methods
The Indiana Animal Disease Diagnostic Laboratory archives at
Purdue University were searched for cases of vascular and non-
vascular neoplasms in domestic cats. Cases included in the
study were biopsy and necropsy specimens received from
October 2000 to February 2010. All tissues had been fixed in
10% neutral buffered formalin, paraffin embedded, and stored
in the dark at room temperature. For the vascular neoplasms,
hematoxylin and eosin (HE)–stained sections were evaluated
by all 3 authors, following established diagnostic criteria,11 and
a consensus diagnosis was attained. Vascular neoplasms were
classified as hemangioma or hemangiosarcoma.
Immunohistochemistry was performed on all formalin-
fixed, paraffin-embedded (FFPE) vascular neoplasms using
antibodies to factor VIII–related antigen (FVIII-ra), CD31, and
CD34 according to established protocols21,23 with minor mod-
ifications (Table 1). Immunohistochemistry using anti-CD34
antibody was also performed on all FFPE nonvascular neo-
plasms. Immunohistochemistry was performed using a Dako
autostainer; antibody incubations were at room temperature.
Canine and feline lymph nodes and canine hemangiosarcoma
samples were initially used as positive controls to standardize
the immunohistochemical technique. The chromogen-
substrate combination of 3,30 -diaminobenzidine-H2O2 was
used to detect immunological labeling. Endothelial cells of nor-
mal tissue vessels were used as internal positive controls in all
neoplasms. Normal lymphatic endothelial cells expressed the 3
endothelial markers examined in this study.
The authors independently evaluated the 61 vascular neo-
plasms for neoplastic cell immunohistochemical labeling
intensity and nonspecific background labeling. Specific immu-
noreactivity and background were graded on a subjective scale
of 0 to 3 (in increments of 0.5), in which 0, 1, 2, or 3 equaled
absent, weak, intermediate, or strong labeling (or background),
respectively. The mean immunoreactivity intensity index and
background index were calculated for each vascular neoplasm
and antibody. A value representing the difference between
labeling intensity and background (labeling index) was calcu-
lated by subtracting the mean background index from the mean
intensity index for each antibody. The labeling index for CD34
was compared with those of FVIII-ra and CD31 using a Stu-
dent’s paired t-test (2-tailed, df ¼ 60, P < .05).
533
Incubation Time, Detection
Antigen Retrievala Dilution min System
Proteinase K; 4 min 1:200 30 LSAB 2
HIER citrate buffer; pH, 1:80 120 LSABþ
6.0
HIER citrate buffer; pH, 1:100 90 LSABþ
6.0
Two authors (RNJ and JAR) independently evaluated the
immunoreactivity to CD34 in the nonvascular neoplasms of
cats (Table 2), and a consensus diagnosis for immunoreactivity
(positive or negative labeling) was made.
Results
Vascular Neoplasms
Sixty-one vascular neoplasms (50 hemangiosarcomas and 11
hemangiomas) from 59 cats were included in the study. Two
of 48 hemangiosarcomas had metastasis (included in this
study). Neoplasms of lymphatic origin were not available for
this study. The sites of the neoplasms were skin (23), subcutis
(21), lymph node (4), liver (3), mesentery (3), lung (2), colon
(1), heart (1), tongue (1), gingiva (1), and 1 of unreported tissue
origin. The endothelial cells of nonneoplastic vessels (internal
positive control) within the histological sections were labeled
with all 3 antibodies in all cases. All neoplasms were positive
for factor VIII-ra, CD31, and CD34; however, variation among
markers and neoplasms was marked (Figs. 1–6). Immunolabel-
ing for CD31 was predominantly membranous, with mild cyto-
plasmic labeling. CD34 and FVIII-ra had diffuse cytoplasmic
labeling. FVIII-ra also had punctate, granular labeling in a few
neoplastic cells. In summary, CD34 had the highest mean
immunolabeling intensity (2.95), followed by factor VIII–
related antigen (2.46) and CD31 (2.1). Factor VIII–related anti-
gen had the highest mean background labeling (1.71), followed
by CD34 (1.22) and CD31 (0.21). The average labeling inten-
sity for FVIII-ra, CD31, and CD34 was 2.43, 2.27, and 2.94,
respectively, in hemangiosarcomas, and 2.62, 1.5, and 2.98,
respectively, in hemangiomas. Background labeling for factor
VIII-ra was generally present within the neoplastic interstitium,
in areas of necrosis, and within neoplastic vascular channels.
Background labeling for CD34 was predominantly within the
neoplastic interstitium and was less intense within vascular
channels. Background labeling for CD31 was generally low,
multifocal, and inconsistently present. The labeling index for
factor VIII-ra, CD31, and CD34 was 0.75, 1.92, and 1.74,
respectively. The labeling index for CD34 was significantly
higher (P < .001) than that for FVIII-ra but not significantly dif-
ferent from that for CD31 (P ¼ .115). The labeling index of the
primary and metastatic neoplasms was similar.
534 Veterinary Pathology 49(3)

Table 2. CD34 Immunoreactivity of Feline Epithelial Tumors, based on established criteria6 and included heterogeneous neo-
Mesenchymal Tumors, Leukocytic Tumors, and Melanomas plasms with mixtures of pleomorphic spindle-shaped cells, his-
CD34
tiocytoid cells, and multinucleated giant cells. Details of the
Tumor Type Reactivitya neoplasm types and CD34 reactivity are in Table 2. In summary,
14 of 62 epithelial neoplasms, 39 of 43 mesenchymal neoplasms,
Epithelial tumors 8 of 23 leukocytic neoplasms, and 2 of 2 melanomas had
Intestinal adenocarcinoma 5/7 CD34 immunoreactivity (Figs. 7, 8). In the sole thymoma, the
Apocrine sweat gland ductular adenoma 2/4
neoplastic epithelial cells labeled strongly with CD34, whereas
Parathyroid adenoma 1/1
Parathyroid carcinoma 1/1 the benign lymphocytic component was CD34-negative. In the
Thymoma 1/1 apocrine gland ductular adenoma, more basilar than luminal
Cutaneous squamous cell carcinoma 1/3 epithelial cells were positive for CD34, although this distinction
Oral squamous cell carcinoma 1/3 was lost in more solid regions. Approximately 1% of the hema-
Trichoblastoma 1/3 topoietic cells in the myelolipoma were positive for CD34 (and
Mammary carcinoma 1/14 probably represented hematopoietic stem cells).
Pulmonary adenocarcinoma 0/3
Pulmonary squamous cell carcinoma 0/1
Pulmonary adenosquamous carcinoma 0/1 Discussion
Pulmonary bronchogenic carcinoma 0/1
Pancreatic adenocarcinoma 0/3 Immunohistochemistry is useful in definitive diagnosis of
Salivary adenocarcinoma 0/1 vascular neoplasms in poorly differentiated cases. Immunohis-
Apocrine sweat gland carcinoma 0/2 tochemical markers commonly used to identify neoplastic
Basal cell carcinoma 0/2 endothelial cells include factor VIII–related antigen, CD31,
Biliary carcinoma 0/2
and CD34. In this study of vascular neoplasms in cats, CD34
Meibomian carcinoma 0/1
Thyroid follicular carcinoma 0/1 was the marker with the highest labeling intensity (2.95) for
Hepatocellular carcinoma 0/1 neoplastic endothelial cells and had a moderate background
Hepatic neuroendocrine carcinoma 0/1 (1.22). FVIII-ra had the highest background (1.71) with a mod-
Transitional cell carcinoma 0/3 erate labeling intensity (2.46). CD31 had the lowest back-
Trichoepithelioma 0/1 ground (0.21) and also had a moderate labeling intensity
Trichofolliculoma 0/1 (2.1). A labeling index was calculated by subtracting the back-
Total 14/62
ground scores from the labeling intensity scores for each
Mesenchymal tumors
Pleomorphic sarcoma 10/10 endothelial marker. The endothelial cell labeling index for
Vaccination site sarcoma 8/8 CD34 was significantly higher than that of FVIII-ra and not dif-
Leiomyosarcoma 8/10 ferent from that of CD31. A large difference between specific
Undifferentiated sarcoma 4/4 labeling intensity and nonspecific background labeling is char-
Peripheral nerve sheath tumor 2/2 acteristic of an ideal immunohistochemical marker,22 so based
Osteosarcoma 2/3 on this feature, CD34 may be particularly useful when there is
Sarcoid 1/1
significant background staining with factor VIII-ra in the diag-
Myelolipoma 1/1
Fibrosarcoma 1/1 nosis of vascular neoplasms in cats.
Myxosarcoma 1/1 CD31 is expressed in nearly all human vascular neoplasms31
Fibromatous epulis of periodontal ligament origin 1/2 and was expressed in all cat vascular neoplasms in this study.
Total 39/43 Well-differentiated neoplasms, such as hemangiomas, might
Leukocytic tumors be expected to label equally or more intensely with a specific
Plasma cell tumor 2/2 marker than less well-differentiated neoplasms. However, the
Lymphoma 2/14
average labeling intensity score for CD31 in the hemangiomas
Mast cell tumor 2/5
Histiocytic sarcoma 1/1 (1.5) was less than that in hemangiosarcomas (2.27). This
Cutaneous histiocytosis 1/1 unexpected difference may be due in part to the low number
Total 8/23 of hemangiomas examined (11 vs 50 hemangiosarcomas) and
Additional tumors may not be representative. For FVIII-ra, the labeling intensity
Melanoma 2/2 score was slightly lower in hemangiosarcoma (2.43) than in
a
Number of positive cases / total number of cases examined.
hemangioma (2.62). In human angiosarcomas, inconsistent
FVIII-ra antibody labeling has been reported.16 Alternatively,
overexpression of some genes has been reported in some canine
Nonvascular Neoplasms
vascular neoplasms.26
One hundred thirty nonvascular neoplasms in cats were selected Despite the sensitive labeling of neoplastic endothelial
from the archives. Sixty-two were epithelial neoplasms, 43 were cells with antibodies to CD34, many nonvascular (epithelial,
mesenchymal neoplasms, 23 were leukocytic neoplasms, and 2 mesenchymal, leukocytic, and melanocytic) neoplasms also
were melanomas. The diagnosis of pleomorphic sarcoma was had positive labeling for CD34. CD34 labeling has been
Jennings et al 535

Figure 1. Lymph node; cat. Hemangiosarcoma. Aggregates of neoplastic endothelial cells (arrowheads) label strongly positive for factor
VIII–related antigen, with moderate to marked background labeling of the tumor interstitium (I) and plasma (p). Inset: Detail of the cytoplasmic
labeling. Immunoperoxidase with diaminobenzidine (DAB) chromogen and hematoxylin counterstain. Figure 2. Lymph node; cat.
Hemangiosarcoma; same area as in Fig. 1. Labeling of neoplastic cells for CD34 is stronger than for factor VIII-ra. Moderate background label-
ing is in the neoplastic interstitium and plasma. Inset: Detail of the cytoplasmic labeling. Immunoperoxidase–DAB, hematoxylin counterstain.
536 Veterinary Pathology 49(3)

well-characterized in human endothelial cell neoplasms31 and
in nonvascular neoplasms, such as human precursor lymphoid
and myeloid neoplasms,29,31 human and canine gastrointestinal
stromal neoplasms,8,9,17,19,32 and canine and feline meningeal
neoplasms.23 CD34 labeling has also been sporadically
described in human fibroblastic, myofibroblastic, lipomatous,
smooth muscle, perivascular wall, melanocytic, follicular, and
peripheral nerve sheath neoplasms.1,3,4,13,17,27,28 In our study,
CD34 labeling was observed in most intestinal adenocarcino-
mas (5/7) and parathyroid neoplasms (2/2) and sporadically
in apocrine gland neoplasms (2/6), squamous cell carcinomas
(2/7), follicular neoplasms (1/5), and mammary carcinomas
(1/14). In the sole thymoma, CD34 labeling was restricted to
the neoplastic thymic epithelial cells (confirmed by immuno-
histochemistry for pancytokeratin). CD34-positive labeling has
not been reported in human thymomas.
About one-third of the leukocytic neoplasms in this study
were CD34-positive. Lymphoma derived from precursor lym-
phocytes may be CD34-positive,29,31 so the 2 (of 14) CD34-
positive lymphomas in this study may have been derived from
precursor lymphocytes. Two of 5 mast cell neoplasms had
CD34 labeling. CD34 labeling has been described in mature
murine mast cells,7 although to our knowledge, CD34 labeling
of mast cell neoplasms in domestic species has yet to be eval-
uated. All histiocytic (2/2) and plasmacytic (2/2) neoplasms
evaluated were positive for CD34.
Ninety-one percent of the mesenchymal neoplasms
expressed CD34. Nonvascular spindle cell neoplasms may be
considered in the differential diagnosis for otherwise ambigu-
ous or poorly differentiated vascular neoplasms. The wide-
spread labeling of nonvascular spindle cell neoplasms with
CD34 antibodies in our study suggests that CD34 is not a dis-
tinguishing immunomarker for hemangiosarcoma, in particular
when vascular formation is not apparent and other spindle cell
neoplasms are considered in the differential diagnosis.
Both the melanomas in this study were CD34-positive.
CD34-positive melanomas have been occasionally reported
in humans,2 although CD34 immunoreactivity was not
observed in a recent evaluation of the immunohistochemical
phenotype of canine oral melanoma.25
The difficulty in evaluating the immunoreactivity of the
nonvascular neoplasms is in interpreting the specificity of the
CD34 immunolabeling. Polyclonal antibodies are considered
to be more sensitive markers because of recognition of
multiple epitopes. However, with this increased sensitivity
comes inherent epitope cross-reactivity. In addition, formalin
fixation–associated cross-linking of proteins modifies anti-
genic properties and can affect antigen availability and immu-
noreactivity,20 so a component of nonspecific CD34 labeling
in the examined nonvascular neoplasms cannot be excluded.
Many of the CD34-positive neoplasms in this study have not,
to our knowledge, been reported to be CD34-positive in
human counterparts, making it difficult to interpret positive
immunolabeling as true CD34 protein expression in the
neoplastic cells. Evaluation for CD34 mRNA expression may
better characterize the specificity of CD34 immunoreactivity
in these nonvascular neoplasms. Finally, neoplastic cells are
well-known for their aberrant and varied expression of nonna-
tive proteins, and aberrant CD34 protein expression within
these nonvascular tumor cells is possible.
In a similar study of CD34, CD31, and FVIII-ra expression
in human vascular and nonvascular neoplasms,16 the authors
concluded that a combination of CD34 and CD31 antibodies
provided the best sensitivity and specificity in the diagnosis
of human vascular neoplasms. Our study results are similar and
suggest that although CD34 is a sensitive label for vascular
neoplasms of cats, low specificity may hinder the sole use of
this marker in the diagnosis of feline vascular neoplasms, in
particular when nonvascular spindle cell sarcomas are included
in the differential diagnosis. In summary, the broad expression
of CD34 in neoplasms of cats does not warrant its use as a mar-
ker of vascular neoplasms in this species.
Acknowledgements
We thank Dee Dusold and the Animal Disease Diagnostic Laboratory
histology laboratory staff for technical assistance. Preliminary results
from this study were presented at the 2010 annual meeting of the
American College of Veterinary Pathologists.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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