Veterinary Pathology

48(2) 443-450
Immunohistochemical Identification ª The American College of
Veterinary Pathologists 2011
Reprints and permission:
of Canine Melanocytic Neoplasms sagepub.com/journalsPermissions.nav
DOI: 10.1177/0300985810382095
With Antibodies to Melanocytic http://vet.sagepub.com

Antigen PNL2 and Tyrosinase:

Comparison With Melan A

J. A. Ramos-Vara1 and M. A. Miller1

Abstract
The immunoreactivity of PNL2 and antityrosinase in formalin-fixed, paraffin-embedded canine melanocytic neoplasms (n ¼ 101)
was compared with that of Melan A. Of the 113 samples overall, 106 were positive for PNL2, 101 for Melan A, and 90 for
tyrosinase. Six melanomas that were positive for PNL2 were negative for Melan A; 1 melanoma that was negative for PNL2 was
positive for Melan A. Eighty tumors were positive for all 3 markers; 111 reacted with at least 1 the 3 antibodies. Decalcification
with formic acid for up to 1 week did not affect immunoreactivity of any of the markers; however, decalcification with HCl for
1 day or 1 week notably decreased or completely abrogated immunoreactivity for Melan A and PNL2. There was only minor loss
of immunoreactivity for tyrosinase in tissues decalcified with HCl for 1 week. Prolonged fixation (up to 2 months) did not affect
PNL2 or tyrosinase immunoreactivity; however, Melan A immunoreactivity was reduced after 1 month of fixation. PNL2 was not
expressed in 120 nonmelanocytic tumors (carcinomas, sarcomas, steroid-producing tumors, and leukocytic tumors). In summary,
antibody PNL2 is slightly more sensitive than Melan A and more sensitive than tyrosinase in the identification of canine melano-
cytic neoplasms. Furthermore, PNL2 does not appear to cross-react with nonmelanocytic neoplasms. PNL2 is resistant to
prolonged fixation but sensitive to strong decalcification. Results indicate that PNL2 is an excellent marker in the identification
of canine melanomas and that the sensitivity is close to 100% when used in conjunction with Melan A and tyrosinase.

Keywords
canine, immunohistochemistry, Melan A, melanoma, PNL2, tyrosinase

The diagnosis of melanoma may be challenging, particularly in
tumors without appreciable melanin.27,35 In addition, the vari-
ety of histologic appearances of canine melanocytic neoplasms
makes them difficult to distinguish from sarcomas, carcinomas,
or leukocytic tumors.27 Immunohistochemistry is widely used
to confirm a diagnosis of melanoma in human and veterinary
medicine.5,8,10,16,18,24,27,28,31,35 There are numerous melanoma
markers with variable sensitivity and specificity.10 Commonly
used melanocytic markers in humans include Melan A, PNL2,
S100 protein, and tyrosinase.5,10,35
S100 protein, a calcium-binding protein with an unknown
function in melanogenesis, is a widely used melanocytic mar-
ker with high sensitivity (defined as percentage of positive mel-
anomas) for human melanomas (97 to 100%) and with slightly
lower sensitivity (76%) for canine melanomas.24,27 S100 has
been the standard melanoma marker for many years, but its
specificity is poor in human and animal tissues; numerous non-
melanocytic tumors and normal tissues express this marker,
including but not limited to nerve sheath cells, myoepithelial
cells, adipocytes, and leukocytes, thereby making it unsuitable
as a sole test for the diagnosis of melanoma.24,27
Melan A is a cytoplasmic protein recognized by cytotoxic
T cells.24 It is a specific melanocytic differentiation antigen
(defined as percentage of Melan A–negative nonmelanocy-
tic tumors examined; 95 to 100% in human melanomas)
and highly sensitive (75 to 92%).24 Melan A has been used
for more than a decade in the diagnosis of melanomas from
animals—particularly, dogs but also other species.8,15,18,27,28
Its sensitivity in the dog is high (positive in more than 92%
of melanomas),27 but it is also expressed in nonmelanocytic
steroid-producing tumors of the adrenal gland and gonads.26
PNL2 is a monoclonal antibody originally produced against
a subtype of human somastotatin receptor; its function in
1
Animal Disease Diagnostic Laboratory and Department of Comparative
Pathobiology, Purdue University, West Lafayette, Indiana
Corresponding Author:
Dr José A. Ramos-Vara, Animal Disease Diagnostic Laboratory and Department
of Comparative Pathbiology, Purdue University, 406 South University,
West Lafayette, IN 47907
Email: ramosja@purdue.edu

443
444 Veterinary Pathology 48(2)

Table 1. Antibody Reagents, Antigen Retrieval, and Detection Systems Used in Immunohistochemistry

Antibody (Clone) Dilution Incubation Pretreatmenta Detection System Source of Antibody

CD18 (CA16.3C10) 1:100 60 min HIER citrate buffer; pH, 6.0 EnVisionþ Dr P. F. Moore, Davis, CA
Cytokeratins (AE1/AE3) 1:100 60 min HIER citrate buffer; pH, 6.0 LSABþ Dako, Carpenteria, CA
Melan A (A103) 1:160 90 min HIER EDTA buffer; pH, 8.0 EnVisionþ Dako, Carpenteria, CA
Melanoma antigen (PNL2) 1:150 120 min HIER citrate buffer; pH, 6.0 EnVisionþ Santa Cruz, Santa Cruz, CA
Multiple myeloma antigen 1:200 90 min HIER EDTA buffer; pH, 8.0 ImmPress Dako, Carpenteria, CA
(MUM1)
S100 (polyclonal) 1:800 30 min HIER citrate buffer; pH, 6.0 LSAB 2 Dako, Carpenteria, CA
Tyrosinase (SPM360) 1:25 120 min HIER citrate buffer; pH, 6.0 EnVisionþ Affinity Bioreagents, Golden, CO
Vimentin (SP20) 1:100 30 min Proteinase K, 4 min LSAB 2 LabVision, Freemont, CA
a
HIER, heat-induced epitope retrieval with decloaker, 20–24 psi, 125 C for 30 seconds.

melanogenesis is unknown.30 However, this monoclonal
antibody recognizes a fixative-resistant melanocytic anti-
gen,6,30 the exact structure of which has yet to be identified.24
In humans, PNL2 also labels angiomyolipomas and mature
myeloid cells, including neutrophils but not other nonmelano-
cytic tumors, including various carcinomas and sarcomas.6 The
melanocytic antigen targeted by monoclonal PNL2 can still be
detected after melanin bleaching or decalcification.30 The sen-
sitivity of PNL2 in human melanomas is comparable to that of
Melan A, particularly in the epithelioid variants, but it is very
low for desmoplastic melanomas.6,30 In one study,6 PNL2
detected metastatic melanomas in a higher percentage than did
Melan A, although when compared with the primary tumor, the
number of positive cells was somewhat lower.30 In another
study, PNL2 was less sensitive than Melan A or HMB-45, an
antibody recognizing the premelanosomal glycoprotein
100.22,24 Human mucosal melanomas appear to be more readily
detected with antibodies to PNL2 than to Melan A.21
Tyrosinase is a cytoplasmic melanocyte differentiation pro-
tein, with an essential role in the synthesis of melanin.4,7,23 Tyr-
osinase is present in human cells and tumors of melanocytic
lineage.16 Tyrosinase sensitivity for human melanomas is very
high (84 to 94%) but diminishes with increasing clinical stage
and in metastatic lesions.24 The most commonly used monoclo-
nal antibody to tyrosinase (clone T311) is poorly reactive in des-
moplastic and spindle cell variants of human melanomas.5,16,24
The aims of this study were to (1) document the immunor-
eactivity of monoclonal antibody to melanocytic marker
PNL2 and a monoclonal antibody to tyrosinase in canine mel-
anomas; (2) compare the immunoreactivity with that of Melan
A; (3) determine PNL2 cross-reactivity with nonmelanocytic
tumors; (4) examine the effects of prolonged formalin fixation
on the immunoreactivity of PNL2, Melan A, and tyrosinase
antigens; (5) determine the effects of mild and strong decalci-
fication on the immunohistochemical detection of Melan A,
PNL2, and tyrosinase.
Methods
The Purdue University Animal Disease Diagnostic Laboratory
database was searched for canine melanocytic neoplasms; 122
cases were selected, and histologic sections of each case were
reviewed by one author (J.A.R.-V.) to confirm the diagnosis.
The number of cases selected, based on the diagnosis and the
presence of enough tissue for immunohistochemical proce-
dures, was 101 (total number of samples, 113). The samples
in this study were mostly from the oral cavity (n ¼ 53) or the
skin (n ¼ 26). Fewer melanomas were in other organs, includ-
ing bone (n ¼ 7), heart (n ¼ 1), eye (n ¼ 8), lip (n ¼ 9), lung
(n ¼ 2), and lymph node (n ¼ 7). Nineteen specimens—mostly,
melanomas from the foot or oral cavity—had been decalcified;
in most instances, the exact type of decalcifying solution and
duration of decalcification were unknown.
Three melanocytic markers were used (Table 1): mouse
monoclonal antibody anti-melanoma marker, clone PNL2
(Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclo-
nal antibody anti–Melan A, clone A 103 (Dako, Carpinteria,
CA); mouse monoconal antibody anti-tyrosinase, clone SPM360
(Affinity Bioreagents, Golden, CO). Antibody PNL2 was diluted
1:150 and incubated for 2 hours; anti-Melan A antibody was
diluted 1:160 and incubated for 90 minutes; and antibody to
tyrosinase was diluted 1:25 and incubated for 2 hours. All
reagent incubations were at room temperature. Heat-induced
epitope retrieval with a decloaker was used for all 3 markers
(citrate buffer [pH, 6.0] for PNL2 and tyrosinase; EDTA buffer
[pH, 8.0] for Melan A). To avoid day-to-day variations in inten-
sity of the reaction, all 3 markers were simultaneously tested
for a given sample. A non-avidin-biotin immunoperoxidase-
diaminobenzidine (DAB) detection method (EnVisionþ, Dako)
was used for all markers. Optimization and validation of
Melan A in canine tissues have been reported.26,27 For PNL2 and
tyrosinase, we optimized the immunohistochemical technique
following a previously reported protocol.25 In addition to these
3 melanocytic markers, one or more of the following markers
were used to confirm or rule out a diagnosis of melanoma in
specific cases: S100, vimentin, cytokeratins, CD18, MUM1.25,29
Counterstaining with Azure B was done in cases with moderate
to abundant melanin that was difficult to distinguish from DAB
precipitate. Melanin stained green and DAB remained brown,
although it darkened. Cells containing melanin and DAB stained
dark brown to black.17,27 Normal tissues and 120 nonmelanocy-
tic tumors, including various carcinomas, sarcomas, leukocytic
tumors, and steroid-producing tumors, were also evaluated with
monoclonal antibody PNL2.

444
Ramos-Vara and Miller 445

When possible, the immunoreactivity was compared between Table 2. Immunohistochemical Results by Phenotypea
nondecalcified and decalcified samples from the same tumor
Cell Type n Melan A PNL2 Tyrosinase
(10 cases). To further evaluate the effects of decalcification
on immunoreactivity for the 3 melanocytic markers, formalin- Balloon 3 3 (100) 2 (66) 2 (66)
fixed samples from 2 large melanomas were used. Five paraf- Desmoplastic 7 6 (86) 6 (86) 4 (57)
fin blocks were produced from each tumor; one was not Epithelioid/polygonal 47 41 (87) 45 (96) 40 (85)
exposed to decalcifying solution, and the other 4 were Mixed 32 30 (94) 31 (97) 27 (84)
Spindle 24 21 (88) 21 (88) 16 (67)
immersed in decalcifying solution (either formic acid or HCl
Melanotic tumors 88 85 (97) 87 (99) 69 (78)
solutions; US BioTex Corporation, Webbville, KY) for 1 day Amelanotic melanomas 25 16 (64) 19 (75) 21 (84)
or 1 week. Total 113 101 (89) 106 (94) 90 (80)
The exact fixation time for each sample in this case series a
was unknown, although based on our experience, it ranged Number of positive tumors (percentage).
from 10 hours to 3 days. To determine possible effects of pro-
longed fixation on immunoreactivity, samples from one mela- intensity of reaction for tyrosinase was weaker than for PNL2
noma were fixed in 10% neutral buffered formalin for 1 day and, in some heavily pigmented neoplasms, difficult to distin-
and 1, 2, 4, 6, 8, and 10 weeks. Paraffin sections from each sam- guish from melanin, even after counterstaining with Azure
ple were simultaneously reacted with the 3 melanocytic mar- B (Fig. 1C, inset). The number of samples positive for at least 1
kers used in this study.27 of the 3 melanocytic markers was 111 (98%); the 2 negative
Immunoreactivity was semiquantitatively evaluated in a melanomas were amelanotic, of spindle cell type, and S100
4-grade scale (0 ¼ < 5% positive cells; 1 ¼ 5 to 10% positive positive. Of 88 melanotic tumors, 85 (96.6%), 87 (98.8%), and
cells; 2 ¼ 11 to 50% positive cells; 3 ¼ > 50% positive cells) 69 (78.4%) were positive for Melan A, PNL2, and tyrosinase,
modified from a previous study.27 This grading system was respectively; of 25 amelanotic melanomas, 16 (64%), 19
used to compare reactivity among different melanocytic mar- (75%), and 21 (84%) were positive for Melan A, PNL2, and
kers in the same tumor and the effects of decalcifying solutions tyrosinase, respectively.
and duration of formalin fixation. When immunohistochemical results were grouped by
phenotype (Table 2), Melan A was positive in 41 (87%), 30
(94%), and 21 (88%) of epithelioid/polygonal, mixed, and spin-
Results dle melanomas, respectively; PNL2 was positive in 45 (96%),
The melanocytic tumors in this study were clasified by the 31 (97%), and 21 (88%) samples of epithelial/polygonal,
prevalent cell type.13 A desmoplastic melanoma type was mixed, and spindle melanomas, respectively. The most com-
added to this classification, based on the tumor definition in mon positive phenotype for tyrosinase was epithelioid/polygo-
human pathology. Desmoplastic melanoma is characterized nal, with 40 (85%) samples, followed by mixed (27 samples,
by the presence of abundant stroma admixed with neoplastic 84%) and spindle (16 samples, 67%) cell types. The number
cells.12 The most common phenotype in this series was epithe- of spindle cells positive for Melan A was, in many spindle or
lioid/polygonal (47 samples), followed by mixed (32 samples) mixed melanomas, lower than that for PNL2; tyrosinase reac-
and spindle (24 samples). Less common subtypes included des- tivity in spindle cells was higher than that for the other markers
moplastic (7 samples) and balloon (3 samples). Of the 113 sam- (Fig. 2). In 1 mixed melanoma, the spindle cell component was
ples, 88 (77.7%) were melanotic melanomas and 25 (22.3%) negative for all 3 melanocytic markers. In addition, in compar-
were amelanotic. Twenty-three melanocytic tumors were con- ison of the number and/or intensity of labeling between the
sidered benign; the rest were malignant on the basis of nuclear spindle and polygonal phenotypes of a given mixed melanoma,
features and mitotic index or the presence of metastasis.32 spindle cells were labeled more weakly than polygonal cells.
Of 7 desmoplastic melanoma samples, 6 (86%) were positive
for Melan A and PNL2 and 4 (57%) for tyrosinase. When the
Immunoreactivity of Melanomas for Melan A, PNL2,
cell type of desmoplastic melanomas was considered, those
and Tyrosinase with the polygonal type (2 samples) were always positive for
All 3 melanocytic antigens were located in the cytoplasm. all markers, whereas the spindle cell type was positive in
For PNL2, immunoreactivity was usually granular and diffuse; 2 samples (66%) for Melan A and PNL2 and in 1 sample
for Melan A, granular and diffuse or polar;27 for tyrosinase, dif- (33%) for tyrosinase. In most melanomas, there was no signif-
fuse and usually dull (Fig. 1). Immunoreactivity was strong icant difference in the labeling for any of the 3 melanocytic
(3þ) in 77 (68%), 62 (55%), and 57 (50%) samples for PNL2, markers based on the location of neoplastic cells (superficial
Melan A, and tyrosinase, respectively. Overall, the number of or deep); in only 2 samples, cells in the deepest portion of the
positive cells and intensity of reactivity was higher for PNL2 mass were nonreactive or less reactive than those near the
than for Melan A, although in some cases, Melan A reactivity surface (Fig. 3).
was stronger than for PNL2. In most melanocytic tumors, the Although the majority of tumors were from the skin or the
number of immunoreactive cells for PNL2 was similar and/or oral cavity, there were no apparent differences in the immunor-
lower than that for tyrosinase; however, with few exceptions, eactivity of examined markers based on organ affected (Fig. 4).

445
446 Veterinary Pathology 48(2)

Figure 1. Lip; dog. Polygonal cell–type melanoma. Comparison of immunoreactivity with melanocytic markers: A, Melan A; B, PNL2;
C, tyrosinase. Labeling is more intense with PNL2 than with the other 2 markers. Immunoperoxidase-DAB, hematoxylin counterstain.
Inset: Tyrosinase-positive cells (light brown) contain variable number of melanin granules (green). One melanophage is deeply stained
green. Immunoperoxidase-DAB, Azure B counterstain. Figure 2. Skin; dog. Mixed cell–type melanoma. Immunoreactivity varies, with the
polygonal cells (black circle) more strongly labeled than the spindle cells (asterisk), particularly with Melan A. A, Melan A; B, PNL2; C, tyrosinase.

446
Ramos-Vara and Miller 447

Tyrosinase immunohistochemistry resulted in nonspecific Table 3. PNL2 Immunohistochemistry in Nonmelanocytic

background staining of the epidermis, adnexal epithelium, Neoplasms
and mucosal epithelium. This background staining was some- Tumor Type Positive/Totala
times as intense as the specific immunoreactivity of melano-
cytic tumors (Fig. 5). Adrenocortical carcinoma 0/5
Anal sac carcinoma 0/4
Apocrine adenoma 0/1
Apocrine carcinoma 0/4
Decalcification Effects on Immunoreactivity Basal cell carcinoma 0/1
The effects of decalcification on immunoreactivity was Basal cell tumor 0/6
evaluated in 10 cases in which both decalcified and nondecal- Fibroleiomyoma 0/2
Fibrosarcoma 0/3
cified tumor samples were available. In 6 of 10 cases, there was
Hemangiosarcoma 0/6
reduced immunoreactivity in decalcified samples for Melan Histiocytic sarcoma 0/2
A and PNL2; of these 6 cases, 2 were negative for Melan A. For Histiocytoma 0/12
tyrosinase, 4 of 8 cases had mild reduction in immunoreactiv- Interstitial (Leydig) cell tumor 0/10
ity. Two samples of melanomas were used in a controlled Leiomyoma 0/1
experiment of decalcification effects. When formic acid was Leiomyosarcoma 0/2
used as decalcifying solution, there were no negative effects Malignant peripheral nerve sheath tumor 0/1
Mast cell tumor, grade 3 0/10
in immunoreactivity for any of the 3 melanocytic markers,
Mycosis fungoides 0/11
except for a slight decrease in reactivity for PNL2-labeled tis- Liver 0/1
sues in 1 sample decalcified for 1 week. There was major Spleen 0/1
immunoreactivity reduction for Melan A and PNL2 in samples Osteosarcoma 0/5
decalcified with HCl after 1 day; samples were nonreactive Plasmacytoma 0/5
after 1 week of decalcification (both samples for Melan A and Sarcoma 0/1
1 sample for PNL2) (Fig. 6). Loss of immunoreactivity was Sebaceous adenocarcinoma 0/1
Sebaceous epithelioma 0/3
mild to moderate for tyrosinase and observed after only 1 week
Sertoli cell tumor 0/7
of decalcification in HCl. In addition, the intensity for tyrosi- Soft tissue sarcoma 0/10
nase was higher in tissues decalcified with HCl for 1 day than Squamous cell carcinoma 0/5
in nondecalcified tissue from the same specimen. Total 0/120
a
Number of positive cases / total number of cases examined.

Effects of Prolonged Fixation on Immunoreactivity

Slight to moderate Melan A immunoreactivity reduction (2þ)
was observed in tissues fixed for 1 month or longer and was
more apparent (1þ) at 10 weeks’ fixation. Prolonged fixation
(up to 10 weeks) did not affect immunoreactivity for PNL2.
Tyrosinase immunoreactivity was slightly reduced in a mela-
noma fixed for 10 weeks.
PNL2 and Nonmelanocytic Neoplasms
None of the normal tissues (except for epidermal melanocytes
and late spermatids of seminiferous tubules) or nonmelanocytic
neoplasms (including steroid-producing tumors) reacted with
antibody PNL2 (Table 3). These tumors were not examined
with the other 2 melanocytic markers. Canine neutrophils were
not labeled with PNL2 antibody.
Discussion
In this study, antibody PNL2 was highly sensitive and specific
for canine melanocytic neoplasms. Although PNL2 antibody
has been successfully used for human and rodent melanocytic
proliferations,6,19,30 to our knowledge this is the first report
of PNL2 immunohistochemistry in canine melanocytic neo-
plasms. The number of melanomas that were positive for PNL2
was equal to, or slightly higher than, that of Melan A for the
different phenotypes. PNL2 reactivity is low or undetectable
in human spindle and desmoplastic melanomas.6,30 In our
study, although spindle cell melanoma was less reactive to
PNL2 than were other phenotypes, the number of positive cases
for this phenotype was high (86%) and identical to that for
Melan A. Other authors have not noticed a significant reduction
of PNL2 reactivity in rodent spindle cell melanomas.19,20

Figure 2 (continued). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 3. Oral cavity; dog. Polygonal cell–type melanoma. Strong
labeling of junctional melanoma cells as well as through the entire thickness of the tumor. Inset: Detailed labeling of mucosal melanocytes.
Immunoperoxidase-DAB for PNL2, hematoxylin counterstain. Figure 4. Eye; dog. Mixed cell–type melanoma. Retina (asterisk).
Immunoperoxidase-DAB for PNL2, hematoxylin counterstain. Figure 5. Lip; dog. Polygonal cell–type melanoma. Strong labeling of
melanoma cells in the dermis and background labeling of the epidermis (asterisk). Immunoperoxidase-DAB for tyrosinase, hematoxylin
counterstain. Figure 6. Oral mass; dog. Spindle cell–type melanoma. Effects of decalcification. Major loss of immunoreactivity occurs after
decalcifying with HCl. A, nondecalcified sample; B, formic acid decalcification for 1 day; C, formic acid decalcification for 1 week; D, HCl
decalcification for 1 day; E, HCl decalcification for 1 week. Immunoperoxidase-DAB for PNL2, hematoxylin counterstain.

447
448
None of the nonmelanocytic tumors or normal tissues
(except for normal melanocytes and late spermatids of the
seminiferous tubules) reacted with antibody PNL2. Based
on these results, the specificity of PNL2 for canine melanocy-
tic tumors appears to be 100%. In contrast, Melan A is
expressed in canine steroid-producing tumors.26 No attempt
was made to examine cross-reactivity of tyrosinase with non-
melanocytic neoplasms.
Melan A is one of the most commonly used melanocytic dif-
ferentiation markers for human and canine melanomas.16,27 As
with other melanocytic markers, Melan A is poorly reactive
with many human spindle or desmoplastic tumors.16 Melan
A sensitivity in this study was slightly lower (89%) than
previously reported and lower than that for PNL2.27 However,
Melan A was positive in one sample that was negative for
PNL2 and tyrosinase. Melan A reactivity for spindle and
desmoplastic melanomas was identical to that for PNL2 and
higher than that for tyrosinase.
The reported sensitivity of tyrosinase in human melanomas
is comparable to that for other melanocytic markers (84 to
97%) but slightly lower in metastatic lesions and poorly reac-
tive in desmoplastic and spindle melanomas.4,5,9,11,14,16,22,24
A monoclonal antibody to tyrosinase commonly used in human
studies (clone T311) has been used in canine melanomas with
poor results owing to cross-reactivity with normal mucosal
epithelium, epidermis, adnexal structures, and skeletal mus-
cle.27 In the current study, a different anti-tyrosinase monoclo-
nal antibody was used, one reported by the manufacturer to be
reactive on human, canine, and feline samples, and the number
of positive melanomas was lower than that for Melan A or
PNL2. In addition, tyrosinase immunoreactivity was usually
less intense than that for the other markers and sometimes dif-
ficult to distinguish from background reaction or melanin,
even after Azure B counterstain. Also, we experienced
cross-reactivity issues similar to those previously reported.27
In this study, the main advantage of using tyrosinase as a mel-
anocytic marker was that decalcification with HCl apparently
did not reduce its antigenicity, as opposed to that for the
other 2 markers. Interestingly, decalcification, particularly
with HCl, seemed to increase the intensity of the reaction for
tyrosinase when compared with that of the nondecalcified
samples. Moreover, the percentage of amelanotic melanomas
positive for tyrosinase was higher than that for melanotic
tumors (23 of 25, 92%) and higher than that for Melan A or
PNL2 combined (20 of 25, 80%), which makes tyrosinase a
valuable marker in amelanotic melanomas. However, the
apparent background produced by the tyrosinase antibody
will require evaluation of nonmelanocytic tumors with this
marker to validate its use in melanocytic neoplasms. The
reduced number of melanotic melanomas positive for tyrosi-
nase might have resulted from difficulties evaluating the reac-
tivity in heavily pigmented tumors.
The combined use of antibodies to Melan A, PNL2, and
tyrosinase in this study provided a high sensitivity (98%) for
melanocytic tumors. The combined use of Melan A and PNL2
slightly increased the sensitivity when compared with that of
448
Veterinary Pathology 48(2)
PNL2 alone (95% versus 94%). Based on the phenotype
(spindle amelanotic) of the 2 neoplasms negative for these 3
markers and their positivity for S100, we cannot completely
rule out other tumor types, including malignant peripheral
nerve sheath tumor.
The antigen recognized by monoclonal antibody PNL2 is
considered resistant to melanin bleaching with KMnO4 and
nitric acid decalcification, although the duration of this treat-
ment was not indicated; in the same study, Melan A immunor-
eactivity was abolished after these treaments.30 We attempted
to bleach several melanotic melanomas, but during the immu-
nohistochemical procedure, most of the tissue was lifted off the
slide or was severely damaged, precluding interpretation of
results. Two different decalcifying solutions were used in this
study—one solution considered to have mild decalcifying
action with good preservation of morphology and hematoxylin
and eosin staining (formic acid) and one solution with HCl con-
sidered to be a strong decalcifying agent with alterations in the
hematoxylin and eosin staining. Results of the controlled
experiment with decalcifying solutions indicate that the anti-
gens recognized by all 3 melanocytic markers did not lose
immunoreactivity after 1 day of treatment with formic acid,
and only PNL2 had minor loss of immunoreactivity after
1 week of decalcification. However, when HCl was used, there
was major loss of immunoreactivity for Melan A and PNL2 and
complete loss after 1 week of decalcification. Immunoreactiv-
ity for tyrosinase was better preserved than it was for the other
2 antigens after decalcification with HCl. These results suggest
that when stringent decalcification is used in a suspected mel-
anoma, tyrosinase should be included in the diagnostic panel
because it appears to be more resistant than Melan A or PNL2
to decalcification-induced antigenicity loss. Because decalcifi-
cation with nitric acid is not a regular procedure in our labora-
tory, we did not examine its effects in this study.
In most samples, location of the tumor cells (superficial
or deep) within the mass did not significantly affect the
labeling intensity of any of the 3 melanocytic markers. Only
in 2 cases were cells in the deep portion of the mass either
not labeled or only weakly labeled. Immunoreactivity for
antibody HMB45, commonly used to characterize human
melanomas, is progressively lost with increasing depth from
the tumor surface.24
Prolonged fixation results in mild to moderate reduction in
immunoreactivity for Melan A.27 To our knowledge, the effects
of prolonged fixation on PNL2 and tyrosinase immunoreactiv-
ity have not been reported. In this study, fixation up to 10 weeks
did not affect the immunoreactivity for PNL2. Tyrosinase
immunoreactivity was slightly affected after 8 weeks of
fixation; Melan A immunoreactivity was slightly or moderately
affected after 4 weeks of fixation. Historically, prolonged for-
malin fixation was believed to reduce tissue antigenicity owing
to the formation of cross-links,1-3 altering or hiding epitopes
and preventing immune recognition.33 However, we evalu-
ated 82 cellular and infectious disease antigens of various ani-
mal species and found that 93% of the antigens were
adequately preserved after 7 weeks of fixation.33,34 Results
Ramos-Vara and Miller
of the current study corroborate those findings and support the
idea that for most antigens, with optimal antigen retrieval pro-
cedures, prolonged formalin fixation does not significantly
alter immunoreactivity.
Given the results of this study, we conclude the following:
First, immunohistochemistry on formalin-fixed, paraffin-
embedded tissues with antibody to melanoma marker PNL2
is highly sensitive and specific for canine melanocytic neo-
plasms. Second, antibody PNL2 sensitivity is slightly superior
to that of Melan A or tyrosinase. Third, decalcification with
HCl markedly reduces immunoreactivity for Melan A and
PNL2. Fourth, prolonged formalin fixation does not affect
PNL2 immunoreactivity and only slightly reduces Melan A and
tyrosinase immunoreactivities. Fifth, the combined use of anti-
bodies to PNL2, Melan A, and tyrosinase provides almost
100% sensitivity for canine melanocytic neoplasms.
Acknowledgements
We appreciate the technical assistance of Dee DuSold and the Animal
Disease Diagnostic Laboratory histology staff. Preliminary results
from this study were presented as a poster in the 2009 annual meeting
of the American College of Veterinary Pathologists (abstract No. 83).
Declaration of Conflicting Interests
The authors declared no potential conflicts of interests with respect to
the authorship and/or publication of this article.
Financial Disclosure/Funding
The authors received no financial support for the research and/or
authorship of this article.
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