Received: 3 March 2022 Revised: 26 April 2022 Accepted: 3 May 2022

DOI: 10.1111/vco.12827

REVIEW

Diagnosis and histopathologic prognostication of canine

melanocytic neoplasms: A consensus of the
Oncology-Pathology Working Group

Rebecca C. Smedley1 | Laura Bongiovanni2,3 | Cynthia Bacmeister4 |

Craig A. Clifford5 | Neil Christensen6,7 | Jennifer M. Dreyfus8,9 | Joy M. Gary10,11 |
Alana Pavuk12 | Peter H. Rowland13 | Christine Swanson14 | Chelsea Tripp15 |
J. Paul Woods16 | Philip J. Bergman17
1
Veterinary Diagnostic Laboratory, Michigan State University, Lansing, Michigan, USA
2
Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
3
Faculty of Veterinary Medicine, Department of Biomolecular Sciences, Utrecht University, Utrecht, The Netherlands
4
Anatomic Pathology, Antech Diagnostics, Fountain Valley, California, USA
5
Oncology Service, Hope Veterinary Specialists/BluePearl, Malvern, Pennsylvania, USA
6
Oncology Service, Veterinary Specialty Hospital Hong Kong, Wan Chai, Hong Kong
7
Veterinary Medical Teaching Hospital, University of Wisconsin, Madison, Wisconsin, USA
8
Anatomic Pathology, Dreyfus Veterinary Pathology Consulting, Madison, Wisconsin, USA
9
School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin, USA
10
Neuropathology, StageBio, Frederick, Maryland, USA
11
Comparative Biomedical Training Program, Molecular Pathology Unit, NCI, NIH, Bethesda, Maryland, USA
12
Anatomic Pathology, Antech Diagnostics, Durham, North Carolina, USA
13
Biopsy Service, Histopath Consulting, Worcester, Massachusetts, USA
14
Oncology Service, BluePearl Specialty and Emergency Pet Hospital, Grand Rapids, Michigan, USA
15
Oncology Service, Bridge Animal Referral Center, Edmonds, Washington, USA
16
Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
17
Clinical Studies, VCA, Los Angeles, California, USA

Correspondence
Rebecca C. Smedley, Veterinary Diagnostic Abstract
Laboratory, Michigan State University, Lansing, One of the primary objectives of the Oncology Pathology Working Group (OPWG) is
MI 48910, USA.
Email: [email protected] for oncologists and pathologists to collaboratively generate consensus documents to
standardize aspects of and provide guidelines for veterinary oncologic pathology. Con-
sensus is established through review of relevant peer-reviewed literature relative to a
subgroup's particular focus. In this article, the authors provide a critical review of the
current literature for the diagnosis of, and histopathologic prognostication for, canine
cutaneous and oral/lip melanocytic neoplasms, suggest guidelines for reporting, provide
recommendations for clinical interpretation, and discuss future directions. This

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.

Vet Comp Oncol. 2022;20:739–751. wileyonlinelibrary.com/journal/vco 739

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740 SMEDLEY ET AL.

document represents the opinions of the working group and the authors and does not
constitute a formal endorsement by the American College of Veterinary Pathologists,
American College of Veterinary Internal Medicine or the Veterinary Cancer Society.

KEYWORDS
cancer, dogs, melanoma, oncology, pathology, prognosis

1 | I N T RO DU CT I O N paper addressed any diagnostic or prognostic markers. If it was uncer-

tain, the chairs (PJB and RCS) scanned the articles to determine if any
In 2011, Smedley et al., published “Prognostic Markers for Canine previous or new diagnostic or prognostic parameters were evaluated as
Melanocytic Neoplasms: A Comparative Review of the Literature and a minor portion of the study. Articles related to treatment or prognostic
Goals for Future Investigation”, a critical review of canine melanocytic clinical criteria of canine cutaneous and oral/lip melanocytic tumours
neoplasm studies published prior to February 2011, as an initiative of were not included in this review but were saved for a subsequent con-
the American College of Veterinary Pathologists' (ACVP) Oncology sensus report. The focus of this consensus statement is on the histologi-
Committee.1 The authors based that review on the criteria described cal features of, and molecular markers for, cutaneous and oral/lip
in the consensus by Webster et al. “Recommended Guidelines for the melanocytic neoplasms. Three subgroup members were assigned to each
Conduct and Evaluation of Prognostic Studies in Veterinary Oncol- article for critical review, avoiding assignment of articles they authored.
ogy”.2 The main goal of the 2011 review was to report which publi- Each member used an Excel table template that was the same as the
shed parameters “had the most statistically supported validity for one used by Smedley et al.1 and was based on the standards outlined by
prognostic use in canine melanocytic neoplasia.”1 Similarly, as an Webster et al.2 This table provided a summary of the objective/hypothe-
update of the 2011 review, this current manuscript provides a critical sis, study design, materials and methods, statistical soundness, conclu-
consensus review of canine melanocytic neoplasm diagnostic and sions by the article's authors, and conclusions of the reviewing subgroup
prognostic pathology studies that have been published from 2011 to member, in addition to other criteria. The co-chair (RCS) then combined
2021 based on the same guidelines published by Webster et al.2 This the reviews from each author into one review per article in the same
consensus is based on the work of the canine melanoma subgroup as Excel table format. To expedite review of an additional 6 articles, includ-
part of the Oncology Pathology Working Group (OPWG), a joint ini- ing one published in 201220 and 5 published from July 2017 to March
tiative of the Veterinary Cancer Society and the ACVP that was 2019,21–25 the co-chair (RCS) completed the review of these articles and
formed from the ACVP Oncology Committee. We provide recommen- distributed the reviews to the subgroup for comments and discussion.
dations for the diagnosis, and histopathologic prognostication, of Next, the co-chair (RCS) summarized and condensed the reviews and
canine cutaneous and oral/lip melanocytic neoplasms in a diagnostic drafted the initial consensus report. Additional articles were referenced
setting, suggest guidelines for reporting, provide recommendations in the report as needed but were not critically reviewed.26–35 The
for clinical interpretation, and discuss future directions. This document reviews and the consensus report were distributed to all subgroup
represents the opinions of the working group and the authors and members for edits, discussion and comments and then submitted to
does not constitute a formal endorsement by the ACVP or the Veteri- the OPWG Executive Committee for review. The consensus was
nary Cancer Society. made available to OPWG membership at large, which approved the
report by popular vote, and then it was made available online on
the OPWG webpage (http://vetcancersociety.org/vcs-members/vcs-
2 | METHODS groups/oncology-pathology-working-group/) in August 2020. While
preparing the current manuscript, 14 additional articles36–49 were identi-
2.1 | Development of the consensus report fied that were published from November 2019 to August 2021. Again,
to expedite review of these 15 additional articles, the co-chair (RCS)
The canine melanoma subgroup of the OPWG, which includes all coau- completed the reviews and distributed them to the subgroup for com-
thors of this article, reviewed recent canine melanocytic literature. The ments and discussion. All authors agreed on inclusion of these references
canine melanoma subgroup includes 6 board-certified veterinary oncolo- here, but they were not present in the OPWG consensus statement and,
gists (PJB, CS, CT, CAC, NC, PW) and 7 board-certified veterinary therefore, were not voted on by the OPWG membership.
pathologists (RCS, LB, CB, JG, PR, JD, AP). The chairs (PJB and RCS) ini-
tially selected the articles for review, which included the 2011 review
paper by Smedley et al.1 and subsequent diagnostic or prognostic articles 2.2 | Review of the literature
3–19
from February 2011 to December 2017. Articles were searched in
https://pubmed.ncbi.nlm.nih.gov/ using the phrase “canine melanoma”. Since 2011, there have been few published studies that have identi-
The abstracts were then evaluated to determine whether or not the fied novel diagnostic or prognostic markers for canine melanocytic
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SMEDLEY ET AL. 741

neoplasms that have enough statistical soundness, and that can easily origin, demonstration of immunohistochemical (IHC) labelling for
be used in a diagnostic setting. The conclusions in most of these stud- specific melanocytic markers is needed (Table 1).
ies still require further validation via additional corollary studies. Melanocytic neoplasms in veterinary species have been shown to
Unfortunately, there have only been rare published prospective prog- label for several different IHC markers, but each marker has different
nostic studies for canine melanocytic neoplasms and those particular sensitivities and specificities among species, including humans, thereby
studies have not yielded established prognostic markers at this limiting the use of a single antibody for diagnosing amelanotic
19
point. While not the major goal, some of the new studies do provide melanocytic neoplasms. The most sensitive and specific markers to
further support for the use of the parameters recommended in the detect melanocytic neoplasms in veterinary species are still Melan-A and
2011 review.1 The studies that provide the most potentially useful PNL2,34,46 which are antigens that are found on melanocytes. In dogs,
data, support previous recommendations, and have future utility are antibodies against tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-
discussed below. 2) have also been shown to be highly sensitive and specific.34 A diagnos-
tic melanoma cocktail that contains antibodies against Melan-A, PNL2,
TRP-1 and TRP-2 has been shown to have 100% specificity and 93.9%
3 | RESULTS sensitivity in detecting canine oral melanocytic neoplasms compared to
soft tissue sarcomas in one study, and is commercially available.34 This
A total of 37 articles were originally selected, of which 24 contained cocktail has been shown to have a sensitivity that is greater than the
prognostic criteria suitable for the consensus document. A total of individual sensitivities of each individual antibody and to result in a
23 additional articles were identified after the consensus document greater labelling intensity.34 Thus, this cocktail makes it easier to identify
was published online. Of these, 14 contained prognostic criteria that labelling in small samples and in tumours that only exhibit a small amount
were deemed important to include here. of labelling with the individual markers and it is considered to be the cur-
rent gold standard for diagnosing canine amelanotic malignant melano-
mas (MM).34,46 The most common cells to label with melanocytic
3.1 | Diagnostic markers for melanocytic markers are intraepithelial neoplastic cells, which are the most differenti-
neoplasms ated of the neoplastic cells, as the growth of the neoplasm begins in the
epithelium.34 Thus, it is extremely important for clinicians to submit non-
The first step in prognostication, and to decide the correct therapeutic ulcerated portions of the mass, as well as wide lateral margins that
approach, is to obtain an accurate diagnosis. This should also be the include intact lateral flanking epithelium, in order to increase the likeli-
first step when performing and reviewing prognostic studies. If hood of identifying intraepithelial nests of neoplastic melanocytes and to
melanocytic origin has not been definitively confirmed for every improve likelihood of complete excision. Small nests can sometimes be
neoplasm in a study population, the results of that study cannot be difficult to discern with routine histology, but they are easily identified
interpreted accurately. Diagnosis is based on specific histologic with IHC labelling.
features of cutaneous and mucosal melanocytic tumours. However, IHC for S-100 and for microphthalmia-associated transcription
when histologic features alone are not confirmatory of melanocytic factor (MITF) have also been explored.34 These markers showed

TABLE 1 Diagnostic molecular markers for canine melanocytic tumours

Marker(s) Samplesa-tumour locationb Methodsc Application utility References

Melan-A T-CM and OM IHC Confirm melanocytic origin 34,46
T-OM ICC Confirm melanocytic origin 16
PNL2 T-CM, OM IHC Confirm melanocytic origin 34,46
TRP-1 and TRP-2 T-CM, OM IHC Confirm melanocytic origin 34
SOX-10 T-OM IHC Differentiate from soft tissue sarcomas (90% specificity) 46
TYR, CD34 T-OM (spindloid amelanotic) RT-qPCR Differentiate from soft tissue sarcomas 46
and CALD1
MITF T-CM IF Confirm melanocytic origin 4
IHC 89.8% sensitivity but only 30% specificity when used to 34
differentiate from soft tissue sarcomas
Metabolite profiled P-OM GC–MS Distinguish dogs with melanomas from healthy control dogs 13
a
Samples: T, tissue; P, plasma.
b
Tumour location: CM, cutaneous melanoma; OM, oral melanoma.
c
Methods: IHC, immunohistochemistry; ICC, immunocytochemistry; IF, immunofluorescence; RT-qPCR, quantitative reverse transcription PCR; GC–MS,
gas chromatography–mass spectrometry.
d
Metabolite profile: Citric acid, lactic acid, oleic acid, linoleic acid, palmitoleic acid, octadecenoic acid and glycerol.
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
742 SMEDLEY ET AL.

81.6% and 89.8% sensitivity respectively, but only showed 20% and morphology.16 It still may be difficult to differentiate between meta-
30% specificity, respectively, in differentiating melanocytic tumours static neoplastic melanocytes and draining melanocytes, however.
34
from soft tissue sarcomas. Campagne et al. used immunofluorescent
labelling for MITF to identify neoplastic and non-neoplastic melano-
cytes.4 In that study, the immunofluorescent method showed 100% 3.2 | Prognostication of melanocytic neoplasms
sensitivity and 100% specificity. In contrast, the authors stated that
IHC labelling for MITF did not identify tumuoral cells in all of the An accurate prognosis is critical for appropriate recommendations
samples.4 for primary and/or adjunct therapy. Assessment of a combination of
SOX-10 is a marker that has been used to diagnose melanocytic highly reliable parameters will provide the most accurate prediction of
neoplasms in humans26,33 but until recently, there had been no publi- prognosis. In terms of prognostication of canine melanocytic
shed studies of its use in dogs. In humans, it is not specific to melano- neoplasms, the recommendations in the review paper by Smedley
cytes and has been shown to be consistently expressed in benign et al.1 were judged to still be the most diagnostically and
Schwann cell tumours of soft tissue and the gastrointestinal tract and prognostically useful, with the addition of assessing tumour thickness
to be variably present in malignant peripheral nerve sheath tumours.33 for non-oral cutaneous melanocytic neoplasms.
It has also been shown to label myoepithelial cell origin tumours, gran-
ular cell tumours, histiocytes, occasional alveolar rhabdomyosarcomas
and some epithelial tumours, including rare squamous cell carcinomas 3.2.1 | Tumour location of cutaneous, oral and lip
of the head and neck and pulmonary small cell carcinomas.26,29,30,33 In melanocytic neoplasms
addition, SOX-10 can be expressed in entrapped non-neoplastic
Schwann cells or melanocytes in various neoplasms and this has to be Historically, canine oral melanocytic tumours have been regarded as
considered when diagnosing SOX-10-positive tumours.33 In humans, malignant and cutaneous melanocytic tumours have been regarded as
soft tissue sarcomas are uncommon and often are not a differential benign. While, in general, digital/subungual and lip tumours have been
for a melanoma. In contrast, soft tissue sarcomas, including peripheral shown to have increased recurrence and metastasis compared to
nerve sheath tumours, in dogs are very common and are the primary tumours at other cutaneous sites.20,35 Other studies have shown that
differential for a spindloid melanoma. this is not always the case and that location alone cannot predict
The diagnostic melanoma cocktail has the lowest sensitivity in prognosis.23,27,28,31,35 In one study, 92% of oral and 74% of feet & lip
34
spindloid variants of amelanotic MM. Thus, a recent study exam- melanocytic neoplasms were originally classified as malignant but only
ined additional IHC markers and gene expression patterns in these 59% of oral and 38% of feet & lip neoplasms showed malignant
tumours to improve the ability to differentiate between oral behaviour, and a subset of cutaneous melanocytic neoplasms showed
amelanotic spindloid MMs and oral soft tissue sarcomas.46 IHC label- malignant behaviour that would have been predicted to be benign
ling for SOX-10 was also examined in that study. Tsoi et al. examined based on location and current microscopic criteria for prognostica-
20 oral MMs and 20 soft tissue sarcomas for SOX-10 immuno- tion.35 Recently, a significant correlation between histologic diagnosis
expression and found that all 20 oral MMs and 2 out of 20 soft tissue of cutaneous melanocytic neoplasms and location was found when
sarcomas labelled for SOX-10, resulting in 100% sensitivity but only the diagnostic criteria described by Smedley et al.1 were used.40 Simi-
46
90% specificity for that antibody. In addition, the authors examined lar to Spangler and Kass,35 these authors found that cutaneous MMs
various gene expression patterns and determined that mRNA levels were more likely to be on the digit compared to melanocytomas. They
of TYR, CD34, and CALD1 can be used to differentiate between also found that there were fewer MMs than melanocytomas on the
canine oral spindloid amelanotic MMs and soft tissue sarcomas that abdomen.40
are negative for specific melanocytic markers (Melan-A, PNL2, TRP-1 Overall, the challenge is to identify benign oral/lip melanocytic
and TRP-2) with 100% specificity and 65%, 95% and 60% sensitivity, neoplasms and malignant cutaneous ones. At this time, histologic
respectively.46 Availability of gene expression analysis for canine criteria and Ki67 labelling, by use of commercially available antibodies,
melanomas in a diagnostic setting would greatly improve a patholo- are evaluated for this purpose.
gist's ability to more confidently distinguish between these two
tumour types.
Cytologic +/ immunocytochemical (ICC) examination of poten- 3.2.2 | Histological criteria
tial melanocytic neoplasms should not be forgotten as a potential use-
ful preoperative diagnostic tool.16 In one study, the authors found In terms of histologic criteria, nuclear atypia, mitotic count (MC),
that ICC using anti-Melan-A, anti-Vimentin, and anti-cytokeratin degree of pigmentation, level of infiltration/invasion, and vascular
improved the ability to diagnose canine amelanotic oral melanomas invasion have been shown to be statistically relevant for predicting
with cytology. The diagnosis reached with ICC matched the histologic prognosis of cutaneous and oral melanocytic neoplasms in dogs in
16
diagnosis. The study also showed that ICC for Melan-A can cor- studies prior to and after 2011.20,23,25,27,28,31,35,39–42,47 Histologically
rectly identify metastatic amelanotic melanoma in regional lymph evaluated ulceration1,25,31 and macroscopically assessed tumour
nodes, especially when they are in low numbers or have a round cell thickness20,25 are only predictive of prognosis for cutaneous tumours.
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SMEDLEY ET AL. 743

While assessment of nuclear atypia can result in significant inter- with ≥4 mitoses showing shorter survival times.27 A marked
observer variation, this parameter continues to show statistical rele- difference in survival at 1 year between the two groups created by
vance when correctly applied. Only one paper reported a lack of this cut-off value has been demonstrated with a sensitivity of 90%
association between nuclear atypia and survival in dogs with oral and a specificity of 84% in one oral/lip study.27
48
MMs. Nuclear atypia should be assessed according to the strict For pigmentation, it is difficult to measure objectively with vali-
criteria outlined by Spangler and Kass.35 Using those criteria, a thresh- dated cutoff points, but a high degree of pigmentation does suggest a
old value of 20% atypical nuclei for cutaneous melanocytic neoplasms favourable clinical outcome for cutaneous and oral/lip melanocytic
and a threshold of 30% atypical nuclei for canine oral/lip melanocytic neoplasms.1,25,27,31,42 Oral/lip neoplasms with ≥50% of pigmented
neoplasms have shown statistical significance for predicting survival cells have been shown to have longer survival times.1,27 However,
times.25,27,35,47 The most accurate way to determine the percentage outcome is not predictable in oral/lip or cutaneous neoplasms with
of atypical nuclei is to count the number of atypical nuclei among moderate, low, or no pigmentation.1,27,31 Pigmentation should be
200 cells. evaluated but should not be used as a sole predicting factor.
Despite the inter- and intraobserver variation of MC, it has still In addition, ulceration is another prognostic marker that can be used
shown statistical relevance in several studies of melanocytic neoplasms for cutaneous melanocytic neoplasms. Ulceration of cutaneous melanocytic
at various locations, including cutaneous studies published since 2011, neoplasms has been associated with significantly shorter survival times and
and is more objective than nuclear atypia.1,20,23,25,31,37,39,41,42 It should was shown to be an independent prognostic factor in two studies.25,31
be noted that most veterinary studies incorrectly refer to MC as mitotic
index. Mitotic index is the number of mitotic figures/total number of
cells in a defined area or volume of tumour and this has never been 3.2.3 | Molecular markers
done in veterinary pathology. MC is the number of mitotic figures in a
defined square mm area. The area 2.37 mm2 was proposed for animal Several molecular markers have been investigated for their potential
tumours because this is the area in 10 fields of view with a 40 objec- as prognostic markers for canine melanocytic neoplasms (Table 2).
tive and a 10 ocular that has field number (FN) of 22 engraved in the Currently, immunohistochemical evaluation of Ki67, as the Ki67 index
eyepiece.32 An ocular with an FN 22 is the most common ocular man- or Ki67 count, is the only established prognostic molecular marker,
ufactured by commercial sources for pathologists. For this consensus and it has been shown to be highly predictive of behaviour for both
statement we have defined the MC as the number of mitoses in 10 fields oral and cutaneous melanocytic tumours in dogs in multiple studies,
at 400 magnification/40 objective (which we will refer to as hpf including four that were published after 2011.1,4,12,20,27,31,37 Whereas
throughout this text) because that is how earlier studies performed the MC only accounts for cells in the M phase of the cell cycle, Ki67 is a
MC. Therefore, these terms and definition will be used throughout this nuclear protein that is expressed in all phases of the cell cycle, except
report, regardless of how it is stated in the cited reference. the resting phase; therefore, it is a measure of growth fraction. The
The MC threshold for cutaneous melanocytic tumours was level of Ki67 expression is much more objective and has been shown
established by counting mitoses in 10 random high power fields.31 to have a similar or higher predictive value as traditional histologic
However, in order to decrease the rate of underestimation of malig- criteria for both cutaneous and oral/lip melanocytic neoplasms.27,31
nant neoplasms, MC should be determined in the area of highest Some neoplasms with histological criteria of malignancy, but a low
mitotic activity, similar to how the threshold for oral/lip melanocytic level of Ki67 expression have longer survival times than expected by
neoplasms was determined. Therefore, first scan the neoplasm at histology.27,31 It is also much easier to identify the areas with the
100 magnification/10 objective to locate areas of potentially high most proliferation by looking for red nuclear labelling than it is to
mitotic activity. Locate a field containing one or more mitotic figures, identify an area of high mitotic activity when scanning a tumour. Thus,
if possible, and begin counting mitoses in that area at 400 this marker is more objective than MC. Nuclei with weak to strong dif-
magnification/40 objective. Count mitotic figures in 10 consecutive fuse labelling and nuclei with only nucleolar labelling are counted
hpf. Avoid areas of ulceration, necrosis, and inflammation when cou- while avoiding areas of ulceration and inflammation. For heavily
nting mitoses. For heavily pigmented neoplasms bleaching may be pigmented neoplasms, bleaching the sections after immunohisto-
needed to better assess the MC. Ideally, MCs should now be reported chemical labelling may be needed to better assess Ki67 labelling. If
per 2.37 mm2. For cutaneous neoplasms, an MC of 3 or greater in sections are bleached before immunohistochemistry, Ki67 labelling
10 hpf has been associated with more aggressive behaviour and does not work. In cutaneous melanocytic neoplasms, the Ki67 index is
shorter survival times.1,31 Neoplasms with less than 3 mitoses in determined as a percentage of positive nuclei in 500 cells.31 Thus, the
10 hpf generally exhibit benign behaviour. One study showed that number of cells evaluated is standardized. Assistance of a 1 cm2 opti-
50% of dogs with neoplasms with an MC of ≥3 in 10 random hpf were cal grid reticle is very helpful. The grid reticle simply helps the pathol-
alive for <7 months while 90% of dogs with neoplasms with an MC ogist keep track of which cells have been counted already. For oral/lip
<3 in 10 random hpf were still alive at 2 years.31 For oral/lip melanocytic neoplasms, the Ki67 count is determined as the average
melanocytic neoplasms, a cut-off of 4 mitoses per 10 hpf is a statisti- number of positively labelled neoplastic cell nuclei per area of a 1 cm2
cally determined threshold value for MC, with dogs having tumours optical grid reticle at 400x magnification/40x objective (5 grid areas
 TABLE 2 Prognostic molecular markers for canine melanocytic tumours
744

Marker(s) Samplesa-tumour locationb Methodsc Type of cells marked Prognostic significanced References
Ki67 (Ki67 index T-CM, OM IHC Proliferating tumour cells Strong 1,4,12,20,27,31,37
and Ki67 count)
Survivin T-CM, OM IHC, RT-qPCR Proliferating/resisting To be confirmed 3,37
to apoptosis tumour cells
H2AFZ T-CM, OM RT-qPCR Proliferating tumour cells To be confirmed 37
RACK1 T-CM, melanocytoma IHC Tumour cells To be confirmed 4
FoxP3, IDO, and CTLA-4 T-CM, OM IHC, flow cytometry Treg (regulatory T cells) To be confirmed 24,41,48
CD20+ T-CM, OM IHC TILs (B cells) To be confirmed 40
KIT, c-Kit T-CM, OM IHC, mutation status Tumour cells Absent 8,10,14,18,36
S100A4 T-CM IHC Tumour cells Absent 9
E-cadherin T-CM, OM IHC, RT-qPCR Tumour cells To be confirmed 3,11,43
β-catenin
COX-1 and COX-2 T-CM, OM, OcM IHC Tumour cells To be confirmed 15
P-glycoprotein 1 T-CM, OM Tumour cells To be confirmed 5
PDGFR-α and -β T-OM IHC Tumour cells To be confirmed 12
KMO, STAT3, pSTAT3 T-CM, OM, OcM, DM IHC Tumour cells To be confirmed 39
Somatic focal amplification T-OM qPCR Tumour cells To be confirmed 42
of chromosome 30(CFA 30)
PCNA and Connexins T-OM (melanotic IHC, IF, RT-qPCR, Tumour cells To be confirmed 19
(Cx26 and Cx43) & amelanotic variant) & Western blot
MCAM/CD146 T-OM, CM, OcM, AM IHC Tumour cells Absent 21
activation of the MAPK and T-OM Micro-array Tumour cells Not a prognostic study 6
PI3K/AKT pathways & mutational analysis
nBAP1 T-OM IHC Tumour cells Absent 22
Cyclin D1 T-OM IHC Proliferating tumour cells Possible 49
Galectin-3 T-OM IHC Tumour cells resistant to apoptosis Possible 47
BCL-2 T-OM IHC Tumour cells resistant to apoptosis Absent 47
Caspase 3 T-OM IHC Tumour apoptotic cells Absent 47
MAGE-A T-OM IHC Tumour cells Absent 38
a
Samples: T, tissue.
b
Tumour location: CM, cutaneous melanoma; OM, oral melanoma; OcM, ocular melanoma; AM, anal melanoma; DM, digit melanoma.
c
Methods: IHC, immunohistochemistry; ICC, immunocytochemistry; IF, immunofluorescence; RT-qPCR, quantitative reverse transcription PCR; qPCR, quantitative PCR.
d
Prognostic significance: Strong: several studies demonstrated the correlation with the levels of expression of the marker with main prognostic factors (clinical and pathological). To be confirmed: authors show a
statistically significant association of the marker with more than one (clinical and pathological) factor. Possible: the authors observed a significant correlation of the marker's expression with one (clinical or
pathological) parameter or a tendency towards the association (closer to the statistical significance). Absent: no (statistically significant) correlation of the levels of expression of the marker has been reported by
more than one author.
SMEDLEY ET AL.

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SMEDLEY ET AL. 745

counted) in the highest labelling area.27 This grid method standardizes study, none of the dogs with a tumour thickness ≤0.95 cm died; thus,
the area assessed so that it is the same no matter what microscope is the 1-year estimated survival probability was 100% ± 0%, while it was
used. It should be noted that previous references refer to this method only 45.0% ± 18.8% for dogs with tumour thickness >0.95 cm.25 To pre-
for oral/lip melanocytic tumours as “Ki67 index”. However, similar to dict which tumours would be more likely to develop recurrence/metas-
the incorrect use of the term “mitotic index”, it may be more accurate tasis, a thickness cutoff of 0.75 cm was determined (sensitivity = 86%,
to refer to this method of evaluating Ki67 labelling for oral/lip specificity = 81%; ROC curve analysis, AUC = 0.886; 95% CI, 0.795–
melanocytic tumours as “Ki67 count”, as it is not reported as a percent- 0.977; P = .005).25 Those that were >0.75 cm were associated with a
age of cells. A threshold value of 19.5 was statistically determined using shorter disease-free time than those that were ≤0.75 (P < .001).25 Dogs
27
a receiver operator characteristic (ROC) curve. Kaplan–Meier survival with tumours that were >0.75 cm thick had a 1-year estimated probabil-
analysis showed that the survival curves for dogs with a Ki67 count ity of not developing recurrence/metastasis that was 54.7% ± 15.4%,
<19.5 and dogs with a Ki67 count ≥19.5 are significantly different based whereas it was 97.2% ± 2.7% for dogs with tumours that were
on a one-year survival period (P < .0001).27 In cutaneous melanocytic ≤0.75 cm.25 One must remember that false positives and false negatives
neoplasms, a threshold value of 15% had been previously empirically can still occur with these thresholds for individual cases, and the prog-
determined and has been evaluated in regard to survival with Kaplan– nostic significance of tumour thickness was not able to be confirmed
31
Meier survival curves. Statistically significant lower survival rates were with multivariable analysis in this study, nor was it compared to Ki67
reported for dogs with neoplasms with a Ki67 index ≥15% in that study, index.25 In addition, the tumour thickness cutoff of 0.45 cm was deter-
20,31
as well as in a more recent study. In one study, none of the mined by ROC curve analysis to distinguish cutaneous MMs from
behaviourally benign cutaneous melanocytic neoplasms had a Ki67 melanocytomas as defined by histopathology with a sensitivity of 87%
31
index greater than or equal to 15%. The percentage of correctly classi- and a specificity of 64%.25 However, the authors stated that these
fied neoplasms using the Ki67 index (97%) was higher than that of MC values are not optimal due to the possibility of false-positive results. The
(91%) and histological criteria (93%) in one study.31 Thus, a threshold of authors used both an ocular micrometre as well as a standard ruler to
15% should be used to predict prognosis of cutaneous melanocytic neo- measure tumour thickness and found that these two methods had excel-
1,20,31
plasms. Assessment of Ki67 value is especially helpful for lent agreement. Thus, it is recommended for routine diagnostics to mea-
melanocytic neoplasms that exhibit both prognostically favourable and sure tumour thickness by simply applying a ruler to the surface of a glass
poor histological parameters, or so called “grey zone” cases, but it is slide perpendicularly to the epidermis and measuring the largest thick-
never wrong to perform Ki67 immunolabelling for added confidence. ness of the tumour.25 This study also examined the usefulness of a modi-
fied Clark level measurement for predicting prognosis; however, this
measurement was not associated with clinical outcome or presence of
3.2.4 | Newly examined prognostic parameters for recurrence/metastasis and did not show prognostic significance.25 Stud-
canine cutaneous melanocytic neoplasms ies that compare tumour thickness to other established parameters, such
as Ki67 index, are still needed to further evaluate this parameter and to
Recently, tumour thickness has been shown to be a useful prognostic determine if this parameter adds value to the other parameters.20,25 In
marker for cutaneous melanocytic neoplasms in two studies, although conjunction with the other described prognostic parameters, patholo-
the authors of those studies state that additional studies with larger case gists should begin to report tumour thickness for cutaneous melanocytic
numbers are needed to further support its use as an independent prog- neoplasms and use the cut-offs of 0.95 and 0.75 cm as one means of
nostic marker.20,25 Nonetheless, the study by Silvestri et al. 2019 pro- predicting survival times and risk of recurrence/metastasis, respectively.
vides convincing statistical evidence that a greater tumour thickness is This will allow for greater data collection regarding this parameter.
associated with shorter overall survival and disease-free time and pro- Tumour symmetry and growth pattern (expansive vs. infiltrative) are two
vides an easy method to measure tumour thickness with established additional parameters that have shown prognostic significance in one
cut-off values that can be used in a diagnostic setting.25 ROC curve anal- study but still require further evaluation with larger case numbers.20
ysis and Youden Index identified cutoffs of 0.95 and 0.75 cm which A few studies have evaluated various molecular markers as
were associated with a higher hazard for an unfavourable outcome and potential prognostic markers for cutaneous melanocytic neoplasms
to develop recurrence/metastasis, respectively.25 Kaplan–Meier survival and some may hold future promise. However, most have low sample
curves and log-rank tests were used to compare overall survival sizes, limited or no follow-up, or other limitations that prevent them
according to diagnosis.25 Via univariate analysis, dogs with greater from being used in a diagnostic setting at this time. One such marker
tumour thickness had an approximately 10 times higher hazard of death that requires further investigation is survivin. In one study, nuclear
and a greater than 5 times higher hazard to develop recurrence/ survivin expression was significantly greater in malignant cutaneous
metastasis than dogs with thinner tumours.25 The cutoff of 0.95 cm dis- melanomas compared to melanocytomas, and increased expression
criminated between favourable and unfavourable (tumour-related death) (≥8% of neoplastic cells) was related to the presence of metastasis
clinical outcomes (sensitivity = 100%, specificity = 86%; ROC curve and death of the animal due to melanoma.3 In a recent study, survivin
analysis, AUC = 0.886; 95%CI, 0.795–0.977; P = .005). A shorter overall gene expression from formalin fixed-paraffin embedded (FFPE)
survival time was seen for dogs with tumours that were >0.95 cm thick tumour tissue was assessed in both cutaneous and oral melanocytic
compared to those with a tumour thickness ≤0.95 cm (P < .001).25 In this tumours, and confirmed a correlation of this marker with tumour
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
746 SMEDLEY ET AL.

related death and Ki67 index.37 In the same study, gene expression lack of correlation of either labelling extension or intensity with other
from FFPE tissue of another marker, H2AFZ, showed promising morphologic grading factors does not suggest this will be an important
results as a new prognostic marker, being correlated with MC, Ki67 prognostic marker or diagnostic tool. The Gramer et al. study only
index, presence of metastasis, and death due to melanoma.37 included three melanomas and two of the three showed KIT expres-
RACK1 is another marker that has been investigated as a poten- sion.8 One of these tumours had a missense, or non-synonymous,
tial diagnostic, as well as a potential prognostic, marker.4 In one study, mutation, but this was not a prognostic study and the sample size was
RACK1 distribution differed between benign and malignant canine too small to draw any significant conclusions.8
melanocytic neoplasms and a RACK1 homogeneous labelling pattern Other studies that included canine cutaneous melanocytic neo-
was highly correlated with other criteria such as classic histologic fea- plasms could not demonstrate prognostic utility for the markers they
tures of malignancy, Ki-67 index and MC.4 However, sample size was evaluated, may have found promising statistical significance but were
very small and included both cutaneous and mucosal neoplasms, and too preliminary to make any significant conclusions, were not designed
there was a lack of follow-up data in this study; thus, additional test- as prognostic studies or only used melanoma cell lines.5,7,9,11,15,17,21,43
ing is needed before this marker can be used in a diagnostic setting. These included expression of: S100A4, E-cadherin/β-catenin, COX-1
Even then, evaluation of this marker in a diagnostic setting may prove and COX-2, P-glycoprotein 1, and MCAM/CD146.3,5,9,11,15,17,21,43
to be too complicated and it has not been shown to add additional
value over Ki67 index and MC. In addition, other neoplasms can also
label for RACK 1, so this marker cannot be used as a standalone 3.2.5 | Newly examined prognostic parameters for
marker for diagnosis of melanocytic origin. canine oral/lip mucosal melanocytic neoplasms
Several papers have been published in recent years on the prognostic
significance of tumour infiltrating lymphocytes (TILs) in canine melanocytic There are a few new prognostic markers that have been investigated
neoplasms. Several markers used to identify different TIL subtypes, such for canine oral melanocytic neoplasms since 2011 and a few of them
as regulatory T cells (Treg) or B cells, have been investigated. FoxP3, IDO show potential usefulness. For example, Iussich et al.12 provides good
and CTLA-4 expression, markers of Treg, have been shown to hold promise statistical support for considering platelet-derived growth factor
as prognostic markers for canine cutaneous and oral melanocytic neo- receptors (PDGFR)-α and -β as prognostic markers in oral MM of
plasms, but also require further evaluation with larger sample sizes, with dogs. The purpose of this study was to evaluate the expression of
comparisons to other established prognostic markers, besides MC, and by PDGFR-α and -β in stage II and III canine oral MMs and to correlate it
24,41
separating cutaneous from oral melanomas. Ideally, this should be with prognosis. The neoplasms in this study were confirmed as
done with a prospective study. melanocytic in origin via IHC labelling for PNL-2 and pathologists
A high number of CD20+ TILs (B cells) has been shown to recorded nuclear atypia, MC, pigmentation and Ki67 values for each
be associated with tumour-related death, presence of metastasis/recur- tumour.12 This study suggests that PDGFRs may play a role in the path-
rence, shorter overall and disease-free survival, increased hazard of ogenesis of oral canine MM and the co-expression of both PDGFRs-α
death, and of developing recurrence/metastasis.40 No associations and -β should be considered as a negative prognostic marker. In addition,
were found related to infiltrating CD3+ TILs (T cells). This study com- despite an unclear methodology for determining the Ki67 value that is
pared the degree of CD20+ TILs with other histologic parameters not consistent with the published method by Bergin et al. (2011),27 sta-
including MC, tumour thickness and modified Clark level for cutaneous tistical analysis showed that co-expression of PDGFRs and Ki67 values
tumours, nuclear atypia, pigmentation, ulceration, necrosis, and cellular were both associated with worse prognosis, further supporting the prog-
pleomorphism. There was a statistically significant association between nostic importance of Ki67 assessment. PDGFRs were detected not only
the quantity of CD20+ TILs and the MC. Tumour size was significantly in tumour tissue but also in the stroma, suggesting a potential role in
lower in tumours that lacked CD20+ TILs compared to those with mild, matrix remodelling and tumour invasion.12 The authors stated that fur-
moderate, or severe infiltrates, in terms of quantity. An association was ther prospective studies on a greater number of cases are warranted to
also seen between the quantity of CD20+ TILs and the percent pig- confirm these findings before they are used in a diagnostic setting.12
mentation or the cellular pleomorphism. Based on these results, a high Higher protein expressions of Kynurenine 3-monooxygenase (KMO),
quantity of CD20+ TILs appears to be a new potential negative prog- signal transducer and activator of transcription 3 (STAT3), and STAT3/
nostic factor for both oral and cutaneous melanocytic tumours. How- phosphorylated (pSTAT3) have recently been shown to be associated with
ever, the authors state that they need to be confirmed using larger case reduced survival times in canine melanoma patients in one study by Liu
numbers and by studying cutaneous and oral melanocytic tumours et al.39 The authors used IHC to evaluate expression of these proteins in
40
separately. 85 cases of canine melanoma that included mostly oral tumours (58 oral,
A few studies have evaluated the role of c-Kit in canine cutaneous, as 4 lip, 8 skin, 7 digit, and 8 other sites). KMO expression was more common
well as oral, melanocytic neoplasms but no significant association between in oral tumours than tumours at other sites. The authors also found a cor-
KIT labelling and survival time has been demonstrated.8,10,14,18,36 Gomes relation between high MC (≥4 mitoses per 10hpf) and oral location, meta-
et al. demonstrated decreased expression of KIT protein in cutaneous static cases, those that were >2 cm, and higher stage. KMO, STAT3 and
MMs compared to cutaneous melanocytomas.10 The decreased num- pSTAT3 expressions were all significantly higher in tumours with an MC
ber of cells labelled in malignant versus benign tumours may give some ≥4 mitoses per 10 hpf. A combination of MC and KMO had a sensitivity
insight into the role of c-Kit in melanocytic tumour progression, but the of 74.4%, a specificity of 63%, and a diagnostic accuracy of 68.2%. A
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SMEDLEY ET AL. 747

combination of MC and STAT3 had a sensitivity of 94.9%, a specificity of lactic acid, oleic acid, linoleic acid, palmitoleic acid, octadecenoic acid,
43.5%, and a diagnostic accuracy of 67.1%.39 and glycerol; E-cadherin/β-catenin expression; COX-1 and COX-2
Another group demonstrated that somatic focal amplification of expression; P-glycoprotein 1 expression; expression of MCAM/
chromosome (Canis familiaris [CFA]) 30, but not CFA10, was signifi- CD146; activation of the MAPK and PI3K/AKT pathways; the expres-
cantly associated with an amelanotic phenotype, a high MC, and sion pattern of Cx26 and Cx43; nBAP1 protein expression; E-cadherin
shorter survival times in a set of 73 canine oral MM.42 expression; Cyclin D1 index; expression of Galectin-3; expression of
Thus, like KMO, STAT3 and pSTAT3, these parameters may prove B-cell lymphoma (BCL) 2 and caspase (CASP) 3; and expression of
to be useful prognostic parameters in the future, but they first should MAGE-A.3,5,6,11,13,15,17,19,21,22,38,43,47,49
be compared to other established prognostic parameters, such as
Ki67 and nuclear atypia, to see if they add value.
The presence of TILs and the levels of expression of their markers 3.3 | Recommendations for prognostication
in the tumour tissue has been suggested to have a prognostic signifi-
cance in oral melanomas as well. The expression of the markers RACK1, Tables 3 and 4 can be used as guides for prognostication. For each of
FoxP3, and IDO24 and the quantity of CD20+ TILs40 have been exam- the evaluated parameters, a favourable prognosis relates to expected
ined in both cutaneous and oral melanocytic neoplasms and discussed survival times longer than 1 year and a poor prognosis relates to an
above under cutaneous melanocytic neoplasms. Furthermore, the pres- expected death due to melanocytic neoplasia within less than 1-year
ence and distribution of TILs, including the frequency of CD8+ T cells, post-diagnosis for all melanocytic neoplasms. These predictions are
CD4+ T cells, and Treg cells by histopathology and flow cytometry have based solely on publications that met our strict criteria for inclusion
been investigated in a retrospective study of 50 canine oral MMs, con- for each single parameter. These predictions do not take into account
firming their potential utility as prognostic markers.48 Higher survival stage of disease or treatment strategies.
rates were seen in dogs that had higher TIL scores, specific TIL patterns Ideally, using the criteria in Table 3, a cutaneous melanocytic neo-
termed “brisk” and “nonbrisk” (compared to those with no TILs), and an plasm should be categorized as a cutaneous melanocytoma (benign
increased frequency of CD8+ T lymphocytes infiltrating the tumour.48 melanocytic tumour) or a cutaneous MM. Typically, a cutaneous mel-
Evaluation of TILs is emerging as a potential prognostication method anocytoma is non-ulcerated, often raised, generally small (<2 cm diameter
for canine melanocytic tumours and further evaluation is warranted. and ≤0.45 cm thickness), limited to the dermis, heavily pigmented, has
Also discussed above under cutaneous melanocytic neoplasms was very bland appearing nuclei, and has a low MC and very low Ki67
the role of c-Kit. Murakami et al. evaluated KIT labelling in canine oral index.20,25,31,35 In some cases, a cutaneous MM may have several of the
14
MMs and found no association with overall survival or WHO stage. In same features as a melanocytoma but may have only one or two parame-
addition, no significant mutations were identified in exon 11 of c-Kit.14 ters that suggest malignant behaviour, such as high nuclear atypia, a MC
While this study did have some limitations, such as relatively low sample above the threshold value, a tumour thickness >0.95 cm, extension
size, lack of complete detail regarding diagnosis and follow-up times, and beyond the dermis, or a Ki67 index above the threshold value. Clear-cut
the inclusion of only MMs, this study concluded that KIT expression does cutaneous MMs are often ulcerated, poorly pigmented, exhibit marked
not appear to be a prognostic factor for canine melanoma.14 Brocca et al. nuclear atypia, have a high MC and a high Ki67 index, are >0.95 cm thick,
similarly did not find a correlation between KIT protein expression and extend beyond the dermis, and may exhibit vascular invasion.20,25,35
mutation status, as they did not find any mutations in 14 canine oral Ideally, using the criteria in Table 3, a mucosal lip or oral neoplasm
melanocytic neoplasms.36 Another recent study by Smedley et al, should be able to be categorized as an oral MM or a histologically
completely sequenced the c-Kit gene in canine oral melanocytic neo- well-differentiated melanocytic neoplasm (HWDM),28 also known as a
plasms. The authors identified nine nonsynonymous mutations that melanocytic neoplasm of low malignant potential.18 The term mel-
resulted in amino acid changes predicted to affect protein function. anocytoma should be avoided for oral and lip mucosal melanocytic
These mutations were more common in MMs than in those of low malig- neoplasms as the long term (>2 years) behaviour of these neoplasms,
nant potential but the authors did not find any correlation between if not excised, is still uncertain. Thus, they should be treated as neo-
mutation status, KIT labelling or histologic features.44 Both of the above plasms of low malignant potential. HWDMs are usually raised, non-
studies concluded that there are no current documented indications, in ulcerated, <2 cm diameter, heavily pigmented, lack cellular atypia, do
regards to c-Kit mutation status or KIT labelling, to suggest tyrosine not invade bone, and have rare mitoses, a very low Ki67 index, and
kinase inhibitors (TKIs) would be beneficial for these tumours.14,44 How- abundant collagenous stroma.18,28 They also often lack junctional
ever, a recent single case report described a dog with a gingival MM that activity and lateral surface epithelial spread which improves the
had a novel deletion mutation c.1725_1733del within KIT, which was chance of complete excision and likely plays a role in the longer sur-
considered to be an oncogenic driver mutation.45 vival time of these tumours.18 A mean survival time of 23.4 months
Other oral melanocytic neoplasm studies that were reviewed could has been reported for HWDMs in one study.28 These features are in
not demonstrate prognostic utility for the markers that they evaluated, strict contrast to oral MMs which often show junctional activity, lat-
may have found promising statistical significance but were too preliminary eral epithelial spread, poor pigmentation, bone invasion, and marked
to make any significant conclusions, were not designed as prognostic nuclear atypia and often have a very high MC and Ki67 index.27,35
5–7,11,13,15,17,19,21,22,38,43,47,49
studies, or only used melanoma cell lines. When there are mixed results, results for each parameter should
These markers included: a metabolite profile that included citric acid, be reported, but Ki67 index should be used for final interpretation in
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
748 SMEDLEY ET AL.

TABLE 3 Prognostication of canine cutaneous and oral/lip melanocytic neoplasms (modified from Smedley et al1)

†
Parameter was not specifically examined for neoplasms of the digit.
‡
For this consensus, the mitotic count (reported as mitotic index, in the literature reviewed) is obtained by counting the absolute number of mitoses in 10
high‐power fields (400 magnification/40 objective or, ideally, in an area of 2.37mm2) in the region with highest mitotic activity, as determined initially
on a low power scan (100 magnification/10 objective) of the specimen.
§
Parameter should be assessed in epithelioid predominant neoplasms and in spindloid neoplasms with sufficiently observable nuclear detail.
¶
Tumor thickness is measured with a ruler by placing the ruler on the glass slide perpendicular to the epidermis or mucosal epithelial surface and measuring
the largest thickness of the tumor.25
¥
A favorable prognosis relates to expected survival times longer than one year and a poor prognosis relates to an expected death due to melanocytic
neoplasia within less than one‐year post‐diagnosis for all melanocytic neoplasms. These predictions are based solely on publications that met our strict
criteria for inclusion for each single parameter. These predictions do not take in to account stage of disease or treatment strategies. When there are mixed
results, results for each parameter should be reported, but Ki67 index should be used for final interpretation in most cases.

most cases.1 In cases that are still ambiguous, due to all parameters in this consensus are based on the results of the studies that adhere
being at or very near the threshold values, a diagnosis of melanocytic to most of those standards. The first step in any prognostic study is to
neoplasm should be made and each parameter should be discussed.1 ensure an accurate diagnosis of melanocytic origin for each case in
the study population. This involves the use of specific histologic
criteria and IHC labelling for highly sensitive and specific markers, if
4 | DISCUSSION those specific histologic criteria are lacking.1,34,46 Gene expression
may also be used to differentiate between amelanotic spindloid MMs
4.1 | Future directions and soft tissue sarcomas in the near future.46 Once melanocytic origin
is established, neoplasms should be classified based on location as
None of the published prognostic studies meet all of the standards well as established histomorphologic and molecular prognostic
defined by the Webster et al.2 white paper but the recommendations parameters.
 14765829, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vco.12827 by Readcube (Labtiva Inc.), Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SMEDLEY ET AL. 749

TABLE 4 Summary of recommendations for diagnosis and histopathologic prognostication of canine melanocytic neoplasms

Summary of recommendations for diagnosis and histopathologic prognostication of canine melanocytic neoplasms
1. Confirm melanocytic origin by identifying classic histologic features of melanocytic neoplasms or by demonstrating labelling of neoplastic cells in
amelanotic neoplasms for melanocytic specific immunohistochemical (IHC) markers
 Classic histologic features: intracytoplasmic melanin, variable cell morphology in the same tumour, junctional activity, pagetoid growth, presence
of neoplastic cells at the mucosal‐submucosal (epidermal‐dermal) junction even in the absence of junctional activity, and finely stippled to
vesiculate nuclei with a prominent central nucleolus (“owl's eye”).
 If amelanotic or otherwise poorly differentiated, demonstrate IHC labelling for Melan‐A, PNL2, TRP‐1 or TRP‐2.
2. Determine prognosis of cutaneous and lip/oral melanocytic neoplasms by evaluating the specific histologic and molecular parameters described in
Table 3. For heavily pigmented neoplasms, bleaching may be necessary in order to accurately evaluate nuclear features and mitotic count (MC). To
evaluate Ki67 labelling in heavily pigmented neoplasms, bleaching should be performed AFTER IHC labelling for Ki67.
 For lip/oral melanocytic neoplasms:
• If there is marked nuclear atypia (≥30% atypical nuclei), a high MCa (≥4/2.37 mm2), or evidence of vascular invasion or metastasis, the
neoplasm is diagnosed as a malignant melanoma and evaluation of the Ki67 count is offered for further prognostication (pathologists may
indicate that Ki67 count is not necessary for neoplasms that greatly surpassb the nuclear atypia and MC thresholds and/or show evidence of
vascular invasion or metastasis).
• If the histologic parameters give mixed results, are at or near the threshold values, or if all the histologic parameters indicate a favourable
prognosis, evaluate the Ki67 count for further prognostication.
• Ki67 count is the most useful parameter for prognostication due to high specificity and objectivity.
• Provide the results of all parameters examined in the pathology report.
 For cutaneous melanocytic neoplasms:
• If there is marked nuclear atypia (≥20% atypical nuclei), a high MCa (≥3/2.37 mm2), a tumour thickness >0.95 cm, or evidence of vascular
invasion or metastasis, the neoplasm is diagnosed as a malignant melanoma and evaluation of the Ki67 index is offered for further
prognostication (pathologists may indicate that Ki67 index is not necessary for neoplasms that greatly surpassb the nuclear atypia and MC
thresholds and/or show evidence of vascular invasion or metastasis).
• If the histologic parameters give mixed results, are at or near the threshold values, or if all the histologic parameters indicate a favourable
prognosis, evaluate the Ki67 index for further prognostication.
• Ki67 index is the most useful parameter for prognostication due to high specificity and objectivity.
• Provide the results of all parameters examined in the pathology report.
3. Some general rules for prognostication of melanocytic neoplasms:
 It is impractical to accurately predict, on an individual basis, the biological behaviour of melanocytic neoplasms.
 More than one parameter should be used to classify melanocytic neoplasms histologically as benign, of low malignant potential, or malignant
because of the inherent subjectivity in histological evaluation.
 If histologic prognostic factors conflict with one another, a neoplasm should be diagnosed as a melanocytic neoplasm and prognostic factors
should be discussed.
 Evaluation of nuclear atypia and MC in combination with Ki67 expression level and clinical features will maximize the correctly classified
neoplasms.1
a
For this consensus, the mitotic count (reported as mitotic index, in the literature reviewed) is obtained by counting the absolute number of mitoses in
10 high‐power fields (400× magnification/40× objective) in the region with highest mitotic activity, as determined initially on a low power scan
(100× magnification/10× objective) of the specimen. Future studies which evaluate mitotic count as a prognostic parameter should also adopt this
methodology, while also defining and standardizing the microscopic area evaluated to 2.37 mm2.
b
There are no established values for “greatly surpassing” the cut‐offs, but pathologists will use their experience to subjectively make this recommendation.
In general, the subgroup agrees that Ki67 count is not necessary for tumours with ≥50% atypical nuclei or oral/lip tumours with an MC ≥40/2.37 mm2 and
cutaneous tumours with an MC ≥30/2.37 mm2, which are 10 times higher than the respective threshold values.

Another major requirement for a sound prognostic study is new potential prognostic markers should be evaluated in conjunction
follow-up data that includes disease free survival times, progres- with, and compared to, the current statistically proven prognostic param-
sion free survival times, overall survival, cause of death, and post- eters, such as nuclear atypia, MC, and Ki67 index.
mortem results. Collection of follow-up data is one of the most Much of the recent literature has focused on the genetic features
challenging tasks in veterinary medical research studies and post- of canine melanocytic neoplasms, and this field holds promise for bet-
mortem examination of a significant number of cases is often not ter diagnosis, prognostication and prediction of metastasis of these
possible. However, researchers should always have an initial study tumours, as well as for identification of potential targets for therapy.
design, ideally prospective, that will allow for as much follow-up In the future, genetic analysis may prove to be the gold standard for
data as possible. diagnosis and prognostication but, at this point, the most accurate
There are very few published studies that are true prognostic stud- diagnostic methods are the immunohistochemical markers Melan-A,
ies of canine melanocytic neoplasms and only one of these evaluated PNL2, TRP-1 and TRP-2 and the most accurate predictors of progno-
prognostic studies19 is prospective. While prospective studies are very sis include the histologic features described above and Ki67 index.
challenging studies to conduct in veterinary medicine, ideally an effort Clinical parameters for prognostication, other than location (cuta-
should be made to conduct such studies in order to verify the recom- neous versus oral/lip), are not addressed here, but are slated for
mendations made by the referenced retrospective studies. Additionally, future evaluation and consensus. It is hoped that a combination of
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750 SMEDLEY ET AL.

histologic, molecular, and clinical parameters will further improve our 11. Han JI, Kim Y, Kim DY, Na KJ. Alteration in E-cadherin/β-catenin
ability to prognosticate these neoplasms, in order to better determine expression in canine melanotic tumors. Vet Pathol. 2013;50(2):274-
280. †.
appropriate therapy for each case.
12. Iussich S, Maniscalco L, DiSciuva A, et al. PDGFRs expression in dogs
affected by malignant oral melanomas: correlation with prognosis. Vet
ACKNOWLEDGEMEN TS Comp Oncol. 2017;15(2):462-469. †.
The authors would like to thank the Oncology Pathology Working 13. Kawabe M, Baba Y, Tamai R, et al. Profiling of plasma metabolites in
canine oral melanoma using gas chromatography-mass spectrometry.
Group, the American College of Veterinary Pathologists and the
J Vet Med Sci. 2015;77(8):1025-1028. †.
Veterinary Cancer Society for overseeing this work. 14. Murakami A, Mori T, Sakai H, et al. Analysis of KIT expression and
KIT exon 11 mutations in canine oral malignant melanomas. Vet Comp
CONF LICT OF IN TE RE ST Oncol. 2011;9(3):219-224. †.
15. Pires I, Garcia A, Prada J, Queiroga FL. COX-1 and COX-2 expression
Philip J. Bergman is a paid consultant and speaker for Boerhinger
in canine cutaneous, oral and ocular melanocytic tumours. J Comp
Ingelheim Animal Health and also receives a minority royalty stream Pathol. 2010;143(2–3):142-149. †.
payment for the Oncept melanoma vaccine. 16. Przezdziecki R, Czopowicz M, Sapierzynski R. Accuracy of routine
cytology and immunocytochemistry in preoperative diagnosis of oral
amelanotic melanomas in dogs. Vet Clin Pathol. 2015;44(4):597-
ORCID
604. †.
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