Canine melanoma diagnosis: RACK1 as a potential biological marker

Oncology (Research)

Canine Melanoma Diagnosis: RACK1 as a Potential Biological Marker

Veterinary Pathology 50(6) 1083-1090 ª The Author(s) 2013 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985813490754 vet.sagepub.com

C. Campagne1,2, S. Jule´1,2,3, C. Alleaume4, F. Bernex1,2,4,5, J. Ezagal1,2, S. Chaˆteau-Joubert4, M. Estrada6, G. Aubin-Houzelstein1,2,7, J.-J. Panthier1,2,7, and G. Egidy1,2,7

Abstract Melanoma diagnosis in dogs can be challenging due to the variety of histological appearances of canine melanocytic neoplasms. Markers of malignancy are needed. Receptor for activated C-kinase 1 (RACK1) was found to characterize melanomas in other mammals. We investigated the value of RACK1 detection in the classification of 19 cutaneous and 5 mucosal melanocytic neoplasms in dogs. These tumors were categorized as melanocytomas or benign and melanomas or malignant after evaluation of their morphology, mitotic index, and Ki-67 growth fraction. Using immunofluorescence, we confirmed microphthalmia-associated transcription factor (MITF) as a marker of normal and transformed melanocytic cells in dog tissues. All control (n ¼ 10) and tumoral (n ¼ 24) samples stained positively for MITF (34/34, 100%). Whereas RACK1 was not detected in healthy skin melanocytes, melanocytic lesions were all positive for RACK1 signal (24/24, 100%). RACK1 cytoplasmic staining appeared with 2 distinct distribution patterns: strong, diffuse, and homogeneous or granular and heterogeneous. All melanoma samples (13/13, 100%) stained homogeneously for RACK1. All melanocytomas (11/11, 100%) stained heterogeneously for RACK1. Immunohistochemistry was less consistent than immunofluorescence for all labelings in melanocytic lesions, which were often very pigmented. Thus, the fluorescent RACK1-MITF labeling pattern helped to distinguish melanomas from melanocytomas. Furthermore, RACK1 labeling correlated with 2 of 11 morphological features linked to malignancy: cell and nuclear size. These results suggest that RACK1 may be used as a marker in dog melanomas. Keywords RACK1, diagnosis, dog, immunofluorescence, melanoma, melanocytoma, MITF Canine melanomas account for 3% of dog neoplasms and 7% of malignant tumors.30 Preferential sites are, in descending order, the oral cavity, lips, skin, and toes.30 Cellular pleomorphism is the main characteristic of these tumors.22 Differential diagnosis should first include melanocytomas.12 Herein, the term melanoma refers to a melanocytic malignancy, and melanocytoma indicates a benign tumor of melanocytic origin regardless of its topologic location, according to the current World Health Organization nomenclature.10 Setting a definitive diagnosis based on anatomopathological examination alone can still be challenging. Biomarkers of malignancy are needed to confirm diagnosis to adapt therapy to improve prognosis. Early identification of malignant melanocytic lesions is crucial for dog survival since melanomas possess high metastatic capacities.4 Immunodetection of RACK1 protein could be considered one such marker. Indeed, RACK1 is strongly and homogeneously detected in melanoma cells of primary tumors and metastases developed in human patients, MeLiM minipigs,7 and horses.5 This pattern of RACK1 detection is specific for malignant melanocytic tumors. Nonetheless, RACK1 protein is ubiquitously expressed, but its levels are cell type dependent.1 Functionally, RACK1 is regarded as a signaling hub allowing crosstalk between various pathways contributing to

both physiological and tumorigenic cellular functions.1,19,27 The aim of this study was to assess the value of RACK1 detection in canine melanoma diagnosis.

1 INRA, UMR955 de Ge´ne´tique fonctionnelle et me´dicale, Ecole Nationale Ve´te´rinaire d’Alfort, Maisons-Alfort, France 2 Universite´ Paris Est, Ecole Nationale Ve´te´rinaire d’Alfort, Maisons-Alfort, France 3 Laboratoire Physiopathologie Orale et Mole´culaire, Centre de Recherche des Cordieliers, INSERM UMRS 872, Paris, France 4 Ecole Nationale Ve´te´rinaire d’Alfort, Service d’Anatomie pathologique, Maisons-Alfort, France 5 RHEM-Re´seau d’Histologie Expe´rimentale de Montpellier, Institut de Recherche en Cance´rologie de Montpellier, INSERM U896, Montpellier, France 6 Laboratoire IDEXX Alfort, Alfortville, France 7 Institut Pasteur, Unite´ de Ge´ne´tique Fonctionnelle de la Souris; CNRS URA 2578, De´partement de Biologie du De´veloppement, USC INRA, Paris, France

Supplementary material for this article is available on the Veterinary Pathology website at http://vet.sagepub.com/supplemental. Corresponding Author: Giorgia Egidy, INRA, UMR955 de Ge´ne´tique fonctionnelle et me´dicale, Ecole Nationale Ve´te´rinaire d’Alfort, 7 avenue du Ge´ne´ral de Gaulle, Maisons-Alfort, F-94704 France. Email: [email protected]

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Material and Methods

Table 1. Epidemiological Data From Dogs With the Melanocytic Neoplasms Examined.

Dog Tissues Dog (Canis familiaris) tissues in paraffin blocks came from biopsy specimens at the Alfort Veterinary Medicine School (n ¼ 17) (Maisons-Alfort, France) and at the IDEXX Diagnosis Laboratory (n ¼ 17) (Alfortville, France). Samples consisted of previously diagnosed cutaneous (n ¼ 8) and mucosal melanomas (n ¼ 5), including 2 nonpigmented melanomas; cutaneous melanocytomas (n ¼ 11); and normal skin (n ¼ 5) and lip tissues (n ¼ 5) as controls. Plasmocytomas (n ¼ 2), cutaneous histiocytoma (n ¼ 1), cutaneous nonepitheliotropic T-cell lymphoma (n ¼ 1), peripheral nerve sheath tumor (n ¼ 1), and low-grade fibrosarcoma (n ¼ 1) were included as control cutaneous canine tumors. Routine diagnosis between melanocytoma and melanoma relied on morphologic criteria such as invasiveness and architectural symmetry of the tumor as well as cellular characteristics: anisocytosis, anisokaryosis, nucleolar prominence, and atypical mitoses. Cases for which differentiation between melanocytomas and melanomas was difficult were excluded. Mitotic index and growth fraction were later assessed as they have been demonstrated to provide a strong indication of eventual outcome of the disease for melanocytic tumors in general.3,31 No information on the history of tumors was available. Dark skin and dark mucous membrane dogs were overrepresented. Mean age was 9.5 years. Ten melanomas of 13 (77%) were localized in the oral cavity or at the lips or toes. In contrast, 8 melanocytomas of 11 (73%) were localized on furry parts of the body (Table 1). Epidemiological analysis of our samples indicated that our cohort was representative of reported dog melanoma.8,12,20,21,31

Immunostaining, ApoTome Microscopy Analysis of RACK1 Distribution, and Ki-67 Index Immunostaining was performed without bleaching the sections. The immunofluorescence protocol for microphthalmia-associated transcription factor (MITF) and receptor for activated C-kinase 1 (RACK1) was previously described.5 Briefly, mouse monoclonal antibodies anti-RACK1 (Transduction Laboratories, 1:150; BD Biosciences, Franklin Lakes, NJ) and anti-MITF (Zymed, around 3 mg/ml; Invitrogen, Carlsbad, CA) and rabbit polyclonal antibody anti–cytokeratin 5 (Covance; 1:1000; Eurogentec, Seraing, Belgium) were incubated on 4-mm sections after antigen retrieval in Tris-EDTA (pH 9) for 30 minutes in a water bath at 98 C to 100 C. Primary antibodies were incubated overnight at 4 C. Respective secondary antibodies were Alexa Fluor labeled (1:400; Invitrogen). Alternatively, a biotinylated anti–mouse IgM antibody (Vector Labs; Eurobio Abcys, Courtaboeuf, France) was used for RACK1 and revealed with Alexa Fluor–labeled streptavidin (1:400). 40 ,6-Diamidino-2phenylindole (DAPI) was used for nuclear counterstaining. Immunofluorescence for Ki-67 was performed on serial sections with mouse monoclonal MIB-1 antibody (Dako-

Samples

Age, y Sex Breed

Melanocytomas 1 5 2 7 3 13 4 6 5 14 6 8 7 7 8 6 9 — 10 12 11 12 Melanomas 12 — 13 9 14 15 15 — 16 17 18 19 20

7 17 11 9 7

21 22 23 24

9 8 12 10

M F M M NM NF F M F F M

Dogue de Bordeaux Mongrel Shetland Sheepdog Labrador Retriever Westie Golden Retriever Mongrel Boxer Mongrel Fawn Brittany Griffon Mongrel

M NF M M

Labrador Retriever German Shepherd Beauceron American Staffordshire Terrier NF Shar Pei F Mongrel F Labrador Retriever M Mongrel M American Staffordshire Terrier NM Rottweiler F Rottweiler M Poodle F Golden Retriever

Localization Head Head Head Head Trunk Trunk Trunk Trunk Toe Toe Toe Oral cavity Oral cavity Lips Lips Lips Head Head Trunk Toe Toe Toe Toe Toe

F, female; M, male; NF, neutered female; NM, neutered male; —, unavailable data.

Cytomation, 1:75; Dako, Glostrup, Denmark) after antigen retrieval in citrate buffer (pH 6.0) at 97 C for 20 minutes in a decloaking chamber (Biocare Medical; Eurobio-Abcys). Sections were examined with a Zeiss Axio Observer Z1 M ApoTome microscope (Carl Zeiss S.A.S., Le Pecq, France) and processed with the AxioVision 4.6 computer program (Carl Zeiss S.A.S.). All images shown are individual sections of zseries stacks. RACK1 staining distribution was analyzed at the tissue and cellular levels. Distribution within the cytoplasm was graded 0 when homogeneous and 1 when heterogeneous. Samples were analyzed blindly and independently by 2 authors (CC, SL). The Ki-67 index was calculated as the percentage of labeled nuclei after counting 500 DAPI-positive nuclei in the areas of heaviest Ki-67 staining, avoiding areas of inflammation and necrosis. A Ki-67 index >15 is correlated with a low survival rate.3,15,29 The immunohistochemistry protocol used the same reagents as immunofluorescence except for the avidin-biotin peroxidase kit (Vector Labs) with a 3,30 diaminobenzidine-nickel kit (Vector Labs) for MITF, which stains positive cells dark gray, and the 3-amino-9ethylcarbazol kit (Vector Labs) for RACK1, which stains positive cells red. Sections were then counterstained with Gill’s hematoxylin. The incubation of MITF and RACK1 antibodies

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was sequential with MITF incubated for 30 minutes and RACK1 overnight or separately. Negative controls without the first antibody showed no background staining.

Analysis of Histological Features

Samples were graded blindly and independently by 2 authors (CC, FB) without reference to previous pathology reports. Tumor size and completeness of excision had not been clinically assessed.

Statistical Analysis

Mitotic index. Four-micron sections were first bleached by immersion in 0.25% potassium permanganate solution for 1 hour followed by 5% oxalic acid for 5 minutes. Then sections were stained with hematoxylin-eosin-saffron (HES) according to standard histological procedures in a Leica ST 5020 multistainer (Leica Microsystems S.A.S., Nanterre, France). The mitotic index (MI) was the number of mitotic figures in 10 high-power fields (HPF), in areas with the highest mitotic activity. For cutaneous melanocytic tumors, an MI >3 is considered to indicate poor prognosis and hence is associated with melanoma diagnosis.15 The equivalent value for oral melanoma is MI 4.3 Elementary histomorphological features. Complementary histopathologic evaluations were carried out on routinely stained HES examined at 3 different magnifications (10, 20, and 40 objectives). Sections were observed with a Leica DMLB microscope (Leica Microsystems S.A.S.). Images were processed with the MetaVue Imaging System computer program (Molecular Devices, Sunnyvale, CA). Lesions were graded according to 11 histomorphometric criteria as previously defined in the classification of human and horse melanocytic tumors.5,33 The criteria analyzed were the ulceration, the upper scatter of intraepidermal melanocytes, the pigmentation of melanocytes, nest formation of intraepidermal melanocytes, the lateral circumscription, the contour of the epidermis, and the size and shape of cells, nuclei, and nucleoli.5,33 Briefly, the upper scatter of intraepidermal melanocytes was the proportion of intraepidermal melanocytes present above the basal layer, graded as absent (essentially all melanocytic cells situated at the dermoepidermal junction), slight (when up to 75% of melanocytes were at the dermoepidermal junction), medium (when melanocytic cells were equally distributed between the basal and upper epidermal layers), or prominent (when >50% of melanocytic cells localized to the upper layers of the epidermis). Lateral circumscription was assessed by examining the transition of the intraepidermal growth portion of the tumor to normal skin at the tumor periphery. The area with the most gradual transition was scored 0 for discontinuous areas of apparently uninvolved epidermis interspersed with tumor, 1 for a gradual but continuous decrease in the number of intraepidermal melanocytes, or 2 for an abrupt transition. The contour of the epidermis involved by the melanoma was compared with the adjacent normal epidermis and scored 0 for atrophic epidermis with effacement of the rete ridges, 1 for thinned epidermis with partial attenuation of the rete ridges, 2 for normal epidermis, 3 for up to 2-fold thickened epidermis by a moderate hyperplasia, or 4 for marked epidermal hyperplasia resulting in a greater than 2-fold increase in epidermal thickness.

Statistical differences between means taken in pairs were evaluated by the Student’s t-test. The test was adapted for a number of samples below 30 as previously described.5 A P value less than .05 was considered statistically significant.

Results MITF Is a Sensitive Immunofluorescence Marker to Identify Melanocytic Cells in Dogs To identify melanocytic cells within tissue sections, we used an MITF in vitro diagnostic monoclonal antibody.5 MITF-positive cells were identified at the basal layer of healthy skin and mucosa by a moderate to strong immunofluorescent nuclear signal (Fig. 1a). By immunofluorescence, tumoral cells were identified as MITF-positive cells in all melanocytic neoplasms regardless of the pigmentation of the lesion (Figs. 2a–5a). In contrast, by immunohistochemistry with a 30-minute incubation of the primary antibody, tumoral cells were labeled but not in every sample. Canine neoplasms considered in the differential diagnosis of melanocytic neoplasms, such as plasmocytomas, cutaneous histiocytomas, cutaneous nonepitheliotropic T-cell lymphoma, peripheral nerve sheath tumors, or low-grade fibrosarcoma, were all negative for MITF by immunofluorescence (not shown).

RACK1 Protein Distinguishes Melanoma From Melanocytoma in Dogs In control healthy skins, RACK1 protein was cytoplasmic in keratinocytes (Fig. 1b,c). By contrast, MITF-positive melanocytes were negative for RACK1 (Fig. 1b,c). Triple immunofluorescence of RACK1-MITF together with cytokeratin 5 (KRT5), a marker of basal keratinocytes, showed that MITFpositive cells were RACK1-KRT5 negative, letting us exclude the RACK1 signal on melanocyte membranes of healthy skin (Suppl. Fig. S1). In every melanocytic lesion examined, RACK1 was detected in all tumoral MITF-positive cells. The RACK1 signal appeared with 2 distinct distribution patterns. RACK1 was either heterogeneously (Fig. 2b) or homogeneously (Fig. 3b–5b) distributed over the lesion. The RACK1 heterogeneous pattern consisted of a granular cytoplasmic staining of low to moderate intensity. In sharp contrast, the RACK1 homogenous pattern was intense and diffuse, cytoplasmic, and perinuclear. RACK1 signal was detected by immunofluorescence regardless of the pigmentation load of the sample. Samples were arbitrarily graded 0 when homogenous and 1 when heterogeneous. When tested in samples of tumors considered in the melanocytic tumor differential

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Figure 1. Control skin; canine No. 1. Specific nuclear microphthalmia-associated transcription factor (MITF) labeling is observed in normal melanocytes (a), whereas receptor for activated C-kinase 1 (RACK1) is not detected in melanocytes but in surrounding keratinocytes (arrows in c). The dotted lines indicate the epidermis-dermis boundary. ApoTome microscopy analysis of immunofluorescent double labeling of MITF (red) and RACK1 (green): (a) MITF with DAPI nuclear counterstaining (blue), (b) RACK1, and (c) merge of the 3 channels. Figure 2. Toe melanocytoma; canine No. 2. Specific nuclear MITF labeling is observed across the melanocytic proliferation (arrows in a). RACK1 signal is low to moderate in intensity (b) in the cytoplasm of melanocytic cells with a granular distribution termed heterogeneous (arrows in c). ApoTome microscopy analysis of immunofluorescent labeling of MITF (a, red with blue nuclear counterstaining), RACK1 (b, green), and merge (c). Figure 3. Nonpigmented melanoma from trunk; canine No. 3. A nuclear MITF signal is detected in the lesion with pronounced anisokaryosis (a). RACK1 signal appears intense and diffuse (b) and is cytoplasmic and perinuclear in all MITF-positive cells (c). ApoTome microscopy analysis of immunofluorescent labeling of MITF (a, red with blue nuclear counterstaining), RACK1 (b, green), and merge (c). Figure 4. Poorly pigmented melanoma from lip; canine No. 4. Dermal cells with enlarged nuclei present MITF nuclear labeling (a) and an intense, diffuse, and homogeneous cytoplasmic and perinuclear RACK1 signal (b, c). ApoTome microscopy analysis of immunofluorescent labeling of MITF (a, red with blue nuclear counterstaining), RACK1 (b, green), and merge (c). Figure 5. Pigmented melanoma from toe; canine No. 5. Tumoral cells are MITF positive (a) even in the presence of melanin. The lesion displays an intense RACK1 signal (b) that is homogeneous, cytoplasmic, and perinuclear on MITFpositive cells (c). ApoTome microscopy analysis of immunofluorescent labeling of MITF (a, red with blue nuclear counterstaining), RACK1 (b, green), and merge (c). Bar: 25 mm.

diagnosis, RACK1 was detected in MITF-negative cells with variable intensity (not shown). To correlate RACK1 distribution with prognosis markers of our tumoral samples, 2 authors independently estimated the mitotic index and Ki-67 index (CA, GE). Mitotic index values per 10 HPF were either below 1 or higher than 4, with few exceptions (Table 2, column 1). Applying the prognostic cutoff according to the recent literature, which distinguishes melanocytic neoplasms with cutaneous or oral origin,3,15 data were concordant with the pathology reports except for sample 22. The Ki-67 index, also known as the growth fraction, displayed values either below 7 or higher than 15 with few exceptions (Table 2, column 2). The accepted cutoff of 15% for canine melanocytic neoplasms3,15,28,29 supported pathology reports,

with the exception of samples 16, 20, and 23, which displayed an otherwise high mitotic index. Conversely, sample 22 displayed a high Ki-67 index. Overall, these data confirmed pathology reports with statistical significance (P < .01, Student’s t-test). Hence, subsequently, melanocytic neoplasms were grouped according to the histopathologic reports (ie, melanocytomas vs melanomas). Comparison of RACK1 grading between both groups resulted in a statistically significant difference (P < .001, Student’s t-test) (Table 2, last column). All samples histologically classified as melanomas (n ¼ 13) stained homogeneously for RACK1, with a strong diffuse expression, localized in perinuclear and cytoplasmic compartments. In sharp contrast, all samples classified as melanocytomas (n ¼ 11) stained heterogeneously for RACK1, with a low to moderate cytoplasmic signal

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— 4.2 10.6 2.4 0.5 1.2 5.4 6.7 8.3 5 1.5

18.5 16.3 16.2 24.1 7.2 22.4 — — 12 — 22.6 10 16.7