Identification of a Splice Variant of gp100 Expressed in Canine Melanoma Tumours

Introduction:  The melanoma‐associated antigen (MAA) gp100 is a glycoprotein expressed by melanocytes and is considered to be an important target for anti‐tumour immunity in human melanoma. The aim of the current study was to characterise the canine gp100 gene with a view to its inclusion in a DNA vaccine for treatment of canine oral malignant melanoma.
Methods:  RNA was extracted from eight canine oral melanomas and seven control samples (pigmented oral mucocutaneous tissue) and cDNA synthesised. PCR was performed using human gp100 primers designed to generate a 421base pair (bp) fragment. In addition to the expected pcr product, a smaller amplicon (∼260 bp) was present in all tumours and control tissues. Both PCR amplicons were cloned and sequenced. The gp100 genomic DNA sequence was identified from the dog genome resource (NCBI).
Results:  The 421 bp fragment of canine gp100 shared 90.8% identity with human gp100. This partial sequence was located on dog chromosome 10 (CFA10_contig 2982). Further analysis, using the human gp100 gene as a reference, allowed the complete canine gp100 coding sequence to be identified from the dog genome. Sequence analysis of the small PCR product showed it to be a splice variant of gp100 with exon 5 deleted.
Conclusion:  The canine gp100 sequence has been identified and this will allow DNA vaccine constructs containing this MAA to be developed. The splice variant of gp100 with exon 5 deleted was not tumour‐specific and was expressed in both melanomas and normal pigmented tissue.     

Use of immunocytochemical techniques in canine melanoma

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

p53 expression and apoptosis in melanomas of dogs and cats

Twenty-seven melanocytic tumours from 20 dogs and four cats were examined for p53 expression and apoptosis. They included tumours that were histologically classified as benign (BM), primary malignant (PMM) and metastatic malignant melanomas (MMM). For all cases clinical follow-up was available. p53 expression was examined immunohistochemically using different monoclonal and polyclonal antibodies. Apoptosis was detected using the TUNEL technique. The tissue sections were analysed using a quantitative image analysing system. A p53 index (p53I) and an apoptotic index (AI) were determined. p53 over-expression was found infrequently in these canine and feline melanocytic tumours. Apoptosis was observed in some of the malignant tumours. In one feline case of malignant melanoma, p53 accumulation together with apoptosis was seen in three metastases but not in the primary tumour. p53I and AI were not significantly correlated with survival. These results are similar to those reported for human cutaneous melanomas.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

The immune response to disialoganglioside GD3 vaccination in normal dogs: a melanoma surface antigen vaccine

As a result of its metastatic potential, canine malignant melanoma like its human counterpart like its human counter part, has a poor response to conventional treatment protocols. This prompted us to investigate the possibility of enhancing the immune response against the melanoma cell surface antigen, disialoganglioside GD3. Initially a flow cytometric study was designed in which the incidence of GD3 on the cell surface, recognized by the monoclonal antibody Mel-1 (R24), was established in canine melanoma cell lines. Results from the flow cytometry found GD3 to be highly expressed (94.2%) in six out of seven canine melanoma cell lines. Since it was thus potentially a good target, a study in which normal dogs were vaccinated intradermally with a vaccine containing GD3 plus adjuvants was designed. The adjuvant included CpG oligodeoxynucleotide (CpG-ODN) sequences and RIBI-adjuvant, which are known to target toll-like receptors (TLR) of the innate immune system. From a cohort of 10 dogs, 4 were vaccinated 3 times, at 4 weekly intervals with GD3 plus adjuvant, and 4 received only RIBI-adjuvant, and 2 phosphate buffered saline. Caliper measurements were collected to assess skin reaction at the vaccination site and sera assayed for IgM and IgG antibodies against GD3 and cell-mediated cytotoxicity against a melanoma cell line. Results from the study found significant differences (P<0.05) in the vaccine site reactions, IgM/IgG levels and cell-mediated cytotoxicity in the vaccinated versus unvaccinated dogs. The addition of CpG-ODN sequences and increasing GD3 concentration in the vaccine increased the inflammation response at the injection site. GD3 IgG and IgM antibodies in vaccinated dogs showed increasing titers over time and achieved significance at weeks 9 and 12, respectively. Cell-mediated cytotoxicity was only detected in peripheral blood mononuclear cells from vaccinated dogs. In conclusion, by combining the tumor antigen GD3 (a known weak self-antigen) and an adjuvant, tolerance was overcome by an innate and adaptive immune response in this population of normal dogs.     

Cytogenetic analysis of three oropharyngeal malignant melanomas in dogs.

Three cases of histologically confirmed oropharyngeal malignant melanomas in dogs are presented including clinical examinations and cytogenetic analysis. Case one showed a hyperdiploid karyotype. Case two, a recurrent tumour, had a highly hypodiploid karyotype with supernumerary meta- and submetacentric chromosomes in all metaphases analysed. In the third case, a clonal fusion of chromosome 1 and 25 was observed. Comparing these results with another case of canine cutaneous melanoma as well as with human malignant melanomas reported in the literature, these tumours obviously often show cytogenetic aberrations like aneuploidy and centric fusions.     

Mast cells and angiogenesis in canine melanomas: malignancy and clinicopathological factors

The biological significance of mast cells and angiogenesis in canine melanomas is unclear. Eighty canine melanomas (56 malignant and 24 benign), investigated to determine the relationship between mast cell count (MCC), microvessel density (MVD) and clinicopathology, revealed significantly higher MCC and MVD counts in malignant melanomas. Evaluation of the prognostic significance of MCC and MVD in malignant melanomas showed a significant correlation between MCC and MVD both within and at the edges of the tumour. Multivariate analysis indicated that MCC and MVD were independent predictors of survival but the former was a significantly better prognostic marker. Greater numbers of mast cells and microvessels were found in malignant melanomas of poor prognosis. The findings demonstrate a prognostic significance of MCC and MVD in canine melanocytic tumours.     

Feline non-ocular melanoma: a retrospective study of 23 cases (1991-1999).

Non-ocular melanoma is considered to be a rare neoplasm in cats; however, more than 150 cases have been reported in the literature since 1961. The objective of this study was to characterise this tumour better by evaluating case outcome and survival data for cats with melanoma and to compare clinical and histopathological findings with those of previous reports. Twenty-three feline non-ocular melanomas were identified, the most common locations being the nose, digit and pinna. Cats with digital melanomas had survival rates similar to their canine counterparts. Histological assignation of benignity, malignancy or junctional activity was not found to be an accurate predictor of clinical behaviour. Melanoma should be considered as a differential diagnosis for cats presenting with pigmented or non-pigmented masses and histopathology is essential for definitive diagnosis, as other tumours may clinically appear quite similar. Regular follow-up examinations are recommended indefinitely for benign or malignant feline melanomas.     

Immunoexpression of epithelial membrane antigen in canine meningioma: Novel results for perspective considerations

Epithelial membrane antigen (EMA) is one of the most widely used diagnostic immunohistochemical markers for human meningioma. To date, no published study on EMA expression in formalin‐fixed paraffin‐embedded (FFPE) tissue samples of canine meningioma is available. Here, we describe the results of an immunohistochemical study on 25 FFPE canine meningiomas using a monoclonal anti‐human EMA antibody. All meningiomas showed positive staining for EMA with cytoplasmic pattern, in nine cases associated with membranous staining. Area and intensity of staining were highly variable among cases. No clear relationships between tumour subtype/grade and area/intensity of staining were found. However, epithelial‐like patterns showed a higher affinity for EMA compared to the mesenchymal one. The present study provides the basis to explore the potential diagnostic application of this marker in canine meningioma. To investigate EMA expression in other central nervous system tumours of dogs are necessary to assess the specificity of this marker.     

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.     

Fascin‐1 expression in canine cutaneous and oral melanocytic tumours

Fascin‐1 expression was examined in 9 cutaneous melanocytomas and 47 oral melanomas. The cases were scored on the basis of extent and intensity of staining, and combined scores were calculated. Fascin‐1 expression was observed in 5/9 (56%) melanocytomas and 46/47 (98%) melanomas. The combined score for fascin‐1 was significantly greater in stage III/IV melanomas than in stage I/II melanomas (P < 0.05). In addition, strong fascin‐1 staining was associated with a significantly shortened survival time (P < 0.05). The results of this study suggest that fascin‐1 overexpression correlates with the malignancy of canine melanoma and has the potential to be a new immunohistochemical marker to predict the clinical course of canine melanoma. In addition, targeted therapy for fascin‐1 may represent a new strategy for the treatment of canine melanoma.     

Comparison of tyrosinase-related protein-2, S-100, and Melan A immunoreactivity in canine amelanotic melanomas

Tyrosinase-related protein-2 (TRP-2) is a highly conserved melanogenic enzyme expressed in both pigmented and unpigmented melanomas of the mouse. To determine whether TRP-2 would be a good diagnostic marker for amelanotic melanomas of the dog, we performed immunohistochemistry for TRP-2, S-100, and Melan A on 21 canine tumors identified as amelanotic melanomas based on routine histopathologic examination. Thirteen of the tumors were TRP-2 positive, 10 were Melan A positive, and 19 were S-100 positive. TRP-2 was expressed in the cytoplasm of tumor cells in both primary and metastatic melanomas. S-100 staining was positive in all of three schwannomas and two of three gastrointestinal stromal tumors (one fibrosarcoma and one leiomyosarcoma) tested. Neither Melan A nor TRP-2 antibodies reacted with these tumors. Our findings indicate that staining for TRP-2 is a sensitive and specific method for confirming the diagnosis of amelanotic melanoma in dogs.     

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer‐related signalling pathways despite differences in activating mutations.     

MIB‐1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma

The growth fraction of 68 canine cutaneous melanomas was determined by immunostaining with MIB‐1, a monoclonal antibody to a Ki‐67 epitope that recognizes all proliferating cells. Fifty tumours were classified histologically as benign and 18 as malignant. The Ki‐67 proliferative index (percentage of positive cells over 500 neoplastic cells) was low (< 15%) in 55 cases and high ( 15%) in 13 cases. High Ki‐67 proliferative index and histological malignancy were both associated with significantly poorer 2‐year survival (P < 0.0001). However, the predictive value of the Ki‐67 proliferative index (97%) was higher than the predictive value of classical histology (91%). The evaluation of the growth fraction by the Ki‐67 proliferative index is highly predictive of the biological behaviour of canine cutaneous melanoma.     

Malignant fibrous histiocytomas in dogs and cats: an immunohistochemical study.

Immunohistochemical staining was performed on seven canine and 10 feline soft tissue tumours histologically diagnosed as malignant fibrous histiocytomas (MFHs) or MFH-like tumours, and eight other histologically specified tumours (non-MFH). This was done to determine if commercially available antibodies that are used routinely in human diagnostic pathology for MFHs would express the same immunohistochemical patterns in canine and feline MFHs and MFH-like tumours. The antibodies were directed against human alpha 1-anti-trypsin (AT), human alpha 1-anti-chymotrypsin (ACT), human lysozyme, bovine S-100 protein and human desmin. AT did not show any immunoreactivity in the tissues investigated. Except for one MFH, all canine MFHs and other soft tissue tumours with a 'histiocytic' character stained for lysozyme and not for S-100. Six out of seven canine MFHs and MFH-like tumours stained positive for desmin as did most non-MFH sarcomas. Most of the canine and feline MFHs and MFH-like tumours were positive for ACT. These findings for ACT staining in canine and feline MFHs and MFH-like tumours are in agreement with the findings in human MFHs. The immunohistochemical results of canine MFHs and MFH-like tumours were different from those in cats. Feline MFHs differed from canine MFHs for both lysozyme and desmin staining.     

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.     

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.