Oral Melanoma Research Articles

Pilot study to evaluate isolation by size of circulating tumour cells in canine oral melanoma.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

MELANOMA OF ORAL CAVITY: A CASE REPORT OF AN UNUSUAL PRESENTATION

Oral mucosal melanoma is a rare malignancy with the tendency to metastasize and locally invade tissues more readily than other malignant tumor of the oral cavity. It occurs approximately four times more frequently in the oral mucosa of the upper jaw usually on the palate or alveolar gingiva. The chameleonic presentation of malignant melanoma, its asymptomatic condition, rarity of the lesion, poor prognosis and the necessity of a highly specialized treatment are factors that should be seriously considered by the involved health care provider. We report a rare and interesting case of oral malignant melanoma of the upper gum, which was clinically and histopathologically diagnosed with a brief review of literature, has been discussed.

Targeting canine mammary neoplastic epithelial cells with a reengineered anthrax toxin: first study.

Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.

Checkpoint Inhibitors in Dogs: Are We There Yet?

Immune checkpoint inhibitors (ICI) have revolutionised cancer treatment in people. Immune checkpoints are important regulators of the body's reaction to immunological stimuli. The most studied immune checkpoint molecules are programmed death (PD-1) with its ligand (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) with its ligands CD80 (B7-1) and CD86 (B7-2). Certain tumours can evade immunosurveillance by activating these immunological checkpoint targets. These proteins are often upregulated in cancer cells and tumour-infiltrating lymphocytes, allowing cancer cells to evade immune surveillance and promote tumour growth. By blocking inhibitory checkpoints, ICI can help restore the immune system to effectively fight cancer. Several studies have investigated the expression of these and other immune checkpoints in human cancers and have shown their potential as therapeutic targets. In recent years, there has been growing interest in studying the expression of immune checkpoints in dogs with cancer, and a few small clinical trials with ICI have already been performed on these species. Emerging studies in veterinary oncology are centred around developing and validating canine-targeted antibodies. Among ICIs, anti-PD-1 and anti-PD-L1 treatments stand out as the most promising, mirroring the success in human medicine over the past decade. Nevertheless, the efficacy of caninized antibodies remains suboptimal, especially for canine oral melanoma. To enhance the utilisation of ICIs, the identification of predictive biomarkers for treatment response and the thorough screening of individual tumours are crucial. Such endeavours hold promise for advancing personalised medicine within veterinary practice, thereby improving treatment outcomes. This article aims to review the current research literature about the expression of immune checkpoints in canine cancer and the current results of ICI treatment in dogs.

A ten-year literature review of oral malignant melanoma cases: A meta-analysis study

AimOral malignant melanoma (OMM) is a rare and aggressive malignant tumour arising from the proliferation of melanocytes and accounts for only 0.5 % of all oral malignancies. It is invasive and tends to metastasise. The aim of this study is to determine the patient profile of OMM and to provide information about the clinical features and treatment plans of the disease. Materials and MethodsThe Pubmed database was searched for OMM cases published as case reports in the last 10 years. The search was limited to English and open-access case reports. A total of 49 OMMs in 45 patients from 43 case reports were analyzed. In addition to the age, gender, smoking and alcohol consumption habits of the patients, data on the signs and symptoms of OMM, location of the lesion, imaging modalities used in diagnosis and treatment, lymph node involvement, presence of metastasis, survival time, and treatment modalities were recorded. ResultsIn OMM cases, 18 (40 %) of the patients were female, 27 (60 %) were male, and the mean age was 53.13 ± 16.09 years. The most common symptom was "swelling" (n = 23, 33.8 %) and the most common finding was "hyperpigmentation" (n = 40, 24.4 %). The most common site of OMM was the maxilla (n = 21, 46.7 %). According to the rate of use of imaging modalities in the cases, it was determined that "advanced imaging modalities" were mostly used (n = 24, 53.3 %). It was determined that 22.2 % (n = 10) of the patients died within the first 5 years. Combined treatment (n = 21, 46.5 %) were mostly applied to the patients. ConclusionOMM is more common in the maxilla, in males and in the fifth decade of life, and advanced imaging modalities are most commonly used to detect the disease. Because of its poor prognosis, knowing the patient profile and common clinical features of OMM will increase the clinical awareness of physicians.

Local intralesional talimogene laherparepvec therapy with complete local response in oral palatine mucosal melanoma: a case report

BackgroundMucosal melanoma, an aggressive type of malignancy different from the cutaneous melanomas commonly seen in the head and neck region, represents < 1% of all malignant melanomas. The pathogenesis of mucosal melanoma is unknown. Targetable mutations commonly seen in cutaneous melanoma, such as in the BRAF and NRAS genes, have a lower incidence in mucosal melanoma. Mucosal melanoma carries a distinct mutational pattern from cutaneous melanoma. Surgery with negative margins is the first-line treatment for mucosal melanoma, and systemic therapy is not well defined. Talimogene laherparepvec, an oncolytic viral immunotherapy, is United States Food and Drug Administration approved for the treatment of advanced malignant cutaneous melanoma, with local therapeutic benefits. Mucosal melanoma was initially excluded from talimogene laherparepvec’s initial phase III clinical trial.Case presentationWe present the case of a white female patient in her 40s with past medical history of systemic lupus erythematous, scleroderma, and estrogen-receptor-positive invasive ductal breast carcinoma. Following a bilateral mastectomy, the patient was found to have BRAF-negative mucosal melanoma of her hard palate with a soft palate skip lesion. Owing to the presence of a skip mucosal lesion as well as the anticipated defect and need for free-flap reconstructive surgery, nonsurgical management was considered. The patient was referred to medical oncology, where—based on the patient’s complicated medical history and the risk of immunotherapy possibly worsening her prior autoimmune diseases—local talimogene laherparepvec injections were chosen as the primary therapy for her mucosal lesions. Though talimogene laherparepvec is approved for the treatment of cutaneous melanoma, there are limited data available on the use of talimogene laherparepvec in mucosal melanomas.ConclusionThe patient had a complete local tumor response at both the primary lesion as well as the skip lesion with the local injections. She had no side effects and maintained a high quality of life during treatment.

Canine Oral Melanoma: Questioning the Existing Information through a Series of Clinical Cases.

Twelve dogs with oral malignant melanomas (MM) were evaluated in this study, with demographic details indicating a balanced distribution of gender, age, and weight among various breeds. Tumor locations varied, with diverse surgical procedures being performed, including mandibulectomies and maxillectomies. Lymphadenectomies were conducted, revealing a 16.66% metastatic rate in regional lymph nodes. At the time of surgery, clinical staging identified stages I, II, and III, with most cases having non-infiltrated margins and a high mitotic index. Follow-up revealed local recurrences and metastases, prompting additional surgeries and affecting survival rates. This study reports varying outcomes, with some dogs completing one year without recurrence, while others experienced progressive disease, leading to six oral melanoma-related deaths. The characteristics of melanotic melanoma and amelanotic melanoma are observed in order to study differences between them, the degree of aggressiveness, the mortality rate and the possibility of future therapeutic targets. Although high pigmentation has been correlated with a better outcome, we could not find any significant correlation between survival and achromia. Oral benign melanomas might exist, and this could justify variabilities between stage and survival; however, carefulness is required due to their unpredictable behavior. The findings underscore the complexity of oral melanoma cases and highlight the need for further research on effective management strategies.

Salivary metabolomic identification of biomarker candidates for oral melanoma and oral squamous cell carcinoma in dogs.

Oral melanoma (OM) and oral squamous cell carcinoma (OSCC) are frequently diagnosed in dogs, presenting a challenge in distinguishing them from benign oral tumors (BN). Salivary metabolomic biomarkers offer a practical solution because of saliva's direct contact with tumors and the noninvasive nature of collection. Assess the diversity and abundance of the salivary metabolome in dogs with BN, OM, and OSCC using amine/phenol submetabolome analysis and high-performance chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS). Study included 11 BN, 24 OM, 10 OSCC, and 20 healthy control dogs. Case-control cross-sectional study was conducted to assess salivary submetabolic profiles in dogs with BN, OM, and OSCC and healthy dogs. Samples were labeled with 12C-dansyl chloride and analyzed using CIL LC-MS targeted to amine- and phenol-containing metabolites for amine/phenol submetabolome analysis. Distinct clusters and significant differences in metabolite concentrations were observed among the oral cancer, BN, and control groups. A total of 154 and 66 metabolites showed significantly altered concentrations, particularly in OM and OSCC, respectively, when compared with BN (Padj < .05). Potential metabolic biomarkers were identified for each cancer, including decreased concentrations of seryl-arginine and sarcosine in OSCC. Moreover, high-confidence putative metabolites were identified, including an increase in tryptophyl-threonine and a decrease in 1,2-dihydroxynapthalene-6-sulfonic acid and hydroxyprolyl-hydroxyproline for OM. We identified high coverage of the amine/phenol submetabolome, including seryl-arginine, and sarcosine, in OSCC. Our findings emphasize the potential of these biomarkers for distinguishing between oral OSCC and BN in dogs.

Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

SOX-10 and TRP-1 expression in feline ocular and nonocular melanomas.

In felines, ocular and nonocular melanomas are uncommon tumors that represent a diagnostic challenge for pathologists, especially when amelanotic. To date, the immunohistochemical diagnostic panel in cats is based on specific melanocytic markers (Melan-A and PNL2) and a nonspecific but sensitive marker (S100). In human medicine, SOX-10 is reported to be a sensitive antibody for the detection of melanoma micrometastasis in the lymph node. TRP-1, an enzyme involved in melanogenesis, has recently been used in humans and dogs as a specific melanocyte marker. The aim of this study was to evaluate the cross-reactivity and the expression of SOX-10 and TRP-1 antibodies in feline normal tissue and melanocytic tumors. Thirty-one cases of ocular, cutaneous, and oral melanomas were retrospectively evaluated and confirmed by histopathological examination and by immunolabeling with Melan-A and/or PNL2. SOX-10 nuclear expression in normal tissues was localized in epidermal, subepidermal, hair bulb, and iridal stromal melanocytes and dermal nerves. In melanomas, nuclear expression of SOX-10 was detected in ocular (11/12; 92%), oral (6/7; 86%), and cutaneous sites (12/12; 100%). TRP-1 cytoplasmic immunolabeling in normal tissue was observed in epidermal and bulbar melanocytes and in the lining pigmented epithelium of the iris and in its stroma. Its expression was positively correlated to the degree of pigmentation in the tumor and was observed in 75% of ocular (9/12), 43% of oral (3/7), and 33% of cutaneous melanomas (4/12). This study demonstrated the cross-reactivity of SOX-10 and TRP-1 antibodies in feline non-neoplastic melanocytes and their expression in ocular and nonocular melanomas.

Mutational landscape of oral mucosal melanoma based on comprehensive cancer genomic profiling tests in a Japanese cohort

ObjectivesOral mucosal melanoma (OMM) is a rare but aggressive melanoma subtype. Due to its rarity, the genomic landscape of OMM remains unknown despite a relatively thorough understanding of the genetic profile of cutaneous melanoma (CM). In this study, we analyzed the genomic mutational profiles of Japanese patients with OMM and compared them with those of patients with nose/sinuses mucosal melanoma (NMM) and CM to identify potential therapeutic targets. Materials and MethodsWe extracted clinical and genomic information of patients with OMM (n = 15), NMM (n = 63), and CM (n = 413) who underwent comprehensive genomic profiling tests under the National Health Insurance between June 2019 and November 2023 from the Center for Cancer Genomics and Therapeutics database. ResultsThe most frequent genomic alteration identified in OMM was RICTOR (40%) followed by CDK4 (33.3%), MDM2 (33.3%), KDR (30%), KIT (26.7%), and NF1 (26.7%). CDK4 and MDM2 were co-amplified. Gene alterations in MYC and NRAS were the highest in patients with NMM, followed by those with CM, and no MYC alteration was observed in patients with OMM. BRAF V600 mutation, which is frequently observed in patients with CM (23.2%) were only present in 1.6% of patients with NMM and none in patients with OMM. ConclusionThis study clarified the genetic differences between OMM and NMM, and the first to report the frequent occurrence of RICTOR amplification in OMM. This analysis offers insights into the development of personalized therapeutics for OMM.

Melanoma of the dog and cat: consensus and guidelines.

Melanoma of the dog and cat poses a clinical challenge to veterinary practitioners across the globe. As knowledge evolves, so too do clinical practices. However, there remain uncertainties and controversies. There is value for the veterinary community at large in the generation of a contemporary wide-ranging guideline document. The aim of this project was therefore to assimilate the available published knowledge into a single accessible referenced resource and to provide expert clinical guidance to support professional colleagues as they navigate current melanoma challenges and controversies. Melanocytic tumors are common in dogs but rare in cats. The history and clinical signs relate to the anatomic site of the melanoma. Oral and subungual malignant melanomas are the most common malignant types in dogs. While many melanocytic tumors are heavily pigmented, making diagnosis relatively straightforward, melanin pigmentation is variable. A validated clinical stage scheme has been defined for canine oral melanoma. For all other locations and for feline melanoma, TNM-based staging applies. Certain histological characteristics have been shown to bear prognostic significance and can thus prove instructive in clinical decision making. Surgical resection using wide margins is currently the mainstay of therapy for the local control of melanomas, regardless of primary location. Radiotherapy forms an integral part of the management of canine oral melanomas, both as a primary and an adjuvant therapy. Adjuvant immunotherapy or chemotherapy is offered to patients at high risk of developing distant metastasis. Location is the major prognostic factor, although it is not completely predictive of local invasiveness and metastatic potential. There are no specific guidelines regarding referral considerations for dogs with melanoma, as this is likely based on a multitude of factors. The ultimate goal is to provide the best options for patients to extend quality of life and survival, either within the primary care or referral hospital setting.

RAC1 pathway in canine oral melanoma progression and prognosis

RAC1 pathway in canine oral melanoma progression and prognosis

Primary Malignant Melanoma of Tongue: Rare and Aggressive Malignant Tumor.

Primary melanoma of the oral cavity is extremely rare, accounting for 0.2-8% of all melanomas. Lesions arising from mucosal melanocytes occur most frequently on the gingiva or palate. Mucosal melanomas carry a worse prognosis than cutaneous melanomas. Very few studies have been published due to rarity of disease. 46 year old lady presented with black colored lesion over left side of her tongue for 6 weeks. On examination there was 3 × 2.5cm black colored patch over left lateral part of tongue in middle 1/3rd, not crossing midline. Tip and base of tongue were free. Biopsy was suggestive of malignant melanoma of tongue. Patient underwent surgery (wide local excision of left lateral border of tongue lesion + left selective neck dissection). Final Histopathological report revealed malignant melanoma of tongue with all margins free, Depth of invasion 3mm, Lympho-vascular invasion present, and no perineural invasion, left level III cervical lymph node metastases. Patient thus received adjuvant RT. Immunotherapy was also advised in multidisciplinary clinic, but patient was non-compliant. Early diagnosis will be promoted by careful oral examination and early biopsy of pigmented and non-pigmented masses. Early diagnosis and treatment will improve the prognosis of patients with oral malignant melanoma.

Limited Clinical Efficacy with Potential Adverse Events in a Pilot Study of Autologous Adoptive Cell Therapy in Canine Oral Malignant Melanoma.

Adoptive cell therapy (ACT) has been studied in several human and canine cancers with some promising clinical outcomes but not in canine oral malignant melanoma (OMM). Our manuscript aimed to explore one kind of ACT, the ex vivo-expanded autologous immune cell infusion in canine OMM, as this tumor remains a treatment dilemma. The study recruited dogs with histopathological diagnoses of oral malignant melanoma, generated their peripheral blood mononuclear cells, expanded them into predominantly non-B non-T cells via stimulations of IL-15, IL-2, and IL-21, and then re-infused the cells into tumor-bearing dogs. Ten dogs were enrolled; three dogs did not report any adverse events; three had a mildly altered appetite; one had a mildly increased liver index, while the other three developed suspected anaphylaxis at different levels. The median progression-free interval was 49 days. Dogs with progressive disease during treatment had a shorter survival. This pilot study indicates limited efficacy with potential adverse events of this ACT. Most recruited patients were in a later stage and had macroscopic disease, which might affect the treatment efficacy. Further exploration of this cell therapy in an adjuvant setting, with adequate protocol modification and standardization, could still be considered.

Abstract 3736: Bridging the gaps: A cross-species approach to PD-1 and OX40 combination immunotherapy

Abstract Monoclonal antibodies (mAbs) targeting immune checkpoints, such as PD-1, PD-L1, and CTLA4, have provided unprecedented clinical benefits to human cancer patients. Novel strategies such as combination therapy with OX40 or 4-1BB agonists are currently under investigation in various pre-clinical studies to enhance the therapeutic benefits of immune checkpoint inhibitors. While syngeneic mouse tumor xenograft models offer valuable insights in pre-clinical testing, they fall short in capturing the nuanced heterogeneity within and between tumors and the dynamic tumor-immune interface. In contrast, canine patients with spontaneous neoplasms possess a closely analogous and intact immune system. These canine patients, who receive sophisticated and advanced medical therapy like their human counterparts, can serve as a vital intermediate pre-clinical model system between rodent studies and human clinical trials, potentially reducing clinical trial failures. In this study, we have developed a novel bispecific fusion protein, denoted BsOXPD, that combines OX40 agonism and PD-1 blockade into a single therapeutic. Our bispecific fusion protein employs an anti-PD-1 nanobody (Nb) to bind and block the PD1 pathway and the extracellular domain (ECD) of the OX40 ligand to stimulate the OX40 receptor. We have developed murinized, caninized, and humanized versions of this protein. All versions of the bispecific fusion protein retain their biochemical and functional properties following the murinization, caninization, and humanization processes. Importantly, all versions effectively disrupt the PD-1/PD-L1 axis in murine, canine, and human systems while concurrently acting as potent OX40 agonists. To further improve the humanized version of the BsOXPD, we partially humanized the anti-PD1 Nb by introducing amino acid substitutions in various framework regions. The humanized anti-PD1 Nb still completely inhibited the binding of human PD-L1 to the PD-1 receptor. The caninized version of BsOXPD exhibits no toxicities or immune-related adverse events (irAEs) following intravenous injection at doses of 1 mg/kg or 3 mg/kg body weight in healthy beagle dogs. In summary, we have designed a cross-species Nb-based fusion protein for PD1 blockade and OX40 agonism. Our next goal is to evaluate the therapeutic efficacy of caninized BsOXPD in pet dogs with oral melanoma in neoadjuvant settings. Therefore, our cross-species approach will offer comprehensive insights into the safety, pharmacokinetic (PK) profile, and clinical benefits associated with the concurrent application of PD1 blockade and OX40 agonism. Citation Format: Maninder Sandey, Damien Ruiz, Jonathan Marable, Deepa Bedi, Payal Agarwal. Bridging the gaps: A cross-species approach to PD-1 and OX40 combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3736.

Assessment of SOX10 expression in 437 canine neoplasms of different embryologic origins.

Several members of the SRY-related HMG-box (SOX) protein family are implicated in tumorigenesis, metastasis, and regulation of the tumor microenvironment. SOX10, which is involved in neural crest cell migration and differentiation, has long been recognized a sensitive and specific immunohistochemical (IHC) marker in the diagnosis of melanoma in humans. However, expression of SOX10 in other tumor types has infrequently been evaluated in humans until recently and has not been thoroughly investigated in the dog. Our aim was to characterize the expression of SOX10 in canine neoplasms to objectively assess its value as a diagnostic IHC marker. Immunohistochemistry for SOX10 was performed on 437 archived, formalin-fixed paraffin-embedded tissues from representative canine neoplasms of ectodermal (15 tumor types), mesodermal (13 tumor types), endodermal (8 tumor types), and mixed/unknown (7 tumor types) embryologic origin. Oral and cutaneous tumors of melanocytic origin were used as positive controls. Intense SOX10 immunolabeling was observed in most tumors of ectodermal origin, including consistent expression in mammary carcinomas, and gliomas. Embryonal and hair follicle neoplasms inconsistently exhibited strong nuclear immunolabeling. Oral fibrosarcomas and undifferentiated oral sarcomas both inconsistently exhibited moderate to strong nuclear immunolabeling. Neoplasms of mesodermal and endodermal origin lacked immunolabeling. Salivary carcinomas, representing an unknown/mixed embryologic origin, were strongly labeled. SOX10 expression is not limited to melanomas, but is expressed by canine tumors of diverse tissues and embryologic derivation. Importantly, expression of SOX10 by a subset of oral sarcomas impairs its value as a marker for spindle cell oral melanomas.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

Single institution study of the immune landscape for canine oral melanoma based on transcriptome analysis of the primary tumor.

Understanding a tumor's immune context is paramount in the fight against cancer. Oral melanoma in dogs serves as an excellent translational model for human immunotherapy. However, additional study is necessary to comprehend the immune landscape of dog oral melanomas, including their similarity to human melanomas in this context. This retrospective study utilizes formalin-fixed paraffin-embedded (FFPE) tissue samples to analyze RNA sequences associated with oral melanoma in dogs. Nanostring Technologies was used for conducting RNA sequencing. The focus is on understanding the differences between melanoma tumors restricted to the oral cavity (OL) and the same primary oral tumors with a history of metastasis to the lymph nodes or other organs (OM). Normal buccal mucosa samples are also included as a normal tissue reference. In the OM patient group, gene signatures exhibit significant changes relative to the OL patient group, including significantly decreased expression of S100, BRAF, CEACAM1, BCL2, ANXA1, and tumor suppressor genes (TP63). Relative to the OL tumors, the OM tumors had significantly increased expression of hypoxia-related genes (VEGFA expression), cell mobility genes (MCAM), and PTGS2 (COX2). The analysis of the immune landscape in the OM group indicates a shift from a possible "hot" tumor suppressed by immune checkpoints (PDL1) to significantly heightened expression not only of those checkpoints but also the inclusion of other immune blockades such as PD1 and IDO2. In addition, the OM group had significantly reduced expression of Toll-like receptors (TLR4) and IL-18 relative to the OL group, contributing to the tumor's immune escape. Additionally, signs of immune cell exhaustion are evident in both the OM and OL groups through significantly increased expression of TIGIT relative to normal tissue. Both the OM and OL groups had significantly increased expression of the immune cell marker CD4 expression relative to normal tissue. Further, CD4 expression significantly decreased in OM relative to OL; however, this study cannot determine the specific cell types expressing CD4 in OM and OL tumors. This preliminary study reports significant changes in gene expression for oral melanoma between canine patients with localized disease relative to those with metastatic disease. In the future, a more in-depth investigation involving immunohistochemistry analysis and single-cell RNA expression is necessary to confirm our findings.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.