Foiani Greta, Melchiotti Erica, Capello Katia, Porcellato Ilaria, Brachelente Chiara, Iussich Selina, Giacobino Davide, Morello Emanuela, Martano Marina, Buracco Paolo, Vascellari Marta
Histopathology Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy.
Epidemiology and Biostatistics Unit, Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy.
Vet Comp Oncol. 2025 Jun;23(2):141-151. doi: 10.1111/vco.13039. Epub 2025 Jan 9.
Canine oral melanoma (OM) exhibits poor prognosis and limited treatment options. The success of immune checkpoint inhibitors (ICIs) in human melanoma has driven interest in similar therapeutic approaches in the dog, although the immunosuppressive mechanisms adopted by canine OM remain unclear. This study aimed to evaluate the expression of the immune checkpoints PD-1/PD-L1 and CTLA-4 by RNAscope in situ hybridization (ISH) in canine OM, to investigate their expression pattern and explore their potential role in melanoma progression. Twenty-four formalin-fixed, paraffin-embedded canine OM were included in the study. PD-L1 expression by tumour cells was detected in 100% melanomas (score 1-3), especially at the host-tumour interface. PD-1 and CTLA-4 expression by tumour cells was detected in 13/24 (54%, score 1-2) and 18/24 (75%, score 1) melanomas, respectively. Dual ISH-immunohistochemistry with Melanoma Triple Cocktail, CD3, CD20 and Iba1 demonstrated the expression of tested immune checkpoints in neoplastic and immune cells. Notably, PD-1 and CTLA-4 were predominantly expressed by tumour-infiltrating T lymphocytes, while PD-L1 was primarily expressed by tumour-associated macrophages. PD-1 expression in neoplastic cells was significantly correlated with mitotic count (p < 0.05), while no associations were found between immune checkpoint expression and disease-free interval or overall survival. Whole tumour PD-L1 and PD-1 expression, assessed by image analysis, correlated to PD-L1 scores in neoplastic cells and the grade of tumour-infiltrating lymphocytes, respectively. Collectively, PD-L1, PD-1 and CTLA-4 likely contribute to immunosuppression in canine OM. Further studies are warranted to investigate whether ISH can serve as a biomarker for selecting patients suitable for ICI treatment.
犬口腔黑色素瘤（OM）预后较差且治疗选择有限。免疫检查点抑制剂（ICI）在人类黑色素瘤治疗中的成功促使人们对犬类的类似治疗方法产生兴趣，尽管犬OM所采用的免疫抑制机制仍不清楚。本研究旨在通过RNAscope原位杂交（ISH）评估犬OM中免疫检查点PD-1/PD-L1和CTLA-4的表达，研究其表达模式并探索它们在黑色素瘤进展中的潜在作用。本研究纳入了24例福尔马林固定、石蜡包埋的犬OM。在100%的黑色素瘤中检测到肿瘤细胞表达PD-L1（评分1-3），尤其是在宿主-肿瘤界面。在13/24（54%，评分1-2）和18/24（75%，评分1）的黑色素瘤中分别检测到肿瘤细胞表达PD-1和CTLA-4。使用黑色素瘤三联鸡尾酒、CD3、CD20和Iba1进行双重ISH-免疫组织化学检测，证实了所检测的免疫检查点在肿瘤细胞和免疫细胞中的表达。值得注意的是，PD-1和CTLA-4主要由肿瘤浸润性T淋巴细胞表达而PD-L1主要由肿瘤相关巨噬细胞表达。肿瘤细胞中PD-1的表达与有丝分裂计数显著相关（p<0.05），而在免疫检查点表达与无病间期或总生存期之间未发现相关性。通过图像分析评估的全肿瘤PD-L1和PD-1表达分别与肿瘤细胞中的PD-L1评分和肿瘤浸润淋巴细胞的分级相关。总体而言，PD-L1、PD-1和CTLA-4可能在犬OM的免疫抑制中起作用。有必要进一步研究ISH是否可作为选择适合ICI治疗患者的生物标志物。
