No abstract

Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain an insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with chemotherapy (n = 12) or chemoimmunotherapy with APAVAC (n = 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity.

Expression of telomerase reverse transcriptase (TERT) and telomerase activity (TA) is a main feature of cancer, contributing to cell immortalization by causing telomeres dysfunction. BIBR1532 is a potent telomerase inhibitor that showed potential anti-tumor activities in several types of cancer, by triggering replicative senescence and apoptosis. In a previous work, we detected, for the first time, TERT expression and TA in preclinical models of feline oral squamous cell carcinoma (FOSCC); therefore, we aimed at extending our investigation by testing the effects of treatment with BIBR1532, in order to explore the role of telomerase in this tumor and foreshadow the possibility of it being considered as a future therapeutic target. In the present study, treatment of FOSCC cell lines SCCF1, SCCF2, and SCCF3 with BIBR1532 resulted in successful inhibition of TA, with subsequent cell growth stoppage and decrease in cell viability. Molecular data showed that up-regulation of cell cycle inhibitor p21, unbalancing of Bax/Bcl-2 ratio, and down-regulation of survival gene Survivin were mostly involved in the observed cellular events. Moreover, BIBR1532 diminished the expression of TERT and its transcriptional activator cMyc, resulting in the down-regulation of epidermal growth factor receptor (EGFR), phospho-ERK/ERK ratio, and matrix metalloproteinases (MMPs)-1/-2 and−9, likely as a consequence of an impairment of TERT extra-telomeric functions. Taken together, our data suggest that BIBR1532 exerts multiple anti-cancer activities in FOSCC by inhibiting telomerase pathway and interfering with signaling routes involved in cell proliferation, cell survival, and invasion, paving the way for future translational studies aimed at evaluating its possible employment in the treatment of this severe tumor of cats.

Telomerase activity contributes to cell immortalization by avoiding telomere shortening at each cell division; indeed, its catalytic subunit telomerase reverse transcriptase (TERT) is overexpressed in many tumors, including human oral squamous cell carcinoma (hOSCC). In these tumors, matrix metalloproteinases (MMPs), a group of zinc-dependent endopeptidases involved in cell migration, contribute to invasive potential of cancer cells. A proportion of hOSCC is associated with infection by high-risk human papillomavirus (HR-HPVs), whose E6 oncogene enhances TERT and MMPs expression, thus promoting cancer progression. Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor with highly invasive phenotype; however, studies on telomerase activity, TERT, and MMPs expression are scarce. In this study, we demonstrate telomerase activity, expression of TERT, and its transcriptional activator cMyc along with expression of MMP-1,-2, and-9 in FOSCC-derived cell lines SCCF2 and SCCF3, suggesting a contribution by these pathways in cell immortalization and invasion in these tumors. Recent studies suggest that a subgroup of FOSCC as well as SCCF2 and SCCF3 are associated with Felis catus PV type-2 (FcaPV-2) infection. However, in this work, FcaPV-2 E6 gene knock-down caused no shift in either TERT, cMyc, or MMPs levels, suggesting that, unlike its human counterpart, the viral oncogene plays no role in their regulation.

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes (ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0–50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone (n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid neoplasm in dogs, being characterized by a remarkable degree of clinical heterogeneity not completely explained by molecular data. This poses a major barrier to understanding the disease and response to therapy or when using dogs with DLBCL in clinical trials. We performed an integrated analysis of genome, exome and RNA sequencing in a cohort of 77 canine DLBCL to define the genetic landscape of this tumor. A wide range of signaling pathways and cellular processes were found in common with human DLBCL, but the frequencies of the most recurrently mutated genes were different overall. We developed a prognostic model integrating clinical, exonic variants and transcriptomic features to predict outcome in dogs with DLBCL. These results comprehensively define the genetic drivers of canine DLBCL and can be prospectively utilized to identify new therapeutic opportunities.

Canine diffuse large B-cell lymphoma (cDLBCL) is characterized by high mortality and clinical heterogeneity. Although chemo-immunotherapy improves outcome, treatment response remains mainly unpredictable. To identify a set of immune-related genes aberrantly regulated and impacting the prognosis, we explored the immune landscape of cDLBCL by NanoString. The immune gene expression profile of 48 fully clinically characterized cDLBCLs treated with chemo-immunotherapy was analyzed with the NanoString nCounter Canine IO Panel using RNA extracted from tumor tissue paraffin blocks. A Cox proportional-hazards model was used to design a prognostic gene signature. The Cox model identified a 6-gene signature ( IL2RB, BCL6, TXK, C2, CDKN2B, ITK) strongly associated with lymphoma-specific survival, from which a risk score was calculated. Dogs were assigned to high-risk or low-risk groups according to the median score. Thirty-nine genes were differentially expressed between the 2 groups. Gene set analysis highlighted an upregulation of genes involved in complement activation, cytotoxicity, and antigen processing in low-risk dogs compared with high-risk dogs, whereas genes associated with cell cycle were downregulated in dogs with a lower risk. In line with these results, cell type profiling suggested the abundance of natural killer and CD8+ cells in low-risk dogs compared with high-risk dogs. Furthermore, the prognostic power of the risk score was validated in an independent cohort of cDLBCL. In conclusion, the 6-gene-derived risk score represents a robust biomarker in predicting the prognosis in cDLBCL. Moreover, our results suggest that enhanced tumor antigen recognition and cytotoxic activity are crucial in achieving a more effective response to chemo-immunotherapy.

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo‐like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c‐Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self‐renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c‐Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c‐Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c‐Myc and PLK1‐overexpressing cOSA cell line (D17). PLK1 and c‐Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c‐Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT‐qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5–15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G2/M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c‐Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c‐Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c‐Myc.