Canine B-cell Lymphoma Research Articles

Cannabinoid receptor-2 expression in canine multicentric diffuse large B-cell lymphoma: An immunohistochemical, digital pathology and clinical analysis

The cannabinoid 2 receptor (CB2R) is a crucial element of the endocannabinoid system (ECS), which is predominantly expressed on cells of the reticuloendothelial system. Alterations in CB2R expression have shown a prognostic role in various human neoplastic diseases and its expression has been studied in canine mast cell tumours (MCT). Canine diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in dogs and has a variable clinical behaviour. Expression of CB2R was assessed by means of immunohistochemistry in fifteen dogs with proven histological diagnosis of DLBCL. A semiquantitative and quantitative assessment of immunoreactivity (IR) by digital analysis was performed in all cases. Our results indicate that CB2R expression is conserved in canine DLBCL but does not correlate with clinical outcome.

MicroRNAs implicated in canine diffuse large B-cell lymphoma prognosis.

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin lymphoma (NHL) in domestic dogs, with many similarities to its human counterpart. The progression of the disease is rapid, and treatment must be initiated early to achieve cancer remission and extend life. This study examined the relationship between progression-free survival (PFS) and microRNA (miRNA) expression in dogs with DLBCL. miRNAs are small non-coding RNA molecules that typically regulate gene expression post-transcriptionally. They are involved in several pathophysiological processes, including the growth and progression of cancer. Based on the analysis of small RNA sequencing (sRNA-seq) data, we validated a group of miRNAs in lymph nodes from 44 DLBCL-affected dogs with known outcomes. We used quantitative PCR to quantify their expression and report a specific subset of miRNAs is associated with decreased PFS in dogs with DLBCL. The miR-192-5p and miR-16-5p expression were significantly downregulated in dogs with increased PFS. These results indicate that miRNA profiling may potentially identify dogs with DLBCL that will experience poor outcomes following treatment. Identifying specific miRNAs that correlate with the progression of canine DLBCL could aid the development of individualized treatment regimens for dogs.

Thinking Outside the Box: Indirect Myc Modulation in Canine B-Cell Lymphoma.

B-cell lymphomas (BCL) is the most frequent hematological cancer in dogs. Treatment typically consists of chemotherapy, with CHOP-based protocols. However, outcome remains generally poor, urging the exploration of new therapeutic strategies with a targeted approach. Myc transcription factor plays a crucial role in regulating cellular processes, and its dysregulation is implicated in numerous human and canine malignancies, including canine BCL (cBCL). This study aims to evaluate the efficacy of indirectly inhibiting Myc in cBCL using BI2536 and MZ1 compounds in two in vitro models (CLBL-1 and KLR-1201). Both BI2536 and MZ1, alone and combined, affected cell viability in a significant concentration- and time-dependent manner. Western Blot revealed an upregulation of PLK1 expression in both cell lines upon treatment with BI2536, in association with a reduction in c-Myc protein levels. Conversely, MZ1 led to a decrease in its primary target, BRD4, along with a reduction in c-Myc. Furthermore, BI2536, both alone and in combination with MZ1, induced larger transcriptomic changes in cells compared to MZ1 alone, primarily affecting MYC target genes and genes involved in cell cycle regulation. These data underscore the potential role of Myc as therapeutic target in cBCL, providing a novel approach to indirectly modulate this molecule.

Effects of Cannabidiol, ∆9-Tetrahydrocannabinol, and WIN 55-212-22 on the Viability of Canine and Human Non-Hodgkin Lymphoma Cell Lines.

In our previous study, we demonstrated the impact of overexpression of CB1 and CB2 cannabinoid receptors and the inhibitory effect of endocannabinoids (2-arachidonoylglycerol (2-AG) and Anandamide (AEA)) on canine (Canis lupus familiaris) and human (Homo sapiens) non-Hodgkin lymphoma (NHL) cell lines' viability compared to cells treated with a vehicle. The purpose of this study was to demonstrate the anti-cancer effects of the phytocannabinoids, cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), and the synthetic cannabinoid WIN 55-212-22 (WIN) in canine and human lymphoma cell lines and to compare their inhibitory effect to that of endocannabinoids. We used malignant canine B-cell lymphoma (BCL) (1771 and CLB-L1) and T-cell lymphoma (TCL) (CL-1) cell lines, and human BCL cell line (RAMOS). Our cell viability assay results demonstrated, compared to the controls, a biphasic effect (concentration range from 0.5 μM to 50 μM) with a significant reduction in cancer viability for both phytocannabinoids and the synthetic cannabinoid. However, the decrease in cell viability in the TCL CL-1 line was limited to CBD. The results of the biochemical analysis using the 1771 BCL cell line revealed a significant increase in markers of oxidative stress, inflammation, and apoptosis, and a decrease in markers of mitochondrial function in cells treated with the exogenous cannabinoids compared to the control. Based on the IC50 values, CBD was the most potent phytocannabinoid in reducing lymphoma cell viability in 1771, Ramos, and CL-1. Previously, we demonstrated the endocannabinoid AEA to be more potent than 2-AG. Our study suggests that future studies should use CBD and AEA for further cannabinoid testing as they might reduce tumor burden in malignant NHL of canines and humans.

Effect of proteasome inhibitors on canine lymphoma cell response to CHOP chemotherapy in vitro.

The standard treatment for canine lymphoma is the CHOP chemotherapy regimen. Proteasome inhibitors have been employed with CHOP for the treatment of human haematological malignancies but remain to be fully explored in canine lymphoma. We identified an association between poor response to CHOP chemotherapy and high mRNA expression levels of proteasomal subunits in a cohort of 15 canine lymphoma patients, and sought to determine the effect of proteasome inhibitors on the viability of a canine B-cell lymphoma cell line (CLBL-1). The aim of this study was to investigate whether proteasome inhibitors sensitize these cells to the CHOP agents doxorubicin, vincristine and cyclophosphamide (as 4-hydroxycyclophosphamide/4-HC). CLBL-1 cells were sensitive to proteasome inhibition by bortezomib and ixazomib. The IC50 of bortezomib was 15.1 nM and of ixazomib was 59.14 nM. Proteasome inhibitors plus doxorubicin had a synergistic effect on CLBL-1 viability; proteosome inhibitors plus vincristine showed different effects depending on the combination ratio, and there was an antagonistic effect with 4-HC. These results may have clinical utility, as proteasome inhibition could potentially be used with a synergizing CHOP compound to improve responsiveness to chemotherapy for canine lymphoma patients.

Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Hematological ratios and indices in canine large B-cell lymphoma.

Canine lymphoma is the most common hematopoietic cancer in dogs. Numerous studies have evaluated the prognostic value of hematological abnormalities and ratios in both humans and dogs with lymphoma. To compare hematological parameters and complete blood count ratios between a population of dogs affected by lymphoma and healthy dogs to identify potential prognostic markers for lymphoma. This retrospective case-control study compares hematological parameters and complete blood count ratios between a population of 114 dogs affected by multicentric large B-cell lymphoma (LBCL) and 60 healthy dogs. The study found several statistically significant differences between the hematological indices of LBCL dogs and healthy dogs, but no correlation between these parameters and the survival times of 78 dogs treated with chemotherapy Madison Wisconsin protocol. In addition, hematological alterations were evaluated such as anemia, leukocytosis, and thrombocytopenia. Hematological ratios have been suggested as potential prognostic markers for canine LBCL but their real prognostic value remains controversial and requires future investigation.

Abstract B170: The in vitro anti-tumor activity of the multi-kinase inhibitor nemtabrutinib (ARQ-531, MK-1026) is seen across multiple B-cell lymphoma subtypes, only partially overlapping with what achieved by single BTK inhibition

Abstract Background. Nemtabrutinib is a reversible, ATP competitive tyrosine kinase inhibitor of BTK and of several other kinases, including LYN and MEK1 (Reiff et al, Cancer Discov 2018). Nemtabrutinib has shown preclinical and clinical activity in chronic lymphocytic leukemia (CLL), also against tumors bearing mutations affecting the C481 amino acid of BTK (Reiff et al, Cancer Discov 2018; Eradat et al, ICML 2023), which is commonly mutated in patients treated with 1st and 2nd generation inhibitors such as ibrutinib, acalabrutinib, zanubrutinib (Thompson & Tam, Blood 2023). Based on these data, a phase 3 trial is comparing the drug against chemo-immunotherapy in untreated-CLL patients (NCT05624554). Phase 1/2 studies are currently exploring nemtabrutinib in various B-cell lymphomas as single-agent (NCT05673460, NCT04728893) and combined with the ROR1-targeting antibody drug conjugate zilovertamab vedotin (NCT05458297). Due to the ability of nemtabrutinib to block multiple kinases, we exposed a large series of lymphoma cell lines to nemtabrutinib to understand the lymphomas that might most benefit. Methods. Nemtabrutinib (MedChemExpress) was assessed for its anti-proliferative activity at 72h across 54 cell lines, comprising germinal-center B-cell like (GCB) DLBCL (n.=18), activated B-cell like (ABC) DLBCL (n.=8), mantle cell lymphoma (MCL) (n.=10), marginal zone lymphoma (MZL) (n.=6), CLL (n.=2), plus other two derived from B- and eight from T-cell (n.=8) lymphomas. Results. The median IC50 for nemtabrutinib was 1.4 µM (95%CI, 0.8-2.3 µM) across all 54 lymphoma cell lines tested. Higher activity was found in B (median-IC50, 1.1 µM) compared to T (median IC50, 22.6 µM) cell lymphomas. Strong anti-lymphoma activity was observed in CLL cell lines (median IC50, 389 nM), MCL (median IC50, 627 nM), in one primary mediastinal B cell lymphoma (815 nM) and one canine diffuse large B-cell lymphoma cell line (389 nM). No difference was seen between ABC (median IC50, 1 µM) and GCB (median IC50, 1.2 µM) DLBCL. In DLBCL, nemtabrutinib anti-tumor activity was not affected by the presence of inactive TP53, BCL2 and/or MYC translocations. Taking advantage of publicly available ibrutinib data, we observed that nemtabrutinib and the 1st generation BTK inhibitor behaved similarly across 45 lymphoma cell lines (Pearson correlation r=0.5, P=0.001), especially in MCL (r=0.8, P=0.005) and ABC-DLBCL (r=0.75, P=0.039), but not in GCB-DLBCL, in which only nemtabrutinib was active. Further analyses integrating baseline transcriptome and mutational data are underway. Conclusions. Nemtabrutinib showed anti-tumor activity across B-cell lymphomas, higher in some histotypes (including MCL, CLL). The pattern of anti-tumor activity was only partially overlapping by what achieved by the BTK inhibitor ibrutinib: while MCL and ABC-DLBCL responded similarly, activity in GCB-DLBCL was seen only with nemtabrutinib. Citation Format: Giulio Sartori, Filippo Spriano, Luciano Cascione, Chiara Tarantelli, Alberto J. Arribas, Luca Aresu, Davide Rossi, Giovanna Damia, Massimo Broggini, Francesco Bertoni. The in vitro anti-tumor activity of the multi-kinase inhibitor nemtabrutinib (ARQ-531, MK-1026) is seen across multiple B-cell lymphoma subtypes, only partially overlapping with what achieved by single BTK inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B170.

Canine diffuse large b-cell lymphoma downregulates the activity of CD8 + T-cells through tumor-derived extracellular vesicles

BackgroundTumor-derived extracellular vesicles (EVs) have been proposed as the essential mediator between host immunity and cancer development. These EVs conduct cellular communication to facilitate tumor growth, enable invasion and metastasis, and shape the favorable tumor microenvironment. Lymphoma is one of the most common hematological malignancies in humans and dogs. Effective T-cell responses are required for the control of these malignancies. However, the immune crosstalk between CD8 + T-cells, which dominates anti-tumor responses, and canine lymphoma has rarely been described.MethodsThis study investigates the immune manipulating effects of EVs, produced from the clinical cases and cell line of canine B cell lymphoma, on CD8 + T-cells isolated from canine donors.ResultsLymphoma-derived EVs lead to the apoptosis of CD8 + T-cells. Furthermore, EVs trigger the overexpression of CTLA-4 on CD8 + T-cells, which indicates that EV blockade could serve as a potential therapeutic strategy for lymphoma patients. Notably, EVs transform the CD8 + T-cells into regulatory phenotypes by upregulating their PD-1, PD-L1, and FoxP3 mRNA expression. The regulatory CD8 + T-cells secret the panel of inhibitory cytokines and angiogenic factors and thus create a pro-tumorigenic microenvironment.ConclusionIn summary, the current study demonstrated that the EVs derived from canine B cell lymphoma impaired the anti-tumor activity of CD8 + T-cells and manipulated the possible induction of regulatory CD8 + T-cells to fail the activation of host cellular immunity.

Panobinostat-loaded folate targeted liposomes as a promising drug delivery system for treatment of canine B-cell lymphoma.

Cancer is a major public health problem with over 19 million cases reported in 2020. Similarly to humans, dogs are also largely affected by cancer, with non-Hodgkin's lymphoma (NHL) among the most common cancers in both species. Comparative medicine has the potential to accelerate the development of new therapeutic options in oncology by leveraging commonalities between diseases affecting both humans and animals. Within this context, in the present study, we investigated the potential of panobinostat (Pan)-loaded folate-targeted PEGylated liposomes (FA-PEG-Pan-Lip) for the treatment of canine B-cell lymphoma, while contributing to new perspectives in comparative oncology. Two formulations were developed, namely: PEG-Pan-Lip and FA-PEG-Pan-Lip. Firstly, folate receptor expression in the CLBL-1 canine B-cell lymphoma cell line was assessed. After confirming receptor expression, both Pan-loaded formulations (PEG-Pan-Lip, FA-PEG-Pan-Lip) demonstrated dose-dependent inhibitory effects on CLBL-1 cell proliferation. The FA-PEG-Pan-Lip formulation (IC50 = 10.9 ± 0.03 nM) showed higher cytotoxicity than the non-targeted PEG-Pan-Lip formulation (IC50 = 12.9 ± 0.03 nM) and the free panobinostat (Pan) compound (IC50 = 18.32±0.03 nM). Moreover, mechanistically, both Pan-containing formulations induced acetylation of H3 histone and apoptosis. Flow cytometry and immunofluorescence analysis of intracellular uptake of rhodamine-labeled liposome formulations in CLBL-1 cells confirmed cellular internalization of PEG-Lip and FA-PEG-Lip formulations and higher uptake profile for the latter. Biodistribution studies of both radiolabeled formulations in CD1 and SCID mice revealed a rapid clearance from the major organs and a 1.6-fold enhancement of tumor uptake at 24 h for 111In-FA-PEG-Pan-Lip (2.2 ± 0.1 %ID/g of tumor) compared to 111In-PEG-Pan-Lip formulation (1.2±0.2 %ID/g of tumor). In summary, our results provide new data validating Pan-loaded folate liposomes as a promising targeted drug delivery system for the treatment of canine B-cell lymphoma and open innovative perspectives for comparative oncology.

MiRNome expression analysis in canine diffuse large B-cell lymphoma.

Lymphoma is a common canine cancer with translational relevance to human disease. Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype, contributing to almost fifty percent of clinically recognized lymphoma cases. Identifying new biomarkers capable of early diagnosis and monitoring DLBCL is crucial for enhancing remission rates. This research seeks to advance our knowledge of the molecular biology of DLBCL by analyzing the expression of microRNAs, which regulate gene expression by negatively impacting gene expression via targeted RNA degradation or translational repression. The stability and accessibility of microRNAs make them appropriate biomarkers for the diagnosis, prognosis, and monitoring of diseases. We extracted and sequenced microRNAs from ten fresh-frozen lymph node tissue samples (six DLBCL and four non-neoplastic). Small RNA sequencing data analysis revealed 35 differently expressed miRNAs (DEMs) compared to controls. RT-qPCR confirmed that 23/35 DEMs in DLBCL were significantly upregulated (n = 14) or downregulated (n = 9). Statistical significance was determined by comparing each miRNA's average expression fold-change (2-Cq) between the DLCBL and healthy groups by applying the unpaired parametric Welch's 2-sample t-test and false discovery rate (FDR). The predicted target genes of the DEMs were mainly enriched in the PI3K-Akt-MAPK pathway. Our data point to the potential value of miRNA signatures as diagnostic biomarkers and serve as a guideline for subsequent experimental studies to determine the targets and functions of these altered miRNAs in canine DLBCL.

Dissecting the immune landscape of canine diffuse large B-cell lymphoma by NanoString technology

Dissecting the immune landscape of canine diffuse large B-cell lymphoma by NanoString technology

Predicting outcome in dogs with diffuse large B-cell lymphoma with a novel immune landscape signature.

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.

Predictive value of topoisomerase IIα immunoreactivity in canine diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) treated with a doxorubicin-based chemotherapy protocol

Topoisomerase IIα (TOPIIα) plays a key role in DNA replication and is a molecular target for TOPIIα inhibitor chemotherapy. A doxorubicin-based chemotherapy remains the most effective treatment for canine lymphoma, but there are no clear indications for its use in individual patients. The aim of this study was to assess the predictive value of TOPIIα expression in the treatment of canine lymphomas with TOPIIα inhibitors. Samples of formalin-fixed paraffin-embedded lymph nodes from 14 dogs with diffuse large B-cell lymphoma (DLBCL) and 5 dogs with peripheral T-cell lymphoma (PTCL) treated with a CHOP protocol were immunohistochemically labelled with anti-TOPIIα. Progression-free survival time (PFS) and overall survival time (OS) were estimated and compared with TOPIIα expression. Dogs with DLBCL had a higher level of TOPIIα expression compared to dogs with PTCL. The median PFS for dogs with DLBCL was significantly longer (260 days) than it was for dogs with PTCL (60 days). In DLBCL dogs with a high TOPIIα expression level, the median PFS was significantly longer (310 days) then it was in dogs with week or medium TOPIIα expression (51 days). The results of the study indicate that the immunohistochemical assessment of TOPIIα expression may be a valuable predictive indicator for anthracycline-based treatment of dogs with malignant lymphoma.

Prognostic significance of CD25 expression in dogs with a noninvasive diagnosis of B-cell lymphoma treated with CHOP chemotherapy.

Prior studies have identified high CD25 expression in canine diffuse large B-cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B-cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B-cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196-348 days) and median PFS was 196 days (95% CI, 172-220 days). Higher percentage of B-cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p=.02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed.

P-Glycoprotein Activity at Diagnosis Does Not Predict Therapy Outcome and Survival in Canine B-Cell Lymphoma.

Simple SummaryClinical experience in human and canine clinics shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and eventually, most tumors become resistant to all available therapies. The most straightforward cause of therapy resistance is linked to cellular alterations that prevent drugs from acting on their target. Drug efflux mediated by the ABC transporter P-glycoprotein (P-gp) contributes to unfavorable treatment outcome in several human malignancies. Here, we characterize a large cohort of canine B-cell lymphoma patients followed for over 7 years. We show that the intrinsic P-gp activity of tumor cells characterized at the time of diagnosis is not predictive for therapy outcome. Our results highlight the complexity of clinical drug resistance mechanisms and suggests that the relevance of P-gp in acquired resistance should be further investigated by the continuous monitoring of tumor cells during treatment.Various mechanisms are known to be involved in the development of multidrug resistance during cancer treatment. P-glycoprotein (P-gp) decreases the intracellular concentrations of cytotoxic drugs by an energy-dependent efflux mechanism. The aim of this study was to investigate the predictive value of P-gp function based on the evaluation of P-gp activity in tumor cells obtained from canine B-cell lymphoma patients at diagnosis. P-gp function of 79 immunophenotyped canine lymphoma samples was determined by flow cytometry using the Calcein assay. Dogs were treated with either the CHOP or the L-CHOP protocol, a subset of relapsed patients received L-asparaginase and lomustine rescue treatments. Among the 79 dogs, the median overall survival time was 417 days, and the median relapse-free period was 301 days. 47 percent of the samples showed high P-gp activity, which was significantly higher in Stage IV cancer patients compared to Stage II + III and V. Whereas staging was associated with major differences in survival times, we found that the intrinsic P-gp activity of tumor cells measured at diagnosis is not predictive for therapy outcome. Further studies are needed to identify the intrinsic and acquired resistant mechanisms that shape therapy response and survival in B-cell canine lymphoma patients.

POT1 mutations are frequent and associated with Ki-67 index in canine diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents one of the most frequent and deadliest neoplasia in dogs worldwide and is characterized by a remarkable degree of clinical heterogeneity, with poor chances to anticipate the outcome. Even if in the last years some recurrently mutated genes have been identified, the genetic origin of canine DLBCL (cDLBCL) is not yet completely understood. The aim of the present study was to assess the prevalence of POT1 mutations in cDLBCL and to elucidate the role of such gene in the pathogenesis of this tumor. Mutations in POT1 were retrieved in 34% of cases, in line with previous reports, but no significant associations with any clinico-pathological variable were identified. Likewise, POT1 mutations are not predictive of worse prognosis. Interestingly, Ki-67 index was significantly higher in dogs harboring POT1 mutations compared to wild-type ones. These results suggest that POT1 mutations may exert their pathogenic role in cDLBCL by promoting cellular proliferation.

The genomic landscape of canine diffuse large B-cell lymphoma identifies distinct subtypes with clinical and therapeutic implications.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid neoplasm in dogs and in humans. It is characterized by a remarkable degree of clinical heterogeneity that is not completely elucidated by molecular data. This poses a major barrier to understanding the disease and its response to therapy, or when treating dogs with DLBCL within clinical trials. We performed an integrated analysis of exome (n = 77) and RNA sequencing (n = 43) data in a cohort of 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 (TRAF3, SETD2, POT1, TP53, MYC, FBXW7, DDX3X and TBL1XR1) differed. We developed a prognostic model integrating exonic variants and clinical and transcriptomic features to predict the 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.

IL-1R8 Downregulation and Concomitant TLR7 and TLR9 Upregulation Are Related to the Pathogenesis of Canine Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of the NF-κB pathway as a fundamental underlying pathogenetic mechanism. In humans, the downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 control lymph nodes obtained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analyzed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be involved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs compared to control lymph nodes (p < 0.001), while a higher expression of TLR7, TLR9, and MYC was observed in DLBCLs (p < 0.01). Immunohistochemistry confirmed the gene expression results, revealing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p < 0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through the dysregulation of the Toll-like/interleukin receptors signaling cascade and the aberrant activation of the classical NF-κB pathway.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.