Canine Diffuse Large B-cell Lymphoma Research Articles

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.

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.

Baseline tumor gene expression signatures correlate with chemoimmunotherapy treatment responsiveness in canine B cell lymphoma.

Pet dogs develop spontaneous diffuse large B cell lymphoma (DLBCL), and veterinary clinical trials have been employed to treat canine DLBCL and to inform clinical trials for their human companions. A challenge that remains is selection of treatment to improve outcomes. The dogs in this study were part of a larger clinical trial evaluating the use of combinations of doxorubicin chemotherapy, anti-CD20 monoclonal antibody, and one of three small molecule inhibitors: KPT-9274, TAK-981, or RV1001. We hypothesized that significant differential expression of genes (DEGs) in the tumors at baseline could help predict which dogs would respond better to each treatment based on the molecular pathways targeted by each drug. To this end, we evaluated gene expression in lymph node aspirates from 18 trial dogs using the NanoString nCounter Canine Immuno-oncology (IO) Panel. We defined good responders as those who relapsed after 90 days, and poor responders as those who relapsed prior to 90 days. We analyzed all dogs at baseline and compared poor responders to good responders, and found increased CCND3 correlated with poor prognosis and increased CD36 correlated with good prognosis, as is observed in humans. There was minimal DEG overlap between treatment arms, prompting separate analyses for each treatment cohort. Increased CREBBP and CDKN1A for KPT-9274, increased TLR3 for TAK-981, and increased PI3Kδ, AKT3, and PTEN, and decreased NRAS for RV1001 were associated with better prognoses. Trends for selected candidate biomarker genes were confirmed via qPCR. Our findings emphasize the heterogeneity in DLBCL, similarities and differences between canine and human DLBCL, and ultimately identify biomarkers that may help guide the choice of chemoimmunotherapy treatment in dogs.

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.

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.

Evaluation of 41 single nucleotide polymorphisms in canine diffuse large B-cell lymphomas using MassARRAY

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma in dogs with a multicentric form. This study aimed to assemble 41 variants of the previously reported genes and to investigate these variants in canine DLBCL using the Agena MassARRAY platform. These variants were chosen based on the high prevalence observed in canine B- and T-cell lymphomas, their significance for target therapy, and compatibility for multiplex PCR amplification. Lymph node biopsy was performed from 60 dogs with B-cell lymphoma comprising 47 purebred and 13 crossbred dogs. All dogs presented single nucleotide polymorphisms (SNPs) at HYAL4 and SATB1 genes. The lesser mutual SNPs were observed at SEL1L, excluding a cocker spaniel, and c-Kit, with the exception of a pug and a French bulldog. Even though no statistical association was noted between each SNP and dog breed, purebreds were 3.88 times more likely to have a SNP at FLT3 rs852342480 (95%CI 0.50–45.03, p = 0.26), 3.64 times at TRAF3 F306X (95%CI 0.58–42.50, p = 0.43) and 2.66 times at TRAF3 E303EX (95%CI 0.56–13.12, p = 0.31). Also, DLBCL dogs (CHOP-based treatment) with c-Kit T425= had a poorer prognosis with shorter median overall survival times (OST) than dogs with the wild type. Dogs treated with COP chemotherapy and contained 3–5 variants at SEL1L were associated with decreased median OST. Therefore, this SNP’s lymphoma panel provides valuable information that we can use to outline a prognosis and develop a treatment plan for the targeted therapy of each dog.

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells.

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

Prognostic Value of PD-L1, PD-1 and CD8A in Canine Diffuse Large B-Cell Lymphoma Detected by RNAscope.

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.

Fascin Inhibition As a Novel Therapeeutic Strategy in Diffuse Large B-Cell Lymphoma

Fascin is an actin bundling protein used to form cellular projections (e.g. filipodia) involved in cell motility and invasion. Cancer cells often upregulate fascin, and in many cancer types this is associated with poor prognosis, likely due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block breast cancer metastasis in mice. Here we explored the potential role of fascin inhibition in lymphoma. We have shown that fascin is upregulated in 44% of canine B-cell (n=62) and 57% of T-cell lymphoma cases (n=28), and that increased fascin expression is correlated with worse overall survival (p=0.03). B-cell lymphoma cases had a median survival time of 388 days while that of T-cell cases was 263 days. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine diffuse large B-cell lymphoma (DLBCL) cells in vitro, and in vivo in mice bearing canine DLBCL xenografts, with GI50s ranging from 5 to 200uM. Animal toxicology studies (performed by Charles River Laboratories) show no hematologic or other toxicity in dogs and rats at concentrations up to 1,000 mg/kg/day for 28 days; these doses are much higher than the doses needed to achieve concentrations that kill lymphoma in vivo . Therefore, we are planning clinical studies in pet dogs with lymphoma to establish proof of principle prior to a human clinical trial. Collectively, our results demonstrate that fascin is a potential new therapeutic target in DLBCL. DisclosuresHuang:Novita: Equity Ownership, Patents & Royalties: Cornell University and Novita Pharmaceutical has patient on the fascin described here. .

Genome-wide DNA methylation and RNA-seq analyses identify genes and pathways associated with doxorubicin resistance in a canine diffuse large B-cell lymphoma cell line.

Doxorubicin resistance is a major challenge in the successful treatment of canine diffuse large B-cell lymphoma (cDLBCL). In the present study, MethylCap-seq and RNA-seq were performed to characterize the genome-wide DNA methylation and differential gene expression patterns respectively in CLBL-1 8.0, a doxorubicin-resistant cDLBCL cell line, and in CLBL-1 as control, to investigate the underlying mechanisms of doxorubicin resistance in cDLBCL. A total of 20289 hypermethylated differentially methylated regions (DMRs) were detected. Among these, 1339 hypermethylated DMRs were in promoter regions, of which 24 genes showed an inverse correlation between methylation and gene expression. These 24 genes were involved in cell migration, according to gene ontology (GO) analysis. Also, 12855 hypermethylated DMRs were in gene-body regions. Among these, 353 genes showed a positive correlation between methylation and gene expression. Functional analysis of these 353 genes highlighted that TGF-β and lysosome-mediated signal pathways are significantly associated with the drug resistance of CLBL-1. The tumorigenic role of TGF-β signaling pathway in CLBL-1 8.0 was further validated by treating the cells with a TGF-β inhibitor(s) to show the increased chemo-sensitivity and intracellular doxorubicin accumulation, as well as decreased p-glycoprotein expression. In summary, the present study performed an integrative analysis of DNA methylation and gene expression in CLBL-1 8.0 and CLBL-1. The candidate genes and pathways identified in this study hold potential promise for overcoming doxorubicin resistance in cDLBCL.

Peripheral blood cell ratios as prognostic factors in canine diffuse large B-cell lymphoma treated with CHOP protocol.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic tumour in dogs and recognized as clinical model for its human counterpart. Recently, neutrophil-to-lymphocyte (NLR) and lymphocyte-to-monocyte (LMR) ratios have been shown to predict time-to-progression (TTP) and lymphoma-specific survival (LSS) in dogs with DLBCL treated with CHOP-based chemotherapy. We retrospectively evaluated in 59 dogs diagnosed with DLBCL the prognostic value of haematological parameters and derived ratios: NLR, LMR, platelet-to-lymphocyte (PLR) and platelet-to-neutrophil (PNR) ratios for TTP, LSS and associated secondary end-points (time-to-progression-rate [TTPR] and lymphoma-specific survival-rate [LSSR]) as rates at 180 and 365 days. PNR is an independent prognostic marker (p ≤ .001) for TTPR/180 and 365 days, dogs with a PNR above 0.032 were more likely to progress before 180 days (sensitivity 46.5%, specificity 87.5%, p=.004). On univariate analysis, NLR showed a prognostic significance for LSSR/180 (p=.006) and LSSR/365 (p=.009). A baseline NLR value below 7.45 was positively associated with survival at 180 days (sensitivity 52%, specificity 85.3%, p=.025). The presence of substage b, was associated with early progression and decreased survival at 180 days (p=.031). Anaemia significantly reduced LSSR at 365 days (p=.028). This is the first study evaluating PLR and PNR in canine DLBCL and demonstrates that PNR could be a predictor of early lymphoma progression. Since peripheral blood cell composition can be affected by several non-oncological causes, the development of larger multicenter studies with homogeneous inclusion criteria could help to better determine the true predictive values of blood cell ratios in dogs' DLBCL treated with CHOP chemotherapy.

Are B-symptoms more reliable prognostic indicators than substage in canine nodal diffuse large B-cell lymphoma.

In humans B‐symptoms refer to systemic symptoms of lymphoma such as fever, weight loss, and night sweats and influence the prognosis of patients. In canine lymphoma, substage B is used to describe any clinical sign observed. Aim of the retrospective study was to compare the prognostic value of substage B with B‐symptoms to predict treatment response and survival in canine nodal diffuse large B‐cell lymphoma. Affected dogs treated with CHOP chemotherapy between 2008 and 2019 were included. B‐symptoms were defined by weight loss greater than 10% of normal weight, fever and the occurrence of unexplained resting tachypnoea substituted human night sweats. Substage B was defined as any symptoms but lymphadenopathy. Fifty‐five cases were included. B‐symptoms were present in 20/55 (36%) and substage B in 40/55 (74%) patients. No significant associations between B‐symptoms or substage B and weight, sex, breed, WHO stage and lymphoma grade were found. Treatment response was negatively associated with both substage B (P = .02) and B‐symptoms (P = .001). B‐symptoms significantly decreased progression free survival (PFS) (95 vs 330 days, P = .001) and lymphoma specific survival (LSS) (160 vs 462 days, P = .001). Data showed that B‐symptoms might be a more reliable prognostic indicator than substage B in canine nodal diffuse large B‐cell lymphoma. Prospective studies assessing B‐symptoms in a larger cohort of patients and in other common lymphoma types are warranted. The abstract was presented at the fourth meeting of the European Canine Lymphoma Network Group in Lugano, 22 June 2019 and published in the proceeding of the meeting on the page 26.

Abstract IA46: Molecular and genetic profiling in canine lymphoma unravels targets for the human counterpart

Abstract Canine lymphoma represents an excellent cancer model for the human counterpart and is the most widely investigated tumor both in veterinary and comparative oncology. Knowledge of the diagnosis, molecular biology, genetic, and epigenetic has progressively grown in recent years, but prognosis in both B- and T-cell lymphoma remains poor. Several lymphoma subtypes are recognized in canine species and diffuse large B-cell lymphoma (DLBCL) is the most frequent, followed by marginal zone lymphoma (MZL) and a peculiar T-cell malignancy named T-zone lymphoma. DLBCL resembles in many elements the human counterpart, including frequency, molecular abnormalities, and similar therapeutic responses to chemotherapy. However, many other aspects are still scarcely known, and a complete characterization becomes fundamental when including dogs with this lymphoma in clinical trials for new molecules. Indeed, the inability to accurately predict outcome poses several limitations when testing efficacy of new drugs. With NGS advent and the dramatic reduction of costs, researchers in veterinary medicine have started to describe canine lymphoma in a similar fashion to rodent models. The aims of this presentation are to provide an overview of the most significant results in canine lymphoma that have been published in the last decade, focusing on molecular and therapeutic aspects of this tumor. About B-cell lymphoma, transcriptomic studies in canine DLBCL, MZL, and follicular lymphoma (FL) have revealed similarities with the human histotypes. Genes involved in B-cell receptor signaling were found to be enriched both in canine DLBCL and MZL, and several Toll-like receptors were overexpressed, suggesting mechanisms associated with human activated B-cell (ABC)-like subtype DLBCL pathogenesis. The aberrant expression of these genes seems to contribute to enhancing proliferation and protection of canine B cells from apoptosis by stimulating NF-κB activity. The biologic role of NF-kB was tested in dogs with DLBCL using NEMO-binding domain peptide in two clinical trials and the compound resulted in safe blocking of NF-kB activity. Previously, treatment with Ibrutinib was found effective in dogs with B-cell lymphoma. Also, genes coding for proteins involved in MYC signaling and PI3K/AKT/mTOR pathway were found to be overexpressed in both DLBCL and MZL. A peculiar MYC deregulation controlled by LIN28B/Let-7 axis was identified only in DLBCL and LIN28B resulted the most upregulated gene both in vivo and in vitro, revealing an important functional relevance and opening new scenarios for treatment of this tumor in dogs. Within this context, the pan-BET inhibitor OTX015, targeting BRD4, was able to reduce proliferation and expression of MYC-associated genes in vitro. When investigating the cell of origin of canine DLBCL, a study from our group showed a segregation of this lymphoma in two transcriptomic subgroups that were further characterized by distinct methylation profiles and associated with survival. The dogs with inferior survival showed a higher expression of transcripts involved in T-cell and macrophage regulation (CD163, CD96, PD-1, PDL-1, CTLA4, CD8a, CD4). Recently, a study explored the mutation profile of canine B- and T-cell lymphoma in three pure breeds. B-cell lymphomas were categorized based on their mutation profile, and TRAF3, BIRC3, and MAP3K14 were found to be frequently mutated. For TRAF3, a combination of somatic frameshift mutations and truncating SNVs in causing down-expression was identified and germ-line mutations were retrieved in some dogs, possibly indicating that inherited mutations in this gene contribute to genetic predisposition to the tumor. In future, we expect an increasing number of preclinical trials including data from dogs with different lymphoma histotypes, but a detailed pathologic and molecular classification of the tumors is definitively needed to avoid biases related to the heterogeneity of this disease. Inclusion of dogs with lymphoma as part of preclinical testing or in parallel with human trials will be a serious advantage both for human and veterinary medicine. However, limitations, including that dogs are from private owners and toxicities, should always be considered. Citation Format: Luca Aresu. Molecular and genetic profiling in canine lymphoma unravels targets for the human counterpart [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA46.