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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., Competing Interests: Author NR was employed by Hastim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fanelli, Marconato, Licenziato, Minoli, Rouquet and Aresu.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011., Methods: To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety., Results: Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P <  0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P <  0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P <  0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events., Conclusions: Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.

Purpose: Active immunotherapy is a promising antitumoral strategy; however its use in combination with chemotherapy in dogs with large B-cell lymphoma (DLBCL) remains largely untested. Heat shock proteins (HSP) bind the small peptides they chaperone (HSPPC), allowing for immunization of the host against a large repertoire of tumor-associated antigens. Hydroxylapatite vehicles HSPPCs and acts as an immunologic adjuvant. The aim of this study was to show that an autologous vaccine with hydroxylapatite and tumor-derived HSPPCs is safe and therapeutically effective in dogs with DLBCL., Experimental Design: Nineteen dogs with naturally occurring DLBCL were entered into a prospective randomized placebo-controlled double-blinded trial of HSPPCs-hydroxylapatite plus chemotherapy versus chemotherapy alone. Endpoints included time to progression (TTP), lymphoma-specific survival (LSS), and incidence of toxicoses., Results: Median first TTP after randomization to the vaccine arm was 304 days versus 41 days for the control arm (P = 0.0004). There was also a statistically significant difference in duration of second remission between the two groups (P = 0.02). Median LSS was 505 days for the vaccinated dogs versus 159 days for the unvaccinated dogs (P = 0.0018). Six vaccinated dogs achieved molecular remission, as shown by clonal immunoglobulin H (IgH) rearrangement. Toxicoses were comparable between the two treatment arms., Conclusions: The results of this trial demonstrate that the autologous vaccine tested here is safe and efficacious in prolonging TTP and LSS in dogs with DLBCL when used in combination with dose-intense chemotherapy. On the basis of these results, additional evaluation of this novel therapeutic strategy is warranted in human DLBCL., (©2013 AACR.)

Experimental models of sepsis using endotoxin challenges, including studies with sensitized animals with D-galactosamine, have largely contributed to the basic rationale for innovative clinical trials in human septic shock, which have, to date, failed. The ability of these models to reproduce human disease has been highly discussed. We report here that the widely used D-galactosamine/LPS model does not account for septic shock. Treatment with YVAD-CMK, a potent tetrapeptide inhibitor of caspases of the interleukin (IL)-1beta converting enzyme (ICE) family, protects from LPS-induced liver apoptosis and mortality in D-galactosamine-sensitized mice when administered either before or up to 2 h after the lethal challenge. This curative effect is related to complete inhibition of caspase-3 activity in the liver. However, YVAD-CMK does not affect LPS-induced release of IL-1beta and does not protect from a lethal dose of LPS in unsensitized mice. These experiments demonstrate the difference between these two widely recognized experimental models of sepsis. LPS toxicity in D-galactosamine-treated mice, leading to blocked gene transcription, results from tumor necrosis factor (TNF)-alpha-induced caspase-3-dependent liver injury, not from the systemic inflammatory response. These results provide evidence that inhibitors of the ICE caspase family can prevent or even overcome the ongoing hepatic injury induced by TNF-alpha during sepsis, ischemia-reperfusion, or severe hepatitis.

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.