Multicentric aggressive unclassified hematopoietic neoplasm involving the placenta in a pregnant bitch.

Hematopoietic neoplasms are common in dogs; however, their association with pregnancy has not been previously reported in veterinary medicine. This rare occurrence presents a variety of diagnostic, therapeutic, prognostic, and ethical challenges. We report a case of a 3-year-old pregnant Bernese Mountain Dog diagnosed with multicentric aggressive unclassified hematopoietic cancer associated with paraneoplastic hypercalcemia during pregnancy. The dog died 7 days after diagnosis, and at Day 36 of pregnancy before any treatment decision could be made. Post-mortem evaluation, including histology, immunohistochemistry, and clonality analysis, led to the diagnosis of an unclassified hematopoietic cancer affecting the uterus and placenta, with no evidence of fetal involvement. The placenta likely acted as a barrier, preventing neoplastic involvement of the fetuses. Alternatively, the pregnancy might have been too early for the hematopoietic neoplasm to affect the labyrinth zone of the placenta and the fetuses. The dramatic disease progression could be explained by compromised cell-mediated immunity during pregnancy. This immunodeficient state is induced by embryonic, maternal, and hormonal factors, which suppress the response to mitogens to prevent rejection of the placenta and the conceptuses. Thus, pregnant dogs might exhibit increased vulnerability to cancer and infectious diseases that rely on cell-mediated immunity for host defense.

PI3Kδ activation, IL6 overexpression, and CD37 loss cause resistance to naratuximab emtansine in lymphomas.

CD37-directed antibody and cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562, IMGN529) is an antibody-drug conjugate (ADC) incorporating an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload, with activity as a single agent and in combination with rituximab in lymphoma patients. We studied naratuximab emtansine and its free payload in 54 lymphoma models, correlated its activity with CD37 expression, characterized two resistance mechanisms, and identified combination partners providing synergy. The activity, primarily cytotoxic, was more potent in B- than T-cell lymphoma cell lines. After prolonged exposure to the ADC, one diffuse large B-cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the CD37 gene biallelic loss. After CD37 loss, we also observed upregulation of IL6 and related transcripts. Recombinant IL6 led to resistance. Anti-IL6 antibody tocilizumab improved the ADC's cytotoxic activity in CD37+ cells. In a second model, resistance was sustained by PIK3CD activating mutation, with increased sensitivity to PI3Kδ inhibition and a functional dependence switch from MCL1 to BCL2. Adding idelalisib or venetoclax overcame resistance in the resistant derivative and improved the cytotoxic activity in the parental cells. In conclusion, targeting B-cell lymphoma with the naratuximab emtansine showed vigorous anti-tumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as MYC translocations and TP53 inactivation or R-CHOP resistance. Resistant DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL6, PI3Kδ, and BCL2.

Predictive value of TP53 RNAscope®in situ hybridization and p53 immunohistochemistry for TP53 mutational status in canine diffuse large B-cell lymphoma.

TP53 mutations are associated with short survival and poor treatment response in canine diffuse large B-cell lymphoma (cDLBCL). The expression of TP53 by RNAscope® in situ hybridization and p53 by immunohistochemistry (IHC) was investigated in 37 formalin-fixed paraffin-embedded cDLBCL, to assess their correlation with TP53 mutational status and to evaluate their prognostic value. TP53 was detected in all samples by RNAscope®. Ten of 37 (27%) cases expressed p53 by IHC, with highly variable percentage of positive cells. TP53 RNAscope® scores and p53 IHC results were not correlated. The expression of TP53 by RNAscope® was not influenced by its mutational status. Conversely, p53 IHC and TP53 mutations were significantly associated. p53 IHC predicted TP53 genetic mutations with high accuracy (97.3%). All TP53-mutated samples carrying missense mutations exhibited p53 expression by IHC, while all wild-type cases and a single case with frameshift insertion were negative. In univariable analysis, p53 IHC was associated with shorter time to progression (TTP) and lymphoma-specific survival (LSS). Nevertheless, in multivariable analysis, only treatment significantly affected TTP and LSS. These findings suggest p53 IHC is an accurate, cost-effective tool for predicting TP53 mutations in cDLBCL, unlike TP53 RNAscope®, though its prognostic value requires further validation.

Comprehensive Analysis of Microsatellite Instability in Canine Cancers: Implications for Comparative Oncology and Personalized Veterinary Medicine.

Microsatellite instability (MSI) is a crucial feature in cancer biology, yet its prevalence and significance in canine cancers remain largely unexplored. This study conducted a comprehensive analysis of MSI across 10 distinct canine cancer histotypes using whole-exome sequencing data from 692 tumor-normal sample pairs. MSI was detected in 64% of tumors, with prevalence varying significantly among cancer types. B-cell lymphomas exhibited the highest MSI burden, contrasting with human studies. A novel "MSI-burden" score was developed, correlating significantly with tumor mutational burden. MSI-high (MSI-H) tumors showed elevated somatic mutation counts compared to MSI-low and microsatellite stable tumors. The study identified 3632 recurrent MSI-affected genomic regions across cancer types. Notably, seven of the ten cancer types exhibited MSI-H tumors, with prevalence ranging from 1.5% in melanomas to 37% in B-cell lymphomas. These findings highlight the potential importance of MSI in canine cancer biology and suggest opportunities for targeted therapies, particularly immunotherapies. The high prevalence of MSI in canine cancers, especially in B-cell lymphomas, warrants further investigation into its mechanistic role and potential as a biomarker for prognosis and treatment response.

Circulating nucleosomes as a potential cancer biomarker in dogs with splenic nodular lesions.

Splenic nodular lesions in dogs can be either benign or malignant. They might be discovered incidentally or, in case of rupture, they may lead to hemoabdomen. Nevertheless, splenectomy followed by histopathology is essential for diagnosis and to prevent rupture. Yet, this invasive procedure might be postponed for dogs with benign splenic nodular lesions. Conversely, owners may opt for euthanasia over surgery for malignancies with poor prognosis like hemangiosarcoma. Thus, anticipating diagnosis with non-invasive biomarkers is crucial for proper patient management. In this prospective study, plasma samples were collected from 66 dogs with histologically confirmed splenic nodular lesions. A canine-specific ELISA kit was applied to assess nucleosome concentration, with histopathology of the spleen serving as the gold standard. Nucleosome concentration was found to be significantly higher in dogs with malignant splenic nodular lesions, particularly in those with hemangiosarcoma and other malignancies. The presence of hemoabdomen, more prevalent in dogs with splenic malignancy, also resulted in increased plasmatic nucleosome concentrations. Plasma nucleosomes could serve as a biomarker for detecting malignant splenic nodular lesions in dogs. More research is needed to understand how nucleosome concentration relate to disease stage and prognosis in dogs with hemangiosarcoma.

Histologic, Phenotypic, and Molecular Characterization of Feline Hodgkin-Like Lymphoma With Classical Reed-Sternberg Cells.

Hodgkin-like lymphoma (HLL) is a rare neoplasm in cats that shares characteristics with the human disease. The hallmark of HLL is the presence of Reed-Sternberg (RS) cells expressing CD30 and CD20. This study aimed to elucidate the clinicopathologic features, immunophenotype and clonality patterns of feline HLL. A comprehensive retrospective review of clinicopathologic and molecular data of nodal lymphomas over a 6-year period was conducted in MyLav laboratory. Twenty-four cases were identified. All cats presented with submandibular or retropharyngeal lymphadenopathy. Histopathologic examination revealed a multifocal to diffuse proliferation of medium-to-large lymphoid cells with low mitotic activity, interspersed RS cells, and a heterogeneous inflammatory infiltrate comprising T-cells, plasma cells and neutrophils. In addition, extensive necrosis was a consistent finding. Immunohistochemistry showed a variable membranous CD20 and nuclear PAX5 expression in neoplastic cells, while RS cells displayed only mild to moderate CD20 positivity and were negative to PAX5. In 21/24 cases (87.5%), RS cells were diffusely CD30-positive. PARR analysis demonstrated clonal B-cell expansion in 60% of cases, with the remaining 40% exhibiting polyclonality. For the 10 cats with available follow-up, the prognosis was generally favourable, with only two cats succumbing to progressive disease. In conclusion, diagnosing feline HLL is challenging. The expression of CD30 and CD20 by RS cells should be considered a hallmark of the disease, but only after excluding differential diagnoses such as anaplastic B-cell lymphoma and granulomatous lymphadenopathy.

The K9 lymphoma assay allows a genetic subgrouping of canine lymphomas with improved risk classification.

We present here the K9 lymphoma assay, a novel 31-gene targeted next-generation sequencing panel designed for genomic profiling of canine lymphoid neoplasms. Addressing the growing demand for advanced diagnostics in veterinary oncology, this assay enables sensitive identification of known and actionable mutations specific to canine lymphomas, while evaluating its prognostic potential to facilitate diagnosis and prognosis. Our analysis, spanning several B- and T-cell lymphoma histotypes, unveiled distinct mutational landscapes distinguishing tumors derived from immature versus mature lymphocytes. Clustering analysis revealed a shared genetic origin between diffuse large B-cell lymphoma and marginal zone lymphoma, aligning with findings in human lymphomas, with TRAF3 emerging as the most frequently mutated gene across B-cell lymphoma subtypes. Significantly, TP53 mutations demonstrated universal adverse prognostic implications across B-cell lymphomas. Additionally, SETD2 mutations contributed to shorter time-to-progression, underscoring the role of epigenetic dysregulation in B-cell tumors. In T-cell lymphomas, SATB1 and FBXW7 were frequently mutated, warranting further investigation in larger cohorts. Our findings advocate for tailored therapeutic approaches based on the genetic profile, impacting treatment decisions and outcomes in canine lymphoma management. This study provides pivotal insights bridging veterinary and human oncology, paving the way for comprehensive genomic diagnostics and therapeutic strategies in comparative oncology.

Exploring the dynamics of Programmed Death-Ligand 1 in canine lymphoma: unraveling mRNA amount, surface membrane expression and plasmatic levels.

Introduction: Programmed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated. Methods: Samples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration. Results: Considering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21-2.03, range 1.01-3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07-1.32, range 1.02-2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03-1.46, range 1.02-6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas (p = 0.023). Discussion: PD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations.

Renal alterations in cats (Felis catus) housed in shelters and affected by systemic AA-amyloidosis: Clinicopathological data, histopathology, and ultrastructural features.

AA-amyloidosis is frequent in shelter cats, and chronic kidney disease is the foremost cause of death. The aims were to describe kidney laboratory and microscopic findings in shelter cats with AA-amyloidosis. Cats were included if kidney specimens were collected post-mortem and laboratory data were available within 6 months before death. Renal lesions were evaluated with optical and electron microscopy. Mass spectrometry was used to characterize amyloid. Nine domestic short-hair cats were included; 4 females and 5 males with a median age of 8 years (range = 2-13). All cats had blood analyses and urinalyses available. Serum creatinine concentrations were increased in 6 cats and symmetric dimethylarginine was increased in all of the cats. All of the cats had proteinuria. Eight of 9 cats had amyloid in the medulla, and 9 had amyloid in the cortex (glomeruli). All cats had amyloid in the interstitium. Six cats had concurrent interstitial nephritis and 1 had membranoproliferative glomerulonephritis. All cats had extrarenal amyloid deposits. Amyloid was AA in each case. In conclusion, renal deposition of amyloid occurs in both cortex and medulla in shelter cats and is associated with azotemia and proteinuria. Renal involvement of systemic AA-amyloidosis should be considered in shelter cats with chronic kidney disease. The cat represents a natural model of renal AA-amyloidosis.

The diagnostic relevance of mesenteric lymph node biopsy in small intestinal lymphoma in cats.

BACKGROUND: Regional lymph nodes are frequently sampled in cats with suspected intestinal lymphoma; however, their diagnostic value has not been explored. OBJECTIVES: To investigate whether histologic and immunohistochemical analysis of mesenteric lymph nodes correlates with the diagnosis of intestinal lymphoma in cats. ANIMALS: One hundred 2 client-owned cats diagnosed with intestinal lymphoma. METHODS: Retrospective study. The inclusion criteria required a full-thickness biopsy of the small intestine and concurrent excision of mesenteric lymph nodes. Histologic and immunophenotypic analyses were performed on intestinal biopsies and corresponding lymph nodes. Selected nodal samples diagnosed with reactive lymph nodes underwent clonality testing. RESULTS: Transmural T-cell lymphomas, encompassing small and large cell types, were predominant (64 cases, 62.7%), with large B-cell lymphomas being more frequently transmural (68.8%) than mucosal (31.2%). Among all lymph nodes examined, 44 (43.1%; 95% CI: 33.9%-52.8%) exhibited neoplastic infiltration. Among cases of small cell lymphoma, 51 out of 72 (70.8%; 95% CI: 59.4%-80.1%) showed no nodal involvement. Clonality results correctly identified 19/30 (63.3%; 95% CI: 45.5%-78.2%) reactive lymph nodes. Concerns were raised regarding clonal identification in the remaining cases and potential misdiagnoses based on phenotypic characteristics. CONCLUSION AND CLINICAL IMPORTANCE: The study underscores the potential drawbacks of relying solely on mesenteric lymph nodes for diagnosing intestinal lymphomas in cats, particularly small cell subtypes. It emphasizes the importance of full-thickness biopsies for assessing transmural infiltration and recommends caution when utilizing mesenteric lymph nodes for histologic, immunohistochemical and clonality evaluations in mucosal lymphomas. Despite limitations, this research highlights the need for comprehensive diagnostic strategies in cats with intestinal lymphoma.

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.

Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

PI3Kδ activation, IL6 over-expression, and CD37 loss cause resistance to the targeting of CD37-positive lymphomas with the antibody-drug conjugate naratuximab emtansine.

Purpose: The transmembrane protein CD37 is expressed almost exclusively in lymphoid tissues, with the highest abundance in mature B cells. CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562, IMGN529) is an antibodydrug conjugate (ADC) that incorporates an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload. Naratuximab emtansine has shown activity as a single agent and in combination with the anti-CD20 monoclonal antibody rituximab in B cell lymphoma patients. Experimental Design: We assessed the activity of naratuximab emtansine using in vitro models of lymphomas, correlated its activity with CD37 expression levels, characterized two resistance mechanisms to the ADC, and identified combination partners providing synergy. Results: The anti-tumor activity of naratuximab emtansine was tested in 54 lymphoma cell lines alongside its free payload. The median IC 50 of naratuximab emtansine was 780 pM, and the activity, primarily cytotoxic, was more potent in B than in T cell lymphoma cell lines. In the subgroup of cell lines derived from B cell lymphoma, there was some correlation between sensitivity to DM1 and sensitivity to naratuximab emtansine (r=0.28, P = 0.06). After prolonged exposure to the ADC, one diffuse large B cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the biallelic loss of the CD37 gene. After CD37 loss, we also observed upregulation of IL6 (IL-6) and other transcripts from MYD88/IL6-signaling. Recombinant IL6 led to resistance to naratuximab emtansine, while the anti-IL6 antibody tocilizumab improved the cytotoxic activity of the ADC in CD37-positive cells. In a second model, resistance was sustained by an activating mutation in the PIK3CD gene, associated with increased sensitivity to PI3K δ inhibition and a switch from functional dependence on the anti-apoptotic protein MCL1 to reliance on BCL2. The addition of idelalisib or venetoclax to naratuximab emtansine overcame resistance to the ADC in the resistant derivative while also improving the cytotoxic activity of the ADC in the parental cells. Conclusions: Targeting B cell lymphoma with the CD37 targeting ADC naratuximab emtansine showed vigorous anti-tumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as MYC translocations and TP53 inactivation or resistance to R-CHOP. Resistance DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL6, PI3K δ , and BCL2. Despite notable progress in recent decades, we still face challenges in achieving a cure for a substantial number of lymphoma patients (1,2). A pertinent example is diffuse large B cell lymphoma (DLBCL), the most prevalent type of lymphoma (3). More than half of DLBCL patients can achieve remission, but around 40% of them experience refractory disease or relapse following an initial positive response (3). Regrettably, the prognosis for many of these cases remains unsatisfactory despite introducing the most recent antibody-based or cellular therapies (3,4), underscoring the importance of innovating new therapeutic strategies and gaining insights into the mechanisms of therapy resistance. CD37 is a transmembrane glycoprotein belonging to the tetraspanin family, primarily expressed on the surface of immune cells, principally in mature B cells but also, at lower levels, in T cells, macrophages/monocytes, granulocytes and dendritic cells (5) (6-8). CD37 plays a crucial role in various immune functions, including B cell activation, proliferation, and signaling, although its precise role still needs to be fully elucidated. CD37 interacts with multiple molecules, including SYK, LYN, CD19, CD22, PI3K δ , PI3K γ , and different integrins, among others (6-8). In mice, the lack of CD37 is paired with reduced T cell-dependent antibody-secreting cells and memory B cells, apparently due to the loss of CD37-mediated clustering of α 4 ß 1 integrins (VLA-4) on germinal center B cells and decreased downstream activation of PI3K/AKT signaling and cell survival (5). Reflecting the expression pattern observed in normal lymphocytes, CD37 exhibits elevated expression in all mature B-cell lymphoid neoplasms, including most lymphoma subtypes, and absence in early progenitor cells or terminally differentiated plasma cells (6,8-14). In DLBCL, CD37 expression has been reported between 40% and 90% of cases across multiple studies performed using different antibodies (10,14-16). CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical (7,10-14,17-23) and early promising clinical activity (24-32). Among the CD37-targeting agents, naratuximab emtansine (Debio 1562, IMGN529) is an antibody-drug conjugate (ADC) that incorporates the anti-CD37 humanized IgG1 monoclonal antibody K7153A conjugated to the maytansinoid DM1, as payload, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (10). Based on the initial in vitro and in vivo evidence of anti-tumor activity in lymphoma and chronic lymphocytic leukemia (CLL) (7,10), naratuximab emtansine entered the clinical evaluation as a single agent. The phase 1 study exploring naratuximab emtansine enrolled 39 patients with relapsed/refractory B cell lymphoma (27). The overall response rate (ORR) was 13% across all patients and 22% in DLBCL patients, including the only observed complete remission (CR) (27). In preliminary results of a phase 2 trial exploring the combination of naratuximab emtansine with the anti-CD20 monoclonal antibody rituximab (18), based on positive preclinical data (18), the ORR was 45% in 76 patients with DLBCL with 24 CRs (32%), 57% in 14 patients with follicular lymphoma (five CR), 50% in four MCL patients (2 CR) (31). Here, we studied the pattern of activity of naratuximab emtansine across a large panel of cell lines derived from DLBCL and other lymphoma subtypes and characterized two resistance mechanisms to the ADC.

The microtubule-targeted agent lisavanbulin (BAL101553) shows anti-tumor activity in lymphoma models.

Microtubules are major components of the cellular cytoskeleton, ubiquitously founded in all eukaryotic cells. They are involved in mitosis, cell motility, intracellular protein and organelle transport, and maintenance of cytoskeletal shape. Avanbulin (BAL27862) is a microtubule-targeted agent (MTA) that promotes tumor cell death by destabilization of microtubules. Due to its unique binding to the colchicine site of tubulin, differently from other MTAs, avanbulin has previously shown activity in solid tumor cell lines. Its prodrug, lisavanbulin (BAL101553), has shown early signs of clinical activity, especially in tumors with high EB1 expression. Here, we assessed the preclinical anti-tumor activity of avanbulin in diffuse large B cell lymphoma (DLBCL) and the pattern of expression of EB1 in DLBCL cell lines and clinical specimens. Avanbulin showed a potent in vitro anti-lymphoma activity, which was mainly cytotoxic with potent and rapid apoptosis induction. Median IC50 was around 10 nM in both ABC and GCB-DLBCL. Half of the cell lines tested showed an induction of apoptosis already in the first 24 h of treatment, the other half in the first 48 h. EB1 showed expression in DLBCL clinical specimens, opening the possibility for a cohort of patients that could potentially benefit from treatment with lisavanbulin. These data show the basis for further preclinical and clinical evaluation of lisavanbulin in the lymphoma field.

Subcutaneous mast cell tumours: A prospective multi-institutional clinicopathological and prognostic study of 43 dogs.

BACKGROUND: Canine subcutaneous mast cell tumours (ScMCTs) reportedly have a good prognosis. However, biomarkers that can be used to predict outcome are currently limited. METHODS: A multicentre prospective study was conducted to identify new prognostic markers. Dogs with a first occurrence of ScMCT were enrolled upon primary tumour removal and regional lymphadenectomy. In the absence of metastasis, dogs were monitored, while dogs with overtly metastatic lymph nodes (histological node 3, HN3) received adjuvant vinblastine. RESULTS: Forty-three dogs were enrolled: 15 (34.9%) had at least one HN3 lymph node and received vinblastine, and 28 (65.1%) were monitored. Three tumours harboured exon 8 and 9 c-kit mutations. Eight (18.6%) dogs experienced tumour progression, and five (11.6%) died of MCT-related causes. The 1- and 2-year survival rates were 90% and 77%, respectively. Variables significantly associated with an increased risk of progression included high cytograde, a mitotic count (MC) greater than 4/10 high-power fields (hpf) and Ki67-index greater than 23. An MC greater than 4/10 hpf was also associated with an increased risk of tumour-related death. LIMITATIONS: Regional rather than sentinel lymphadenectomy was performed in these dogs. Dogs were enrolled in oncology referral centres, constituting a different population compared to previous studies. CONCLUSIONS: ScMCTs have a good prognosis. However, the metastatic rate at admission was higher in this study than previously reported, and a subset of tumours were associated with a fatal outcome despite multimodal treatment. Proliferative activity and cytograding may predict more aggressive behaviour in ScMCTs.

Bridging clinic and wildlife care with AI-powered pan-species computational pathology.

Cancers occur across species. Understanding what is consistent and varies across species can provide new insights into cancer initiation and evolution, with significant implications for animal welfare and wildlife conservation. We build a pan-species cancer digital pathology atlas (panspecies.ai) and conduct a pan-species study of computational comparative pathology using a supervised convolutional neural network algorithm trained on human samples. The artificial intelligence algorithm achieves high accuracy in measuring immune response through single-cell classification for two transmissible cancers (canine transmissible venereal tumour, 0.94; Tasmanian devil facial tumour disease, 0.88). In 18 other vertebrate species (mammalia = 11, reptilia = 4, aves = 2, and amphibia = 1), accuracy (range 0.57-0.94) is influenced by cell morphological similarity preserved across different taxonomic groups, tumour sites, and variations in the immune compartment. Furthermore, a spatial immune score based on artificial intelligence and spatial statistics is associated with prognosis in canine melanoma and prostate tumours. A metric, named morphospace overlap, is developed to guide veterinary pathologists towards rational deployment of this technology on new samples. This study provides the foundation and guidelines for transferring artificial intelligence technologies to veterinary pathology based on understanding of morphological conservation, which could vastly accelerate developments in veterinary medicine and comparative oncology.

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.

Development of Monoclonal Antibodies Targeting Canine PD-L1 and PD-1 and Their Clinical Relevance in Canine Apocrine Gland Anal Sac Adenocarcinoma.

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.

The mitotic regulator polo-like kinase 1 as a potential therapeutic target for c-Myc-overexpressing canine osteosarcomas.

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.