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Abstract Background Polymerase chain reaction for antigen receptor rearrangement (PARR) is a molecular diagnostic tool used for discrimination of lymphoid malignancies in dogs from benign processes. Assay variations have been described and are commercially available, but performance metrics are not uniformly reported. Objectives To describe performance (accuracy, sensitivity, specificity) and rigorous benchmarking of a PARR protocol (ePARR) in clinically relevant samples. Animals One hundred eighty-one client-owned dogs. Methods Lymphoma and benign tissues representative of the clinical spectrum with gold standard histopathologic and immunohistochemical diagnoses were collected. Assay development and benchmarking were performed on fresh frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tissue, flow cytometry pellets, and air-dried fine-needle aspirates (FNA). Assay performance was determined for FFPE from 56 dogs (18 B-cell lymphoma, 24 T-cell lymphoma, and 14 non-lymphoma), 80 frozen flow cytometry pellets (66 B-cell lymphoma, 14 T-cell lymphoma, 0 non-lymphoma), and 41 air-dried FNA slides (23 lymphoma, 18 non-lymphoma). Results For discrimination of lymphoma versus non-lymphoma, ePARR had 92% and 92% sensitivity and specificity on FFPE with 92% accuracy, 85% sensitivity from flow cytometry pellets (non-lymphoma was not evaluated to calculate specificity) with 85% accuracy, and 100% and 100% sensitivity and specificity for FNA with 100% accuracy. Stringent quality control criteria decreased assay success rate without significant performance improvement. Performance metrics were lower in most cases for discrimination of B- or T-cell versus non-B- or non-T-cell samples than for lymphoma versus non-lymphoma. Conclusions and Clinical Importance These benchmarking data facilitate effective interpretation and application of PARR assays in multiple sample types.

Abstract Background Cytopathology is a minimally invasive and convenient diagnostic procedure, often used as a substitute for histopathology to diagnose and characterize lymphoma in dogs. Objectives Assess the diagnostic performance of cytopathology in diagnosing lymphoma and its histopathological subtypes in dogs. Animals One-hundred and sixty-one lymph node samples from 139 dogs with enlarged peripheral lymph nodes. Methods Based only on cytopathology, 6 examiners independently provided the following interpretations on each sample: (a) lymphoma vs nonlymphoma; (b) grade and phenotype; and (c) World Health Organization (WHO) histopathological subtype. Histopathology and immunohistochemistry (IHC) findings were used as reference standards to evaluate diagnostic performance of cytopathology. Clinical, clinicopathologic, and imaging data also were considered in the definitive diagnosis. Results Classification accuracy for lymphoma consistently was >80% for all examiners, whereas it was >60% for low grade T-cell lymphomas, >30% for high grade B-cell lymphomas, >20% for high grade T-cell lymphomas, and <40% for low grade B-cell lymphomas. Interobserver agreement evaluated by kappa scores was 0.55 and 0.32 for identification of lymphoma cases, and of grade plus immunophenotype, respectively. Conclusions and Clinical Importance Cytopathology may result in accurate diagnosis of lymphoma, but accuracy decreases when further characterization is needed. Cytopathology represents a fundamental aid in identifying lymphoma and can be used as a screening test to predict grade and phenotype. However, these results must be confirmed using other ancillary techniques, including flow cytometry, histopathology, and immunohistochemistry (IHC).

Abstract 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.

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.

Background Liquid biopsies have gained increasing recognition as minimally invasive ways to monitor treatment response in cancer patients. They carry circulating DNA, RNA, and proteins in and outside of extracellular vesicles (EVs). We have demonstrated that Cell Division Cycle 6 (CDC6), which is involved in controlling cell proliferation, was detected in circulating EVs and increased in the plasma of canine cancer patients compared to healthy control dogs. Here, we investigated whether plasma CDC6 mRNA levels can be used to monitor treatment response in dogs diagnosed with non-Hodgkin lymphoma, and we aimed to determine whether plasma expression is related to the systemic release of EVs. Methods We performed longitudinal proof-of-concept studies and collected platelet-free plasma (PFP) samples of 16 canine lymphoma patients before and during their chemo treatments at various time points, as well as PFP samples of 15 healthy control dogs. The EVs were isolated from PFP by size exclusion chromatography, further purified by density gradient ultracentrifugation, and quantified by high-resolution flow cytometry. The CDC6 mRNA abundance in both the unfractionated plasma and the EVs samples was measured by digital droplet PCR (ddPCR). Results This study demonstrates that, in dogs with lymphoma, circulating CDC6 levels are increased compared to healthy controls. We show that CDC6 levels decline significantly in lymphoma patients that undergo remission in response to chemotherapy. Moreover we show that CDC6 mRNA levels correlate with the number of circulating EVs. Conclusions Overall, our findings provides compelling evidence that plasma CDC6 mRNA expression can be used as a liquid biomarker to monitor treatment efficacy in dogs with lymphoma.

Abstract Background Acute tumor lysis syndrome (ATLS) is caused by the rapid breakdown of tumor cells, leading to electrolyte imbalances and renal dysfunction. The risk of ATLS is particularly high in lymphoma, and therefore it is crucial to consider this risk when initiating chemotherapy. However, risk factors associated with ATLS in dogs remain largely unexplored. Hypothesis/Objectives Identify potential risk factors for ATLS in dogs with multicentric lymphoma. Animals A retrospective analysis was conducted on 24 dogs diagnosed with B-cell high-grade multicentric lymphoma that received chemotherapy. Methods Blood samples were collected before treatment and on days 3, 5, and 8 after treatment. Serum concentrations of uric acid, potassium, phosphorus, calcium, and creatinine were measured based on the Cairo-Bishop Criteria to classify cases as laboratory tumor lysis syndrome (LTLS) or clinical tumor lysis syndrome (CTLS). Clinical variables, including signalment, clinical signs, comorbidities, stage, chemotherapy agents, hematologic and biochemical findings, chemotherapy response, and clinical outcomes were compared between two groups: normal and LTLS/CTLS. Results Of 24 dogs, LTLS occurred in 5 (20.8%), whereas CTLS occurred in 2 (8.3%). The development of LTLS/CTLS was strongly associated with the initial administration of L-asparaginase, presence of chronic kidney disease (CKD), weight loss, and metabolic acidosis. However, no significant differences were observed between the normal and LTLS/CTLS groups regarding signalment, clinical signs, stage, CBC abnormality, and clinical outcomes. Conclusions and clinical importance Evaluating the four risk factors at the initiation of chemotherapy may help establish personalized prevention strategies for ATLS in dogs with multicentric lymphoma.

Background Canine breeds may be considered good animal models for the study of genetic predisposition to cancer, as they represent genetic clusters. From epidemiologic and case collection studies it emerges that some breeds are more likely to develop lymphoma or specific subtypes of lymphoma but available data are variable and geographically inconsistent. This study was born in the context of the European Canine Lymphoma Network with the aim of investigating the breed prevalence of canine lymphoma in different European countries and of investigating possible breed risk of lymphoma overall and/or different lymphoma subtypes. Results A total of 1529 canine nodal lymphoma cases and 55,529 control cases from 8 European countries/institutions were retrospectively collected. Odds ratios for lymphoma varied among different countries but Doberman, Rottweiler, boxer and Bernese mountain dogs showed a significant predisposition to lymphoma. In particular, boxers tended to develop T-cell lymphomas (either high- or low-grade) while Rottweilers had a high prevalence of B-cell lymphomas. Labradors were not predisposed to lymphoma overall but tended to develop mainly high-grade T-cell lymphomas. In contrast with previous studies outside of Europe, the European golden retriever population did not show any possible predisposition to lymphoma overall or to specific subtypes such as T-zone lymphoma. Conclusion Further prospective studies with more precise and consistent subtype identification are needed to confirm our retrospective results and to create the basis for the investigation of possible genes involved in different predispositions.

Background High-grade lymphoma in dogs is a chemotherapy-responsive neoplasia with remission rates exceeding 80% under combination chemotherapy protocols. Usually these protocols are intensive and 24 + weeks. The objective of the present study was to investigate if a shorter protocol combined with an oral lomustine maintenance treatment (3 × in 8 weeks) would present an acceptable result, both for B- and T-cell lymphomas, and for the different types of lymphomas normally encountered in private veterinary practice. Results 144 dogs entered the study. Lymphoma types included multicentric (n = 123), alimentary (n = 13), miscellaneous (n = 7), and mediastinal lymphoma (n = 1). Overall response rate was 83.3% (B-cell: 86.6%, T-cell: 79.4%). Complete remission (CR) was achieved in 72.2% (B-cell: 77.3%, T-cell: 67.6%) and partial remission (PR) in 11.1% (B-cell: 9.3%, T-cell: 11.8%) of the dogs. Median duration of first CR amounted to 242 days (B-cell: 263 d, T-cell: 161 d). Median survival in dogs with CR was 374 days (B-cell: 436 d, T-cell: 252 d), and median overall survival time was 291 days (B-cell: 357d, T-cell: 210d). Immunophenotype demonstrated an independent significant influence on duration of remission and survival in the whole group. Findings of splenic and hepatic cytology were not significant associated with patient outcome. Treatment was well tolerated; the majority of adverse events were classified as grade 1 or 2. Conclusions Short-term chemotherapy followed by lomustine consolidation leads to compara-ble remission and survival times compared to conventional protocols with cyclophosphamide, doxorubicin, vincristine and prednisolone with acceptable toxicosis in dogs with both B-cell and T-cell lymphoma.

We report a rare case of a spontaneous composite tumour with the histopathological characteristics of B‐cell lymphoblastic lymphoma and histiocytic sarcoma in a 104‐week‐old male C57BL/6 mouse. It appeared to be a large, well‐circumscribed intra‐abdominal mass, presumably originating from a mesenteric lymph node. Histological examination revealed two distinct neoplastic cell populations, including small‐ to intermediate‐sized CD20‐ and Pax5‐positive lymphoid cells and large pleomorphic histiocytes expressing Iba1. Both cell types were strongly stained by proliferating cell nuclear antigen, which is indicative of high proliferative activity. No evidence of metastases was observed in other organs. While single cases of similar composite neoplasms have been reported in humans, this is the first documented case in a laboratory mouse. These findings expand the known spectrum of spontaneous hematopoietic tumours in aged mice and highlight the importance of comprehensive histopathological and immunophenotypic evaluation in diagnosing atypical lesions. This study describes a rare concurrent tumour composed of B‐cell lymphoblastic lymphoma and histiocytic sarcoma in a laboratory mouse. The two neoplastic components were clearly distinguished by distinct histopathological features and immunophenotypes, with CD20 and Pax5 expression in the lymphoblastic population and Iba1 positivity in the histiocytic component. This case highlights the diagnostic and interpretative challenges of composite hematopoietic neoplasms in ageing laboratory mice used in toxicologic pathology studies.

This case series assessed CT findings of pulmonary lymphoma in a dog and two cats. The dog (Case 1) showed defined consolidation and nodules with enlarged sternal and mediastinal lymph nodes. Air bronchogram and distinct pulmonary vessels were observed within the lesion. Several nodules were also found in the kidneys. Pulmonary and kidney lesions were diagnosed as B cell lymphoma. One cat (Case 2) showed well‐defined consolidation with an enlarged tracheobronchial lymph node. Air bronchogram and distinct pulmonary vessels were also observed in the lesion. No additional lesions were detected. The pulmonary lesion was diagnosed as B cell lymphoma. Another cat (Case 3) showed a nasopharyngeal mass and multiple well‐defined pulmonary nodules without lymphadenomegaly. Pulmonary nodules and the nasopharyngeal mass were diagnosed as lymphoma. In Case 3, T/B classification was not performed. Of three pulmonary lymphoma cases, the distinguishing imaging feature was a well‐defined lesion with distinct air bronchograms, pulmonary vessels and homogeneous enhancement. This study was retrospective case series analysis of CT findings of pulmonary lymphoma in a dog and two cats. Of three pulmonary lymphoma cases, the distinguishing CT feature was a well‐defined lesion with distinct air bronchograms, pulmonary vessels, and homogeneous enhancement.