Stage Migration in Canine Multicentric Lymphoma: Impact of Diagnostic Techniques on Assessing Disease Extent.

BACKGROUND/AIM: Stage migration, a phenomenon triggered by technological advancements allowing more sensitive tumor spread detection, results in alterations in the distribution of cancer stages within a population. Canine multicentric lymphoma is staged I to V based on the affected anatomic site(s) and substage a or b depending on the presence of tumor-related clinical signs. The primary objective of this study was to assess the influence of various diagnostic techniques on staging accuracy and determine whether multiple staging methods lead to significant stage migration, impacting the reliability of disease stage assignments. MATERIALS AND METHODS: Dogs cytologically diagnosed with multicentric lymphoma were staged using four different staging methods (A-D): A (physical examination, hemogram, blood smear), B (A plus thoracic X-ray, abdominal ultrasound), C (B plus liver and spleen cytology) and D (C plus bone marrow cytology). RESULTS: Twenty-three dogs were enrolled: 16 females (70%) and seven males (30%). Regarding immunophenotype, 21 dogs (91.3%) were B-cell and two dogs (8.7%) were T-cell. Stage migration was observed between all staging methods. Between A and B, 12 animals migrated from stage III to stage IV. Between B and C, four animals migrated, three to a higher stage (stage III to IV) and one to a lower stage (stage IV to III). Between C and D, one animal migrated from stage IV to V. The differences between staging methods A and B were statistically significant (p≤0.001). CONCLUSION: Stage migration in canine multicentric lymphoma depends on the diagnostic methods used and reinforces the need to use standardized staging methods to avoid it.

Hematological and blood biochemistry parameters as prognostic indicators of survival in canine multicentric lymphoma treated with COP and L-COP protocols.

Background and Aim: Hematological and blood chemistry parameters are crucial for evaluating and monitoring canine multicentric lymphoma during chemotherapy. Pre-treatment hematological and blood chemistry parameters can be used as prognostic survival outcomes for this disease. Therefore, this study aimed to investigate the effect of hematological and blood chemistry parameters pre-treatment and 4 weeks post-treatment on the survival outcomes of dogs treated with either a combination of cyclophosphamide, vincristine, and prednisolone (COP) or a combination of COP with L-asparaginase (L-COP) protocols. Materials and Methods: We conducted a retrospective study. Medical records and hematological and blood chemistry parameters of 41 dogs with multicentric lymphoma treated with L-COP (n = 26) and the COP protocols (n = 15) were obtained from the hospital information system. Most cases were classified as high-grade lymphoma based on the Kiel cytological classification. The effects of hematological and blood chemistry parameters on survival outcomes were investigated using the Cox proportional hazard regression model. The median survival time (MST) for each hematological and blood chemistry parameter affecting survival outcome was established and compared using the Kaplan-Meier product limit method with the log-rank test. Results: Dogs with high-grade multicentric lymphoma that were treated with the COP protocol and had monocytosis at pre-treatment had a significantly shorter MST than dogs with normal monocyte counts (p = 0.033). In addition, dogs with azotemia, both pre-treatment and 4 weeks post-treatment, had a significantly shorter MST than dogs with normal serum creatinine levels (p = 0.012). Dogs with high-grade multicentric lymphoma treated with the L-COP protocol who had hypoalbuminemia (serum albumin concentration <2.5 mg/dL) at both pre-treatment and 4 weeks post-treatment had a significantly shorter MST than dogs with normal serum albumin levels (p < 0.001). Furthermore, dogs with leukocytosis at 4 weeks post-treatment had a significantly shorter MST than those with a normal total white blood cell count (p = 0.024). Conclusion: Serum albumin level can serve as a simple negative prognostic indicator of survival outcomes in dogs with high-grade multicentric lymphoma treated with the L-COP protocol. Dogs with hypoalbuminemia pre-treatment and 4 weeks post-treatment tended to have a shorter MST than those with normal serum albumin concentrations.

Environmental radon, fracking wells, and lymphoma in dogs.

BACKGROUND: Multicentric lymphoma (ML) in dogs resembles non-Hodgkin lymphoma (NHL) in humans. Human NHL is associated with multiple environmental exposures, including to radon and volatile organic compounds (VOCs). HYPOTHESIS/OBJECTIVES: The objective of this study was to determine whether ML in dogs was associated with environmental radon or proximity to horizontal oil and drilling (fracking), a source of VOC pollution. METHODS: We identified dogs from the Golden Retriever Lifetime Study that developed ML (n = 52) along with matched controls (n = 104). Dog home addresses were categorized by Environmental Protection Agency radon zone and average residential radon by county, as well as by distance from fracking and associated wastewater wells. RESULTS: We found no significant differences in county level radon measurements. Individual household radon measurements were not available. There was no difference in residential proximity to active fracking wells between dogs with ML and unaffected dogs. While dogs with ML lived closer to wastewater wells (123 vs 206 km; P = .01), there was no difference in the percentage of cases vs controls that lived in close proximity (20 km) to a fracking well (11.5% for cases, 6.7% for controls; OR 1.81, 95% CI 0.55 to 5.22; P = .36), or a wastewater well (6.7% for cases, 4.4% for controls; P > .99). CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest that more proximate sources of chemical exposures need to be assessed in dogs with ML, including measurements of individual household radon and household VOC concentrations.

Construction and validation of a scoring system to predict resistance to chemotherapeutic drugs using gene expression profiles in canine lymphoma.

The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.

Hematological and biochemical profiles of canine CD45- T lymphomas are different from other immunophenotypes.

BACKGROUND: Canine multicentric lymphomas are lymphoproliferative malignancies that have increased in recent decades. The patient's treatment and prognosis are determined by the grade, histological type, and lymphoma immunophenotyping. AIM: To investigate the paraclinical signs and survival time in canines with different lymphoma immunophenotypes. METHODS: Over 2 and a half years, 47 untreated dogs were diagnosed with multicentric lymphoma at the Veterinary School Hospital of Uruguay. The disease was clinically and cytologically diagnosed, and immunophenotyping was determined by flow cytometry. After the immunophenotyping, most of the patients were grouped into the following: B (LB), T aggressive (LTCD45+), or T-zone lymphoma (LTCD45-). The patients' haematological values, calcemia, lactate dehydrogenase (LDH) levels, and plasmatic electrophoretic profiles were all determined immediately after that. RESULTS: Of all canine lymphomas, 55.3% were B, 31.9% were LTCD45+, and 10.6% were TCD45-. Only 2.2% were classified as nonB/nonT, and survival time differed between groups. Patients with LTCD45- lymphomas had a mean life span of 641 days after diagnosis, followed by LB (166 days) and LTCD45+ (62 days). Red blood cell count, hematocrit, and hemoglobin levels did not differ between groups. However, the LTCD45- group had significantly higher lymphocyte levels than the LTCD45+ and LB groups (p = 0.01 and 0.006, respectively). Levels of albumin, alpha-1, and alpha-2 globulins did not differ between groups. On the other hand, gamma globulins levels in the LTCD45- were higher than in the other lymphoma groups. The presence of hypercalcemia and high plasma LDH levels were associated with patient severity. Only the TCD45+ group had hypercalcemia although both the LB and TCD45+ groups had elevations in LDH activity. Interestingly, there was a direct relationship between high LDH values (greater than 500 IU/l) and lower survival in TCD45+ lymphomas. CONCLUSION: Survival time and hematological and biochemical patterns differed among canine lymphomas immunophenotypes. Patients of LTCD45- phenotype showed higher lymphocyte counts and gamma globulin levels and more prolonged survival. Serum LDH activity may provide additional prognostic information in high-grade T-cell lymphoma.

Canine multicentric lymphoma exhibits systemic and intratumoral cytokine dysregulation.

Non-Hodgkin Lymphoma (NHL) is among the most common neoplasias in dogs and humans. Owing to remarkable similarities with its human counterpart, the canine lymphoma (cNHL) model has been proposed as a powerful framework for rapid and clinically relevant translation of novel immunotherapies. However, the establishment of cNHL as a predictive preclinical model has been hampered by the limited characterization of the canine immune system. Cytokines are key players of the interaction between tumor and its microenvironment. In human NHL, multiple cytokines have been linked to the development of lymphoma and are relevant biomarkers for treatment response and prognosis. In contrast, few studies have investigated cytokines in cNHL. Within this context, this study aimed to investigate cytokine regulation in cNHL. A multicentric cNHL biobank was successfully constructed. Cytokine mRNA profiles in tumor tissue and circulating PBMC were analyzed by qRT-PCR and compared to a healthy control group. Specific primers were used to evaluate Th1, Th2 and Th17 responses. Systemic cytokine concentrations were measured using a commercial canine multiplex assay which included IL-2, IL6, IL-10 and TNF-α, and compared to a healthy control group. Our results demonstrated a dysregulation of cytokine mRNA expression, representative of the tumor microenvironment and systemic response in cNHL. Intratumoral cytokine response revealed a significant downregulation of humoral and Th1 responses. The systemic response demonstrated a distinct mRNA pattern, however immunosuppression also prevailed. Cytokine serum quantification showed a significant increase of IL-10 concentration in cNHL. Significant differences in hematological parameters were described and a correlation between IL-6 protein serum levels and neutrophil count was shown. Finally, data analysis demonstrated that baseline pretreatment IFN-γ tissue mRNA levels were correlated to survival outcome, predicting a favorable response to chemotherapy. Altogether, these results revealed that cNHL presents a local and systemic dysregulation in cytokine response. By confirming and extending previous research, our work contributed for the evaluation of potential cytokine candidates for diagnostic, prognostic purposes and therapeutic intervention, therefore adding value to comparative oncology.

Expression of blood hepatocyte-derived microRNA-122 in canine multicentric lymphoma with hepatic involvement.

This study was performed to evaluate the hepatocyte-derived microRNA (miR)-122 as novel diagnostic biomarker in canine lymphoma. Fifteen dogs were enrolled in this study. Dogs presented at Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Cairo University. Dogs were divided into 8 clinically healthy dogs act as control and 7 clinically ill dogs. All dogs were subjected to clinical, ultrasonographic, hemato-biochemical and ultrasound-guided fine-needle biopsy for cytological and histopathological investigations. On the basis of these results, 7 dogs were found to be suffering from multicentric lymphoma involving liver. Serum hepatocyte-derived miRA-122 was determined by real-time quantitative polymerase chain reaction in all dogs. Multicentric lymphoma involving liver manifested by inappetance for several days, depression and peripheral lymphadenopathy. Hematological examination showed significant lymphocytosis. Serum biochemical analysis revealed significant increase in ALT, AST, ALP compared to control dogs. Ultrasonography revealed hypoechoic lymphoid aggregation at area of "porta hepatis" and circumscribed hypoechoic nodule interrupt liver parenchyma. Cytology revealed infiltration of liver tissue by lymphoblast cells and histopathology revealed diffuse infiltration of hepatic sinusoids and portal area by uniform population of small lymphocytes. Serum miRNA-122 analysis showed a significant increase represented as 9.00 fold in canine multicentric lymphoma involving liver. Serum hepatocyte-derived miRNA-122 is of diagnostic value, non invasive, stable and easily measurable blood biomarker for the detection of hepatocellular injury in dogs with multicentric lymphoma involving liver.

Intestinal parasite infections in dogs affected by multicentric lymphoma and undergoing chemotherapy.

Prevalence and species composition of intestinal parasites were evaluated in dogs affected by high-grade multicentric lymphoma and undergoing chemotherapy and in control healthy dogs. Obtained data were statistically analyzed. The overall prevalence of intestinal parasite infections was 33.3%. In lymphoma dogs, the prevalence of protozoa infections (46.7%) was significantly higher (p < 0.05) than that of helminth infections (6.7%) and Giardia duodenalis, Cryptosporidium spp., Neospora caninum, Cystoisospora ohioensis-complex, Entamoeba sp. and Spirocerca lupi were identified. In the control group, only 3/15 dogs (20%) were found positive and no statistically significant differences emerged regarding helminth (hookworms and Toxocara canis) and protozoa (G. duodenalis) infections. Results from this study may suggest a potential higher prevalence of opportunistic intestinal protozoa, including some potentially zoonotic species, in dogs affected by high-grade multicentric lymphoma, emphasizing the need to monitor lymphoma-affected dogs for these protozoa, especially those undergoing chemotherapy.

Corneoconjunctival manifestations of lymphoma in three dogs.

An 8-year-old Shih Tzu, a 5-year-old Maltese, and a 10-year-old Maltese presented with conjunctival hyperemia and peripheral corneal edema. Severe conjunctival thickening with varying degrees of corneal extension was observed. Cytological examination showed many large lymphocytes with malignant changes in the conjunctiva which was consistent with findings in fine-needle aspiration samples taken from regional lymph nodes. They were diagnosed as having Stage V multicentric lymphoma. When conjunctival thickening is observed in canine patients with multicentric lymphoma, conjunctival metastasis with infiltration of neoplastic lymphoid cells should be included in the differential diagnosis.

Corneoconjunctival manifestations of lymphoma in three dogs

An 8-year-old Shih Tzu, a 5-year-old Maltese, and a 10-year-old Maltese presented with conjunctival hyperemia and peripheral corneal edema. Severe conjunctival thickening with varying degrees of corneal extension was observed. Cytological examination showed many large lymphocytes with malignant changes in the conjunctiva which was consistent with findings in fine-needle aspiration samples taken from regional lymph nodes. They were diagnosed as having Stage V multicentric lymphoma. When conjunctival thickening is observed in canine patients with multicentric lymphoma, conjunctival metastasis with infiltration of neoplastic lymphoid cells should be included in the differential diagnosis.

Aspectos epidemiológicos, clínicos e anatomopatológicos do linfoma folicular em cães / Epidemiological, clinical and pathological aspects of follicular lymphoma in dogs

Linfomas foliculares são uma rara forma de distúrbio linfoproliferativo descrita em medicina veterinária. Juntamente com a não reconhecida ocorrência dos linfomas de Hodgkin em cães, essa é a maior diferença acerca de linfoma entre humanos e cães. O objetivo deste artigo é descrever os achados epidemiológicos, clínicos e anatomopatológicos vistos em cinco cães com linfoma folicular. Destes, dois eram machos (40%) e três eram fêmeas (60%). A idade dos cães afetados variou de 11 a 13 anos. Quatro dos cinco (80%) cães eram de raça pura e um (20%) não tinha raça definida. Todos os cães apresentaram linfadenomegalia generalizada e esplenomegalia, o que incluiu os casos como linfoma multicêntrico. Na necropsia, os linfonodos e o baço demonstraram um padrão nodular à superfície de corte, caracterizado por dezenas a centenas de nódulos brancos, multifocais ou coalescentes e de tamanhos variáveis. Na superfície natural do baço, frequentemente (4/5, 80%), havia miríades de pontos brancos, multifocais ou coalescentes, de tamanhos variáveis. Na histopatologia, os tumores foram confirmados como linfomas foliculares. Todos os casos eram Grau III, sendo dois (40%) incluídos como IIIa e outros três (60%) como IIIb. Em um caso (1/5, 20%), o linfoma folicular foi considerado como IIIb variante de pequenos centroblastos semelhantes aos linfócitos neoplásicos vistos no linfoma de Burkitt. Os linfomas foram validados como tendo origem em células B através da imuno-histoquímica, utilizando anticorpos anti-CD20. Os casos de linfomas foliculares descritos comportaram-se de forma agressiva e levaram os pacientes à morte.(AU)

Follicular lymphomas are a rare form of lymphoproliferative disorder described in veterinary medicine. Together with the probable non-existence of Hodgkin's lymphomas in dogs, this is the biggest difference about lymphoma between humans and dogs. The aim of this article is to describe the epidemiological, clinical and anatomopathological findings observed in five dogs with follicular lymphoma. Of the five dogs with follicular lymphoma, two were male (40%) and three were female (60%). The age of affected dogs ranged from 11 to 13 years. Four of the five (80%) dogs were purebred and one (20%) had no defined breed. All dogs presented generalized lymphadenomegaly and splenomegaly, which included cases as multicentric lymphoma. At necropsy, the lymph nodes and the spleen demonstrated a nodular pattern at the cut surface, characterized by tens to hundreds of white nodules of variable size, multifocal or coalescing. On the natural surface of the spleen, often (4/5, 80%), there were myriads of white, multifocal or coalescing dots of varying sizes. In histopathology, the tumors were confirmed as follicular lymphomas. All cases were Grade III, two (40%) included as IIIa and three (60%) as IIIb. In one case (1/5, 20%), follicular lymphoma was considered as a IIIb variant of small centroblasts similar to the neoplastic lymphocytes seen in Burkitt's lymphoma. Lymphomas were validated as having origin in B cells through immunohistochemistry, using anti-CD20 antibody. The cases of follicular lymphomas described behaved aggressively and led the patients to death.(AU)

Comparison of a CHOP-LAsp-based protocol with and without maintenance for canine multicentric lymphoma.

The recommendation to treat canine lymphoma with a discontinuous protocol is based on small case numbers and mostly historic controls. This study compares duration of first remission (DFR) and overall survival time (ST) with a discontinuous protocol to the same protocol with maintenance phase. 408 dogs were treated with a CHOP-LAsp (C=cyclophosphamide; H=hydroxydaunorubicin; O=Oncovin; P=prednisolone; LAsp=l-asparaginase)-based 28-week induction protocol. In 75 dogs (cohort 1), this was followed by a maintenance phase consisting of vincristine, chlorambucil and actinomycin-D with a total treatment duration of two years. In the subsequent 333 dogs, therapy was discontinued after induction (cohort 2). Median DFR and ST in cohort 1 were 216 and 375 days and 184 and 304 days in cohort 2. 6-Month, 1-year and 2-year survival rates in cohort 1 were 73 per cent, 50 per cent, 24 per cent and 67 per cent, 39 per cent, 21 per cent in cohort 2. There was no significant difference between the two protocols (P=0.291 for ST, P=0.071 for DFR). On multivariate analysis, corticosteroid pretreatment (P=0.005), thrombocytopenia at diagnosis (P=0.019), stage (P=0.009), substage b at relapse (P<0.001), age (P=0.002) and incomplete or unstable remission necessitating intensification of therapy (P=0.004) were negatively correlated with ST in both groups. This study supports the use of a discontinuous protocol for canine multicentric lymphoma.

Conformity and controversies in the diagnosis, staging and follow-up evaluation of canine nodal lymphoma: a systematic review of the last 15 years of published literature.

Diagnostic methods used in the initial and post-treatment evaluation of canine lymphoma are heterogeneous and can vary within countries and institutions. Accurate reporting of clinical stage and response assessment is crucial in determining the treatment efficacy and predicting prognosis. This study comprises a systematic review of all available canine multicentric lymphoma studies published over 15 years. Data concerning diagnosis, clinical stage evaluation and response assessment procedures were extracted and compared. Sixty-three studies met the eligibility criteria. Fifty-five (87.3%) studies were non-randomized prospective or retrospective studies. The survey results also expose variations in diagnostic criteria and treatment response assessment in canine multicentric lymphoma. Variations in staging procedures performed and recorded led to an unquantifiable heterogeneity among patients in and between studies, making it difficult to compare treatment efficacies. Awareness of this inconsistency of procedure and reporting may help in the design of future clinical trials.

Regulatory T cells in dogs with multicentric lymphoma: peripheral blood quantification at diagnosis and after initial stage chemotherapy / Células T reguladoras em cães com linfoma multicêntrico: quantificação, em sangue periférico, no momento do diagnóstico e após etapa inicial do tratamento quimioterápico

Lymphoma is the most common hematopoietic malignancy in dogs and one of the most frequent among all neoplastic diseases in this species. It can occur in several anatomical locations with distinct histological and immunophenotypes. Depending on the host immune response towards the tumor, prognosis information could be collected. Because its well established immunosuppressant, antitumor activity, the function of regulatory T cells (Tregs) in canine neoplasias has been investigated. In this study, we sought to quantify, using flow cytometry, the Tregs subpopulation in peripheral blood of healthy dogs (10) and in those diagnosed with type-B (14) and type-T (8) multicentric lymphoma before (at diagnosis) and after the first cycle (5-week) of 19-week Madison-Wisconsin (MW) protocol of chemotherapy. Our results indicated that dogs with lymphoma showed higher percentage of Tregs (18,84±2,56) when compared to healthy dogs (4,70±0,50) (P<0,01). In addition, 5-week chemotherapy treatment reduced the Tregs subpopulation (7,54±1,08) to levels similar to control (4,70±0,50) (P>0,05). There was no difference in Tregs percentage between B-type (17,45±2,77) and T-type (21,27±5,27) lymphoma (P>0,05). With this, we conclude that canine lymphoma increases Tregs in the peripheral blood and the MW protocol of chemotherapy reduces this cell subpopulation to control values.

O linfoma é a neoplasia hematopoiética mais comum nos cães e uma das mais frequentes, dentre todas as neoplasias, nesta espécie. Apresenta-se em diversas localizações anatômicas e pode apresentar classificações histológicas e imunofenotipos distintos. Dependendo da resposta imune do paciente frente à instalação de um tumor, algumas informações sobre o prognóstico podem ser obtidas. Atualmente, as células T reguladoras (Tregs) vêm sendo estudadas em algumas neoplasias caninas, por seu comprovado potencial imunossupressor, principalmente por inibir a resposta antitumoral. Neste contexto, o presente trabalho teve como objetivos quantificar, por citometria de fluxo, as células Tregs em sangue periférico de cães com linfoma multicêntrico de imunofenótipos B e T, respectivamente 14 e 8 cães, no momento do diagnóstico e após o primeiro ciclo de quimioterapia antineoplásica com o protocolo Madison-Wisconsin (MW) de 19 semanas adaptado, e comparar com cães saudáveis (n=10), buscando quantificá-las em cães com linfoma de diferentes imunofenótipos antes e após a 5ª semana do protocolo MW. Os resultados mostraram que cães com linfoma apresentaram uma porcentagem significativamente maior de Tregs (18,84±2,56) quando comparada aos cães sem neoplasia (4,70±0,50) (P<0,01). Além disso, após a quinta semana de tratamento houve uma significante redução da população de Tregs (7,54±1,08), atingindo valores semelhantes a dos cães controle (4,70±0,50) (P>0,05). Não houve diferença nas Tregs em relação aos imunofenotipos B (17,45±2,77) e T (21,27±5,27) (P>0,05). Concluiu-se que o linfoma em cães leva a um aumento de células Tregs e que o tratamento com o protocolo quimioterápico MW reduz significativamente as células Tregs em sangue periférico, atingindo valores próximos aos dos cães saudáveis.