V. Martini

Canine indolent and aggressive lymphoma: clinical spectrum with histologic correlation

Sixty-three dogs with newly diagnosed lymphoma underwent complete staging and received the same chemotherapy. Diffuse large B-cell lymphoma was the leading histotype (44.4%), followed by peripheral T-cell lymphoma (20.6%). Indolent lymphomas accounted for 30.2% of cases. Most dogs with aggressive B-cell lymphoma had stage IV disease. Dogs with indolent and aggressive T-cell lymphoma had more often stage V disease and were symptomatic. Liver and bone marrow were predominantly involved in B-cell and T-cell lymphoma, respectively. The clinical stage was significantly related to substage, sex and total lactic dehydrogenase (LDH) levels. Aggressive B-cell lymphomas were more likely to achieve remission. Median survival was 55 days for aggressive and indolent T-cell lymphoma, 200 and 256 days for indolent and aggressive B-cell lymphoma, respectively. The prognosis of advanced indolent lymphoma does not appear to be appreciably different from that of aggressive disease. Familiarity with the various histotypes is critical to make the correct diagnosis and drive therapy.

Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia.

BACKGROUND Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy. OBJECTIVES The aim of the study was to determine if the immunophenotype of neoplastic cells predicts survival in canine CLL. DESIGN Retrospective study. ANIMALS Forty-three dogs with CLL. PROCEDURES Records of dogs with a final diagnosis of CLL were reviewed. For each included dog, a CBC, blood smear for microscopic reevaluation, and immunophenotyping data had to be available. Data on signalment, history, clinical findings, therapy, follow-up, as well as date and cause of death were retrieved. RESULTS Seventeen dogs had B-CLL (CD21+), 19 had T-CLL (CD3+ CD8+), and 7 had atypical CLL (3 CD3- CD8+, 2 CD3+ CD4- CD8-, 1 CD3+ CD4+ CD8+, and 1 CD3+ CD21+). Among the variables considered, only immunophenotype was associated with survival. Dogs with T-CLL had approximately 3-fold and 19-fold higher probability of surviving than dogs with B-CLL and atypical CLL, respectively. Old dogs with B-CLL survived significantly longer than did young dogs, and anemic dogs with T-CLL survived a significantly shorter time than dogs without anemia. CONCLUSIONS Although preliminary, results suggested that immunophenotype is useful to predict survival in dogs with CLL. Young age and anemia are associated with shorter survival in dogs with B-CLL and T-CLL, respectively.

Flow‐cytometric detection of phenotypic aberrancies in canine small clear cell lymphoma

Histopathology and immunohistochemistry are mandatory to solve the differential between canine low-grade lymphoma and reactive hyperplasia. However, clinicians and owners often show reluctance toward these invasive tests. However, molecular biology techniques are still not sensitive and specific enough to be regarded as a reliable tool for final diagnosis. In humans, flow cytometry (FC) allows a definitive diagnosis of T-cell lymphoma based on high prevalence of antigen aberrancies. We describe here the immunophenotype of 26 cases of suspect canine small-clear cell lymphoma, determined by multi-colour FC. All cases showed antigen aberrancies and therefore neoplasia was always confirmed. As a consequence, we argue that the combined use of cytology and FC allows solving the differential diagnosis between small clear cell lymphoma and non-neoplastic reactive conditions when histopathology is not available. Further studies are needed to establish if any aberrancy can be considered indicative of specific histotypes.

Assessment of bone marrow infiltration diagnosed by flow cytometry in canine large B cell lymphoma: Prognostic significance and proposal of a cut-off value.

The aims of this study were to assess the prognostic significance of bone marrow (BM) infiltration in canine large B cell lymphoma (LBCL) and to establish cut-off values for designating the BM as infiltrated by lymphoid blasts. The degree of BM infiltration by large CD21 positive cells in dogs with LBCL was assessed by flow cytometry (FC) and related to time to progression (TTP) and lymphoma-specific survival (LSS). Forty-six dogs were prospectively enrolled, staged and treated with a dose-intense chemotherapeutic protocol. BM infiltration was directly correlated with peripheral blood infiltration (P=0.001), high lactate dehydrogenase activity (P=0.0024) and substage b disease (P<0.001). In the univariate analysis, there was a significant association between BM infiltration diagnosed by FC and both TTP (P=0.001) and LSS (P<0.001). Substage was the only factor associated with TTP in the multivariate analysis (P=0.002), whereas substage (P<0.001) and anaemia (P=0.008) were associated with LSS. A cut-off of 3% BM infiltration had the strongest prognostic value, since it discriminated between dogs with a poorer prognosis (median TTP 69 days; median LSS 155 days) and dogs with a better prognosis (median TTP 149 days; median LSS 322 days). BM analysis is an essential step in the staging of LBCL. The presence of BM infiltration by FC at diagnosis is a negative prognostic indicator in canine LBCL.

Canine small clear cell/T-zone lymphoma: clinical presentation and outcome in a retrospective case series

Published studies, taken together, suggest the existence of a single canine lymphoma entity, with a small clear cell appearance by cytological evaluation, a histopathological T-zone pattern and an aberrant CD45-negative T-cell phenotype, mostly characterized by long-term survival. We describe clinical presentation and outcome in a retrospective case series of canine small clear cell/T-zone lymphoma. Despite the reported predisposition of Golden retriever, this breed was not represented in our case series. Most dogs presented with stage V disease, whereas only few had clinical signs or peripheral cytopenias. Blood was almost always more infiltrated than bone marrow. Median survival confirmed the favourable prognosis described in literature, but a few dogs died within a short time. Also, a subgroup of dogs developed second malignancies, eventually leading to death. We did not investigate possible prognostic factors because of the wide variety in treatments, and further studies are needed to identify high-risk animals.

Minimal residual disease detection by flow cytometry and PARR in lymph node, peripheral blood and bone marrow, following treatment of dogs with diffuse large B-cell lymphoma

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed. Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 64.3% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR(+); conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL.

Peripheral blood abnormalities and bone marrow infiltration in canine large B-cell lymphoma: is there a link?

Official guidelines do not consider bone marrow (BM) assessment mandatory in staging canine lymphoma unless blood cytopenias are present. The aim of this study was to find out if blood abnormalities can predict marrow involvement in canine large B-cell lymphoma. BM infiltration was assessed via flow cytometry. No difference was found between dogs without haematological abnormalities and dogs with at least one. However, the degree of infiltration was significantly higher in dogs with thrombocytopenia, leucocytosis or lymphocytosis and was negatively correlated to platelet count and positively to blood infiltration. Our results suggest that blood abnormalities are not always predictive of marrow involvement, even if thrombocytopenia, leucocytosis or lymphocytosis could suggest a higher infiltration. BM evaluation should therefore be included in routine staging in order not to miss infiltrated samples and to improve classification. However, its clinical relevance and prognostic value are still not defined and further studies are needed.

Prognostic factors in canine acute leukaemias: a retrospective study

Canine acute leukaemias (ALs) have a poor prognosis, with reported survival times (ST) of only a few weeks or months. Also, clinical studies assessing prognostic factors are lacking. This study aims to retrospectively assess variables that predict ST in dogs with AL, and to identify correlations between outcome and therapeutic protocols. Diagnosis and sub-classification into AL subtypes was made based on haematological findings, morphological assessment and flow cytometric immunophenotyping. Clinical-pathological features of AL subtypes at presentation concurred with those described in the literature. A normal neutrophil count at presentation significantly prolonged ST (P = 0.027). Additionally, there was a trend for anaemic dogs to have shorter survival compared with those without anaemia, and the incorporation of cytosine in the chemotherapy protocol produced a moderate but not significant increase in median ST for dogs with AL. Further prospective studies with standardized treatments are needed to confirm and improve our results.

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.