Utility of polymerase chain reaction for analysis of antigen receptor rearrangement in staging and predicting prognosis in dogs with lymphoma

In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions with unique sequences, resulting from recombination of the V, D, and J genes. The purpose of this study was to determine the utility of polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) for molecular staging and predicting prognosis in canine lymphoma. We hypothesized that the PARR assay would offer a sensitive method for detecting neoplastic cells in blood, and that the presence of such cells would be a negative prognostic finding compared with dogs with no detectable circulating tumor cells. Eighty-six patients with histologically or cytologically confirmed lymphoma were studied from initial diagnosis until death or euthanasia. All patients had PARR assays of a representative tumor-infiltrated lymph node and peripheral whole blood. Sixty-two patients had clonal rearrangements detected in the lymph node and were able to be staged by PARR. Seventeen patients (27%) had no detectable tumor in their blood and 45 (73%) were blood positive. Our findings showed that (1) PARR correlated with clinical stage in that the PARR assay was more likely to detect tumor cells in blood in stage 5 lymphomas, (2) PARR was more sensitive for detecting circulating tumor cells than visual assessment of blood or bone marrow because 80% of stage 3 lymphomas were blood-PARR-positive, and (3) PCR stage was not prognostic for disease-free interval (DFI) or survival.     

Melting curve analysis in canine lymphoma by calculating maximum fluorescence decrease

PARR is widely used in the diagnostics of canine lymphoma. In human and veterinary medicine, melting curve analysis (MCA) has successfully been introduced to facilitate the process. Since visual interpretation of melting curves can be rather subjective, the purpose of this study was to develop an objective interpretation of melting curves by calculating the maximum fluorescence decrease (dF_max) within a defined rise of temperature. Lymph node aspirates and blood of 34 dogs with lymphoma and 28 control dogs were tested. 27/34 lymphoma cases were correctly detected to be monoclonal (sensitivity 79%). 2/28 control dogs showed a monoclonal rearrangement (specificity 93%). B‐ and T‐cell neoplasia were still detectable using DNA amount as low as 10 ng. In serial dilutions of tumor DNA with DNA of normal tonsils, the detection limit was 25% for B‐cell lymphomas and 100% for T‐cell lymphoma, suggesting that PCR conditions could still be optimized.     

A case of hemophagocytic syndrome progressing into large granular lymphoma in a dog

A 12‐year‐old castrated male mixed breed dog was presented with anorexia, lethargy, intermittent vomiting, diarrhea, and weight loss. Clinicopathologic and imaging abnormalities included pancytopenia, icterus, and splenomegaly with multiple minute hypoechogenic nodules. Bone marrow (BM) smears revealed 2.5% hemophagocytic macrophages. In addition, an increased number of small to intermediate lymphocytes (16.3%) and plasma cells (3.2%) were recognized in the BM smears. More than 80% of the lymphocytes contained multiple small intracytoplasmic magenta granules. Histopathologic findings of the spleen revealed hemophagocytosis. Large granular lymphocytes (LGLs) were not found on the liver cytology or splenic histopathology at this time. PCR for antigen receptor rearrangement (PARR) analysis showed a clonal reaction in the T‐cell receptor ? (TCR?) gene in the BM sample. The dog was diagnosed with hemophagocytic syndrome (HPS). The dog was maintained in good condition with immunosuppressive therapy. However, the dog developed hepatic LGL lymphoma 7 months later. At this time, PARR analysis showed a clonal TCR? gene rearrangement in the hepatic LGL lymphoma samples. The BM and liver sample clonal rearrangements showed 100% homology, indicating that the small to intermediate granular lymphocytes in the BM at the HPS stage had progressed to hepatic LGL lymphoma. To our knowledge, this is the first report of canine secondary HPS caused by the occurrence of a BM LGL lymphoma clone that progressed to hepatic LGL lymphoma.     

Integrated immunohistochemical and DNA copy number profiling analysis provides insight into the molecular pathogenesis of canine follicular lymphoma

Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti‐apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B‐cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour‐initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL.     

Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide, and Doxorubicin in dogs with lymphoma by measuring the number of neoplastic lymphoid cells with real-time polymerase chain reaction

Background: The cytoreductive efficacy of the individual components of multidrug chemotherapy for canine lymphoma is difficult to evaluate after complete remission. Objectives: To compare the cytoreductive efficacy of vincristine (VCR), cyclophosphamide (CPA), and doxorubicin (DXR) in dogs that received a 6‐month modified version of the University of Wisconsin‐Madison chemotherapy protocol (UW‐25). Animals: Twenty‐nine dogs with high‐grade B‐cell multicentric lymphoma. Methods: Rearranged immunoglobulin heavy chain gene fragments from lymphoma cells were amplified by polymerase chain reaction (PCR) and sequenced to prepare clone‐specific primers and probes for real‐time PCR. The number of lymphoma cells in peripheral blood was measured from diagnosis to week 11 of UW‐25. Results: The number of lymphoma cells after the 1st administration of VCR, CPA, and DXR in weeks 1–4 was decreased in 29/29 (100%), 15/29 (51.7%), and 26/27 (96.3%) dogs, respectively. The cytoreductive efficacy of CPA was less than that of VCR and DXR. VCR, CPA, and DXR administered in weeks 6–9 were effective in 5/26 (19.2%), 5/20 (25.0%), and 14/19 (73.7%) dogs, respectively, indicating the sustained cytoreductive efficacy of DXR. CPA nonresponders were heavier and exhibited a shorter 1st remission than CPA responders. Conclusion and Clinical Importance: When using UW‐25 for treatment of canine lymphoma, CPA was found to have less cytoreductive efficacy than VCR and DXR. Real‐time PCR‐based quantification of tumor cells is an objective marker of the efficacy of chemotherapeutic agents.     

Diagnosis of canine renal lymphoma by cytology and flow cytometry of the urine

Lymphoma is a common hematopoietic neoplasm of dogs. A definitive diagnosis typically requires the collection of samples via fine-needle aspirate or biopsy. A unique case of canine renal T-cell lymphoma diagnosed using urine sediment microscopy with flow cytometry and PCR for Antigen Receptor Rearrangement (PARR) is presented. A fresh urine sample was collected via a urinary catheter and immediately prepared for cytologic examination, flow cytometry, and PARR. The flow cytometric study revealed that 83% of the cells were large CD3⁺CD8⁺ T cells, while PARR identified a clonally rearranged T-cell receptor gene, supporting the flow cytometry findings. Despite supportive care, the patient progressed to anuric renal failure and was humanely euthanized. A necropsy was performed, and tissues from the upper and lower urinary tracts were collected. Histologically, the right and left kidneys were infiltrated by a neoplastic round cell population effacing the cortex and medulla. Immunohistochemistry for the T- and B-cell antigens CD3 and CD20, respectively, revealed that the neoplastic population within the kidney demonstrated diffuse, strong, membranous to intracytoplasmic CD3 expression while lacking CD20 expression. These results confirmed the diagnosis of renal T-cell lymphoma. This is the first known report of canine lymphoma diagnosed using either urine flow cytometry or clonality testing. Therefore, in select cases, urine flow cytometry and/or PARR are feasible to perform on urine-derived cells as a quick and cost-effective means to aid in the diagnosis of urinary tract lymphoma.     

Evaluation of Pax5 expression and comparison with BLA.36 and CD79αcy in feline non‐Hodgkin lymphoma

Paired box gene 5 (Pax5) is a widely used B‐cell marker for human and canine non‐Hodgkin's lymphoma (nHL); however, in the literature there is only one case report using Pax5 in a cat B‐cell lymphoma. The purposes of this study were to investigate the expression and detection of B‐cell specific activator protein (BSAP) using a monoclonal anti‐Pax5 antibody in feline nHL (FnHL) tissue samples to evaluate its diagnostic relevance as a B‐cell marker. A total of 45 FnHL samples in 45 cats were evaluated. B‐cell lymphoma was the most common immunophenotype (51.1%) for all the samples and T‐cell the most common immunophenotype (64.3%) for the gastrointestinal (GI) form. Pax5 stained 82.6% of all B‐cell lymphomas and no expression was found in any of the T‐cell lymphomas. Anti‐Pax5 antibody staining in FnHL is similar to that reported in human and canine counterparts and may offer an excellent B‐cell marker in cats.     

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high‐grade B‐cell lymphoma

Ki67 can discriminate between high‐ and low‐grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67‐positive cells), evaluated by flow cytometry, in 40 dogs with high‐grade B‐cell lymphoma, treated with a modified Wisconsin–Madison protocol (UW‐25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1–40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high‐grade B‐cell lymphoma.     

The Utility of PCR for Antigen Receptor Rearrangement (PARR) in Staging and Predicting Prognosis in Canine Lymphoma

Introduction:  In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions that are unique in sequence, resulting from recombination of the V, D, and J genes. Based on the belief that malignancies are clonal, the presence of a single, clonal rearrangement can indicate neoplasia. In humans with lymphoid malignancies, PCR for antigen receptor rearrangement has been studied as a molecular staging test and a therapeutic monitoring tool. The purpose of this study was to determine the utility of PARR for molecular staging and predicting prognosis in canine lymphoma.
Methods:  Patients with lymphoma who had complete clinical staging were included in the study. All had PARR of pre treatment lymph node, blood and/or bone marrow. All were assigned a clinical stage (WHO 1–5) and a PCR stage (PS) where 0 indicated no clonal expansion in the blood, and 5 indicated the presence of a clonal expansion. In addition to stage, other factors that could affect prognosis were evaluated.
Results:  Eighty six patients were included. Clinical stage was distributed as follows: 3 = 44, 4 = 21, and 5 = 21. Sixty two patients had clonal rearrangements detected in the lymph node and were able to be PCR staged. Seventeen were PS 0 and 45 were PS 5. PCR stage was not prognostic for DFI or survival.
Conclusion:  PARR is sensitive at detecting blood/bone marrow infiltration in canine lymphoma. It is not a useful prognostic tool in the diverse population in this study.     

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.     

Immunohistochemical characterization of estrogen and progesterone receptors in lymphoma of horses

Abstract: Immunohistochemical techniques were used to examine 29 cases of equine lymphoma for estrogen receptor (ER) and progesterone receptor (PR) expression. The lymphomas examined included T-cell-rich large B-cell lymphomas, B-cell neoplasms, and T-cell lymphomas. The individual cases were also classified according to the anatomic location of the tumors. One normal equine lymph node was also examined for ER and PR expression. All of the cases of equine lymphoma and the normal lymph node were negative for ER. A total of 16/29 (55%) PR-positive lymphomas were identified. Seven of the 12 (58%) T-cell-rich large B-cell lymphomas were positive, 7/11 (64%) B-cell tumors were positive, and 2/6 (33%) T-cell neoplasms were positive. Anatomically, 6/9 (66%) subcutaneous lymphomas were PR positive, 3/5 (60%) intrathoracic lymphomas were positive, 1/4 (25%) intra-abdominal lymphomas were positive, 2/5 (40%) intra-abdominal/intrathoracic lymphomas were positive, 1/2 (50%) upper airway lymphomas were positive, and 3/3 (100%) splenic lymphomas were positive. One case involving abdominal and thoracic tumors and leukemia was negative for PR expression. The normal lymph node contained a low percentage (1.9%) of PR-positive lymphocytes. The presence of PR in neoplastic equine lymphoid tissue indicates that these tumors may be responsive to serum progesterone. Also, identification of PR-positive cells in the normal lymph node suggests that PR may be constitu-tively expressed in normal equine lymphocytes. Further studies are needed to quantify PR levels in normal and malignant equine lymphoid tissue and to determine the usefulness of either progestin or antiprogestin drugs in the management of equine lymphoma.     

Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry

Flow cytometry may be a useful tool to analyze lymphoma samples that are obtained from fine needle aspirations (FNA). This study aimed to determine if flow cytometric analysis add more objective and standardized information on the cellularity and morphology of lymphoma cells to conventional cytology. The typical immunophenotype of different lymphoma subtypes was assessed and leukocyte marker expression was evaluated to determine which antigens were more frequently over- or under-expressed in these lymphoma subtypes. Fifty FNA lymph node samples were evaluated from canine lymphomas. Thirty-one samples were identified to be of B-cell origin, sixteen were identified to be of T/NK-cell origin and three cases were classified as acute lymphoblastic leukaemia with lymph nodes involvement. The most common B-cell lymphoma subtypes were centroblastic lymphomas, whereas three cases were atypical and classified as B-large cell pleomorphic lymphomas. Among the T/NK lymphomas, small clear cells, large and small pleomorphic mixed cells, large granular lymphocytic cells and small pleomorphic cells were identified. Aberrant phenotypes and/or antigen under/over regulation was identified in thirty out of forty-seven lymphoma cases (64%; 18/31 B-cell=58% and 12/16 T-cell=75%). In B-cell lymphomas the most frequent finding was the diminished expression of CD79a (45%). CD34 expression was also observed in four cases (13%). Among T-cell lymphomas the prevalent unusual phenotype was the under-expression or absence of CD45 (25%). These findings reveal flow cytometry may be useful in confirming the diagnosis of lymphoma, as the technique allows one to add useful information about morphology of the neoplastic cells and identify antigenic markers and aberrant phenotypes.     

Evaluation of a 15‐week CHOP protocol for the treatment of canine multicentric lymphoma

Dose intense CHOP protocols have been shown to improve outcome for people with non‐Hodgkin's lymphoma, but evaluation of dose intense CHOP protocols for canine lymphoma is currently limited. The hypothesis of this retrospective study was that a 15‐week dose intense CHOP protocol would have shorter treatment duration with similar efficacy to other doxorubicin‐based multidrug protocols. Thirty‐one client owned dogs with multicentric lymphoma were treated with a 15‐week CHOP chemotherapy protocol with an overall response rate of 100% and a median progression‐free interval (PFI) of 140 days [95% confidence interval (CI) 91–335 days]. Dogs that had two or more treatment delays had significantly prolonged PFI and overall survival in multivariate analysis. Dose intensity did not correlate with patient outcome. Dogs experiencing multiple treatment delays secondary to adverse events may receive their individual maximally tolerated dose while dogs with no adverse events may be underdosed. Future studies should focus on individual patient dose optimization.     

T-cell-derived malignant lymphoma in the boxer breed

The boxer breed of dog is at high risk for a variety of neoplasms including lymphoma. In this observational study, tissue sections from boxer dogs with lymphoma were immunostained for T and B lymphocyte distinction, and the results compared with similar studies carried out on lymphoma tissues from temporally selected cohorts of golden retriever and rottweiler dogs. The frequency of T‐cell lymphomas was significantly (P < 0.001 for all comparisons) higher in the boxers than in the rottweilers or golden retrievers. We are unaware of other reports linking immunotype of canine lymphoma with breed; whether other brachycephalic breeds of dogs have a similar preponderance of T‐cell lymphoma awaits further study.     

Diagnostic evaluation and treatment recommendations for dogs with substage‐a high‐grade multicentric lymphoma: results of a survey of veterinarians

The goal of this study was to survey veterinarians regarding their current initial diagnostic and treatment recommendations for dogs with substage‐a high‐grade multicentric lymphoma. A written survey was conducted at the 2009 Veterinary Cancer Society conference asking veterinarians to provide demographic information, initial staging diagnostics and treatment recommendations for canine lymphoma. The most commonly recommended staging diagnostics were complete blood count (100%), chemistry panel (100%), urinalysis (85%), lymph node cytology (88%), thoracic radiographs (84%), immunophenotyping (76%) and abdominal ultrasound (75%). The most commonly used first‐line B‐cell protocols combined the drugs L ‐asparaginase, cyclophosphamide, doxorubicin, vincristine and prednisone (L ‐CHOP, 51%). CHOP (30%) and other CHOP‐based protocols (12%) were used as well. Thirty‐one percent of responders treated B‐ and T‐cell lymphomas differently. Protocol lengths varied from ≤16 weeks to >2 years. Current staging and treatment recommendations for canine lymphoma are varied. Efforts to standardize recommendations should be considered.     

Rapid diagnosis of retina and optic nerve abnormalities in canine patients with and without cataracts using chromatic pupil light reflex testing

Objective To develop fast and reliable testing routines for diagnosing retina and optic nerve diseases in canine cataract patients based on chromatic properties of the pupillary light reflex response. Procedures Seventy‐seven canine patients with a history of cataract and decreased vision (43 patients with cataracts and no evidence of retina or optic nerve disease, 21 patients with cataracts and retinal degeneration [RD], 13 patients with cataracts and retinal detachment [RDT]), 11 canine patients with optic neuritis (ON) and 23 healthy dogs were examined using chromatic pupillary light reflex (cPLR) analysis with red and blue light and electroretinography. Results Electroretinography analysis showed statistically significant deficits in a‐ and b‐wave amplitudes in dogs with cataracts and RD, or cataracts and RDT, when compared to dogs with cataracts without evidence of retinal abnormalities. Evaluation of b‐wave amplitudes showed that presence of 78.5‐μV (or lower) amplitudes had high sensitivity of 100% (95% CI: 87.2–100%) and high specificity of 96.7% (95% CI: 88.4–100%) in RD and RDT. Evaluation of cPLR responses using red light showed that presence of the pupil end constriction diameter of 5.5 mm (or higher) had moderately high sensitivity of 76.5% (95% CI: 50.1–93.2%) and high specificity of 100% (95% CI: 91.2–100%) in detecting RD and RDT. Optic neuritis patients had absent cPLR responses, regardless of the visual status. Conclusions and Clinical Relevance Chromatic evaluation of the pupillary light reflex is a rapid and accurate test for diagnosing retina and optic nerve diseases in canine patients.     

Cytologic–histologic concordance in the diagnosis of neoplasia in canine and feline lymph nodes: a retrospective study of 367 cases

Lymph nodes are frequently sampled in dogs and cats for the diagnosis of primary and metastatic neoplasia. We determined the accuracy of cytologic diagnosis in lymph nodes using histology as the gold standard. Lymph node reports (2001–2011) were retrospectively evaluated and diagnoses were categorized as neoplastic or non‐neoplastic. Lymph nodes from 296 dogs and 71 cats included 157 (42.7%) non‐neoplastic lesions, 62 (16.9%) lymphomas and 148 (40.3%) metastatic neoplasms. Cytology had a sensitivity of 66.6% [95% confidence interval (CI) 60.0–72.8%], specificity of 91.5% (CI 86.3–95.2%), and accuracy of 77.2% (CI 72.6–81.3%) for neoplasia. Likelihood of malignancy with a positive cytologic diagnosis of neoplasia was 93.0%. High proportions of false‐negative results were found in mesenteric T‐cell lymphoma (22/35, 63%, mainly cats), metastatic sarcoma (8/14, 57%) and metastatic mast cell tumour (15/48, 31%, mainly dogs). Factors contributing to discrepancies included well‐differentiated lymphocyte morphology, focal distribution of metastases and poorly defined criteria for metastatic mast cell tumours.     

GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas

The diagnosis of canine lymphoma is achieved using morphological and immunological methods. In a certain percentage of cases, difficulties in making a definitive diagnosis of lymphoproliferative disorders may occur despite extensive immunophenotyping. Therefore, additional diagnostics, such as molecular assessment of Ig/TCR gene rearrangements clonality, may confirm the final diagnosis. Polyacrylamide gel electrophoresis and heteroduplex analysis have already been proven to be suitable for detecting clonality but are cumbersome and labor-intensive. In the present study, GeneScanning analysis of PCR products originating from different primer sets targeting different regions of Ig and TCR was validated in improving sensitivity as well as in reducing the turnaround time of gene rearrangement assays. GeneScanning exploits 5' fluorescently labelled primers for the automated and fast analysis of PCR products either as singleplex or multiplex runs. Initially, the assay was set up using DNA purified from normal tissues (n=6), hyperplastic/reactive tissues (n=10) and a small set of immunophenotyped lymphoma samples (n=12). The optimized methods were then used in a large set of 96 canine lymphoma samples. Normal and hyperplastic/reactive lymphoid tissues showed typically polyclonal or, occasionally, oligoclonal PCR products. Lymphoma samples showed monoclonal peaks arranged as a single or, occasionally, a double narrow base peak sometimes embedded in a polyclonal background. In all immunophenotyped cases, an Ig or TCR clonal finding corresponded to B- and T-cell lymphomas, respectively. Overall, 94/96 (97.9%) samples showed clonal Ig/TCR clonal rearrangements among which clonal Ig was found in 61/96 (63.5%) of samples and clonal TCR in 33/35 Ig negative samples (34.4% of all cases). In one out of ten randomly chosen cases, both Ig and TCR clonal gene rearrangements were found. Among the factors affecting assay accuracy, DNA quality has been shown to be critical and the amplification of DNA controls of different size are recommended to evaluate DNA integrity. Frozen material such as that which remained inside the hub of the needle used for diagnostic procedures is optimal for the analysis herein described. In conclusion, GeneScanning represents a versatile tool for routinely assessing Ig/TCR clonal rearrangements and supporting the diagnostic protocol of canine lymphomas.     

Clinical and pathological classification of canine intraocular lymphoma

The objectives of this retrospective study of 100 dogs with intraocular lymphoma were to describe the histomorphologic and immunohistochemical features of canine intraocular lymphoma, determine the proportion of cases with presumed solitary ocular lymphoma (PSOL) compared to multicentric disease, and assess the clinical outcomes of these patients. Selected cases from Penn Vet Diagnostic Laboratory and Comparative Ocular Pathology Lab of Wisconsin (2004–2015) were evaluated and subtyped using the WHO classification system. Peripheral T‐cell lymphoma and diffuse large B‐cell lymphoma were the two most common subtypes. Questionnaires were distributed to the referring veterinarians and veterinary ophthalmologists inquiring about clinical signs at time of enucleation, staging, patient outcome, treatment, and disease progression. Cases were categorized as PSOL if only ocular involvement was noted at the time of diagnosis based on the clinical staging criteria. The majority of cases (61%) did not have systemic involvement at the time of diagnosis, and these cases did not progress postoperatively. Median survival time (MST) was significantly higher for the presumed solitary intraocular cases: 769 vs. 103 days, hazard ratio of 0.23 (95% CI: 0.077–0.68). The subtype of lymphoma did not affect survival time. The results of this study suggest two significant points of clinical interest: the majority of dogs (61%) presented without signs of systemic involvement of lymphoma at the time of enucleation, and dogs with only ocular involvement showed no disease progression postenucleation.