Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Hypercortisolism is caused by a cortisol‐secreting adrenocortical tumour (ACT) in approximately 15%‐20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol‐secreting ACTs were included from 40 dogs, of which follow‐up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate‐high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT‐PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour‐transforming gene‐1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor‐1 (SF‐1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.     

Multiple endocrine neoplasias in a dog: Corticotrophic tumour,bilateral adrenocortical tumours,and pheochromocytoma

Summary

In a 10‐year‐old ovariohysterectomized standard Schnauzer, the finding of dexamethasone‐resistant hypersecretion of cortisol, the results of computed tomography, and elevated plasma concentrations of ACTH suggested the presence of both adrenocortical tumour and pituitary‐dependent hyperadrenocorticism. The dog made an uneventful recovery after bilateral adrenalectomy and remained in good health for 31/2 years with substitution for the induced hypoadrenocorticism. Then the enlarged pituitary caused neurological signs and eventually euthanasia was performed. The surgically excised right adrenal contained a well‐circumscribed tumour of differentiated adrenocortical tissue and in the left adrenal there were two adrenocortical tumours and a pheochromocytoma. The unaffected parts of the adrenal cortices were well developed and without regressive transformation. At necropsy there were no metastatic lesions. The cells of the pituitary tumour were immunopositive for ACTH and had characteristics of malignancy.

The present combination of corticotrophic tumour, adrenocortical tumours, and pheochromocytoma may be called ‘multiple endocrine neoplasia’ (MEN), but does not correspond to the inherited combinations of diseases known in humans as the MEN‐1 and the MEN‐2 syndromes. It is suggested that the co‐existence of hyperadrenocorticism and pheochromocytoma may be related to the vascular supply of the adrenals. Some chromaffin cells of the adrenal medulla are directly exposed to cortical venous blood, and intra‐adrenal cortisol is known to stimulate catecholamine synthesis and may promote adrenal medullary hyperplasia or neoplasia.     

Inter‐ and intra‐rater reliability and agreement in determining subcutaneous tumour margins in dogs

The objective of this prospective study was to evaluate agreement and reliability of calliper‐based measurements of locally invasive subcutaneous malignant tumours in dogs. Four raters measured the longest diameter of 12 subcutaneous tumours (7 soft tissue sarcomas and 5 mast cell tumours) from 11 client‐owned dogs during 3 randomized, blinded measurement trials, both pre‐ and post‐sedation. Inter‐ and intra‐rater reliability was evaluated using intra‐class correlation coefficient (ICC) and agreement was evaluated using Bland‐Altman plots. Inter‐ and intra‐rater reliability was good (ICC range of 0.8694‐0.89520) and excellent (ICC range of 0.9720‐0.9966), respectively. For agreement calculations, an a priori clinically relevant limit of agreement of 10 mm was set. Inter‐ and intra‐rater agreement was unacceptable with inter‐rater limits of agreement ranging from 15.9 to 55.6 mm and intra‐rater limit of agreement ranging from 11.9 to 28.1 mm. Review of the measurement trial photographs revealed that calliper orientation changes were frequent, occurring in 9/12 (75%) and 8/12 (67%) pre‐ and post‐sedation cases. No significant correlation was found between inter‐rater measurement standard deviations and calliper orientation changes or dog body condition score. These findings suggest veterinarians may have poor agreement in determining the gross edge of tumours, which is expected to introduce bias and inconsistency in tumour staging, assessing response to therapy, and surgical margin planning. Due to the potential consequences for veterinary cancer patients, future studies are needed to validate the present findings.     

Association of Ki67 index with prognosis for intermediate‐grade canine cutaneous mast cell tumours*

Intermediate‐grade mast cell tumours (MCT) represent a heterogeneous population of tumours. The prognosis for the majority of dogs is excellent following surgical excision, but a minority die because of their disease. A previous study identified Ki67 expression as a predictor of prognosis in all three grades of MCT. The purpose of this study was to validate those results in a new group of dogs, with intermediate‐grade MCT only. Ki67 immunohistochemistry was performed on intermediate‐grade MCT from 163 dogs with known outcome. Digital microscopy images were taken from each tumour, and an index calculated of Ki67‐positive cells. Ki67 index as a binary variable with a cut‐off value of 1.8% was confirmed to be associated with prognosis (hazard ratio = 19.1, P < 0.0001) for this cohort of dogs. The 1‐year, 2‐year and 3‐year survival probabilities (with standard errors) of 127 dogs with a Ki67 index ≤1.8% were [0.95 (0.024), similar for all] and for 36 dogs with a Ki67 index >1.8% were 0.54 (0.100), 0.45 (0.101) and 0.33 (0.104), respectively.     

Assessment of biomarkers influencing treatment success on small intestinal lymphoma in dogs

This study aimed to determine a reliable therapeutic biomarker for localized small intestinal lymphoma (SIL) in dogs based on clinical and histopathological features. We retrospectively investigated 84 dogs with localized SIL, including 36 dogs receiving surgery and 48 dogs receiving chemotherapy. The dogs receiving surgery were divided into two subgroups: 18 dogs (group 1) with overall survival (OS) <120 days (median OS) and 18 dogs (group 2) with OS ≥120 days. Correspondingly, the dogs receiving chemotherapy were divided into 24 dogs (group 3) with OS <98 days (median OS) and 24 dogs (group 4) with OS ≥98 days. Clinical, haematological, histopathological and immunohistochemical analyses were comparatively evaluated among the four subgroups. There was no significant difference in OS between the surgery and chemotherapy groups. In dogs receiving surgery, the rate of Ki67‐positive cells was significantly increased in group 1 compared to group 2 and showed no significant difference between groups 3 and 4. In dogs receiving chemotherapy, the rate of O6‐methylguanine‐DNA methyltransferase (MGMT) was significantly higher in group 3 than in group 4 and showed no significant difference between groups 1 and 2. Additionally, our data showed that OS in dogs with higher Ki67 expression might be significantly increased by chemotherapy than by surgery, that of those with higher MGMT expression might be significantly increased by surgery than by chemotherapy, and Ki67 and MGMT were independent of each other. Indices of Ki67 and MGMT are suggested therapeutic biomarkers to determine the optimal first‐line treatment for localized SIL in dogs.     

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%–20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log₂ fold change was >2 or < −2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans.     

Comparison of mitotic index and Ki67 index in the prognostication of canine cutaneous mast cell tumours

Proliferation markers are commonly used for prognostication of mast cell tumours. The aim of the study is to compare the relative abilities of Ki67 and mitotic index to predict survival in the same cohort of dogs with cutaneous MCTs. Histological grade, mitotic index and Ki67 index were performed in all samples and clinical information was obtained by a follow‐up questionnaire. Ninety‐five dogs were included in the study with a median follow‐up of 1145 days. Survival times varied significantly between categories of histological grade, mitotic index and Ki67 index. Multivariable analyses showed that the risk of dying due to MCT was similar in dogs with increased Ki67 index [hazard ratio, HR: 3.0 (95% CI 1.3–6.8)] or increased mitotic index [HR: 2.7 (95% CI 1.1–6.5)]. In conclusion, both mitotic index and Ki67 index were able to independently differentiate MCTs with worse prognosis. This distinction is particularly meaningful in selecting intermediate grade MCTs that may benefit from more aggressive local or systemic treatment.     

Serum Inhibin Concentration in Dogs with Adrenal Gland Disease and in Healthy Dogs

Background

Studies in humans identified the synthesis and secretion of inhibin from adrenocortical tumors, but not pheochromocytoma (PHEO). Inhibin has not been examined in dogs as a serum biomarker for adrenal gland tumors.

Objective

To determine serum inhibin concentration in dogs with adrenal gland disease and in healthy dogs.

Animals

Forty‐eight neutered dogs with adrenal disease including pituitary‐dependent hyperadrenocorticism (PDH, 17), adrenocortical tumor (18), and PHEO (13), and 41 healthy intact or neutered dogs.

Methods

Prospective observational study. Dogs were diagnosed with PDH, adrenocortical tumor (hyperadrenocorticism or noncortisol secreting), or PHEO based on clinical signs, endocrine function tests, abdominal ultrasound examination, and histopathology. Inhibin concentration was measured by radioimmunoassay in serum before and after ACTH stimulation, and before and after treatment.

Results

In neutered dogs, median inhibin concentration was significantly higher in dogs with adrenocortical tumors (0.82 ng/mL) and PDH (0.16 ng/mL) than in dogs with PHEO and healthy dogs (both undetectable). Median inhibin concentration was significantly higher in dogs with adrenocortical tumors than in those with PDH and decreased after adrenalectomy. Median inhibin concentration was significantly higher in intact than in neutered healthy dogs and was similar in pre‐ and post‐ACTH stimulation. Sensitivity, specificity, and accuracy of serum inhibin concentration for identifying an adrenal tumor as a PHEO were 100, 88.9, and 93.6%, respectively.

Conclusions and Clinical Importance

Adrenocortical tumors and PDH but not PHEOs are associated with increased serum inhibin concentration; undetectable inhibin is highly supportive of PHEO in neutered dogs with adrenal tumors.     

Laparoscopic adrenalectomy for treatment of unilateral adrenocortical carcinomas: technique, complications, and results in seven dogs

Objective— To investigate the feasibility of, and outcome after, laparoscopic adrenalectomy in dogs with unilateral adrenocortical carcinoma.
Study Design— Case series.
Animals— Dogs (n=7) with Cushing's syndrome caused by unilateral adrenocortical carcinoma.
Methods— Laparoscopic adrenalectomy with the dog in lateral recumbency on the unaffected side. Three 5-mm portals (1 laparoscopic portal, 2 instrument portals) were placed in the paralumbar fossa. A fourth instrumental portal (5–12 mm) was placed above the kidney. After dissection and hemostatic control of the phrenicoabdominal vein, the adrenal gland was carefully dissected or when there was capsule fragility, necrotic content was partially aspirated. The remaining glandular tissue was removed through the 12-mm trocar site.
Results— Dogs with unilateral adrenocortical carcinoma (3 right-sided, 4 left-sided) without invasion of the caudal vena cava were successfully operated by laparoscopic approach. There were no significant intraoperative complications; 2 dogs died within 48 hours of surgery because of respiratory complications. Five dogs were discharged 72 hours after surgery, and signs of hyperadrenocorticism disappeared thereafter (survival time ranged from 7 to 25 months).
Conclusions— Laparoscopic adrenalectomy is feasible in dogs with either right- or left-sided adrenocortical carcinoma not involving the caudal vena cava.
Clinical Relevance— When performed by experienced surgeons, laparoscopic adrenalectomy offers a minimally invasive alternative to open laparotomy or retroperitoneal surgery for the treatment of unilateral adrenocortical carcinoma in dogs.     

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR‐2, PDGFRs and c‐Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c‐Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.     

Elective and emergency surgical management of adrenal gland tumors: 60 cases (1999-2006)

Sixty-one adrenal gland tumors were surgically removed from 60 dogs. Fifty-two dogs underwent elective adrenalectomy and 8 dogs underwent emergency adrenalectomy for acute adrenal hemorrhage. Size of adrenal tumors ranged from 10 mm to 80 mm. Histopathology confirmed a diagnosis of adrenocortical tumor in 47 dogs, 26 of which were malignant. Pheochromocytoma was diagnosed in 11 dogs. Six dogs had tumor invasion of the caudal vena cava. Of the seven dogs that did not survive the perioperative period, four underwent emergency adrenalectomy. No dogs with tumor invasion of the caudal vena cava died perioperatively. Perioperative morality rates were 5.7% for dogs that underwent elective adrenalectomy and 50% for dogs that underwent emergency adrenalectomy for acute adrenal hemorrhage. Median survival time was 492 days for the 53 dogs that survived the perioperative period. Of the factors analyzed, only adrenal tumor size and the presence of acute adrenal hemorrhage had predictive values for perioperative mortality. Those dogs that survived the perioperative period had extended survival times of up to 1,590 days. The mortality rate associated with elective adrenalectomy in dogs may be lower than previously reported. Dogs with very large tumors or acute adrenal hemorrhage may have a more guarded prognosis.     

A retrospective review of treatment and response of high‐risk mast cell tumours in dogs

This retrospective case series evaluates survival outcome of 94 dogs with high metastatic risk mast cell tumours (MCT). Patients were treated with a cytotoxic chemotherapy protocol or the tyrosine kinase inhibitor masitinib, in the presence of gross disease or as an adjunct to surgical resection of the primary tumour. In patients presenting with metastatic disease, surgical resection of the primary tumour with adjunctive therapy with any chemotherapy incurred a significant survival advantage [median survival time (MST): 278 days] compared to patients receiving chemotherapy without surgical excision of the primary tumour (MST: 91 days, P < 0.0001). Patients with a surgically excised Patnaik grade II tumour and high Ki‐67 in the absence of metastatic disease treated with vinblastine and prednisolone showed a significantly longer survival (MST: 1946 days) than those treated with masitinib (MST: 369 days, P = 0.0037). Further prospective case‐controlled clinical trials of high‐risk MCTs are required to make precise evidence‐based treatment decisions for individual patients.     

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki‐67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki‐67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet‐fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki‐67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki‐67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki‐67 were positively correlated with metastasis, death from neoplasia, low disease‐free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki‐67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki‐67 index in canine mammary tumors by using wet‐fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki‐67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome.     

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.     

Developing and testing prognostic bio‐scoring systems for canine mammary gland carcinomas

Canine mammary carcinomas (CMC) represent a range of histolopathological subtypes with diverse biological behaviours. Several individual factors, including stage, grade, subtypes and presence of invasion, predict outcome. Less is known how these factors interact and impact prognosis. The purpose of this work was to develop and test comprehensive bio‐scoring systems in CMCs. Clinical and histopathological data from 127 dogs with MCs treated through two prospective studies were obtained. All dogs underwent standardized pre‐surgical staging, treatments and regular follow‐up visits. All tumours were evaluated, classified and graded according to published guidelines. Time to primary metastasis was the main endpoint in this study. Two bio‐scoring systems were developed: The multivariate scoring (MVS) was based on traditional statistical analysis where only factors significant in the multivariate analysis (tumour size and grade) were kept for the final model. The refined flexible scoring (RFS) system was based on results from subgroup analysis, which guided the development of a flexible system. Progressive worsening prognosis was observed with increasing bio‐scores in both systems. MVS: Median primary metastasis‐free survival (TTM1 days) was not reached in dogs with bio‐scores 0 to 5, 10, 15 and 648, 149, 317, in MVS groups 25, 30, 40, respectively. Similarly, TTM1 was not reached in dogs with RFS 0, 1, 2 and 374, 407 and 149, in dogs with bio‐scores 3, 4, 5, respectively. However, a more distinct separation between dogs with high risk vs low risk for metastasis was observed with RFS, suggesting superior overall prognostication regarding the risk for metastasis.     

TSLC1 tumour-suppressor gene expression in canine mast cell tumours

Tumour suppressor in lung cancer-1 (TSLC1) is a tumour-suppressor gene coding for an adhesion molecule that is expressed by mast cells. Reduced TSLC1 expression is associated with a poor prognosis in several human tumours, and this study sought to investigate if TSLC1 expression could be used to predict outcome in dogs with mast cell tumours (MCTs). Sections of MCTs of different tumour grades from 45 dogs (Group 1) were immunohistochemically assessed for TSLC1 and Ki67 expression. In addition, 35 intermediate-grade MCTs (Group 2) from dogs with known clinical follow-up were immunohistochemically stained for TSLC1 and Ki67. The TSLC1 staining intensity was found to strongly inversely correlate with tumour grade for Group 1 (P = 0.002857). For Group 2 there was a trend towards dogs with lower TSLC1 scores being more likely to die from MCT-related disease (P = 0.058). The intensity of TSLC1 staining inversely correlated with Ki67 expression for both groups.     

Concentration of extracellular vesicles isolated from blood relative to the clinical pathological status of dogs with mast cell tumours

Extracellular vesicles (EVs) are membrane‐enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki‐67 proliferative index, KIT‐staining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT‐staining pattern and Ki‐67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT‐staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations.     

Pituitary–adrenal axis function in dogs with neoplasia*

Adrenocorticotropic hormone (ACTH) stimulation tests were done in healthy and tumour‐bearing dogs. In the tumour‐bearing dogs, plasma endogenous ACTH (eACTH) concentration was measured and adrenal gland size was assessed ultrasonographically. Measurements in the tumour‐bearing dogs were taken prior to therapy. No difference existed in basal or ACTH‐stimulated cortisol concentration between tumour‐bearing and healthy dogs. No difference existed in eACTH concentration between dogs with non‐haematopoietic neoplasia (NHN) and lymphoma. However, of 20 dogs with lymphoma, 15% had increased basal serum cortisol concentration, 5% had an exaggerated response to ACTH and 5% had an increased eACTH concentration. Of 15 dogs with NHN, 20% had increased basal cortisol concentration, 7% had an exaggerated ACTH response and no dogs had an increased eACTH concentration. Of the dogs with lymphoma and NHN, 5 and 13%, respectively, had decreased basal cortisol concentrations; 20% of dogs with lymphoma and 13% with NHN had a subnormal ACTH response. eACTH levels were below the reference range in 10% of dogs with lymphoma and 7% with NHN. Overall, 10 adrenal glands were enlarged in seven dogs, five with lymphoma and two with NHN. The clinical significance of these findings remains to be determined.     

Clinical,histopathological and immunohistochemical characterization of canine indolent lymphoma

Indolent lymphoma comprises up to 29% of all canine lymphoma; however, limited information exists regarding the subtypes and biological behaviour. This retrospective study describes the clinical characteristics, histopathological and immunohistochemical features, treatment, outcome and prognostic factors for 75 dogs with indolent lymphoma. WHO histopathological classification and immunohistochemistry (IHC) for CD79a, CD3, Ki67 and P‐glycoprotein (P‐gp) was performed. The most common histopathological subtype was T‐zone, 61.7%, (MST 33.5 months), followed by marginal zone, 25%, (MST 21.2 months), P = 0.542. The addition of IHC to preliminary histopathological classification resulted in a revised diagnosis in 20.4% of cases. The use of systemic treatment did not influence survival, P = 0.065. Dogs treated with chlorambucil and prednisone did not reach a MST, compared with a MST of 21.6 months with CHOP‐based chemotherapy, P = 0.057. The overall MST of 4.4 years confirms that this is indeed an indolent disease. However, the effect of systemic treatment must be determined through prospective trials.     

Reproducibility of a semiquantitative method to assess cutaneous cytology

Background – Cutaneous cytology is used in veterinary dermatology to assess bacteria and yeast on the skin surface and in the ears for diagnostic purposes and to monitor treatment success. A number of methods were used in reported studies to quantify micro‐organisms on cytology, but evaluation of the intra‐ and interobserver reliability of the methods is rare. Objective – The aim of this study was to evaluate the intra‐ and interobserver reliability of a semiquantitative cytology assessment method frequently used in practice. Methods – A total of 60 experienced and inexperienced veterinarians and veterinary students were asked to evaluate 10 glass slides and 18 photographs of cutaneous cytology twice. Cocci, rods, yeast, neutrophilic and eosinophilic granulocytes and macrophages were graded from 0 to 4+. Results – The intra‐observer reproducibility for evaluating the slides in the experienced group was 84.3%; in the inexperienced group it was 82.6%. For the photographs, the intra‐observer reproducibility was 92.1% in both groups. The interobserver reproducibility for evaluating the slides was 81.6 and 81.0% in the experienced and inexperienced group, respectively; corresponding values for the photographs were 91.0 and 90.0%. There was no significant difference between different participants or between the first and second evaluation by each participant for any of the parameters graded. Conclusion and clinical importance – Based on these results, this semiquantitative method of grading can be recommended for evaluating and monitoring of antimicrobial therapy in daily practice.