Duncan S. Russell

A phase II study to evaluate the toxicity and efficacy of alternating CCNU and high-dose vinblastine and prednisone (CVP) for treatment of dogs with high-grade, metastatic or nonresectable mast cell tumours.

Safety and efficacy of a protocol of alternating 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; 70 mg m(-2)) and vinblastine (3.5 mg m(-2)), and prednisone (1-2 mg kg(-1); CVP) in dogs with mast cell tumours (MCT) were evaluated. A total of 17 dogs had nonresectable MCTs and 35 received CVP as adjunctive treatment to locoregional control of metastatic MCTs or grade III MCTs. Neutropenia with fever occurred in 8% of dogs after treatment with vinblastine and in 2% after treatment with CCNU. Persistent elevation of serum alanine transaminase, suggestive of hepatotoxicity, occurred in 9% of the dogs. Response rate in dogs with nonresectable MCTs was 65%; five achieved a complete response (median, 141 days) and six achieved a partial response (median, 66 days). Overall median progression-free survival (PFS) time in dogs treated in the adjuvant setting was 489 days. Dogs with grade III MCTs had shorter PFS compared with dogs with metastatic grade II MCTs (190 days versus 954 days; P < 0.001). Phase III studies are needed to provide reliable information about the comparative efficacy of this protocol.

Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib in Canine Models of B-Cell Non-Hodgkin Lymphoma.

Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In this study, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy similar to human diffuse large B-cell lymphoma (DLBCL). First, we demonstrated that acalabrutinib potently inhibited BTK activity and downstream effectors in CLBL1, a canine B-cell lymphoma cell line, and primary canine lymphoma cells. Acalabrutinib also inhibited proliferation in CLBL1 cells. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).

Disseminated Canine Herpesvirus-1 Infection in an Immunocompromised Adult Dog

An 8-year-old, spayed-female Golden Retriever was evaluated at the Cornell University Hospital for Animals for complaints of lethargy, inappetence, and fever 4 days after treatment with doxorubicin. Fifteen weeks before this presentation, the dog had generalized lymphadenomegaly. Ultrasonography revealed diffuse mesenteric lymphadenomegaly, a mildly enlarged liver with multiple small hypoechoic nodules, and small hypoechoic splenic nodules. Lymph node biopsy revealed high-grade lymphoma consistent with a B-cell lineage. The diagnosis was World Health Organization stage IV B-cell lymphoma. Treatment with a CHOP-based protocol was initiated. The dog achieved a complete remission by week 2 of the protocol. During weeks 2 through 15 of the chemotherapy protocol, the dog had nausea, vomiting, and prolonged inappetence, and two documented bouts of afebrile neutropenia, which necessitated treatment delays, dose reductions of vincristine and doxorubicin, as well as symptomatic therapy (metoclopramide, maropitant citrate, and cyproheptadine) and intermittent hospitalization. Approximately 15 weeks after the start of chemotherapy, the dog received the 3rd treatment of doxorubicin (1mg/kg IV). She was discharged with maropitant citrate (4mg/kg PO q24h), cyproheptadine (0.5mg/kg PO q12h), and metoclopramide (0.5mg/kg PO q8h). Despite pre-emptive supportive care, the dog developed persistent anorexia and lethargy. Three days after treatment with doxorubicin she was normothermic, lethargic, and mildly dehydrated. There was anemia (PCV 36%; reference range, 37–55%), leukopenia (5.6 10 cells/mL; reference range, 5.7–14.2 10 cells/mL), lymphopenia (0.1 10 cells/mL; reference range, 0.9–4.7 10 cells/ mL), and thrombocytopenia (168 10 cells/mL; reference range, 186–545 10 cells/mL). The dog was treated with lactated Ringer’s Solution, SQ (1,000mL) and maropitant citrate (1mg/kg SQ), and was discharged with instructions to continue metoclopramide, oral maropitant, and mirtazepine (0.5mg/kg PO q24h). Fluids were dispensed to be continued at home (lactated Ringer’s Solution, 500mL SQ q24h). Four days after treatment with doxorubicin, the dog had worsening lethargy, persistent inappetence, and shaking. She was depressed, dehydrated, and febrile (40.41C). There was progressive nonregenerative anemia (HCT 33%; reference range, 41–60%), neutropenia (2.5 10 cells/uL; reference range, 2.7–9.4 10 cells/uL), and elevation in serum ALP (418U/L; reference range, 12–122) and ALT (1,514U/L; reference range, 25–106 U/L) activity. The dog was treated with PlasmaLyte-A (120mL/kg/day IV), supplemented with potassium chloride (10 mEq KCl/L), ampicillin sodium/sulbactam sodium (22mg/kg IV q8h), enrofloxacin (10mg/kg q24h), famotidine (0.5mg/kg IV q12h), and maropitant sodium (1mg/kg SQ q24h). On hospitalization day 2, she remained febrile (40.51C) and lethargic. Mild chemosis and mucoid discharge were noted in both eyes and melena was present. The dog was anemic (PCV 23%). Metronidazole (10mg/kg IV q12h) was administered. Fresh frozen plasma (10mL/kg) and packed red blood cells (10mL/kg) were infused IV separately over 4 hours. On hospitalization day 3, the dog remained febrile (40.51C) and depressed. Petechiae and ecchymoses were noted in her oral cavity, inner pinnae, and ventrum. There was neutropenia (0.1 10 cells/mL) and thrombocytopenia (7 10 cells/mL). Thoracic radiographs revealed consolidation of the right middle lung lobe and air bronchograms. Cytology of an endotracheal lavage revealed suppurative inflammation with both intracellular and extracellular, Gram-negative bacteria through aerobic culture revealed only fastidious organisms. Nebulization with sterile saline was instituted 4 times daily. Because of the persistent pyrexia, ampicillin sodium/ sulbactam sodium and enrofloxacin were discontinued and cefazolin (22mg/kg IV q8h) and amikacin (20mg/ kg IV q24h) were administered. Additional fresh frozen plasma was administered (10mL/kg). There was persistent elevation of serum ALP (1,545U/L), and ALT (992U/L) activity. Total bilirubin was also elevated (0.9mg/dL; reference range 0.0–0.3mg/dL). Abdominal ultrasonography revealed mild hepatomegaly. Complete ophthalmic examination, including slit-lamp biomicroscopy and indirect ophthalmoscopy, revealed bilateral severe ulcerative conjunctivitis associated with conjunctival From the Departments of Clinical Sciences (Malone, Ledbetter, Rassnick), Biomedical Sciences (Russell), and Population Medicine and Diagnostic Sciences (Kim), College of Veterinary Medicine, Cornell University, Ithaca, NY. Corresponding author: Dr Erin K. Malone, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; e-mail: ekm24@cornell.edu. Submitted September 23, 2009; Revised February 11, 2010; Accepted March 1, 2010. Copyright r 2010 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2010.0512.x Abbreviations:

Calcitriol (1,25‐dihydroxycholecalciferol) enhances mast cell tumour chemotherapy and receptor tyrosine kinase inhibitor activity in vitro and has single‐agent activity against spontaneously occurring canine mast cell tumours*

Calcitriol potentiates the effect of multiple chemotherapy agents in a variety of tumour models. In this study, we examine whether calcitriol increases chemotherapy or tyrosine kinase inhibitor in vitro cytotoxicity in canine mastocytoma C2 cells. We also evaluate the in vivo effect of DN101, a highly concentrated oral formulation of calcitriol designed specifically for cancer therapy, as a single-agent therapy in dogs with mast cell tumours (MCTs). Calcitriol exhibits synergistic, antiproliferative activity when used in combination with CCNU, vinblastine, imatinib or toceranib in vitro. The concentrations required for 50% growth inhibition were generally two- to six-fold lower when the drugs were used in combination than when used individually. High-dose oral calcitriol induced remission in 4 of 10 dogs (one complete remission, three partial remissions), although the majority experienced toxicity, necessitating discontinuation of the trial. Further evaluation of calcitriol in combination therapy for dogs with MCTs is warranted.

Surgical margins in the veterinary cancer patient

In veterinary oncologic specimens, histopathology is the gold standard for determining adequacy of excision. Despite limitations of this technique, the pathologists interpretation of margin status significantly impacts patient management, including indications for adjuvant therapy. This article aims to summarize peer-reviewed literature as it relates to histologic margin evaluation in veterinary cancer patients. The value of histologic tumour-free margins and technical factors influencing histopathologic margin outcomes are also discussed. We review alternative strategies for determining excisional status, and discuss how an evolving understanding of tumour biology might inform clinical and research perspectives on surgical margins. In doing so, we aim to provide context and a stimulus for future investigations into this important yet incompletely understood topic.

A novel hydrogel-coated polyester mesh prevents postsurgical adhesions in a rat model.

BACKGROUNDnThe specific aim of this study was to determine the whether a novel, hydrogel-coated polyester mesh (Scout) can be used to reduce the incidence and severity of adhesion formation in vivo.nnnMETHODSnAn established rat model of post-surgical adhesion formation was used in which adhesions are generated through surgical trauma to the surfaces of the cecum and the adjacent abdominal wall. Thirty-seven rats were randomly allocated either to a control group (no intervention; n=14 rats) or to one of two treatment groups in which the abraded surfaces were separated with either the Scout material (n=11 rats) or an FDA-approved form of expanded polytetrafluorethylene (PTFE) (PRECLUDE Vessel Guard; n=12 rats). Animals were euthanized 7 d after surgery and gross necropsy examinations were performed. Mechanical testing was used to measure the strength of any adhesions that were identified, and histology was used to characterize within the adhesion tissue and on the surface(s) of the barrier materials.nnnRESULTSnFive animals were excluded because of surgical failure (1 control; 2 PRECLUDE Vessel Guard; 2 Scout). Adhesions were seen in 10 of 13 control animals (77%). There were no adhesions in any of the animals treated with either PRECLUDE Vessel Guard or Scout material. Histology demonstrated mild cellular adhesion to both the PRECLUDE Vessel Guard and the Scout material. Although there was a sub-acute to chronic inflammatory response to the surgical trauma, there was no evidence of delamination, shearing, or degradation of either the Scout material or PRECLUDE Vessel Guard.nnnCONCLUSIONSnThe hydrogel-coated Scout material was as effective as the approved predicate material in this model. Both materials were well tolerated. Further testing of the Scout material is now warranted.

Shaved margin histopathology and imprint cytology for assessment of excision in canine mast cell tumors and soft tissue sarcomas

OBJECTIVEnTo determine the feasibility and agreement of margin assessment by imprint cytology, shaved margin histopathology, and radial section histopathology in canine cutaneous and subcutaneous mast cell tumors (MCT) and soft tissue sarcomas (STS).nnnSTUDY DESIGNnProspective clinical study.nnnSAMPLE POPULATIONnThree hundred and forty margins from 72 excised tumors (52 MCT and 20 STS) in 54 client-owned dogs.nnnMETHODSnImprint cytology samples were acquired by pressing glass slides to the cut surgical margin of the freshly excised surgical specimen. Shaved margin samples were obtained from the patient wound bed using a scalpel immediately prior to closure. Radial section histopathology was performed as part of routine histopathologic processing. All margins were assessed as either positive or negative for presence of tumor cells at the surgical margin. Agreement among methods was calculated using Fleiss Kappa coefficients and an association of method, margin direction, and tumor type with positive margin status was evaluated using a general linear mixed model.nnnRESULTSnPositive margin detection rates differed for MCT (imprint cytology 21%, radial section histopathology 9%, and shaved margin histopathology 3%; Pu2009<u2009.0001) but not for STS. Intermethod agreement was poor (Fleiss Kappau2009=u20090.051 and 0.176 for MCT and STS, respectively). Margin direction did not influence margin status for either tumor type.nnnCONCLUSIONnImprint cytology and shaved margin histopathology are feasible, but their results are frequently disparate from routine radial section histopathology. Future studies are needed to evaluate the correlation of each method with local recurrence rates.

Extravasation injury associated with parenteral nutrition in a cat with presumptive gastrinomas

OBJECTIVEnTo describe the pathologic consequences of parenteral nutrition (PN) extravasation into the mediastinum of a cat.nnnCASE SUMMARYnAn 8-year-old domestic short hair cat with persistent vomiting and anorexia was initiated on PN for nutritional support. PN was being administered at a rate of 12.9 mL/h when inadvertent jugular catheter migration resulted in thrombophlebitis and cellulitis and 40-80 mL of PN extravasated into the SC and mediastinal tissues. The cat was euthanized 36 hours after the extravasation of PN due to poor prognosis related to the gastric complications associated with a presumed primary gastrinoma. Grossly there was excessive mediastinal lymphatic drainage and pronounced edema in the cervical SC and mediastinal tissue. Histopathologic examination of the PN-extravasated area revealed a severe mixed inflammatory reaction, represented by a severe neutrophilic and mild histiocytic infiltrate with lymphoplasmacytic perivascular cuffing. No bacterial agents were observed or cultured from this area.nnnUNIQUE INFORMATION PROVIDEDnThis is the first case report of a foreign body-type reaction due to extravasation of PN (extravasation injury) in a cat. Extravasation of PN is not without pathologic consequence, and can result in a severe inflammatory reaction in affected tissues.Objective – To describe the pathologic consequences of parenteral nutrition (PN) extravasation into the mediastinum of a cat. n n n nCase Summary – An 8-year-old domestic short hair cat with persistent vomiting and anorexia was initiated on PN for nutritional support. PN was being administered at a rate of 12.9xa0mL/h when inadvertent jugular catheter migration resulted in thrombophlebitis and cellulitis and 40–80xa0mL of PN extravasated into the SC and mediastinal tissues. The cat was euthanized 36 hours after the extravasation of PN due to poor prognosis related to the gastric complications associated with a presumed primary gastrinoma. Grossly there was excessive mediastinal lymphatic drainage and pronounced edema in the cervical SC and mediastinal tissue. Histopathologic examination of the PN-extravasated area revealed a severe mixed inflammatory reaction, represented by a severe neutrophilic and mild histiocytic infiltrate with lymphoplasmacytic perivascular cuffing. No bacterial agents were observed or cultured from this area. n n n nUnique Information Provided – This is the first case report of a foreign body-type reaction due to extravasation of PN (extravasation injury) in a cat. Extravasation of PN is not without pathologic consequence, and can result in a severe inflammatory reaction in affected tissues.

Comparison of histologic margin status in low‐grade cutaneous and subcutaneous canine mast cell tumours examined by radial and tangential sections

BACKGROUNDnRadial sections are widely used to estimate adequacy of excision in canine cutaneous mast cell tumours (MCTs); however, this sectioning technique estimates only a small fraction of total margin circumference. This study aimed to compare histologic margin status in grade II/low grade MCTs sectioned using both radial and tangential sectioning techniques.nnnMATERIALS AND METHODSnA total of 43 circumferential margins were evaluated from 21 different tumours. Margins were first sectioned radially, followed by tangential sections. Tissues were examined by routine histopathology.nnnRESULTSnTangential margin status differed in 10 of 43 (23.3%) margins compared with their initial status on radial section. Of 39 margins, 9 (23.1%) categorized as histologic tumour-free margin (HTFM) >0 mm were positive on tangential sectioning. Tangential sections detected a significantly higher proportion of positive margins relative to radial sections (exact 2-tailed P-value = .0215). The HTFM was significantly longer in negative tangential margins than positive tangential margins (mean 10.1 vs 3.2 mm; P = .0008). A receiver operating characteristic curve comparing HTFM and tangentially negative margins found an area under the curve of 0.83 (95% confidence interval: 0.71-0.96). Although correct classification peaked at the sixth cut-point of HTFM ≥1 mm, radial sections still incorrectly classified 50% of margins as lacking tumour cells. Radial sections had 100% specificity for predicting negative tangential margins at a cut-point of 10.9 mm.nnnCONCLUSIONnThese data indicate that for low grade MCTs, HTFMs >0 mm should not be considered completely excised, particularly when HTFM is <10.9 mm. This will inform future studies that use HTFM and overall excisional status as dependent variables in multivariable prognostic models.

Analysis of stress factors associated with KHV reactivation and pathological effects from KHV reactivation

Koi herpesvirus (KHV) is a highly pathogenic virus of common carp and koi. KHV becomes latent in recovered koi or exposed koi without symptoms, and the latent infection can reactivate under stress conditions. KHV reactivation from latency often occurs when water temperature rapidly rises above 17°C. Dissolved O2 is lower at ≥17°C than at non-stress temperatures ≤15°C. To determine whether reduced dissolved O2 level has a role in KHV reactivation during temperature stress, KHV reactivation was investigated in KHV latently infected koi (KHV+ koi) under stress temperatures by maintaining dissolved O2 consistent with the O2 level at 15°C. There was no significant difference in the amount of reactivated virus between KHV+ koi maintained with and without O2 supplementation during temperature stress. Both handling and sampling were found to be stressful to koi and can contribute to KHV reactivation from latency. There was an increase in KHV genome within white blood cells (WBC) during KHV reactivation, which is about 3-4 fold higher than the amount of KHV genome detectable in WBC during the latency stage. At day 15 post-temperature stress (PTS), inflammation and necrosis were observed in multiple tissues, especially in the gills, eye, intestine, skin and kidney. KHV DNA was also detectable in multiple tissues on days 6, 9 and 15 PTS. Following day 3 PTS, the plasma cortisol levels were higher than that observed in koi before temperature stress, suggesting that KHV reactivation is associated with physiological stress in KHV+ koi.