Proteomic profiling using SELDI‐TOF mass spectrometry: a diagnostic tool for canine B‐cell lymphoma

Introduction: Surface enhanced laser desorption ionization time of flight (SELDI‐TOF) mass spectrometry is a powerful new tool for biomarker discovery and diagnostic test development. In this process, proteins in biological samples are selectively bound to chip surfaces that have various chemistries and then subjected to mass spectrometry. Selectively bound proteins are separated by mass and the relative quantities of each protein compared among samples. Unlike conventional diagnostic test formats that commonly use single biomarkers, SELDI‐TOF technology allows multiple biomarkers to be used in a single assay to improve sensitivity and specificity. This technology has been used to improve diagnostic tests for human prostate and ovarian cancers. The objective of this study is to investigate the utility of this technology for the proteomic profiling of canine B‐cell lymphoma. Developing a diagnostic serum screening test for B‐cell lymphoma in dogs would be valuable as early diagnosis and treatment may confer a more favorable outcome. Methods: Serum samples were collected from 26 dogs diagnosed with B‐cell lymphoma prior to treatment and from 26 apparently healthy dogs that were similar in age, breed, and sex to the test group. Serum proteins were selectively bound to weak cationic, strong anionic, and nickel chelating chip surface chemistries under optimized buffer conditions, and subjected to mass spectrometry. The protein profiles were then compared using Biomarker Wizard and classification trees developed using Biomarker Patterns software from Ciphergen Biosystems, Inc (Fremont, CA). Results: To date several putative biomarkers have been identified. These putative biomarkers have been used to build classification tree models that predict disease in test samples. Preliminary results using cross‐validation of the test and control sample sets indicate that trees built with two or more biomarkers have greater than 90% sensitivity and specificity. Conclusions: Proteomic profiling using SELDI‐TOF technology in canine serum is feasible. Further testing is planned to confirm these initial results.     

Oral melphalan for the treatment of relapsed canine lymphoma

Oral melphalan has been included in multi‐agent rescue protocols for canine lymphoma but its activity as a single‐agent for this purpose has not been established. Inexpensive cost, ease of administration and tolerability make oral melphalan an attractive candidate for single‐agent rescue therapy of canine lymphoma. Retrospective evaluation of 19 cases of relapsed canine lymphoma treated with oral melphalan was performed. Melphalan was primarily administered (n = 16) via a high dose protocol (HDM ) with a median dosage of 19.4 mg m^?2. Fifteen dogs (78.9%) were treated concurrently with corticosteroids. Response evaluation was possible for all dogs with a calculated overall clinical benefit (partial response [PR ] + stable disease [SD ]) of 31.6% (PR 3/19; SD 3/19). Times to progression following melphalan (TTP ‐M) were 14, 24 and 34 days for responders and 20, 28 and 103 days for dogs experiencing SD . Twelve of 17 dogs evaluable for toxicity experienced an adverse event (AE ) with only 3 dogs experiencing a grade III or higher AE . Haematologic toxicity was common (11/17) while gastrointestinal toxicity was rare (1/17). Although treatment resulted in limited clinical benefit and non‐durable responses, oral melphalan was well‐tolerated and may be a reasonable rescue option in cases where minimal effective agents remain.     

Efficacy and toxicity of BOPP and LOPP chemotherapy for the treatment of relapsed canine lymphoma*

Mechlorethamine (Mustargen^®, Oncovin^® (vincristine), procarbazine and prednisone (MOPP) chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of MOPP after substitution of CCNU (lomustine, LOPP protocol) or BCNU (carmustine, BOPP protocol) for mechlorethamine in 60 dogs with relapsed lymphoma. Seven of 14 (50%) dogs treated with BOPP responded, for a median of 129.5 days for complete responders (range 9–354 days) and a median of 140 days for partial responders (range 4–276 days). Twenty‐three of 44 (52%) dogs treated with LOPP responded for a median of 112 days for complete responders (range 48–250 days) and a median of 84.5 days for partial responders (range 69–290 days). Two dogs receiving a combination of LOPP and BOPP partially responded for 28 and 163 days, respectively. With BOPP chemotherapy, nine dogs (20.5%) and seven dogs (50%) had one or more episodes of Grade II or higher neutropenia and thrombocytopenia, respectively. Seven dogs (50%) had one or more episodes of Grade II or higher gastrointestinal toxicity. While receiving LOPP chemotherapy, 28 dogs (63.6%) and 17 dogs (38.6%) had one or more episodes of Grade II or higher neutropenia and thrombocytopenia, respectively. Seventeen dogs (38.6%) had one or more episodes of Grade II or higher gastrointestinal toxicity. Overall, there were 17 non‐fatal treatment‐related episodes of sepsis requiring hospitalization. Eight dogs (13%) died or were euthanized because of treatment‐related sepsis and/or chemotherapy‐related complications. Severe haematologic toxicity, coupled with the improved response duration observed in dogs receiving reduced doses during B/L‐OPP rescue, underscores the need for protocol optimization.     

Rabacfosadine for relapsed canine B‐cell lymphoma: Efficacy and adverse event profiles of 2 different doses

Rabacfosadine (RAB), a novel double prodrug of the acyclic nucleotide phosphonate PMEG, preferentially targets neoplastic lymphocytes with reduced off target toxicity. Historical studies have suggested that every 21‐day dosing is effective with acceptable toxicity. The purpose of this study was to evaluate RAB’s safety and efficacy at 2 different doses every 21 days in dogs with relapsed B‐cell lymphoma. Dogs that had failed 1 doxorubicin‐based chemotherapy protocol were eligible for inclusion in this prospective trial. Once enrolled, dogs were randomized to receive RAB at either 0.82 mg/kg or 1.0 mg/kg as a 30‐minute IV infusion every 21 days for up to 5 treatments. Response assessment and adverse event (AE) evaluation were performed every 21 days via VCOG criteria. Fifty dogs were enrolled, with 16 treated at 0.82 mg/kg and 34 treated at 1.0 mg/kg. The overall response rate was 74%, with 45% of dogs experiencing a complete response (CR). The median progression free intervals (PFIs) were 108 days, 172 days and 203 days for all dogs, all responders, and all CRs, respectively. Response rates and PFIs were similar in both treatment groups. The incidence of AEs, dose delays, dose reductions and withdrawals were not statistically different between the 2 groups. The AEs observed were similar to those previously reported and included hematologic, gastrointestinal, dermatologic and pulmonary AEs. One dog had grade 5 pulmonary fibrosis; otherwise, AEs resolved with supportive treatment. Rabacfosadine is a generally well tolerated, effective chemotherapy option for dogs with relapsed B‐cell lymphoma.     

Evaluation of a discontinuous treatment protocol (VELCAP-S) for canine lymphoma

Eighty-two dogs with lymphoma received a single 15-week course of chemotherapy, after which treatment was ceased until relapse. Fifty-six dogs (68%) achieved complete remission for a median 1st remission duration of 20 weeks. Forty-eight dogs relapsed, of which 30 repeated the induction cycle. In 22 of these dogs, 1st remission had been short, and they received maintenance chemotherapy; the other 8 dogs received 2 or 3 cycles of induction chemotherapy. Second remission rate for these 30 dogs was 87% (26 dogs). Overall disease control for the 38 dogs that remained on protocol was 44 weeks, which was not markedly shorter than for dogs treated with a previously reported protocol in which maintenance chemotherapy was instituted in all dogs after an identical 1st induction (VELCAP-L). Dogs that were febrile and dogs that were dyspneic were less likely to achieve a complete remission to induction chemotherapy. Of dogs that achieved a complete remission, those that were thrombocytopenic at entry had a shorter 1st remission, and dogs that were anorexic at entry had shorter overall disease control. There was a correlation between 1st remission duration and length of any subsequent remission obtained. The incidence of toxicity was high, particularly after the combination of doxorubicin and vincristine. Dose reductions because of toxicity did not markedly reduce remission duration. We conclude that discontinuous chemotherapy may reduce patient visits in a small number of patients because of long-term disease control. Delaying maintenance chemotherapy until after 2nd remission is achieved does not markedly affect overall disease control.     

Correlation of histologic grading of canine mast cell tumors with Ki67/PCNA/AgNOR/c‐Kit scores: 38 cases (2002–2003)

Introduction: The Patnaik grading system for canine cutaneous MCT is currently one of the best determinants of prognosis; however, clinical outcome does not always correlate with histologic grade. The development of molecular markers offers a potential advantage and may complement subjective grading. The primary purpose of this study was to correlate histologic grading to Ki67/PCNA/AgNOR/c‐Kit scores. Methods: Thirty‐eight dogs with cutaneous MCT underwent surgical resection. Tumors were graded, with expansion of grade II MCT to low, medium (or II only) and high. For statistical purposes, MCT grade I, II (low, medium, high) and III were assigned a score of 1, 2, 3, 4, or 5, respectively. Sections were processed for AgNOR staining and expression of PCNA, Ki67 and c‐Kit as previously described (modified biotin‐strepavidin with DAB substrate). Paraffin‐embedded canine tissue arrays were used as positive and negative controls (primary antibody replaced with pre‐immune sera). Parametric statistical testing was performed using Statview statistical software with P ≤ .05 as significant. Results: There were 12, 20, 5 and 1 grade II low, grade II medium, grade II high and grade III MCT, respectively. The mean Ki67 score was 9.114 (median 8.0, range 1–28), mean PCNA score was 26.25 (median 24.0, range 5–65), mean AgNOR score was 1.499 (median 1.35, range 1.02–2.76) and c‐Kit scores were +1 (9/37), +2 (19/37) and +3 (9/37). With parametric statistical testing, significantly positive correlations were found for Ki67/Grade, PCNA/Grade, AgNOR/Grade, Ki67/PCNA, Ki67/AgNOR and PCNA/AgNOR (all P < .0001). No significant correlation was found for c‐Kit and grade; however, +3 c‐Kit scores had statistically significantly higher grades than +2 c‐Kit scores (P = .0458). Conclusions: Ki67/PCNA/AgNOR scores are positively correlated to grade in dogs with MCT. Further studies to correlate Ki67/PCNA/AgNOR/c‐Kit scores with clinical variables are ongoing.     

Clinical outcomes of 30 cases (1997-2004) of canine gastrointestinal lymphoma

In 30 cases of canine gastrointestinal lymphoma, each case was localized to the stomach, small intestine, large intestine, or two or more sites. Treatments consisted of surgery alone (n=4), surgery with chemotherapy (n=8), chemotherapy alone (n=15), or supportive care (n=3). Four dogs died, 24 were euthanized, and two are currently alive. Median survival time for all cases was 13 days. In both surviving dogs, the site of lymphoma was the large intestine. Canine gastrointestinal lymphoma is a severe disease that warrants a poor to grave prognosis. However, cases of colorectal origin may have longer survival times.     

Efficacy and toxicity of carboplatin and cytarabine chemotherapy for dogs with relapsed or refractory lymphoma (2000–2013)

Medical records of 22 dogs treated with carboplatin (n = 8) or carboplatin and cytarabine (n = 14) chemotherapy for relapsed or refractory lymphoma between 2000 and 2013 were retrospectively reviewed. The clinical response rate was 18.2% (4/22). Median time to progression was 18 days (56 for responders; 12 for non‐responders, P = 0.0006). Median overall survival time was 28 days (109 for responders; 21 for non‐responders, P = 0.0007). Thrombocytopenia and neutropenia occurred in 84.2% (16/19) and 52.6% (10/19), respectively. Grade IV thrombocytopenia and neutropenia occurred in 56.3% (9/16) and 60.0% (6/10), respectively. Dogs that received both drugs were more likely to become neutropenic (P = 0.022) or thrombocytopenic (P = 0.001) than dogs receiving carboplatin alone. All responders received both drugs giving a 28.6% (4/14) response rate for the combination. Although some dogs responded to the combination, toxicity was high and the responses were not durable. With adequate supportive care, this protocol may be an acceptable rescue option for some dogs.     

Efficacy and toxicity of paclitaxel (Taxol) for the treatment of canine malignant tumors

Paclitaxel (Taxol) was administered to 25 dogs with histologically confirmed malignant tumors at a dosage of 165 mg/m2 i.v. over 3-6 hours every 3 weeks. Dogs received premedication with antihistimines and corticosteroids to reduce hypersensitivity reactions. However, 64% of the dogs still experienced allergic reactions. Six dogs (24%) had grade 3 or 4 neutropenia, 6 dogs (24%) required hospitalization and 3 dogs (12%) died of sepsis. Five dogs (20%) had a partial response (osteosarcoma [2 dogs] mammary carcinoma [2 dogs] and malignant histiocytosis [1 dog]) for a median duration of 53 days. The overall toxicity was unacceptable at the 165 mg/m2 dose. Therefore, subsequent evaluations of paclitaxel in tumor-bearing dogs should a starting dose of 132 mg/m2 i.v. every 3 weeks.     

Mechlorethamine,vincristine, melphalan and prednisone (MOMP) for the treatment of relapsed lymphoma in dogs

Eighty‐eight dogs with relapsed lymphoma were treated with the MOMP (mechlorethamine, vincristine, melphalan and prednisone) protocol on a 28‐day treatment cycle. The overall response rate (ORR) to the MOMP protocol was 51.1% for a median of 56 days (range 7–858 days). Twelve percent of dogs experienced a complete response for a median of 81 days (range 42–274 days) and 38.6% experienced a partial response for a median of 49 days (range 7–858 days). Dogs with T‐cell lymphoma had an ORR of 55% for a median of 60 days (range 49–858 days) while those with B‐cell lymphoma had an ORR of 57% for a median of 81 days (range 7–274 days) (P = 0.783). The overall survival time for all dogs was 183 days (range 17–974 days). Fifty‐four percent of dogs experienced toxicity with the majority classified as grade I. The MOMP protocol seems well‐tolerated and is an option for dogs with relapsed lymphoma.     

Treatment of canine mast cell tumours with vinblastine, cyclophosphamide and prednisone: 35 cases (1997–2004)

The purpose of this retrospective study was to determine the efficacy and toxicity of a combined protocol of vinblastine, cyclophosphamide and prednisone (VCP) in 35 dogs with mast cell tumours (MCTs). Eleven dogs had measurable disease (group 1) and 24 dogs had incompletely excised MCT or were at high risk for metastasis (group 2). Five patients in group 1 achieved complete response, two partial responses, two stable diseases and two progressive diseases. The median progression‐free survival time (PFST) for group 1 and 2 dogs was 74 and 865 days, respectively. The median overall survival time (OST) for group 1 and 2 dogs was 145 and >2092 days, respectively. Significant negative multivariate prognostic factors included macroscopic disease and reduced vinblastine (VBL) treatments for PFST, and presence of MCT in bone marrow analysis, Patnaik grade III MCT and reduced VBL treatments for OST. Toxicity was infrequent and self‐limiting. This study suggests that the VCP protocol should be considered as an option in the treatment of MCT in dogs.     

CHOP chemotherapy for the treatment of canine multicentric T-cell lymphoma

Dogs with multicentric T-cell lymphoma are commonly treated with CHOP chemotherapy protocols that include cyclophosphamide, doxorubicin, vincristine and prednisone. The purpose of this study was to evaluate the use of CHOP chemotherapy for dogs with multicentric T-cell lymphoma. Identification of prognostic factors in this specific subset of dogs was of secondary interest. Twenty-three out of 24 dogs responded to CHOP chemotherapy and these dogs remained on the protocol for a median of 146 days. No variable was associated with progression free survival (PFS) including stage, substage, hypercalcemia or radiographic evidence of a cranial mediastinal mass. The median overall survival time (OST) for all dogs was 235 days. Dogs that were thrombocytopenic at presentation experienced a significantly longer OST (323 versus 212 days, P=0.01).     

Evaluation of a multidrug chemotherapy protocol with mitoxantrone based maintenance (CHOP-MA) for the treatment of canine lymphoma

The purpose of this study was to evaluate the efficacy of adding mitoxantrone to a cyclophosphamide, doxorubicin, vincristine, l -asparaginase and prednisone containing protocol. Sixty-five dogs with multicentric lymphoma were evaluated for overall remission and survival times. Remission and survival time versus stage, substage, pretreatment hypercalcaemia and pretreatment steroid administration were also evaluated. Overall median remission for dogs with multicentric lymphoma was 302 days and overall median survival was 622 days. Of the dogs with multicentric lymphoma, 23 (35%) received all scheduled mitoxantrone doses. Only median survival versus substage was found to be significant (substage a median survival was 679 days and substage b median survival was 302 days, P = 0.025). Increasing the total combined dose of doxorubicin and mitoxantrone may improve remission times when compared with historical controls, and further studies are needed to determine how best to utilize mitoxantrone in multidrug chemotherapy protocols for canine multicentric lymphoma.     

A combination chemotherapy protocol with MOPP and CCNU consolidation (Tufts VELCAP-SC) for the treatment of canine lymphoma

A chemotherapy protocol using a consolidation phase of alkylating agents was used for treating 94 dogs with lymphoma. Fifty‐seven percent of dogs were in stage V, 63% were ill and 38% had T‐cell lymphoma. The complete remission (CR) rate was 70% and is comparable to results achieved with previously published chemotherapy protocols. Anorexia predicted the remission; of the 40 dogs without anorexia, 35 (88%) achieved CR whereas of 52 dogs with anorexia, 30 (58%) achieved CR. Median first CR duration was 168 days and 1‐ and 2‐year CR rates were 17.4 and 15.5%, respectively. Platelet count affected length of first CR, with a 53.2% reduced chance of coming out of remission with each log increase in platelet count. Median survival time was 302 days. One and 2‐year survival rates were 44 and 13%, respectively. Anorexia and no dose reduction of any drug were independent negative variables. Of 93 dogs with toxicity data, 65 dogs (70%) required a dose reduction. Cyclophosphamide was most commonly reduced with reductions in 31 (38%) of 82 dogs. A dose reduction was significantly more likely in dogs with B‐cell lymphoma than in those with T‐cell lymphoma.     

Response of canine cutaneous epitheliotropic lymphoma to lomustine (CCNU): a retrospective study of 46 cases (1999-2004)

BACKGROUND: Epitheliotropic lymphoma (ELSA) is an uncommon cutaneous canine malignancy of T lymphocytes. A consensus regarding the therapeutic standard of care is lacking, warranting evaluation of chemotherapeutic agents traditionally employed against canine nodal lymphoma in the treatment of ELSA. HYPOTHESIS: The purpose of this retrospective, multi-institutional study was to evaluate the efficacy of 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in the treatment of ELSA. ANIMALS: Forty-six dogs with adequate follow-up and treatment response information. METHODS: All cases were diagnosed histopathologically. Immunohistochemisty (CD3, CD79a) was performed on 42/46 samples. RESULTS: Presenting skin lesions included generalized scales (25/46), plaques or nodules (22/46), mucocutaneous lesions (14/ 46), and corneal involvement (1/46). Lymph node involvement and Sézary syndrome were documented in 7 and 2 dogs, respectively. The median number of CCNU treatments was 4 (range, 1-11), with a median starting dose of 60 mg/m(2) (range, 30-95). Of the 46 dogs, 15 achieved complete remission, 23 achieved partial remission, 5 had stable disease, and 3 had progressive disease, for an overall response rate of 83%. The median number of treatments to achieve a response was 1 (range, 1-6). The overall median duration of response was 94 days (range, 22-282). Sixteen dose reductions were required because of neutropenia (10/46), thrombocytopenia (1/46), anemia (1/46), increased liver enzyme activity (3/46), or unspecified reasons (1/46). CONCLUSIONS AND CLINICAL IMPLICATIONS: Given the high response rate and well tolerated protocol, prospective studies are warranted to investigate the utility of CCNU alone or in multi-agent protocols for the treatment of ELSA.     

Efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: 70 cases (1989–1992)

Summary: In this retrospective study, postoperative ileus was studied in horses having resection of the small intestine followed by a jejunojejunal (n=35) or a jejunocaecal (n=35) anastomosis. Twenty-six horses received no metoclopramide, 27 received metoclopramide as an intermittent intravenous infusion and 17 horses received metoclopramide as a continuous intravenous infusion (0.04 mg/kg/hour). Horses receiving a continuous infusion of metoclopramide had a reduced total volume (P< 0.001), shorter duration (P< 0.001), and a slower rate (P< 0.001) of postoperative gastric reflux, and a shorter postoperative hospital stay (P< 0.01) when compared to horses receiving no metoclopramide and horses receiving metoclopramide as an intermittent infusion. Horses having jejunocaecostomy performed had a larger volume (P< 0.05), longer duration (P< 0.05), and a greater rate (P< 0.05) of postoperative gastric reflux, and a longer postoperative hospital stay (P< 0.001) than horses undergoing jejunojejunostomy. Horses that had an abdominal drain placed during surgery had a longer length of intestine resected (P< 0.05) and a longer postoperative hospital stay (P< 0.05) than horses without an abdominal drain. Horses that died or were euthanased during the postoperative period had a greater total volume (P< 0.05), longer duration (P< 0.05), and greater rate of postoperative gastric reflux (P< 0.01), a longer length of small intestine resected (P< 0.01), and a shorter postoperative hospital stay (P< 0.05) than horses that survived until discharge from the hospital. In a multivariate regression analysis the continuous infusion of metoclopramide was the only variable that contributed significantly to predicting the total volume (P< 0.001; r²= 0.24), duration (P< 0.001; r²= 0.24) and rate (P< 0.001; r²= 0.25) of postoperative gastric reflux. We concluded that metoclopramide given as a continuous intravenous infusion decreased the incidence and severity of ileus following small intestinal resection and anastomosis in horses.