Brenda S. Phillips

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses.

Clostridium novyi-NT targets aberrant tumor physiology and can produce a precise, robust, and reproducible antitumor response. Fighting Cancer with Clostridium Tumors are composed of necrotic, hypoxic, and well-oxygenated regions. Hypoxic tumor regions are more resistant to systemic anticancer agents and radiotherapy. However, they provide a fertile ground for the growth of anaerobic bacteria. Roberts et al. use an attenuated strain of the anaerobic, spore-forming bacterium Clostridium novyi (C. novyi-NT) and demonstrate precise, robust, and reproducible antitumor responses when C. novyi-NT spores were injected into tumors of rats, dogs, and one human patient. These results support the further development of intratumoral injections of C. novyi-NT spores as a therapeutic for patients with locally advanced, nonresectable cancers. Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were observed in 6 of 16 dogs (37.5%), with three complete and three partial responses. On the basis of these encouraging results, we treated a human patient who had an advanced leiomyosarcoma with an intratumoral injection of C. novyi-NT spores. This treatment reduced the tumor within and surrounding the bone. Together, these results show that C. novyi-NT can precisely eradicate neoplastic tissues and suggest that further clinical trials of this agent in selected patients are warranted.

Preliminary evidence for biologic activity of toceranib phosphate (Palladia®) in solid tumours†

The purpose of this study was to provide an initial assessment of the potential biologic activity of toceranib phosphate (Palladia®, Pfizer Animal Health, Madison, NJ, USA) in select solid tumours in dogs. Cases in which toceranib was used to treat dogs with apocrine gland anal sac adenocarcinoma (AGASACA), metastatic osteosarcoma (OSA), thyroid carcinoma, head and neck carcinoma and nasal carcinoma were included. Clinical benefit (CB) was observed in 63/85 (74%) dogs including 28/32 AGASACA [8 partial response (PR), 20 stable disease (SD)], 11/23 OSAs (1 PR and 10 SD), 12/15 thyroid carcinomas (4 PR and 8 SD), 7/8 head and neck carcinomas [1 complete response (CR), 5 PR and 1 SD] and 5/7 (1 CR and 4 SD) nasal carcinomas. For dogs experiencing CB, the median dose of toceranib was 2.8 mg kg(-1) , 36/63 (58.7%) were dosed on a Monday/Wednesday/Friday basis and 47/63 (74.6%) were treated 4 months or longer. Although these data provide preliminary evidence that toceranib exhibits CB in dogs with certain solid tumours, future prospective studies are necessary to define its true activity.

Use of single-agent carboplatin as adjuvant or neoadjuvant therapy in conjunction with amputation for appendicular osteosarcoma in dogs.

Survival following amputation and administration of single-agent carboplatin for treatment of appendicular osteosarcoma (OSA) in dogs was retrospectively examined. Records of 155 dogs with appendicular OSA treated with amputation and single-agent carboplatin were included from 14 centers. Any carboplatin dosage, number of doses, and protocol schedule were eligible for inclusion. The median disease-free interval (DFI) was 256 days. The median overall survival time was 307 days. Similar prognostic survival factors were identified in this study as reported in prior studies of canine appendicular OSA. Median DFI and survival were comparable to those reported in the original Bergman et al publication. Carboplatin treatment improves the survival probability in dogs with appendicular OSA compared to amputation alone and remains an acceptable alternative to adjuvant treatment with cisplatin.

Apoptotic and proliferation indexes in canine lymphoma

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.

A Randomized Trial Investigating the Efficacy and Safety of Water Soluble Micellar Paclitaxel (Paccal Vet) for Treatment of Nonresectable Grade 2 or 3 Mast Cell Tumors in Dogs

BACKGROUND Effective treatments for dogs with advanced stage mast cell tumors (MCT) remain a pressing need. A micellar formulation of paclitaxel (paclitaxel [micellar]) has shown promise in early-phase studies. HYPOTHESIS/OBJECTIVES The objective was to demonstrate greater activity for paclitaxel (micellar) compared with lomustine. The null hypothesis was μ(p) = μ(L) (ie, proportion of responders for the paclitaxel [micellar] and lomustine groups, respectively). ANIMALS Two hundred and fifty-two dogs with advanced stage nonresectable grade 2 or 3 MCT. METHODS Prospective multicenter randomized double-blind positive-controlled clinical trial. The primary endpoint was confirmed overall response rate (CORR) at 14 weeks. A secondary endpoint, biologic observed response rate (BORR), also was calculated. Safety was assessed by the characterization and grading of adverse events (AE). RESULTS Overall CORR (7% versus 1%; P = .048) and BORR (23% versus 10%; P = .012) were greater for paclitaxel (micellar) compared with lomustine. Paclitaxel (micellar)-treated dogs were 6.5 times more likely to have a confirmed response and 3.1 times more likely to experience a biologic observed response. The majority of AE with paclitaxel (micellar) were transient and clinically manageable. Twenty-seven dogs (33%) receiving lomustine were discontinued because of hepatopathy compared with 3 dogs (2%) receiving paclitaxel (micellar) (P < .0001; odds ratio 26.7). CONCLUSIONS AND CLINICAL IMPORTANCE Paclitaxel (micellar)s activity and safety profile are superior to lomustine. The addition of an active and novel taxane to the veterinary armamentarium could fill a substantial need and, as its mechanism of action and AE profile do not overlap with currently available TKI, its availability could lead to effective combination protocols.

Phase II open‐label study of single‐agent hydroxyurea for treatment of mast cell tumours in dogs*

This prospective study evaluated the efficacy and safety of hydroxyurea (HU) in dogs with measurable mast cell tumours (MCTs). Dogs were treated with HU at 60 mg kg(-1)per os q24h for 14 days then 30 mg kg(-1) q24h thereafter or until MCT recurrence. Forty-six dogs were enrolled. The overall response rate was 28%. Two dogs had a complete response (CR) for 256 and 448 days, respectively. Eleven dogs had a partial response for a median duration of 46 days (range, 28-189 days). Grade 2 to 4 neutropenia occurred in eight dogs and grade 4 thrombocytopenia in two. Grade 3-4 anaemia occurred in seven dogs; overall, there was a significant decrease in haematocrit after treatment with HU. The median drop in haematocrit was 10%. This study demonstrated that HU has activity in the treatment of MCTs with mild anaemia being the primary adverse event.

Impact of Toceranib/Piroxicam/ Cyclophosphamide Maintenance Therapy on Outcome of Dogs with Appendicular Osteosarcoma following Amputation and Carboplatin Chemotherapy: A Multi- Institutional Study

Background We hypothesized that the addition of toceranib to metronomic cyclophosphamide/piroxicam therapy would significantly improve disease-free interval (DFI) and overall survival (OS) in dogs with appendicular osteosarcoma (OSA) following amputation and carboplatin chemotherapy. Methods and Findings This was a randomized, prospective clinical trial in which dogs with OSA free of gross metastatic disease (n = 126) received carboplatin chemotherapy (4 doses) following amputation. On study entry, dogs were randomized to receive piroxicam/cyclophosphamide with or without toceranib (n = 63 each) after completing chemotherapy. Patient demographics were not significantly different between both groups. During or immediately following carboplatin chemotherapy, 32 dogs (n = 13 toceranib; n = 19 control) developed metastatic disease, and 13 dogs left the study due to other medical conditions or owner preference. Following carboplatin chemotherapy, 81 dogs (n = 46 toceranib; n = 35 control) received the metronomic treatment; 35 dogs (n = 20 toceranib; n = 15 control) developed metastatic disease during the maintenance therapy, and 26 dogs left the study due to other medical conditions or owner preference. Nine toceranib-treated and 11 control dogs completed the study without evidence of metastatic disease 1-year following amputation. Toceranib-treated dogs experienced more episodes of diarrhea, neutropenia and weight loss than control dogs, although these toxicities were low-grade and typically resolved with supportive care. More toceranib-treated dogs (n = 8) were removed from the study for therapy-associated adverse events compared to control dogs (n = 1). The median DFI for control and toceranib treated dogs was 215 and 233 days, respectively (p = 0.274); the median OS for control and toceranib treated dogs was 242 and 318 days, respectively (p = 0.08). The one year survival rate for control dogs was 35% compared to 38% for dogs receiving toceranib. Conclusions The addition of toceranib to metronomic piroxicam/cyclophosphamide therapy following amputation and carboplatin chemotherapy did not improve median DFI, OS or the 1-year survival rate in dogs with OSA.

Evaluation of factors associated with second remission in dogs with lymphoma undergoing retreatment with a cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy protocol: 95 cases (2000-2007).

OBJECTIVE To evaluate factors associated with second remission in dogs with lymphoma retreated with a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) protocol after relapse following initial treatment with a first-line 6-month CHOP protocol. DESIGN Retrospective case series. ANIMALS 95 dogs with lymphoma. PROCEDURES Medical records were reviewed. Remission duration was estimated by use of the Kaplan-Meier method. Factors potentially associated with prognosis were examined. RESULTS Median remission duration after the first-line CHOP protocol was 289 days (range, 150 to 1,457 days). Overall, 78% (95% confidence interval [CI], 69% to 86%) of dogs achieved a complete remission following retreatment, with a median second remission duration of 159 days (95% CI, 126 to 212 days). Duration of time off chemotherapy was associated with likelihood of response to retreatment; median time off chemotherapy was 140 days for dogs that achieved a complete remission after retreatment and 84 days for dogs that failed to respond to retreatment. Second remission duration was associated with remission duration after initial chemotherapy; median second remission duration for dogs with initial remission duration ≥ 289 days was 214 days (95% CI, 168 to 491 days), compared with 98 days (95% CI, 70 to 144 days) for dogs with initial remission duration < 289 days. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that retreatment with the CHOP protocol can be effective in dogs with lymphoma that successfully complete an initial 6-month CHOP protocol.

Alternating Rabacfosadine/Doxorubicin: Efficacy and Tolerability in Naïve Canine Multicentric Lymphoma

Background Standard of care treatment for multicentric lymphoma in dogs remains doxorubicin (DOX)‐based combination chemotherapy, but owners may hesitate to commit the time and financial resources to complete such a protocol, typically requiring 12–16 visits. Rabacfosadine (RAB), a double prodrug of the nucleotide analog 9‐(2‐phosphonylmethoxyethyl) guanine, has substantial single‐agent activity in dogs with lymphoma, and a different mechanism of action than DOX. Hypothesis/Objectives Our objective was to evaluate the efficacy and adverse effect (AE) profile of alternating doses of RAB and DOX in dogs with naïve multicentric lymphoma. Animals Fifty‐four dogs with previously untreated lymphoma. Methods Open‐label, multicenter prospective clinical trial. Dogs received alternating RAB (1.0 mg/kg IV weeks 0, 6, 12) and DOX (30 mg/m2 IV weeks 3, 9, 15). Dogs that achieved complete response (CR) were followed by monthly evaluations. Complete clinicopathological evaluation and assessment of remission and AEs were performed every 21 days. Results The overall response rate was 84% (68%; CR; 16%; partial response [PR)]. The overall median progression‐free interval (PFI) was 194 days (216 for CR and 63 for PR). Most AEs were mild and self‐limiting: gastrointestinal and hematologic AEs were most common. Thirteen dogs experienced dermatologic AEs, and 2 dogs developed grade 5 pulmonary fibrosis. Conclusions and Clinical Importance Alternating RAB/DOX generally was well tolerated and resulted in PFIs comparable to standard DOX‐based multi‐agent protocols, with fewer treatment visits. Most adverse events were mild or moderate and self‐limiting. Further studies are warranted to explore long‐term outcome and other RAB chemotherapy combinations.