Bone marrow-derived dendritic cell vaccination of dogs with naturally occurring melanoma by using human gp100 antigen

Canine malignant melanoma (CMM) is a common and aggressive form of cancer in dogs. Established therapeutic approaches such as surgery, chemotherapy, and radiation therapy (RT) have not proven curative. As a coadjuvant of RT and to enhance the antimelanoma immune response, we characterized dendritic cells (DCs) from the bone marrow (BM) of dogs with CMM, ex vivo, for use in therapeutic vaccines. BM mononuclear cells from 3 dogs with melanoma and from 1 healthy dog were cultured for 12 days in media supplemented with recombinant human granulocyte-macrophage colony stimulating factor, stem cell factor, tumor necrosis factor, and Flt-3 ligand. On day 11, DCs were transduced with an adenovirus vector encoding a xenoantigen, human melanoma antigen gp100. Each dog received 3 subcutaneous vaccinations over a 4-month period. Phenotypic analysis of the expanded DC population demonstrated expression of CD11c/CD18 and major histocompatibility complex class II surface markers, and ultrastructural features characteristic of DCs were observed on electron microscopy. On functional analysis, these DCs were able to stimulate allo-reactivity and capture and express gp100. One dog demonstrated antigen-specific cytotoxic T lymphocyte (CTL) activity in peripheral blood lymphocytes. This dog has displayed no clinical signs, either locally or systemically, of recurrent melanoma 48 months after initial DC injection. However, another dog, which was CTL negative, relapsed 22 months after vaccination. Ex vivo DC expansion is feasible for immunotherapy of spontaneous cancers in outbred dogs.     

Development of Immunologic Assays to Measure Response in Horses Vaccinated with Xenogeneic Plasmid DNA Encoding Human Tyrosinase

Xenogeneic plasmid DNA constructs have been developed and optimized for immunotherapies targeting cancer in both humans and dogs. Specifically, plasmid vectors containing the tumor antigen tyrosinase have demonstrated immunoreactivity and clinical benefit in the treatment of melanocytic tumors in these species. Overexpression of tyrosinase has also been noted in equine melanocytic tumors, supporting its role as a valid tumor antigen in the horse. Vaccination with plasmid constructs containing tyrosinase may thus have translational immunoreactivity in the treatment of equine melanomas. Here, we describe a methodology that is highly sensitive and specific for the detection of both humoral and cell-mediated immunoreactivity against tyrosinase in equine patients. These antigen-specific immunoassays are used to measure the humoral and cell-mediated responses in a cohort of horses vaccinated with xenogeneic plasmid DNA encoding human tyrosinase. Serum humoral responses were measured using standard enzyme-linked immunosorbent assay technique against the full-length recombinant human tyrosinase protein. Peripheral blood mononuclear cells were collected from vaccinated horses and stimulated with tyrosinase-specific peptides. Cell-mediated responses were then measured using a novel quantitative real-time-polymerase chain reaction technique to determine resultant interferon-γ expression. All horses developed significantly positive humoral and cell-mediated immune responses compared with their individual prevaccination values. No adverse reactions or signs of autoimmunity were detected. Vaccination with xenogeneic plasmid DNA expressing tyrosinase appears to elicit tumor antigen-specific reactivity and should be evaluated in a larger cohort of horses with melanocytic tumors.     

Multimodality treatment including ONCEPT for canine oral melanoma: A retrospective analysis of 131 dogs

Canine oral melanoma (OM) is an aggressive cancer with a high rate of metastasis. Surgery and/or radiotherapy (RT) are effective local treatments, yet many dogs succumb to distant metastasis. Immunotherapy represents an attractive strategy for this potentially immunogenic tumor. The objective of this multi‐institutional retrospective study was to examine the clinical outcome of dogs with OM treated with ONCEPT melanoma vaccine. Most dogs also underwent surgery and/or RT (8 Gy × four weekly fractions). Dogs with distant metastasis at diagnosis and those receiving concurrent chemotherapy were excluded. One hundred thirty‐one dogs treated with ONCEPT were included: 62 had adequate local tumor control defined as complete tumor excision or irradiation of residual microscopic disease; 15 were treated in the microscopic disease setting following an incomplete excision without adjuvant RT; and 54 had gross disease. Median time to progression, median progression‐free survival, and median tumor‐specific overall survival were 304, 260, and 510 days, respectively. In multivariable analysis, presence of gross disease correlated negatively with all measures of clinical outcome. Other negative prognostic indicators were primary tumor ≥2 cm, higher clinical stage (stages 2 and 3), presence of lymph node metastasis at diagnosis, and caudal location in the oral cavity. Radiotherapy had a protective effect against tumor progression. To date, this is the largest reported series of dogs with OM treated with ONCEPT. Several previously reported prognostic indicators were confirmed.     

Prolongation of survival of dogs with oral malignant melanoma treated by en bloc surgical resection and adjuvant CSPG4‐antigen electrovaccination

Reported post‐surgery 1‐year survival rate for oral canine malignant melanoma (cMM) is around 30%; novel treatments are needed as the role of adjuvant chemotherapy is unclear. This prospective study regards adjuvant electrovaccination with human chondroitin sulfate proteoglycan‐4 (hCSPG4)‐encoded plasmid in 23 dogs with resected II/III‐staged CSPG4‐positive oral cMM compared with 19 dogs with resected only II/III‐staged CSPG4‐positive oral cMM. Vaccination resulted in 6‐, 12‐, 18‐ and 24‐month survival rate of 95.6, 73.9, 47.8 and 30.4%, respectively [median survival time (MST) 684 days, range 78–1694, 8 of 23 dogs alive] and 6‐, 12‐, 18‐ and 24‐month disease‐free interval (DFI) rate of 82.6, 47.8, 26.1 and 17.4%, respectively (DFI 477 days, range 50–1694). Non‐vaccinated dogs showed 6‐, 12‐, 18‐ and 24‐month survival rate of 63.2, 26.3, 15.8 and 5.3%, respectively (MST 200 days, range 75–1507, 1 of 19 dogs alive) and 6‐, 12‐, 18‐ and 24‐month DFI rate of 52.6, 26.3, 10.5 and 5.3%, respectively (DFI 180 days, range 38–1250). Overall survival and DFI of vaccinated dogs was longer in those <20 kg. In vaccinated and non‐vaccinated dogs local recurrence rate was 34.8 and 42%, respectively while lung metastatic rate was 39 and 79%, respectively.     

Use of adjuvant carboplatin for treatment of dogs with oral malignant melanoma following surgical excision

Melanoma is the most common oral malignancy in dogs. This retrospective study evaluated adjuvant carboplatin chemotherapy (with or without radiation therapy) in 17 dogs with malignant oral melanoma following surgical resection. The median dosage and number of doses of carboplatin administered to the 17 dogs was 300 mg m^?2 (range, 150–300 mg m^?2) and 4 (range, 2–11), respectively. The overall median progression‐free survival for all dogs was 259 days [95% confidence interval (CI₉₅), 119–399 days]. The first progression‐free survival event was local recurrence in seven dogs (41%) and metastases in seven dogs (41%). The median overall survival for all dogs was 440 days (CI₉₅, 247–633 days). The tumour was the cause of death in 10 dogs (59%). On the basis of this study, systemic therapy with carboplatin may be an appropriate adjunct to local treatment for canine malignant melanoma, although future prospective controlled studies are needed to compare treatment modalities for this aggressive neoplasia.     

A retrospective analysis of the efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma

Oral malignant melanoma (OMM) in the dog is often locally aggressive with a high metastatic potential and there are few treatment options that have been demonstrated to improve outcome of this disease. The purpose of this study was to determine whether adjunctive treatment with the Oncept melanoma vaccine affected the outcome of dogs with OMM that had achieved loco‐regional cancer control. Medical records from 45 dogs that presented to the Animal Cancer and Imaging Center were reviewed, including 30 dogs with stage II and III disease. Dogs that received the vaccine did not achieve a greater progression‐free survival, disease‐free interval or median survival time than dogs that did not receive the vaccine.     

Hypofractionated radiation therapy for the treatment of malignant melanoma and squamous cell carcinoma in dogs and cats

This study describes the experience with hypofractionated radiation therapy of squamous cell carcinoma and melanoma in dogs and cats. A total dose of 32-48 Gray (Gy) was delivered once a week in 8 Gy fractions. 34 animals in which a complete surgical excision was impossible were treated. There was no tumor detectable macroscopically in 14 patients at the beginning of radiation therapy. In 20 animals the median volume of the tumor was 9.9 cm3. The median survival times and the local tumor control of squamous cell carcinoma of the oral and nasal cavities and of the body are comparable to results which were reached with a Monday-Wednesday-Friday scheme. For the treatment of Melanoma the hypofractionated radiation therapy is first choice. There are no significant side effects. Late side effects did not occur. 88% of the owners are satisfied with this kind of treatment and would choose it again.     

Computed tomographic‐lymphography as a complementary technique for lymph node staging in dogs with malignant tumors of various sites

Locoregional lymph nodes are routinely examined in order to define the spatial extent of neoplastic disease. As draining patterns of certain tumor types can be divergent from expected anatomical distribution, it is critical to sample the lymph nodes truly representing the draining area. The aim of this bicenter prospective pilot study was to describe the technique of computed tomographic (CT)‐lymphography for primary draining lymph node mapping in tumor staging in dogs. Forty‐five dogs with macro‐ or microscopic tumors in specified localizations were evaluated. Depending on body weight, 0.8–2 ml contrast agent (iohexol) was injected into four quadrants around the tumor, and CT‐images were obtained at 1, 3, 6, 9, and 12 minutes post‐injection. Attenuation of chosen regions of interest (Hounsfield units (HU)) and patterns of enhancement were assessed for 284 lymph nodes in the precontrast study with median HUs of 31.1 (Interquartile range (IQR) = 18.4) and for 275 in the intravenous postcontrast study with 104.3 HU (IQR = 31.2) (paired Wilcoxon test, P < 0.001). In the CT‐lymphography study, 45 primary draining lymph nodes with a significantly higher median HU value of 348.5 (IQR = 591.4) (one‐sample t‐test, P < 0.001) were identified. Primary draining lymph nodes were found to be clearly visible after 1–3 minutes after local injection, often concurrent with a good visibility of the lymphatic vessel system. The herein described technique of peritumorally injected CT‐contrast agent followed by subsequent CT‐lymphography for primary draining lymph node mapping works well in a majority of cases in all investigated sites and warrants further validation for different tumor entities.     

The basic biology of malignant melanoma: molecular mechanisms of disease progression and comparative aspects

Malignant melanoma (MM) is a life-threatening disease characterized by a highly aggressive biologic behavior in both humans and dogs. Despite improvements in diagnosis and patient care, most deaths from MM are due to metastases that are resistant to conventional treatment modalities. To ultimately reduce the mortality associated with metastatic disease, it is necessary to better define the fundamental molecular mechanisms of malignant tumor progression. The progression of disease is a consequence of the complex interactions between malignantly transformed cells and host factors. Characterization of the stages of tumor progression and the changes occurring in highly malignant cells is important for the development of effective treatment regimens. The dys-regulated molecular mechanisms of transformed melanocytes are presently being characterized. In this review, we summarize the current understanding of the molecular phases in the progression of MM, which include genetic instability, dysregulated proliferation of melanocytes, increased invasion and metastasis, and angiogenesis.     

A case of malignant melanoma,a possible primary site in the digit,with systemic metastasis in a mini-Rex

We report a case of systemic metastasis of malignant melanoma in a mini-Rex (Oryctolagus cuniculus). The animal presented with lameness of the right hind limb, swelling of popliteal lymph node, and a black mass on the first digit. Paralysis of hindlimbs and forelimbs, dysuria, and dysphagia progressed over time, and the rabbit died on day 35 from the first visit. At necropsy, many black lesions were observed in multiple organs including the marrow of most bones. Histopathologically, the tumor cells had highly atypical nuclei of various sizes and an abundant eosinophilic cytoplasm, and some cells contained melanin granules. These cells were positive for PNL2 and S-100, melanoma markers, by immunohistochemistry. This is the first report of malignant melanoma in a mini-Rex with severe malignancy and systemic metastasis including the bone marrow.     

Use of carboplatin for treatment of dogs with malignant melanoma: 27 cases (1989-2000)

OBJECTIVE: To evaluate response rate and duration of malignant melanomas in dogs treated with carboplatin. DESIGN: Retrospective study. ANIMALS: 27 client-owned dogs with spontaneously occurring measurable malignant melanomas. PROCEDURE: Records of dogs with melanomas treated with carboplatin from October 1989 to June 2000 were reviewed. Carboplatin was administered IV at doses of 300 or 350 mg/m2 of body surface area. Response to treatment and evidence of drug toxicity were determined. RESULT: Response to treatment could be evaluated in 25 dogs. Of those, overall response rate was 28%. One dog had a complete response, 6 (24%) dogs had a partial response (> 50% reduction in tumor burden). Median duration of partial response was 165 days. Eighteen dogs had stable disease (n = 9; 36%) or progressive disease (9; 36%). Response to treatment was significantly associated with carboplatin dose on a milligram per kilogram basis (15.1 mg/kg 16.9 mg/lb] of body weight vs 12.6 mg/kg [5.7 mg/lb]). Evidence of gastrointestinal toxicosis could be assessed in 27 dogs. Mean body weight of 5 dogs that developed gastrointestinal toxicosis was significantly less than that of 22 dogs without gastrointestinal toxicosis (9.9 kg [21.8 lb] vs 19.3 kg [42.5 lb]). CONCLUSIONS AND CLINICAL RELEVANCE: Carboplatin had activity against macroscopic spontaneously occurring malignant melanomas in dogs and should be considered as an adjunctive treatment for microscopic local or metastatic tumors. Gastrointestinal toxicosis was associated with body weight. Because small dogs are more likely to have adverse gastrointestinal effects, gastrointestinal protectants should be considered for these patients.     

Evaluation of cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral squamous cell carcinoma in dogs

OBJECTIVE: To determine the maximum tolerated dose (MTD) of cisplatin administered with piroxicam, the antitumor activity and toxicity of cisplatin combined with piroxicam in dogs with oral malignant melanoma (OMM) and oral squamous cell carcinoma (SCC), and the effects of piroxicam on the pharmacokinetics of cisplatin in dogs with tumors. DESIGN: Prospective nonrandomized clinical trial. ANIMALS: 25 dogs. PROCEDURE: Dogs were treated with a combination of cisplatin (escalating dose with 6 hours of diuresis with saline [0.9% NaCI] solution) and piroxicam (0.3 mg/kg 10.14 mg/lb], PO, q 24 h).The initial cisplatin dose (50 mg/m2) was increased by 5 mg/m2 until the MTD was reached. Tumor stage and size were determined at 6-week intervals during treatment. The pharmacokinetics of cisplatin were determined in dogs receiving a combination of cisplatin and piroxicam during the clinical trial and dogs that were treated with cisplatin alone. RESULTS: 11 dogs with OMM and 9 dogs with SCC were included in the clinical trial. The MTD of cisplatin when administered in combination with piroxicam was 50 mg/m2. Tumor remission occurred in 5 of 9 dogs with SCC and 2 of 11 dogs with OMM. The most common abnormality observed was renal toxicosis. Clearance of cisplatin in dogs that were treated with cisplatin alone was not significantly different from that in dogs treated with a combination of cisplatin and piroxicam. CONCLUSIONS AND CLINICAL RELEVANCE: Cisplatin administered in combination with piroxicam had antitumor activity against OMM and SCC. The level of toxicity was acceptable, although renal function must be monitored carefully.     

Indirect computed tomography lymphangiography with aqueous contrast for evaluation of sentinel lymph nodes in dogs with tumors of the head

Sentinel lymph node evaluation is widely used in human medicine to evaluate the first lymph node(s) to which a tumor drains. Sentinel lymph node biopsy allows avoidance of extensive lymphadenectomies in cases where the sentinel lymph node is negative for metastasis, thereby reducing patient morbidity. It has been shown that regional lymph nodes are not always the sentinel lymph node, thus identification and sampling of sentinel lymph nodes allows for more accurate staging, which is critical for treatment and prognostication in dogs with cancer. The objective of this prospective, pilot study was to determine if indirect computed tomography (CT) lymphangiography with aqueous contrast agent would successfully allow identification of sentinel lymph nodes in dogs with masses on the head. Eighteen dogs underwent CT lymphangiography. The sentinel lymph node was successfully identified within 3 min of contrast injection in 16 dogs (89%). Compression of lymphatic vessels from endotracheal tube ties and/or the patient's own body weight delayed or prevented identification of sentinel lymph nodes in two dogs (11%). Computed tomography lymphangiography with aqueous contrast can be used successfully to rapidly identify sentinel lymph nodes in dogs with masses on the head.     

Primary malignant melanoma in the oesophagus of a foal

A 2‐month‐old filly was evaluated for severe colic. Ultrasound, abdominocentesis and physical examination findings prompted an abdominal exploratory surgery. Perforation of the stomach was discovered during the surgery. The filly was humanely subjected to euthanasia under anaesthesia and post mortem examination was performed. In addition to gastric and duodenal ulceration, a thickened, black area of the proximal oesophagus was discovered. Histopathology of the lesion revealed primary malignant melanoma. Although rare, primary melanoma can occur in noncutaneous locations.