Oncolytic Sendai Virus Therapy of Canine Mast Cell Tumors (A Pilot Study)

Introduction
Mast cell tumors (MCTs) are common skin cancers in dogs, accounting for up to 20% of canine skin malignancies. They are most often diagnosed through fine needle aspiration and are known for their variable response to treatment, with advanced MCTs often being fatal. MCTs tend to occur in older dogs, particularly breeds like Beagles, Boxers, and Golden Retrievers, with some breeds developing more aggressive tumors than others. These tumors can present as raised or deep lesions, and can be solitary or multiple.

Mast cells play important roles in immune defense and wound healing, but dysfunctional release of their granules can cause significant damage. The prognosis for MCT patients depends on tumor grade, stage, surgical margin status, and mitotic index. There are two main grading systems for MCTs: the Patnaik system (grade 1 to 3) and the Kiupel system (high-grade vs low-grade), with high-grade tumors associated with shorter survival times. Genetic markers, such as c-KIT mutations, are also linked to prognosis.

Treatment for MCTs often involves surgery, which is most effective for well-differentiated tumors. However, high-grade tumors tend to recur even with wide margins. There is no standard treatment protocol, and the FDA-approved drug Toceranib (Palladia) is used along with radiation and chemotherapy. However, the treatment failure rate for advanced MCTs is high, and tumors with low mitotic indices or lacking c-KIT mutations are less responsive to these treatments.

Oncolytic virotherapy, particularly using Sendai virus, is an emerging option. Studies have shown that genetically engineered Sendai virus can target and kill tumor cells while sparing normal tissue. Research on this approach in canine MCTs has shown promising results, with some dogs experiencing tumor clearance or stabilization following viral treatment. However, more research is needed to fully assess its effectiveness and safety in veterinary oncology.

Tumor Grading and Staging
Tumors were graded based on criteria from Patnaik et al. (16) by a veterinary pathologist, considering factors like cell arrangement, shape, cytoplasmic boundaries, granules, mitotic index, and presence of multinucleated cells, edema, necrosis, and hemorrhage. For cases 1 and 6, histological examination was not performed due to the animals not undergoing surgery, so tumor grade was determined by cytology, which was less precise. Tumor staging for these cases was not done. Primary diagnosis was established via fine-needle aspiration, with confirmation by histology if surgery was performed. Mitotic index was evaluated per 10 high-power fields (400X). Tumor staging included histology of excised masses and fine-needle aspiration of lymph nodes, though staging was imprecise due to the lack of additional d
iagnostic methods like radiography, ultrasound, and biopsies.

Viral Preparation
Sendai virus was prepared with modifications as described (64). In brief, virus seed (100 µL) was inoculated into the allantoic cavity of 10-day embryonated eggs, and the virus-containing allantoic fluid was harvested after 3 days of incubation at 37°C. The virus titer w
as determined by serial dilutions and expressed as 50% Egg Infective Dose (EID50/ml).

RESULTS
Six canine patients were treated with oncolytic Sendai virus, either as monotherapy or in combination with surgery. The treatment was well tolerated, with minor, temporary side effects including fever, fatigue, and pain at the injection site. These side effects were classified as grade 1 according to the Veterinary Cooperative Oncology Group's common terminology criteria for adverse events. The summary of side effects is shown in Table 2. The efficacy of the virotherapy treatment is described below.

Case 1
A 7-year-old male mixed-breed dog presented with a 35mm ulcerated skin tumor near its anal gland. The tumor, which grew to 35mm in three weeks, was poorly differentiated based on fine-needle aspiration. Due to the tumor's size and location, complete surgical excision was not possible. The dog was treated with oncolytic Sendai virus (1ml, 10^7 EID50/ml) every two weeks. The tumor was cleared after two treatments, and two additional treatments were given to ensure effectiveness. Three years later, there has been no recurrence. This dog was the first to receive virus treatment at the Veterinary Clinic of the Herzen Oncology Research Institute, and the lack of side effects led to more frequent weekly treatments for subsequent patients.

Case 2
A 9-year-old male German shorthaired pointer had a subcutaneous mast cell tumor (MCT) in the elbow region. Fine-needle aspiration revealed an intermediate-grade tumor. The tumor was excised, but the margins were not clear, resulting in tumor debulking rather than complete removal. Histological examination showed a mitotic index of 5 and confirmed the tumor's intermediate differentiation. It was staged as regional (stage 2) because it had invaded the underlying muscle. Two weeks after surgery, multiple secondary lesions, likely of the same tumor grade, appeared at the surgical site and fused into a mass 40mm in diameter. Two viral treatments, administered 1 week apart, cleared the secondary growth, followed by three additional weekly treatments to ensure the effect. Three years later, the dog remains alive with no signs of disease recurrence.

Case3
A 10-year-old male mixed-breed dog presented with a subcutaneous mast cell tumor (MCT) in the abdominal region, along with multiple palpable lymph nodes and cutaneous metastases. The primary tumor grew rapidly from 30mm to 120mm in three weeks. Fine-needle aspiration revealed that both the primary tumor and some metastases were poorly differentiated, and the tumor was staged as "distant" or stage 4. Since radical surgical excision of all metastases was not possible, a debulking surgery was performed. Histological examination showed a mitotic index of 9 and confirmed the tumor grade. Following surgery, multiple secondary MCT lesions appeared at the surgical site. Three weekly Sendai virus treatments completely cleared all tumors at the surgical site. Each treatment involved 1ml of virus (10^7 EID50/ml), injected in multiple small doses directly around and under the tumor. Additional virus was injected into visible metastatic nodules. Despite eight treatments, metastatic nodules continued to appear and grow, and the dog was eventually euthanized two weeks after the last treatment. The dog was also treated with daily oral prednisone (1mg/kg) to improve quality of life before euthanasia.

Case4
An 8.5-year-old female mixed-breed dog presented with a 30mm cutaneous mass on the left thigh. Fine-needle aspiration revealed an intermediate-grade mast cell tumor (MCT). The tumor was staged as likely "localized" or stage 1, but regional lymph nodes were not tested. The tumor underwent virotherapy with 10 weekly virus applications. While the treatment did not clear the tumor, it stopped growing, and its size remained unchanged during the treatment period. After the first round of virotherapy, the tumor was surgically removed with curative intent. Histological examination confirmed the initial diagnosis and showed at least 5mm clean surgical margins, with a mitotic index of 0. Despite the low mitotic index, localized stage, and large clean margins, the tumor relapsed 6 weeks after surgery. Resumed virotherapy with two weekly Sendai virus treatments completely cleared the recurrent tumor. Each treatment involved 1ml of the virus (10^8.6 EID50/ml). One and a half years later, the dog is alive with no signs of disease recurrence.

Case5
A 13-year-old female mixed-breed dog presented with a 15mm cutaneous interdigital mass. Fine-needle aspiration revealed an intermediate-grade mast cell tumor (MCT). Due to the tumor's location, radical surgery with clean margins was not possible, and the owner rejected amputation of the foreleg. The dog received 12 weekly applications of Sendai virus therapy, but the tumor did not shrink, remaining the same size during treatment and for 6 months afterward. After secondary fine-needle aspiration, the tumor was confirmed as intermediate-grade MCT with extensive necrosis. Nine months after diagnosis, a small trauma triggered painful degranulation, which was treated with prednisone. One week later, the tumor was surgically debulked, and two consecutive weekly virus treatments (1ml, 10^7 EID50/ml) were given to the remaining tumor mass. The tumor ultimately regressed, and the dog remained tumor-free for two years before dying from unrelated causes. Autopsy showed no MCT cells at the former tumor site.

Case6
A 3-year-old male golden retriever presented with a 20mm cutaneous tumor in the abdominal region, which was mildly inflamed with visible ulceration at the center. Fine-needle aspiration revealed a highly differentiated mast cell tumor (MCT). Two months prior, another highly differentiated MCT (30mm) near the stifle had been surgically removed. The stage of the abdominal tumor was unclear, as it was uncertain whether the new mass was a separate primary tumor or a metastasis from the previous one. Virotherapy with Sendai virus was administered at 2-week intervals (1ml, 10^7 EID50/ml), and the tumor was completely cleared after three virus treatments. Two additional treatments were given to ensure the effect. One and a half years later, the dog remains alive with no signs of disease recurrence.

Conclusion

In summary:

Sensitivity Variability: Canine mast cell tumors (MCTs) show varying levels of sensitivity to the oncolytic effects of Sendai virus.
Therapeutic Outcomes: The virus produces a range of antitumor responses, including complete tumor clearance without surgery, clearing of recurring tumors after surgery, and partial disease stabilization.
Effectiveness in Localized Disease: Animals with localized disease and smaller tumors have a better chance of achieving complete tumor clearance.
Side Effects: Side effects from the oncolytic virotherapy are generally minor and temporary.
Combination Potential: The Sendai virus's mechanism of action does not overlap with existing treatments for MCT, suggesting potential for developing combination therapy protocols with conventional treatments.
Surgical Implications: The virus may allow for less invasive surgeries, preserving affected limbs by clearing remaining tumor tissue after debulking surgery.
Limitations: The therapy has limitations, as seen in case 3, where virus injections were ineffective against metastases at advanced disease stages. In cases 4 and 5, multiple virus treatments only stabilized the disease before surgery was needed. However, cases 1 and 6 show that the virus can sometimes clear MCTs without surgery, indicating a promising direction for further research.

Link Web: Frontiers | Oncolytic Sendai Virus Therapy of Canine Mast Cell Tumors (A Pilot Study)

Oncolytic Sendai Virus Therapy of Canine Mast Cell Tumors (A Pilot Study)

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