Safety Evaluation and Preliminary Flow Cytometrical Analysis in Dogs With Oral Melanoma Treated with An Immunotherapeutic Dendritic Cell Fusion Vaccine and Concurrent Radiotherapy

Combination of immunotherapy and radiation therapy has been regarded as a new trend in treating cancers. The purpose of this study was to evaluate safety and peripheral blood lymphocyte changes in dogs diagnosed with oral malignant melanoma who received an immunotherapeutic dendritic cell vaccine and concurrent radiotherapy. The vaccine was conducted by fusing allogeneic dendritic cells and autologous tumor cells and was administrated subcutaneously every two weeks. Concurrent hypofractionated radiotherapy was given weekly. Six dogs were enrolled in this pilot clinical study. The treatment was well-tolerated, with only mild radiotherapy-related side effects reported. The median progression-free survival was 146.5 days. Flow cytometrical analysis of the peripheral blood lymphocyte revealed a decreased CD4/CD8 ratio in only one dog, who was also the only long-term survivor. The study provides insights into immune cell changes during immuno-radiotherapy and also supports further expansion of this kind of treatment, investigation of treatment efficacy, and exploration of correlations between immune cell changes and outcome.

Introduction

Management of canine oral malignant melanoma (OMM) remains difficult because of its aggressive nature of local invasion and distant metastasis. Effective treatments in canine OMM are mostly local therapies now, including curative-intent wide-margin surgery and radiation therapy (RT), while systemic treatments, especially immunotherapies, are currently under development [1]–[4].

Currently, in Taiwan, radiation therapy and the commercial immunotherapy Oncept® are currently unavailable, highlighting the need for alternative approaches in canine OMM. Our team has developed a kind of immunotherapy, that utilizes allogeneic dendritic cells (DCs) from healthy dog donors, and autologous tumor cells from cancer-bearing dogs, cultured together in vitro to conduct a fusion cell production [5]. In experimental dogs who were implanted with transmissible venereal tumors, the fusion vaccine could slow down tumor growth without causing significant adverse events. When clinically applied to dogs with oral malignant melanoma, the DC vaccine could generate a significantly longer survival compared to dogs who only received surgery [6]. This DC vaccination treatment is currently under further investigation in our department, through rigorous clinical trials and combinations of the DC vaccine and other treatments.

Recently, studies have focused on enhancing treatment efficacy by combining immunotherapy and radiation therapy. In theory, although radiation therapy is a local treatment, it can also elicit systemic immune responses, including both immune-activating and immune-suppressive responses [7]. Therefore, it is reasonable that combining RT and immunotherapy may provide a synergetic effect when appropriate protocols are used, which are being worked on in both pre-clinical and clinical studies [8]–[11]. In canine OMM, radiation can generate an overall response rate of 81%–100% [1]. However, limited studies on combining RT and immunotherapy in canine OMM exist currently. Deguchi’s team reported a 55.6% clinical benefit rate in end-stage OMM dogs who were treated with anti-programmed-cell death receptor-ligand 1 (PD-L1) antibody and hypofractionated RT [9]. Therefore, based on the previously proven general safety and the potential efficacy of the DC fusion vaccine, we started to investigate the treatment combination of DC vaccine and radiation therapy in canine OMM.

In addition to the treatment outcome, immune-related prognostic factors were also studied in human and canine cancers, such as the T cell populations. The CD4+ T cells, or the T helper cells, were important in orchestrating immune responses, while the CD8+ T cells were mainly involved in the direct enemy-killing process. It has been reported that the CD4+, CD8+, regulatory T cells (Tregs), and the relative CD4/8 ratio, were prognostic in canine cancers [12]–[14]. In canine mammary gland cancer, higher infiltrating CD4+ T lymphocytes, lower infiltrating CD8+ T cells, and a higher CD4+/CD8+ ratio of tumor-infiltrating lymphocytes, were associated with a higher metastasis rate [15]. While in canine melanoma, decreased CD4+ T cells and increased CD4+FoxP3 T cells were discovered in peripheral blood from melanoma-bearing dogs compared to healthy controls [13]. In a study conducted by Garcia and colleagues, in dogs with oral melanoma treated with experimental immunotherapeutic anti-tumor vaccine alone or with metronomic chemotherapy, the Treg frequency was significantly related to the progression-free interval, while the CD4+/CD8+ ratio had no impact on survivals [14]. There were no abundant studies investigating the relationship between the CD4+/CD8+ ratio and clinical outcomes in canine oral melanoma.

Therefore, the objectives of the current study are to report a preliminary result of dogs receiving dendritic cell/tumor cell fusion vaccine and concurrent radiation therapy and to track the changes in peripheral blood lymphocyte composition before and after treatment, with a special focus on T cells, to see what influence would be caused by the DC fusion vaccine combined with RT, to the patient’s lymphocyte, and to find possible correlations.

Materials and Methods

Patient Recruitment

Dogs with histopathological diagnosis of oral malignant melanoma attending the National Taiwan University Veterinary Hospital Animal Cancer Treatment Center from 2019 to 2020 were offered the treatment options, including the allogeneic dendritic cell and autologous tumor cell fusion vaccine combined with radiation therapy. Dogs meeting the following inclusion and exclusion criteria were recruited with owners’ consent: dogs should be in clinical stage 1–3 based on the WHO clinical staging system [1] for canine oral melanoma; dogs should be treatment naïve or have failed previous therapy; dogs should have macroscopic disease before enrollment to obtain a tumor biopsy for dendritic cell vaccine preparation; basic blood tests, including complete blood count (CBC) and biochemistry, should be obtained before enrollment, and dogs with severe liver or renal insufficiency or autoimmune disease were excluded; concurrent use of steroids was to be avoided. The study was reviewed and approved by the National Taiwan University Institutional Animal Care and Use Committee (Approval No. NTU-109-EL-00106). Informed consents from all the owners were obtained. The project was stopped after 2020 due to the COVID-19 pandemic.

Dendritic Cell (DC) Vaccine Manufacture

The oral tumor biopsy was performed by surgeons at the National Taiwan University Surgical Department. The biopsied sample was suspended by antibiotics (5% Penicillin-Streptomycin-Amphotericin B, P/S/A, Simply, GeneDireX, Taipei, Taiwan)-added phosphate-buffered saline (PBS, Simply, GeneDireX, Taipei, Taiwan) and was processed into single cells. The autologous single tumor cells were then frozen until use.

Allogeneic dendritic cells were obtained from healthy dog donors’ peripheral blood mononuclear cells (PBMCs). In detail, the PBMCs were isolated by gradient centrifugation using Ficoll-Hypaque (density 1.077, Cytiva, Uppsala, Sweden), and cultured in RPMI 1640 medium (Simply, GeneDireX, Taipei, Taiwan) with 10% donor dog serum and 1% P/S/A for one day. On day 2, the culture medium was changed to RPMI 1640 with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Massachusetts, USA), 1% P/S/A, IL-4, GM-CSF, and Flt-3L (all from R&D System, Minnesota, USA), and the medium was changed every three days. On day 8, lipopolysaccharide (LPS, Merck KGaA, Darmstadt, Germany) was added to the culture medium to facilitate the dendritic cell maturation. And on day 11, the matured dendritic cells were harvested, counted, and then frozen until use.

For the fusion vaccine manufacture, 1 × 10⁷ tumor cells and 1 × 10⁷ dendritic cells were recovered and fused under the condition of 1 mL polyethyleneglycol (PEG, Jena Bioscience GmbH, Jena, Germany) and concurrent stirring for two minutes. The fusion product was then cultured in RPMI 1640 with 10% FBS, 1% P/S/A, IL-4, and GM-CSF for three days. On day 4, 15 μg/mL mitomycin (BOC Science, New York, USA) was used to treat the fusion cells for one hour, and the fresh fusion product was washed and finally suspended in 400 μL 0.9% normal saline for treatment use on the same day.

The above procedures followed the referred references [5], [6].

Radiation Therapy

Radiotherapy was performed under general anesthesia as a hypofractionated treatment with 8–8.5 Gy per fraction, for a total of 40 to 42.5 Gy. A cone-beam CT (Discovery CT 590, GE, 16 slices) imaging was obtained for treatment planning, and the radiation was delivered by a 6 MV linear accelerator (Synergy, 500 MU/min, Elekta, Stockholm, Sweden) one week after planning. A follow-up CT scan was arranged one month after treatment completion.

Treatment Schedule and Protocol

For radiotherapy, the CT planning started on the same week as the tumor biopsy, and the radiation treatment started one week later. Because of the time required for DC culture, the DC vaccine was administrated two days after the second radiation treatment. RT was planned to be five weekly fractions, while the DC vaccine was given subcutaneously every two weeks for four doses.

Peripheral Blood Flow Cytometry

Two to three mL of peripheral blood from the treated dog was collected in an EDTA tube before the first vaccination and one month after vaccination. The blood was centrifugated and the plasma was removed. Red blood cell (RBC) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added to the remaining blood cells to remove RBCs. The remaining white blood cells were preserved at −80°C until analysis.

Before analysis, the white blood cells were thawed and recovered in RPMI with 10–20% FBS for one hour. FACS buffer (2% bovine serum albumin in PBS) was used to re-suspend and mix the cell pellet. A final cell count of 1 × 10⁶ cells/100 μL FACS buffer was used for flow cytometry antibody staining. Propidium iodide (PI) stain was used for cell viability test after cell recovery, and only viable cells were used for further analysis. Fluorescein-conjugated monoclonal antibodies of anti-CD3, CD4, and CD8, as well as corresponding isotype antibodies, were used for immunophenotyping of the lymphocytes. Anti-CD21 stain for B cells was not used because the conjugated fluorescein was similar to the PI stain and would affect the results. PI was chosen prior to anti-CD21 antibody because of the necessity of viability confirmation before immunophenotyping. The cells were incubated with antibodies at 4°C for 30 minutes and were then washed twice with FACS buffer. The cells were re-suspended with PBS and were analyzed using LSR Fortessa Flow Cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed by FlowJo v10 software (BD Biosciences).

Response and Adverse Event Evaluation

Tumor responses were evaluated according to the Veterinary Cooperative Oncology Group Response Evaluation Criteria in Solid Tumors v1.0 [16] and were recorded as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). If the best response was SD, the duration should be at least four weeks. Radiation therapy associated toxicity was assessed according to the Toxicity Criteria of The Veterinary Radiation Therapy Oncology Group [17]. Dendritic cell fusion vaccine associated adverse events were assessed according to the Veterinary Cooperative Oncology Group—Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 [18]. Clinical signs were monitored by the owners, while physical examination was performed by clinicians at each recheck. CBC and biochemistry tests, and 3-view chest radiographs were performed before the first and the third DC vaccinations.

Statistical Analysis

Patients’ characteristics and tumor information were recorded. Progression-free interval (PFI) and overall survival (OST) were also calculated. PFI was defined as the duration from the day treatment started to the day of disease progression. If no disease progression was confirmed, the data would be censored from PFI analysis. OST was defined as the duration from the day treatment started to the day of death caused by any reason. The percentages of the CD3+, CD4+, and CD8+ T cells, as well as a CD4/8 ratio change during the treatment course, were recorded. A log-rank test was utilized for comparison between the CD4/8 ratio change (decrease or not) and survivals (PFI and OST).

Results

Patient’s Characteristics, Treatment Response, and Adverse Events

Six dogs were enrolled in this pilot clinical study. The median age of the dogs was 12.5 years old (range, 8–14). The median body weight was 5.65 kg (range, 2.64–16.5). There were two Miniature Poodles and one each of Maltese, Shiba, Welsh Corgi and Dachshund. Four dogs were intact males, one was a castrated male, and one was a spayed female. Four dogs had left maxillary tumors, the other two had mandibular tumors. Two dogs were stage II (33.3%), and four were stage III (66.7%). Patient No. 1 received metronomic chemotherapy for two weeks before enrollment without clinical benefit. Patient No. 5 received other immunotherapies after tumor progression. All the dogs had macroscopic disease when they received DC vaccination and radiation therapy.

All dogs responded to radiation therapy, with two achieving complete remission and four partial remission. Radiation-related adverse events were all mild and self-limited, including grade 1 alopecia and grade 2 mucositis. DC vaccination-related side effects were only reported in one dog, who developed a tiny subcutaneous nodule which was self-recovered then. All dogs received five fractions of radiation treatments. Five dogs received four doses of DC vaccination as planned, while patient No. 6 received only two doses of DC vaccine because of rapid tumor progression, and the owner declined further vaccination doses but continued radiotherapy for pain management. Patients’ information is presented in Table I.

Table I. Characteristics, Treatments, and Outcome Information of the Six Dogs

Patient No.	1	2	3	4	5	6
Signalment
Breed	Maltese	Shiba	Welsh corgi	Dachshund	Miniature poodle	Miniature poodle
Age (y/o)	13	14	8	11	12	13
Sex	Fs	Mi	Mc	Mi	Mi	Mi
Weight (kg)	2.64	13	16.5	5.8	4.3	5.5
Tumor information
Diagnosis	AMM	MM	MM	MM	MM	MM
Location	Lt. maxilla	Lt. mandible	Rt. mandible	Lt. maxilla	Lt. maxilla	Lt. maxilla
Tumor size1	3.4 cm	3.3 cm	2.1 cm	2.5 cm	4.1 cm	4.5 cm
Mitotic index	2/HPF	6/10 HPF	1/HPF	8/10 HPF	NA	2-5/HPF
Clinical stage2	II	III	III	III	II	III
RLN3 metastasis	Yes	Yes	Yes	Yes	No	Yes
Treatment
Radiation (oral mass)	8.5 Gy*5	8 Gy*5	8 Gy*5	8 Gy*5	8 Gy*5	8 Gy*5
RLN RT	Not included	Included 7.5 Gy*5	Included 7 Gy*5	Included 8 Gy*5	Included 7 Gy*5	Not included
DC fusion vaccination	4 doses, regular schedule	4 doses, regular schedule	4 doses, regular schedule	4 doses, started after 3rd RT	4 doses, regular schedule	2 doses, regular schedule
Tumor response	CR	PR	CR	PR	PR	PR
Outcome
PFI (days)	79	62	630	101	214	32
OST (days)	113	114	630	101	288	41
Local progression	Yes 79 days	Yes 102 days	No	Unknown	Yes 214 days	Yes 32 days
Distant metastasis	No	Yes 56 days	Unsure4	Unknown	No	Yes 32 days
Cause of death	Local disease	Lung metastasis	Liver failure	Sudden death	Local disease	Lung metastasis
Median PFI (days)		146.5 (patients No. 3 and 4 were censored)
Median OST (days)		113.5

Flow Cytometry Analysis

Four (patients No. 1, 2, 3, 6) out of six dogs had sufficient peripheral white blood cells for flow cytometry analysis after cell recovery. All but patient No. 6 had blood samples collected as planned. Patient No. 6 only had two vaccination doses and only had blood samples before the first and second DC vaccinations, due to rapid tumor progression; therefore, the blood sample after the second DC vaccination was used as a post-treatment sample in this patient. All the samples were successfully stained by the antibodies. An example of a flow cytometry result from patient No. 2 is presented in Fig. 1. Basically, the CD3 % = P3_sample − P3_ISO, the CD4 % = (Q2 +Q4) _sample − (Q2 + Q4) _ISO, the CD8 % = (Q1 + Q2) _sample − (Q1 + Q2) _ISO.

Fig. 1. A graphic example of the flow cytometry result before and after treatment (A, B, C, D): (A) Initial identification of peripheral blood lymphocytes (P1); (B) PI stain for cell viability test, only viable cells (P2) were used for further analysis; (C) CD3+ T cell population identification (P3); (D) CD4+ (Q2+Q4) and CD8+ (Q1+Q2) T cell populations identification.

The percentages of CD3+, CD4+, and CD8+, as well as the CD4/8 ratio, are summarized in Table II. The mean percentage of CD3+ T cells before treatment in the analyzed dogs was 69.6%, and 48.1% post-treatment; the mean percentages of CD4+ T cells before and after treatment were 33.1% and 38.2%, while the mean percentages of CD8+ T cells before and after treatment were 30.6% and 30.2%, respectively. The average CD4/CD8 ratios were 2.2 and 1.8 before and after treatment. Three dogs had decreased CD3%, three had increased CD4%, two had increased CD8%, and one dog had decreased CD4/8 ratio. No significant correlation was observed between changes in these different T cell subtypes and tumor response or patient survival. However, the one dog with a decreased CD4/8 ratio (decreased CD4+ and nearly doubled CD8+ T cells) was the only one who survived more than a year, while the other three dogs lived less than six months.

Table II. Flow Cytometrical Analysis of the Six Dogs Before and After Treatment

T cell percentage	CD3%		CD4%		CD8%		CD4/8 ratio
Patient	Before	After	Before	After	Before	After	Before	After
No. 1	57.8	16.1	37.3	43	35.1	22.6	1.06	1.90
No. 2	49.4	58.4	18.2	26.7	35.4	53.8	0.51	0.50
No. 3	95	86.4	40	38	6.1	11.2	6.56	3.39
No. 6	76.3	31.3	36.8	45.1	45.9	33	0.80	1.37
Mean	69.625	48.05	33.075	38.2	30.625	30.15	2.234	1.79

Outcome

All the six dogs died at the time of data analysis. Two dogs died of local tumor progression, two had fatal distant metastasis, and the other two (including patient No. 3, the only long-term survivor) died of undetermined causes. Patient No. 3 was alive 22 months after treatment, and developed liver failure and neurological signs which led to the death. Whether the liver failure or the neurological disease was related to oral melanoma was unknown because no necropsy was performed. However, at the time of death, no local tumor recurrence nor pulmonary metastasis was observed. Patient No. 4 had a sudden death around 3 months after treatment. Some gastrointestinal signs, as well as occasional coughing, were noted several days before death, but no confirmed diagnosis could be reached. Although no local recurrence or distant metastasis was detected at the last re-visit, tumor-related death (e.g., tumor emboli) could not be totally excluded because no necropsy was performed. Detailed outcomes are summarized in Table I. Patients No. 3 and No. 4 were censored from PFI analysis because no disease progression was confirmed at the time of death, resulting in a median PFI of 146.5 days. The median OST (death caused by any reason) was 113.5 days for all six dogs.

Discussion

This study described preliminary results of the clinical presentation and outcome of the dogs that received the immunotherapeutic dendritic cell/tumor cell fusion vaccine combined with hypofractionated radiation therapy, as well as the flow cytometry analysis of the changes in peripheral blood lymphocyte composition before and after treatment.

Clinically, the outcome of the six dogs in this study was not comparable to previous studies involving dogs that received curative-intent surgery alone [19], radiation therapy [20], or multimodal therapy including the immunotherapy Oncept® [21]. However, the results should not be compared directly due to differences in study objectives, inclusion criteria, and patients’ characteristics. Being a pilot study, the current project did not enroll a concurrent control group, making treatment efficacy evaluation less accurate. Several factors might have contributed to the ordinary outcome observed, such as the presence of macroscopic disease, which was also associated with a worse prognosis in patients receiving RT [20]. In addition, the treatment sequence was also possibly inadequate, as discovered in research conducted by Deguchi et al. [9], dogs in the previous RT group achieved significantly better outcomes. Whether to include regional lymph nodes (RLNs) in the radiation field has also been discussed, with recent evidence suggesting that RLN-sparing RT should be considered for head and neck cancers [22]. However, despite the above considerations, assessing efficacy was not the primary objective of this study, so detailed explanations would be omitted here. Overall, the combination of dendritic cell and tumor cell fusion vaccine and radiation therapy was found to be safe, which was one of the key findings in the current study.

The second aim of the study was to investigate the peripheral blood lymphocyte changes during the treatment course. Normally in healthy dogs, the average percentages of CD3+, CD4+, and CD8+ T cells were 54.5%, 22.1%, and 9%, respectively [23]. It has also been reported that the CD8+ T cells would increase with age, the CD4/CD8 ratio decreases, and no significant change in CD4+ T cells [24]. In dogs with melanoma, as was mentioned before, the regulatory T cells would increase [13], which is reasonable because a loss of equilibrium between the immune system and tumor growth happens, resulting in an immune-suppressive environment, which then favors tumor growth [25]. In the current study, our team combined immunotherapy and radiation therapy to see whether the immune balance could be possibly restored after treatment. However, because of the COVID-19 pandemic, RT has been unavailable in our area and has not recovered yet, limiting the study recruitment to only six dogs, and only four of them had sufficient blood samples for analysis. No significant changes were discovered in these four dogs, although we found that the one long-term survivor was the only dog that had decreased CD4/CD8 ratio after treatment. This dog had mildly decreased CD4+ T cells and nearly doubled the CD8+ T cells after treatment. It is possible that a small subset of Tregs existed within the CD4+ T cell population, but a FoxP3 immunophenotyping was not performed because of the limited samples we had and the specific technique requirement. The other possible correlation between the survival and immune status in this dog was the increased CD8+ T cells, from 6.1%–11.2%. Increased cytotoxic T cells might allow the host immunity to attack those microscopic tumor cells, possibly resulting in a better prognosis. It is to be noted that no conclusion could be drawn from the current results due to the small sample size. However, the reported findings could be further investigated in the future, about how the immune status changes during an immunotherapy treatment course.

Several limitations exist in the current study, primarily due to the small sample size. No control group was recruited concurrently, thus the efficacy of combining DC vaccination and RT could not be conclusively determined. Furthermore, there were some deviations in the treatment process, such as patients receiving other treatments before enrollment or after tumor progression (patients No. 1 and No. 5), and starting the DC vaccine after the third radiotherapy (patient No. 4). Whether those deviations would affect the patient’s outcome could not be confirmed within the scope of this study. Moreover, only four dogs had sufficient blood samples for flow cytometry analysis, as the other two dogs’ samples lost viability after recovering from freezing. The freezing procedure might have influenced the overall immunophenotype expression percentage if cells died during thawing. Therefore, a fresh blood sample should be used for flow cytometry in future trials. Despite the limitations, the general safety of concurrent DC vaccination and RT supports further expansion of the clinical trials, with appropriate protocol and blood sample analysis modifications.

Conclusion

The current study reported preliminary results of the combination of dendritic cell fusion vaccine and radiation therapy in treating canine oral malignant melanoma. The treatment was found to be safe, while the blood lymphocyte flow cytometry analysis revealed increased CD8+ T cells and a decreased CD4/CD8 ratio in the only long-term survivor. These results support further exploration and modification of this kind of immuno-radiotherapy in canine cancer treatment.