Canine Melanoma Vaccine:
Where are we now?
International (EU&SA) follow-up study
Author: K.E. Hosman, 3588688
Supervisors: Dr. S.A. van Nimwegen, Prof. J. Kirpensteijn
Utrecht University
Department of Clinical Sciences of Companion Animals
22 weeks (extended) research, 25-08-2014
Table of Contents
INTRODUCTION
2
MATERIALS AND METHOD
6
RESULTS
8
- Canine Malignant Melanoma
- Treatment
- Patient Data
- Tumor Data
- Treatment Data
- Statistics
- Recurrence rates
DISCUSSION AND CONCLUSIONS
20
ACKNOWLEDGEMENT
24
LITERATURE
25
- Data collection
- Statistical outcomes
- Improvements for this study
1
ABSTRACT
Malignant melanoma is the most common oral malignancy in dogs with a high
chance for metastasis. This study evaluated adjuvant immunotherapy (using the
Oncept melanoma vaccine) in 164 dogs with all stages and locations of Canine
Malignant Melanoma following standard treatment. The median number of doses
administered these dogs was 4. Death in 82 dogs (50%) was caused by the tumor.
The median overall survival time (OST) for all dogs was 426 days with a 95%
confidence interval range of 298-554 days and a mean of 763 days. We also
analyzed our stage 2 and 3 patients separately, with a median OST of 418 days (95%
CI 316-519 days) and mean of 617 days. Also, more specific subgroups based on
castration status, location and type of surgery have been identified to have
significant differences in survival time.
Based on the outcome of this study, immunotherapy with the Oncept vaccine may be
an appropriate adjunct to local treatment for canine malignant melanoma, although
more controlled studies are needed to compare treatment outcomes, identify the
influence on certain subgroups and prove its efficacy.
2
INTRODUCTION
Canine melanoma is the most common malignant oral neoplasm in the dog and is
generally considered to be locally aggressive with a high chance for metastasis (up
to 80% of oral CMM and 32-40% of digital CMM) (6) Small breeds, especially the
Cocker Spaniel, and dogs with dark pigmented oral mucosa have a higher risk of
developing oral melanoma (13). Other breeds that seem more likely to develop
melanoma tumors include poodles, dachshunds, Scottish terriers and golden
retrievers. Possible differential diagnoses for CMM include squamous cell carcinoma,
fibrosarcoma, epulides and odontogenic tumors (15). The prognosis for CMM patients is
variable, but can partially be based on stage, clinical behavior, and location of the
tumor (17, 21). The median survival time (MST) found in literature for oral CMM
patients in stage II or III is only two months without treatment. Fortunately, surgery
seems to extend their MST to 5-10 months and a combination of surgery and
radiation therapy will give them about 11 months survival time. (3, 4, 14)
DIAGNOSIS
Malignant melanomas can present in different locations (mostly oral, but they can
also be found on the digits or skin) and appearances. They are usually firm, poor
defined and can be either pigmented (melanotic) or non-pigmented (amelanotic). In
some cases they may have become ulcerated. They can grow rapidly and metastasize
to the draining lymph nodes and other organs including lungs and liver. (14)
A confirmed diagnosis can be made by cytology or histology of biopsy samples of the
primary tumor. (21, 22) Biopsies of the lymph nodes should also be performed in
combination with further imaging (radiographs, CT-scan, ultrasound) to determine if
and how far the disease has spread.
Finding metastatic cells in the lymph nodes will not only give prognostic information
but can actually also help identify the primary tumor. (10) In an ideal situation, both
left and right-sided draining lymph nodes should be aspirated since rostral tumors
and tumors that cross midline will drain bilaterally. However, because of
melanoma’s highly variable histological and cytological patterns diagnosis can still
be a challenge (22). (See figure 1)
Figure 1: Photomicrograph of a suspected malignant melanoma
in the lip of a dog. Cells vary in size and shape, and are poorly
pigmented. Nuclei are large and mitotic rate is low in this field of
view. Hematoxylin and eosin, 400x. (21)
3
Poorly pigmented, or amelanotic melanomas can be especially challenging to
diagnose. Cytology and histology with special stains can be helpful in some of these
cases. The immuno-histochemical markers Melan A, vimentin, and S-100 protein can
be used in amelanotic variants as well as in poorly differentiated tumors to confirm
the diagnosis. (23)
TREATMENT
Several studies have been performed over the past twenty years evaluating various
options for treatment of this disease. The standard approach includes surgery and
radiation, which contribute to a good local control and an increase in survival time
compared to untreated patients. However, overall response rates of canine
melanoma patients remain low due to distant metastasis.(5)
Conservative surgery, for example, has recurrence rates of > 70% and median
survival times in the range of 3 to 4 months (6). More rigorous surgery options such
as maxillectomy or mandibulectomy have resulted in extended median survival
times (9-10 months), but local recurrence (22-48%) is still a significant problem.
(16) Surgery on dogs with CMM of the digits alone (digit amputation or
lumpectomy) have survival times of approximately 12 months (27). The 1-year
survival rates are 42–57% and 2-year survival rates are 11–36% (27). According to
Merial’s website for veterinarians, dogs with WHO stage II or stage III oral
melanoma in their database have survival times of less than five to six months when
treated with surgery alone. (2) Bergman et al reported a MST of 1-5 months with
conventional treatment. (3, 4)
According to Proulx et al, systemic chemotherapy had no impact on the development
of metastatic disease, time to first event, or survival, although it has to be noted the
dosages used in this study were suboptimal. (2) They do think external beam
radiation therapy is effective for inoperable tumors and as an additional treatment
of incompletely removed oral melanomas. (2)
In 2007 the farmaceutical company Merial received a full licence for a new form of
active immunity treatment. This vaccine, called the Oncept Melanoma Vaccine, uses
xenogeneic DNA expressing the human tyrosinase gene (figure 2) and is homologous
to canine tyrosinase. It has been shown to stimulate an immune response in dogs
after vaccination against human tyrosinase, but also against the very similar canine
tyrosinase. This tyrosinase glycoprotein is involved in melanin production and
therefore present in all melanoma cells, which means these cells should be
destroyed as well. (10)
Figure 2 Plasmid map of pING plasmid used for generation of human tyrosinase DNA vaccine
4
At first the producer of the vaccine only received a ‘conditional license’ in 2007, after
a report by Berman et al in 2003. (4). The USDA may issue a conditional product
license to meet an emergency condition, a limited market or other special
circumstance. To obtain conditional licensure, purity and safety must be assured as
well as a reasonable expectation of efficacy. During the period of the conditional
license, additional research will be done to further support the safety and efficacy of
the vaccine. (1) Since 2010 the vaccine is fully licensed by the USDA but it is still
only available to veterinary oncology specialists.
The vaccine should be administered every two weeks for a total of four doses.
Thereafter, booster vaccinations are recommended every six months for the
remainder of the dogs’ life. According to the producer it should only be administered
to dogs with stage 2 and 3 melanomas after following the standard procedure of
CMM treatment (surgery and/or radiation and/or chemotherapy). (1) However,
some veterinarians have also tried the vaccine on patients that did not meet these
criteria. Probably because the prognosis of CMM patients is very poor and no
negative side effects have been reported yet. These patients have therefore also been
included in this study.
5
MATERIALS AND METHOD
In this study 32 dogs from the faculty of Utrecht were included and the medical
records of 132 dogs diagnosed with CMM from other countries across Europe and
South Africa were reviewed. Although they were only included when the diagnosis
for CMM had been confirmed and the use of the vaccine was the same in all dogs,
there were no specific EU guidelines towards the diagnosis and treatment. At
Utrecht University the standard treatment protocol included:
1.
Confirm Tumor with histology or Thick Needle Aspiration Biopsy
(TNAB)(figure 1)
2. Full blood work analysis to exclude any other health issues
3. FNAB of lymph nodes to screen for metastasis
4. CT scan to screen for metastatic disease elsewhere in the body
5. Surgically remove or debulk primary tumor and any affected lymph nodes
6. Radiation therapy within 7-10 days post surgery (6x6 Gy)
7. Application of the Oncept Melanoma vaccine (4x every 2 weeks).
Administered intramuscularly using a transdermal device (in biceps
femoris, semitendinosus or semimembranosus muscle).
8. Checkup 1,3 and 6 months after last vaccine.
9. Possibility to start second vaccination round after 6 months
10. Register and update data including Survival time (ST), Recurrence Free
Interval (RFI), Metastasis Free Interval (MFI), Disease Free Interval (DFI)
and side effects. General data were collected as well including age, breed,
weight and sex of the dogs, tumor location, size, type
(melanotic/amelanotic) and excision margins, stage (18) and any
metastasis or other diseases.
The age was calculated by distraction of the date of birth from the date of first
vaccination. The veterinarian or owner estimated the date of first clinical signs and
the date of diagnosis was on the day the diagnosis was actually confirmed
histologically. The tumor size was calculated by the length x width x depth.
Survival time was measured as the time between the date of first vaccination until
death or last follow up. We also recorded whether death was most likely caused by
CMM (group 1) or a different reason (group 0). Dogs that were still alive were added
to the same group of dogs that died of another cause (group 0).
Based on the following WHO guidelines we were able to stage most of our cases:




Stage 1 melanoma: tumor diameter < 2 cm, without lymph node metastasis
or distant metastasis
Stage 2 melanoma: tumor diameter 2-4 cm, without lymph node metastasis
or distant metastasis
Stage 3 melanoma: tumor diameter >4 cm, or with lymph node metastasis,
without distant metastasis
Stage 4 melanoma: distant metastasis (e.g. in the lungs), independent of
tumor size or lymph node metastasis
To be able to collect the data from other cases in Europe and South Africa (EMOS+),
the pharmaceutical company Merial provided the names and contact details of those
that have been administering the Oncept vaccine to CMM patients. We contacted
them with the request for data mentioned above. After a positive response an excel
file had to be filled in by the local veterinarian and could then be added to our
database. Afterwards, data analysis was performed with the SPSS 22 statistical
software package. Since most studies only include stage 2 and 3 patients oral CMM,
this group will be discussed and analyzed separately as well.
6
This report includes data up until 15-02-2014, which means that patient updates
after this date have not been included.
7
RESULTS
PATIENT DATA
Of the 164 collected cases; most were provided by the UK (n=42 /26%), South
Africa (n=36 /22%) and the Netherlands (n=32 /20%), followed by Portugal (n=24
/15%), Austria (n=19 /12%), France (n=7 /4%) and Spain (n=3 /2%). There were
over 50 different breeds, of which mixed breeds (17.7%), Golden Retrievers
(12.8%), Cocker Spaniels (9.7%) and Labrador retrievers (6.7%) were most
common. The mean age of the dogs was 9.2 years (+/- 3.9; range, 0.7-17.5 yrs). In
two cases the age was unknown. Most of our patients were over 10 years of age
(n=72). (Table 1) Thirty-seven percent was female (of which 29% spayed) and 63%
male (of which 34% castrated). (Table 2)
Most of our cases (N=130) had stage 2 and 3 type melanoma. Details about the
collected tumor and treatment data are described in the next paragraphs.
Censored
AgeCategory
Total N
N of Events
N
Percent
age <5
12
5
7
58.3%
age 5-8
23
11
12
52.2%
age 8-10
55
24
31
56.4%
age >10
72
41
31
43.1%
Overall
162
81
81
50.0%
Table 1: Distribution of patients in age categories.
Frequency
Valid
Percent
Valid Percent
Cumulative Percent
F
13
7.9
7.9
7.9
FC
48
29.3
29.3
37.2
M
48
29.3
29.3
66.5
MC
55
33.5
33.5
100.0
164
100.0
100.0
Total
Table 2: Distribution of patients in intact male, castrated male
TUMOR DATA
The most common location of all melanoma was oral (N=123), followed by digital
(n=22) and cutaneous (n=17). Out of all oral CMM cases, 104 dogs had stage 2 or 3
disease. The most common exact oral locations (e.g. mandibular, maxillar, tongue)
were mandibula (n=34 /32,7%), maxilla (n=29 /27,9%) and lip (n=21 / 20,2%).
(Table 3)
8
Oral location
Valid
Gingiva
Lip
Mandibula
Maxilla
Tongue
Tonsil
Unknown
Total
Frequency
3
21
34
29
4
1
12
104
Percent
2.9
20.2
32.7
27.9
3.8
1.0
11.5
100.0
Valid Percent
2.9
20.2
32.7
27.9
3.8
1.0
11.5
100.0
Cumulative
Percent
2.9
23.1
55.8
83.7
87.5
88.5
100.0
Table 3: Distribution of patients in exact oral tumor location
The overall mean value for size was 25.760 mm3 (+/- 60.004 mm3; range 8-512.000
mm3). The mean size for oral stage 2/3 tumors was 26.415 mm3 (+/- 50320 mm3;
range 12-343.000 mm3).
CT-scans and/or radiographs were taken to determine the stage of disease of the
dogs. In 75 cases (46%) a CT was performed and in 104 cases (63%) radiographs
were taken, of which metastasis was found in 5 cases prior to treatment.
Also, 50 (32%) of the 156 biopsied lymph nodes were positive for CMM. In 8 cases
no biopsy was taken. These patients could therefore not be staged either, because
the stage was based on the WHO guidelines for canine oral tumors (18). The
remaining group contained 130 stage 2/3 dogs and 26 stage 1/4 dogs. (Table 4)
Looking at the stage 2/3 oral CMM patients only, a similar ratio was found for lymph
node status (34% positive).
Stage
Cumulative
Frequency
Valid
Unknown
Percent
Valid Percent
Percent
8
4.9
4.9
4.9
1
21
12.8
12.8
17.7
2
76
46.3
46.3
64.0
3
54
32.9
32.9
97.0
4
5
3.0
3.0
100.0
164
100.0
100.0
Total
Table 4: Distribution of all cases between WHO stages.
The type of tumor was based on histologic examination. Of the 164 tumors, 116
were melanotic (70%), 46 (28%) were amelanotic and 2 undetermined (2%). Some
tumors contained mostly amelanotic cells and were therefore categorized in the
“amelanotic” group for statistical analysis. These results will be described in the
next chapter.
Exactly half of all our patients died due to their primary disease (CMM). The other
half either died from another cause or is still alive. In the stage 2/3 group just under
half (49,6%) died of CMM. Separating the stage 2/3 oral cases, 60 patients (57,7%)
9
died of their primary disease. This is statistically interesting since a median ST can
be calculated.
TREATMENT DATA
Surgery was not performed in 8 cases and in 4 cases tumors were only debulked
(and therefore categorized as “not performed”). Of all surgically removed tumors,
62 were completely removed, 73 marginal and in 17 cases dirty. Ten dogs received
carboplatin chemotherapy, 2 metronomic and 1 palladia therapy additionally.
Thirty-nine dogs received radiotherapy (23,8 %).
After following the standard treatment procedure as much as possible, all 164 dogs
received vaccinations. Sixteen dogs received less than 4 doses, 106 received 4 doses,
38 between 5-8 doses, and four dogs > 8 doses.
In our patients no systemic toxic effects were observed from the vaccine, except in
the case of one beagle named Dino who received 20 doses. His nose and footpads
have started to show signs of depigmentation.
10
STATISTICS
Cox regression analysis
All potential variables were entered in a univariate cox regression analysis for the
overall survival time (OST). Variables that had a p value of < 0.15 where then used
to build a multivariate cox regression analysis.
Exactly 50% of the dogs died caused by the disease, so we were able to calculate the
median OST. For all 164 cases that median was 426 days with a 95% confidence
interval range of 298-554 days and a mean of 763 days. We also analyzed our stage
2 and 3 patients separately, with a median OST of 418 days (95% CI 316-519 days)
and mean of 617 days. Afterwards median ST’s for all stages were calculated
separately as well, to be able to compare them with other studies. Stage 1, 2, 3 and 4
had median ST’s of 789, 468, 301 and 309 days, respectively.
Another group we looked at separately were those with oral tumors, since most
studies have focused on this group only. The median ST for stage 1 patients was 789
days, for stage 2 patients 426 days, for stage 3 patients 289 days and stage 4
patients 242 days with a significance of 0.092 between these stages.
Although we only had 22 cases with digit CMM, statistical analysis was performed. A
mean of 1388 days was found, but the median could not be calculated. However,
separating stage 3 digit CMM cases resulted in a median of 441 days.
Radiotherapy did not seem to have significant influence on the ST for our patients,
even after separating stage 2 and 3 cases dividing groups by tumor location.
Chemotherapy was not significant either in these groups. We therefore also took a
closer look into the UK patients, since they administered chemotherapy to more
than half (n=26; 56%) of their 42 patients. This did not result in a significant
outcome either.
Figure 1 KM for ST, without discriminating between case groups
11
Figure 2 KM for ST in the stage 2/3 group, leaving any possible influencing factors out.
Figure 4 KM for ST in the stage 2/3 group with oral CMM, leaving any possible influencing factors
out.
The first KM gives an overview of the survival time of all our patients (figure 2). In
figure 3 only stage 2 and 3 patients were included and in figure 4 the group was
reduced to oral stage 2/3 only (figure 4). In the next part of this report the
significant factors will be illustrated in separate KM diagrams, and results will be
discussed afterwards.
12
Kaplan-Meier Analysis
Additional Kaplan Meyer Product analysis was performed and where necessary the
variable was recoded into groups. Sex was recoded in intact male (M), castrated
male (MC), intact female (F) and castrated female (FC). For the Kaplan-Meier curve
for surgical outcome, the groups were divided in incomplete (dirty)
margins/unoperated (0) and marginal/wide complete excision (1). The number of
doses was also recoded in groups: less than 4 doses (group 1), 4 doses (group 2) and
> 4 doses (group 3).
Figure 5 KM analysis for the sexes separated into male/female and castration status.
Castrated males have a significant worse survival time than intact dogs or castrated
females. (Figure 5) The P-value was 0.052. Their median ST was about half of the ST
for intact males (301 and 546 days resp.) and was also shorter compared to
castrated females (with a median of 441 days).
Figure 6 KM analysis for the sexes separated into male/female and castration status in stage 2/3
group
13
Similar significant (p=0.016) results were found after separating the stage 2/3
patients from the group (figure 6). Castrated males had a median ST of 289 days and
intact males 653 days.
Figure 7 KM analysis for castration status; without differentiating between male and female
The KM, where no difference was made between male and female, shows that the ST
of castrated dogs is significantly (p=0.097) shorter than intact dogs. (Figure 7)
Figure 8 KM analysis for castration status; without differentiating between male and female in
stage 2 and 3
This was even more significant (P=0.040) in the stage 2/3 group with median
survival times of 327 days for castrated and 653 days for intact animals. (Figure 8)
14
Figure 9 KM analysis of digital, cutaneous and oral location
Another KM analysis was performed to find possible significant differences in
survival time depending on the location of the primary tumor (figure 9). The P-value
was 0.017. The median ST of dogs with oral tumors was significantly shorter (330
days) than cutaneous tumors (963 days). The median for digit tumors could not be
calculated, but their mean ST was 1388 days.
Figure 10 KM analysis of digital, cutaneous and oral location in the stage 2/3 group
Again, similar results were found after leaving out the stage 1 and 4 cases, with a
median ST of 327 days for oral tumors. (Figure 10)
15
Figure 11 KM of dirty/un-operated (value 0) versus marginal/complete surgery (value 1)
Tumors had been removed surgically at the University or by their own/local vets
and margins were determined afterwards by histological examination. The KM
shows a significant advantage (P=0.067) for marginal/complete surgery with a MST
of 441 days versus unoperated/incomplete surgical removal with a MST of 273
days. (Figure 11)
Figure 12 KM of dirty/unoperated (value 0) versus marginal/complete surgery (value 1) for the
stage 2/3 group
This was also the case after separating our stage 2 and 3 cases, with a median ST of
264 days versus 441 days for marginal/complete surgery (Figure 12) and after
separating stage 2/3 oral cases (p=0.042) the median ST for unoperated/incomplete
surgery remained 264 days and a slightly lower number of 418 days for
marginal/complete surgery.
16
Figure 13 KM of the number of vaccinations affecting OST (1= <4 doses; 2= 4 doses; 3= >4 doses)
In order to analyze the effect of the number of vaccinations on the OST, patients
were divided in dose groups. Dose group 1 received less than 4 doses; group 2
completed the first course of 4 doses; group 3= received more than 4 doses. With a
P-value of 0.000, the number of vaccinations significantly affected the OST. (Figure
13) The median ST for dose group 1 was 55 days, for dose group 2 it was 309 days
and for dose group 3 it went up to 944 days. Separating stage 2/3 patients did not
make any difference.
Figure 14 KM of the presence of metastasis or recurrence affecting the OST.
Significance (P=0.000) was found in ST for dogs with metastasis or recurrence after
conventional treatment and after or during vaccination rounds. (Figure 14) Dogs
with metastasis prior to treatment were not included. This is of course an obvious
finding, but it does give more insight in the prognosis for future patients by looking
17
at the actual numbers (Table 5). With a mean of 292, 483, 315 days for metastasis,
recurrence or both respectively compared to the mean of 1455 days for patients
without metastatic or recurrent disease, they have at least a 60-80% decreased ST.
Figure 15 KM of the presence of metastasis or recurrence affecting the OST in stage 2/3 cases
For the stage 2/3 group this was equally important for the median ST. (Figure 15)
Figure 16 KM for the influence of lymph node status on MST
Lymph node status was another significant factor for OST (p=0.010) with a median
ST of 546 days if they were found negative and 299 days for dogs with positive
lymph nodes. (Figure 16) This also counted for the stage 2/3 group with a
significance of 0.068 and MST 468 days for negative lymph nodes and 301 days for
positive nodes.
18
Similar outcomes were found after reducing this group to only oral cases (p=0.018)
with MST’s of 426 days and 289 days, respectively.
Sex and weight of our dogs, the size of their tumors (p=0.421) and pathologic results
(melanotic versus amelanotic) (p=0.956) were statistically not significant, so these
were not further analyzed.
No significant effect on survival was found for chemotherapy either due to the small
amount of treated animals. Radiotherapy also appeared to be not significant
(p=0.173) in our group. Even for the stage 2 and 3 cases there did not seem to be a
significant increase in survival time with these forms of therapy.
RECURRENCE RATES
In the total group of 164 dogs we had 72 dogs (43,9%) that remained disease free.
The others (N=92) were diagnosed with either recurrence or metastasis, or both.
(Table 5) In some cases only metastasis was reported, but they probably had
recurrence as well.
Metastasis and/or Reccurence in all patients
Valid
none
metastasis
recurrence
both
Total
Frequency
72
Percent
43.9
Valid Percent
43.9
Cum. Percent
43.9
34
20.7
20.7
64.6
29
17.7
17.7
82.3
29
164
17.7
100.0
17.7
100.0
100.0
Table 5: Distribution between cases with metastasis, recurrence, both or free of any metastasis or
recurrence. All cases were included (n=164).
For our stage 2 and 3 cases (n=130) just 52 dogs (40%) remained disease free,
whilst 30 dogs (23%) developed metastatic disease and 23 dogs (18%) had
recurrence. The remaining 25 patients (19%) were classified in a separate group
that had both metastasis and recurrence.
Metastasis and/or Reccurence in stage 2/3 oral cases
Valid
none
metastasis
recurrence
both
Total
Frequency
35
Percent
33.7
Valid Percent
33.7
Cum. Percent
33.7
24
23.1
23.1
56.7
23
22.1
22.1
78.8
22
104
21.2
100.0
21.2
100.0
100.0
Table 6 Distribution between cases with metastasis, recurrence, both or free of any metastasis or
recurrence. Only stage 2/3 oral melanoma cases (n=104) were included.
Analyzing only the stage 2/3 oral cases (n=104) the fraction of patients that
remained disease free decreased to 34% (n=35). Twenty three percent (n=24)
19
developed metastatic disease, 22% (n=23) had recurrence and 22% (n=22) had
both. Not enough data were available to calculate the recurrence free intervals.
(Table 6)
DISCUSSION AND CONCLUSIONS
The aim of the study was to look into the long-term effects and collect more data on
the Oncept DNA vaccine by including patients from across Europe and South Africa.
By collecting more than 160 cases, which is considerably more than any individual
canine melanoma study at present, we were able to increase statistical power. We
found several significant factors influencing MST after melanoma treatment in dogs,
but some doubts remain towards the efficacy of the vaccine.
DATA COLLECTION
It was difficult to collect enough data from Europe since 1) not a lot of patients
received the Oncept Melanoma Vaccine and 2) some veterinarians were not able or
willing to provide data in time. Therefore, we requested the Veterinary Hospital in
Pretoria (South Africa) for information on their CMM patients to expand the
database.
Although we needed basic data about our patients, we came across some issues to
standardize the datasheet. A standard procedure is usually followed for CMM
patients at our faculty, but could vary depending on the individual cases and
owners. This was due to the fact that 1) some owners had financial constraints or 2)
an operation or other treatment was not possible in some cases (depending on the
patients’ status or tumor) or 3) the patient had been partially treated by their own
veterinarian, which influenced the standard procedure or 4) some had to be
operated more than once because of incomplete margins or 5) for cases from abroad
no standard procedure was followed, or by a different protocol.
The ony criteria we had for inclusion of patients was diagnosis of CMM and
administration and vaccinated with the Oncept Vaccine at least once. This could
have resulted in incorrect staging. Also, different clinicians performed work-up and
surgery/radiotherapy, which could also have made a difference in staging and even
OST.
We also doubted some records on tumor sizes, since in some cases the entire piece
of removed tissue was measured and sometimes the actual tumor. Another issue
was that several pathologists from different institutions performed the histological
examination, which might have caused an inconsistency in the diagnosis. However,
if we were to exclude all exceptional patients the group would become too small for
statistical analysis.
We were aware of the fact that the vaccine is not registered for stage 1 and 4
patients, but we decided to include this group in parts of our study because it
represents a significant population of dogs with CMM. Since the vaccine does not
seem to have any negative side effects, veterinarians tend to use it on stage 1 and 4
cases too. However, to be able to compare our results with other studies, we
separated the stage II and III oral CMM patients for parts of our statistical analyses.
20
Most of our patients received their first vaccination directly after their operation,
but since there were no specific guidelines when to start vaccination, some were
treated a while after surgery. The reason often was because the owners were not
able to decide whether they wanted to start additional (and experimental)
treatment. This is why we chose the time between first vaccination and survival
date as the ST.
Chemotherapy was not used often, probably because it is proven not to be very
effective against CMM. (7, 20) However, especially in the UK it is used as a common
additional treatment. Veterinarians in the Netherlands and Austria often treat their
CMM patients with radiotherapy. At the University of Pretoria (South Africa) they
have not used either of these treatments. It is unclear whether this is due to the
limited availability, the high expenses, or if it was not a standard procedure for them
or another reason.
STATISTICAL OUTCOMES
The first interesting significant outcome was the relation between sex/castration
status and MST. Castrated animals had a significant shorter ST and especially the
castrated males. There was no literature about this phenomenon for CMM, but it has
been mentioned in studies about several other cancer types, including
osteosarcomas and mast cell tumors. (9, 24, 25) It is still unclear why this occurs,
but it seems plausible that alterations in gonadal hormones might influence cell
growth.
Another significant finding was the difference in MST for the three basic tumor
locations (digit, skin or oral). According to our results, digit and skin melanomas
have a significantly longer ST than oral melanomas. This seems to be slightly
inconsistent with other studies since digit melanomas are often described to display
a high degree of local invasiveness and high metastatic rates as well (29). However,
the cutaneous melanomas that are not in proximity to mucosal margins have also
been described to behave in a more benign manner, like ours. (29) In fact, even the
prognosis for oral malignant melanoma depends on the location of the tumor and
metastatic rate, but overall the oral melanomas are considered to be extremely
malignant tumors because of a high degree of local invasiveness and high tendency
to metastasize. Unfortunately, oral melanomas are difficult to notice in time by dog
owners, but early diagnosis is especially important for the prognosis of these
patients. (26)
The third significant outcome, the type of surgical excision, was not very surprising,
but does show the importance of complete excision. In the case of suspected CMM, it
has always been highly recommended to remove the tumor with as wide margins as
possible. Unfortunately, some clinicians are not aware of the importance of surgical
margins or are restricted by the wishes of their clients (cosmetic reasons). For these
reasons, some of the patients needed a second operation that could have been
avoided. Other reasons for multiple operations were removal of lymph nodes
(affected or suspected) or recurrences.
Another finding was the drastic decrease of 60-80% for the MST of dogs where
recurrence or metastatic disease was found after or during treatment. This is an
important point of concern since 57% of our cases developed metastasis and/or
recurrence despite receiving immunotherapy. This does bring up the question
whether and for whom the vaccine actually seems to be working. It would be
expected that if the immune system was properly activated against melanotic cells
21
in all the CMM patients, spread and regrowth should be less common. However, in a
study with nine dogs by Liao et al the actual antibody titer was measured after
vaccination and only three of these dogs developed an immune response against
human tyrosinase and in only two cases a cross reaction with canine tyrosinase (and
thus the melanoma cells) was found. (17) Another explanation could be that it takes
some time for the immune system to respond, which might be too late for some
patients with progressed disease. (17)
As mentioned earlier, chemotherapy does not seem to be very effective against
CMM. (7, 20) Our results show no significance either, but since only 33 of our
patients received this type of treatment, our results are not very reliable. By
analyzing the UK cases separately, we tried to correct for this problem. However, the
results were still not significantly different from dogs that did not receive
chemotherapy. This seems consistent with the poor results of chemotherapy from
several other studies (7, 20).
Besides having looked at several subgroups, we also compared our results with
control groups. Unfortunately, even though we collected more cases than other
studies, all of our patients had been vaccinated. Therefore, instead of our own
control group we compared our results to historical controls from literature.
The overall median ST of dogs treated with the Oncept vaccination found in the
study by Bergman is 389 days (4). For all 164 cases in our study the median was
426 days. More specifically, according to Bergman, stage 1 had a median ST of 12-14
months; stage 2 less than 5 months, stage 3 or 4 had 2-3 months. (4) In our data
stage 1, 2, 3 and 4 had median ST’s of 789, 468, 301 and 309 days, respectively,
which is much longer than Berman reported. (4)
Manley et al. studied 58 dogs with digit CMM tumors only. (29) Their dogs with
stage 1 disease had an MST of 952 days (median not yet reached), dogs with stage 2
disease had an MST of 1093 days (median not yet reached), dogs with stage 3
disease had an MST of 321 days, and those with stage IV disease had an MST of 76
days. Unfortunately, our study only included 22 cases with digit CMM, so our results
could be biased. Stage 1 (n=8) had an MST of 1244 days (median not yet reached),
stage 2 (n=10) an MST of 875 days (median not yet reached) and stage 3 (n=4) 441
days (median). We did not have any data on stage 4 digit CMM patients. However,
the results for stage 1, 2 and 3 are similar to Manley’s findings. (29)
Furthermore, in stage 2 and 3 oral patients conservative surgery has median
survival times in the range of 90 to 120 days (2, 6). The median ST of historical
stage 2 and 3 control dogs in the study by Grosenbauch was 324 days. (14) Our
vaccinated stage 2 or 3 oral melanoma patients have survival times of 337 days,
which is very close to Grosenbauch’s outcome.
Surgery on dogs with CMM of the digits alone (digit amputation or marginal
excision) has survival times of approximately 12 months (27). Looking at the
median of our stage 3 digit tumors of 441 days and a mean of 1388 days for all
stages, does support clinical efficacy.
In the case of one of our atients, Dino, where depigmentation of the skin was visible,
it is not an uncommon finding. According to Yee et al (2000) these lesions provided
direct evidence that depigmentation (also called vitiligo) after immunotherapy
could be attributed to melanocyte-specific CD8+ cytotoxic T cells. (28)
22
It seems that the Oncept vaccine cannot cure the disease (either too slow immune
response, no response or too weak), since 50% of our cases died of the disease
despite the vaccine. The outcomes also differed depending on the chosen group
criteria. This resulted in a large spread in the outcomes of our cases, so our
conclusions remain reserved. Hopefully, by improving the approach of this disease
in the future by standardizing the protocol and finding a solution for the financial
constraints, we will have better insight in the best way to treat dogs with CMM and
the exact benefits of the Oncept vaccine for each case. Also, collecting more data and
a longterm follow-up of patients remains important.
Some research has been going on, especially for the treatment of Human Malignant
Melanoma, to try adjusting the vaccine or trying completely different forms of
immunotherapy. In the case of CMM it could be interesting to find out if adjustments
could be made to create a stronger immune response (e.g. using a different adjuvant,
or bacteria/viruses as a carrier instead of only a bacterial plasmid). However, these
are usually accompanied by more side effects, which could have an impact on the
quality of life. In my opinion it would therefore be better to get more insight in the
actual immune response on the current Oncept Vaccine by measuring the antibody
titers, like Liao did in his study. (17)
IMPROVEMENTS FOR THIS STUDY
Given that our study did not contain a control group, we had to compare our
outcomes with other studies, which made our results less powerful. However, a lot
of research has been done on conventional therapies and their results were similar,
but having our own control group would be a great improvement.
As mentioned before we would also like to carry out additional diagnostics (e.g.
ELISA) to measure the post-vaccinal humoral response and compare these results
with non-vaccinated patients. Bergman et al already found a positive correlation
between post-vaccinal humoral response and long-term survival and the study by
Liao et al showed interesting results, although that study lacked adequate statistical
power. (3, 17)
Additionally, histologic characterization of the tumor was not further specified than
being malignant melanoma and was made by several pathologists on several
institutions, which may have introduced additional variability. Histological grading
and immunohistochemistry may provide further prognostic information.
There are still a lot more patients that have been treated with the vaccine but have
not been included in this study (as mentioned above). Collecting data on these cases
would obviously strengthen the statistical outcomes for this vaccine, but also give
more insight in the dogs’ general prognosis. It would be strongly recommended to
contact the EU and South African faculties again for a follow up on their cases. A
worldwide collection of data could be interesting too, but very difficult to realize.
23
ACKNOWLEDGEMENT
Special thanks go out to all the owners who provided us with the necessary
information, especially after their dogs had passed away. It has given us valuable
insights in the efficacy of the vaccine.
I would also like to thank professor Kirpensteijn and doctor Van Nimwegen for
giving me this research opportunity and clinical work experience. I have very much
enjoyed working with you.
I am also thankful for the help of Samantha ten Hoope, colleague during part of this
research. She especially contributed to the completion of the data of our Dutch
patients.
24
LITERATURE
(1) Title 9 Animals and Animal Products Part 102. Licenses for Biological Products. 2014:Part
102.
(2) Pet Cancer Vaccine. Available at: www.petcancervaccine.com. Accessed February 5, 2014.
(3) Bergman PJ, Camps-Palau MA, McKnight JA, Leibman NF, Craft DM, Leung C, et al.
Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the
Animal Medical Center. Vaccine 2006 May 22;24(21):4582-4585.
(4) Bergman PJ, McKnight J, Novosad A, Charney S, Farrelly J, Craft D, et al. Long-term survival of
dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human
tyrosinase: a phase I trial. Clin Cancer Res 2003 Apr;9(4):1284-1290.
(5) Boria PA, Murry DJ, Bennett PF, Glickman NW, Snyder PW, Merkel BL, et al. Evaluation of
cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral
squamous cell carcinoma in dogs. J Am Vet Med Assoc 2004 Feb 1;224(3):388-394.
(6) Bostock DE. Prognosis after surgical excision of canine melanomas. Vet Pathol 1979
Jan;16(1):32-40.
(7) Brockley LK, Cooper MA, Bennett PF. Malignant melanoma in 63 dogs (2001-2011): the effect
of carboplatin chemotherapy on survival. N Z Vet J 2013 Jan;61(1):25-31.
(8) Chapman PB, Einhorn LH, Meyers ML, Saxman S, Destro AN, Panageas KS, et al. Phase III
multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with
metastatic melanoma. J Clin Oncol 1999 Sep;17(9):2745-2751.
(9) Cooley DM, Beranek BC, Schlittler DL, Glickman NW, Glickman LT, Waters DJ. Endogenous
gonadal hormone exposure and bone sarcoma risk. Cancer Epidemiol Biomarkers Prev 2002
Nov;11(11):1434-1440.
(10) Coyle VJ, Garrett LD. Finding and treating oral melanoma, squamous cell carcinoma, and
fibrosarcoma in dogs. VetMed 2009 June 1.
(11) Dank G, Rassnick KM, Sokolovsky Y, Garrett LD, Post GS, Kitchell BE, et al. Use of adjuvant
carboplatin for treatment of dogs with oral malignant melanoma following surgical excision. Vet
Comp Oncol 2014 Mar;12(1):78-84.
(12) Food and Drug Administration, HHS. International Conference on Harmonisation; choice of
control group and related issues in clinical trials; availability. Notice. Fed Regist 2001 May
14;66(93):24390-24391.
(13) Goldschmidt MH. Histologic Classification of Epithelial and Melanocytic Tumors of the Skin
of Domestic Animals. 2nd ed.: Armed Forces Institute of Pathology; 1998.
(14) Grosenbaugh DA, Leard AT, Bergman PJ, Klein MK, Meleo K, Susaneck S, et al. Safety and
efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for
oral malignant melanoma in dogs following surgical excision of the primary tumor. Am J Vet Res
2011 Dec;72(12):1631-1638.
(15) Harvey HJ, MacEwen EG, Braun D, Patnaik AK, Withrow SJ, Jongeward S. Prognostic criteria
for dogs with oral melanoma. J Am Vet Med Assoc 1981 Mar 15;178(6):580-582.
(16) Kosovsky JK, Matthiesen DT, Marretta SM, Patnaik AK. Results of partial mandibulectomy for
the treatment of oral tumors in 142 dogs. Vet Surg 1991 Nov-Dec;20(6):397-401.
25
(17) Liao JC, Gregor P, Wolchok JD, Orlandi F, Craft D, Leung C, et al. Vaccination with human
tyrosinase DNA induces antibody responses in dogs with advanced melanoma. Cancer Immun
2006 Apr 21;6:8.
(18) Owen LN. TNM Classification of Tumors in Domestic Animals. 1st ed. Geneva: World Health
Organization; 1980.
(19) Proulx DR, Ruslander DM, Dodge RK, Hauck ML, Williams LE, Horn B, et al. A retrospective
analysis of 140 dogs with oral melanoma treated with external beam radiation. Vet Radiol
Ultrasound 2003 May-Jun;44(3):352-359.
(20) Rassnick KM, Ruslander DM, Cotter SM, Al-Sarraf R, Bruyette DS, Gamblin RM, et al. Use of
carboplatin for treatment of dogs with malignant melanoma: 27 cases (1989-2000). J Am Vet
Med Assoc 2001 May 1;218(9):1444-1448.
(21) Schultheiss PC. Histologic features and clinical outcomes of melanomas of lip, haired skin,
and nail bed locations of dogs. J Vet Diagn Invest 2006 Jul;18(4):422-425.
(22) Smith SH, Goldschmidt MH, McManus PM. A comparative review of melanocytic neoplasms.
Vet Pathol 2002 Nov;39(6):651-678.
(23) Sulaimon S, Kitchell B, Ehrhart E. Immunohistochemical detection of melanoma-specific
antigens in spontaneous canine melanoma. J Comp Pathol 2002 Aug-Oct;127(2-3):162-168.
(24) Torres de la Riva G, Hart BL, Farver TB, Oberbauer AM, Messam LL, Willits N, et al. Neutering
dogs: effects on joint disorders and cancers in golden retrievers. PLoS One 2013;8(2):e55937.
(25) White CR, Hohenhaus AE, Kelsey J, Procter-Gray E. Cutaneous MCTs: associations with
spay/neuter status, breed, body size, and phylogenetic cluster. J Am Anim Hosp Assoc 2011 MayJun;47(3):210-216.
(26) Withrow SJ, Vail DM, Page R. Small Animal Clinical Oncology. 5th ed.: Saunders; 2012.
(27) Wobeser BK, Kidney BA, Powers BE, Withrow SJ, Mayer MN, Spinato MT, et al. Diagnoses
and clinical outcomes associated with surgically amputated canine digits submitted to multiple
veterinary diagnostic laboratories. Vet Pathol 2007 May;44(3):355-361.
(28) Yee C, Thompson JA, Roche P, Byrd DR, Lee PP, Piepkorn M, et al. Melanocyte destruction
after antigen-specific immunotherapy of melanoma: direct evidence of t cell-mediated vitiligo. J
Exp Med 2000 Dec 4;192(11):1637-1644.
(29) Manley CA, Leibman NF, Wolchok JD, Riviere IC, Bartido S, Craft DM, and Bergman PJ.
Xenogeneic Murine Tyrosinase DNA Vaccine for Malignant Melanoma of the Digit of Dogs. J Vet
Intern Med 2011;25:94–99
26