The effect of Cimetidine on the number of melanomas on 41 grey horses
Lettinga C., Helle N. M., Liu N., Sha S.,. x, x. LoomansJ.B.A.
Author: C.Lettinga
Tutor: J. Loomans
Location: Heilan International Equestrian centre, Xin Qiau, China
University of Utrecht, faculty of veterinary medicine
1
Introduction
Melanomas are one of the most common skin tumors in horses. The prevalence is 4-15%3,4
and it constitutes 3,8% of the total number of diagnosed equine neoplasms.6,7 This tumor
affects mainly grey horses at the age of 5 years and older. In addition, it has been reported that
80% of all grey horses older than 15 years old will develop melanomas.4,7,9
Melanomas can be either benign or malignant. Most of them are benign especially in grey
horses. Non-grey horses, in which melanomas are very rare, often develop a more aggressive
version.7,9 The tumor arises from melanocytes, found in the skin. The lesions are well defined,
black nodules and can appear either individual or multiple.7 It can affect all parts of the body,
but they frequently appear in the perineal- and/or perianal area, at the external genitalia, the
ventral surface of the tail, the lips and eyelids.4,7,9 Most equine melanomas present a slow
growth rate and rarely spread. Eventual metastatic spread can be seen after a few years by
haematogenic or lymphatic pathways and can affect almost all organs of the body.4
Etiology
Different theories exist about the true cause of these tumors. Some authors suggest a genetic
factor as a cause of melanomatosis in grey horse. Since the prevalence and disease history of
melanomatosis is quite opposite in grey horses versus non-grey horses this seems to be an
acceptable theorie.7,8,9 Several studies have tried to detect this genetic correlation.
Seltenhammer et al. 20037 described an estimated heritability of 0,36. However, a
corresponding gene has not been found yet.8,9 Given the fact that melanomas occur mostly in
older, grey horses, some suggest that a storage disorder during the process of depigmentation
plays a role in the onset of melanomatosis. Thereby investigators tried to find out whether
vitiligo, a pigment disorder causing white spots on the hairless parts of the body, is associated
with the development of melanomas. Sölkner et al 2004 recorded melanomas, graying level,
vitiligo and heritability for several years and did not find a genetic correlation between speed
of greying and melanomas in horses.7,9 Though, other relevant studies haven’t resulted in any
significant outcomes yet. Nowadays, no unambiguous cause of melanomatosis has been
found.
Clinical relevance
The main disadvantage for the individual horse lies in the fact that the tumor masses can
interfere with the normal function of the urogenital- and/or digestion tract or other organs
such as genitals, eyes etc,depending of the location of the melanoma. Thereby the malignant
form can spread to different organs. Also it can interfere with the normal use of the horse
since it can be located on places where the riding tack covers the skin, hence, horses cannot be
used for riding/driving purposes anymore.
Therapy
An effective therapy has not been found yet. Surgery is an option in some cases although
extensive tumor masses in the perineal/perianal region are invasive and hard to remove
completely. Cryo-therapy and local chemotherapy can be used, sometimes in combination
with surgery, but they often seem to be unsuccessful due to relapses. However, treatment with
the H2 receptor antagonist Cimetidine has been found to regress growth of the primary tumor
2
and preventing disease progression, by acting as an immunomodulator.6,14 These
immunomodulatory effects result in an increase of natural killer cell activity. Research
showed us that human melanoma cells contain Histamine H2 receptors. Cimetidine therefore
is believed to be effective in at least three different ways: antagonism of the H2 receptor
leading to direct inhibition of tumor cell proliferation, activation of the local immune response
and blocking the effects of histamine on T-suppressor cells.6 Now that Ranitidine, another H2antagonist, did not lead to the latter described result, some other mechanisms of cimetidine
could be involved. However, the opinion about the effectiveness of cimetidine on melanomas
is still strongly contrasting.6,10, 12,13 Therefore, more research is necessary to demonstrate the
true effect of cimetidine on tumors.
Experiment
The Heilan International Equestrian Club (HIEC) in China owns about 300 horses of
different breeds for riding, show and competition purposes. This includes 60 grey horses of
Spanish and Portuguese origin. At clinical examination of these horses, melanoma suspected
skin proliferations were found on 41 of the 63 grey horses. There was a large variety in size
and number of these tumors on the horses and in some horses the size, number and location
hampered their wellbeing or interfered with the riding. Given the location and also often the
large amount of tumors (cryo) surgery was not an option. It was decided to use cimetidine on
these horses in order to try to stop the melanoma’s from growing in size and number. Since
this was a private club and melanoma’s normally do not go in regression by themselves it was
decided to treat all horses and not work with a control group. Knowing the results of this
treatment are disputed, we decided to keep proper records and publish them. Given the
cooperation of HIEC and Utrecht University, this was part of a study for veterinary students
from the Netherlands together with Chinese equine veterinarians under the supervision of a
senior equine veterinarian from Utrecht University stationed at the HIEC in China. This paper
is about the influence of the cimetidine treatment on the total number of melanoma’s on the
horse. Another paper will describe the effect of the cimetidine treatment on the size of the
melanoma’s.
Experimental design
A total of 41 grey horses with ages ranging between 5 and 19 years where used in this study.
The horses belonged to the Heilan International Equestrian centre in China, and included 22
Andalusian-, 6 Lusitano horses and 13 crossbreeds.. These horses are all in full training and
perform in the weekly Heilan horse show. They are all in good condition and their health
status is monitored and registered by the own Equine Veterinary Clinic at Heilan. In all these
horses the skin tumors where categorized as melanomas.
Cimetidine
The exact dose of cimetidine was calculated on the basis of the weight of the horses. The
cimetidine was administered as a watery solution. The exact amount of powder for 42 horses
was weighed on a digital scale. Every 8 hours the horses received 2,5 mg/kg, this dosage is
determined by previous similar research.4,6 Therefore the horses were classified in five
groups, the scale ranges from 400 until 650 kilograms. Five groups were made: 400-450; 450500; 500-550; 550-600; 600-650 kg.
3
125 gram cimetidine was dissolved in 0,5 liter water. The groups received respectively 4,5 ml, 5 ml,
5,5 ml, 6 ml and 6,5 ml of this concentration. Using a syringe the exact volume was administered to
the forage and given to the horse three times a day at the following times: 08:00, 16:00, 23:00 for 90
days.
Measuring
The first monitoring (t0) took place before the first administration of the cimetidine started. In
this session, all the horses were subjected to a general clinical examination. Inspection and
palpation of skin surface was carried out with special attention to the areas predisposed to
melanomas. A rectal examination was performed in each horse to determine whether
melanomas were present in the abdomen.
To diagnose whether the lesions were actual melanomas, cytological samples of the masses
were taken from every horse. The samples were obtained by fine needle aspiration with a 21
G needle and send to the diagnostic laboratory of the University of Utrecht, Faculty of
veterinary medicine. Once there, the samples where stained with May-Gruenwald Giemsa and
observed with a light microscope. At the same time the number, location and size of each
melanoma was recorded. Penile and preputial masses where mapped after sedation with
xylazine or detomidine. Special forms where used to draw and describe the location of each
melanoma and made it possible to number each. Direct measurement of every individual
melanoma was done using a caliper measuring the biggest length and the length
perpendicular to this. The size of the melanomas was noted on the form as well. Only
melanomas bigger than 0,4 x 0,4 mm where included in the measurements since the smallest
melanomas made it impossible to count and to proceed a reliable measurement. Thereafter, a
photo of the area containing the tumors was made with a digital camera. The caliper and the
number of the horse where shown on every photo as well.
After 30 (t1), 60 (t2) and 90 (t3) days the measurements were carried out again. The number,
location and size were recorded and new photo’s where made.
After every measurement the data written on the forms where processed in Microsoft ®office
exel (every horse had its own tab-page with a description of its melanomas, their sizes and
locations per time interval). It was important to relate every melanoma on the form to the
same melanoma on the form of the previous measurement. After the last measurement all
information was collected, processed using excel and statistically tested by using SPSS.
Statistical significance was tested with a Wilcoxon-signed test.
Results
41 horses with melanomas were selected from a group of 63 horses, meaning that 65% of this
population suffer from melanomas. Most of the horses did not show any clinical signs, with
the exception of one horse which had difficulties during defecation, thereby few horses
captured with problems during urination because of the disability to fully extend their penis.
Because the horses were purchased only a few years ago nothing is known about duration,
severity or growth of their melanomas in the past. Every horse included in this study was
tested positive for melanomas using the fine needle aspiration biopsy. Unfortunately, horse
H43 was not measured at t3, hence it was excluded from this experiment. The number of
melanomas per time interval of the remaining 40 horses is presented in table 1.
4
Tabel 1. Overview of the number of melanomas per horse per time interval
number of
melanomas
horse
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
t0
9
2
7
7
8
9
10
7
4
39
30
10
1
28
11
4
7
1
7
5
13
8
11
9
8
12
10
6
1
12
18
4
3
2
4
1
2
9
6
2
t1
11
4
12
10
10
12
17
9
5
18
17
10
1
19
21
7
11
1
8
5
13
11
7
13
9
12
10
5
1
18
19
4
3
2
5
4
3
11
7
3
gender
t2
14
4
9
11
10
16
18
9
4
23
20
7
1
23
14
6
12
1
14
5
17
8
9
15
14
12
9
5
1
17
19
4
3
2
4
3
3
13
7
2
t3
25
6
24
10
16
23
18
12
5
38
35
8
1
25
30
7
8
1
15
5
15
7
7
17
11
22
10
5
1
23
25
4
1
2
4
1
2
14
9
3
stallion
stallion
stallion
stallion
gelding
stallion
stallion
stallion
stallion
gelding
stallion
stallion
stallion
gelding
stallion
stallion
stallion
stallion
stallion
gelding
stallion
gelding
stallion
stallion
gelding
gelding
stallion
gelding
gelding
stallion
stallion
gelding
gelding
stallion
gelding
stallion
stallion
stallion
stallion
stallion
age
breed
(years)
17
Pura raza espanol
13
Pura raza espanol
10
half Lusitano crossbreed
17
Pura raza espanol
15
Pura raza espanol
10
Pura raza espanol
10
Pura raza espanol
11
Crutada
10
Pura raza espanol
14
Crutada
19
Lusitana
15
Pura raza espanol
15
Pura raza espanol
10
half Lusitano crossbreed
10
Pura raza espanol
10
Pura raza espanol
15
Pura raza espanol
9
crossbreed
12
Pura raza espanol
10
crossbreed
11
Pura raza espanol
11
Pura raza espanol
10
crossbreed
14
Pura raza espanol
15
Pura raza espanol
10
Lusitana
11
Lusitana
15
Cruzado portugues
14
Pura raza espanol
8
Lusitana
9
Lusitana
11
Pura raza espanol
13
Pura raza espanol
9
Cruzado portugues
13
crossbreed
7
Lusitana
10
Pura raza espanol
11
crossbreed
11
crossbreed
5
crossbreed
5
Because this study focuses on the question whether cimetidine has an effect on the number of
melanomas after administration of cimetidine for three months, only t0 and t3 were used in
the statistical tests. At t0 the number of melanomas per horse varied from a minimum of 1 and
a maximum of 39, the mean number of melanomas was 8,63 with a standard deviation of
7,95. T3 has a minimum of 1 and a maximum number of melanomas of 38, the mean is 12,40
with a standard deviation of 10,06. More interesting is the difference between t3 and t0 to
determine whether there is a decreased or increased number of melanomas after three months
of cimetidine. This difference had a minimum of -3 and a maximum of +20, that means that
the maximum reduction in the amount of melanomas was 3 and the maximum growth counted
20 melanomas. The mean of this difference between t3 and t0 was +3,78 and the standard
deviation 5,70. Figure 1 shows the difference in number of melanoma’s for each individual
horse. In figure 2 these differences are grouped to get an overview of the differences in
number of melanomas of the horses.
Fig 1. Differences in number of melanomas
comparing t0 and t3 for each individual horse
.
Fig 1. difference in the number of melanomas comparing t0
with t3
6
Figure 2: differences in number of melanomas,
grouped.
By taking a look at figure 1 and 2, man can see that 7 horses showed a regression in number
of melanomas and 23 horses had an actual increased number of melanomas and 10 horses
remained the same. Which means that 17,5% of the 40 horses had less melanomas after the
four months of cimetidine administration, 57,5 % had more and 25% remained the same.
Eventually, the distribution between every measurement is pointed out in a graph (figure 3).
This graph is mainly created to show the distribution between each measurement and not to
get an overview of the growth pattern of the melanomas. It is very obvious that there is a
notable difference between each measurement per horse. This will be explained in the
discussion later on.
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Fig 3. An overview about the distribution of the number of melanomas
measured on each horse and between the horses
‘
Statistics
Now that it can be concluded that in most horses the number of melanomas seems to be
increased, a statistical test is needed to determine whether this difference is significant. A test
was required to check whether this population was normally distributed. The Kolmogorovsmirnov test showed that this population of horses with melanomas was not normally
distributed. Due to this result the Wilcoxon-signed test was used to find out whether there was
a significant difference in the number of melanomas before and after the administration of
cimetidine. The null hypothesis says that there is no difference between the two
measurements , the alternative hypothesis says that there is a difference. After completing the
Wilcoxon-signed test it can be concluded that there is a significant increase in the number of
melanomas on most horses after the three months of cimetidine treatment (p = 0,05).
Conclusion and discussion
Given the results of our study we cannot confirm the positive results of cimetidine on the
number of melanoma’s in this group of horses as described in previous scientific research.
2,4,5,11,14
We discovered an actual increasing number of melanomas after three months of
administering cimetidine on these horses.
8
This study does not include a control group, so theoretically it is possible that horses not
treated with cimetidine during this period of time would have got an even bigger increase of
melanoma’s.
From a practical and managerial point of view oral administration of cimetidine to the horses
three times a day was not easy. After some testing the solution of the cimetidine powder in
water in a workable concentration and adding it to some forage with a syringe turned out to be
the most practical. There were no problems regarding the uptake of the forage with the diluted
cimetidine at all, the horses even liked it. Since the horses where in a tight training schedule,
some horses might have had the medication at a slightly different interval.
Counting melanoma’s on a horse is easier said than done given the locations, the character of
the horses and the pliability of the skin. Using the caliper it is hard to measure exactly always
the same way, sometimes one melanoma was measured smaller by pushing harder on the
caliper or larger by keeping it loose. In our study the measurements were carried out by a
group of three persons and despite instructions there can be personal difference as well. Since
the inclusion criteria for the melanoma’s is that they should be > 4 mm x 4 mm this may also
influence the number of melanoma’s present.
The preputium and penis are important predilection areas. In our study, four horses were
found to have penile and/or preputial melanomas at t0. Unfortunately, two horses of these
horses were not counted at t3 and one of them did not fully extend his penis so we could not
count all of the melanomas. Therefore, only one case of penile/preputial melanomatosis is
included in this study.
The photos were made with a digital camera in the clinic on location. Whereby the photos
could be used to count and measure again by using the caliber present on the photos and/or by
using special techniques such as applications of Adobe Photoshop®. Because of the diversity
in ways of measuring it could be useful to find out whether a melanoma is larger or smaller
than 0,4x0,4mm by using the photographs, processed by only one person. However, these
photos were taken by hand, which means that there was no standard distance between the
object and the camera what made it impossible to measure these melanomas afterwards. In
addition, the distance between the caliper and the horse was unknown as well so that
measuring by using the caliper on the photo was unreliable. Thereby, in most of the photos
the light was not optimal, therefore some melanomas were not visible on the photo due to
overexposing or underexposing. Given this we had to adhere to the measurements performed
by the different persons on the forms. Afterall, the photos were not completely useless since
we used them in some cases to see if multiple melanomas were counted as one unit or as few
little melanomas together.
In following studies about the effect of cimetidine on tumor growth we would strongly
recommend to make use of a control group. Also we would advise to make sure with every
measurement that no melanoma has been forgotten. In addition, for a complete count of
melanomas, melanomas in the digestive tract and melanomas smaller than 0,4x0,4 mm should
be included in the study. To optimize the image storing it could be useful to use a tripod and a
good light source. This makes it possible to maintain equal and known distances between
object and camera. In this way it is possible to calculate the scale on the photo and measure
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the size of the melanomas using applications on specialized computer programs. Although it
remains difficult because the tail, for example, is not always kept in the same angle.
In conclusion, although our results confirm that the use of cimetidine in dermal
melanomatosis is not effective in our study, we believe that studies about the effectiveness of
cimetidine on tumor growth should be continued to determine whether the positive effects
found in previous literature can be proved and more importantly, used in practice.
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