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\begin{document}

\title{Retrospective study: Laser excision versus combined laser, cryotherapy
and intralesional 5-fluorouracil in the treatment of equine sarcoids.}



\author[1]{Katie Offer}%
\author[1]{David Sutton}%
\affil[1]{University of Glasgow College of Medical Veterinary and Life Sciences}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\emph{Background} Laser excision is used routinely in the treatment of
sarcoids but may be ineffective in cases where complete excision cannot
be achieved. A multimodal approach is warranted in these cases. 5-FU may
improve the lethal effect of cryotherapy as an adjunct to laser
excision. \emph{Objectives} To compare two treatment protocols for
equine sarcoids: laser excision alone versus a combination protocol of
laser excision, cryotherapy, and 5-FU chemotherapy. Factors associated
with sarcoid recurrence are also investigated. \emph{Study Design}
Retrospective case controlled study. \emph{Results} Eighty-four horses
with 168 histologically confirmed sarcoids were included, with a median
follow up time of 39 months (IQR 21-62 months). Sarcoid recurrence at
the treated site was reported in 38\% of cases and in 23\% of any
individual sarcoids. No significant difference was demonstrated between
treatment categories in either rate of sarcoid recurrence (p=0.45 for
any treated horse, p=0.63 for individual sarcoids) or time to sarcoid
recurrence (p=0.73). Sarcoid recurrence was higher in horses with a
greater number of sarcoids (OR 1.2 (1.0-1.5), p=0.03); when treatment
had been received prior to admission (OR 7.6 (2.0-33), p=0.004); for
individual sarcoids [?]100mm in diameter (OR 5.6 (1.1-30), p=0.03); and
for treatment under general anaesthesia (OR 5.0 (1.4-19), p=0.01).
Horses with urogenital sarcoids and \textgreater{}1 mixed sarcoid
experienced more rapid recurrence (HR 3.6 (1.3-10), p=0.02 and HR 9.9
(3.3-30), p\textless{}0.001) and recurrence was less rapid following the
treatment of a horse's first sarcoid (HR 0.3 (0.1-0.7), p=0.009).
\emph{Main Limitations} Significant differences in case populations in
each treatment category. Treatment selection was neither blinded nor
randomised and missing data and recall bias limit the study's power.
Sarcoid recurrence was owner reported. \emph{Conclusions} When assessing
the likelihood of sarcoid recurrence, characteristics of the individual
patient and sarcoid(s) are likely to be more significant than selection
of any individual treatment protocol.%
\end{abstract}%



\sloppy


\emph{Retrospective study: Laser excision versus combined laser,
cryotherapy and intralesional 5-fluorouracil in the treatment of equine
sarcoids.}

\textbf{Running Title: Treatment of equine sarcoids}

\textbf{Katie S Offer, David G M Sutton}

School of Biodiversity, One Health and Veterinary Medicine, College of
Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

Corresponding author email: katie.offer1@gmail.com

\subsection*{Summary}

{\label{summary}}

Background

{\label{background}}

Laser excision is used routinely in the treatment of sarcoids but may be
ineffective in cases where complete excision cannot be achieved. A
multimodal approach is warranted in these cases. 5-FU may improve the
lethal effect of cryotherapy as an adjunct to laser excision.

Objectives

{\label{objectives}}

To compare two treatment protocols for equine sarcoids: laser excision
alone versus a combination protocol of laser excision, cryotherapy, and
5-FU chemotherapy. Factors associated with sarcoid recurrence are also
investigated.

Study Design

{\label{study-design}}

Retrospective case controlled study.

Results

{\label{results}}

Eighty-four horses with 168 histologically confirmed sarcoids were
included, with a median follow up time of 39 months (IQR 21-62 months).

Sarcoid recurrence at the treated site was reported in 38\% of cases and
in 23\% of any individual sarcoids. No significant difference was
demonstrated between treatment categories in either rate of sarcoid
recurrence (p=0.45 for any treated horse, p=0.63 for individual
sarcoids) or time to sarcoid recurrence (p=0.73).

Sarcoid recurrence was higher in horses with a greater number of
sarcoids (OR 1.2 (1.0-1.5), p=0.03); when treatment had been received
prior to admission (OR 7.6 (2.0-33), p=0.004); for individual sarcoids
[?]100mm in diameter (OR 5.6 (1.1-30), p=0.03); and for treatment under
general anaesthesia (OR 5.0 (1.4-19), p=0.01). Horses with urogenital
sarcoids and \textgreater{}1 mixed sarcoid experienced more rapid
recurrence (HR 3.6 (1.3-10), p=0.02 and HR 9.9 (3.3-30),
p\textless{}0.001) and recurrence was less rapid following the treatment
of a horse's first sarcoid (HR 0.3 (0.1-0.7), p=0.009).

Main Limitations

{\label{main-limitations}}

Significant differences in case populations in each treatment category.
Treatment selection was neither blinded nor randomised and missing data
and recall bias limit the study's power. Sarcoid recurrence was owner
reported.

Conclusions

{\label{conclusions}}

When assessing the likelihood of sarcoid recurrence, characteristics of
the individual patient and sarcoid(s) are likely to be more significant
than selection of any individual treatment protocol.

\subsection*{Keywords}

{\label{keywords}}

Horse, sarcoid, recurrence, cryotherapy, chemotherapy

\subsection*{Clinical Relevance}

{\label{clinical-relevance}}

\begin{itemize}
\tightlist
\item
  In a referral population consisting of horses with multiple and/or
  invasive sarcoids, sarcoid recurrence may be expected in 38\% of cases
  and 23\% of individual sarcoids.
\item
  Sarcoid and patient characteristics are likely to be more significant
  in determining the likelihood of sarcoid recurrence than selection of
  any particular treatment protocol.
\item
  A multimodal approach employing diode laser excision, cryotherapy and
  intralesional 5-fluorouracil may be employed in cases where complete
  sarcoid excision is difficult to obtain.
\end{itemize}

\subsection*{Introduction}

{\label{introduction}}

The equine sarcoid is the most common equine cutaneous neoplasm,
responsible for approximately 46\% of neoplastic cutaneous biopsy
samples. (Schaffer et al., 2013; Taylor \& Haldorson, 2013) Although
tumours are rarely metastatic, aggressive local invasion and secondary
infection/ulceration may have a significant impact on equid welfare.
(Ireland et al., 2013; Taylor \& Haldorson, 2013) Currently no uniformly
effective therapy for its treatment has been reported despite
publication of numerous treatment protocols. (Lane, 1977; Klein et al.,
1986; McConaghy et al., 1994; Carstanjen et al., 1997; Knottenbelt \&
Kelly, 2000; Martens et al., 2001; McCauley et al., 2002; Byam-Cook et
al., 2006; Stewart et al., 2006; Theon et al., 2006; Stadler et al.,
2011; Tamzali et al., 2012; Compston et al., 2016; Hollis \& Berlato,
2018; Hollis, 2020; Knottenbelt et al., 2020) Laser excision, either by
CO\textsubscript{2} or diode laser, is widely used for the surgical
removal of sarcoids. (Carstanjen et al., 1997; A. Martens et al., 2001;
McCauley et al., 2002; Compston et al., 2016) As with any surgical
excision, one of the major limitations is that surgical margins must
still be obtained. This is frequently difficult with equine sarcoids,
depending on sarcoid type, size and anatomic location. (Knottenbelt,
2019) Though thermal injury secondary to laser excision extends beyond
the surgical field, this is variable and difficult to predict.
(Knottenbelt, 2019; Tate, 2019) A multimodal approach to sarcoid
treatment has frequently been suggested to reduce sarcoid recurrence
rates, (Klein et al., 1986; Spoormakers et al., 2003; Tamzali et al.,
2012; Knottenbelt, 2019) likely due to greater destruction of remaining
neoplastic cells in situations where complete tumour excision has not
been achieved.

Cryotherapy, the freezing of cells in this case by the application of
liquid nitrogen, kills cells directly due to ice crystal formation and
microcirculatory failure. (Baust \& Gage, 2005) Papers investigating
cryotherapy as a treatment modality suggest success rates (Klein et al.,
1986)may be as low as 9\% when used as a sole therapy. (Knottenbelt \&
Kelly, 2000) Recent molecular research has focussed on tissues in the
periphery of the cryotherapy treated zone where cell death is delayed
and via apoptosis. (Hollister et al., 1998) It has been suggested that
further therapy promoting apoptosis in this region may be beneficial in
improving the lethal effect of cryotherapy. (Baust \& Gage, 2005)
(Clarke et al., 1999)

5-fluorouracil (5-FU) is a fluoropyrimidine antimetabolite used in the
treatment of a range of cancers, particularly breast and colorectal
cancers. (Longley et al., 2003) It exerts its effects via incorporation
of the fluoronucleotides into cellular RNA and DNA, leading to
subsequent cellular dysregulation and apoptosis. (Longley et al., 2003)
In horses, its use has been reported in the treatment of ocular or
periocular squamous cell carcinoma, (Pucket \& Gilmour, 2014; Offer et
al., 2022) and as a topical agent for the treatment of periocular
sarcoids with variable success. (Knottenbelt \& Kelly, 2000)
Intralesional 5-FU alone has a reported sarcoid resolution rate of
61.5\%, though rates may be lower in large or `resistant' tumours.
(Stewart et al., 2006) Its use as an adjunct to cryotherapy warrants
further investigation since it has been suggested that agents, such as
5-FU, may be beneficial in promoting the apoptotic cell death at the
periphery of cryotherapeutic sites. (Clarke et al., 2001; Baust \& Gage,
2005)

Objectives and hypotheses

This study aimed to compare two treatment protocols for the treatment of
equine sarcoids: laser excision alone versus a combination protocol of
laser excision, cryotherapy, and 5-FU chemotherapy. As a secondary aim,
factors associated with sarcoid recurrence were investigated. The
authors hypothesise that the combination protocol will result in lower
sarcoid recurrence rates than the laser excision protocol.

\subsection*{Materials and methods}

{\label{materials-and-methods}}

Records were reviewed for horses referred to Glasgow Equine Hospital for
the treatment of sarcoids between 2013 and 2022. Criteria for case
inclusion were horses with histologically confirmed sarcoids treated
either by laser excision via diode laser, or by the below combination
therapy protocol. Cases were excluded if histopathological confirmation
was not available, treatment was incomplete, or any other combination
treatment was employed.

The standard combination therapy protocol consisted of initial excision
of the mass(es) via diode laser with 1-2cm surgical margins where
possible. The surgical site was then treated with 3 freeze- thaw cycles
of cryotherapy followed by administration of 5-FU into the margins.
Cryotherapy was performed using the
CryoPro\selectlanguage{ngerman}\textsuperscript{®}cryosurgery unit (Cortex Technology, Denmark).
The unit was held approximately 10cm from the desired site and liquid
nitrogen was applied to spray freeze the site until firm. Care was taken
to avoid excessive freezing of the underlying tissues. The tissues were
allowed to thaw, before repeating a further 2 times. Thereafter, 50
mg/ml 5-FU (Medac) was injected into the resultant wheal to an estimated
dose of 1 ml/cm\textsuperscript{3} target tissue. Appropriate personal
protective equipment (two layers of nitrile gloves, full length gown and
face shield) was used during the application, and the horse handled only
when wearing nitrile gloves for 3 days thereafter. The horse was
re-examined between 2-5 weeks thereafter, and the cryotherapy and 5-FU
application repeated. This was repeated as required by clinical
progress, for a median of 3 total treatments (IQR 2-3).

Data obtained from medical records included: case history and
signalment, details of the lesion(s) (sarcoid type, location, number,
estimated tumour dimensions and volume), treatment protocol, associated
adverse effects and duration, response to treatment and sarcoid
regrowth/ new lesion occurrence. For the purpose of recording sarcoid
location, the body was split into 7 anatomic regions: periocular, head
and neck, dorsum (above the level of the olecranon process), ventrum
(below the level of the olecranon process), upper limb (above the level
of the tarsocrural/ radiocarpal joint), lower limb (below the level of
the tarsocrural/ radiocarpal joint) and urogenital (affecting the penile
sheath, penis, mammary glands or vulva). Sarcoid type was recorded using
the currently accepted convention. (Knottenbelt, 2005) Previous
histopathological findings were recorded, including the presence or
absence of confirmed surgical margins. The volume of sarcoid was
estimated using the ellipsoid volume formula proposed by Tomayko and
Reynolds. (Tomayko \& Reynolds, 1989)

Following initial correspondence, owners were contacted via telephone
between August and October 2022 to determine treatment outcome. An
example of the telephone questionnaire is included in Supplementary
material 1. Data were collected regarding the horse's history of sarcoid
growth and treatment, outcome following sarcoid treatment, regrowth of
treated sarcoids and growth of any new sarcoids.

Data analysis

Data were analysed both by case, and then by individual sarcoid.
Statistical analysis was performed in R studio (R version 4.2.0). (R
Core Team, 2022) Normality was assessed for continuous variables using
the Shapiro-Wilk test. Thereafter, univariate analysis was performed by
chi-squared test or Fisher's exact test for categorical variables,
Wilcoxon's rank sum for continuous variables and Cochrane Armitage test
(two sided) for trend, where appropriate.

For variables of p\emph{\textless{}} 0.2 in the univariate analysis,
logistic regression was performed with sarcoid recurrence as the
dependant variable. Variables with p\emph{\textless{}} 0.2 in the
univariate analysis, n \textgreater{} 10 and low collinearity with other
independent variables, were used to perform multinominal logistic
regression, again with recurrence as the control- dependent variable.
Variables were removed from the model in a manual backwards elimination
manner with the Akaike information criterion (AIC) used to assess the
fit of the model at each step. Variables were retained in the final
model despite not fulfilling p\emph{\textless{}} 0.2 when their
inclusion improved the AIC of the model. Variance inflation factors were
used to assess final variables for multicollinearity, and binned
residual plots created to assess normality of the residuals.

All variables with p\emph{\textless{}} 0.2 in the univariate logistic
regression were evaluated for inclusion in a Cox's proportional hazards
model. Data were right censored at either time to recurrence of the
treated sarcoid, time to follow up telephone conversation or time to
death or sale of the horse. Where multicollinearity existed, the value
with the lowest p value was included. Numerical covariates were
transformed to categorical due to violation of the linearity assumption
of the Cox's proportional hazards model. The model was refined using a
manual backwards elimination approach, with threshold for inclusion in
the model at p\emph{\textless{}} 0.2 whilst maximising concordance of
the model. A plot of scaled Shoenfeld residuals was used to assess the
proportional hazards assumptions of the model. Time to sarcoid
recurrence was used to create a Kaplan-Meier plot, and differences
between treatment protocols compared with a log rank test. Statistical
significance was considered at p\textless{}0.05.

\subsection*{}

{\label{section}}

\subsection*{Results}

{\label{results-1}}

One hundred and eighty equids were referred to Glasgow Equine Hospital
for the treatment of sarcoids during this period, of which 84 met the
inclusion criteria. Histological confirmation was available for 168
individual sarcoids in these 84 horses. Of these, 8 (5\%) were
categorised clinically as occult sarcoids, 26 (15\%) as verrucose, 80
(48\%) as nodular, 24 (14\%) as fibroblastic and 26 (15\%) as mixed.
None were classed as `malevolent,' and sarcoid type was not recorded in
4 cases (2\%). Seventeen different breeds were represented, of which 17
horses were crossbreeds (20\%), 11 Irish Sport Horses (13\%), 10
warmbloods (12\%), 6 Thoroughbreds (7\%) and 5 cobs (6\%). Four donkeys
were included in the study (5\%), and the remaining horses were of
various breeds. Thirty-two of included equids were mares (38\%), 51
geldings (61\%) and 1 stallion (1\%). Ages ranged from 2-20 years
(median 8 years (IQR 6-14 years)). Owner follow up was available for
69/84 individuals, and regrowth ascertained from medical records in a
further 4. Horses for which follow up was not available were excluded
from further analysis. Sixty two horses had multiple sarcoids at the
time of presentation, with a median of 3(IQR 2-7).The overall incidence
of recurrence of any sarcoid on an equid at the same previously treated
site was 38\% (28/73) and time to follow up ranged from 0.75-132 months
(median 39 months (IQR 21-62 months)). For any individual treated
sarcoid, total rate of recurrence was 23\% (29/128) with a median length
of follow up of 48 months (IQR 24-61 months).

In total, 28 equids were treated by the combination treatment protocol
(33\%) and 56 by diode laser excision alone (67\%). Complications
occurred in 14 horses following either treatments (17\%). These were
short lived and comprised: oedema surrounding the treated site (5/14,
36\%), owner reported delayed healing (3/14, 21\%) or infection (3/14,
21\%) of the treated site, and individual cases of myiasis, facial nerve
neuropraxia and excessive scarring (1/14, 7\% each). Overall, 60/67
owners (90\%) said they were happy with the final cosmetic result of the
treatment. Of the 16 horses reported to have been euthanised prior to
follow-up, four of these were for sarcoid- related reasons, resulting in
a fatality rate of 4/69 (5.8\%) over the duration of the follow up
period in this referral population.

Sarcoid recurrence at any treated site was reported in 20/47 (43\%) of
horses receiving laser excision alone and 8/26 (31\%) treated by
combination therapy for which follow up was available. For any
individual sarcoid, eventual recurrence at the same site occurred in
23/95 (24\%) of those treated by laser excision and 6/33 (18\%)
receiving the combination protocol. These differences were not
statistically significant in either case (p=0.32 and p=0.48
respectively). Further differences in the case populations between
treatment categories were examined to aid comparison between groups, and
results are presented in Supplementary Material 2. Briefly, equids in
the combination category had larger sarcoids (median width 59 mm versus
30 mm, p\textless{}0.001) but were less likely to have any incision
closed at the time of excision (p \textless{} 0.001).

Postoperative complications were more frequent in horses treated by
combination protocol (9/25 (36\%) versus 5/45 (11\%), p=0.03) and length
of follow up significantly shorter (20months (14-25 months) versus 52
months (39-72 months), p\textless{}0.001).

\subsection*{Likelihood of Sarcoid
Recurrence}

{\label{likelihood-of-sarcoid-recurrence}}

A forest plot of variables significant in the logistic regression on
both a whole case and individual sarcoid basis is shown in Figure 1.
Full results are available in Supplementary material 3.

Recurrence was more likely if the ventrum was affected (OR 3.9 (1.5-11
p\textless{}0.001), particularly by verrucose (OR 1.9 (1.2-3.4), p=0.01)
or mixed (OR 1.9 (1.3-3.5), p=0.002) sarcoids. Nineteen horses and 20
individual sarcoids had received attempted sarcoid treatment prior to
presentation, and this was significantly associated with subsequent
sarcoid recurrence (13/19 (68\%), OR 7.7 (2.5-27) p\textless{}0.001 and
9/20 (45\%), OR 4.9 (1.5-16) p=0.004). A greater total number of
sarcoids at presentation was also associated with sarcoid recurrence (OR
1.3 (1.1-1.5), p\textless{}0.001) as was the surgical closure of any
incision at the time of initial treatment (OR 4.0 (1.5-11.6), p=0.007).
Sarcoid chronicity was also associated with recurrence both for affected
animals (`cases') and individual sarcoids (p=0.03 and \textless{}0.001)
and any individual sarcoid treated under general anaesthesia was more
likely to recur (OR 6.3 (2.5-17) p\textless{}0.001) than if treated
standing under sedation.

The final multinominal logistic regression models for variables
predictive of sarcoid recurrence are presented in \emph{Table 1} . The
effect of treatment category on recurrence rate whilst accounting for
significant population differences between treatment category (largest
sarcoid width (mm), closure of any incision, occurrence of complications
following treatment and length of case follow up (days)) was examined.
Treatment category remained non-significant in this model (OR 1.3
(0.14-13), p=0.82).

Time to sarcoid recurrence

Data were available regarding time to sarcoid recurrence were available
for 72 horses. Variables retained as significantly associated with time
to sarcoid recurrence in the Cox's Proportional Hazards model are
presented in \emph{Table 2} and the final Kaplan- Meier plot of sarcoid
recurrence for each treatment category is presented in \emph{Figure 2} .
The log-rank test comparing the survival curves again indicated no
significant difference between treatment categories (p=0.73).

\section*{Discussion}

{\label{discussion}}

\subsubsection*{Treatment protocols}

{\label{treatment-protocols}}

The rate of recurrence of any sarcoid in this study was 23\%, with a
median length of follow up of 48 months. There was no significant
difference in recurrence between treatment category (laser 24\% (23/95),
combination 18\% (6/33)). The lack of routinely accepted standardised
outcome measure for the treatment of sarcoids makes comparison between
studies very difficult. (Offer et al., 2024) Though these recurrence
rates are marginally higher than those reported in the literature
(sarcoid regression rates of 83- 89\% with sole laser excision),
(Martens et al., 2001; Compston et al., 2016) this is highly likely to
be influenced by numerous factors, including greater length of follow up
period in this study, and differences in case selection and treatment
populations. These horses were all referred to the hospital for further
evaluation and treatment, and by definition were positioned at the more
severe end of sarcoid phenotype presentation.

Employing a multimodal approach to the treatment of sarcoids has been
suggested by previous authors as a method to improve sarcoid regression
rate, (Knottenbelt, 2019) and was highlighted by this author's recent
systematic review as likely to be advantageous over single treatment
modalities. (Offer et al., 2024) There are several confounding factors
that may have prevented the demonstration of a significant difference
between treatment categories in this retrospective study. Firstly, there
exist several significant differences in the case populations between
treatment groups. Sarcoids treated by the combination protocol were
significantly larger (p\textless{}0.001) and were less likely to have
been treated under general anaesthesia (p=0.04). Both these factors may
prevent attainment of an adequate surgical margin, depending on the
location of the sarcoid and the temperament of the horse. Sarcoids in
the combination treatment category were also on significantly older
horses. Whilst rare, spontaneous remission does occasionally occur in
younger horses, and so this may have further confounded the results.
(Berruex et al., 2016) Conversely, equids treated by laser therapy alone
had a significantly longer time to follow up than those treated by the
combination protocol (median 52 months versus 20 months) due to the
distribution of cases and historical clinician preference. Longer term
recurrence may therefore be underestimated in the combination protocol
treatment category.

However, when a multivariable model was constructed to account for
differences in case population between categories, treatment category
remained non-significant in the model (p= 0.82). Since sarcoid
recurrence is dependent on numerous variables, (Knottenbelt, 2019) case
selection is crucial in determining its likelihood. A true comparison of
these treatment techniques would therefore require a fully randomised
and blinded controlled trial.

\subsubsection*{Risk factors for
recurrence}

{\label{risk-factors-for-recurrence}}

The total number of sarcoids on the individual at the time of
presentation was significant in both the univariate and multivariate
logistic regression. This supports increased susceptibility of certain
individuals to the development of sarcoids. This is well recorded in the
literature- the presence of multiple sarcoids has been reported by
several authors as predictive of an increased likelihood of sarcoid
recurrence, (Lane, 1977; Compston et al., 2016) and it has been shown by
numerous authors that the susceptibility to sarcoids has at least some
genetic basis. (Broström et al., 1988; Lazary et al., 1994; Christen et
al., 2014) The presence of equine leukocyte antigen (ELA) W13 allele has
been correlated with susceptibility to sarcoid growth, although horses
without the allele may also develop sarcoids. (Goodrich et al., 1998)
Candidate genes within specific chromosomal regions also have been
associated with increased susceptibility to sarcoid growth, (Jandova et
al., 2012) and a polygenic mode of inheritance with 21\% heritability
has been demonstrated. (Christen et al., 2014) Presentation at the time
of first sarcoid growth was similarly retained in the survival analysis
as predictive of an increased time to subsequent sarcoid regrowth, i.e.
those individuals with increased susceptibility to sarcoid growth who
present with multiple sarcoids spanning several years have a shorter
time to regrowth than those with solitary sarcoids at first
presentation.

Treatment of any sarcoid prior to presentation was associated with
sarcoid recurrence and was retained in the whole case multivariable
model. The reason for this is two-fold; presentation following prior
treatment indicates that any previous intervention has failed to resolve
the sarcoid. The sarcoid itself is therefore likely to be highly locally
invasive and/or anatomically difficult to remove in its entirety. In
addition, any traumatic intervention to a sarcoid may cause accelerated
growth or increased malignancy, leading to subsequent difficulty in
achieving full sarcoid resolution. (Knottenbelt et al.) Similarly,
recurring sarcoids are likely to be more aggressive and infiltrative
than those treated on first presentation. (Taylor \& Haldorson, 2013)

Further variables associated with sarcoid recurrence in this study may
be associated with the ability to achieve surgical margins at initial
laser excision. Sarcoid chronicity and size are likely correlated and
affect the ability to achieve complete tumour excision. There is likely
to be conscious reluctance to create huge skin deficits in horses with
the largest sarcoid masses. Similar difficulties occur when removing
sarcoids on the ventrum, particularly when attempted under standing
sedation, as limited by patient tolerance and staff safety. Verrucose
and, to a lesser extent mixed sarcoids (often with verrucose
boundaries), have often been reported to have a poorer outcome than
other sarcoid morphological types. (Knottenbelt \& Kelly, 2000; Compston
et al., 2016) It has been hypothesized that these sarcoids may extend
further into the dermal layer than other types, and gross margins are
less defined, potentially influencing the attainment of surgical
margins. (Martens et al., 2000; Compston et al., 2016) Complete excision
of neoplastic cells logically is often stated as a significant factor
contributing to sarcoid resolution, (Carstanjen et al., 1997;
Knottenbelt, 2019) though the desirable surgical margins for equine
sarcoids have not been well defined. A margin of between 1 and 2cm is
often stated as desirable as BPV DNA has been demonstrated in 33\% of
cases with a surgical margin of 16mm, (Martens et al., 2001; Compston et
al., 2016; Knottenbelt, 2019) and histopathological evidence of sarcoid
infiltration has been demonstrated at 2cm from the sarcoid margin
removed via laser excision. (Mair \& Fews, 2016) Coagulative necrosis
extends beyond the surgical site when laser excision is employed but
this may be insufficient to prevent sarcoid recurrence in verrucose or
mixed sarcoids where neoplastic cells extend beyond this region of
necrosis. (Knottenbelt \& Kelly, 2000; Compston et al., 2016) However,
the presence of papillomaviral DNA within the surgical margin has
inconsistently been associated with sarcoid recurrence, as has any
association between width of surgical margin and recurrence. (Martens,
et al., 2001)

The treatment of a sarcoid under general anaesthesia was identified as a
factor positively associated with recurrence in this study. Whilst
general anaesthesia and patient recumbency allow for greater and more
precise access to the sarcoid sites, this is confounded in this study by
case selection and clinician preference. This should therefore be
interpreted with extreme caution, and restraint via general anaesthesia
should remain the preferred option for equids presenting with numerous,
large, or infiltrative masses, or where patient temperament does not
allow surgical excision whilst standing.

\subsubsection*{Limitations}

{\label{limitations}}

There are a number of limitations to this study. Firstly, as with any
retrospective study, missing data, and loss of cases to follow up limits
its power. Owner recall bias may skew results, though answers were
corroborated with written clinical records where possible. The data
described here apply only to a referral hospital with one case
population. With the treatment of equine sarcoids, case selection plays
a significant role in the likelihood of tumour recurrence. Though
populations in each treatment group were compared, they were not
identical and treatment assignation was neither randomised nor blinded.
Similarly, there was no untreated or placebo control population in this
study, and so findings should be interpreted with caution. A significant
limitation with the survival analysis was the differing follow up times
between treatment categories, although it should be noted that both
exceeded the 12-month period used in many previous studies. This may
have prevented any demonstration of differences in time to sarcoid
regrowth between groups.

\subsubsection*{Conclusion}

{\label{conclusion}}

No significant difference in sarcoid recurrence rate or time to sarcoid
recurrence has been demonstrated with the use of a
cryotherapy/chemotherapy protocol following laser excision versus laser
excision alone, though this is limited by available time to follow up
and differences in case selection. When assessing the likelihood of
sarcoid recurrence, characteristics of the individual patient and
sarcoid(s) are likely to be more significant than selection of treatment
protocol. Regardless of treatment protocol, approximately 25\% sarcoid
recurrence may be expected at 24 months following hospital discharge.

\subsection*{Declarations}

{\label{declarations}}

\textbf{Authorship}

K. Offer was responsible for data collection and analysis. Both authors
were involved in preparation of the manuscript.

Authors declarations of interest

The authors have no competing interests to declare.

\textbf{Source of funding}

No funding was required for the study.

\textbf{Ethical animal research}

Ethical approval was obtained from the University of Glasgow Research
Ethics Committee (reference EA42/21).

\textbf{Acknowledgements}

The authors would like to acknowledge the Joana Devesa and Amie McGarva
for their help in data gathering.

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\subsection*{Figure/ Table Legends}

{\label{figure-table-legends}}

\emph{Table 1: Multinominal logistic regression models for variables
associated with sarcoid recurrence in 84 horses with histopathological
confirmation of sarcoid(s).}

\emph{Table 2: Cox's proportional hazards model of variables associated
with time to sarcoid recurrence showing Hazard Ratios (HR) and 95\%
confidence intervals {[}CI{]} for included variables.}

\emph{Figure 1: Forest plot showing Odds Ratios (OR) and 95\% confidence
intervals for variables significantly associated with likelihood of
sarcoid recurrence within an individual and for individual treated
sarcoid lesion. The red dashed line indicates an OR of 1.}

\emph{Figure 2: Kaplan-Meier plot showing time to sarcoid recurrence in
both treatment categories. The table shows the number of included horses
remaining with no sarcoid recurrence at each time point and in each
treatment group, and therefore remaining for inclusion in analysis.}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

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