• Open Access

Treatment of Clinically Diagnosed Equine Sarcoid with a Mistletoe Extract (Viscum album austriacus)

Authors


  • This work was done as a field study in Western Switzerland.

Corresponding author: Ophélie Christen-Clottu, Research Institute of Organic Agriculture (FiBL), Ackerstrasse, CH-5070 Frick, Switzerland; e-mail: ophelie.christen@fibl.org.

Abstract

Background: Equine sarcoids (ES) are common, difficult to treat, and have high recurrence rates. Viscum album extracts (VAE) are used in human cancer treatment.

Hypothesis: That therapy with VAE (Iscador P) is effective in the treatment of ES.

Animals: Fifty-three horses (444 ES); 42 were treated with VAE or placebo as monotherapy; 11 were treated with VAE or placebo after selective excision of ES.

Methods: Prospective, randomised, blinded, clinical trial. Horses were randomly assigned to treatment (VAE; n = 32) or control group (Placebo; n = 21). One milliliter of VAE (Iscador P) in increasing concentrations from 0.1 to 20 mg/mL or physiological NaCl solution was given SC 3 times a week over 105 days. Number, localization, and type of the ES were documented over 12 months. A subset of 163 clinically diagnosed equine sarcoid (CDES) lesions (95 VAE, 68 Placebo) was evaluated in detail, considering clinical findings and tumor volume.

Results: No undesired adverse effects were observed except for mild edema at the injection site in 5 of 32 horses (16%). Complete or partial regression was observed in 13 horses of the VAE group (41%) and in 3 of the control horses (14%; P < .05). After VAE treatment, 48 of 95 CDES (67%) showed an improvement compared with 17 of 68 CDES in the control group (40%; P < .01). Twenty-seven CDES had disappeared completely in the VAE group (38%) compared with 9 CDES in the control group (13% NS).

Conclusions and Clinical Importance: VAE (Iscador P) represents a safe and effective treatment for CDES.

Abbreviations:
CDES

clinically diagnosed equine sarcoid

ES

equine sarcoid

RTV

relative tumor volume

TV

tumor volume

VAE

Viscum album extract

Sarcoid is the most common skin tumor in equids.1 Various therapeutic measures have been described. Common surgical approaches are conventional surgical excision,2,3 cryosurgery with liquid nitrogen,3 and laser surgery.4 Other physical treatment methods are thermotherapy,5 radioactive implants with Iridium-192,6,7 and photodynamic therapy.8 Local topical or intralesional chemotherapy is used in the form of ointments such as 5-fluorouracil9 or imiquimod10 and water in oil emulsions containing cisplatin.11 Immunotherapy with the goal to stimulate auto-defensive mechanisms represents another therapeutic strategy.12 Veterinary immunomodulatory preparations include exogenous immunostimulants derived from bacterial (BCG-vaccine),3,13–15 viral,16,17 or plant12 sources and endogenous immunostimulants such as tumor necrosis factor,18 interleukin,19 or autogenous vaccine.20 Finally, complementary and alternative treatment methods complete the variety of therapeutic concepts used to treat equine sarcoid (ES).21 None of these methods appears to be universally effective.1 Spontaneous regression of ES has been observed in young horses,16 in horses with low numbers of tumors,22 and in untreated affected individuals.19

European mistletoe, Viscum album L., aqueous extracts (VAE) are used as adjuvant in the treatment of human cancer.23 The antitumoral properties of the VAE are mainly attributed to mistletoe lectins, which are glycoproteins, and viscotoxins, a class of polypeptides.24 These two main components showed cytotoxic and growth-inhibiting effects on a variety of human tumor cell lines in vitro and in mouse models.25,26 However, their immune modulating activity, in vitro and in vivo, can represent the relevant effect in cancer therapy.27 Administration of Iscador P, manufactured by extracts of pine mistletoes (Viscum album ssp. austriacus), has been advocated in the treatment of human skin tumors.28 The goal of the present study was therefore to evaluate the effects of a VAE (Iscador P)a on clinically diagnosed equine sarcoid (CDES) of horses.

Materials and Methods

Horses and Study Design

Fifty-three horses with CDES were recruited in Western Switzerland for the study, which was approved by the Swiss animal use committee. In 42 cases (79%), a histological examination was performed on the excised tumors (n = 10) or from biopsied material (n = 32). In all cases, histological investigation confirmed the clinical diagnosis of ES. The distribution of sex, age, breed, coat color, and number of CDES per horse is shown in Table 1. Forty-two horses were treated solely with the trial preparations, VAE or placebo, as monotherapy. In 11 horses the VAE or placebo treatment protocol was started 2 weeks after selective surgical excision. In these horses, 1 (n = 7), 2, 3, 8, and 9 sarcoids were each selected for excision by the treating clinician. In these horses, 3–45 CDES remained as clinically apparent tumors after surgery. Horses with other ES related therapies within 8 weeks before treatment or with signs of acute systemic diseases were not included.

Table 1.   Sex, age, breed, coat color, and number of tumors per horse in the study population.
 LevelVAEControlTotal
N%N%N%
  • *

    Significant difference (P < .05) between treatment groups (χ2 test).

SexMare14449432343
Stallion1341959
Gelding17538382547
Age1–5 years14448382242
6–9 years103110482038
>9 years8253141121
BreedFreiberger6195241121
Swiss Warmblood154711522649
Thoroughbred413210611
Other7223141019
ColorChestnut9286291528
Bay165013622955
Other722210917
Equine sarcoids frequency1–2 per horse7226291325
3–9 per horse123813622547
>9 per horse1341*210*1528
Total 32 21 53 

Sarcoids

In all horses, the number of sarcoids was recorded before the start of the treatment and during the follow-up investigations. A subset of selected tumors of foremost clinical importance according to morphology, size, or both (1–7 per horse; n = 163) was observed and evaluated by the same investigator (O.C.), recording localization (head, neck, chest, axillary region, abdomen, prepuce and udder, inner hind thigh and distal leg), demarcation (yes, no, ie diffuse), type (occult, verrucous, nodular, fibroblastic, mixed), and status (dry, ulcerated, sanguineous, infected). Tumor volume (TV) was calculated based on the formula29:

image(1)

where TVi is the estimated volume (mm3) of a single tumor, a and b are the length of 2 maximum perpendicular diameters, and h, the maximal height above normal skin level. The TV values were classified into 4 categories (<100 mm3, 101–1,000 mm3, 1,001–10,000 mm3, and >10,000 mm3). Diagnostic biopsy of single tumors was included into the analysis as a potential factor influencing development of ES.

Treatment Groups and Protocol

Horses were randomly assigned to the treatment (VAE) or control group (placebo). In order to limit the size of the placebo group, randomization was conducted with a ratio of 2 : 1. The resulting distribution was not significantly different from the expected distribution (χ2 test; P= .55). VAE and placebo preparations were coded by the manufacturerb leaving all investigators and the horse owners blinded until the analysis of the results was completed. Thirty-two horses received the VAE formulation and 21 the placebo (Fig 1). All horses were treated for 15 weeks. They received 3 subcutaneous injections per week (Monday, Wednesday, and Friday) each consisting of 1 mL of the formulation. The VAE group received an aqueous extract of fermented Viscum album austriacus (Iscador P). The basic extract was diluted with sterile saline solution to concentrations between 0.01 and 20 mg/mL (Table 2). This dose-escalating scheme followed the recommendations of the manufacturer28 based on human treatment schedules. It had been evaluated for undesired adverse effects in a previous tolerance test in 7 healthy horses using escalating doses (1, 5, 10, 20, and 30 mg/per dose, respectively). The dose of 20 mg/mL extract was the maximum tolerated dose. Horses of the control group received injections of 1 mL of 0.9% saline solution with the same frequency as described for the VAE group. All injections were administered SC in the pectoral region. A clinical examination focusing on adverse effects and local edema from previous treatments was performed before all injections.

Figure 1.

 Flow chart of study population; number of horses (H) and tumors (T) by subgroups; number of subjects remaining for investigation 1 year after start of treatment is given in brackets. Horses lost to follow-up were categorized as “therapy failure.”

Table 2.   Treatment protocol of the Viscum album extract group (VAE); administered concentrations.
Treatment WeekVAE Dosage Per Injection (mg/mL)
MondayWednesdayFriday
Week 10.10.10.1
Week 20.11.01.0
Week 31.01.010.0
Week 410.010.010.0
Week 510.010.0
Week 61.01.01.0
Week 71.010.010.0
Week 810.010.020.0
Week 920.020.020.0
Week 1020.020.0
Week 11–1520.020.020.0

Follow-Up and Final Assessment

During the observation time after treatment, a detailed examination of the selected tumors was performed every 3 months. To fit observed values to normal distribution, the calculated volume was logarithmically transformed. Then, the relative tumor volume (RTV) compared with the base-line value (1) was calculated with the formula:

image(2)

where RTVt is the relative tumor volume after time t, TVt is the tumor volume after time t and TV0 the base-line tumor volume before the start of the treatment.

In order to analyse effects between treatment groups, the outcome was assessed after the start, and every 3 months up to 1 year (month 3, 6, 9, and 12). Outcome was grouped in 4 effect levels: sarcoids were classified as completely regressed (CR), if they had disappeared, as partially regressed (PR), if they showed a decrease of volume by 50% or more, and as stable (S), if the TV changed <50%. When tumor growth exceeded 50%, sarcoids were classified as deteriorated. For group comparison, the percentage of CR, improvement (including completely and partially regressed sarcoids; CR + PR), and stabilization (including improved and stable sarcoids; CR + PR + S) was calculated.

Statistical Methods

Standard descriptive methods were used to summarize data. Percentage of complete and partial regression and stabilization was presented for both groups every 3 months. Comparison between outcome distributions after 12 months was conducted by Chi-square and Fisher exact tests. If univariate analysis of factorial variables (demarcation, type, status, TV category, and biopsy) showed a P < .10, the variable was included into a logistic regression model reporting odds ratio (OR) and 95% confidence intervals (CI). This was optimized stepwise by omitting the variable with the highest P-value if P≥ .05.

Relative tumor growth was compared after logarithmic transformation as described above. Since data were not normally distributed even after transformation, comparisons between treatment groups were performed by nonparametric rank-sum-tests (Wilcoxon). All analyses were performed by statistical package R ver. 2.9.1. Significance level was set at P < .05.

Results

Horses

A total of 53 horses entered the study. Of these, 32 were assigned to the VAE group (Iscador P) and 21 to the control group (placebo). The average age of the horses was 7.2 (±3.9) with a range from 3 to 17 and a median of 6 years. In the VAE group the horses were on average 7.4 years (±4.2) old compared with 6.8 (±3.4) in the control group. A significant difference between VAE (10.4 ± 10.2) and control groups (5.2 ± 3.2) was found for the initial number of CDES per horse (P < .05) (Table 1). A total number of 444 sarcoids were recorded in 53 horses (1–45 sarcoids per horse, mean 8.4 ± 8.5).

The VAE therapy was well tolerated by the horses. None of the horses developed systemic signs. In 5 horses (16%) treated with VAE, a mild edema at the injection site was observed after a VAE concentration of 10 mg/mL (n = 3) and 20 mg/mL (n = 2). This edema disappeared within 2–3 days without treatment. Two mares of the VAE group were pregnant for >3 months and had a normal pregnancy and subsequent parturition.

At the end of the trial, all sarcoids of 9 horses (91 CDES) in the VAE group (28%) and of 3 horses (9 CDES) in the control group (14%; NS) had clinically disappeared. Another 4 horses in the VAE group showed significant tumor reduction (17/29 CDES) defined as partial regression in at least half of the tumors, resulting in 13 horses (41%) with complete or partial regression compared with 3 horses in control group (14%; P < .05). Of the 11 horses with selective ES excision, 8 had received VAE and 3 placebo. Histological examination of 7 sarcoids (5 in the VAE group and 2 in the placebo group) that were thought to have been excised completely based on gross inspection revealed that excision was incomplete. No recurrence was observed in 6 horses in the VAE group and 1 horse in the placebo group at 12 months after starting treatment.

Seventeen horses with a total of 48 CDES investigated in detail (9 horses, 23 CDES in the VAE group, 8 horses, 25 CDES in the placebo group) were lost to follow-up at month 12, because of decisions by owners to try other treatments. These were all categorized as therapeutic failures.

Sarcoids

A subset of 163 sarcoids was selected according to morphology and/or size (3.20 ± 1.40 per horse; VAE n = 95, control n = 68) for detailed tumor observation. The most frequently affected body areas were the ventral (54 ES; 33.1%), the axillary, and the thoracic regions (each 23 ES; 14.1%), followed by head and neck (each 20; 12.3%), prepuce and inner hind thigh (each 10; 6.1%), and the rest of the extremities (3 ES; 1.8%). Eighty-one sarcoids were of verrucous (49.7%), 55 of occult (33.7%), 14 of mixed (8.6%), 8 of fibroblastic (4.9%), and 5 of nodular type (3.1%). While 134 (82.2%) of all sarcoids were dry, 26 (16.0%) were ulcerated and 3 (1.8%) were of sanguineous type.

After 12 months of observation time, 27 CDES showed a complete regression (37.5%) and 48 complete or partial regression (66.7%) in the VAE group, compared with 9 CDES showing complete regression (13.2%; NS) and 17 CDES (39.5%; P < .01) complete or partial regression in the control group (Table 3). After 1 year, sarcoid volumes were significantly smaller in the VAE group. RTV was 57% compared with 86% in the control group (P < .01).

Table 3.   Outcome on sarcoid level in proportion (%) assessed at 3, 6, 9, and 12 months after treatment start.
 Outcome during Observation (Every 3 Months after Start) N (%)
3 months6 months9 months12 months
VAEControlVAEControlVAEControlVAEControl
  • Treatment group Viscum album extract group (VAE) n = 95, control group (placebo) n = 68.

  • **

    Significant difference (P < .01) between treatment groups (χ2 test).

Outcome
Complete regression (CR)63146169279
(6.3)(4.7)(15.4)(8.8)(17.6)(16.7)(37.5)(20.9)
Partial regression (PR)211124142513218
(22.1)(17.2)(26.4)(20.6)(27.5)(24.1)(29.2)(18.6)
Stabilization (S)522947289117
(54.7)(45.3)(4.4)(10.3)(30.8)(11.7)(15.3)(16.3)
Deterioration1621494122231319
(16.8)(32.8)(53.8)(60.3)(24.2)(42.6)(18.1)(44.2)
Cumulative outcome
CR + PR2714382041224817
(28.4)(21.9)(41.8)(29.4)(45.1)(40.7)(66.7)**(39.5)**
CR + PR + S7943422769315924
(83.2)(67.2)(46.2)(39.7)(75.8)(57.4)(81.9)**(55.8)**
Total equine sarcoids considered9564916891547243
Missing (lost to follow-up)444142325

Of the potential factors influencing treatment effects (Table 4), type (verrucous) and biopsy (yes) remained as significant variables in the final multivariate models. Verrucous CDES showed higher complete regression compared with other types with OR of 2.38 (95% CI 1.03–5.48; P < .05). Diagnostic biopsy affected improvement (complete or partial regression) negatively with an OR of 0.24 (95% CI 0.08–0.73) (P < .05) and stabilization with OR of 0.30 (95% CI 0.10–0.88; P < .05) compared with CDES with no manipulation. VAE treatment significantly increased the chance for tumor improvement (OR 2.83, 95% CI 1.26–6.35, P < .05) and stabilization (OR 3.32, 95% CI 1.39–7.93, P < .01: Table 5).

Table 4.   Univariate logistic regression analysis of factors influencing monotherapy effects on complete regression (CR), improvement (CR + PR), and stabilization (CR + PR + S) of sarcoids after 1 year (n = 115).
VariableLevelCR N (%)Sig. PCR + PR N (%)Sig. PCR + PR + S N (%)Sig. P
  1. Reference (Ref.) levels grouped for type, status and volume.

  2. Significant differences between groups if P < .05. Effects with P < .1 considered for multivariate analysis.

DemarcationDemarcated (55)23 (41.9)<.0537 (67.3)<.0544 (80.0)<.1
Diffuse (60)13 (21.7)Ref.28 (46.7)Ref.39 (65.0)Ref.
TypeVerrucous (59)24 (40.7)<.0538 (64.4)<.145 (76.3)NS
Occult (39)9 (23.1)Ref.19 (48.7)Ref.26 (66.7)Ref.
Nodular (5)1 (20.0)Ref.2 (40.0)Ref.3 (60.0)Ref.
Fibroblastic (2)2 (100)Ref.2 (100)Ref.2 (100)Ref.
Mixed (10)0Ref.4 (40.0)Ref.7 (70.0)Ref.
StatusUlcerous (14)6 (42.9)NS11 (78.6)<.113 (92.9)<.1
Dry (100)30 (30.0)Ref.54 (54.0)Ref.70 (70.0)Ref.
Other (1)0Ref.0Ref.0Ref.
Volume<100 mm3 (39)15 (38.5)NS23 (59.0)NS26 (66.7)NS
101–1,000 mm3 (39)13 (33.3)NS24 (61.5)NS31 (79.5)NS
1,001–10,000 mm3 (29)7 (24.1)Ref.15 (51.7)Ref.20 (69.0)Ref.
>10,000 mm3 (8)1 (12.5)Ref.3 (37.5)Ref.6 (75.0)Ref.
BiopsyNo (96)32 (33.3)NS60 (62.5)<.0174 (77.1)<.01
Yes (19)4 (21.1)Ref.5 (26.3)Ref.9 (47.4)Ref.
Table 5.   Final logistic regression models including significant variables (P < .05; LR-test) and trial group as fixed variable. Odds ratios (OR) represent the effect chance of the respective level on complete regression (CR), improvement (CR + PR) and stabilization (CR + PR + S) of the sarcoids (n = 115).
VariableLevelORCI 95%P
CR
Trial groupVAE2.110.875.16NS
Control1    
TypeVerrucous2.381.035.48<.05
other1    
Log-likelihood =−67.5386
CR + PR
Trial groupVAE2.831.266.35<.05
Control1    
BiopsyYes0.240.080.73<.01
No1    
Log-likelihood =−71.2222
CR + PR + S
Trial groupVAE3.321.397.93<.01
Control1    
BiopsyYes0.300.100.88<.05
No1    
Log-likelihood =−61.1117

Discussion

This double-blind placebo-controlled study showed efficacy of VAE (Iscador P) in the treatment of CDES. After 1 year of observation time, 15 weeks of VAE monotherapy resulted in a significantly higher percentage of improved horses and tumors.

ES can affect horses of any age,22 but an increased incidence in younger horses (3–6 years of age) has been reported.1,30,31 In our study population, the majority of horses were aged 3–9 years; only 20% were 10 years of age and older. The number of ES per horse (8.4/horse, 94% of the horses with >1 tumor) was higher compared with data from the literature.3,16,22,31 In accordance with findings in other field studies,16 sarcoids of occult and verrucous type predominated in this investigation.

The results on horse level in the VAE group with 28% of complete remission and 41% of complete or partial regression are similar to those of a study on immunotherapeutic Nomagen (BCG-Vaccine) for treatment compared with cryosurgery.14

Spontaneous complete regression (after administration of placebo) was observed solely in horses aged between 3 and 7 years and predominantly in horses with 1–2 CDES. In contrast, complete remission in the VAE group was observed in mildly (1–2 CDES), moderately (3–9 CDES), and severely (>9 CDES) affected individuals of all ages. The occurrence of spontaneous tumor regression (14.3% of the horses and 13.2% of the sarcoids) in the placebo control group is in accordance with results of other studies.16,19,22

The observed improvement on sarcoid level after VAE (66.7%) is similar to those reported with intratumoral administration of BCG-Vaccine, which showed partial or complete regression in 57.5%,14 70%,3 and 77%13 of the sarcoids. The percentage of completely resolved tumors (37.5%) was lower than the rates reported for local CO2 laser surgery (62%),4 (71%),3 chemotherapy (60%),10 (87%),11 and radioactive implants (86.7%).6 These methods, however, are focused on treatment of single tumors rather than on the entire horse in a systemic way. Consequently, these measures are not directly comparable to systemic approaches such as immunotherapies.

The curative effects were higher in verrucous CDES. Diagnostic biopsy decreased the chance for improvement of the treated tumors significantly. It has been proposed that any invasive manipulation can result in an activation of the ES followed by more aggressive tumor growth2,32–34 and diagnostic confirmation by taking biopsy samples may be specifically contraindicated in certain ES localizations such as the periocular region.35 After these recommendations, in 11 horses no biopsy was taken because of critical localization of ES, risk of stimulation growth particularly of occult type,33 and decision of the owner. In the other 42 cases the clinical diagnosis of ES was confirmed by histological examination. While clinical diagnosis of single lesions may be uncertain,32 the 11 horses lacking biopsy suffered from multiple tumors (2–29 CDES) displaying the characteristic features of >1 type of ES. Nevertheless, it is important to note that the majority of CDES were not confirmed by histopathology, due to the limitations of this field study, as mentioned above.

The exact mechanism of action by VAE in initiating tumor regression is not completely known. The immune modulating activity can represent the relevant effect in cancer therapy.27 In vitro and in vivo studies have demonstrated activation of macrophages, natural killer cells, granulocytes, and B- and T-lymphocytes, which is associated with the release of interleukins, tumor necrosis factor-α, and interferon γ.24,27,36 Therefore, VAE induces activation of both specific and nonspecific components of the immune system.27 The effects of VAE in our study might be attributed to the stimulation of the immune system.

One advantage of VAE compared with locally administered treatments is the systemic effect, which was observed in horses independently of tumor count per horse. The relatively high efficacy and the systemic effect together with absence of important adverse effects, such as the severe local reactions seen after local chemotherapy,10 BCG-vaccination,13 or radiotherapy,35 suggest that VAE is an appropriate alternative to common methods. Follow-up results of 10 of the 13 horses with complete or partial regression, after at least 3 years, that showed no recurrence or new tumor growth (data not shown) indicate that the regression in these responders is sustainable over a long time.

The disadvantage of VAE therapy was that the response was slow and in some cases the tumors remained stable but showed no regression. Therapeutic effects did not become apparent until after the end of the 15-week treatment protocol in many cases. Only 6% of CDES were cured within or immediately after treatment period, while 37% had disappeared within 1 year (see Fig 2a–c). Such a delayed treatment effect is also observed after other immunotherapy protocols such as autovaccination, BCG vaccination, and Nomagen.13,14,20 A 15-week protocol, applying 3 subcutaneous injections per week in a dose-escalation scheme, was in accordance with schemes used in human cancer treatment to reduce VAE adverse effects. While treatment termination is not defined in human VAE therapy protocols, for horses it has to be taken into account that compliance of the owners could decrease after further continuation of VAE therapy, particularly if horses fail to show a rapid response. Prolongation of the treatment period could possibly further improve results.

Figure 2.

 Swiss Warmblood horse, mare, 1997, 7 occult, verrucous, and mixed sarcoids on head, neck, lower abdomen, and inner side of hind leg, 1 lesion confirmed by histopathology. Primary therapy with VAE (Iscador P). (a) April 4, 2005: verrucous periocular sarcoid before VAE therapy (day 0). (b) August 15, 2005: day 102 after therapy start, end of VAE therapy, beginning of posttherapy observation time, (c) May 4, 2006: day 395 after therapy start (observation time).

In conclusion, VAE proved effective for treatment of CDES in this study. It can be recommended particularly when excision is not indicated as the primary therapy, tumors near the eye, and in cases with multiple CDES where complete surgical removal or local medical treatment is impractical.

Footnotes

aWeleda AG, CH-4144, Arlesheim, Switzerland

bSociety for Cancer Research, Institute Hiscia, Arlesheim CH, Switzerland

Acknowledgments

The authors gratefully acknowledge the funding and support of the Society for Cancer Research (Arlesheim) and Weleda AG (Arlesheim) and Dr med. vet. Claudia von Tscharner for the histological examination of biopsy samples.

Ancillary