Animal Reproduction Science 216 (2020) 106358

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Vincristine and ivermectin combination chemotherapy in dogs

T
with natural transmissible venereal tumor of different cyto-
morphological patterns: A prospective outcome evaluation
Laiane Ferreira Bulhosaa, Alessandra Estrela-Limaa,b, Manuela da Silva Solcàc,
Gabriel Saraiva Diniz Gonçalvesa, Daniela Farias Larangeiraa,b,
Flaviane Alves de Pinhoa,b, Stella Maria Barrouin-Meloa,b,*
a
Teaching Hospital of Veterinary Medicine, Federal University of Bahia (UFBA), Salvador, Bahia, 40170-110, Brazil
b
Department of Veterinary Anatomy, Pathology and Clinics of the School of Veterinary Medicine and Zootechny, UFBA, Salvador, Bahia, 40170-110,
Brazil
c
Department of Preventive Veterinary Medicine and Animal Production of the School of Veterinary Medicine and Zootechny, UFBA, Salvador, Bahia,
40170-110, Brazil

A R T IC LE I N F O ABS TRA CT

Keywords: Vincristine is the first-line drug for the chemotherapy of canine transmissible venereal tumor
Cytomorphology (CTVT). Drug resistance is related to tumor cyto-morphological patterns of CTVT. There are anti-
Chemotherapy cancer properties of ivermectin, thus, a combination of ivermectin and vincristine could be an
Neoplasia effective chemo-therapeutic treatment regimen for CTVT. Study aims, therefore, were to (1)
Canine
assess the frequency of CTVT cyto-morphologies, and (2) evaluate treatment efficacy and possible
p-Glycoprotein
adverse reactions to vincristine compared with a combination vincristine and ivermectin. Dogs (n
Genital system
= 41) with CTVT were characterized by tumor cyto-morphology and disease severity and of
those, 20 were randomly allocated into two groups. There was a control group (G-Vin; n = 10) in
which there was treatment with vincristine; and an experimental group (G-Iv/Vin; n = 10) in
which there was treatment with the ivermectin/vincristine combination. Although dogs in the G-
Iv/Vin group had more severe disease at the beginning of the study (P = 0.0031), the number of
weeks and chemotherapy sessions until tumor remission were similar among dogs of the two
groups, indicating both treatments were effective. There was a decrease in the leukocyte counts
(P = 0.0020), related to neutropenia (P = 0.0371) in the G-Vin but not the G-Iv/Vin treatment
group. There was no tumor resistance that developed during the study regardless of the treatment
regimen used or tumor cytomorphology. In summary, the use of the vincristine/ivermectin
combination was well tolerated and efficacious for CTVT treatment.

1. Introduction

The canine transmissible venereal tumor (CTVT) affects the genital system of dogs and compromises the general clinical condition
(Ganguly et al., 2016; Ferreira et al., 2017). The CTVT disease is globally distributed (Baez-Ortega et al., 2019). Among 90 countries
throughout all continents of the globe, there was reporting that CTVT prevalence varies from 0.5 % in Australia to 10 % in Argentina,

⁎
Corresponding author at: Laboratório de Infectologia Veterinária, Hospital-Escola de Medicina Veterinária, UFBA, Av. Adhemar de Barros 500,
Salvador, Bahia, 40170-110, Brazil.
E-mail address: barrouin@ufba.br (S.M. Barrouin-Melo).

https://doi.org/10.1016/j.anireprosci.2020.106358
Received 25 September 2019; Received in revised form 25 March 2020; Accepted 26 March 2020
Available online 06 April 2020
0378-4320/ © 2020 Elsevier B.V. All rights reserved.
L. Ferreira Bulhosa, et al. Animal Reproduction Science 216 (2020) 106358

and there is a tendency for a lesser incidence in Europe and a greater incidence in South America (Strakova and Murchison, 2014).
In recent years, CTVT has been studied as a model for the comprehension of neoplastic diseases, owing to its peculiar im-
munological and metabolic properties associated with transmission and spontaneous regression in the natural host (Gonzalez et al.,
2000; Ostrander et al., 2016; Ujvari et al., 2016; Fassati, 2018; Frampton et al., 2018).
Cytology assessments of tumor samples collected using fine-needle aspiration biopsy procedures is the routine method for con-
firmation of a clinical presumptive CTVT diagnosis and for cyto-morphological classification as lymphocytic, plasmacytic, or of mixed
cell types (Amaral et al., 2007; Lima et al., 2013; Ganguly et al., 2016; Setthawongsin et al., 2016; Mascarenhas et al., 2017).
Plasmacytic CTVTs are clinically more aggressive and considered resistant to chemotherapy (Flórez et al., 2017; Ballestero Fêo et al.,
2018).
Vincristine sulfate is the first-line drug for CTVT treatment (Calvert et al., 1982; Nak et al., 2005), and is usually administered at
weekly intervals until tumor remission (Scarpelli et al., 2010). As occurs with the use of other anti-neoplastic drugs, treatment with
vincristine can result in adverse reactions affecting the bone marrow (Mealey et al., 2008; Hantrakul et al., 2014), or the gastro-
intestinal system (Ramadinha et al., 2016). Additionally, cases of CTVT resistance to vincristine have been reported (Brandão et al.,
2002; Gaspar et al., 2010).
Treatments with ivermectin can result in a reversing of the resistance to antineoplastic drugs by inhibiting p-Glycoprotein (P-gp)
production, with this being a protein that promotes the efflux of xenobiotics from within the tumor cell (Didier and Loor, 1996;
Pouliot et al., 1997; Griffin et al., 2005; Lespine et al., 2006; Jiang et al., 2019). Ivermectin, therefore, has been proposed to be a
therapeutic agent as an anticancer drug (Jiang et al., 2019; Markowska et al., 2019). Both vincristine and ivermectin are substrates
for P-gp (Didier and Loor, 1996; Mealey and Fidel, 2015), and results from at least one clinical study indicate there is an association of
ivermectin with vincristine in promotion of earlier remission of CTVT when compared with vincristine as a single drug (Lapa et al.,
2012). More recently, diverse direct anti-cancer activities also have been listed as a result of the newly studied ivermectin properties
(Crump, 2017; Dominguez-Gomez et al., 2018; Juarez et al., 2018).
It, therefore, was hypothesized that a chemotherapy based on the use of the combination of vincristine and ivermectin for
treatment of dogs with CTVT could be effective. Considering that clinical studies on this subject are few, the aim of the present study
was to evaluate the efficacy and occurrence of adverse reactions to a combined ivermectin/vincristine therapy. The dogs diagnosed
with natural CTVT had categorization of the cyto-morphological patterns as plasmacytic, lymphocytic and mixtures of these cell
types. The dogs had been admitted to a teaching veterinary hospital located in an endemic area for the disease.

2. Materials and methods

2.1. Animals, experimental design and ethics

Dogs of different breeds, ages and both sexes with genital tumors were examined at the Teaching Hospital of Veterinary Medicine
of the Federal University of Bahia, in Salvador, Brazil. The clinical diagnosis of CTVT was confirmed by cytological examination of
tumor smears from samples collected with fine-needle aspiration biopsy. Inclusion criteria for dogs in the study were a confirmed
CTVT diagnosis when there was a cytological examination of the tumor using procedures that have been previously described
(Amaral et al., 2007; Setthawongsin et al., 2016). There was owners’ consent to conduct the study as indicated by them signing an
informed consent form. The main exclusion criterion was a negative result when there was diagnosis for CTVT based on the tumor
cytology. There were additional exclusion criteria for dogs to undergo chemotherapy in either study group. These were a failure in
determining the cyto-morphological type of the CTVT when there was conducting of the cytological examination, co-morbidities, and
dogs for which owners did not comply with the conditions proposed in the study or when owners failed to return for subsequent
clinical evaluations after inclusion of their dogs in the study.
There were treatment evaluations in a clinical prospective study in which there was random assignment of dogs to two groups for
chemotherapy prior to the dogs being returned to the teaching hospital for initiation of the study. There was comparative analysis of
the cyto-morphological data from dogs that had imposed on them the specified treatment regimens until the end of the entire clinical
study. The dogs of the group treated with Vincristine (n = 10; G-Vin) were intravenously administered vincristine sulfate at weekly
doses of 0.5 mg/m² of body surface area diluted in 10 mL of 0.9 % sodium chloride to reduce the risk of phlebitis. The dogs of the
experimental group (n = 10) were treated with the combination of Ivermectin and Vincristine (G-Iv/Vin). The dogs of this group
were administered 0.5 mg/kg of ivermectin 1.0 % subcutaneously, a dosage based in pharmacokinetic and pharmacodynamic studies
using ivermectin treatments of dogs (Gokbulut et al., 2006; Eraslan et al., 2010) 24 h prior to administering vincristine (vincristine
was administered at the same dosage as G-Vin). There was subsequent treatments with ivermectin at the same dose at 2-week
intervals until tumor remission. All dogs were observed for at least 1 h after vincristine administrations for any immediate adverse
reactions. With both the G-Vin and G-Iv/Vin treatments, weekly vincristine treatments were administered until 1 week after complete
tumor remission, defined in the present study as the end of the treatment period.
Clinical examination, tumor cytology and hemogram assessments occurred weekly before each chemotherapy session to evaluate
the extent of tumor remission and the general health status of the dogs. There was cessation of the treatment if neutrophil counts
decreased to the lesser values of the reference range or to values less than the minimum of the reference range. Treatments were
resumed when leukogram assessments indicated that neutrophil counts had returned to those in the mid to upper portion of the
reference range for dogs (Thrall et al., 2012). Weekly chemotherapy with vincristine occurred until there was detection that tumor
remission had occurred.
All procedures were approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Zootechny of the

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Table 1
Criteria used to determine the clinical score for severity of canine transmissible venereal tumor (CTVT) in dogs with spontaneous
disease attended at the Teaching Hospital of Veterinary Medicine at the Federal University of Bahia, Salvador, Brazil.
Clinical features Score based on the severity of the clinical signs

Systemic signs
Attitude active (0) apathetic (3)
Appetite normal (0) hyporexia (1)
Hydration normal (0) dehydration (1)
Nutritional status normal (0) thin (1)
Mucosal membrane colour normal (0) pale (1)
Nasal secretion absence (0) mucous (1) with blood (2)

Tumour characteristics
Genital secretion absence (0) mucous (1) mucopurulent (5) necrotic (10)
Tumor location genital (1) genital and extragenital (5) extragenital (10)
Number of nodules per site one (1) two (2) three (3) four (4) more (5)
Myiasis absence (0) presence (5)
Local pain absence (0) presence (5)
Haemorrhage absence (0) discrete (1) patent (3)
Metastasis absence (0) to one site (10) > two sites (20)
Anatomical deformity absence (0) presence (5)

Federal University of Bahia (Permit number CEUA-68/2017). The procedures used in this study were consistent with the guidelines of
the Brazilian Council of Animal Experimentation and there was strict adherence to the Brazilian law for “Procedures for the Scientific
Use of Animals” (No. 11.794/2008).

2.2. Clinical examination and hemograms

All dogs underwent clinical examination for disease assessments. The anamnesis included past medical history; time lapse of
tumor development/regression assessments and other health alterations perceived by the owners; and the habitat for the dogs,
including if they were domiciled or semi-domiciled. The physical examination included the evaluation of nutritional state, mucosal
coloration, body temperature, tumor size and presentation, lesion severity, reactive lymph nodes, cardiopulmonary auscultation and
abdominal palpation. Peripheral blood was collected and hemogram analyses were performed in an automated blood counter
(Sysmex pocH-100iVDiff, Roche®) which included erythrocyte counts (n x 106/μL); hemoglobin (g/dL); hematocrit (%); platelets (n x
103/mm3) and white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils, and basophils (n x 103/μL). The results
from the blood tests were compared to the reference values for the canine species as previously described by Thrall et al. (2012).
There were chest and skull X-rays and abdominal ultrasonography conducted in cases of respiratory discomfort, extra-genital neo-
plasms and/or slow responses to treatment.
A clinical score classification system was developed to estimate the severity of the clinical conditions and to assess the response of
the dogs to the treatment regimens with classification criteria being described in Table 1. To assess the severity of the CTVT, the total
disease score determined for each dog was categorized as being a mild (≤ 10 points), moderate (11–25), severe (26–40) or terminal
(> 41 points) disease. Adverse reactions to chemotherapy, if there were such occurrences, were categorized as previously described
(VCOG-CTCAE, 2016; Cunha et al., 2017).

2.3. Cytology and histopathology

There were cytological examinations of smears of tumor samples that were collected using fine-needle aspiration (Cowell and
Tyler, 1989) for CTVT diagnosis and there was classification in cyto-morphological patterns as being lymphocytic, plasmacytic or of
the mixed cell types (Amaral et al., 2007). There was CTVT severity categorizations using the TNM classification system for tumors in
domestic animals (Owen, 1980). When genital tumors were no longer visible after chemotherapy, samples were collected using
gynecological brushes at the site the tumor was originally detected. Smears were stained using a modified Romanowsky method
(Panotic Rapid, Laborclin®, Brazil) and analyzed using optical microscopy. There were histopathological exams of tumor fragments
fixed in 10 % buffered formalin and embedded in paraffin for routine histological processing and staining with hematoxylin and eosin
(H-E). Histopathology evaluations were conducted to reassess and complement the diagnosis previously determined using cytology
evaluations (Ganguly et al., 2016; Mascarenhas et al., 2017), when owners agreed to the procedure.

2.4. Statistical analysis

Statistical analyses were performed using Graph Pad Prism v.5.0 (Graph Pad Prism Inc., USA) and STATA 12 (StataCorp LP, USA)
software. For sample size calculations, there was a dichotomous endpoint for two independent samples (80 % power and 0.05 alpha)
with an expected size of 20 individuals per group, and the primary endpoint was that there had been CTVT remission based on
clinical and cytological assessments. There was use of the Fisher’s exact test to evaluate the homogeneity of the dog populations in

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Table 2
General characteristics, tumor aspects and clinical scores of 41 dogs diagnosed with canine transmissible venereal tumor in the
Teaching Hospital of Veterinary Medicine – Federal University of Bahia, Salvador, Brazil, from January 2017 to August 2018.
Variable N (%)

Sex
Male 16/40 61.0
Female 25/40 39.0
Breed
Purebred 8/40 19.5
Mixed-breed 33/40 80.5
Tumor localization
Genital 32/40 78.0
Extragenital 4/40 9.8
Genital and extragenital 5/40 12.2
Nodules
Single 32/40 78.0
Multiple 9/40 22.0
Presence of metastases
Yes 4/40 90.2
No 37/40 9.8
Cytomorphological pattern (Cytology)
Lymphocytic (L) 8/40 19.5
Plasmacytic (P) 21/40 51.2
Mixed (L + P) 5/40 12.2
Undetermined 7/40 17.1
Hematological changes
Anemia 10/37 27.0
Leukopenia 3/37 8.1
Neutropenia 1/37 2.7
Lymphopenia 8/37 21.6
Thrombocytopenia 8/37 21.6
Clinical Score (Points)
Mild disease 13/41 31.7
Moderate disease 23/41 56.1
Severe disease 4/41 9.76
Terminal disease 1/41

both groups with respect to categorical variables. There was assessment for data normality of distribution using the Shapiro-Wilk and
Skewness/Kurtosis tests for all quantitative variables, which were expressed as mean values ± standard deviations (SDs) for para-
metrical variables or median values ± interquartile ranges (IRs) for non-parametrical variables. There was analysis of values for
quantitative variables that were a normally distributed using the Student’s t-test; otherwise, there was use of the Mann-Whitney test.
For all analyses, there were considered to be differences in values when there was a P ≤ 0.05.

3. Results

3.1. CTVT frequency and cyto-morphological patterns

From January 2017 to August 2018, 472 dogs with neoplasias were admitted to the Teaching Hospital of Veterinary Medicine of
the Federal University of Bahia, of which 51 (10.8 %; 51/472) were diagnosed with CTVT using cytology procedures. There were ten
of these 51 dogs excluded for subsequent study because the owners chose not to participate in the study.
The characteristics of the 41 dogs with CTVT are described in Table 2. The mean age of the dogs was 5.0 years ± 3.11 SD. Four
dogs had genital CTVT with metastasis, one had spleen nodules suggestive of TVT when there were evaluations using ultra-
sonography, however, these spleen nodules were not assessed for CTVT using cytology procedures, due to the risks of an invasive
sampling. The clinical scoring of 37 dogs that had values for all variables available for evaluation indicated there was a median value
of 12 points ± 8 IR, indicating that most dogs had moderate to severe clinical diagnoses of the CTVT disease (Table 1).
The diagnosis of CTVT using cytological examination that was conducted by two pathologists using blinded procedures was
conclusive in all cases; however, the cyto-morphological pattern of the tumors was characterized only in 34 cases because there were
technical limitations in seven (17.1 %; 7/41) of the samples. In these seven samples, the presence of isolated intact cells allowed for
the diagnosis of CTVT, but cell type overlap prevented adequate cyto-morphological characterizations. The cyto-morphological
confirmation of CTVTs occurred in the 34 dogs there being 61.8 % (21/34) plasmacytic, 23.5 % (8/34) lymphocytic, and 14.7 % (5/
34) of mixed plasmacytic/lymphocytic cell types (Fig. 1).
The most frequent malignancy characteristics in the cytological smears are detailed in Table 3. Tumors with plasmacytic and
mixed cell patterns had a greater incidence of malignancy features than those of a lymphocytic pattern. In two cases (5.9 %; 2/34),
cytoplasmic vacuoles were not microscopically visible in the smears; thus, other microscopic aspects were used to make a conclusion
for the diagnosis, such as large and central nucleoli, in addition to the typical macroscopic characteristics of the CTVTs, including a

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L. Ferreira Bulhosa, et al. Animal Reproduction Science 216 (2020) 106358

Fig. 1. Clinical presentation and cytological characteristics of the different cyto-morphological types of canine transmissible venereal tumor (CTVT);
(A) Genital plasmacytic CTVT lesion in a male dog: photographic image of a mass in the penile region with subcutaneous nodules in the inguinal
area (black arrows); (B) Plasmacytic cyto-morphological pattern characterized by moderate cell pleomorphism (Diff-Quik stain; Original magni-
fication, ×400; Scale bars 50 μm), with abundant cytoplasm, eccentric and ovoid nuclei, and a small nucleus:cytoplasm ratio (black arrow); high
power view in inset (Diff-Quik stain; Original magnification, ×1000. Scale bars 20 μm); (C) Genital presentation of the lymphocytic CTVT lesion in a
male dog: photographic image of a mass at the base of the penis (black arrow); (D) Lymphocytic cyto-morphological pattern characterized by
discrete cell pleomorphism (Diff-Quik stain; Original magnification, ×400; Scale bars 50 μm), with central and round nuclei and high nucleus:
cytoplasm ratio (black arrow); high power view in inset (Diff-Quik stain; Original magnification, ×1000; Scale bars 20 μm); (E) Genital presentation
of the mixed CTVT lesion in a female dog: photographic image of a mass in the vulva and vagina (black arrow); (F) Mixed cyto-morphological
pattern characterized by intense cell pleomorphism (Diff-Quik stain; Original magnification, ×400. Scale bars 50 μm) with central round (white
arrow) to ovoid nuclei (red arrow); high power view in inset (Diff-Quik stain; Original magnification, ×1000; Scale bars 20 μm).

vegetating appearance, friable consistency and tumor location (Fig. 1).

There were histo-pathological examinations (Fig. 2) of samples from 16 dogs (39.02 %; 16/41) because the owners of the other 25
dogs (60.97 %; 25/41) did not authorize the procedure. Among these 16 dogs, 13 (75 %; 13/16) were diagnosed as having CTVT with

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Table 3
Criteria of malignancy found in the different cytomorphological patterns of canine transmissible venereal tumor (CTVT) in 41 dogs examined by
cytology at the Teaching Hospital of Veterinary Medicine – Federal University of Bahia, Salvador, Brazil, from January 2017 to August 2018.
Malignancy criteria Cytomorphological patterns Total n = 34

Plasmacytic n = 21 Lymphocytic n = 8 Mixed n = 5 %

Anisocytosis 100 % (21/21) 62.5 % (5/8) 80 % (4/5) 88.23 % (30/34)

Anisokaryosis 100 % (21/21) 50 % (4/8) 60 % (3/5) 82.35 % (28/34)
Binucleation 14.28 % (3/21) 25 % (2/8) 40 % (2/5) 20.59 % (7/34)
Coarse chromatin 95.23 % (20/21) 62.5 % (5/8) 60 % (3/5) 82.35 % (28/34)
Cellular moulding 0% (0/21) 25 % (2/8) 20 % (1/5) 8.82 % (3/34)
Cytoplasmic basophilia 38.09 % (8/21) 0% (0/8) 40 % (2/5) 29.41 % (10/34)
Phagocytosis 9.52 % (2/21) 12.5 % (1/8) 20 % (1/5) 11.76 % (4/34)
Macrocariosis 9.52 % (2/21) 0%(0/8) 40 % (2/5) 11.76 % (4/34)
Macrocytosis 28.57 % (6/21) 0%(0/8) 0% (0/5) 17.65 % (6/34)
Atypical Mitosis 90.48 % (19/21) 37.5 % (3/8) 40 % (2/5) 70.59 % (24/34)
Reniform nucleus 4.76 % (1/21) 12.5 % (1/8) 20 % (1/5) 8.82 % (3/34)

the cyto-morphological pattern being consistent with the cytological diagnosis.

3.2. Outcome of chemotherapy with vincristine with or without ivermectin

There were 20 of 41 dogs (48.7 %; 20/41 with confirmed CTVT) in which there was completion of the chemotherapy treatment
regimen. There were 18 of 41 dogs (43.9 %; 18/41) excluded from the study, because seven of these dogs did not had a CTVT cyto-
morphological classification and the 11 remaining dogs had the treatment regimen interrupted as a result of owner decisions. There
were three of 41 dogs (7.3 %; 3/41) in which treatments were not initiated; one of these as a result of a decision by the dog owner, a
second had a very poor clinical condition and died of peritonitis, and the third dog had metastasis to the central nervous system and
was euthanized because of severe neurological symptoms, including blindness and difficulty swallowing.
There was completion of the chemotherapeutic protocols in 20 dogs, with 10 (50 %; 10/20) having been randomly assigned to the
G-Vin, and 10 (50 %; 10/20) to the G-Iv/Vin treatment regimens. There were 13 of these 20 dogs that had a history of having access
to a free-range type of habitat, including access to roadways. The main complaint of the owners when the dogs were admitted to the
veterinary clinic was an increased size of the genital region and blood excretions. Most of the dogs were of mixed breeding in both
groups: 90 % (9/10) in the G-Vin and 80 % (8/10) in the G-Iv/Vin treatment groups. The ages ranged from 1 to 12 years (mean ± SD
= 6.2 ± 3.3; median = 5.5) in the G-Vin group and from 1 to 11 years (4.8 ± 2.6; median = 5) in the G-Iv/Vin group. There were no
differences in outcomes due to sex (P = 0.35), breed (P = 1.0) or age (P = 0.30) between the two groups. Disease severity, based on
clinical scores of dogs that were assigned to the G-Iv/Vin group (mean = 19.1 ± 10.6; median 17.5 ± 13.5) were greater (P =
0.0439) than those assigned to the G-Vin group (mean = 10.0 ± 3.43; median 10.5 ± 4.25) before treatment (Fig. 3).
Information in Table 4 summarizes clinical characteristics, tumor descriptions, including TNM assessments, and response to
chemotherapy of the 20 treated dogs. There were single nodules in 100 % (10/10) and 80 % (8/10) of dogs in the G-Vin and G-Iv/Vin
groups, respectively. There were tumors with unique characteristics in the genital area in 90 % (9/10) and 60 % (6/10) of the G-Vin
and G-Iv/Vin dogs, respectively (Fig. 1).
Two dogs in the G-Iv/Vin (20 %; 2/10) group had genital neoplasia and metastasis, both with mixed cyto-morphology. One dog
had a genital tumor with metastasis to the inguinal lymph nodes; the other dog had metastasis in peritoneal lymph nodes, spleen,
liver, mediastinum and skin but no visible mass in the genitals, however, when swab samples were collected, the samples from the
apparently normal vaginal mucosa contained CTVT cells.
Three dogs had no genital neoplasia but had an extra-genital CTVT. Two of these dogs were in G-Iv/Vin group with one having a
lymphocytic pattern of CTVT in the perianal region, and the other having a plasmacytic CTVT in the nasal cavity. One dog in the G-
Vin group had a single plasmacytic CTVT as an atypical cutaneous ulcerated, adhered, nonfriable nodule with a small amount of
bloody excretion from the lateral region of the neck.
Altogether, among dogs of the G-Iv/Vin group, 60 % (6/10) had plasmacytic, 30 % (3/10) mixed cell types, and only 10 % (1/10)
had a CTVT categorized as a lesser aggressive lymphocytic type. In the G-Vin group, 50 % (5/10) of the dogs had plasmacytic, 20 %
(2/10) mixed, and 30 % (3/10) lymphocytic cyto-morphological cell types (Table 4).
There was comparison of treatment outcomes in both groups by evaluating values for clinical and laboratory variables for each
group and between groups before and at the end of chemotherapy. From the beginning to the end of the treatment period, values for
clinical variables within the groups decreased in dogs from both the G-Vin (P = 0.002) and G-Iv/Vin (P = 0.002) groups (Fig. 3). All
treated dogs from both the G-Vin and G-Iv/Vin groups had clinical and cytological values indicating there was tumor remission as a
response to chemotherapy (Table 4).
For the number of chemotherapy sessions until complete tumor remission, there was no difference in values between the G-Vin
(mean 4.1 ± 1.8) and G-Iv/Vin (mean 4.3 ± 1.8) groups (P = 0.80). Values from evaluation of the overall treatment duration in
weeks required for tumor regression to occur for dogs in the G-Vin group were 3–12 week (median 5.2 ± 2.74), while for the dogs of
the G-Iv/Vin group these values were 2–8 weeks (median 4.3 ± 1.76). These ranges in durations and time taken for tumor regression,

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Fig. 2. Photomicrography of different cytomorphological types using histo-pathological examination of canine transmissible venereal tumor
(CTVT); (A) Plasmacytic pattern characterized by moderate cell pleomorphism (H-E stain; Original magnification, ×400; Scale bars 50 μm); (B)
Detail of the plasmacytic CTVT with the oval and eccentric nuclei (black arrow) and small nucleus:cytoplasm ratio (H-E stain; Original magnifi-
cation, ×1000; Scale bars 20 μm); (C) Lymphocytic pattern characterized by the discrete cell pleomorphism (H-E stain; Original magnification,
×400; Scale bars 50 μm); (D) Detail of the lymphocytic CTVT showing its central and round nuclei (black arrows), and high nucleus: cytoplasm ratio
(H-E stain; Original magnification, ×1000; Scale bars 20 μm); (E) Mixed pattern characterized by intense cell pleomorphism (H-E stain; Original
magnification, ×400. Scale bars 50 μm); (F) Detail of the mixed CTVT with the central round (black arrow) or ovoid nuclei (white arrow) (H-E stain;
Original magnification, ×1000; Scale bars 20 μm).

however, were not different in these responses among the dogs of the two treatment groups (P = 0.48).
One case of tumor remission occurred, after a single treatment with vincristine, in a dog in the G-Vin group with plasmacytic
CTVT. Chemotherapy was halted before the second treatment because the dog developed intense leukopenia (1,300/μL; reference
range = 6,000–17,000/μL), neutropenia (676/μL; reference range = 3,000–11,500/μL) and lymphopenia (104/μL; reference range

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Fig. 3. Clinical scores of 20 dogs with natural canine transmissible venereal tumor (CTVT) treated with vincristine (G-Vin; n = 10 dogs) or with the
combination ivermectin/vincristine (G-Iv/Vin; n = 10 dogs); (A) Comparison of clinical scores of dogs of the G-Vin group with those treated with
the combination of G-Iv/Vin before starting the treatment (BT); *P < 0.05 - Unpaired Mann Whitney test; (B) Comparison of clinical scores of dogs in
the G-Vin group with those of the G-Iv/Vin group from before treatment (BT) to the end of the treatment (After treatment = AT); **P < 0.005 -
Wilcoxon matched-pairs signed rank test.

= 1,000–4,500/μL). When cell counts increased to the reference range 4 weeks after cessation of vincristine treatment, the tumor
could no longer be detected.
During the period of treatment, dogs that were treated with only vincristine sulfate (G-Vin) had a decrease in the mean counts of
total leukocytes (P = 0.0027), neutrophils (P = 0.0371), and monocytes (P = 0.0468), while values for all variables remained within
the reference range in the dogs that were treated with the vincristine/ivermectin combination (G-Iv/Vin) (Fig. 4).
Among all 20 treated dogs, only two had vomiting bouts between 1 and 3 days after chemotherapy, and both dogs responded well
to anti-emetic drugs. One of these dogs, from the G-Iv/Vin group, had weight losses and signs of apathy 1 week after the first
chemotherapy session. This dog was in a poor clinical condition (Score 41) and there were metastases detected at the time of the first
consultation. From the time of the second chemotherapy session, the response of this dog was similar to that of the other dogs of the
group, and the tumor eventually was no longer detected.

4. Discussion

The present study spanned 17 months and CTVT comprised 10.8 % (an average frequency of three cases per month) of all canine
neoplasia cases at the largest veterinary hospital of Salvador, a city with an estimated canine population of 443,000 dogs in 2019
(personal communication). The findings in the present study were within the range of reported frequencies of CTVT cases in teaching
hospitals in other areas (De Nardi et al., 2002; Araujo et al., 2016).
Considering the epidemiological nature of the disease, however, the prevalence of CTVT in facilities for veterinary care might be
underestimated. The clinical profile of affected dogs and the clinical characteristics of dogs were similar to that described in other
endemic areas (Brandão et al., 2002; Ganguly et al., 2016). The findings during clinical evaluations of the dogs in the present study
indicate that most owners bring their dogs to veterinary care when there is a moderate to severe disease condition, usually when
there was bleeding from the tumor or the tumor was large enough to be visible. Concerning the response of the CTVT to che-
motherapy with vincristine, this condition is curable in most cases (Gonzalez et al., 2000), and the cost of treatment is not considered
great compared to that of other neoplasias in dogs (Ramadinha et al., 2016). Nevertheless, less than 50 % of the dogs that were
diagnosed with the disease were provided with the complete course of chemotherapy in the present study. This reflects the dogs were
in a habitat where CTVT is endemic, typically in areas where there is a relatively lesser human development index, as described by
the United Nations Development Programme (UNDP, 2018). In this regard, as evidenced by knowledge of CTVT transmission due to
dog-to-dog contact that results for the dog’s freedom of movement in common habitats, the occurrence of CTVT might indicate a risk
factor for other transmissible diseases, including zoonotic diseases. Results in the present study are thus important for One Health
awareness.
Comparison of treatment outcomes, as a result of treatment regimens in the present study, indicated the combination of iver-
mectin/vincristine was at least as effective as vincristine alone. It, however, is emphasized that dogs randomly assigned to the
treatment groups were administered ivermectin prior to vincristine treatments occurred and were more severely diseased than the
dogs administered only vincristine (Fig. 3, Table 4). Dogs assigned to the G-Iv/Vin group had a greater frequency of plasmacytic and

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Table 4
Clinical and tumor characteristics of 20 dogs with canine transmissible venereal tumor accordingly with treatment protocol and clinical outcome.
Dogs Groups Age (years) Sex Tumour localization Cytomorphologic type TNM stage Number of the chemotherapy sessions Tumour size score*

Treatment

Before After

1 G-Vin 10 M E P I [T1, N0, M0] 1 1 0

2 G-Vin 3 F G L II [T2, N0, M0] 4 2 0
3 G-Vin 4 M G P II [T2, N0, M0] 4 2 0
4 G-Vin 5 F G L III [T3, N0, M0] 3 3 0
5 G-Vin 5 F G M III [T3, N0, M0] 4 3 0
6 G-Vin 10 M G P III [T3, N0, M0] 7 3 0
7 G-Vin 6 F G P II [T2, N0, M0] 3 2 0

9
8 G-Vin 1 F G L II [T2, N0, M0] 4 2 0
9 G-Vin 7 M G M II [T2, N0, M0] 4 2 0
10 G-Vin 11 M G P II [T2, N0, M0] 7 2 0
11 G- Iv/Vin 4 F GE M IV [T2, N1, M0] 4 2 0
12 G- Iv/Vin 5 M E P II [T2, N0, M0] 4 2 0
13 G- Iv/Vin 6 F G P II [T2, N0, M0] 3 2 0
14 G- Iv/Vin 3 M G P I [T1, N0, M0] 6 1 0
15 G- Iv/Vin 10 F E L I [T1, N0, M0] 3 1 0
16 G- Iv/Vin 5 F G P II [T2, N0, M0] 5 2 0
17 G- Iv/Vin 2 F G P III [T3, N0, M0] 2 3 0
18 G- Iv/Vin 5 F G P III [T3, N0, M0] 5 3 0
19 G- Iv/Vin 7 F G M III [T3, N0, M0] 3 3 0
20 G- Iv/Vin 1 F E M V [T1, N0, M1] 8 1 0

E = extragenital; G = genital; GE = genital and extragenital; P = plasmacytic; L = lymphocytic; M = mixed cell; T, tumor; N, lymph node; M, metastasis; *tumor size score: 0= total remission; 1= < 3
cm; 2 = 3−5 cm; 3 > 5 cm.
Animal Reproduction Science 216 (2020) 106358
L. Ferreira Bulhosa, et al. Animal Reproduction Science 216 (2020) 106358

Fig. 4. Comparison of hematological biomarkers of drug cytotoxicity, before starting the treatment (BT) and at the end of the treatment (AT) in the
group of dogs treated with vincristine (G-Vin) and those treated with the ivermectin/vincristine combination (G-Iv/Vin); (A) Erythrocytes
(Reference range = 5.5-8.0 × 106/μL). (B) Hemoglobin (Reference range = 12.0-18.0 g/L); (C) Platelets (Reference range = 166-575 × 103/μL);
(D) Leucocytes (Reference range = 6-17 × 103/μL); (E) Neutrophils (Reference range = 3-11.5 × 103/μL); (F) Monocytes (Reference range =
0.15-1.35 × 103/μL); Unpaired Mann Whitney test was used for comparisons amongst groups G-Vin and G-Iv/Vin and Wilcoxon matched-pairs
signed rank test was used for comparisons within each group before (BT) and at the end of treatment (AT), *P < 0.05, **P < 0.005; Reference ranges
as described by Thrall et al., 2012.

mixed CTVT cell types, which are more resistant to chemotherapy when compared dogs with only the lymphocytic cell type
(Setthawongsin et al., 2018).
With the combined G-Iv/Vin treatment, there was 1 less week needed for tumor remission than the other group, with this finding
being inconsistent with the expectation for a longer time needed for tumor remission because of the cyto-morphological-related
prognosis for the dogs of the group treated with the G-Iv/Vin combination. The relative abundance of P-gp is a marker of drug
resistance that occurs with several tumors (Pouliot et al., 1997), and this also explains the resistance to vincristine by CTVT cells
(Montoya Florez et al., 2014). The abundance of the P-gp marker in CTVT cells is large being as great as 80 % in these cells (Gerardi
et al., 2014), and the resistance is greater in the more drug resistant plasmacytic and mixed cyto-morphological cell types (Gaspar
et al., 2010; Gerardi et al., 2014; Setthawongsin et al., 2018). The P-gp marker protein in the cell membrane is a known protein
involved in the mechanism for tumor resistance to chemotherapy, including vincristine (Mealey and Fidel, 2015; Levi et al., 2016;
Mealey et al., 2017; Kim et al., 2018). The P-gp functions as an efflux pump by transporting drugs to the outside of tumor cells,
resulting in relatively lesser drug cytoplasmic concentrations hindering the induction of cell death when there is use of chemotherapy
treatments (Mealey et al., 2001; Linardi and Natalini, 2006; Montoya Florez et al., 2014). In this regard, it is hypothesized that the
approach in the present study, of injecting vincristine when blood concentrations of ivermectin were at the steady state, might have
induced a prior inhibition of P-gp in tumor cells when vincristine was injected, thus possibly resulting in effective vincristine con-
centrations inside tumor cells. Following ivermectin administration in dogs, the plasma concentrations were maximum 1.4 days after
the treatment and the area under the concentration compared with time curve was greater than 300 ng/day/mL (Gokbulut et al.,
2006; Eraslan et al., 2010). Ivermectin was also subsequently administered every 15 days until the end of the treatment period,
maintaining a relatively consistent concentration with this treatment regimen throughout the chemotherapy period.
There are very few reports on the clinical use of the vincristine/ivermectin combination for the treatment of spontaneous CTVT.
The present study seems to be unique in using such a chemotherapeutic combination in a prospective clinical approach focused on the
cyto-morphological types of tumor cells, and in evaluation of the possible adverse reactions to the treatment regimen in dogs with
naturally acquired CTVT. Results from a previous study indicated that the vincristine/ivermectin combination treatment resulted in a
more rapid remission of the CTVT compared with the use of vincristine as the only chemotherapeutic agent (Lapa et al., 2012).
In the veterinary practice, an important concern regarding the safety of ivermectin use in dogs is well-described as being the
central nervous system (CNS) depression that can occur at therapeutical doses as an adverse reaction in dogs of specific breeds, such
as the Collies. The dogs of these breeds are affected with a mutant deletion in the MDR1 gene, which encodes for P-gp (Mealey et al.,
2001). The mutation renders the dogs more sensitive not only to neurological toxicosis by ivermectin and other P-gp substrates, but
also to gastrointestinal and myelo-suppressive effects of anti-cancer drugs including vincristine (Mealey et al., 2008), and this can
occur in dogs of other breeds, including mixed breed dogs (Mealey and Fidel, 2015). Nevertheless, there was no signs of CNS

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L. Ferreira Bulhosa, et al. Animal Reproduction Science 216 (2020) 106358

depression or enhanced adverse reactions attributable to vincristine in any of the dogs that were treated with ivermectin in the
present study.
Interestingly, even though the group of dogs treated with the combination ivermectin/vincristine in the present study had more
individuals with the more clinically aggressive plasmacytic or mixed CTVT cells that contributes to having poorer clinical statuses,
there were less adverse reactions to chemotherapy than the group treated with only vincristine. There is some thought that there is a
need for poly-chemotherapy in resistant tumor cells using drugs that are substrates for P-gp which would result in severe adverse
reactions to the anti-neoplastic drugs, owing to the drug-drug interactions involving P-gp substrates and P-gp inhibitors, potentiating
the mechanisms of action for these drugs (Mealey and Fidel, 2015; Mealey et al., 2017). Only a few dogs in the present study,
however, had leukopenia following the vincristine treatment, and in the group that previously were treated with ivermectin, the
leukopenia condition was less, being no greater than Grade 2 when there was a 1–5 scale used to assess adverse reaction intensity
according to the VCOG-CTCAE (2016). In other studies, there have been adverse reactions to vincristine such as diarrhea, vomiting,
weight loss and lack of appetite (Ramadinha et al., 2016) or hematological disorders including the previously described decrease in
leukocyte counts (Nak et al., 2005; Hantrakul et al., 2014; Furini et al., 2015). Thus, it could be expected that treatment with
vincristine combined with ivermectin might result in more intense adverse reactions in dogs with CTVT (Mealey et al., 2008, 2017),
particularly in those with poorer clinical statuses. This, however, did not occur in the present study; rather, the reverse response was
observed. The clinical results from the present study indicate that adding ivermectin was an effective treatment in dogs when there
was chemotherapy using vincristine. This finding indicates that ivermectin might have enhanced bone marrow “chemoprotection”,
perhaps inducing a desirable retention of vincristine within tumor cells, particularly in the more aggressive plasmacytic and mixed
types of CTVT cells that have greater abundances of P-gp molecules (Gaspar et al., 2010). In this regard, there would be less available
vincristine in the extracellular space, thus less drug to interfere with bone marrow cells, for example. These possibilities should be
explored in molecular studies on Pg-P cell abundance and the pharmacokinetics of both drugs in dogs treated for CTVT.
An anti-cancer effect of ivermectin (Crump, 2017; Dominguez-Gomez et al., 2018; Juarez et al., 2018) also might explain the
effectiveness of the ivermectin/vincristine combination in the present study. The mechanism underlying a possible protective effect
of ivermectin association to the adverse reactions to vincristine, however, should be further investigated. There should be more
clinical studies with greater numbers of dogs that had natural-acquirement of the disease. A limitation of the present study was the
small number of dogs that could be evaluated for the duration of the study, however, the results from the present study were quite
clear.
In conclusion, the frequency of CTVT in a teaching veterinary hospital in a typical area where the disease is endemic can be as
great as 10 % among dogs with neoplasias. The plasmacytic cyto-morphological pattern was the most frequently detected of the cell
types. Comparing the outcomes after the two treatment regimens for the chemotherapy for CTVT were completed, the regimen
including the use of vincristine as a single drug was not as effective as the other regimen in which there was use of combination
ivermectin/vincristine. Ivermectin combined with vincristine induced tumor remission without enhancing adverse reactions as ex-
pected when there is a conventional single vincristine-based treatment. For a more thorough understanding of the mechanisms
involved in the response of different cyto-morphological CTVT patterns to the drug combination, studies on clinical, immunological,
molecular, cytological, histological, epidemiological and pharmacokinetic variables should be developed in dogs with the naturally
acquired disease.

CRediT authorship contribution statement

Laiane Ferreira Bulhosa: Methodology, Investigation, Writing - original draft. Alessandra Estrela-Lima: Conceptualization,
Methodology, Writing - review & editing. Manuela da Silva Solcà: Data curation, Formal analysis, Writing - review & editing.
Gabriel Saraiva Diniz Gonçalves: Methodology, Investigation. Daniela Farias Larangeira: Methodology, Investigation. Flaviane
Alves de Pinho: Methodology, Data curation, Writing - review & editing. Stella Maria Barrouin-Melo: Supervision,
Conceptualization, Resources, Writing - review & editing.

Declaration of Competing Interest

There are no conflicts of interest to report.

Acknowledgements

The study was supported by the Brazilian National Council for Scientific and Technological Development (CNPq - PQ Researcher
scholarship for SMBM: Proc. 307813/2018-5, and for AESL: Proc. 305938/2018-5), by the Bahia Research Foundation (FAPESB -
Grant n. PRONEM: 498/2011-PNE 0002/2011), and by the Coordination for Improvement of Higher Education Personnel – CAPES
(MSc scholarship for LFB n. 1654450). We thank Dr. Aroldo Carneiro (Center for Zoonosis Control, Municipal Health Secretariat -
SMS), Salvador, Bahia, Brazil. We are also grateful to Dr. Emanoel Martins, Dr. Tiago C. Peixoto, Dr. Ana Leonor P.C. Godoy and Dr.
Karine A. Damasceno for their critical review.

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L. Ferreira Bulhosa, et al. Animal Reproduction Science 216 (2020) 106358

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anireprosci.
2020.106358.

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