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JSAP JOURNAL OF SMALL ANIMAL PRACTICE

ORIGINAL ARTICLE

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Toceranib phosphate for the treatment of dogs with high-risk adrenal gland tumours: 16 cases (2019-2023)

C. CHALFON (D *,1, L. MARCONATO (D +, S. GALAC+, A. M. TARDO (D +, M. ZANDVLIET+, F. FRACASSI (D +, F. DEL BALDO (Dt, G. GHISONIt, L. PISONIt AND R. FINOTELLO ID $,1

*Centro Veterinario Torinese, Turin, Italy

*Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, Italy

*Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands

$Polo Oncologico Veterinario, AniCura Italy Holding S.r.l., Bologna, Italy

1Corresponding author email: carmit_yam@hotmail.com; riccardofin@libero.it

OBJECTIVES: The aims of this study were to evaluate the response rate, time to progression (TTP) and survival times of dogs with high-risk adrenal gland tumours (ATs) treated with toceranib phosphate, in both macroscopic and microscopic setting, and to report the adverse event (AE) profiles.

MATERIALS AND METHODS: Medical records of dogs diagnosed with a high-risk adrenocortical carcinoma (ACC) or phaeochromocytoma (PCC), treated with toceranib, were retrospectively reviewed. High-risk ATs were defined as inoperable and/or metastatic ATs or cortical tumours with high Utrecht score. Endpoints were response rate, TTP and overall progression-free survival time (PFST). Adverse events were reported according to VCOG-CTCAE.

RESULTS: Sixteen dogs were included: 10 diagnosed with PCC and six with ACC. All dogs with ACC received adjuvant toceranib due to a high Utrecht score or metastatic disease, while all dogs with PCC were treated with toceranib in the macroscopic setting. A clinical benefit was detected in 80% of dogs with PCC: four achieved stable disease for a median TTP of 176.5 days, and two achieved partial response for 182 and>100 days, respectively. Median PFST for dogs with PCC was 112 days. Among dogs with ACC, 3 (50%) progressed and were euthanized after 237, 364 and 273 days; the remaining 3 (50%) dogs were alive and disease free 382, 508 and 583 days after starting toceranib. Overall, toc- eranib was well-tolerated.

CLINICAL SIGNIFICANCE: Toceranib may offer clinical benefit and improve outcome in dogs with high-risk ATs in both the macroscopic and microscopic disease setting.

Journal of Small Animal Practice (2025); 66, 412-419

DOI: 10.1111/jsap.13840

Accepted: 20 January 2025; Published online: 20 February 2025

INTRODUCTION

Primary adrenal gland tumours (ATs) are relatively infrequent in dogs, with adrenocortical carcinoma (ACC) and phaeochro- mocytoma (PCC) being the most prevalent malignant types (Anderson, 2001; Lang et al., 2011; Lunn & Boston, 2020; Massari et al., 2011; Schwartz et al., 2008).

Adrenocortical carcinoma s may be functional or non-functional, predominantly associated with cortisol secretion, leading to signs of adrenal-dependent hyperadrenocorticism (Barrera et al., 2013; Kyles et al., 2003; Massari et al., 2011). Phaeochromocytomas derive from chromaffin cells in the adrenal medulla and can also be functional by secreting epinephrine and norepinephrine (Galac & Korpershoek, 2017; Lunn & Boston, 2020; Schwartz et al., 2008).

Use of Toceranib for adrenal gland tumours in dogs

Malignant ATs metastasise through haematogenous and lym- phatic routes (Lunn & Boston, 2020). Both vascular and soft tissue invasion have been observed in malignant ATs in dogs (Fontes et al., 2024).

Nevertheless, their early progression primarily involves direct invasion into adjacent veins, including phrenicoabdominal vein, renal vein or caudal vena cava (CVC) (Barrera et al., 2013; Kyles et al., 2003; Pascaline et al., 2022). Intravascular exten- sion has been reported to occur in 16 to 61% of dogs with ATs (Anderson, 2001; Barrera et al., 2013; Herrera, 2008; Kyles et al., 2003; Lang et al., 2011; Massari et al., 2011; Pascaline et al., 2022).

Dogs with ACC often present higher prevalence of metas- tasis (around 50%) at diagnosis, frequently affecting the liver and lungs, compared to dogs with PCC (Labelle, 2004; Massari et al., 2011).

Dogs with PCC typically show metastatic disease in 20% to 40% of cases, affecting regional lymph nodes, liver, spleen, pancreas, lung, heart, central nervous system, kidney and bone (Barthez et al., 1997; Galac & Korpershoek, 2017; Reusch, 2015).

Adrenalectomy is typically recommended for ATs with func- tional hormone secretion to manage hormone overproduction and associated clinical signs. It is also advised for functional or non-secreting ATs larger than 2 cm, or if a known adrenal mass shows progressive growth, as these factors increase the potential for malignancy or metastasis. Additionally, adrenalectomy is also recommended in cases of tumour rupture (Cook et al., 2014; Mayhew et al., 2019; Pagani et al., 2016; Traverson et al., 2023). Median survival time for dogs with ATs undergoing adrenal- ectomy ranges between 360 and 944 days (Anderson, 2001; Harding et al., 2021; Massari et al., 2011), with a 1- and 2-year survival rates of 77 to 95% and 60 to 88%, respectively, sug- gesting that long-term outcome is possible after adrenalectomy (Bokhorst et al., 2023; Cavalcanti et al., 2020; Pitt et al., 2016; Taylor & Monner, 2021).

Recently, a novel histopathological scoring system, the Utrecht score, was developed to predict prognosis in dogs with cortisol-secreting ATs after adrenalectomy, identifying cases with an increased recurrence risk and shorter survival (Sanders et al., 2019). The Utrecht score is calculated by adding the Ki67 labelling index, three points for the presence of necrosis and four points if at least 33% of neoplastic cells have a clears or vacu- olated cytoplasm. Cases were stratified into three groups based on score cut-offs of less than 6, 6 to 10 and greater than 10. This scoring system was correlated with overall survival, with median survival times of more than 60, 51.5, and 14.4 months, respec- tively (Sanders et al., 2019).

Surgery might not be feasible due to large tumour size, extensive vascular invasion, or widespread metastases (Galac & Korpershoek, 2017; Massari et al., 2011). In such cases or if surgery is declined, medical options with trilostane or mito- tane may be considered for cortisol-secreting ACC. Trilostane helps in controlling cortisol production, while mitotane may also have direct antitumoral effects, alongside lowering cortisol levels (Arenas et al., 2014). Dogs with metastatic disease appear to have

less favourable response to mitotane when compared to those without metastatic disease, and the choice of medical treatment (mitotane versus trilostane) does not influence survival time in dogs with primary functional ACC (Arenas et al., 2014). In addi- tion, adjuvant medical treatment after surgical resection might be applied in patients in which the Utrecht score indicates an increased recurrence risk, and in those with local recurrence and metastases (Arenas et al., 2014; Sanders et al., 2019).

A recent retrospective study indicated potential clinical benefits of toceranib phosphate for dogs with inoperable, metastatic or recurrent PCCs (Musser et al., 2018). Toceranib, a licensed multi-targeted tyrosine kinase receptor inhibitor (TKI) in veterinary medicine, shares molecular similarities with sunitinib and sorafenib, effectively targeting vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptor-ß (PDGFRØ), KIT, and fms-like tyrosine kinase-3 (FLT3) (Frezoulis & Harper, 2022; London et al., 2003; Olivares- Sheppard et al., 2020; Olivares-Sheppard et al., 2022).

Human studies have shown that VEGF, VEGFR2 are significantly upregulated in the protein level in a considerable proportion of ACC cases compared to normal adrenal gland. Given the expression of VEGFR2 and its ligand VEGF in ACC, both key target of sunitinib and sorafenib, suggesting that sunitinib and sorafenib are promising candidates for further clinical investigation in ACC treatment (Cerquetti et al., 2021; Kroiss et al., 2011; Kroiss et al., 2012). In a recent study, no overexpression of kit, flt-3, PDGFRØ and VEGFR2 was observed in canine ATs. However, differential receptor expression was observed between cortical tumours and phaeochromocytomas, with cortical tumours showing an increase in tyrosine kinase receptors (Harding et al., 2021).

Based on the above, it may be hypothesized that toceranib phosphate exhibits biological activity in dogs with either PCC or ACC.

The primary aim of this study was to evaluate the response rate, time to progression (TTP) and progression-free survival time (PFST) in dogs with high-risk ATs treated with toceranib in both macroscopic and microscopic setting. A secondary aim was to report the adverse events (AEs) profile associated with toceranib phosphate.

MATERIALS AND METHODS

Study design and inclusion criteria

The electronic medical records of (‘masked for review’) were retrospectively searched between April 1, 2019 and May 2, 2023 to identify dogs diagnosed with phaeochromocytoma or adrenocortical carcinoma that received toceranib.

Three operators independently reviewed medical records using search terms including “dog”, “adrenal gland tumours”, “phaeochromocytoma”, “adrenocortical carcinoma” and “toceranib”.

To be included in the study, dogs were required to (1) have a diagnosis of AT, (2) have diagnostic imaging performed via tho- racic radiographs and abdominal ultrasound and/or total-body

computed tomography (TBCT) before toceranib initiation, (3) be treated with toceranib for at least 10 weeks, (4) have adequate follow-up information including adverse events (Veterinary and Comparative Oncology, 2016) and treatment response docu- mented via serial imaging studies (RECIST; Nguyen et al., 2015).

The conclusive diagnosis of AT relied on (1) histopathology if surgery was performed; (2) cytology alongside imaging findings indicating an adrenal mass documented via ultrasonography or CT; (3) adrenal functional tests (e.g. plasma normetanephrine concentration or urine normetanephrine to creatinine ratio, endogenous plasma ACTH concentration and low-dose dexamethasone suppression test [LDDST]).

Adrenal gland tumours were defined as high-risk if they were deemed as inoperable and/or metastatic (suspected or confirmed), or ACC with an Utrecht score ≥11 (Sanders et al., 2019) when histopathology was available.

According to the Animal Experimentation Ordinance of the Italian National Legislation for animal welfare (DL 14th March, 2014 n.26) and best standard of Veterinary practice (Good Veterinary Practice from the Federation Veterinary Europe- Italian National Federation of the Veterinary’ Orders, 29th January, 2005), there is no requirement for any permission to proceed with this kind of retrospective study. Therefore, we will not provide IREC for the involved institutions. Furthermore, all owners (in all institutions involved in this study) signed a con- sent from which allows us to use (anonymized) patient data for research propose.

Data collection

Data obtained from medical records included signalment (breed, sex, age, weight), clinical signs at diagnosis, duration of clinical signs, comorbidities (if any), blood pressure measurement at diagnosis, adrenal functional testing results, imaging techniques and findings, tumour size, date and type of surgery (if performed), histopathology results (for operated cases), Utrecht score (if performed; Sanders et al., 2019), cytology results (if performed), reason for toceranib use (inoperable AT, metastatic disease, or maintenance after surgery due to negative prognostic factors such as metastatic disease or high Utrecht score), toceranib dose and schedule, duration of toceranib treatment, best treatment response based on previously defined criteria (Nguyen et al., 2015), AEs (VCOG-CTCAE; Veterinary and Comparative Oncology, 2016), reasons for toceranib discontinuation, concomitant medications, response rate to toceranib, progression and progression-free survival.

Follow-up information were collected from the clinical records of each referral institution or referring veterinarians. Owners were contacted as needed to confirm survival status, or date and cause of death.

Statistical analysis

Descriptive statistics were generated to characterise the study population. Data normality was assessed using the Shapiro-Wilk test, and continuous variables were reported as median and range (minimum and maximum value) accordingly. Categorical vari- ables were described with frequencies, proportion or percentages.

The median PFST was estimated using the Kaplan-Meier product limit method. Overall PFST was defined as the interval between the first toceranib treatment to the time of euthanasia or death of any cause. Dogs had censored PFST if alive at the end of the study or lost to follow-up.

Time to progression was defined as the interval between the first toceranib treatment to the occurrence of at least one of the following: local progression, metastasis development or metastatic progression.

Best response to treatment was reported using the Response Evaluation Criteria for Solid Tumours and was categorised as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) (Nguyen et al., 2015). For dogs with macroscopic disease, clinical benefit (CB) was defined as CR or PR of any duration, or SD of at least 10 weeks duration (London et al., 2012; Nguyen et al., 2015).

All statistical analyses were performed using a commercially available software program (MedCalc Software Ltd, Version 20, Ostend, Belgium).

RESULTS

Demographics and clinical presentation

The electronic search identified 20 dogs potentially suitable for inclusion. Three dogs with ACC were excluded as toceranib treatment was discounted one month after initiation due to owner’s financial constraints, and one dog with PCC was excluded because the patient was lost to follow-up 5 weeks after starting toceranib.

A total of 16 dogs with ATs met the inclusion criteria and were ultimately included in the analysis. There were 10 (62.5%) females (four intact) and six (37.5%) males (three intact). There were seven (43.7%) mixed breeds, and one (6.3%) each of the following: Jack Russell terrier, Yorkshire terrier, Cavalier King Charles Spaniel, dachshund, Border collie, poodle, French bulldog, Dobermann, American Staffordshire. The median age was 11 years (range, 1.5 to 15) and median weight was 15kg (range, 7 to 46).

Comorbidities were present in nine (56%) dogs and consisted of stage I mammary carcinoma (n=1), multiple endocrine neoplasia-like syndrome (n= 1), degenerative mitral valve disease (ACVIM-B1; n=1), bilateral otitis (n=1), diabetes mellitus (n=1), chronic kidney disease (n= 1), chronic pancreatitis (n=1), dermatopathy (n=1) and bilateral cataract (n=1).

Fourteen (87.5%) dogs had clinical signs at presentation, among them one dog had one (7.1%) sign, six (42.9%) dogs had two signs, six (42.9%) dogs had three signs, and one (7.1%) dog had four signs. Clinical signs included polydipsia and poly- uria (n=8; 57.1%), polyphagia (n=2; 14.3%), vomiting (n=2; 14.3%), weakness (n=2; 14.3%), syncope (n=2; 14.3%), abdom- inal distension (n=1; 7.1%), weight loss (n=1; 7.1%), diarrhoea (n=1; 7.1%), abdominal pain (n=1; 7.1%), abdominal disten- sion (n=1; 7.1%), tachypnoea (n=1; 7.1%), exercise intolerance (n=1; 7.1%), heavy panting (n=1; 7.1%) and back pain (n=1; 7.1%). Clinical signs were present for a median of 12 days (range,

Use of Toceranib for adrenal gland tumours in dogs

5 to 365) prior to diagnosis. Two (12%) dogs showed no symp- toms related to AT, with the discovery of the tumour occurring incidentally during evaluations for other concurrent health issues and during an ovariohysterectomy, respectively.

Diagnostic investigations and staging

Blood pressure was measured in all dogs; four (25%) had hyper- tension (median 170 mmHg; range 165 to 180). These dogs were treated with phenoxybenzamine (median dose 0.5 mg/kg PO every 12 hours, range 0.4 to 0.8) before any additional intervention was initiated. Throughout the observation period, no cardiac arrhyth- mias were observed. Additionally, a fundic examination was con- ducted in three dogs, all of which showed normal results.

In seven (43.7%) dogs, the results of adrenal functional testing were consistent with cortisol-secreting ACC (n=3; 42.8%) and PCC (n=4; 57.2%). The remaining nine (56.3%) dogs had no endocrinopathy based on preoperative endocrine testing.

For staging purposes, seven (43.7%) dogs underwent abdomi- nal ultrasound and TBCT, five (31.3%) dogs underwent TBCT, and four (25%) dogs underwent abdominal ultrasound and thoracic radiographs. Based on imaging, 10 (62.5%) tumours affected the right adrenal gland and six (37.5%) the left. Median tumour size was 6cm (range, 2.8 to 12). Caval thrombosis was identified in 10 (62.5%) cases.

Five (31%) dogs had suspected metastatic disease. Among these, four dogs had PCC and one had ACC. The metastatic sites in dogs with PCC included abdominal lymph nodes (n=1), liver (n=1), liver and lungs (n=1), spine (n=1). The dog with ACC had metastasis of abdominal lymph node and liver.

Ultrasound-guided fine-needle aspiration of the adrenal mass was performed in 13 (81%) cases and cytology was consistent with PCC in seven (44%) cases and ACC in six (37%) cases (Bertazzolo et al., 2014). Cytology of suspected metastatic disease was performed in 3 out of 5 cases (60%). Two of these dogs had PCC and one had ACC. Cytology confirmed liver metastasis and/or abdominal lymph node metastasis in all three cases.

Six (37.5%) dogs underwent surgery, and a final diagnosis of ACC was reached in all cases based on histopathology; one dog (case number 16) had also lymphadenectomy of an abdominal lymph node and surgical removal of a liver nodule and histopathol- ogy results confirmed metastatic disease in both lymph node and liver. The remaining 10 (62.5%) dogs were diagnosed with PCC either through the presence of an adrenal mass, endocrine testing, and cytology results (n=7) or based on the adrenal mass and endo- crine testing results alone (n=3). None of these dogs underwent surgical removal of PCC due to metastatic disease (n=4), inoper- able disease due to large tumour size and caval thrombosis (n=4), or because owners declined surgery involving vascular invasion which may pose a higher risk of complications (n=2).

Clinical and tumour characteristics are detailed in Table 1.

Toceranib treatment, clinical response and outcome

Reasons for toceranib administration in dogs with ACC included a high Utrecht score (median 13; range 11 to 20.5)

in 5 (31.3%) cases and metastatic disease (liver and abdominal lymph node) in 1 (6.3%) case. In the six dogs with ACC, toceranib was used as a standard treatment and was initiated 2 to 4weeks after surgery, depending on the clinician’s recommendation.

In dogs with PCC, toceranib was administrated at the time of diagnosis to treat inoperable tumour and/or macroscopic metastatic disease.

All dogs received toceranib orally at a starting median dose of 2.4 mg/kg (range, 2 to 2.9), rounded up to the nearest available tablet size on a Monday, Wednesday, and Friday schedule.

Four dogs with functional ACC received concomitant trilostane therapy; additional supportive medications included lactic probiotics (n=6), maropitant (n=2), gabapentin (n=2), amantadine (n=1).

All 10 dogs with PCC were evaluated for response (Table 2): eight (80%) achieved CB, including 2 (25%) PR and 6 (75%) SD. The remaining two dogs showed progression, as determined by abdominal ultrasound (Nguyen et al., 2015). Time to progres- sion for the two dogs showing PR was 100 and 182 days, respec- tively. One dog with PR remained stable for 182 days before being euthanized due to PD 243 days after treatment initiation. The second dog experienced PR for 100 days and was receiving toceranib while in PR at the time of data analysis. Among the six dogs with SD, median TTP was 176.5 days (range, 75 to 365). Median duration of toceranib treatment for all dogs with PCC was 182 days (range, 72 to 682). Toceranib was discontinued in five (50%) dogs with PCC due to progressive disease and owner- perceived bad quality of life.

At data analysis, five (50%) dogs with PCC were dead due to PD and owner-perceived poor quality of life, with a median overall PFST of 112 days (range, 75 to 243). The remaining five (50%) dogs with PCC were alive at the time of data analysis, with a median follow-up time of 310 days (range, 72 to 682 days).

Among the six dogs with ACC, three (50%) experienced disease progression and were humanly euthanized after 237, 273 and 364 days, resulting in a median PFST of 273 days; the remaining three dogs were alive and disease free 382, 508 and 583 days after starting toceranib. Median duration of adjuvant toceranib therapy was 274 days (range, 222 to 404). Toceranib was discontinued in five (83%) dogs with ACC, due to progressive disease (n=3) and due to financial constraints (n=2). For the latter two dogs, which remained disease-free after 508 and 583 days, toceranib was discontinued 385 and 404 days, after treatment initiation, respectively.

Adverse events

There were 17 episodes of AEs reported in 10 (62%) dogs.

Seven dogs were reported with one or more VCOG grade 1 or 2 gastrointestinal AEs including diarrhoea, inappetence and vomiting. Two out of these 7 dogs required dose reduction. One dog had treatment discontinuation due to grade 3 diarrhoea, which resolved one-month after the treatment break; toceranib was subsequently started at a lower dose (2.2mg/kg with the same schedule) with no further AEs recorded.

Table 1. Clinical and tumour characteristics
Case	Comorbidities	Clinical signs	Adrenal functional testing	Diagnostic imaging	Tumour diameter	CVC invasion	Metastasis	Cytology	Histology	Utrecht score
1	MMVD (ACVIM B1)	Abdominal distention, tachypnoea	Endogenous ACTH, LDSST	Abdominal ultrasound, thoracic radiographs	8 cm	Yes	Absent	Medullary neoplasia	ND	ND
2	Nammary gland carcinoma (stage I)	None	Endogenous ACTH, LDSST, plasma NMN	Abdominal ultrasound, thoracic radiographs	4 cm	No	Absent	Medullary neoplasia	ND	ND
3	None	Syncope, exercise intolerance, heavy panting, back pain	Plasma NMN	Abdominal ultrasound, thoracic radiographs	7.5cm	Yes	Spine	ND	ND	ND
4	None	Syncope	Plasma NMN, endogenous ACTH	Abdominal ultrasound, thoracic radiographs	3.6cm	Yes	Absent	ND	ND	ND
5	Chronic pancreatitis	Abdominal pain, vomiting	Plasma NMN, LDSST	Abdominal ultrasound, TBCT	3.5cm	Yes	Absent	ND	ND	ND
6	Bilateral cataract	Polyuria, polydipsia, polyphagia	Endogenous ACTH, LDSST	TBCT	4 cm	Yes	Liver, lung	Medullary neoplasia	ND	ND
7	MEN-1	Polyuria, polydipsia	Endogenous ACTH, LDSST	TBCT	3.3cm	Yes	Absent	Medullary neoplasia	ND	ND
8	None	Polyuria, polydipsia, weakness	Plasma NMN	TBCT	3.1 cm	Yes	Liver	Medullary neoplasia	ND	ND
9	None	None	Endogenous ACTH, LDSST	TBCT	12 cm	Yes	Absent	Medullary neoplasia	ND	ND
10	None	Vomiting, diarrhoea	Endogenous ACTH, LDSST	TBCT	7 cm	Yes	Abdominal lymph node, peritoneal effusion	Medullary neoplasia	ND	ND
11	CKD	Polyuria, polydipsia, weakness	Endogenous ACTH, LDSST	Abdominal ultrasound, TBCT	4.5cm	No	No	Cortical neoplasia	ACC	20.5
12	Bilateral otitis	Polyuria, polydipsia	LDSST	Abdominal ultrasound, TBCT	3.5cm	No	No	Cortical neoplasia	ACC	14.8
13	None	Polyuria, polydipsia, weight loss	LDSST	Abdominal ultrasound, TBCT	2.8cm	No	No	Cortical neoplasia	ACC	12
14	None	Polyuria, polydipsia	LDSST	Abdominal ultrasound, TBCT	3.8cm	No	No	Cortical neoplasia	ACC	11
15	Diabetes mellitus	Polyuria, polydipsia, abdominal distension	Endogenous ACTH, LDSST	Abdominal ultrasound, TBCT	5 cm	Yes	No	Cortical neoplasia	ACC	13
16	Dermatopathy	Polyuria, polydipsia, polyphagia	LDSST	Abdominal ultrasound, TBCT	3.5cm	No	Abdominal lymph node, liver	Cortical neoplasia	ACC and lymph node and liver metastasis	8.5

Journal of Small Animal Practice . Vol 66 . June 2025 . @ 2025 British Small Animal Veterinary Association.

CVC Caudal vena cava; MMVD Myxomatous mitral valve disease; LDDST Low-dose dexamethasone suppression test; NMN Normetanephrine; PCC Pheochromocytoma; ND Not done; TBCT Total-body computed tomography; MEN Multiple endocrine neoplasia; CKD Chronic kidney disease; ACC Adrenocortical carcinoma.

Use of Toceranib for adrenal gland tumours in dogs

Table 2. Response to toceranib and outcome in 10 dogs with macroscopic PCC
Case	Treatment response	Duration of CB (days)	TTP (days)	Reasons for toceranib discontinuation	OST (days)
1	PD	/	70	Bad QoL, PD	75
2	SD	141	141	Ongoing	+141
3	PD	/	56	Bad QoL, PD	112
4	SD	116	116	PD	116
5	SD	75	75	Bad QoL, PD	95
6	SD	283	283	Ongoing	+310
7	SD	212	212	Ongoing	+673
8	PR	182	182	Bad QoL, PD	243
9	SD	365	365	Ongoing	+682
10	PR	100	100	Ongoing	+100

CB Clinical benefit; TTP Time to progression; OST Overall survival time; PD Progressive disease; SD Stable disease; PR Partial response; owner-perceived; QoL Quality of life.

One dog had 3 episodes of VCOG grade 1 and 2 neutropenia, requiring a dose reduction (from 2.8 to 2.4 mg/kg with the same schedule).

One dog had VCOG grade 1 episode of proteinuria. Enalapril was started to treat proteinuria and was subsequently maintained at a stable level throughout the duration of toceranib treatment.

DISCUSSION

The current study was designed to describe the outcome of dogs with high-risk AT treated with toceranib, both in microscopic and macroscopic disease setting.

Clinical responses to toceranib have been documented in dogs with a spectrum of solid tumour types, including neuroendocrine tumours (Alonso-Miguel et al., 2021; Coto et al., 2021; Flesner et al., 2019; Frezoulis & Harper, 2022; Lew et al., 2019; London et al., 2012; Olivares-Sheppard et al., 2020; Olivares-Sheppard et al., 2022). Tyrosine kinase inhibitors are often recommended for directly inhibiting constitutively activated or overexpressed RTKs, significantly enhancing drug effectiveness (London et al., 2003). A recent study demonstrated a substantial decrease in KIT and PDGFR-ß expression in ATs and reduced PDGFR-B expression in the invasive and metastatic areas of PCCs in dogs compared to normal adrenal tissues (Harding et al., 2021). When comparing adrenal cortical tumours with PCCs, the for- mer showed significantly higher tyrosine kinase receptors expres- sion (Harding et al., 2021). Based on this, one might assume that ATs are not ideal candidates for TKI treatment. However, studies indicate that in tumours lacking receptor tyrosine kinase over- expression, response rate may be attributed to toceranib’s effects on the supporting endothelium and other cells in the tumour microenvironment, rather than on the tumour cells themselves (Heaton et al., 2020; London et al., 2003; Olivares-Sheppard et al., 2020).

In the present study, dogs with macroscopic PCC received toc- eranib, and CB was detected in 80% of them. Our results are in line with a recent retrospective case series (Musser et al., 2018) in terms of overall clinical benefit, the rate of SD, PR and dura- tion of response. In that study five dogs with inoperable, meta- static and/or recurrent PCC were treated with toceranib (Musser et al., 2018). CB was observed in all dogs; four (80%) dogs achieved SD with a progression-free interval (PFI) ranging from

11 to 36 weeks, while one (20%) dog achieved PR for at least 61 weeks, suggesting a biological activity of toceranib in dogs with PCCs (Musser et al., 2018).

We hypothesized that toceranib might also exhibit biological activity in dogs with ACC. The rationale behind our hypothesis stemmed from the significant increase in receptors tyrosine kinase expression observed in canine ATs compared to PCC (Harding et al., 2021).

In the current study, dogs with ACC were treated with adjuvant toceranib, due to the presence of a high Utrecht score or metastatic disease. It has been reported that an Utrecht score ≥11 in dogs with cortisol-secreting ATs is associated with a high risk of recurrence and short survival time (Sanders et al., 2019). A median survival of 14.4 months was reported for dogs with Utrecht score ≥11 treated with surgery alone (Sanders et al., 2019). Although further research is necessary to identify prognostic factors for dogs with ACC, our results suggest that toceranib might improve the long-term outcome of dogs with a high Utrecht score or metastatic disease.

Like in previous reports, gastrointestinal AEs were common in this study, occurring in 44% of dogs (Berger et al., 2018; Kim et al., 2017; Lew et al., 2019; London et al., 2003). Most gastrointestinal AEs were of VCOG grade 1 or 2, while one dog had VCOG grade 3 diarrhoea requiring discontinuation of toceranib. Three dogs required dose reduction of tocera- nib due to gastrointestinal toxicity. Additional AEs included VCOG grade 1 or 2 neutropenia and proteinuria. No addi- tional AEs were reported in our study and treatment was gen- erally well-tolerated.

This study has several limitations, largely due to its retrospec- tive nature and small population. First, a definitive diagnosis of PCC requires histological examination of the adrenal gland; however, adrenalectomy was not performed in this study due to the presence of suspected and/or confirmed metastatic disease, inoperable disease (e.g. large-tumour size and/or CVC invasion) or because owners hesitated to pursue surgery due to poor prog- nosis and high risks associated with the procedure. Nevertheless, in most cases the diagnosis of PCC was based on the presence of an adrenal mass, endocrine testing, and cytology. Furthermore, the cytological distinction between adrenocortical neoplasia and PCC is relatively straightforward for most cases with an individual diagnostic accuracy of 90% to 100% (Bertazzolo et al., 2014). Second, follow-up was not standardised, potentially

biasing results. Third, the absence of a control group prevented the comparison of outcome results observed in the current study with a population of dogs with high-risk ATs that were not treated with toceranib.

In conclusion, the present study suggests that toceranib might offer clinical benefit and improve outcome in dogs with high-risk ATs in both the macroscopic and microscopic disease setting. Prospective studies are warranted to investigate the role of toceranib in a larger sample size of dogs with ATs compared with a control group.

Author contributions

C. Chalfon: Conceptualization (lead); data curation (lead); for- mal analysis (lead); investigation (lead); methodology (lead); proj- ect administration (lead); supervision (lead); writing - original draft (lead); writing - review and editing (lead). G. Ghisoni: Data curation (equal); investigation (equal); software (equal). L. Pisoni: Data curation (equal); investigation (equal); visualiza- tion (equal). R. Finotello: Conceptualization (supporting); data curation (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); supervision (support- ing); writing - original draft (supporting); writing - review and editing (supporting). F. Del Baldo: Data curation (equal); inves- tigation (equal); writing - original draft (equal). S. Galac: Data curation (equal); formal analysis (equal); investigation (equal); writing - original draft (equal); writing - review and editing (equal). L. Marconato: Conceptualization (supporting); data curation (supporting); investigation (supporting); methodology (supporting); project administration (equal); supervision (sup- porting); writing - original draft (supporting); writing - review and editing (supporting). A. M. Tardo: Data curation (support- ing); formal analysis (lead); methodology (equal); writing - origi- nal draft (equal); writing - review and editing (equal). F. Fracassi: Data curation (equal); investigation (equal); writing - original draft (equal); writing - review and editing (equal). M. Zandvliet: Data curation (equal); investigation (equal); writing - original draft (equal); writing - review and editing (equal).

Conflict of interest

None of the authors of this article has financial or personal rela- tionship with other people or organisations that could inappro- priately influence or bias the content of the paper.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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