Prospective evaluation of Doppler echocardiography, tissue Doppler imaging
and biomarkers measurement for the detection of doxorubicin-induced
cardiotoxicity in dogs: A pilot study

J. Gallay-Lepoutre, M.C. Belanger, M.E. Nadeau.

PII: S0034-5288(16)30025-X
DOI: doi: 10.1016/j.rvsc.2016.02.001
Reference: YRVSC 3045

To appear in:

Received date: 6 June 2014

Revised date: 3 February 2016
Accepted date: 5 February 2016

Please cite this article as: Gallay-Lepoutre, J., Belanger, M.C., Nadeau., M.E., Prospec-
tive evaluation of Doppler echocardiography, tissue Doppler imaging and biomarkers
measurement for the detection of doxorubicin-induced cardiotoxicity in dogs: A pilot
study, (2016), doi: 10.1016/j.rvsc.2016.02.001

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 ACCEPTED MANUSCRIPT
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Prospective evaluation of Doppler echocardiography, Tissue Doppler Imaging and

biomarkers measurement for the detection of doxorubicin-induced cardiotoxicity in dogs: a

pilot study

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J Gallay-Lepoutre, MC Blanger, ME Nadeau.

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From the Department of Clinical Sciences, School of Veterinary Medicine, University of

Montreal, Saint-Hyacinthe, QC, Canada (Blanger, Nadeau); and the Clinique Vtrinaire

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Olliolis, Ollioules, France (Gallay-Lepoutre).
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This study was conducted at the Veterinary Teaching Hospital of the University of

Montreal, Quebec, Canada.

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Corresponding author: Julie Gallay-Lepoutre,

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Clinique vtrinaire Olliolis,

414A chemin des Canniers

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83190 Ollioules

France

Phone: +334 94 22 03 23

e-mail: gallay@olliolis.com
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Abbreviations

Amax Peak velocity of the late diastolic mitral wave

BNP Brain natriuretic peptide

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BSA Body surface area

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CHOP Cyclophosphamide-doxorubicin-vincristine-prednisone combination chemotherapy

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cTn Cardiac troponin

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DC Doxorubicin-induced cardiomyopathy

E Peak velocity of the pulsed wave TDI-derived early diastolic wave

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Emax Peak velocity of the early diastolic mitral wave

EF Ejection fraction
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EPSS Mitral valve E point to septal separation

ET Ejection time
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E/A Ratio of early to late diastolic left ventricular filling velocities

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FS Fractional shortening

IVCT TDI-derived isovolumic contraction time

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IVRT TDI-derived isovolumic relaxation time

LA/Ao Left atrium to aorta ratio

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LVIDd Left ventricle end-diastolic internal diameter

LVIDs Left ventricle end-systolic internal diameter

MPI Myocardial performance index

NT-proBNP N-terminal proBNP

PEP Pre-ejection period

S Peak velocity of the pulsed wave TDI-derived systolic mitral wave

TDI Tissue Doppler imaging

UW-25 25 weeks multidrug combination protocol from the University of Madison-Wisconsin

VPC Ventricular premature contraction

VT Ventricular tachycardia
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Abstract

The purpose of this pilot study was to evaluate the usefulness of selected echocardiographic

parameters, NT-proBNP and cardiac troponin I (cTnI) in the detection of cardiotoxicity in

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dogs treated with doxorubicin for various malignancies. Echocardiographic studies and

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biomarkers measurements were performed before each administration of doxorubicin, then 1

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and 3 months after completion of therapy. Thirteen dogs were included, with a total

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cumulative dose of doxorubicin ranging from 30 to 150 mg/m. E/A ratio significantly

decreased during doxorubicin administration (p=0.047). cTnI level was also significantly

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affected by treatment (p=0.046), increasing above normal at least at one time point in 11 of

13 dogs.
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The results of this pilot study suggest that monitoring of left ventricular diastolic function and

cTnI level measurement might be useful in the early detection of cardiotoxic signs of
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doxorubicin therapy in dogs.

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Keywords: doxorubicin, dog, cardiotoxicity, Doppler echocardiography, biomarkers

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1. Introduction

Doxorubicin (DOX) is a potent chemotherapeutic agent, widely used in human and veterinary

oncology. In dogs, this drug is part of the standard-of-care for treatment of a variety of

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cancers, including lymphoma, hemangiosarcoma and osteosarcoma (Chun et al., 2007).

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However, its use is limited by its myocardial toxicity, characterized by a delayed-onset

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cardiomyopathy that mimics dilated cardiomyopathy and carries a grave prognosis, through

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the development of refractory congestive heart failure or fatal arrhythmias (Van Vleet et al.,

1980; Mauldin et al., 1992; Singal and Iliskovic, 1998). The reported incidence of

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cardiotoxicity in dogs treated with DOX is 8-64% depending on the chemotherapy protocol
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used and the criteria for diagnosis of toxicity (Mauldin et al., 1992; Sorenmo et al., 2004;

Gillings et al., 2009; Ratterree et al., 2012). The occurrence of doxorubicin-induced

cardiomyopathy (DC) is related to the cumulative dose of DOX administered (Von Hoff et al.,
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1979), leading to the recommendation in dogs that maximum lifetime dose should not exceed
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180 to 240 mg/m of body surface area (BSA) (Mauldin et al., 1992; Chun et al., 2007;
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Ratterree et al., 2012). Nonetheless, sensitivity to the drug varies among individuals, and
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cardiotoxic events have been observed at lower doses in dogs treated with DOX (Susaneck,

1983; Mauldin et al., 1992; Ratterree et al., 2012).

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Guidelines for evaluation and follow-up of DOX-induced cardiotoxicity have not yet been

defined in dogs and current screening methods lack sufficient predictive power (Ratterree et

al., 2012; Tater et al., 2012). In people, serial endomyocardial biopsy has been historically

considered the gold standard for diagnosis of anthracyclines related cardiotoxicity (Mason et

al., 1978; Singal and Iliskovic, 1998). Non-invasive techniques are however preferred in

practice, with ECG monitoring and echocardiographic follow-up of fractional shortening (FS)

and left ventricular ejection fraction (LVEF) used traditionally (Mauldin et al., 1992; van Dalen

et al., 2006; Galderisi et al., 2007). However, this approach is considered insensitive to

detect subtle alterations in myocardial function; heart failure can occur despite a preserved

systolic function and when changes are detected, cardiac dysfunction deteriorates rapidly
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and is irreversible (Barry et al., 2007; Jurcut et al., 2008). To improve early detection of toxic

myocardial injury, new diagnostic tools have been evaluated in people. Assessment of

conventional Doppler echocardiography has shown that changes in diastolic function are

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frequent and often precede systolic dysfunction in patients treated with DOX (Marchandise et

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al., 1989; Tassan-Mangina et al., 2006). In fact, early signs of cardiotoxicity are often related

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more to the filling pressures than to the systolic function (Barry et al., 2007). Evaluation of

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mitral inflow velocity profile by conventional Doppler echocardiography is used to

characterize left ventricular diastolic function in people and dogs (Spirito et al., 1986;

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Nishimura et al., 1989), through measurement of peak velocities of early (E) and late (A)

diastolic mitral waves, and determination of the ratio between these velocities (E/A ratio). In
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people, Tissue Doppler echocardiography (TDI), a newer technique measuring myocardial

velocities, appears sensitive in detecting early ventricular dysfunction secondary to DOX

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therapy (Kapusta et al., 2000; Tassan-Mangina et al., 2006; Nagy et al., 2008; Baysal et al.,
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2010). Myocardial performance index (MPI), a parameter of global left ventricular function,

derived from conventional or tissue Doppler (Hori et al., 2007), has been reported to increase
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in patients treated with DOX (Santin et al., 2007; Dodos et al., 2008). These tools have not

yet been evaluated in the context of canine DC, but early alterations in mitral inflow velocity
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profile, MPI, and TDI parameters, have been reported during the occult phase of dilated

cardiomyopathy (Lee et al., 2002; Chetboul et al., 2004a; 2004b; O'Sullivan et al., 2007).

Cardiac biomarkers have been extensively studied in the last few years to assess their ability

to improve diagnosis of DC in humans. Cardiac troponins (cTn) I and T are highly sensitive

and specific markers of cardiomyocytes injury (Prosek and Ettinger, 2009). Some studies

have suggested a relationship between cTn increase and myocardial dysfunction secondary

to DOX therapy in people (Lipshultz et al., 1997; Cardinale et al., 2000; Mavinkurve-

Groothuis et al., 2008), whereas others failed to show such association (Kismet et al., 2004;

Koseoglu et al., 2005; Soker and Kervancioglu, 2005). In dogs, increase of cTn levels has

been observed following administration of DOX (DeFrancesco et al., 2002; Selting et al.,
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2004a; 2004b). Dogs that developed clinical heart disease had an increase of cTnI that

preceded recognition of cardiac dysfunction. Optimal timing for cTnI measurement after DOX

administration remains controversial since ongoing cardiomyocyte loss continues after DOX

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administration, and therefore monitoring intervals are still exploratory (Selting et al., 2004a;

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2004b).

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B-type natriuretic peptide (BNP) is secreted by ventricles in response to increased workload.

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N-terminal proBNP (NT-proBNP) is an inactive fraction of BNP precursor, more stable in

plasma than the active peptide (Prosek and Ettinger, 2009). Similarly to cTn, research in

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people has yielded conflicting results, but the ability of natriuretic peptides to predict DC has

been observed in several studies (Soker and Kervancioglu, 2005; Dodos et al., 2008;
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Mavinkurve-Groothuis et al., 2008). In one study, an increase in BNP level was seen during

DOX administration in 7 dogs, but of no clinical importance (Alves de Souza and Camacho,
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2006). NT-proBNP has not been evaluated in this setting in dogs, but NT-proBNP
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measurement has proven useful in detecting occult DCM (Chetboul et al., 2004c; Oyama et

al., 2007; 2008).

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The primary objective of this study was to assess the effect of doxorubicin on cTnI, NT-

proBNP levels, and selected Doppler and TDI parameters in dogs.

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2. Materials and methods

2.1. Study population

All dogs presenting to the oncology department of the Veterinary Teaching Hospital of the

University of Montreal between July 2008 and December 2009 to receive a doxorubicin-

based chemotherapy treatment were considered for inclusion in the study. Written informed

consent was obtained from owners before enrolment. Dogs were excluded if significant

cardiac disease (i.e., precluding safe doxorubicin use or requiring specific cardiac treatment)

was detected upon first cardiac evaluation, if DOX had already been administered before
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entering the study, or if the dog was lost to follow-up after initial evaluation and administration

of doxorubicin. The study protocol was approved by the institutional Animal Care Committee.

2.2. Study design

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Dogs were prospectively evaluated for cardiotoxicosis from the first administration of DOX

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until 3 months after the last administration. A complete cardiac evaluation including physical

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examination, 30 minutes ECG recording and complete echocardiography with TDI, was

performed before each administration of the drug, then 1 month and 3 months after the last

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DOX treatment. Blood samples for measurement of cTnI and NT-proBNP were collected at

the same time points.

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2.3. Chemotherapy protocols

Dogs were treated with DOX either alone or in combination with other agents, as indicated by
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their medical condition, according to the protocol defined by a board-certified oncologist

(MEN). Depending on the chemotherapy protocol, DOX was thus administered every 3 to 6
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weeks.
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After administration of diphenhydramine (1 mg/kg IM), doxorubicin was administered through

an IV infusion over 20 minutes, as currently recommended at the authors institution. The

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standard dose of 30 mg/m BSA was used, unless specific dose adjustment was

recommended by the oncologist, i.e. in small dogs (Arrington et al., 1994), or previous grade

3 or 4 adverse reaction to the drug (VCOG, 2004).

Doxorubicin administration was repeated to a maximal cumulative dose of 150 mg/m BSA (5

sessions), depending on the chemotherapy protocol, the response to therapy and the

occurrence of toxicities. However, in case of good response to chemotherapy and in the

absence of adverse events, treatment could be pursued beyond 150 mg/m BSA, if deemed

beneficial to the dog by the oncologist and with the informed consent of the owner.
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2.4. Cardiac evaluation

Cardiac auscultation, ECG recording and complete echocardiography were performed before

each administration of DOX, then 1 month and 3 months after the last treatment. 6-lead

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computerized ECGa was recorded with the dogs in right lateral recumbency during a period

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of 5 minutes. Lead II monitoring was then maintained for arrhythmia detection during

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echocardiography. Heart rate and rhythm or conduction abnormalities were recorded. All

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echocardiographic examinations were performed by the same operator (JGL) on unsedated

dogs, by use of an ultrasound systemb equipped with a multi-frequency 4 MHz phased-array

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transducer. During echocardiography, animals were positioned in lateral recumbency while
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obtaining standard views (Thomas et al., 1993; Blanger, 2009). The following

echocardiographic parameters were specifically evaluated: fractional shortening (FS), mitral

valve E point to septal separation (EPSS), bi-dimensional left atrium to aorta ratio (LA/Ao),
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left ventricular end-diastolic and end-systolic internal diameters (LVIDd and LVIDs,
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respectively) were obtained from the right parasternal short-axis view. Conventional Doppler
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and TDI measurements were obtained from the left apical 4-chambers view. Mitral inflow was
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recorded using pulsed-wave Doppler, with the sample volume positioned at the tip of the

mitral leaflets and E/A ratio was calculated. Free wall mitral annulus motion was recorded
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using pulsed-wave TDI mode, with the filter set to exclude high-frequency signals and gains

minimized to decrease background noise; the sample volume was placed at the level of the

mitral annulus on the left ventricular free wall. Peak myocardial velocities during systole (S)

and early diastole (E), duration of the systolic wave, TDI-derived isovolumic contraction time

(IVCT) and TDI-derived isovolumic relaxation time (IVRT) were recorded. TDI-derived MPI

was then calculated, as described by Hori, according to the following formula: (IVCT +

IVRT) / systolic wave duration (Hori et al., 2007). All echocardiographic measurements were

repeated on 3 consecutive cardiac cycles and the results were averaged for statistical

analysis.
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2.5. Biomarkers measurement

Blood for biomarker measurement was collected prior to each administration of DOX, and

then 1 and 3 months after termination of DOX treatment.

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Five mL of blood were collected and divided between a purple-top EDTA tube and a red-top

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tube for measurement of NT-proBNP and cTnI, respectively. After immediate centrifugation,

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plasma was separated, transferred in a purple-top EDTA glass tube and stored frozen at -

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40C until analyzed. The same procedure was performed with the red-top tube after a delay

of 20 minutes to allow time for blood clotting and subsequent serum separation.

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All samples were analyzed as one batch by an external laboratoryc at the end of the study,
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after overnight shipment on dry ice. Time between blood collection and biomarkers analysis

ranged from 4 to 20 months.

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2.5.1. cTnI measurement

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Serum cTnI concentration was measured using a commercially available human assayd,

validated for the canine species. The detection limit was 0.2 ng/mL and cTnI was considered
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abnormal when it exceeded 0.2 ng/mL.

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2.5.2. NT-proBNP measurement

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Plasma NT-proBNP was measured using a commercially available canine-specific assaye.

As previously described, NT-proBNP concentration was considered abnormal when

exceeding 445 pmol/L (Oyama et al., 2008).

2.6. Statistical analysis

A linear model for the analysis of repeated measurements was used to evaluate the effect of

the cumulative dose of doxorubicin on the mean value of selected parameters (LVID, SF,

LA/Ao, EPSS, Emax, E/A ratio, S, E and MPI, biomarkers levels), with the dose as the

intrasubject variability factor.

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A p-value<0.05 was considered significant. Statistical analyses were performed using a

commercially available softwaref. Unless specifically mentioned, results are presented as

mean values standard deviation.

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3. Results

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3.1. Dogs

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Fifteen dogs were included in this pilot study. Two dogs died of their cancer rapidly after the

first administration of DOX and were thus subsequently excluded from analysis. Thirteen

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dogs completed the study: 7 males (1 intact, 6 neutered) and 6 females (1 intact, 5 spayed)
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aged from 4 to 12 years (median: 9 years), weighing 10.0 to 47.0 kg (mean: 31.0 kg). The

dogs belonged to various breeds, with a predominance of large breed dogs: Labrador

Retrievers (4), Golden Retrievers (3), cross-breed (3), Bernese Mountain (1), Boxer (1), and
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Welsh Corgi (1).

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3.2. Cancers and chemotherapy treatments

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Treatment protocols and types of malignancy are presented in Table 1. Lymphoma was the

most common neoplasia, with 5 of 13 dogs affected, among which one dog was concurrently
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suffering from lymphoma and mammary carcinoma with inguinal lymph node metastasis.

Total cumulative dose of DOX ranged from 30 to 150 mg/m BSA (mean: 94.6 mg/m2;

median 120 mg/m BSA) : 30 mg/m in one dog, 60 mg/m in 3 dogs, 90 mg/m in 2 dogs,

120 mg/m in 6 dogs and 150 mg/m in one dog. 6 of 13 dogs received the intended total

dose according to their prescribed treatment protocol. In the 7 remaining dogs, DOX

administration was discontinued before completion of therapy (dose range 30-120 mg/m)

because of progressive neoplastic disease.

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3.3. Follow-up

Median duration of follow-up was 6.5 months (range 1.5-19). Seven of 13 dogs completed

the follow-up period of 3 months after discontinuation of DOX (total dose 90 mg/m in 2 dogs,

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120 mg/m in 4 dogs, 150 mg/m in one dog). The 6 remaining dogs completed the follow-up

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period of 1 month after the last administration of DOX but died or were euthanized before

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completion of the last evaluation (3 month post-doxorubicin). In 5 of these 6 dogs, death was

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related to disease progression; the remaining dog died in a car accident while in remission.

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3.4. Standard cardiac monitoring

No dog showed any clinical signs of congestive heart failure during follow-up. Resting
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tachycardia was detected in 2 dogs, attributed to anxiety in one dog, and paroxysmal

ventricular tachycardia in the other dog, as described below. Cardiac physical examination
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remained unremarkable in the remaining dogs.

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Results of bidimensional and M-mode echocardiography are presented in Table 2. Statistical

analysis was limited to dogs receiving doses ranging from 30 to 120 mg/m as only one dog
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received a higher total dose. No significant variation of selected echocardiographic

parameters was identified. In one dog, bidimensional echocardiogram showed alteration of

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left ventricular free wall motility one month after the last DOX administration but this

abnormality had resolved when rechecked 2 months later. This dog was followed by the

oncology department for 5 months after this last evaluation, without showing any cardiac

issue.

Four dogs showed significant electrical disturbances during the follow-up period. One dog

showed isolated left VPCs (approximately 30 VPCs per hour) 3 months after the end of DOX

therapy (cumulative dose 150 mg/m). He was lost to follow-up 10 months after this last

cardiac evaluation, without exhibiting any sign of cardiac disease. One dog showed

paroxysmal ventricular tachycardia 20 days after a cumulative dose of 90 mg/m, but no ECG

disturbances were detected when rechecked 2 months later. One dog exhibited
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supraventricular tachycardia with tachycardia-dependent intermittent left bundle branch

block, 3 weeks after a cumulative dose of 60 mg/m. Finally, one Boxer dog showed isolated

right VPCs (10 per minute) one month after a cumulative dose of 60 mg/m. These last 3

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dogs died or were euthanized shortly after the last evaluation due to cancer progression.

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3.5. Conventional Doppler and TDI parameters: Emax, E/A ratio S, E and MPI

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Table 3 summarizes the effect of cumulative DOX administration on Doppler and TDI

parameters. Statistical analysis was limited to dogs receiving doses ranging from 30 to 120

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mg/m, as only one dog received a higher total dose.

Mean E/A ratio decreased significantly with increasing cumulative dose of DOX (p=0.047).
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Conversely, no effect of DOX administration was detected for Emax, S, E and MPI

throughout the follow-up period.

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3.6. Cardiac troponin I

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At baseline, serum cTnI was below 0.2 ng/mL in 11 of 13 dogs (Figure 1). Initial level of cTnI
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was increased to 0.386 ng/mL in one dog (then decreased to 0.2 ng/mL on subsequent
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evaluation) and was unavailable for one dog (but below 0.2 ng/mL on subsequent

evaluation). Mean cTnI increased significantly with cumulative DOX dose (Table 3). Eleven
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of 13 dogs showed increase of cTnI level above normal at one or more occasion. The 2

remaining dogs received a total dose of DOX of 30 and 60 mg/m. In 5 of 7 dogs that had a

measurement of cTnI 3 months after the end of DOX therapy, the highest cTnI value was

detected at this time point (Figure 1).

3.7. NT-proBNP

Baseline NT-proBNP concentration ranged from 50 to 1250 pmol/L. No significant effect of

cumulative doses of DOX on mean plasma NT-proBNP values was found (p=0.50) (Table 3).
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4. Discussion

In this prospective pilot study, some dogs treated with DOX for various malignancies

developed ECG or echocardiographic abnormalities at doses lower than 150 mg/m2, similar

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to what has been reported in previous retrospective studies (Susaneck, 1983; Mauldin et al.,

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1992; Ratterree et al., 2012). A recent study documented the occurrence of electrical

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disturbances (ventricular and atrial premature contractions, supraventricular tachycardia,

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bundle branch block) in dogs treated at lower cumulative doses of DOX, ranging from 30 to

150 mg/m2 (Ratterree et al., 2012). We observed similar ECG abnormalities: VPCs,

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supraventricular tachycardia, bundle branch block. Paroxysmal ventricular tachycardia was
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previously reported in Mauldins case series (1992). In one Boxer dog, right-sided VPCs

were detected after 60 mg/m of DOX. They could indicate early cardiotoxicosis, but occult

arrythmogenic right ventricular cardiomyopathy could not be ruled out. Indeed, the cardiac
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abnormalities detected in our patients were non specific, and could have been related to
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other causes than DOX therapy, particularly cancer progression. However, these data
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highlight the interest for close cardiac monitoring in all dogs treated with DOX during
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treatment, since cardiotoxicosis may occur for low cumulative doses.

In our study, E/A ratio, a marker of left ventricular diastolic function, significantly decreased
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with cumulative doses of DOX. Impaired diastolic function by repeated DOX administration

has been reported in humans and alteration of left ventricular filling is often observed earlier

than systolic dysfunction (Marchandise et al., 1989; Tassan-Mangina et al., 2006; Nagy et

al., 2008; Radulescu et al., 2008). Conversely, in one experimental study in dogs treated with

DOX, changes in mitral inflow velocities were observed only in a few dogs and occurred later

than alterations of systolic function (Hanai et al., 1996). Changes of Doppler transmitral

profile have been reported in the occult phase of dilated cardiomyopathy in Doberman

Pinschers (O'Sullivan et al., 2007). A recent study focused on TDI measurements in 14 dogs

receiving DOX for treatment of lymphoma (Tater et al., 2015): similar to our results, TDI

indices were not significantly affected by DOX therapy, but a slight reduction in early diastolic
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(Emax) to late diastolic myocardial velocities ratio, which is the TDI-derived E/A ratio, was

observed following DOX administration. Decreasing E/A ratio is suggestive of alteration of

left ventricular relaxation and/or compliance. Abnormal left ventricular relaxation occurs when

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interaction between actin-myosin complex and calcium is altered in cardiomyocytes.

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Decreased compliance is associated with myocardial fibrosis or increased chamber stiffness

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due to cardiac remodeling (O'Sullivan et al., 2007). Mechanisms leading to DOX chronic

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cardiotoxicity are complex and not fully understood. However, alteration of myocardial

calcium handling and actin-myosin synthesis has been identified (Minotti et al., 2004).

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Histopathologically, DC is characterized by cardiomyocyte vacuolar degeneration and death,

and some degree of myocardial fibrosis (Van Vleet et al., 1980; Singal and Iliskovic, 1998).
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Thus, alteration of left ventricular diastolic function, identified in our study by decreasing E/A

ratio, could be induced by DOX treatment. However, this change might also be attributed to
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myocardial injury caused by cancer progression itself.

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Cardiac troponins are sensitive and specific markers of cardiomyocyte injury (Burgener et al.,

2006). In the present study, cTnI levels significantly increased with cumulative DOX
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administration, especially when reaching 120 mg/m2, which is in agreement with previous

data in people (Lipshultz et al., 1997; Cardinale et al., 2000; 2002; Dolci et al., 2008) and
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dogs (DeFrancesco et al., 2002; Selting et al., 2004a). Abnormal cTnI was recorded in most

dogs during and/or following DOX treatment, regardless of occurrence of echocardiographic

or ECG abnormalities, with increases remaining minor for doses of less than 120 mg/m.

DOX is known to cause myocardial injury; oxidative stress is thought to play a key role in

DOX toxicity, leading ultimately to cell degeneration and death (Van Vleet et al., 1980; Singal

and Iliskovic, 1998). Similar to what has been found in another canine study (Selting et al.,

2004a), myocardial damage might be ongoing despite termination of DOX therapy, as

highest cTnI values were obtained 3-months after the end of therapy in many dogs of the

present study. Interestingly, Tater et al. (2015) also measured cTnI during DOX treatment for

lymphoma and found no significant modification of this index. However, cTnI measurement
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was not pursued beyond the end of chemotherapy. This could at least partly explain the

difference with our results. In Selting et als retrospective study (2004a), cTnI concentration

did not accurately predict cardiac outcome in dogs treated with DOX. The role of cTnI

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measurement for prediction of cardiotoxicosis could not be evaluated in our pilot study, as no

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case of DC could be confirmed. In people, troponins have been identified as promising

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predictors of ventricular dysfunction in some studies (Lipshultz et al., 1997; Cardinale et al.,

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2000; 2002; Sawaya et al., 2012), whereas others have shown no increase of cTnI

(Koseoglu et al., 2005; Soker and Kervancioglu, 2005) or cTnT (Dodos et al., 2008) following

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anthracycline therapy, despite occurrence of echocardiographic changes. The cause for such

discrepancies is unclear but the timing of cTn measurement is likely to be critical, as the
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heart may compensate for the damage. Another issue lies in the specificity of cTnI increase:

cTnI is a highly specific marker of cardiomyocyte injury, but regardless of the cause of this
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injury. Thus, it cannot be ruled out that the cTnI increase in some dogs was due to cancer
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progression and not to treatment. Indeed, even if no evidence of cardiac involvement was

detected in any dog during echocardiographic follow-up, some degree of cardiac infiltration
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or damage might have occurred in patients with advanced neoplasia. Particularly, in one dog

suffering from both lymphoma and mammary carcinoma, baseline cTnI was high, then
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decreased below 0.2 ng/mL on subsequent measurements. Cardiac damage due to disease,

with improvement following chemotherapy, could be hypothesized in this case.

NT-proBNP has been reported as an interesting predictor of DOX toxicity in several human

studies (Lipshultz et al., 2012; Pongprot et al., 2012; Kittiwarawut et al., 2013). Particularly,

NT-proBNP was reported to rise before evidence of echocardiographic alteration of left

ventricular systolic function (Lipshultz et al., 2012; Kittiwarawut et al., 2013). However,

although criteria and methods used to define and detect cardiotoxicity differed, others found

that NT-proBNP was not effective in detecting cardiac alterations following anthracycline

therapy (Sawaya et al., 2012). We failed to show any correlation between DOX cumulative

dose and NT-proBNP levels and timing of the assessment cannot be incriminated since NT-
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proBNP was measured during and shortly after DOX therapy similarly to what was done in

these human studies.

The main and obvious limitation of this pilot study is the small number of patients included,

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which limits the statistical significance of the obtained results (especially negative findings),

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but guides further investigation.

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Another important issue is that dogs were included regardless of type of cancer and DOX-

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chemotherapy protocol. As a consequence, only one of 13 dogs reached the total dose of

150 mg/m of DOX, which is the clinically used limit above which risks of DC are considered

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to increase significantly (Mauldin et al., 1992; Sorenmo et al., 2004). Thus, our results lack

information on the meaning of parameter changes above this critical dose. Indeed, total
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cumulative dose of DOX, appears as the most important risk factor for cardiotoxicity with

dose intensity potentially serving as a contributor. Additionally, drugs used in combination

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with DOX in this study, although not likely to have contributed to cardiac toxicity in dogs,
TE

could have interacted (Senkus and Jassem, 2011).

Finally, the duration of follow-up was limited to 3 months after the end of chemotherapy,
P
CE

which could have been too short to detect DC, as DC can occur a long time after DOX

exposition, particularly in people (Steinherz et al., 1991). However, in the largest case series
AC

of DOX toxicity in dogs, the median time between initiation of therapy and development of

cardiac abnormalities was 77 days for ECG abnormalities and 105 days for congestive heart

failure (Mauldin et al., 1992).

5. Conclusion

The results of this pilot study suggest that monitoring of left ventricular diastolic function

might be useful as a tool to detect cardiotoxic signs in dogs treated with doxorubicin.

Furthermore, cTnI increased during doxorubicin treatment but NT-proBNP did not,

suggesting cTnI might be of greater value than NT-proBNP in this setting. Larger prospective

studies are warranted to determine if these tools might improve the monitoring of doxorubicin
 ACCEPTED MANUSCRIPT
17

toxicity and should be included in the routine cardiac monitoring of dogs treated with this

drug.

T
Acknowledgements

IP
Guy Beauchamp for statistical assistance

R
Zoetis Clinical Research Fund for financial support (no involvement in study design,

SC
collection, analysis and interpretation of data)

Idexx Laboratories for in-kind grant through measurement of biomarkers

Conflicts of interest statement NU

MA
The study was supported by an in-kind grant from Idexx Laboratories (biomarkers

measurement).
D
TE

Footnotes
a
Marquette Mac 5000 Resting ECG, GE Medical System, Milwaukee, WI.
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CE

b
Vivid 7 Vantage, GE System, Milwaukee, WI.
c
Vita-Tech Canada, Idexx Laboratories, Markham, ON.
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d
Immulite 2000 troponin-I, Siemens Medical Solutions Diagnostics, Deerfield, IL.
e
Canine Cardiopet proBNP, Idexx Laboratories, Markham, ON.
f
SAS System, Version 9.2, SAS Institute, Cary, NC.
 ACCEPTED MANUSCRIPT
18

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Chemotherapy Time interval Dose intensity Number of Type of

protocol between 2 doxorubicin malignancy

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doxorubicin administrations (number of dogs)

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administrations (expected total

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dose)

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DOX (Ogilvie et 3 weeks 10 mg/m/week 4 to 5 Hemangiosarcoma

al., 1996) (120 to 150 (4)

NU mg/m BSA) Lymphoma (2)

Soft tissue
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sarcoma (1)

SCC* (1)
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UW-25 (Garrett 4 weeks 7.5 4 Lymphoma (1)

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et al., 2002) mg/m/week (120 mg/m Lymphoma and

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BSA) MC* (1)

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DOX 6 weeks 5 mg/m/week 4 Osteosarcoma (1)

carboplatin ( (120 mg/m SCC* (1)

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Kent et al., 2004 ; BSA)

Selmic et al., 2014)

DOX 6 weeks 5 mg/m/week 4 Lymphoma with

lomustine** (120 mg/m CNS involvement

BSA) (1)

* SCC = squamous cell carcinoma; MC = mammary carcinoma

** Combination chemotherapy consisting of alternating doses of doxorubicin (30 mg/m IV)

and lomustine (50-60 mg/m PO) every 21 days for a total of 4 cycles

Table 1: Chemotherapy treatment protocols

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LVIDd LVIDs EPSS

Dose (mg/m) (mm) (mm) SF (%) LA/Ao (mm)

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0 39.2 7.3 27.0 5.7 31.5 4.3 1.3 0.2 4.6 1.1

(23.1-46.7) (14.6-34.0) (24.4-37.0) (1.0-1.6) (2.8-6.6)

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N=13 N=13 N=13 N=13 N=13

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30 39.3 6.8 26.6 5.7 32.9 6.1 1.3 0.1 4.5 1.0

(19.1-44.8) (10.8-33.0) (26.2-43.8) (1.0-1.5) (3.0-6.1)

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N=13 N=13 N=13 N=11 N=12
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60 38.5 6.6 25.8 6.6 33.9 8.4 1.3 0.2 4.2 1.8

(23.3-46.6) (11.2-33.5) (21.98-51.8) (7.19-15.64) (0.0-6.4)

N=12 N=12 N=12 N=12 N=11

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90 40.8 4.8 28.2 4.8 31.2 4.8 1.4 0.2 5.1 1.9
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(32.8-49.2) (20.8-35.1) (30.0-36.48) (0.9-1.6) (2.1-7.6)

N=9 N=9 N=9 N=9 N=9

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120 41.3 4.3 28.5 5.1 31.2 5.5 1.3 0.2 5.6 2.1

(0.65-0.84) (0.88-1.53) (10.81-18.39) (7.63-13.93) (0.88-1.35)

N=7 N=7 N=7 N=7 N=7

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P value 0.91 0.56 0.42 0.47 0.15

Results are reported as mean standard deviation (range). N indicates the number of dogs evaluated.

Asterisk indicates a significant difference between the doses. Measurements were obtained before

each DOX administration, i.e. 3 to 6 weeks after previous DOX treatment.

Table 2. Mean values of standard echocardiographic parameters during doxorubicin therapy

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Dose NT-proBNP

(mg/m) E max (m/s) E/A ratio S' (cm/s) E' (cm/s) TDI-MPI cTnI (ng/mL) (pmol/L)

0 0.74 0.16 1.17 0.26 14.66 2.96 11.23 3.23 1.29 0.38 0.215 0.052 396 327

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(0.57-1.03) (0.85-1.64) (10.1-20.59) (5.38-16.59) (0.86-2.2) (0.2-0.382) (50-1250)

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N=11 N=11 N=13 N=13 N=13 N=12 N=12

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30 0.74 0.15 1.21 0.18 14.24 3.68 10.51 3.04 1.22 0.31 0.201 0.004 348 223

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(0.52-1.01) (0.93-1.55) (7.99-19.76) (5.25-15.99) (0.71-1.94) (0.2-0.215) (131-875)

N=13 N=13 N=13 N=13 N=13 N=12 N=13

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60 0.67 0.13 1.03 0.23 14.99 3.28 11.08 2.70 1.18 0.43 0.241 0.071 293 126

(0.48-0.97) (0.81-1.44) (10.6-20.57) (7.19-15.64) (0.63-2.25) (0.2-0.411) (151-576)

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N=12 N=12 N=12 N=12 N=12 N=11 N=11

90 0.72 0.10 1.09 0.27 15.79 3.49 9.92 2.95 1.22 0.32 0.207 0.019 327 207
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(0.56-0.84) (0.91-1.65) (9.97-21.55) (5.08-14.21) (0.83-1.89) (0.2-0.254) (157-804)

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N=9 N=9 N=9 N=9 N=9 N=8 N=8

120 0.68 0.11 1.05 0.23 14.75 2.65 10.68 2.55 1.16 0.16 0.902 1.404 443 250
P

(0.65-0.84) (0.88-1.53) (10.81-18.39) (7.63-13.93) (0.88-1.35) (0.2-4.07) (250-882)

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N=7 N=7 N=7 N=7 N=7 N=7 N=7

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P value 0.21 0.047* 0.90 0.22 0.64 0.046* 0.50

Results are reported as mean standard deviation (range). N indicates the number of dogs evaluated.

Asterisk indicates a significant difference between the doses. Measurements were obtained before

each DOX administration, i.e. 3 to 6 weeks after previous DOX treatment.

Table 3. Mean values of selected echocardiographic and biochemical parameters during

doxorubicin therapy
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Highlights

Echocardiographic or electrocardiographic changes may be observed after a low

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cumulative dose of doxorubicin in some dogs

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Cardiac troponin I and Doppler-derived E/A ratio are affected by doxorubicin

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administration in dogs

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Cardiac troponin I measurement and echocardiographic evaluation of left ventricular

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diastolic function may be of interest to monitor cardiotoxicosis in dogs receiving

doxorubicin
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