* CorrespondE-mail addr

https://doi.org1760-2734/ª 2creativecommo

Journal of Veterinary Cardiology (2021) 36, 77e88

www.elsevier.com/locate/jvc

The predictive value of clinical,radiographic, echocardiographic variablesand cardiac biomarkers for assessing riskof the onset of heart failure or cardiacdeath in dogs with preclinicalmyxomatous mitral valve disease enrolledin the DELAY study

M. Borgarelli, DVM, PhDa,*, L. Ferasin, DVM, PhDb, K. Lamb,PhDc, D. Chiavegato, DVM, PhDd, C. Bussadori, DVM, MD,PhDe, G. D’Agnolo, DVMf, F. Migliorini, DVMg, M. Poggi,DVMh, R.A. Santilli, DVM, PhDi, E. Guillot, DVMj, C. Garelli-Paar, Pharm.D, MSj, R. Toschi Corneliani, DVM, MSk,F. Farina, DVM, MSl, A. Zani, DVMm, M. Dirven, DVMn,P. Smets, DVM, PhDn, C. Guglielmini, DVM, PhDo, P. Oliveira,DVMp, M. Di Marcello, DVM, PhDq, F. Porciello, DVMr,S. Crosara, DVM, PhDs, P. Ciaramella, DVMt, D. Piantedosi,DVM, PhDt, S. Smith, DVMu, S. Vannini, DVMe, E. Dall’Aglio,DVMv, P. Savarino, DVM, PhDw, C. Quintavalla, DVM, PhDx,M. Patteson, DVM, PhDy, J. Silva, DVMe, C. Locatelli, DVM,PhDz, M. Baron Toaldo, DVM, PhDaa

aDepartment Small Animal Clinical Science, Virginia Maryland College of VeterinaryMedicine, Blacksburg, VA, USAb Specialist Veterinary Cardiology Consultancy Ltd, Alton, Hampshire, UKc Lamb Statiscal Consulting and Scientific Writing LLC, West St. Paul, MN, USAdClinica Veterinaria Arcella, Padova, ItalyeClinica Veterinaria Gran Sasso, Milan, ItalyfTrieste, Italy

ing author.ess: mborgarelli@gmail.com (M. Borgarelli).

/10.1016/j.jvc.2021.04.009021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://ns.org/licenses/by-nc-nd/4.0/).

78 M. Borgarelli et al.

gOspedale Veterinario Croce Azzura, Roma, ItalyhCentro Veterinario Imperiese, Imperia, ItalyiClinica Veterinaria Malpensa, Samarate Varese, ItalyjCeva Sante Animale, Libourne, FrancekOspedale Veterinario San Francesco, Milan, ItalylAmbulatorio Veterinario del Parco Margherita, Naples, ItalymClinica Cardiovet, Livorno, ItalynDierenkliniek Rijen, Rijen, the NetherlandsoDipartimento di Medicina Animale, Produzione e Salute, Universita di Padova,Legnaro, Padova, ItalypDavies Veterinary Specialists Ltd, Higham Gobion, Hertfordshire, UKqCentro Veterinario Cellatica, Cellatica, Brescia, ItalyrDipartimento di Medicina Veterinaria, Universita degli Studi di Perugia, Perugia, ItalysClinica Veterinaria CMV, Varese, ItalytDipartimento di Medicina Veterinaria e Produzioni Animali, Universita degli Studi diNapoli Federico II, Naples, Italyu Sarah Smith Cardiology Ivy Court, Willington, UKvClinica Veterinaria Milano Sud, Peschiera Borromeo, Milano, Italyw Facolta di Medicina Veterinaria di Torino, Ospedale Didattico Veterinario dellaFacolta, Sezione Clinica Medica, Grugliasco, Turin, ItalyxDipartimento di Scienze Mediche Veterinarie, Universita di Parma, Parma, ItalyyHeartvets, The Animal Hospital Stinchcombe, Dursley, UKzDipartimento di Medicina Veterinaria, Universita degli Studi di Milano, Milan, ItalyaaDipartimento di Scienze Mediche Veterinarie, Alma Mayor Studiorum, Universita diBologna, Ozzano Emilia, Italy

Received 1 June 2020; received in revised form 27 April 2021; accepted 29 April 2021

KEYWORDSHeart;Dog;Valve;Cardiac ultrasound;NT-proBNP

Abstract Objectives: To identify the predictive value on time to onset of heartfailure (HF) or cardiac death of clinical, radiographic, and echocardiographic vari-ables, as well as cardiac biomarkers N-terminal pro brain natriuretic peptide (NT-proBNP) and cardiac troponin I in dogs with preclinical myxomatous mitral valve dis-ease (MMVD).Animals: One hundred sixty-eight dogs with preclinical MMVD and left atrium toaortic root ratio �1.6 (LA:Ao) and normalized left ventricular end-diastolic dia-meter �1.7 were included.Methods: Prospective, randomized, multicenter, single-blinded, placebo-con-trolled study. Clinical, radiographic, echocardiographic variables and plasma cardi-ac biomarkers concentrations were compared at different time points. Usingreceiving operating curves analysis, best cutoff for selected variables was identifiedand the risk to develop the study endpoint at six-month intervals was calculated.Results: Left atrial to aortic root ratio >2.1 (hazard ratio [HR] 3.2, 95% confidenceinterval [95% CI] 1.9e5.6), normalized left ventricular end-diastolic diameter > 1.9(HR: 6.3; 95% CI: 3.3e11.8), early transmitral peak velocity (E peak) > 1 m/sec (HR:3.9; 95% CI: 2.3e6.7), and NT-proBNP > 1500 rmol/L (HR: 5.7; 95% CI: 3.3e9.5)were associated with increased risk of HF or cardiac death. The best fit model topredict the risk to reach the endpoint was represented by the plasma NT-proBNPconcentrations adjusted for LA:Ao and E peak.Conclusions: Logistic and survival models including echocardiographic variables andNT-proBNP can be used to identify dogs with preclinical MMVD at higher risk to de-velop HF or cardiac death.ª 2021 The Authors. Published by Elsevier B.V. This is an open access article underthe CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

List of abbreviations

95% CI confidence intervalDELAY DELay of Appearance of sYmptoms of

canine degenerative mitral valvedisease treated with spironolactoneand benazepril

E peak early transmitral peak velocityHF heart FailureHR hazard ratioLA:Ao left atrium to aortic root ratioLVEDDn normalized left ventricular end dia-

stolic diameterMMVD mxomatous mitral valve diseaseNT-proBNP N-terminal pro brain natriuretic

peptideROC receiver operating characteristic

Echocardiographic variables and biomarkers in dogs with preclinical myxomatous mitral valve disease79

Introduction

Dogs affected by mitral regurgitation caused bypreclinical myxomatous mitral valve disease(MMVD) represent a heterogeneous group ofpatients with different degree of cardiac remod-eling and variable prognosis [1]. Several studieshave reported the value of clinical, radiographic,and echocardiographic variables in identifyingMMVD dogs at higher risk to develop heart failure(HF) or cardiac death [1e5]. Cardiac biomarkers,such as N-terminal pro brain natriuretic peptide(NT-proBNP) and cardiac troponin I, have also beenassociated with disease severity and increased riskof developing HF in dogs with MMVD [6e12].However, there are only a few controlled studiesthat have prospectively investigated the prog-nostic values of clinical, radiographic, and echo-cardiographic variables and cardiac biomarkerconcentrations in identifying the risk for dogsaffected by preclinical MMVD to develop HF orcardiac death [5]. The ‘DELay of Appearance ofsYmptoms of canine degenerative mitral valvedisease treated with spironolactone and benaze-pril (DELAY) study’ was a prospective, multicenter,single-blinded, randomized, placebo-controlledstudy aimed to assess the efficacy of the com-bined treatment on delaying onset of HF or cardiacdeath in dogs with preclinical MMVD and car-diomegaly [13]. Dogs enrolled in the DELAY studyhad to be rechecked at established time pointsafter initial enrollment, and for this reason, thispopulation was ideal to assess the prognostic valueof clinical and diagnostic variables in identifyingdogs with higher risk to die of cardiac disease or to

progress into HF. The aim of this study was toinvestigate the prognostic value of clinical, radio-graphic, and echocardiographic variables, as wellas NT-proBNP and cardiac troponin I concen-trations to identify the risk to the development ofHF or experience cardiac death at 6, 12, 18, 24, 30,36, and 42 months after enrollment in this pop-ulation of dogs with preclinical MMVD andcardiomegaly.

Animals, material, and methods

Study design

The DELAY study was a prospective, multicenter,single-blinded, randomized, placebo-controlledstudy [13]. Complete and detailed description ofthe study design is reported in the published article[13]. Briefly, dogs with MMVD and left atrial toaortic root ratio (LA:Ao) � 1.6 and normalized leftventricular end diastolic diameter (LVEDDn) � 1.7were included. Dogs were excluded if they hadcardiovascular disease other than MMVD, significantconcurrent disease, systemic or pulmonary hyper-tension, atrial fibrillation, previous treatment withcardiovascular drugs lasting more than 2 weeks or inthe 2 weeks before inclusion. Dogs with hemody-namically insignificant tricuspid regurgitation wereeligible for enrollment in the study. Eight man-datory visits were scheduled every 6 months fromday 0 to 42 months. Additional visits were allowedfor individual medical concerns. At each visit, dogsunderwent a complete clinical evaluation andthoracic radiographs. In addition, a urine sample formeasurement of urinary aldosterone-creatinineratio and a blood sample were collected forassessment of creatinine, NT-proBNP, and cardiactroponin. An echocardiographic examination wasmandatory for visit 1, 3, 5, 7, and 8. However, theinvestigators had the option to perform an addi-tional examination at the other visits, and mostdogs included in the study had these additionalexaminations performed. The population of thisstudy included animals that were enrolled in boththe treatment and placebo group of the DELAYstudy, as there was no significant difference iden-tified on the primary endpoint between groups.

Clinical evaluation

At inclusion, dogs’ characteristics (breed, age,sex, body weight, and body condition score) wererecorded. Clinical history and physical findingswere documented at each visit.

80 M. Borgarelli et al.

Thoracic radiographs

Thoracic radiographs were performed for assessingcardiac size by the vertebral heart scale method[14]. They were also taken, whenever possible, forconfirming the presence of pulmonary edema at thetime dogs presented onset of clinical signs. Radio-graphic evidence of cardiogenic pulmonary edemawas defined by the presence of pulmonary venouscongestion and unstructured interstitial pattern oralveolar pattern that could not be explained byother medical conditions (e.g. pneumonia).

Echocardiography

Echocardiographic examinations were performedon unsedated dogs as per the established standardfor veterinary cardiology [15]. All measurementswere taken from at least three consecutive cardiaccycles, and the mean was recorded. The followingmeasurements were taken from the right para-sternal short-axis view: LA:Ao obtained in two-dimensional view as described by Hanson et al.2002 [16], and left ventricular diameter measuredin M-mode from the short axis with the leadingedge to inner edge method at the level of the tipsof papillary muscles. Left ventricular normalizeddimensions were calculated as described in thestudy by Cornell et al. [17]. Early (E peak) and lateA transmitral inflow velocities were assessed byspectral pulsed-wave Doppler from the left four-chamber apical view with the Doppler gate posi-tioned at the tip of the mitral valve leaflets.

ab PLAN procedure in SAS, SAS Institute, Cary NC, 2016.

Endpoint

The endpoint was the time to cardiac death or firstoccurrence of HF defined by the presence of eitherdyspnea and/or tachypnea (�36 breaths/min atrest) that could not be explained by another dis-ease based on clinical judgment by the inves-tigator [18]. Radiographs were performed at thelast visit if the dog was presented at the inves-tigator clinic and if its health condition allowed it.They were used as a basis to either confirm pul-monary edema or to exclude any other reasonassociated with the observed clinical signs. Toconfirm the first onset of HF, radiographs were firstevaluated by the investigator, and in case ofdetection of pulmonary edema, this had to beconfirmed by the lead investigator (M.B.) within 7days. In case of discrepancy between the twoevaluators (investigator and M.B.), a third opinionfrom a certified boarded radiologist, blinded todog clinical status, had to be requested.

In case where it was impossible to obtainradiographs because of critical health conditionsor refusal by the owner to perform this exami-nation, or if the dog was not directly seen bythe investigator because of a sudden clinicaldeterioration (death at home, visit to theirgeneral practitioner), diagnosis of HF wasdetermined by the investigator based onobserved clinical signs and/or informationreported by the owner or by other veterinarians.Details about validation of the primary endpointwere previously published [13].

Statistical methods

The statistical analyses were composed of fourmain components carried out in order, with onebuilding on the previous model. These are brieflydescribed as (1) ordinary Cox model, (2) extendedlongitudinal Cox model with interval censoring, (3)receiver operating characteristic (ROC) curveanalysis to define categorical variable cutoffsbased on continuous variables used in the longi-tudinal Cox model, and (4) the longitudinal Coxmodel retaining the full follow-up survival time inthe study for all time intervals, but right censoringevents at 6-month intervals, providing event-freesurvival analysis after a specified time period.Analyses were performed with statistical softwar-eab, where a p < 0.05 was deemed significant.

Conventional Cox modelThe conventional Cox model time-to-event analy-ses were carried out exclusively in univariable byway of KaplaneMeier product limit estimates andCox semiparametric regression models used togenerate unadjusted survival curves and hazardratios (HRs). In this approach, the date of studyentry was the time of the enrollment visit on asingle visit as previously published [13], and dataobtained at this time were evaluated in what isreferred to as baseline analyses. The extendedlongitudinal Cox model was built on the ordinaryCox model. While continuing to use the existingendpoints from the ordinary Cox model, theapproach was extended to control for individualchange of variables over time by the way ofinterval censoring. The surveillance time (survival)was the time between the entrance of the study tothe composite endpoint (HF or cardiac death) orcensoring.

ac Allignol A, Latouche A. CRAN task view: survival analysis,http://cran.r-project.org/web/views/Survival.html; 2012.).

Echocardiographic variables and biomarkers in dogs with preclinical myxomatous mitral valve disease81

Extended Cox modelBecause some patients were examined at timesbetween scheduled clinic visits, neither the con-ventional Cox model nor a series of logisticregressions could address varying intervals; hence,an extended Cox model with interval censoringwas used. This model allows for the most up-to-date variable value, while still using instantaneousrisk of the same composite endpoint associatedwith the Cox model approach. For example, incontrast to the fixed variable (e.g. the variable NT-proBNP), in the baseline analysis, the value of thevariable may change over the course of the studyat different visits which may not be measured atconsistent times (e.g. repeated measurements ofthe variable NT-proBNP). Specifically, although inmost cases ‘visit 2’ may be at 12 months exactlyfor most patients, there could be some deviationor missed visits. The extended Cox model (intervalcensoring) was used to accommodate possibledeviation and congruency of visit number to time.To address this nuance of time-varying covariates,two models were constructed, one evaluatinghazards of baseline values (baseline HR) based on asingle visit (previously described) and the currentlydescribed longitudinal approach assessing the HRwith change in time and measured values ofrespective variables (longitudinal HR) for bothunivariable and multivariable in terms of selec-tion, as well as goodness of fit, as described byEriksson [19]. The multiple visits with refreshed orupdated data are hereafter referred to as thelongitudinal analyses, where the covariates are thesame throughout the longitudinal analysis, but theHR takes into account the changes in seriallymeasured covariates and ultimately analyzes themost up-to-date value. In summary, the baselineanalysis only considers the initial baseline visit forsurvival time, whereas the longitudinal considersmultiple visits and associates the survival timewith the respective covariate value. Notably, thebaseline analysis is only measured and presentedin univariable, whereas the longitudinal analysis ispresented as both univariable and multivariable.Covariates found significant in longitudinal uni-variable were used in conjunction with treatment(spironolactone and benazepril) and other candi-date variables for multivariable models. The mul-tivariable models were analyzed using main effectand interaction terms preceded by an iterativeenter method of selection to avoid over-specification of the model which may be a risk incertain stepwise approaches when observationsand events are limited. Models being evaluatedwere subsequently compared with each other byChi-squared goodness of fit 1 degree of freedom

based on the Akaike information criterion when avariable was added or removed from the modelreturning the probability of difference betweenthe models as determined from the central Chi-squared distribution. The Chi-square probabilityfunction P(x, df ) suggests that an observationfrom a Chi-square distribution, with degrees offreedom df, is less than or equal to x. In our case, xis defined as a numeric constant, variable, orexpression that specifies the value of a randomvariable; df is a numeric constant, which is definedas the Akaike information criterion differencebetween the model variable while df defines thedegrees of freedom parameter which in this case is1 as there are only two models (n-1) beingcompared.

Significant covariates from the multivariableCox model were used to generate adjusted survivalcurves presented in concert with unadjusted sur-vival curves for presentation. Specifically, regard-ing adjusted survival curves, the longitudinalpopulation mean value of the significant covariatewas imputed (held constant) over both strata ofthe respective survival curve (e.g. stratified NT-proBNP of �1500 rmol/L vs. <1500 rmol/L incor-porated population mean LA:Ao and E peakvelocity, both statistically significant in multi-variable, to the Cox model generating the adjustedsurvival probability).

Multicollinearity was assessed by varianceinflation factor analysis and deemed acceptable.Covariates for the multivariable model were pre-sented as both continuous and categorical. Testsfor proportionality were carried out by visualinspection of Schoenfeld residuals, negative logestimated survival distribution function, and for-mal hypothesis testing of covariate by log (time)interactions followed by Wald Chi-squared sta-tistics and deemed proportional. Statistical dif-ferences between groups for time to event wereanalyzed by survival time analyses. Time to eventrepresented the time from the first day of enroll-ment in the study to the end date. Cases lost tofollow-up or remaining alive were censored. Theeffect of time-to-event and change in HR with agiven variable was modeled using the splineapproach as previously described.ac

Receiver operating characteristicContinuous demographic and echocardiographicvariables were categorized into two discrete(dichotomized) values by the way of a generalized

82 M. Borgarelli et al.

linear logistic model using ROC curve analysis andby inspection of HR change in the value of a givenvariable exhibited by the spline approach. TheROC curve analysis was based on the interval-censored data set used by the extended Cox modelwhere the last known value before congestive HFor right censoring was used as the value of thegiven variable analyzed for the ROC curve calcu-lation. The optimal cut point was assigned by aYouden statistic as previously described [19].Specifically, a variable such as NT-proBNP, whichmay have demonstrated a range of 200e1700rmol/L, may have been found to have an optimalcut point associated with congestive HF calculatedto be 1500 rmol/L. The same ROC approach wasapplied to remaining echocardiographic andradiographic variables. Dichotomized covariatesdefined by ROC analysis were subject to furtheranalysis in univariable and multivariable in con-junction with other categorized and continuousvariables.

The longitudinal Cox model with right censoringThe longitudinal analysis with updated covariatevalues was explored further by retaining eachpatient’s time in the study in place but investigat-ing a modified calculation of survivorship at 6-month intervals. This univariable analysis was rightcensored for both time and composite endpointevents at months 6, 12, 18, 24, 30, 36, and 42.Briefly, the full population is analyzed, but frozenin ‘snapshots’ of 6-month intervals building on eachother. Specifically, a given variable was analyzed(e.g. LA:Ao) with survival time followed for theduration of the study. Seven right censored evalu-ation periods were constructed (months 6, 12, 18,24, 30, 36, and 42), whereas subsequent Cox mod-els independent of each other were carried outbased on each window, for each variables eval-uated, by right censoring events at 6-month inter-vals to determine the effect of full follow-upcoupled with serially censored events. For exam-ple, in the 6-month censored analysis, if a patientwere to have survival time and corresponding HFevent date of 6 months and 1 day, this event wouldbe censored for the 6-month analysis because thatevent occurs after 6 months and the survival timewould have a maximum time of 6 months. In this 6-month example, while all patients’ comprehensivetime in the study is considered, only events occur-ring in this 6-month window are considered. Incontrast, using the 12-month window analysis, thissame patient’s full follow-up and event of 6 monthsand 1 day would be recorded for the 12-monthwindow. Extending further with the example forthe purpose of completeness, this patient would

not be included for the 18, 30, 36, 42, and fullfollow-up because the patient would be censoredwith event at 6 months and 1 day. In summary,seven different censored windows (months 6, 12,18, 24, 30, 36, and 42) were constructed with therespective set limit of survival time but censoringpotential events if the event occurred after thatgiven period. Pointedly, the 42-month analysiswould be nearly the same as the full follow-up ofthe study because the study was only three years(48 months) long. This approach is hereafterreferred to as the longitudinal right censoredanalysis. The ROC curve dichotomized variableswhich remained statistically significant in the lon-gitudinal multivariable models were further ana-lyzed for survivorship at the specified 6-monthintervals in this longitudinal right censored analysis.

Results

One hundred and sixty-eight dogs constitute theper protocol population and were used for analysisin this study. The median age of patients enrolledper investigator was 7.5 years (range 5e13 years).Eighteen different breeds were included, withmixed-breeds being the most represented (n ¼ 72;40%), followed by Cavalier King Charles spaniels(n ¼ 34; 19%), poodles (n ¼ 12; 7%), dachshunds(n ¼ 12; 7%), Chihuahuas (n ¼ 7; 4%), Jack Russellterriers (n ¼ 5; 3%), and bichon frise (n ¼ 5; 3%).No differences were observed between the twogroups at inclusion.

The median time in the study was 561 days (95%confidence interval [95% CI]: 142e977 days), and75 dogs (44.9%) reached the composite endpoint.Forty-seven dogs developed HF, and 28 died of acardiac cause. The median survival time based onthe KaplaneMeier product limit estimate consid-ering censored events, in the study of dogsreaching the composite endpoint, was 1045 days(95% CI: 766 daysenot available). Ninety-threedogs were censored from the analysis of end-point, including 36 dogs that were still asympto-matic at the end of the study.

In the univariable Cox proportional hazard analysisthat included the baseline (single visit) values ofcovariates, four variables were associated with anincreased hazard to reach the composite endpoint atthe baseline (Fig. 1). Covariates found significantwere used in conjunction with treatment (spi-ronolactone and benazepril) for multivariable mod-els. However, treatment remained non-significant. Inthe univariable longitudinal Cox proportional analy-sis, seven variables, using the most up-to-datecovariate value associatedwith a given time interval,

Fig. 1 Hazard ratios and 95% confidence intervals obtained from univariate Cox proportional hazard analysis at thebaseline. BPM: beats per minute; cTnI: cardiac troponin I; E peak: early transmitral peak velocity; HR: heart rate;LA:Ao: left atrium to aortic root ratio; LVEDDn: normalized left ventricle end-diastolic diameter; LVESDn: normalizedleft ventricular end-systolic diameter; NT-proBNP: N-terminal pro brain natriuretic peptide; SB treatment: spi-ronolactone and benazepril treatment; U A: urine aldosterone; U A:C: urinary aldosterone-creatinine ratio; VHS:vertebral heart score.

Echocardiographic variables and biomarkers in dogs with preclinical myxomatous mitral valve disease83

were associated with an increased risk for reachingone of the composite endpoints (Table 1, Fig. 2). TheROC curve analysis demonstrated optimal Youdenstatistic cut points of LA:Ao > 2.1, LVEDDn > 1.9, Epeak > 1 m/sec, and NT-proBNP > 1.500 rmol/L.Based on the longitudinal data univariable analysis,the risk of reaching the composite endpoint basedsolely on NT-proBNP � 1500 rmol/L (parameterestimate: 1.717, HR: 5.57, 95% CI: 3.3e9.5,p < 0.001) was greater than the risk of reaching thecomposite endpoint for NT-proBNP � 1500 rmol/L(parameter estimate: 0.999, HR: 2.71, 95% CI:1.45e5.1, p ¼ 0.0018) when in the presence of con-tinuous variables LA:Ao*10 (parameter estimate:

Table 1 Probability to reach the primary endpointin Cox longitudinal proportional univariate analysis.

Hazard ratio 95% CI p value

HR > 130 bpm 2.289 1.43e3.66 <0.001VHS > 11.3 2.566 1.57e4.19 <0.001LA:Ao > 2.1 3.253 1.88e5.64 <0.001LVEDDn > 1.9 6.272 3.34e11.76 <0.001E peak > 1 m/sec 3.909 2.27e6.73 <0.001NT-proBNP >1.500 (rmol/L)

5.567 3.26e9.51 <0.001

cTnI > 0.35 (ng/mL) 3.371 1.92e5.91 <0.001

bpm: beats per minute; cTnI: cardiac troponin I; E peak:early transmitral peak velocity; HR: heart rate; LA:Ao: leftatrium to aortic root ratio; LVEDDn: normalized left ven-tricle end-diastolic diameter; NT-proBNP: N-terminal probrain natriuretic peptide; VHS: vertebral heart score.

0.105, HR: 1.1, 95% CI: 1.01e1.2, p ¼ 0.02) and Epeak*10 (parameter estimate: 0.168, HR: 1.18, 95%CI: 1.06e1.3, p ¼ 0.0026). On the aforementionedparameter estimates, the following equation wasused to generate the adjusted survival curves: NT-proBNP � 1500 rmol/L (parameter estimate:0.999)þ LA:Ao*10 * (parameter estimate: 0.105)þ Epeak*10 (parameter estimate: 0.168). Specifically,the adjusted survival curves were generated basedon the significant variables in multivariable in thepresence of NT-proBNP � 1500 rmol/L stratifying onNT-proBNP � 1500 rmol/L coupled with imputing orholding constant the mean longitudinal populationvalue for LA:Ao and E peak. For purposes of mean-ingful clinical parameterization, both LA:Ao and Epeak were multiplied by ten to present a 0.1-unitincrease. Specifically, the actual longitudinal pop-ulation means of LA:Ao and E peak were 1.91 and1.01, respectively, but they were parameterized onvalues of 19.1 and 10.1, to allow for a meaningfulclinical 1-unit increase for the regression whenapplying the model in a clinical setting.

The adjusted model suggests that the risk ofreaching the endpoint for dogs with [NT-proBNP]rmol/L > 1500 rmol/L is 2.71 times higher thanthe risk of dogs with the lower NT-proBNP. In thepresence of the dichotomized NT-proBNP rmol/L > 1500 rmol/L variable, the risk of achievingthe combined endpoint increases by 11% per 0.1-unit increase of LA:Ao and 18.4% per 0.1-unitincrease of E peak. The mentioned unadjustedand adjusted survival curves based on this

Fig. 2 Hazard ratios and 95% confidence intervals obtained from longitudinal univariate Cox proportional hazardanalysis. BPM: beats per minute; cTnI: cardiac troponin I; E peak: early transmitral peak velocity; HR: heart rate;LA:Ao: left atrium to aortic root ratio; LVEDDn: normalized left ventricle end-diastolic diameter; LVESDn: normalizedleft ventricular end-systolic diameter; NT-proBNP: N-terminal pro brain natriuretic peptide; SB treatment: spi-ronolactone and benazepril treatment; U A: urine aldosterone; U A:C: urinary aldosterone-creatinine ratio; VHS:vertebral heart score.

84 M. Borgarelli et al.

longitudinal analysis are presented in Fig. 3. Inobserving the unadjusted survival in Fig. 3,median survival of NT-proBNP � 1500 rmol/L was16 months, whereas NT-proBNP < 1.500 rmol/L

Fig. 3 Unadjusted and adjusted KaplaneMeier survival cplasma concentration (NT-proBNP). The survivor functionunadjusted) by definition does not include the effect of LA/Athese values (NT-proBNP � 1500 rmol/L adjusted), a truer s

was not able to be estimated. However, the 25thpercentile can be found in Fig. 3 to be 10 and 36months. In observing the effect of the populationmean value being imputed for LA:Ao and E peak

urves as per N-terminal pro brain natriuretic peptideof NT-proBNP in univariate (NT-proBNP � 1500 rmol/Lo and peak transmitral E wave velocity. By adjusting forurvival fit is achieved.

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NT-

Echocardiographic variables and biomarkers in dogs with preclinical myxomatous mitral valve disease85

for the adjusted model survival curves, themedian survival for NT-proBNP � 1500 rmol/Lwas 29 months, whereas the NT-proBNP < 1500rmol/L was still non-estimable as indicated tothe very similar survivorship to the unadjustedNT-proBNP < 1.500 rmol/L, suggesting thatimputing the population mean has a minimaleffect on this group. In contrast, imputation ofthe mean value of LA:Ao and E peak improvessurvivorship with the NT-proBNP � 1.500 rmol/Lpopulation, suggesting contribution of the stat-istically significant covariates LA:Ao and E peakto predicting survivorship, as opposed to the NT-proBNP � 1.500 rmol/L being the sole predictor.In a sensitivity analysis with the minimumLA:Ao ¼ 1.14 and E Peak ¼ 0.48 m/sec imputedas opposed to the mean values presented, nei-ther the median nor the 25th percentile survi-vorships could be estimated. In contrast, in asimilar sensitivity analysis using the populationmaximum values of LA:Ao ¼ 3.18 and EPeak ¼ 1.81 m/sec imputed, as opposed to thepresented mean imputation, the median survivalof the NT-proBNP � 1.500 rmol/L population was3 months while the NT-proBNP < 1.500 rmol/Lpopulation was 6 months (minimum and max-imum imputed survivorships not shown on Fig. 3).Although the longitudinal unadjusted survival forNT-proBNP � 1.500 rmol/L presented a larger HRbetween groups, the multivariable longitudinalanalysis stratifying on NT-proBNP � 1.500 rmol/Lin conjunction with LA:Ao and E peak as con-tinuous variables provided the best model fit.

The risk for reaching the composite endpointfor dogs with LA:Ao > 2.1, LVEDDn > 1.9, E peak> 1 m/sec, and NT-proBNP > 1.500 rmol/Lbased on the longitudinal analysis right censoredat 6, 12, 18, 24, 30, 36, and 42 months isreported in Table 2.

Table

2Riskto

reach

theco

mbinedendpointf

through

42months.

Visit

time(m

onths)

NT-proBNP

>1.50

0rmol/L

HR(95%

CI)

p

63.04

(0.96,

9.59

)12

4.28

(1.94,

9.47

)<

185.71

(2.95,

11.03)

<24

5.65

(3.08,

10.38)

<30

5.59

(3.17,

9.85

)<

365.46

(3.14,

9.47

)<

425.53

(3.23,

9.44

)<

CI:co

nfidence

interval;Epeak:

earlytransm

itralpeak

proBNP:N-term

inalpro

brain

natriureticpeptide.

Discussion

This study demonstrates that NT-proBNP andsome selected echocardiographic variables canpredict the risk for the first onset of HF or cardiacdeath in dogs with preclinical MMVD and cardiacremodeling. Previous studies have assessed andreported the value of NT-proBNP to assess the riskfor progression or death in patients with bothpreclinical and clinical MMVD. For example,Moonarmart et al. (2010) [6] reported that a 100rmol/L increase of NT-proBNP was associatedwith an increased risk of all-cause mortality in apopulation of dogs with both preclinical andclinical MMVD. In another study, Wolf et al. (2012)

86 M. Borgarelli et al.

[7] reported that NT-proBNP plasmaconcentrations < 965 rmol/L 7e30 days after ini-tiating treatment of HF were associated with ahigher survival time in a population of dogs withclinical MMVD. Furthermore, Mattin et al. (2019)[20] reported that plasma NT-proBNP > 1.800rmol/L was strongly associated with the higher riskfor clinical progression in dogs affected by pre-clinical MMVD. In this study, NT-proBNP plasmaconcentrations > 1.500 rmol/L represented thebest predictor for reaching the predefined com-posite endpoint. Reynolds et al. (2012) [11]reported that the same cutoff for NT-proBNP wasan independent predictor of onset of HF in apopulation of preclinical MMVD with anLA:Ao > 1.6. In the same study, they also reportedthat, similar to our study, the best predictivemodel included the NT-proBNP concentration andthe left atrial dimension.

In this study, in addition to NT-proBNP concen-tration, left atrial and ventricular dimensions andE peak of transmitral flow were identified as usefulindependent predictors for reaching the endpoint.The prognostic value of echocardiographic varia-bles in preclinical MMVD has been previouslyreported in several studies. Left atrial enlarge-ment assessed by different methods, or LA:Aoincrement by 0.1 unit, represented the mostcommon identified echocardiographic predictorfor the progression of the preclinical disease[1,4,13,21]. An LVEDDn > 1.7 and the rate ofchange of left ventricular end-diastolic dimensionswere reported to be associated with an increasedhazard for dogs with preclinical MMVD [3,4,13,21].In our study, the optimal cutoff values to identifydogs with an increased risk for progression of thedisease were 2.1 for LA:Ao and 1.9 for LVEDDn.These cutoff values are higher than those pre-viously reported [1,3,4,6]. Left atrial to left aorticratio values > 1.6 are generally accepted foridentifying dogs with left atrial enlargement [22].However, a recent study has reported that normaldogs may present an LA:Ao up to 1.9 [23]. Nor-malized end-diastolic left ventricular diameter>1.7 has been found to be a predictor of outcomes[6], and this cutoff is used to identify dogs withleft ventricular enlargement [17,24]. However, itshould be noted that LVEDDn ¼ 1.73 represents theupper 95% percentile for normal dogs in the studyfrom Cornell et al. (2004) [17], whereas the upper97.5% for normal dogs is 1.85. Therefore, thecutoff values reported in this present study iden-tify dogs with significant cardiac enlargement andtherefore that may have a higher risk of develop-ing HF or die for their cardiac disease.

In the present study, transmitral inflow E peakvelocity higher than 1 m/sec was an independentpredictor for progression to a primary endpoint.This cutoff value was higher than normal valuesobserved in normal dogs of similar age [25]. Apossible explanation for this finding is the associa-tion between increased E peak velocity andincreased left atrial pressure [26]. However, inpatients with volume overload and normal systolicfunction, such as dogs with preclinical MMVD,increased E peak velocity may also reflect leftventricular volume overload and not necessarily asole increased atrial pressure [27]. Regardless ofthe cause of its increased value, E peak velocity canstill be considered as an indirect indicator of theseverity of the disease, and it has been reportedbeing independently associated with an increasedhazard in both preclinical and clinical dogs [1,3,28].

In this study, the best predictor model for iden-tifying dogs with increased risk of reaching thecomposite endpoint was a composite of NT-proBNPconcentrations, LA:Ao ratio, and E peak velocity.Normalized left ventricular end-diastolic diameterwas not included in this statistical model becauseof its strong correlation to the left atrial enlarge-ment. However, it is of interest to notice thatLVEDDn was a variable associated with an increasedhazard at 6 months after inclusion in the study.

All echocardiographic variables that were foundto be associated with clinical outcomes and theNT-proBNP showed an increased hazard over time.This result is not unexpected considering the longduration of the preclinical phase of MMVD in dogs[1,13,21]. This finding suggests that monitoring theprogression of the disease by echocardiographicvariables and NT-proBNP may represent an effec-tive way to make appropriate therapeutic deci-sions and to assess the risk for a preclinical dog todevelop HF or to die for the underlying cardiacdisease.

Limitations

The major limitation for this study is representedby the fact that the onset of HF was not con-firmed in all dogs by documenting the presence ofcardiogenic pulmonary edema on thoracic radio-graphs. However, although thoracic radiographyis often considered the gold standard for diag-nosing left-sided congestive HF, interpretation ismostly subjective. Indeed, it is now generallyaccepted that combination of history and clinicalassessment in association with response totreatment represents an acceptable way to

Echocardiographic variables and biomarkers in dogs with preclinical myxomatous mitral valve disease87

identify onset of HF in dogs with preclinical MMVD[20,22]. Another limitation of this study is rep-resented by the presence of a treatment and aplacebo group, which were combined and ana-lyzed as a single population. Because the treat-ment in this study had no effect on primaryendpoints, it is unlikely that combining the twogroups into a single population affected theresults of this study.

Conclusions

This study reports reliable echocardiographic andplasma NT-proBNP cutoff values that can be usedto identify dogs with preclinical MMVD that are athigher risk of developing HF or experiencing car-diac death. Furthermore, the results of this studymay also be useful for designing future studies toevaluate the effect of various interventions aimedat delaying the progression of MMVD.

Conflicts of Interest Statement

This project was funded by Ceva Sante Animale,and two authors, E.G. and C.G-P., are repre-sentatives of Ceva Sante Animale.

Assessment of the N-terminal pro-brain natriu-retic peptide was sponsored by IDEXX BioResearch,Vet Med Labor GmbH, Ludwigsburg, Germany.

All the other authors have received fundingfrom Ceva Sante Animale within the last 5 years forsome or all of the following activities: research,travel, speaking fees, consultancy fees, andpreparation of educational materials.

Acknowledgments

The authors thank Jens Haggstrom and Ingrid Ljung-vall for helping with the handling and interpretationof cardiac troponin I test and Cristina Vercelli andGiovanni Re for performing and helping with theinterpretation and validation of urinary aldosteroneand urinary aldosterone creatinine ratio.

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