NT-proBNP as a tool to stratify disease severity in pulmonary arterial hypertension

Respiratory Medicine (2007) 101, 69 75 NT-proBNP as a tool to stratify disease severity in pulmonary arterial hypertension Rogerio Souza a,b,c,, Carlos Jardim a, Caio Julio Cesar Fernandes a, Monica Silveira Lapa a, Rogerio Rabelo b, Marc Humbert c a Pulmonary Division, Pulmonary Hypertension Unit, Heart Institute, University of São Paulo Medical School, Sao Paulo, Brazil b Fleury Research Institute, Sao Paulo, Brazil c Centre des Maladies Vasculaires Pulmonaires, UPRES EA 2705, Hôpital Antoine Béclère, Université Paris-Sud, Clamart, France Received 23 November 2005; accepted 16 April 2006 KEYWORDS Pulmonary hypertension; Hemodynamics; NT-proBNP; Idiopathic pulmonary arterial hypertension; six-minute walk test; Functional class Summary The recent development of treatment modalities for patients with idiopathic pulmonary arterial hypertension has been based on the evaluation of many different markers such as functional capacity, addressed by NYHA classification, six-minute walk test (6 MWT) and hemodynamic parameters. The aim of this study was to evaluate the correlation of N-terminal fragment (NT-proBNP) with other markers in IPAH and its potential for patient stratification. We studied 42 IPAH patients consecutively evaluated through right heart catheterization in the absence of any specific treatment for pulmonary hypertension. Blood samples, clinical evaluation and 6 MWF distance were collected at baseline. The levels of NT-proBNP showed a high correlation with hemodynamic parameters, such as pulmonary vascular resistance (r ¼ 0:80, Po0.001). A significant difference was found among patients with different functional classes, addressed by NYHA classification (Po 0.02 for all groups comparison). The discriminant analysis reinforced the ability of NT-proBNP to stratify patients according to NYHA functional class. Compared to the other variables studied (hemodynamics and 6 MWT), NTproBNP had the lowest level of overlap in the stratification of IPAH patients. We conclude that NT-proBNP differs among the different functional classes and correlates with other measures of disease severity, although its role in predicting survival still needs to be addressed. & 2006 Elsevier Ltd. All rights reserved. Corresponding author. R. Afonso de Freitas 451 ap 112, São Paulo-SP Brazil. Tel/Fax: +55 11 3069 7202. E-mail address: rgrsz@uol.com.br (R. Souza). 0954-6111/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2006.04.014

70 Introduction Idiopathic pulmonary arterial hypertension (IPAH) is a disorder of unknown etiology characterized by the obstruction of small pulmonary arteries leading to progressive right ventricular failure. 1 Although there was a significant development in therapies in the last decade, 2 the survival rate remains unsatisfactory. 3,4 Among all clinical trials of medical treatment for IPAH, published in the recent years, the six minute walk test (6 MWT) was the most-used end point. 5 The 6 MWT is a submaximal test, highly reproducible, that has been shown to correlate with survival and treatment response not only in IPAH 3,6 but also in other forms of pulmonary hypertension as those related to scleroderma 7 and Eisenmenger. 8 The 6 MWT has been mostly studied in patients with NYHA functional class III and IV; however, its role in less-advanced disease (class I and II) has not been addressed, resulting in the question of whether it would be sensitive enough in this subset of patients. Hemodynamic measurements through right heart catheterization have been shown to reflect prognosis in pulmonary hypertension, allowing the development of a formula based on the NIH registry 9,10 to predict survival. Nevertheless, it requires an invasive procedure and it is not clear if hemodynamics at rest clearly reflect the extent of pulmonary vasculature involvement in IPAH, although a baseline measurement is needed for an accurate diagnosis of the disease. 11 More recently, natriuretic peptides have been used to address heart diseases, mainly left ventricular dysfunction and myocardial infarction. 12,13 Pro-BNP is a pro-hormone produced mainly by the ventricles, which is cleaved into an inactive N- terminal fragment (NT-proBNP) and into its active form BNP (brain natriuretic peptide); eventually both are secreted into circulation by the cardiomyocytes. 14 In pulmonary hypertension, BNP has been shown to correlate to functional status 15 and treatment response, 16 and to be an independent predictor of survival. 17,18 NT-proBNP has a longer half-life in blood and has been shown to be a marker of pulmonary hypertension in scleroderma patients. 19 In IPAH, NT-proBNP presented a good correlation with hemodynamics 20 and also reflected treatment response. 21 However, it has not been demonstrated if natriuretic peptides would be able to accurately stratify patients with IPAH from all different functional statuses. Stratification of patients is based mostly on NYHA functional class. 22 It is certainly easy to use but relies on the patient and/or physician perception of the functional limitations. This could impair its ability to reflect changes mainly after specific interventions. Thus, a non-invasive marker that could reflect the functional capacity and could also be correlated to the hemodynamic status would be a useful tool for the correct stratification and follow-up of IPAH patients. The aim of this study was to evaluate the correlation of NT-proBNP with different response markers in IPAH and its potential for patient stratification according to disease severity as an adjuvant tool to NYHA functional class. Methods Patient group From August 2003 to May 2005, 42 patients with the final diagnosis of IPAH were consecutively enrolled in this study. Patients were on conventional therapy (anticoagulation, diuretics and cardiac glycosides) but without any other specific therapy to pulmonary hypertension. The diagnosis of IPAH was confirmed based on the International Symposium on pulmonary hypertension criteria. 11 Thromboembolic disease was ruled out by ventilation-perfusion scan or spiral CT scan. Lung disease was excluded by pulmonary function tests and CT scans. Connective tissue diseases were excluded by physical examination and serological markers. Patients that presented a mean pulmonary artery pressure lower than 25 mmhg or a pulmonary artery occlusion pressure higher than 15 mmhg at the right heart catheterization were excluded from the study. The study was approved by our institutional ethics committee. Hemodynamics and exercise capacity measurements R. Souza et al. A clinical evaluation, including NYHA functional class and 6 MWT, was performed at baseline. The 6 MWT was performed on the same day of the clinical evaluation, according to the recommendations of the American Thoracic Society 23 ; the distance walked was recorded, regardless of any interruption. Hemodynamic evaluation was performed in all patients while breathing room air, at supine position, within 1 week of the clinical evaluation without any change in treatment or in clinical status. All patients presented arterial oxygen saturation greater than 90% at the beginning of the measurements. A 7F flow-directed pulmonary

NT-proBNP for severity stratification in PH 71 artery catheter (Baxter Healthcare Corporation, Irvine, CA, USA) was used in all patients. Cardiac output (CO) was measured by the standard thermodilution technique. Cardiac index (CI) was calculated as CO divided by body surface area (m 2 ). Indexed pulmonary vascular resistance was calculated by the following formula: 80 (mean pulmonary artery pressure pulmonary artery occlusion pressure)/ci. NT-proBNP measurements Blood samples were collected at the beginning of right heart catheterization. In 12 patients, the blood tests were taken more than 7 days after hemodynamic measurements; therefore, their values were not used in the correlation analysis. The samples were centrifuged within 60 min of collection. The resulting serum samples were stored at 70 1C. The NT-proBNP measurements were performed on an Elecsys 2010 instrument by a sandwich immunoassay (Roche Diagnostics, Basel, Switzerland). Statistical analysis All the results are expressed as mean (SEM). We used a Pearson correlation coefficient to evaluate the correlation between NT-proBNP and the other continuous variables. We have also used analysis of variance (ANOVA) for the evaluation of all continuous variables and NYHA functional class; a Bonferroni post hoc test was applied for multiple comparisons when statistical significance was obtained. A multiple regression model was used to estimate the impact of hemodynamic data and NTproBNP in the NYHA functional class determination. A discriminant function analysis was performed to determine the ability of NT-proBNP to classify patients according to NYHA functional class. Results Baseline hemodynamics and clinical characteristics of the 42 patients enrolled in the study are listed in Table 1. These data are in accordance with previously published data from IPAH patients. Linear regression analysis disclosed a significant association between NT-proBNP and right atrial pressure (r ¼ 0:68, P ¼ 0:004), mean pulmonary artery pressure (r ¼ 0:58, Po0.001), cardiac index (r ¼ 0:70, Po0.001) and indexed pulmonary vascular resistance (r ¼ 0:80, Po0.001) (Fig. 1). Table 1 Baseline clinical and hemodynamic data. Age (yr) 37 (2) Gender (male/female) 10/32 Functional Class I 4 II 15 III 16 IV 7 Six-min walk test distance(m) 427 (21) NT-proBNP (pg/ml) 1074 (201) Right atrial pressure (mmhg) 12 (2) Mean pulmonary artery pressure 67 (5) (mmhg) Pulmonary artery occlusion 9 (2) pressure (mmhg) Cardiac index (L/min/m 2 ) 2.3 (0.2) Indexed pulmonary vascular 2029 (202) resistance (dyn cm 5 sm 2 ) Results are presented as mean (SEM). There was a trend in the association between NTproBNP and the 6 MWT distance (r ¼ 0:31, P ¼ 0:052). Functional class presented significant correlation with 6 MWT (r ¼ 0:49; P ¼ 0:001),with NT-proBNP (r ¼ 0:81; Po0.001) and with hemodynamic data, considering cardiac index (r ¼ 0:70; Po0.001), indexed pulmonary vascular resistance (r ¼ 0:72; Po0.001) and mean pulmonary artery pressure (r ¼ 0:52; P ¼ 0:002). We used analysis of variance to assess the differences between each one of the measured parameters at the different functional classes. The NT-proBNP levels were significantly different at each one of the functional classes (Po0.02 for all group comparison) (Fig. 2). The discriminant analysis evidenced a high significant model (Table 2) that allowed patient stratification into the different NYHA functional classes according to NTproBNP. Cardiac index, although significantly different at global analysis (Po0.001), showed no significant difference among functional classes II, III and IV (P40.26); the same pattern was noted with 6 MWT distance and indexed pulmonary vascular resistance, and the global comparison resulted in a significant difference among groups (P ¼ 0:005 and 0.001, respectively), but the post hoc analysis showed only differences between more preserved (functional classes I and II) and more advanced disease (functional classes III and IV) (Po0.04). The analyses of NT-proBNP, 6 MWT distance and cardiac index in a multiple regression model with NYHA as the dependent variable resulted in a very significant model (r ¼ 0:89; Po0.001) where NTproBNP was the only significant factor (Po0.001).

72 R. Souza et al. Figure 1 Linear regression between NT-proBNP and indexed pulmonary vascular resistance (r ¼ 0:80, Po0.001). Figure 2 Association of different markers and NYHA functional class. (A) CI cardiac index; (B) PVRi indexed pulmonary vascular resistance; (C) 6 MWT 6-min walk test; (D) Ln NT-proBNP. Discussion Our results showed that NT-proBNP not only has a strong correlation with hemodynamic parameters but also differs among the NYHA functional class allowing patients stratification according to disease severity. The small number of patients enrolled should be seen as a limitation of our study, even considering

NT-proBNP for severity stratification in PH 73 Table 2 Discriminant model for stratification in NYHA functional class according to NT-proBNP. NYHA functional class I II III IV Ln NT probnp (a) 2.624 4.438 5.774 7.084 (Constant)(b) 5.272 12.502 20.202 29.714 To classify a patient into the NYHA functional class based on the NT-proBNP level, the obtained level should be log transformed and then multiplied by (a) and added to (b) for each column. The column that determines the highest value will then evidence to which NYHA functional class that level of NT-proBNP is related. that the clinical and hemodynamic data from our population resembled previously described data from IPAH patients. 9 Seven of our patients were included in our previous study about NT-proBNP 20 ; nevertheless, the broader spectrum of functional capacity in this study strengthens our previous findings on the correlations of NT-proBNP and hemodynamic variables in PAH. Hemodynamic measurements are well recognized as prognostic markers since they somehow reflect the progressive obstruction to blood flow that is characteristic of pulmonary arterial hypertension. 5 However, it is not clear if baseline hemodynamics reflect all the heterogeneity of the pulmonary circulation in the presence of the disease. 24,25 Right heart catheterization is still mandatory for diagnostic confirmation, for the acute vasodilator challenge and treatment followup, 26 in spite of its limitation due to invasiveness. Natriuretic peptides have been used as markers of heart disease based on the observation that they are involved in the activation of the cyclic guanylate cyclase system as a counterregulatory mechanism in heart failure, probably with increased cardiac wall stress as the trigger mechanism. 27,28 Natriuretic peptides have also been correlated with hemodynamic parameters and prognosis in patients with heart failure. 29,30 In pulmonary hypertension, NT-proBNP has been correlated to the hemodynamics and to the acute response to nitric oxide. 20 Similar results have been described in systemic sclerosis-related pulmonary hypertension. 31 The same behavior can be found in this study, in a larger population of patients. Furthermore, NT-proBNP demonstrated an ability to stratify patients according to the functional class superior to any other marker studied, even considering patients with functional class I or IV. Previous studies with BNP have addressed disease stratification in patients with pulmonary hypertension, but the limited spectrum of functional classes of these studies did not allow a proper analysis of the ability of BNP to reflect disease severity in such patients, 32 despite its role as a prognostic marker. 18 One of the limitations of the use of the NYHA functional classification is the subjectivity that may be involved in the patients report of their symptoms and physicians interpretation. Even though, the NYHA functional classification has been shown to correlate with prognosis in pulmonary hypertension. 3 In our study, patients functional class was addressed by the same trained physician, blinded to all other variables of study, including the results of NT-proBNP throughout the inclusion period; hence the variability might be lower than that usually reported. This approach was chosen once the main objective was to show that NTproBNP could be used as an adjuvant and not necessarily a substitute tool to functional class. The NT-proBNP was independently associated with functional class in our study; furthermore, the discriminant analysis evidenced a high significant model where levels of NT-proBNP allowed the stratification of patients at least as the NYHA functional class, although this model should be confirmed in another study population in order to be used. This result, together with the high stability of NT-proBNP in the serum and the fact that this test can be performed under routine laboratory conditions, 14,31 indicates that NTproBNP may be a useful test in the clinical setting for patient stratification, potentially better than the 6 MWT, considering patients with less severe disease. The 6 MWT is a highly reproducible test and has been used as the primary end point in most of the clinical trials in pulmonary hypertension. 33 36 Nevertheless, the majority of the patients included in those trials were of functional classes III and IV; there are no data validating the use of 6 MWT in patients with less-advanced disease. In our study, although 6 MWT would perfectly differentiate patients with classes III and IV from patients with functional classes I and II, we found no significant difference within these two subsets, even with a

74 significant correlation with hemodynamics and functional classes when considering the whole population. The lack of survival data could be considered a limitation of the study once the proposition of cutoff values that could determine not only disease severity but also long-term prognosis would be of major impact in the utilization of NT-proBNP in daily practice; however, this would demand for a larger and homogeneous cohort, considering the many therapeutical options available nowadays. In spite of this limitation, our results showed that disease severity can be addressed by NT-proBNP, justifying the long-term studies needed to define true values for risk stratification. Our study showed that NT-proBNP, among the different variables studied, including hemodynamic parameters and 6 MWT, was the marker with lower degree of overlap considering the different functional classes. This brings up the possibility of a more specific and certainly less subjective stratification of patients, which could be of significant importance for the follow-up of patients under different treatment strategies. With the growing interest in pulmonary hypertension pathophysiology and treatment, a better patient stratification is necessary to appropriately address the therapeutic options available and those still to be tested. Our results strengthen the previous findings on the use of NT-proBNP as an independent marker in IPAH. Although its role in predicting survival still needs to be addressed, NTproBNP differs among the different functional classes and correlates with other measures of disease severity. Acknowledgment This study is greatly indebted to an educational grant received from CAPES Ministry of Education Brazil. References 1. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004;351:1425 36. 2. Rubin LJ. 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