A Story of PA/BSA and Biomarkers to Diagnose Pulmonary Hypertension in Patients with Severe Aortic Valve Stenosis—The Rise of IGF-BP2 and GDF-15

(1) Background: Currently, echocardiography is the primary non-invasive diagnostic method used to screen patients with severe aortic valve stenosis (AS) for pulmonary hypertension (PH) by estimating systolic pulmonary artery pressure (sPAP). Other radiological methods have been a focus of research in the past couple of years, as it was shown that by determining the pulmonary artery (PA) diameter, prognostic statements concerning overall mortality could be made in these patients. This study compared established and novel cardiovascular biomarkers with the PA/BSA value to detect PH in patients with severe AS. (2) Methods: The study cohort comprised 188 patients with severe AS undergoing transcatheter aortic valve replacement (TAVR), who were then divided into two groups based on PA/BSA values obtained through CT-angiography. The presence of PH was defined as a PA/BSA ≥ 16.6 mm/m2 (n = 81), and absence as a PA/BSA < 16.6 mm/m2 (n = 107). Blood samples were taken before TAVR to assess cardiovascular biomarkers used in this study, namely brain natriuretic peptide (BNP), cardiac troponin I (cTnI), high-sensitive troponin (hsTN), soluble suppression of tumorigenesis-2 (sST2), growth/differentiation factor 15 (GDF-15), heart-type fatty acid-binding protein (H-FABP), insulin-like growth factor binding protein 2 (IGF-BP2), and soluble urokinase-type plasminogen activator receptor (suPAR). (3) Results: Patients with a PA/BSA ≥ 16.6 mm/m2 showed significantly higher levels of BNP (p = <0.001), GDF-15 (p = 0.040), and H-FABP (p = 0.007). The other investigated cardiovascular biomarkers did not significantly differ between the two groups. To predict a PA/BSA ≥ 16.6 mm/m2, cut-off values for the biomarkers were calculated. Here, GDF-15 (p = 0.029; cut-off 1172.0 pg/mL) and BNP (p < 0.001; cut-off 2194.0 pg/mL) showed significant results. Consequently, analyses of combined biomarkers were performed, which yielded IGF-BP2 + BNP (AUC = 0.721; 95%CI = 0.585–0.857; p = 0.004) as the best result of the two-way analyses and GDF-15 + IGF-BP2 + BNP (AUC = 0.727; 95%CI = 0.590–0.864; p = 0.004) as the best result of the three-way analyses. No significant difference regarding the 1-year survival between patients with PA/BSA < 16.6 mm/m2 and patients with PA/BSA ≥ 16.6 mm/m2 was found (log-rank test: p = 0.452). (4) Conclusions: Although PA/BSA aims to reduce the bias of the PA value caused by different body compositions and sizes, it is still a controversial parameter for diagnosing PH. Combining the parameter with different cardiovascular biomarkers did not lead to a significant increase in the diagnostic precision for detecting PH in patients with severe AS.


Introduction
Patients suffering from severe aortic stenosis (AS) regularly have concomitant pulmonary hypertension (PH), leading to increased all-cause mortality in surgical and interventional AS repair [1,2]. Invasive right heart catheterization has been used in preoperative/pre-interventional diagnostics to assess the additional risk posed by PH. This practice has been largely abandoned in recent years due to new findings regarding the evaluation of PH through echocardiographic measurements such as systolic pulmonary artery pressure (sPAP) [3,4]. Studies have shown that sPAP can not only be used to accurately diagnose PH, but also as an independent risk factor for early and long-term mortality, as well as length of hospital stay [5,6]. However, there is no definitive consensus in the literature concerning the cut-off value of sPAP defining the presence of PH. Current guidelines and studies give sPAP values ranging from ≥40 mmHg to ≥50 mmHg, leading to a lack of standardization [3][4][5][6][7][8][9].
Additionally, CT angiography is utilized to gain information about the great arteries and aortic valve annulus of every patient undergoing TAVR. As per current European Society of Cardiology (ESC) guidelines [10], CT imaging may be used to assess PH in a non-invasive manner [11,12]. Pulmonary artery diameter/body surface area (PA/BSA), with a cut-off value of 16.6 mm/m 2 , was used in this study to possibly diagnose PH.
In addition to imaging, several biomarkers have been found to correlate with important cardiovascular diseases. New biomarkers such as soluble suppression of tumorigenesis-2 (sST2), growth/differentiation factor 15 (GDF-15), heart-type fatty acid-binding protein (H-FABP), insulin-like growth factor binding protein 2 (IGF-BP2), and soluble urokinasetype plasminogen activator receptor (suPAR) are starting to be mentioned together with classical biomarkers such as brain natriuretic peptide (BNP) and cardiac troponin I (cTnI) because of their potential for an adequate diagnosis of cardiovascular diseases, including myocardial infarction [13,14] or heart failure [15,16].
As the literature investigating the predictive value of biomarkers regarding the concurrence of severe AS and PH is rare, this study aims to compare PA/BSA obtained through CT angiographic imaging with biomarkers, to assess the presence of PH in patients with severe AS before TAVR in a non-invasive manner.

Study Population
Data of 188 patients with severe, primary degenerative AS who were supposed to undergo the TAVR procedure were analyzed at Paracelsus Medical University Hospital Salzburg and Kepler University Hospital Linz between 2016 and 2018. Local ethics committees at Paracelsus Medical University Salzburg (415-E/1969/5-2016) and Johannes Kepler University Linz (E-41-16) reviewed and approved the study protocol. All study participants consented in writing to the parameters of this study.

Transthoracic Echocardiography
Guidelines of the European Society for Cardiology were used to classify severe AS, defined as AV Vmax (maximal velocity over aortic valve) of 4.0 m/s, AV dpmean (mean pressure gradient over aortic valve) ≥ 40 mmHg, and an aortic valve area ≤ 1.0 cm 2 . Simpson's method was used to calculate left ventricular ejection fraction (LVEF). Valve regurgitations of mitral, aortic, and tricuspid valves were classified as minimal, mild (I), moderate (II), and severe (III) using spectral and color Doppler images. Continuous wave Doppler was used over the tricuspid valve to obtain maximum tricuspid regurgitant jet velocity (TRV). Using the formula 4 × TRV 2 + estimated right atrial pressure (RAP), the pulmonary artery pressure (PAP) was calculated. Estimated RAP corresponds to the central venous pressure, which is determined by the inferior vena cava (IVC) diameter (Table 1): Finally, sPAP was calculated using the simplified Bernoulli equation (4 × TRV 2 ) + RAP, which was then used to determine PH in accordance with the current literature [3][4][5][7][8][9]. To perform transthoracic echocardiography, common devices (iE33 and Epiq 5; Philips Healthcare) were used.

CTA Protocol and Measurement of PA Diameter for PH Assessment
Before TAVR, aortic annulus size, aortic anatomy, and vascular access were evaluated using ECG triggered CT-angiography (second-generation, multidetector 128-slice dual source CT; Somatom Definition AS+, Siemens Healthcare, Erlangen, Germany) of the aorta and femoral arteries. A 100 mL bolus of non-ionic iodinated contrast media followed by 70 mL saline solution injected at 3.5-5 mL/s were used to visualize the vasculature. Two radiological investigators who had no prior knowledge of the patients' clinical information analyzed the images on a stationary workstation (Impax, Agfa-Gevaert, Mortsel, Belgium). The diameter of the pulmonary artery (PA) was measured within 3 cm of the bifurcation, as can be seen in Figure 1. The quotient of PA/BSA was generated using the DuBois formula (BSA = 0.007184 × Height 0.725 × Weight 0.425 ).

Biomarker Analysis
Biomarker analysis was performed using samples obtained one day before TAVR, as well as 3 and 12 months after the procedure in an outpatient setting using a vacuumcontaining system. The collection tubes were centrifuged to separate plasma from blood components and then frozen at −80 °C. The samples of all 188 patients were measured at similar time points under the same conditions. To avoid bias in the biomarker analyses,

Biomarker Analysis
Biomarker analysis was performed using samples obtained one day before TAVR, as well as 3 and 12 months after the procedure in an outpatient setting using a vacuumcontaining system. The collection tubes were centrifuged to separate plasma from blood components and then frozen at −80 • C. The samples of all 188 patients were measured at similar time points under the same conditions. To avoid bias in the biomarker analyses, patients with end-stage renal disease or requiring dialysis and patients with previous myocardial infarction or coronary intervention with drug-eluting stent implantation <1 month before TAVR were excluded in advance.
The selection of biomarkers was based on extensive literature research as well as preliminary work of our own research group: sST2 has already been described as a potentially valuable biomarker for risk stratification of PH in TAVR patients [17]. IGF-BP2 is considered a general risk predictor in patients with severe AS and TAVR [18]. High plasma levels of suPAR have been associated with increased postoperative mortality and complications after surgical aortic valve replacement [19]; increased mortality after TAVR was found in patients with elevated GDF-15 levels [20]. Finally, H-FABP as a relevant biomarker for acute myocardial injury was shown to be decreased in the course after TAVR, so the hypothesis was raised that falling H-FABP levels after TAVR are associated with improvement of the hemodynamic situation [21].

Statistical Analysis
Statistical analysis was performed using SPSS (Version 25.0, SPSSS Inc., Armonk, NY, USA). GraphPad Prism (Version 8.0.0, GraphPad Software, San Diego, CA, USA) as well as SPSS were used to create figures.
The estimated sample size was calculated using the following formula, where z stands for confidence interval, p for the sample proportion, and ε for the margin of error: This was repeated using different confidence levels and margin errors ( Figure S1). To test variables for normal distribution, the Kolmogorov-Smirnov test was used. If metric data were normally distributed, they were expressed as mean ± standard deviation (SD) and analyzed using the unpaired Student's t-test. For non-normally distributed metric data, the Mann-Whitney U-test was used; median and interquartile range (IQR) were used to convey the data. Categorical data were compared using the chi-square test.
To determine a cut-off value for PA/BSA, area under the receiver operating characteristics (AUROC) curves with area under the curve (AUC) and subsequent Youden Index (YI) analyses were performed at different sPAP values (sPAP 40-45-50 mmHg). Biomarker plasma levels were then statistically compared based on two groups ("No PH": PA/BSA < cut-off and "PH": PA/BSA ≥ cut-off; cut-off = 16.6 mm/m 2 ) using AUROC analyses and YI analyses for each examined biomarker.
Twofold and threefold biomarker combinations by first completing a binary logistic regression for each combination and then submitting the obtained values to an AUROC analysis. All possible combinations of the named biomarkers (sST2, GDF-15, H-FABP, IGF-BP2, suPAR, BNP, hsTN, cTnI) were considered in this study.
A Kaplan-Meier curve with a log-rank test was generated to compare the 1-year survival between patients with a PA/BSA above and below 16.6 mm/m 2 .
Statistical significance was set to a p-value ≤ 0.050.

Sample Size Calculation
Using the aforementioned formula to calculate, it was assessed that in this study, for a 95% confidence interval/5% margin error, a sample size of 385 subjects would have been needed. For a 90% confidence interval/10% margin error, the sample size necessary was calculated to be 69 patients. Averaging the four calculated versions of the estimated sample size in this study equals 206 patients, which is in an acceptable range to our cohort of 188 patients. Supplementary Figure S1 provides further detail and summarizes the sample size calculation.

Study Cohort
Of the 188 patients included in this study, 107 patients (56.9%) had a PA/BSA ratio measured through CT angiography below 16.6 mm/m 2 , which we defined as "No PH". The remaining 81 patients (43.1%) showed a PA/BSA ratio higher or equal to 16.6 mm/m 2 , which was equated to them having PH.

Baseline Characteristics
In Table 2, the baseline characteristics of the overall cohort, as well as the characteristics of the two groups defined as PA/BSA < 16.6 mm/m 2 and PA/BSA ≥ 16.6 mm/m 2 are listed and compared using the statistical methods laid out above. Most of the study population was male (52.70%), and the percentages for the PA/BSA < 16.6 mm/m 2 and the PA/BSA ≥ 16.6 mm/m 2 groups differed significantly (60.7% vs. 42.0%; p = 0.011). The average age of the cohort was calculated to be 82.8 years ± 4.9 years. The two groups also differed in weight (76.0 kg ± 15.5 kg vs. 71.0 kg ± 12.8 kg; p = 0.021), height (168.6 cm ± 8.7 cm vs. 164.7 cm ± 9.0 cm; p = 0.003), and STSScore mean (2.4 ± 1.0 vs. 3.5 ± 1.8; p = 0.005). Concerning clinical characteristics, a difference in mean LVEF (57.5% ± 9.4% vs. 53.4% ± 12.4%; p = 0.015) and sPAP (38.5 mmHg ± 14.3 mmHg vs. 49.0 mmHg ± 14.8 mmHg; p = <0.001) between the two groups was seen. Other basic characteristics did not show any significant difference between the two groups investigated.

AUROC Results-sPAP vs. PA/BSA Ratio
With the goal of calculating a cut-off value for the PA/BSA ratio to accurately diagnose PH, an AUROC analysis was performed, which is shown in Figure 2 and Table 3.

AUROC Results-sPAP vs. PA/BSA Ratio
With the goal of calculating a cut-off value for the PA/BSA ratio to accurately diagnose PH, an AUROC analysis was performed, which is shown in Figure 2 and Table 3.
A rounded PA/BSA of 16.6 mm/m 2 was used as a cut-off value in this study based on these calculations.

Biomarker Concentrations in the Study Cohort
A comparison of the cardiovascular biomarker concentrations regarding the PA/BSA cut-off value determined can be found in Figure 3.
A significant difference in the median biomarker concentrations between patients with a PA/BSA < 16.6 mm/m 2 and patients with a PA/BSA ≥ 16

AUROC Results-PA/BSA vs. Singular Biomarkers
To evaluate the potential of biomarkers to predict PA/BSA ≥ 16.6 mm/m 2 in patients with severe AS before undergoing TAVR, AUROC and YI analyses were again performed on each of the biomarkers (sST2, GDF-15, H-FABP, IGF-BP2, suPAR, BNP, hsTN, cTnI). Figure 4 and Table 4 depict the results of these analyses.

AUROC Results-PA/BSA vs. Singular Biomarkers
To evaluate the potential of biomarkers to predict PA/BSA ≥ 16.6 mm/m 2 in patients with severe AS before undergoing TAVR, AUROC and YI analyses were again performed on each of the biomarkers (sST2, GDF-15, H-FABP, IGF-BP2, suPAR, BNP, hsTN, cTnI). Figure 4 and Table 4 depict the results of these analyses.   sST2, H-FABP, suPAR, hsTN, and cTnI did not show any significant results in their AUROC analyses.

AUROC Results-PA/BSA vs. Dual-Biomarker Combinations
As stated previously, the biomarkers were then combined using a binary logistical regression and AUROC and YI analyses were performed on the resulting data. Firstly, they were performed on all possible dual-biomarker combinations of the biomarkers used in this study up to this point. Results can be seen in Table 5 and are visualized in Figure 5.

AUROC Results-PA/BSA vs. Triple-Biomarker Combinations
All possible three-way combinations of these biomarkers were then analyzed in the same manner as the dual combinations. Findings are depicted in Table 6 and are visualized in Figure 6.

AUROC Results-PA/BSA vs. Triple-Biomarker Combinations
All possible three-way combinations of these biomarkers were then analyzed in the same manner as the dual combinations. Findings are depicted in Table 6 and are visualized in Figure 6. Table 6. Tabular overview of AUROC analyses of a combination of 3 biomarkers for prediction of a PA/BSA value ≥ 16.6 mm/m 2 with concerning Youden Index, sensitivity, and specificity.     Almost all other combinations also reached a significant p-value ≤ 0.050.

Kaplan-Meier Analysis
One-year survival of patients with a PA/BSA < 16.6 mm/m 2 and patients with a PA/BSA ≥ 16.6 mm/m 2 was compared using Kaplan-Meier curves, as shown in Figure 7. Patients with a PA/BSA < 16.6 mm/m 2 showed a lower risk of death during the 1-y follow-up period at 28.3%, whereas patients with a PA/BSA ≥ 16.6 mm/m 2 demonstra a death rate of 34.1%. However, the log-rank test did not show any significant differe with p = 0.452.

Discussion
This study aimed to investigate the relationship between PA/BSA and different omarkers in patients with severe AS and co-existing PH, as there are currently few stud exploring this relationship.

Can a PA/BSA ≥ 16.6 mm/m 2 Be Used as Evidence for PH?
The PA/BSA cut-off value of 16.6 mm/m 2 used in this study was determined us different sPAP values (40-45-50 mmHg). As the current literature does not agree o single diagnostic sPAP cut-off value for PH diagnosis, to calculate a PA/BSA cut-off, A ROC analyses of the different sPAP values were performed and the rounded average the results was used [3][4][5][6][7][8]. This value is also closely reflected in a study conducted Sudo et al. looking at a study population of 770 patients and concluded that a PA/BS 1.68 cm/m 2 (16.8 mm/m 2 ) is associated with pre-and post-TAVR PH as well as right he failure [22]. Other reports also shown that PA dilation is prominent in PH [11,23]. In g eral, these previous studies show that PA/BSA could provide additional information garding the risk of death in patients undergoing TAVR. It should be mentioned, howev that severe AS alone may lead to widening of the PA diameter due to vascular remodel caused by hemodynamic changes. Although PA/BSA tries to consider the variability the PA diameter in different body types by taking BSA into account, this value alone mi not be enough to accurately diagnose PH [23]. Therefore, this study mainly aimed to ev uate the value of different biomarkers for this important task of diagnosing PH pre-TAV Patients with a PA/BSA < 16.6 mm/m 2 showed a lower risk of death during the 1-year follow-up period at 28.3%, whereas patients with a PA/BSA ≥ 16.6 mm/m 2 demonstrated a death rate of 34.1%. However, the log-rank test did not show any significant difference with p = 0.452.

Discussion
This study aimed to investigate the relationship between PA/BSA and different biomarkers in patients with severe AS and co-existing PH, as there are currently few studies exploring this relationship. The PA/BSA cut-off value of 16.6 mm/m 2 used in this study was determined using different sPAP values (40-45-50 mmHg). As the current literature does not agree on a single diagnostic sPAP cut-off value for PH diagnosis, to calculate a PA/BSA cut-off, AUROC analyses of the different sPAP values were performed and the rounded average of the results was used [3][4][5][6][7][8]. This value is also closely reflected in a study conducted by Sudo et al. looking at a study population of 770 patients and concluded that a PA/BSA ≥ 1.68 cm/m 2 (16.8 mm/m 2 ) is associated with pre-and post-TAVR PH as well as right heart failure [22]. Other reports also shown that PA dilation is prominent in PH [11,23]. In general, these previous studies show that PA/BSA could provide additional information regarding the risk of death in patients undergoing TAVR. It should be mentioned, however, that severe AS alone may lead to widening of the PA diameter due to vascular remodeling caused by hemodynamic changes. Although PA/BSA tries to consider the variability of the PA diameter in different body types by taking BSA into account, this value alone might not be enough to accurately diagnose PH [23]. Therefore, this study mainly aimed to evaluate the value of different biomarkers for this important task of diagnosing PH pre-TAVR. Luçon et al. postulated a pre-interventional sPAP ≥ 40 mmHg to be an independent prognostic factor for patients with severe AS undergoing TAVR, regarding their 1-year risk of death [24]. In this work, for an sPAP ≥ 40 mmHg, a PA/BSA cut-off value of 16.6 mm/m 2 was calculated. Compared to the results found in the study investigating the prognostic value of sPAP ≥ 40 mmHg, the Kaplan-Meier calculation conducted in this study did not yield a significant difference in 1-year mortality between patients with a PA/BSA < 16.6 mm/m 2 and patients with a PA/BSA ≥ 16.6 mm/m 2 after TAVR. However, a comparable study conducted by Sudo et al. found a significant difference in mortality between patient groups classified by large or small PA/BSA. Patients with PA/BSA ≥ 16.8 mm/m 2 showed a cumulative 2-year mortality of 30.0%, compared to a 2-year mortality of 13.7% in the other group [22]. Compared to our work, this study evaluated the two-year mortality as opposed to the one-year mortality, with a slightly different PA/BSA cut-off value of 16.8 mm/m 2 as opposed to 16.6 mm/m 2 . Moreover, the patient cohort used was considerably larger than ours, with 770 patients compared to 188 patients. These results lead us to believe that PA/BSA might be useful as a prognostic factor before TAVR; however, to confirm this assumption, more research would be necessary. Due to the unconvincing results of the Kaplan-Meier calculation, we combined the data collected through CT-angiography with certain biomarkers obtained through preinterventional laboratory measurements.
Matching the results of previous studies investigating the levels of BNP or NT-proBNP in patients with PH, our work also found significantly increased levels of BNP/NT-proBNP in patients with PA/BSA ≥ 16.6 mm/m 2 [25][26][27]. In the available literature, however, PH was defined by sPAP values obtained through echocardiography [27,28] or right heart catheter measurements [26]. Considering the known stimuli for the release of BNP, which encompass cardiovascular diseases such as severe AS, which leads to chronic hypertrophy of the left ventricle, the worth of the calculated cut-off value for BNP (1172.0 pg/mL, sensitivity = 0.51, specificity = 0.80) remains questionable [29].
Another significantly elevated cardiovascular biomarker was H-FABP, an organotropic biomarker which is mainly secreted by cardiomyocytes [30]. Compared to a previous study with patients suffering from severe AS, where concomitant PH was defined by an sPAP ≥ 40 mmHg, no significantly different expression level of H-FABP could be observed in patients "with" and "without" PH [31]. In a recently published study of our working group, this apparent contradiction was explained by the ability of CT angiography to assess vascular geometry and therefore give indirect information about right ventricular strain [32].
Other novel cardiovascular biomarkers such as GDF-15 and IGF-BP2 also showed significantly elevated levels in patients with a PA/BSA ≥ 16.6 mm/m 2 . GDF-15 is a stress-induced cytokine which acts at multiple different sites in the body, as well as a cellautonomous regulator associated with senescence and apoptosis. It has been previously found to be a predictive biomarker of all-cause mortality and adverse cardiovascular events in pulmonary critical care [33]. GDF-15 levels have also been previously associated with increased mean pulmonary artery pressure, which would, like the results of our study, mean levels would be elevated in PH [34]. Similarly, IGF-BP2, a biomarker which potentially plays a role in the pathobiology of PH, has been previously associated with worse outcomes in pediatric PH patients. It has even been proposed as a potential mechanistic target in pediatric PH [35].

Conclusions
Although PA/BSA aims to reduce the bias of the PA value caused by different body compositions and sizes, it is still a controversial parameter for diagnosing PH. Combining the parameter with different cardiovascular biomarkers did not lead to a significant increase in the diagnostic precision for detecting PH in patients with severe AS.

Limitation
The present study is based on data from a small cohort of two medical centers in Austria over a circumscribed time period (2016-2018). Biomarker levels were only measured at baseline without statements regarding expression after TAVR procedure. Additionally, technical pitfalls in echocardiographic and radiological measurements which lead to misclassifications should always be conceded, even if examinations (echocardiography and computed tomography measurements) were performed by experienced clinical investigators.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. sPAP systolic pulmonary artery pressure sST2 soluble suppression of tumorigenesis-2 suPAR soluble urokinase-type plasminogen activator receptor TRV tricuspid regurgitant jet velocity YI Youden Index