Predicting clinical response to a given treatment is one of the most difficult challenges in PAH pharmacotherapy. Though upfront combination therapy in PAH has improved outcomes (3, 4), whether this is related to combining therapies with synergistic mechanisms, or because a higher proportion of individuals receive the drug most effective for them, remains unknown. Thus, a treatment-relevant biomarker could improve decision-making, and potentially, outcomes in PAH. Currently the only predictive biomarker used for PAH therapy selection is vasoreactivity, or acute vasodilation in the presence of inhaled NO during cardiac catheterization (13). Our study is the first to assess the impact of mediators along current treatment pathways on clinical outcomes in a concerted, hypothesis-driven manner. We demonstrated that both ET-1 and the cGMP/NT-proBNP ratio associated 6MWD at the same visit. Furthermore ET-1 and the cGMP/NT-proBNP ratio predicted future 6MWD in a larger cohort of patients with pulmonary vascular disease that included patients with CTEPH, as well as PAH patients with concomitant heart or lung disease. Also in the larger cohort, ADMA associated with same visit 6MWD in patients with PAH on an ERA as well as patients with PAH and concomitant lung disease. In addition, SDMA associated with 6MWD at the same visit, regardless of medication class or concomitant heart or lung disease.
Given the complex interactions among mediators within the biological pathways that influence pulmonary vascular remodeling and vasoreactivity (10), the parallel study of putative mediators as biomarkers may have intrinsic advantages and predictive power as compared to their study individually. Illustrating the web of related mediators is the interaction between cGMP and NT-proBNP. While cytosolic cGMP is produced in response to nitric oxide (NO), membrane-derived cGMP is produced in response to members of the natriuretic peptide (including ANP and BNP) family, and thus cGMP elevations may parallel those in BNP rather than in response to NO (8, 14, 15). This could explain the varying and unexpected relationships in treatment-induced changes in cGMP levels to clinical endpoints seen in previous studies (14, 16). Therefore we were particularly interested in the cGMP/NT-proBNP ratio, as a biomarker of treatment response. Our finding that cGMP/NTproBNP ratio predicted 6MWD at a subsequent clinic visit is novel, has potential theranostic value, and will need to be validated in future studies.
ET-1 is a potent vasoconstrictor that also stimulates proliferation of pulmonary artery smooth muscle cells. Benza and colleagues previously identified common genetic polymorphisms in the endothelin pathway that correlated with response to ERA therapy (6), but it is currently unknown whether ET-1 levels could predict therapeutic response. ERA therapy is known to acutely increase ET-1 levels in patients with PAH (17), reflecting a feedback regulation loop. Consistent with this, ET-1 levels in our cohort were the lowest in patients with PAH on PDE-5i, and highest in patients on the combination of ERA and PDE-5i therapies. Our finding that elevations in ET-1 associated with a lower same visit 6MWD is consistent with prior work by Rubens and colleagues (18). A novel finding in the present study is that elevations in ET-1 also predicted a decreased 6MWD at the subsequent clinic visit, in a larger cohort of patients with pulmonary vascular disease. Interestingly, the correlation between ET-1 and same visit or future 6MWD was similar among patients on ERA monotherapy, patients on PDE-5i monotherapy, and patients on a combination of PDE-5i and ERA therapy, though this could be related to insufficient power to detect a difference among the oral therapy classes.
To our knowledge, this is the first prospective cohort study to investigate associations between SDMA and clinical outcomes in patients with PAH. The arginine derivatives SDMA and ADMA effectively interfere with NO synthesis by competing with L-arginine for the active site of NO synthase, and have been proposed as biomarkers in PAH and other cardiovascular diseases (19, 20). Elevated SDMA has previously associated with renal dysfunction (21) and increased mortality in patients with coronary artery disease (22), atrial fibrillation (20), and stroke (23). In the current study, we demonstrated that increased levels of SDMA associated with worsening 6MWD at the same visit after a threshold of log(SDMA + 1) = 6 in the larger cohort of patients with pulmonary vascular disease. A similar inflection point with SDMA and clinical outcomes has also been seen in previous studies in cardiovascular disease (20, 22). This threshold effect might result when a critical SDMA level capable of competing with L-arginine is reached.
A more potent inhibitor of NO synthesis than SDMA (24), ADMA has been shown to associate with hemodynamic indices and survival in prior studies of PAH patients (19, 25). In the current study, we found that that high ADMA levels associated with lower 6MWD in PAH patients on ERAs but not in PAH patients on either PDE-5i or on PDE-5i/ ERA combination therapy. One possible explanation is that the presence of PDE-5i may increase cGMP levels to help compensate for decreased NO signaling in patients with high levels of ADMA. High ADMA levels in patients on ERA therapy might therefore help identify those patients most likely to benefit from initiation of PDE-5i (as opposed to those patients in whom combination therapy adds only exposure risk and no benefit). In our study high ADMA levels also associated with lower 6MWD in PAH patients with concomitant lung disease. This could because hypoxia causes a decrease in expression of dimethyl-arginine dimethylaminohydrolase (DDAH), the enzyme that metabolizes ADMA (26). As PAH patients with concurrent lung disease are likely to have hypoxemia, ADMA levels may be higher in PAH patients with lung disease compared to other PAH patients. As a result, ADMA may play a bigger role in the pathogenesis of PAH in patients with lung disease.
Other mediators evaluated along the NO pathway, including SNO-Hb and NO2−, did not associate with 6MWD in this study. This may be because NO2− can be affected by other factors including diet and glomerular filtration, explaining the variable levels of NO2− in patients with PAH in other studies (27–29).
Important strengths of our study include the concerted study of multiple biomarkers, over time, in patients with PAH. In many previous studies, potentially relevant biomarkers have been examined or at least reported only individually (10, 30), which limits the ability to account for the cross-talk in these biological systems. Another strength of this study is that we successfully employed a protocol ensuring that potentially labile analytes such as NO2−, SNO-Hb, cGMP, and ET-1, were protected from light; preserved in low-nitrite, opaque (foil-covered) tubes on ice; and immediately transported to the analytical lab and processed within 120 minutes. Finally, the inclusion of patients with PAH with concomitant heart and lung disease reflects contemporary, real world practice of PAH treatment.
This study has several important limitations. We studied only prevalent PAH patients, precluding the ability to determine changes in biomarkers before and after oral therapy. As patients are increasingly being started on upfront ERA-PDE5i combination therapy (3), prevalent PAH patients are an important population to study in order to understand how to titrate therapies. Patients on parenteral therapy tend to have more advanced disease, and because we excluded such patients, our cohort was a relatively healthy one with slower progression of disease. We therefore do not know the role these biomarkers might play in predicting such rapid progression. By the same token, rapid deterioration in a PAH patient typically leads to intensification of therapy that often includes addition of intravenous or other parenteral prostanoid therapy. Therefore biomarker data relevant to the choice of oral therapy class in this setting is unlikely to provide additional actionable information.
Another limitation in this study is the heterogeneity of the cohort, and as such the sample size in each therapy class subgroup may have lacked power to detect an interaction. As this is an exploratory study, no adjustment was made for the multiple testing performed and the associations identified must therefore be interpreted cautiously and require validation in a separate cohort. Also, the use of a single variable (6MWD) may limit the ability to determine associations between biomarker and disease. However, other measures of disease severity, including echocardiography or pulmonary hemodynamic measurements were not performed as frequently in this cohort of patients on oral PAH therapies. Finally, routinely performed lab values such as NT-proBNP were available to treating physicians and were likely considered in the clinical management of patients. This may weaken any natural association between the biomarker and clinical outcomes.