WO2024023139A1

WO2024023139A1 - Methods for prognosis and monitoring pulmonary hypertension

Info

Publication number: WO2024023139A1
Application number: PCT/EP2023/070678
Authority: WO
Inventors: Christophe GUIGNABERT; Marc Humbert; Laurent SAVALE; Olivier SITBON; Athénaïs BOUCLY; Ly ieng TU
Original assignee: Institut National de la Santé et de la Recherche Médicale; Université Paris-Saclay
Priority date: 2022-07-27
Filing date: 2023-07-26
Publication date: 2024-02-01

Abstract

In the present invention, inventors used highly sensitive, immunoassay platform, to analyze combined cytokine profiles in serum of patients with Pulmonary Arterial Hypertension (PAH) ((EFFORT cohort). Thereafter, this approach allows to identify a cytokine combinations that represent a reliable biomarker of Pulmonary Arterial Hypertension (PAH) severity and/or mortality, validated in a second independent cohorts of PAH patient (Imperial College of London, UK study) 3-biomarker panel composed of ß-NGF, CXCL9 and TRAIL that was independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after PAH therapy initiation. β-NGF and CXCL9 were predictors of death or transplantation, whereas high levels of TRAIL were associated with a better prognosis. More specifically present invention relates to methods for prognosis and/or monitoring of the severe form of Pulmonary Hypertension (PH) and especially Pulmonary Arterial Hypertension (PAH) through comparison of specific cytokine combinations in PH or PAH patient.

Description

- 1 - METHODS FOR PROGNOSIS AND MONITORING PULMONARY HYPERTENSION 5 FIELD OF THE INVENTION: The present invention relates to methods and kits for prognostic and monitoring Pulmonary Hypertension (PH) and in particular Pulmonary Arterial Hypertension (PAH). More specifically present invention relates to methods for prognosis of Pulmonary Arterial Hypertension (PAH) through detection of a specific of cytokines species in a patient. 10 BACKGROUND OF THE INVENTION: Pulmonary arterial hypertension (PAH) is a heterogeneous group of incurable cardiopulmonary disorders characterized by occlusive remodeling of precapillary pulmonary arteries, leading to right heart failure and premature death. Hemodynamically, PAH is defined 15 by a resting mean pulmonary artery pressure (mPAP) >20 mmHg in the presence of abnormal pulmonary vascular resistance (PVR) ≥3 Wood Units and normal left heart filling pressure (pulmonary artery wedge pressure ≤15 mmHg)¹. Endothelin receptor antagonists, phosphodiesterase type-5 inhibitors, and prostacyclin analogs represent the currently approved PAH medications that have markedly improved overall quality of life, exercise 20 capacity, and long-term outcomes ^2–6. However, the 5-year survival rate for patients suffering from PAH remains low (around 60%)^7–9, and lung transplantation remains an important treatment option for eligible patients with severe PAH if medical treatment fails¹⁰. Risk assessment is of paramount importance in the management of patients with PAH^5,6,11. European guidelines have proposed a risk stratification table based on the 25 evaluation of a panel of clinical, functional, biological and hemodynamic variables to determine the risk of mortality at one year^5,6. Other methods of risk stratification have also been proposed including REVEAL risk scores^12,13. Regardless of the method utilized, repeated assessment of risk is essential to determine treatment strategy and to guide PAH therapy^2–6. The accepted clinical tools to assess severity, determine prognosis and response to 30 therapy include functional class, 6-minute walk test (6MWT), echocardiography, right heart catheterization (RHC) and circulating levels of B-type natriuretic peptide (BNP) or its N- terminal fragment (NT-proBNP)^2–6. Plasma proteins, metabolites and whole blood transcriptomics have been studied for prognostication in PAH patients, as well as circulating endothelial cells, microRNAs and cell-free DNA^14–18. However, none have to date replaced BNP/NT-proBNP as a prognostic marker or in the assessment of treatment effect in routine clinical practice.

Although the exact pathogenic mechanisms of PAH are complex and poorly understood, pulmonary vascular lesions occurring in patients with PAH as well as in animal models of pulmonary hypertension (PH) are characterized by varying degrees of perivascular inflammatory infiltrates, comprising T- and B-lymphocytes, macrophages, dendritic cells and mast cells ^{19 22}. In addition, circulating levels of certain cytokines and chemokines are abnormally elevated in human PAH [i.e., interleukin (IL)-lα and β, IL-6, IL-8, IL-10, IL-12, CCL2, CCL5 and tumor necrosis factor (TNF)-a], and some have been reported to correlate with a worse clinical outcome^{22 24}. More recently, four PAH clusters corresponding to different PAH immune phenotypes with different clinical risks were identified in a cohort of 281 patients with PAH (mostly prevalent) using a panel of 48 circulating factors, including several inflammatory mediators and growth factors²⁵. The cluster of PAH patients with the worst prognosis was characterized by high levels of TNF -factor-related apoptosis-inducing ligand (TRAIL), CC motif chemokine ligand 5 (CCL5), CCL7, CCL4, CXC motif chemokine ligand 9 (CXCL9), IL- 18 and macrophage migration inhibitory factor (MIF). These results were validated in a Sheffield cohort including 104 incident patients with PAH²⁵. However, the heterogeneity of the study population (with incident and prevalent cases from all PAH Group 1) and the absence of follow-up data are the two main obstacles that limited the transfer into clinical practice. To facilitate the incorporation of multiplex immunoassays within routine clinical diagnosis, the new generation of multiplex platforms should offer the possibility to test few samples with robust automation to minimize time and operational costs. Compared to the other commercialized multiplex platforms, the Ella™ microfluidic platform (Protein Simple, CA, USA) offers advantage in ease and time of completion, number of samples per assay, and dynamic concentration range²⁶.

Accordingly, there remains an unmet need in the art for specific and more rapid prognostic test for PH and PAH patient, reflecting directly the dysfunction of the inflammatory component in Pulmonary Arterial Hypertension.

Inventors therefore postulated that focusing on separate cytokine levels might only give a partial view of a complex and interlinked inflammatory response that might not account for the various clinical profiles associated with Pulmonary Hypertension (PH) and in particular Pulmonary Arterial Hypertension.

Inventors therefore hypothesized that the assessment of inflammatory biomarker panels in serum using an Ella™ automated immunoassay platform could represent simple, accessible, and easily measurable biomarkers for the evaluation of the disease at diagnosis as well as the determination of prognosis. We investigated a panel of circulating cytokine/chemokine/adipokine levels in the serum of well-phenotyped PAH patients with idiopathic, heritable, or drug-induced PAH at the time of first presentation (baseline) and at the first follow-up.

The inventors therefore set up a prognostic and monitoring method of Pulmonary Hypertension (PH) and in particular Pulmonary Arterial Hypertension (PAH) that allows direct access to the inflammation status of the patient that is critical for risk stratification and assessment of disease progression.

SUMMARY OF THE INVENTION:

A first object of the invention relates to the present invention relates to an in vitro method for assessing a subject’s risk of having or developing a severe form of Pulmonary Hypertension (PH), comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding

-when the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, is predictive of a high risk of having or developing a severe form of Pulmonary Hypertension (PH) and/or

- when the level of cytokine markers P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value is predictive of a low risk of having or developing a severe form of Pulmonary Hypertension (PH) .

In a particular embodiment, the Pulmonary Hypertension (PH) is Pulmonary Arterial Hypertension (PAH).

An additional object of the invention relates to an in vitro method for monitoring Pulmonary Hypertension (PH) disease comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis- factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: β-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level of cytokine marker β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step ii) is higher than the levels determined at step i) and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is lower than the level determined at step i) .

An additional object of the invention relates to an in vitro method for monitoring the treatment of Pulmonary Hypertension (PH)) comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (betanerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumornecrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject before the treatment, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9) and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject after the treatment”, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the treatment is efficient when the level d the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9) determined at step ii) is lower than the level determined at step i). and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is lower than the level determined at step i)

In a particular embodiment regarding the method for prognosis and monitoring (the disease or the treatment) the Pulmonary Hypertension (PH) is Pulmonary Arterial Hypertension (PAH).

In a particular embodiment regarding the method for prognosis and monitoring (the disease or the treatment) the Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH), heritable Pulmonary Arterial Hypertension (heritable PAH) and drug- and toxin-induced (anorexigen) Pulmonary Arterial Hypertension (PAH).

DETAILED DESCRIPTION OF THE INVENTION:

In the present invention, inventors used highly sensitive, automated immunoassay platform, to analyze combined cytokine profiles in serum of patients with Pulmonary Arterial Hypertension (PAH) [EFORT (Evaluation of prognostic FactORs and Treatment goals in PAH) cohort (NCT 01185730)], at the time of PAH diagnosis and at the first follow-up after PAH therapy initiation. Thereafter, this approach allows to identify the cytokine combinations that represent a reliable biomarker of Pulmonary Arterial Hypertension (PAH) severity and/or mortality validated in a second independent cohorts of PAH patient (UK Research Ethics Committee approval EC Reference 17/LO/0563, Imperial College of London, UK study).

Briefly, risk stratification and assessment of disease progression in patients with pulmonary arterial hypertension (PAH) are challenged by the lack of accurate disease-specific and prognostic biomarkers. To date, B-type natriuretic peptide (BNP) and/or its N-terminal fragment (NT-proBNP) is the only marker for right ventricular dysfunction used in clinical practice in association with echocardiographic and invasive hemodymamic variables to predict outcome in patients with PAH. This study was designed to identify an easily measurable biomarker panel in the serum of 80 well-phenotyped PAH patients with idiopathic, heritable, or drug-induced PAH at baseline and first follow-up. Among the 20 biomarkers studied with the multiplex microfluidic Ella™ platform, inventors identified a 3- biomarker panel composed of [3-NGF, CXCL9 and TRAIL that was independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after PAH therapy initiation. [3-NGF and CXCL9 were predictors of death or transplantation, whereas high levels of TRAIL were associated with a better prognosis. Furthermore, prognostic value of the three cytokines was more powerful for predicting survival than usual non-invasive variables (functional class, 6-minute walking distance and BNP/NT-proBNP level). The results were validated in a fully independent external validation cohort (Cohort from Imperial College of London, UK study). The monitoring of [3-NGF, CXCL9 and TRAIL levels in serum should be considered in the management and treatment of patients with PAH to objectively guide therapeutic options.

Together these data suggest that point of care enumeration of cytokine status might help distinguish patients most likely to benefit or not from at least one approved PAH-specific therapies, including among other an endothelin receptor antagonists (ERAs) therapy, phosphodiesterase type-5 inhibitors therapy, and prostacyclin analogs therapy or lung surgery therapy. This minimal marker set may be used as prognosis tool in combination with usual invasive hemodymamic [including right atrial pressure (RAP), mean pulmonary arterial pressure (mPAP), pulmonary artery wedge pressure (PAWP), cardiac output (CO), cardiac index (CI), pulmonary vascular resistance (PVR), mixed venous oxygen saturation (SvO2)], and non-invasive variables included in risk stratification tools [including echocardiographic parameters, 6-minute walk distance (6MWD), New -York Heart Association (NYHA-FC) and B-Type Natriuretic Peptide (BNP) or N-Terminal pro-B-Type Natriuretic Peptide (NT- proBNP)]. These results thus set-up the basis for the development of a rapid functional specific test for PAH and also improved personalized patient management through cytokine profiling.

Prognostic methods according to the invention

The present invention relates to an in vitro method for assessing a subject’s risk of having or developing a severe form of Pulmonary Hypertension (PH) , comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: |3-NGF (beta-nerve growth factor): CXCL 9 (chemokine (C-X-C motil) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding

-when the level of cytokine marker |3-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motil) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, is predictive of a high risk of having or developing a severe form of Pulmonary Hypertension (PH) and/or

- when the level of cytokine markers |3-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value for each marker is predictive of a low risk of having or developing a severe form of Pulmonary Hypertension (PH) . In another term the present invention relates to an in vitro prognosis method of having or developing a severe form of Pulmonary Hypertension (PH) comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: β-NGF (beta-nerve growth factor): CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding

-when the level of cytokine marker β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, is predictive of a high risk of having or developing a severe form of Pulmonary Hypertension (PH) and/or

- when the level of cytokine markers P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is higher than the reference value is predictive of a low risk of having or developing a severe form of Pulmonary Hypertension (PH).

In a particular embodiment the Pulmonary Hypertension (PH) is Pulmonary Arterial Hypertension (PAH).

In a particular embodiment the Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH), heritable Pulmonary Arterial Hypertension (heritable PAH) and drug- and toxin-induced (anorexigen) Pulmonary Arterial Hypertension (toxin-induced PAH).

The term “prognosis” is a medical term for predicting the likely or expected development of a disease. Prognostic scoring is also used for disease outcome predictions.

In the context of the present invention the “prognosis” is associated with the levels of pro-inflammatory cytokines comprising P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), and/or TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers which in turn may be a risk for developing a severe form of Pulmonary Hypertension (PH) and in particular of Pulmonary Arterial Hypertension (PAH)

The term “subject” as used herein refers to a mammalian, such as a rodent (e.g., a mouse or a rat), a feline, a canine or a primate. In a preferred embodiment, said subject is a human subject. The subject according to the invention can be a healthy subject or a subject suffering from a given disease such as Pulmonary Hypertension (PH) and in particular Pulmonary Arterial Hypertension (PAH).

In particular embodiments, the subject of the present invention suffers from Pulmonary Hypertension (PH) and in particular Pulmonary Arterial Hypertension (PAH) and/or have been previously diagnosed with PH and in particular PAH.

In particular embodiments, a plurality of inflammatory cytokine biomarkers and interferon biomarkers (“Biomarker cytokine”: β-NGF (beta-nerve growth factor): CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand)) may be used in the methods of prognostic /prognostic of survival / classification / monitoring/ treatment response / of the invention. In other words, the methods of the invention may comprise steps of: detecting in the biological sample the level of 1, 2, 3 inflammatory cytokine biomarkers present in the biological sample; and detecting any biomarker of the invention.

In preferred embodiments, the methods of prognostic/ prognostic of survival / classification / monitoring / treatment response are performed using the 3 different inflammatory cytokine biomarkers including a P-NGF, CXCL 9, and TRAIL.

In particular embodiment, the methods of prognostic/ prognostic of survival/ classification / monitoring / treatment response are performed using 1 or 2 of the cytokine biomarkers selected from the list consisting of P-NGF , CXCL 9, and TRAIL.

As used herein, the term “Pulmonary Hypertension” or “PH” and the term “Pulmonary Arterial Hypertension” or “PAH” has its general meaning in the art and refers to Pulmonary hypertension (PH) which defines a group of clinical conditions presenting with abnormal elevation in the pulmonary circulation pressure. Thus, a normal mean pulmonary artery pressure (mPAP) at rest is 14 ± 3.3 mm Hg, and a PH is commonly defined as an increase of mPAP > 20 mm Hg at rest, as assessed by right heart catheterization. The PH diseases are classified into five classes: class 1 to class 5 (Simonneau et al., Eur Respir J. 2019 Jan 24;53(1): 1801913). In particular, pulmonary hypertension diseases include pulmonary arterial hypertension (group 1), PH due to left heart disease (group 2), PH due to lung diseases and/or hypoxia (group 3), chronic thromboembolic pulmonary hypertension (group 4), and other PH conditions with unclear multifactorial mechanisms (group 5) (Simonneau et al., Eur Respir J. 2019 Jan 24;53(1): 1801913).

Among pulmonary hypertension diseases, the pulmonary arterial hypertension is a devastating pulmonary vascular disease-causing breathlessness, loss of exercise capacity and ultimately death. As recently, pointed by the inventors, this disease is characterized by a chronic increase in pulmonary artery pressure (above 20 mmHg), caused by an important remodeling of small pulmonary vessels associated to inflammation, leading to progressive vessel occlusion, ultimately leading to right ventricular failure and death (Humbert et al., Eur Respir J. 2019 Jan 24;53(l):1801887).

There is unfortunately no cure of PAH. The current PAH therapies are essentially focused on decreasing pulmonary vascular resistance by stimulating pulmonary vasodilation (prostacyclin analogues, phosphodiesterase type 5 inhibitors, and endothelin receptor antagonists) (Humbert et al., N. Engl. J. Med. 2004, O’ Callaghan DS, et al. Nat. Rev. Cardiol., 2014). These agents have some anti-remodeling properties, but there is no current anti-remodeling strategy approved for PAH. In spite of these treatments targeting endothelial cell dysfunction that are now available to improve quality of life and survival, in most patients the outcome is very poor. Median survival of PAH (that was 2.8 years in the 1980’s) remains inferior to 5 years and refractory cases are candidates for heart-lung transplantation, a major surgery with current limitations due to shortage of organ donors and severe long-term complications (5-year survival is only 50%). Some hemodynamic and clinical effects of the tyrosine kinase inhibitor imatinib have also been reported in severe PAH, but at the expense of severe side effects.

According to WHO (World Health Organisation) classification there are 5 groups of PH (pulmonary hypertension), where Group I (pulmonary arterial hypertension) is further subdivided into Group I' and Group I" classes (Simonneau G, et al. (2009). Journal of the American College of Cardiology. 54 (1 Suppl): S43-54 and Simonneau G, et al. (2013). Journal of the American College of Cardiology. 62 (25 Suppl): D34-41). The most recent WHO classification system (with adaptations from the more recent ESC/ERS guidelines) can be summarized as follows (Simonneau G, et al. (2013). Journal of the American College of Cardiology. 62 (25 Suppl): D34-41) and Galie N, et al. (2016)., International Society for Heart and Lung Transplantation (ISHLT)". European Heart Journal. 37 (1): 67-119) :

WHO Group I - Pulmonary arterial hypertension (PAH)

• Idiopathic

• PAH with vasoreactivity

• Heritable (BMPR2, ALK1, SMAD9, CAV1, KCNK3 mutations) (Aldred et al, Circ Res. 2022 Apr 29;130(9): 1365-1381) • Drug- and toxin-induced (e.g., methamphetamine, amphetamine, or cocaine use (Kolaitis NA, et al. (2021). Annals of the American Thoracic Society. 18 (4): 613-622.)

• Associated conditions: Connective tissue disease, HIV infection, Portal hypertension, Congenital heart diseases, Schistosomiasis

• PAH with overt features of venous/capillaries (PVOD/PCH) involvement

• Persistent PAH of the newborn syndrome

A “severe form of Pulmonary Hypertension (PH)” or “severe form of Pulmonary Arterial Hypertension (PAH)”, refers to the progression of the disease associated with high mortality rate related to the remodeling of precapillary pulmonary arteries, leading to right heart failure. Patient with severe form of PH or PAH are mostly current therapy refractory cases and are candidates for heart-lung transplantation. According to the 2015 European Society of Cardiology (ESC)ZEuropean Respiratory Society (ERS) PH guidelines (2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir J 2015; 46: 903-975), patients are classified as high risk when the estimated 1-year mortality exceeds 10%. Utilizing risk stratification tools or scores (i.e. REVEAL score >10) or ESC/ERS criteria) may be particularly useful to denote high-risk individuals (Benza RL, Gomberg-Maitland M, Miller DP, et al.. The REVEAL Registry risk score calculator in patients newly diagnosed with pulmonary arterial hypertension. Chest 2012; 141: 354-362) (Galie N, Channick RN, Frantz RP, Griinig E, Jing ZC, Moiseeva O, et al. Risk stratification and medical therapy of pulmonary arterial hypertension. Eur Respir J. 2019 Jan 24;53(1): 1801889)

In particular embodiments, the Pulmonary Arterial Hypertension (PAH) is idiopathic Pulmonary Arterial Hypertension (iPAH) or heritable Pulmonary Arterial Hypertension (heritable PAH) or drug- and toxin-induced (anorexigen) induced Pulmonary Arterial Hypertension (toxin-induced PAH).

As used herein, the term “sample” or "biological sample" as used herein refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy. In a particular embodiment regarding the prognostic method of the PAH according to the invention, the biological sample is a body fluid sample (such blood, serum or plasma) or tissue biopsy of said subject. In preferred embodiments, the fluid sample is a blood sample. In one embodiment, the blood sample to be used in the methods according to the invention is a whole blood sample, a serum sample, or a plasma sample. In a preferred embodiment, the blood sample is a whole blood sample obtained from a subject (e.g., an individual for which it is interesting to determine whether a population of serum biomarkers can be identified).

As used herein, the term " B-NGF, " or “beta NGF” also known as beta Nerve Grow factor has its general meaning in the art refers to a neurotrophic that in humans is encoded by the proNGF gene (gene ID 4803) / UniProtKB P01138). NGF is neurotrophic factor and neuropeptide primarily involved in the regulation of growth, maintenance, proliferation, and survival of certain target neurons. Since it was first isolated by Nobel Laureates Rita Levi- Montalcini and Stanley Cohen in 1956, numerous biological processes involving NGF have been identified, two of them being the survival of pancreatic beta cells and the regulation of the immune system. NGF is initially in a 7S, 130-kDa complex of 3 proteins - Alpha-NGF, Beta-NGF, and Gamma-NGF (2:1:2 ratio) when expressed. This form of NGF is also referred to as proNGF (NGF precursor). The gamma subunit of this complex acts as a serine protease, and cleaves the N-terminal of the beta subunit, thereby activating the protein into functional NGF.

The term nerve growth factor usually refers to the 2.5S, 26-kDa beta subunit of the protein, the only component of the 7S NGF complex that is biologically active (i.e., acting as a signaling molecule).

As its name suggests, NGF is involved primarily in the growth, as well as the maintenance, proliferation, and survival of nerve cells (neurons). In fact, NGF is critical for the survival and maintenance of sympathetic and sensory neurons, as they undergo apoptosis in its absence. However, several recent studies suggest that NGF is also involved in pathways besides those regulating the life cycle of neurons, such as regulation of the immune system: NGF plays a critical role in the regulation of both innate and acquired immunity. In the process of inflammation, NGF is released in high concentrations by mast cells, and induces axonal outgrowth in nearby nociceptive neurons. This leads to increased pain perception in areas under inflammation. In acquired immunity, NGF is produced by the Thymus as well as CD4+ T cell clones, inducing a cascade of maturation of T cells under infection (Lambiase A, et al (1997). The Journal of Allergy and Clinical Immunology. 100 (3): 408-14).

As used herein, the term " CXCL9 " or “Chemokine (C-X-C motil) ligand 9”, has its general meaning in the art and refers to a small cytokine belonging to the CXC chemokine family that is also known as monokine induced by gamma interferon (MIG) and that in humans is encoded by the CXCL9 gene (gene ID 4283 /UniProtKB Q07325). The CXCL9/CXCR3 receptor regulates immune cell migration, differentiation, and activation. Immune reactivity occurs through recruitment of immune cells, such as cytotoxic lymphocytes (CTLs), natural killer (NK) cells, NKT cells, and macrophages. Tumorinfiltrating lymphocytes are a key for clinical outcomes and prediction of the response to checkpoint inhibitors (Fernandez-Poma SM, et al (2017). Cancer Research. 77 (13): 3672- 3684. CXCL9 predominantly mediates lymphocytic infiltration to the focal sites and suppresses tumor growth (Gorbachev, A. V.; et al (2007). The Journal of Immunology. 178 (4): 2278-2286.)

It is closely related to two other CXC chemokines called CXCL10 and CXCL11, whose genes are located near the gene for CXCL9 on human chromosome 4. CXCL9, CXCL10 and CXCL11 all elicit their chemotactic functions by interacting with the chemokine receptor CXCR3 (Tensen CP, et al (1999). The Journal of Investigative Dermatology. 112 (5): 716-22).

CXCL9, -10, -11 have proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the development of adverse cardiac remodeling (Altara R, et al. (2015). PLOS ONE. 10 (10): e0141394.

This chemokine has also been associated as a biomarker for diagnosing Q fever infections (Jansen AF, et al (2017BMC Infectious Diseases. 17 (1): 556)

As used herein, the term "TRAIL" or “TNF-related apoptosis-inducing ligand (TRAIL) “also known as CD253 (cluster of differentiation 253) and TNFSF10 (tumor necrosis factor (ligand) superfamily, member 10) has its general meaning in the art and refers to a cytokine produced by produced and secreted by most normal tissue cells. In humans, the TRAIL protein is encoded by the TNFSF10 gene (gene ID 8743/ UniProtKB - P50591). TRAIL), is a protein functioning as a ligand that induces the process of cell death called apoptosis (Wiley SR, et al (1995). Immunity. 3 (6): 673-82). TRAIL causes apoptosis primarily in tumor cells, [7] by binding to certain death receptors. TRAIL and its receptors have been used as the targets of several anti-cancer therapeutics since the mid-1990s, such as Mapatumumab. However, as of 2013, these have not shown significant survival benefit (Cormier Z (2013). Nature. 494). TRAIL has also been implicated as a pathogenic or protective factor in various pulmonary diseases, particularly pulmonary arterial hypertension (Braithwaite AT, Marriott HM, Lawrie A (2018). "Divergent Roles for TRAIL in Lung Diseases". Frontiers in Medicine. 5: 212.). TRAIL has indeed been suspected to drive the underlying proliferative pulmonary vascular remodeling in rodent models, but TRAIL has also been demonstrated to protects against pulmonary fibrosis in mice models.

TRAIL shows homology to other members of the tumor necrosis factor superfamily. It is composed of 281 amino acids and has characteristics of a type II transmembrane protein. The N-terminal cytoplasmic domain is not conserved across family members; however, the C- terminal extracellular domain is conserved and can be proteolytically cleaved from the cell surface. TRAIL forms a homotrimer that binds three receptor molecules.

The level of the markers of the invention may be determined by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

Standard methods for detecting the level of specific biomarker such as: P-NGF (betanerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumornecrosis-factor Related Apoptosis Inducing Ligand) are well known in the art. Typically, the step consisting of detecting the marker may consist in using at least one differential binding partner directed against the marker.

For example, the serum cytokine level (β-NGF, CXCL 9, and TRAIL ) can be determined using the ELLA Automated Immunoassay System Platform (Protein Simple).

As used herein, the term “binding partner directed against the marker” refers to any molecule (natural or not) that is able to bind the surface marker with high affinity. The binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal antibodies. In another embodiment, the binding partners may be a set of aptamers.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.

The binding partners of the invention such as antibodies or aptamers may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)]) or radioactive molecule or a non-radioactive heavy metals isotopes to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. More particularly, the antibodies are already conjugated to a fluorophore (e.g., FITC-conjugated and/or PE-conjugated).

The aforementioned assays may involve the binding of the binding partners (i.e., antibodies or aptamers) to a solid support. The solid surface could a microtitration plate coated with the binding partner for the surface marker. Alternatively, the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled.

Such methods comprise contacting a biological sample obtained from the subject to be tested under conditions allowing detection of P-NGF, CXCL 9, and TRAIL (inflammatory cytokine) markers. Once the sample from the subject is prepared, the level of PH and PAH biomarkers (“Biomarker cytokine”: (P-NGF, CXCL 9, and TRAIL) may be measured by any known method in the art.

Typically, the high or low level of PH-associated biomarkers (“Biomarker inflammatory cytokine”: (P-NGF, CXCL 9, and TRAIL) is intended by comparison to a control reference value. Said reference control values may be determined in regard to the level of biomarker present in blood samples taken from one or more healthy subject(s) or to the cell surface biomarker in a control population.

In one embodiment, the method according to the present invention comprises the step of comparing said level of PH-associated biomarkers, namely “Biomarker inflammatory cytokine” (P-NGF, CXCL 9, and TRAIL) to a control reference value for each marker wherein a high level of P-NGF and/or CXCL 9 marker(s) and/or a low level of TRAIL marker compared to said respective control reference value is predictive of a high risk of having a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH) and a low level of P-NGF and/or CXCL 9 marker(s) marker(s) and a high level of “TRAIL marker compared to said control reference value is predictive of a low risk of having or developing a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH) .

The control reference value may depend on various parameters such as the method used to measure the level PH-associated biomarker (“Biomarker inflammatory cytokine”: (P- NGF, CXCL 9, and TRAIL) or the gender and the age of the subject.

Typically regarding the reference value using “Biomarker inflammatory cytokine” ((P- NGF, CXCL 9, and TRAIL), as indicated in the Experimental section (see Table 3), for the levels of inflammatory cytokines markers using immunoassay approach identify and quantify inflammatory cytokine markers (P-NGF, CXCL 9, and TRAIL) wherein the blood levels of cytokine markers (P-NGF, CXCL 9) is superior to respectively 3.65 pg/ml, and 625.5 pg/ml and the blood level of TRAIL markers is inferior to 52.65 pg/ml is predictive of having or a high risk of having or developing a severe form of Pulmonary Arterial Hypertension (PAH) . Conversely wherein the levels of cytokine marker (P-NGF, CXCL 9) is inferior to respectively 3.65 pg/ml, and 625.5 pg/ml and the level of TRAIL is superior to 52.65 pg/ml, is predictive of not having or at a low risk of having a severe form of Pulmonary Arterial Hypertension (PAH) .

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of cell surface biomarker or cell death in blood samples previously collected from the patient under testing.

A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the levels of “Biomarker inflammatory cytokine” (β-NGF, CXCL 9, and TRAIL) with a defined threshold value for each marker. In one embodiment of the present invention, the threshold value is derived from the inflammatory cytokine levels (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders (to the method according to the invention). In one embodiment of the present invention, the threshold value may also be derived from inflammatory cytokine level (or ratio, or score) determined in a blood sample derived from one or more subjects or who are non-responders (i.e., asymptomic subject). Furthermore, retrospective measurement of the inflammatory cytokine levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of “Biomarker inflammatory cytokine” (P-NGF, CXCL 9, and TRAIL) in fluids samples previously collected from the patient under testing.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH), and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l- p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH) conversion risk reduction ratios. "Risk evaluation" or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition or asymptomatic form of PAH or symptomic form of PAH to a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH) condition or to one at risk of developing a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH). Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH), such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH), thus prognosing and defining the risk spectrum of a category of subjects defined as being at risk for a severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH). In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for severe form of Pulmonary Hypertension (PH) and/or Pulmonary Arterial Hypertension (PAH).

Prognostic of survival methods of the invention

As demonstrated by the inventors, distinct cytokine profiles are observed in association with PH and/or PAH severity and are differentially predictive of mortality of PH and/or PAH patient. These results warrant new classification of PH and/or PAH patients based on cytokine profiling regarding the need or not of need for instance of lung surgery.

Accordingly, another object of the invention relates to an in vitro method for assessing a PH patient’s risk of having a poor prognostic of survival, comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motil) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding

-when the level of cytokine marker β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, then said PH patient is at high risk of having a poor prognostic of survival; or

- when the level of cytokine markers P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motil) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value then said PH patient is at high risk of having a good prognostic of survival.

In a particular embodiment the Pulmonary Hypertension (PH) is the Pulmonary Arterial Hypertension (PAH).

In a particular embodiment regarding the method for prognostic of survival the Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH); heritable Pulmonary Arterial Hypertension (heritable PAH) and drug and toxin-induced (anorexigen) induced Pulmonary Arterial Hypertension (toxin-induced PAH)

Monitoring methods and Management

After the identification of cytokine subsets that harbour an inflammatory phenotype, inventors highlighted, that the three “Biomarker inflammatory cytokines”: β-NGF, CXCL 9 and TRAIL were independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after PAH therapy initiation. High levels of β-NGF and CXCL9 as well as low levels of TRAIL were predictors of death or transplantation. Accordingly, inventors provided evidence that this cytokine subset may serve as a severity biomarker in PH and/or PAH for prognosis and monitoring purpose (pathology or treatment).

Accordingly, an additional object of the invention relates to an in vitro method for monitoring Pulmonary Hypertension (PH) disease comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step ii) is higher than the levels determined at step i) and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is lower than the level determined at step i) .

An additional object of the invention relates to an in vitro method for monitoring the treatment of Pulmonary Hypertension (PH) comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: β-NGF (betanerve growth factor),: CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumornecrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject before the treatment, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor),: CXCL 9 (chemokine (C-X- C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject after the treatment”, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the treatment is efficient when the level d the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9) determined at step ii) is lower than the level determined at step i). and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is lower than the level determined at step i)

In a particular embodiment regarding the method for prognosis and monitoring (the disease or the treatment) the Pulmonary Arterial Hypertension (PAH is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH); heritable Pulmonary Arterial Hypertension (heritable PAH) and drug and toxin-induced (anorexigen) induced Pulmonary Arterial Hypertension (toxin-induced PAH).

The decrease or increase (depending of biomarkers) can be e.g. at least 5%, or at least 10%, or at least 20%, more preferably at least 50% even more preferably at least 100%.

Therapeutic Method of a specific population

As mentioned, endothelin receptor antagonists, phosphodiesterase type 5 (PDE-5) inhibitors, and prostacyclin derivatives are the current approved treatments for Pulmonary Hypertension (PH) and especially for Pulmonary Arterial Hypertension (PAH). Even if these drugs have markedly improved overall quality of life, exercise capacity, and long-term outcomes ^{2 -6}. the 5 -year survival rate for patients suffering from PH or PAH remains low (around 60%)^{7 9}. and lung transplantation remains an important treatment option for eligible patients with severe PAH if medical treatment fails¹⁰. Multiparametric risk stratification at the time of PH or PAH diagnosis and at follow-up provides useful information for the choice of first-line therapy and for subsequent treatment escalation.

The present study shows that cytokine profiling may have clinical implications for improved personalized treatment. For example, the present invention allows to identify a novel panel of three cytokines (B-NGF, CXCL9, and TRAIL) in serum independently associated with prognosis at both baseline and at the first follow-up after PAH therapy initiation. Subsequent analysis revealed that the prognostic value of this 3-biomarker panel was more powerful for predicting PAH survival than usual clinical and hemodynamic variables. Therefore, it is proposed that serum B-NGF, CXCL9, and TRAIL levels should be considered in the management and treatment of patients with PAH during follow-up to objectively identify patients with a high risk of death to adapt treatment (treatment escalation and/or lung transplantation).

Accordingly, the invention also relates to a method for treating Pulmonary Hypertension (PH) with endothelin receptor antagonist and/or phosphodiesterase type 5 (PDE-5) inhibitor and/or and prostacyclin derivative in a subject wherein the level of at least one cytokine selected from the list consisting of β-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers obtained from said subject, have been detected, by one of the methods of the invention.

Thus, the invention also relates to a method for guiding personalized therapy of Pulmonary Hypertension (PH), with either endothelin receptor antagonist treatment and/or phosphodiesterase type 5 (PDE-5) inhibitor and/or prostacyclin derivative according to the cytokine profile of the subject.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

The term “endothelin receptor antagonists” or “ERA” means a class of a drug that blocks endothelin receptors. Three main kinds of ERAs exist:

• selective ETA receptor antagonists (sitaxentan, ambrisentan, atrasentan, BQ- 123, zibotentan), which affect endothelin A receptors.

• dual antagonists (bosentan, macitentan, tezosentan), which affect both endothelin A and B receptors. [1]

• selective ETB receptor antagonists (BQ-788 and A192621) which affect endothelin B receptors are used in research but have not yet reached the clinical trial stage.

Sitaxentan, ambrisentan and bosentan are mainly used for the treatment of pulmonary arterial hypertension, while atrasentan is an experimental anti-cancer drug.

Edonentan is an endothelin receptor antagonist drug.

In a particular embodiment, endothelin receptor antagonists according to the invention can be Sitaxentan, ambrisentan and bosentan

The term “phosphodiesterase type 5 inhibitors” or “PDE5 inhibitor” is a vasodilating drug which works by blocking the degradative action of cGMP-specific phosphodiesterase type 5 (PDE5) on cyclic GMP in the smooth muscle cells lining the blood vessels supplying various tissues. For instance, these drugs dilate the corpora cavernosa of the penis, are used in the treatment of erectile dysfunction (ED). Accordingly, Phosphodiesterase-5 (PDE5) inhibitors such as sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra) are clinically indicated for the treatment of erectile dysfunction. Because PDE5 is also present in the smooth muscle of the walls of the arterioles within the lungs, two PDE5 inhibitors, sildenafil and tadalafil, are FDA/EMD -approved for the treatment of pulmonary hypertension while tadalafil (Levitra) is also licensed for the treatment of benign prostatic hyperplasia. As of 2019, the wider cardiovascular benefits of PDE5 inhibitors are being appreciated (Tzoumas N, et al. (2019). British Journal of Pharmacology. 177 (24): 5467-5488).

In a particular embodiment, PDE5 inhibitor according to the invention can be: sildenafil and tadalafil.

The term “Prostacyclin” (also called prostaglandin 12 or PGI2) is a prostaglandin member of the eicosanoid family of lipid molecules. It inhibits platelet activation and is also an effective vasodilator. When used as a drug, it is also known as epoprostenol and the terms are sometimes used interchangeably (Kermode J, et al. (1991). " British Heart Journal. 66 (2): 175-178).

Prostacyclin is commonly considered the most effective treatment for PAH. Epoprostenol (synthetic prostacyclin) is given via continuous infusion that requires a semi- permanent central venous catheter. This delivery system can cause sepsis and thrombosis. Prostacyclin is unstable, and therefore has to be kept on ice during administration. Other Prostacyclin derivatives have therefore been developed. Treprostinil can be given intravenously or subcutaneously, but the subcutaneous form can be very painful. An increased risk of sepsis with intravenous Remodulin has been reported by the CDC. Iloprost is also used in Europe intravenously and has a longer half-life. Iloprost was the only inhaled form of prostacyclin approved for use in the US and Europe, until the inhaled form of treprostinil was approved by the FDA in July 2009

In a particular embodiment, Prostacyclin derivatives according to the invention can be: Epoprostenol, Treprostinil Remodulin and Iloprost.

Another object of the present invention is a method of treating Pulmonary Hypertension (PH) in a subject comprising the steps of: a) providing a blood sample from a subject, b) detecting the levels of cytokines selected from the list consisting of P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motil) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers c) comparing the level determined at stet b) with a reference value for each marker and if the level of cytokine markers P-NβF (beta-nerve growth factor) and/or CXCL 9

(chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value then, treating the subject with endothelin receptor antagonists and/or phosphodiesterase type 5 (PDE-5) inhibitors and/or prostacyclin derivatives. if the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, then treating the subject with treatment escalation of endothelin receptor antagonists and/or phosphodiesterase type 5 (PDE-5) inhibitors and/or and prostacyclin derivatives and/or lung transplantation.

In particular embodiments, the Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH), heritable Pulmonary Arterial Hypertension (heritable PAH) and drug- and toxin-induced (anorexigen) Pulmonary Arterial Hypertension (PAH).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

FIGURE 1: Kaplan-Meier survival curves according to levels of P-NGF, CXCL9 and TRAIL at the diagnosis (A, B, C) and at the follow-up (D, E, F): Cytokines are expressed as dichotomous variables according to thresholds determined by ROC curves: 3.65 pg/mL for β- NGF, 625.5 pg/mL for CXCL9 and 52.65 pg/mL for TRAIL. Log rank p<0.005 for both analyses.

FIGURE 2: Kaplan-Meier survival curves according to the number of low-risk status of P-NGF, CXCL9 and TRAIL at the diagnosis (A) and at the follow-up (B) in the French cohort: P-NGF < 3.65 pg/mL; CXCL9 < 625.5 pg/mL, TRAIL > 52.65 pg/mL. Log rank p<0.001 for both analyses.

FIGURE 3: Kaplan-Meier survival curves according to levels of P-NGF, CXCL9 and TRAIL at the diagnosis (A, B, C) and at the follow-up (D, E, F) in the validation cohort: Cytokines are expressed as dichotomous variables according to thresholds determined by ROC curves in the study population: 3.65 pg/mL for P-NGF, 625.5 pg/mL for CXCL9 and 52.65 pg/mL for TRAIL. Log rank p<0.001 for both analyses.

FIGURE 4;: Comparisons of serum cytokines levels concentrations between controls and PAH at the diagnosis, and in PAH between baseline and the first follow-up:

* Significant difference between controls and PAH at the diagnosis

^Λ Significant difference in PAH between baseline and the first follow-up (f-up)

FIGURE 5: ROC curves of transplant-free survival of P-NGF (A), CXCL9 (B) and TRAIL (C) at the diagnosis

EXAMPLE:

Methods:

Cohort data collection This is an ancillary study from the EFORT (Evaluation of prognostic FactORs and Treatment goals in PAH) cohort (NCT 01185730). This study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained for each patient prior to their enrollment.

The ‘EFORT’ study is a prospective study to assess prognostic factors at both baseline and follow up in a French cohort of incident (i.e. , newly diagnosed) patients with PAH. All incident patients entered in the French Registry between January 2011 and December 2013 with a diagnosis of idiopathic, heritable or anorexigen-induced PAH were enrolled in the EFORT study.

Assessments have been performed at baseline (i.e., time of PAH diagnosis), 3-4 months after treatment initiation or treatment change, and then once a year until a 3-year follow up for patients included into the study in the first two years (2011-2012) and until a 2- year follow up for patients included in the last year (2013). Serial assessments included NYHA functional class (FC), non-encouraged 6MWT, right heart catheterization, echocardiography and biomarkers (BNP/NT-pro-BNP, uric acid, creatinine, sodium). An ancillary study was performed in 80 patients with collection of serum samples at both baseline and first follow-up visit for cytokines measurements. These 80 patients constituted the study population.

Sixteen healthy blood donors with serum samples constituted the control population.

Serum samples from 125 incident patients with PAH followed up at Imperial College of London, UK, constituted our validation cohort. A sample was available at the time of diagnosis in all patients. In addition, another sample was withdrawn in 33 of these patients at follow-up.

Ella™ microfluidic platform

Reagents for the SimplePlex Ella™ microfluidic platform (Protein Simple, CA, USA) were custom developed. Twenty biomarkers were selected according to results from previous studies^{22 25} and divided into 6 panels based on relative serum abundance and assay dynamic range. Panel 1 included the following 3 high-abundance biomarkers tested at a dilution of 1:10: leptin, MIF, CCL5. Five other panels included the following low-abundance biomarkers: 1:2: β-NGF, G-CSF, IL-6, IL-10 (Panel 2); 1:2: CXCL9, TRAIL, VEGF-A (Panel 3); 1:2: CCL2, CCL4, CXCL10 (Panel 4); 1:2: IL-12p70, IL-15, IL-18, IL-17 (Panel 5); 1:2: IL-8, IL-la, IL-4 (Panel 6). For the external validation cohort, three panels have been used: 1:2: β-NGF (panel 1); CXCL10, IL-18, IL-6, TRAIL (Panel 2); CXCL9, IL-la (panel 3). Assays were performed in a single center according to the manufacturer's protocol. Briefly, 50 pL of diluted serum was added to the appropriate cartridge, followed by placement into the Ella instrument requiring no further user intervention. Each cartridge included a built- in lot-specific standard curve and samples were run as internal triplicates. Detection and washing steps were automatically performed by the instrument. Raw data were analyzed using the SimplePlex Explorer software Version 3.7.2.0. CA, USA.

Statistical analysis

Data were collected from the web-based French PH Registry (PAHTool®; Inovultus Ltd. Santa Maria da Feira, Portugal). Statistical analysis was performed using SPSS Statistics version 26 (SPSS Inc., Chicago, IL). Continuous variables are expressed as the means ± SD or medians (interquartile range, 25% to 75% [IQR]) according to the data distribution.

Levels of cytokines in PAH patients were compared with the serum of healthy controls (blood donors) by Mann-Whitney U tests. Comparisons between levels of cytokines at baseline and the first follow-up were performed by paired t test or nonparametric test according to the data distribution.

The date of diagnostic RHC was used as the starting point to determine the length of survival. The cutoff date was December 31, 2020. Transplant-free survival was represented using the Kaplan-Meier method. Univariable and multivariable forward stepwise Cox proportional hazards regression models were performed to determine the risk of event (death or transplantation) according to baseline and first follow-up visit variables. A p value threshold of <0.10 was used for entry into the multivariable model, and p>0.05 was the threshold for variable removal. All comparisons were two-sided, and a p value <0.05 was considered statistically significant.

Variables identified in univariable analysis to be significantly associated with the prognosis at both baseline and follow-up were considered cytokines of interest. For each of them, ROC curves were performed at baseline to determine the best threshold of transplant- free survival by Youden’s index.

Patients with idiopathic, heritable or anorexigen-induced PAH from the UK in whom blood samples were collected at the time of PAH diagnosis and during follow-up were used for external validation. In this validation cohort, survival analyses were performed using the Kaplan-Meier method.

Results

Patient Demographics Eighty PAH patients constituted our study population (71% female, mean age 51±19 years, 66% idiopathic PAH, 20% heritable PAH and 14% anorexigen-induced PAH). The baseline characteristics and initial treatment strategies are summarized in Table 1.

Cytokine Measurements

Levels of leptin, G-CSF, MIF, CXCL9, CXCL10, IL-1, IL-4, IL-6, IL-8 and IL- 15 were significantly increased in the PAH group as compared with healthy subjects whereas levels of TRAIL and IL- 10 were significantly decreased. Details of these results are presented in (Figure 4). ).

Levels of TRAIL and IL-17 were significantly increased at follow-up compared to baseline levels, whereas levels of G-CSF, CXCL10, CCL2, CCL4, P-NGF, IL-6, IL-8, IL- 15 and IL-18 were significantly decreased between baseline and follow-up ((Figure 4).).

Survival Analysis

After a median follow-up of 69 (50-81) months, 21 patients had died and 5 underwent lung transplantation. Univariable analysis was performed with the 20 cytokines at baseline and at follow-up. Significant results of univariable analyses are presented in Table 2. At baseline, prognostic cytokines were β-NGF, CXCL9, TRAIL and IL-18, whereas P-NGF, CXCL9, TRAIL, CXCL10 and IL-6 were associated with survival at the first follow-up. In univariable analysis, 3 cytokines were associated with transplant-free survival at both baseline and follow-up: P-NGF, CXCL9, which were both associated with poor prognosis, and TRAIL, which was associated with good outcomes. The relationship between each cytokine and survival persisted in multivariable models adjusted for age and sex.

Determination Of Discriminating Thresholds

For each identified cytokine at both baseline and follow-up (P-NGF, CXCL9 and TRAIL), ROC curve analysis was performed to determine the best threshold of transplant-free survival at baseline: <3.65 pg/mL for P-NGF, <625.5 pg/mL for CXCL9 and >52.65 pg/mL for TRAIL. The ROC curves from which the thresholds were identified are presented in (Figure 5). Kaplan-Meier survival curves according to thresholds of P-NGF, CXCL9 and TRAIL are presented in Figure 1.

In univariable analysis, the hazard ratios of cytokines expressed as dichotomous variables (according to thresholds previously determined) at diagnosis were: P-NGF HR= 9.866 (95%CI 2.906 - 33.494), CXCL9 HR= 6.429 (₉₅%CI 2.531 - 16.335) and TRAIL HR= 0.200 (95%CI 0.069 - 0.581), and at follow-up: P-NGF HR= 10.811 (₉₅%CI 4.266 - 27.396), CXCL9 HR= 3.875 (₉₅%CI 1.615 - 9.299) and TRAIL HR= 0.169 (₉₅%CI 0.064 - 0.450). Determination of the Added Value of Cytokines to Current Risk Stratification

The results of univariable analysis at baseline and follow-up are presented in Table 3.

Several models of multivariable analysis were performed by testing each cytokine separately or together. In multivariable analysis, P-NGF, CXCL9 and TRAIL were more powerful for predicting survival than usual clinical variables (NYHA FC, 6MWD) and biomarkers (BNP and NT-proBNP) (Tables 4 and 5).

Survival According to P-NGF, CXCL9 and TRAIL Status

Transplant-free survival was associated with levels of β-NGF, CXCL9 and TRAIL at baseline and at follow-up. No deaths have occurred in patients with 3 “low-risk” statuses of cytokines (low levels of P-NGF and CXCL9, and high levels of TRAIL). On the other hand, patients without a “low-risk” profile of cytokines had a worse prognosis (Figure 2). Patients achieving 2 “low-risk” statuses of cytokines at the first follow-up had an excellent long-term survival similar to that of patients achieving 3 “low-risk” statuses of cytokines.

Validation Cohort

The London validation cohort comprised 125 incident patients (69% female, mean age 59±17 years, 91% idiopathic PAH, 9% heritable PAH), with a mean pulmonary vascular resistance of 12±6 WU (Table 1). After a median follow-up of 49±29 months, 53 patients had died. Among the 125 patients of the cohort, a collection of serum samples was available at both baseline and follow-up in 33 of them. Kaplan-Meier survival analysis in this cohort according to the status of P-NGF, CXCL9 and TRAIL confirmed the results previously observed in the French cohort (Figure 3).

DISCUSSION

In this study, we used a cohort of incident idiopathic, heritable, or anorexigen- associated PAH patients to examine the prognostic value of multiple cytokine markers in PAH. Twenty biomarkers were selected and measured by a multiplex Ella™ platform. This approach allowed the identification of three cytokines independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after PAH therapy initiation: B-NGF, CXCL9 and TRAIL. P-NGF and CXCL9 were predictors of death or transplantation, whereas high levels of TRAIL were associated with a better prognosis. Furthermore, we observed that the prognostic value of the three cytokines was more powerful for predicting survival than usual clinical and hemodynamic variables. These results were validated in a fully independent external validation cohort. Inflammation and autoimmune disorders are common denominators of all forms of PAH, even in the absence of associated inflammatory or autoimmune disorders^{19 22}. Consistent with this notion, circulating autoantibodies targeting endothelial cells and fibroblasts as well as high levels of certain cytokines, such as IL-6, TNF-a and IL-1β, have previously been described in patients with idiopathic PAH^{23 25}-²⁷-^2S. Inflammation and immune disorders are detected in the circulating blood as well as within the pulmonary vascular lesions that characterize PAH, where they facilitate pulmonary vascular cell survival and growth^19-22. Given the link between inflammation and pulmonary vascular remodeling, several studies have demonstrated the prognostic value of certain inflammatory mediators in PAH. However, these studies are limited by the fact that they mostly integrated incident and prevalent cases of PAH and different forms of PAH, including those associated with another disease or condition that can strongly influence the inflammatory profiles. In addition, data on the evolution of these biomarkers during follow-up are also lacking. As a result, the integration of inflammatory biomarkers into risk assessment tools and treatment response assessment has never been performed.

The ability to predict PAH patients at risk of adverse outcomes is of value to clinicians to improve risk stratification. Currently, BNP and/or NT-proBNP are the only two biomarkers incorporated into several PAH risk stratification tools and screening algorithms to detect and monitor PAH^{5 6}, even if other potential candidates have been identified such as growth and differentiation factor (GDF)-15²⁹, red cell distribution width (RDW)^{30 33}, uric acid (UA)³⁴, creatinine³⁵, IL-6^23-25,27,28 and angiopoietins³⁶. While most of these individual biomarkers are closely linked with the level of right ventricular (RV) dysfunction, it is possible that characterizing risk assessment by profiling multiple factors involved in different disease components may be a more robust approach than only measuring impairment of cardiac function. Therefore, novel biological markers to predict response to therapies and outcome are needed to accelerate the development of precision medicine tools for PAH. In this context, the use of objective biomarkers and clinical trial end points are crucial as well as the selection of a reproducible, accurate, sensitive, and appropriate multiplex immunoassay platform. Since the past few years have seen significant progress and innovation and several multiplex assays that are now commercially available, we have chosen to use the Ella™ platform. Indeed, this microfluidic-based system is an easy-to-use and fully automated platform allowing the acquisition of both highly sensitive and reproducible results with minimal sample handling²⁶. In contrast to the other multiplex platforms that are designed to analyze a large number of samples in a short time frame, the Ella™ microfluidic platform has the capacity to test fewer samples with fast turn-around (approximately 1.25 hours), making this platform effective for clinical use. This high-throughput proteomic approach allows accurate measurement of different biomarkers that can have various circulating levels of expression and concentration. Our analysis confirmed the known association between PAH development and high levels of various cytokines and inflammatory mediators. As previously reported, some of these individual cytokines are predictors of survival at diagnosis and/or at first follow-up. To identify cytokines with the most powerful prognostic value, we chose to select only those that exhibited a circulating level independently associated with transplant-free survival at diagnosis and during follow-up.

Even if this 3-biomarker panel detected in the peripheral blood could be useful in clinical settings, it remains unclear how they reflect what is occurring in PAH lungs. Recent studies have emphasized the importance of B-NGF, CXCL9 and TRAIL in the development and progression of PAH, but much remains to be done in this area. It has been previously demonstrated that the increased expression of NGF and its receptor in human and experimental PAH promotes pulmonary vascular cell proliferation and migration, pulmonary arterial hyperreactivity, and secretion of proinflammatory cytokines³⁷. Recently, the prognostic value of NGF was identified for the first time in a discovery cohort of 121 incident and prevalent PAH patients and a validation cohort of 76 patients³⁸. High levels of NGF were associated with worse Mayo and Stanford scores independent of pulmonary vascular resistance or pressure in both cohorts³⁸. The chemokine CXCL-9 (also known as MIG) is an interferon-inducible members of the CXC chemokine family that lack the tripeptide structure/function motif Glu-Leu-Arg (ELR) that is important in the chemoattraction of mononuclear cells including of activated T cells, B cells and natural killer (NK) cells. Thus, CXCL-9 could play a role in the infiltration of inflammatory cells into the perivascular area of pulmonary vessels in PAH. CXCL9 also demonstrates angiogenic activity via the receptor CXCR3³⁹. However, its potential implication in PAH development has never been studied. Recently, CXCL-9 was identified as a prognostic factor in the subpopulation for patients with chronic thromboembolic pulmonary hypertension (CTEPH)⁴⁰. TRAIL, also known as Apo2L, is a member of the TNF superfamily of cytokines that that can bind five different receptors to induce several biological processes including cell survival, migration and proliferation via kinase signaling pathways⁴¹. In our study, high circulating TRAIL levels were associated with better prognosis. Paradoxically, previous experimental studies reported that TRAIL was upregulated in idiopathic PAH and could be involved in PAH pathophysiology by inducing migration and proliferation of pulmonary artery smooth muscle cells (PA-SMCs)^42,43. Accordingly, administration of an anti-TRAIL antibody or its genetic deletion have been reported to prevent and reverse vascular remodeling in various models of PAH. However, the role of TRAIL in the pathogenesis of lung diseases can be divergent⁴¹. TRAIL also has the ability to function as either a pro-apoptotic or pro-survival signal depending on the cell types and receptor expression on local tissues to mediate either protective or pathogenic mechanisms. The exact mechanism by which TRAIL modulates these functions is not fully understood, although regulation of TRAIL, and its cleavage, as well as the expression of receptors by specific cell types, are clearly important in determining its effects. Further work is required to fully elucidate the divergent roles of TRAIL to gain a better understanding its role in underlying processes of lung disease and its potential as a therapeutic agent — or target — depending on disease context.

Our study confirmed the importance of noninvasive parameters in risk stratification, specifically at the first follow-up after PAH therapy initiation. As expected, NYHA functional class, 6MWT, and BNP/NTproBNP were independent predictive factors of transplant-free survival. BNP and NTproBNP are the only biomarkers that are currently used for risk stratification according to the ERS/ESC method^5,6. eGFR is also listed in the REVEAL score^12,13, but cardiorenal syndrome is more frequently indicative of advanced PAH with severe right heart failure. Other candidate prognostic biomarkers have been identified^{23 25 27} ³⁶, but most often fail to demonstrate a sufficient power to replace or bring added value to the use of BNP or NT-proBNP alone. Our study clearly demonstrated that the prognostic value of B-NGF, CXCL9, and TRAIL remains strong and independent after adjustment for all other variables included in risk stratification. In addition, the multivariable Cox regression analysis demonstrated a higher predictive value of this 3 -biomarker panel than BNP or NT-proBNP in our cohort. Interestingly, this multimarker approach allowed us to distinguish a subgroup of patients with a very good prognosis when the threshold established by the ROC analysis reached at least 2 biomarkers. These results suggest that the use of these three biomarkers should be useful for predicting patient survival and evaluating response to treatment independent of commonly used variables.

Our results emphasize the importance of including multiple inflammatory markers in PAH risk assessment. This is a large incident cohort that has been prospectively phenotyped at both baseline and during follow-up after PAH therapy initiation. The detailed clinical and biological data captured allow for multivariable modeling to adjust for key confounders. Moreover, the prognostic value of the three identified biomarkers was validated in an external cohort of PAH patients. The main limitation is the relatively small number of patients included in the discovery cohort (n=80). Additional studies are mandated to confirm the usefulness of these new biomarkers in real clinical settings. In this first study, we chose to analyze PAH patients without confounding inflammatory conditions. It will be interesting in the future to extend the analysis to other types of PAH associated with inflammatory diseases, such as connective tissue disease, HIV or portal hypertension.

In conclusion, our study identified a novel panel of three cytokines (B-NGF, CXCL9 and TRAIL) in serum independently associated with prognosis at both baseline and at the first follow-up after PAH therapy initiation. Subsequent analysis revealed that the prognostic value of this 3-biomarker panel was more powerful for predicting PAH survival than usual clinical and hemodynamic variables. The results were validated in a fully independent external validation cohort. Therefore, we propose that serum B-NGF, CXCL9 and TRAIL levels should be considered in the management and treatment of patients with PAH during followup to objectively identify patients with a high risk of death to adapt treatment (treatment escalation and/or lung transplantation).

Results are expressed as mean ± SD, median (IQR25-75%) or n (%). All assessments were performed before any treatment was initiated.

Oral dual combination therapy was defined by the association of endothelin receptor antagonist (ERA) and phosphodiesterase-5 inhibitor (PDE5-i). Triple combination therapy was defined by the association of ERA, PDE5-i and parenteral prostacyclin or selexipag.

Abbreviations: 6MWD: 6-minute walk distance; BNP: brain natriuretic peptide; CI: cardiac index; CO: cardiac output; mPAP: mean pulmonary arterial pressure; NTproBNP: N- Terminal pro-B-Type Natriuretic Peptide; NYHA: New-York Heart Association; PAH: pulmonary arterial hypertension; PAWP: pulmonary artery wedge pressure; PVR: pulmonary vascular resistance; RAP: right atrial pressure; RHC: right heart catheterization; SvO2: mixed venous oxygen saturation; WU: Wood unit

Table 2: Identification of cytokines associated with transplant-free survival by univariable analysis at diagnosis (A) and at first follow-up (B): Cytokines are expressed as continuous variables

Abbreviations: P-NGF: beta-nerve growth factor; CI: confidence interval; CXCL: chemokine (C-X-C motif) ligand; IL interleukin; TRAIL: tumor-necrosis-factor related apoptosis inducing ligand; VEGFR2: vascular endothelial growth factor receptor-2;

Table 3: Univariable Cox regression analysis of usual low risk variables and three cytokines at diagnosis (A) and at first follow-up (B): Cytokines are expressed as dichotomous variables (according to thresholds determined by ROC curves: P-NGF high: > 3.65 pg/mL; CXCL9 high: > 625.5 pg/mL, TRAIL high: > 52.65 pg/mL).

Abbreviations: 6MWD: 6-minute walk distance; BNP: brain natriuretic peptide; CI: cardiac index; CI: confidence interval; NTproBNP: N-Terminal pro-B-Type Natriuretic Peptide; NYHA: New-York Heart Association; RAP: right atrial pressure; β-NGF: beta-nerve growth factor; CXCL: chemokine (C-X-C motif) ligand; TRAIL: tumor-necrosis-factor related apoptosis inducing ligand. Table 4: Multivariable Cox regression analysis including the 3 non-invasive low risk variables and the three selected cytokines assessed at diagnosis in discovery EFORT cohort.

Table 5: Multivariable Cox regression analysis including the 3 non-invasive low risk variables and the three selected cytokines assessed at follow-up in discovery EFORT cohort

- Model 1 : NYHA I-II, 6MWD <440m, BNP<50 et β-NGF high

- Model 2 : NYHA I-II, 6MWD <440m, BNP<50 et CXCLhigh

- Model 3 : NYHA I-II, 6MWD <440m, BNP<50 et TRAIL high

- Model 4 : NYHA I-II, 6MWD <440m, BNP<50, β-NGF high, CXCL9 high et TRAIL high

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1 An in vitro method for assessing a subject’s risk of having or developing a severe form of Pulmonary Hypertension (PH) comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motil) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding

- when the level of cytokine markers P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value for each marker is predictive of a low risk of having or developing a severe form of Pulmonary Hypertension (PH).

2. The in vitro method according to claim 1, wherein the sample is a blood sample.

3. An in-vitro method for assessing a Pulmonary Hypertension (PH) patient’s risk of having a poor prognostic of survival, comprising the steps of i) determining in a sample obtained from the subject the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) ii) comparing the level determined in step i) with a reference value for each marker and iii) concluding -when the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, then said PH patient is at high risk of having a poor prognostic of survival; or

- when the level of cytokine markers β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value then said PH patient is at high risk of having a good prognostic of survival.

4. An in vitro method for monitoring Pulmonary Hypertension (PH) disease comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X- C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step ii) is higher than the levels determined at step i) and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is lower than the level determined at step i)

5. An in vitro method for monitoring the treatment of Pulmonary Hypertension (PH)comprising the steps of i) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject before the treatment, ii) determining the level of at least one cytokine marker selected from a group of gene consisting of: P-NGF (betanerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9) and TRAIL (Tumor- necrosis-factor Related Apoptosis Inducing Ligand) in a sample obtained from the subject after the treatment”, iii) comparing the levels determined at step i) with the levels determined at step ii) and iv) concluding that the treatment is efficient when the level of cytokine marker P-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motil) ligand 9) determined at step ii) is lower than the level determined at step i). and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) determined at step ii) is higher than the level determined at step i)

6. The in vitro method according to any one of claim 3 to 5, wherein the sample is a blood sample.

7. The in vitro method according to any one of claim 1 to 6, wherein the Pulmonary Hypertension (PH) is Pulmonary Arterial Hypertension (PAH).

8. The in vitro method according to claim 7, wherein the Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH), heritable Pulmonary Arterial Hypertension (heritable PAH) and drug- and toxin-induced (anorexigen) Pulmonary Arterial Hypertension (toxin-induced PAH)..

9. A method for treating Pulmonary Hypertension (PH) with endothelin receptor antagonist and/or phosphodiesterase type 5 (PDE-5) inhibitor and/or and prostacyclin derivative in a subject wherein the level of at least one cytokine selected from the list consisting of P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers obtained from said subject, have been detected, by one of the methods of claim Ito 8.

10. A method of treating Pulmonary Hypertension (PH) in a subject comprising the steps of: a) providing a blood sample from a subject, b) detecting the levels of cytokines selected from the list consisting of P-NGF (beta-nerve growth factor), CXCL 9 (chemokine (C-X-C motif) ligand 9), and TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) markers c) comparing the level determined at stet b) with a reference value for each marker and if the level of cytokine markers β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are lower than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) are higher than the reference value then, treating the subject with endothelin receptor antagonists and/or phosphodiesterase type 5 (PDE-5) inhibitors and/or and prostacyclin derivatives. if the level of cytokine marker β-NGF (beta-nerve growth factor) and/or CXCL 9 (chemokine (C-X-C motif) ligand 9), determined at step i) are higher than the reference value for each marker and/or the level of TRAIL (Tumor-necrosis-factor Related Apoptosis Inducing Ligand) marker determined at step i) is lower than the reference value, then treating the subject with treatment escalation of endothelin receptor antagonists and/or phosphodiesterase type 5 (PDE-5) inhibitors and/or and prostacyclin derivatives and/or lung transplantation.

11. The method according to claim 9 or 10, wherein the Pulmonary Hypertension (PH) is Pulmonary Arterial Hypertension (PAH).

12. The method according to claim 11, wherein Pulmonary Arterial Hypertension (PAH) is selected from the list consisting of: idiopathic Pulmonary Arterial Hypertension (iPAH), heritable Pulmonary Arterial Hypertension (heritable PAH) and drug- and toxin- induced (anorexigen) Pulmonary Arterial Hypertension (toxin-induced PAH).