Novel methods for heart failure diagnosis and treatment

Abstract

An in vitro method for detecting or diagnosing a Heart Failure subtype defined by left ventricular ejection fraction (EF) in a subject, comprises: (a) detecting or measuring at least one extracellular

Description

FIELD OF THE INVENTION

[001] The present invention relates to novel diagnostic means for the diagnosis, prevention, or treatment of heart diseases, such as heart failure. BACKGROUND OF THE INVENTION

[002] Heart failure (HF) is a clinical syndrome heterogeneous disease, encompassing a wide range of symptoms, including breathlessness, excessive fatigue, reduced exercise tolerance, body and limb swelling, and signs of fluid retention such as peripheral oedema. These symptoms are a consequence of impaired myocardial function, which causes an inability to maintain cardiac output in response to metabolic demand. Heart failure affects l%-3% of people worldwide (Savarese G et al., 2023), with specific subpopulations having an increased risk of being affected (e.g., more than 10% of those over the age of 70 are affected) and with a risk of death due to HF that is 50-75% over 5 years. As indicated in official clinical guidelines (McDonagh T et al., 2021), there is a consensus that many patients are undiagnosed and the true prevalence of HF is likely higher, and that HF prevalence is projected to increase as a consequence of the ageing population or as a consequence of lower socioeconomic conditions (Bragazzi N et al., 2021; Wei S et al., 2023).

[003] HF is caused by an impaired capacity of the heart to fill with and pump blood. HF is a complex disease due to multifactorial pathophysiological mechanisms and patient heterogeneity. The diagnosis of HF risk and the characterization of HF is established on the basis of major generic symptoms as discussed above, although the percentage of blood the heart can pump, measured by the left ventricular ejection fraction (EF) before or after a cardiovascular event (such as HF), is the functional feature of the heart that is most commonly used by clinicians. In healthy individuals, the average EF value is approximately 55%, a percentage that is variably reduced in symptomatic individuals who are defined as HF candidates (due to any cardiovascular or other affection) in three main groups: at highest HF risk having a reduced EF (HFrEF, presenting EF <40%), at high HF risk having a mildly reduced EF (HFmrEF, EF 41-49%), or at high HF risk having a preserved EF (HFpEF, EF >50%), whereby HFmrEF and HFpEF share spontaneous or inducible increased left ventricular filling pressures (Santulli S et al., 2022; Golla M and Shams P, 2024).

[004] Unlike HFrEF, where impaired contractility and ventricular remodelling predominate, HFpEF pathophysiology typically involves comorbidities and risk factors such as hypertension, obesity, diabetes, hyperlipidemia, and obstructive sleep apnea. The clinical consensus is that the risk of adverse cardiovascular events is lower in patients with HFmrEF or HFpEF than in those with HFrEF, while the risk of non-cardiovascular adverse events is greater in patients with HFmrEF or HFpEF than in those with HFrEF. Standard-of-care treatments for individuals with HFrEF lack efficacy for HFpEF patients. Moreover, HFpEF and HFrEF present major differences (Simmonds S et al., 2020): HFpEF is preceded by chronic comorbidities (such as hypertension, type 2 diabetes mellitus, obesity, and renal insufficiency), whereas HFrEF is often preceded by the acute or chronic loss of cardiomyocytes due to ischemia, a genetic mutation, myocarditis, or valvular disease. HFmrEF can progress into either HFrEF or HFpEF. Indeed, patients who present HFpEF are more likely to be older and female compared to those presenting HFrEF and HFmrEF.

[005] HF-specific intervention should be implemented early to prevent mortality, morbidity and poor patient-reported outcomes. The clinical characteristics and management of HFrEF, HFmrEF and HFpEF have been described in major HF randomized clinical trials (McDonagh T et al., 2021; Savarese G et al., 2022). Treatment options for the three HF types differ considerably. HFrEF and HFmrEF can be treated by a specific compound or combinations of compounds chosen among ACE inhibitors, beta-blockers, dapaglifozin / empaglifozin (SGLT2 inhibitors), mineralocorticoid receptor antagonists, GLP-1 agonists, and loop diuretics for fluid retention. Most of these treatments lack efficacy for HFpEF management. HFpEF is usually treated with diuretics and treatment of the cardiovascular and extra-cardiovascular comorbidities, such as hypertension, obesity, and Type 2 Diabetes (McDonagh T et al., 2021; Jasinska-Piadlo A and Campbell P, 2023).

[006] HFrEF, HFmrEF and HFpEF present differences in both the development and progression of the disease that are secondary to changes at the cellular and molecular level. Indeed, the correct diagnosis for HFpEF remains a challenge. Although there have been attempts at using a systematic combination of objective HF testing of symptoms, signs, echocardiography findings and biomarkers, with score-based algorithms showing varying diagnostic performance, these approaches have not adequately integrated the use of a diverse range of biomarkers. The N-terminal brain natriuretic peptide (NT-proBNP) is presently considered as the benchmark clinical biomarker (Von Haeling S et al., 2024; Jasinska-Piadlo A and Campbell P, 2023; Formiga F et al., 2024; Javed N et al., 2024). However, there is no gold standard for correctly diagnosing and assigning patients to HF subtypes that are related to ejection fraction in order to apply the most appropriate treatment. Therefore, new sensitive biological indicators and molecular signatures are needed to help early diagnosis, to differentiate the subtypes of the disease, and to improve their prognosis or prevention.

[007] Blood samples are routinely analysed in medical practice as a starting point to define clinical status in individuals in a non-invasive manner. Cells and other biological or inorganic components can be identified and precisely measured, even at very low concentrations, using a variety of technologies that allow for performing "liquid" or "fluid" biopsies that are suitable for determining the presence of intracellular components that are released into the bloodstream from dying cells. Among such components, chromatin is of particular interest. Chromatin is the complex of histones and DNA in the cell nucleus. The nucleosome is the basic repeating unit of chromatin, made up of 146 base-pairs of DNA wrapped around an octameric core of proteins called histones (H2A, H2B, H3, H4), while linker Hl allows for compacting clusters of nucleosomes. Histone composition, isoforms and post-translational modifications allow regulating genome compaction and gene expression (Mansisidor A and Risca V. 2022; Kirkiz E et al., 2023).

[008] Upon cell death, cell-free DNA (cfDNA) and histones are constantly released into the bloodstream both in healthy and in pathological conditions. In particular, histones, nucleosomes, and cfDNA, separately or still complexed in nucleosomes, have been shown to trigger inflammation at local and systemic levels (Marsman G et al., 2016; Storci G et al., 2018; Church M et al., 2021). Such extracellular, circulating nucleosomes and other subcellular biological entities that contain histones (as post-transcriptionally modified, sequence variant, or wild-type forms) together with or without DNA, have been studied as cell by-products of diagnostic interest that can be detected in liquid biopsies, in particular as biomarkers of cancer, inflammation, metabolic diseases, sepsis, or stroke (TulanovO and Tamkovich S, 2022; Buzova D et al., 2022; Chen Z et al., 2023; Tsoneva D et al. 2023; Sinictyn P et al., 2023; Su F et al., 2024). A number of technologies have been developed for detecting circulating nucleosomes and histones and / or DNA (Everitt M et al., 2021; Garcia-Gimenez J et al., 2023; Shahriari S and Ravi Selvaganapathy P, 2024; WO2013 / 030579; WO2016 / 067011; WO2017 / 068359; WO2021 / 038010).

[009] However, the relevance for diagnostic use in HF remains poorly defined and understood. In a clinical study involving HFrEF patients, cfDNA levels were associated with morbidity and mortality (Salzano A et al., 2021). Other studies have generically evoked the possibility of using circulating nucleosomes, histones modifications and related epigenetic features for evaluating heart failure (Hamdani N et al., 2021; Rabkin S and Wong C, 2023). Accordingly, there is a need for methods of effectively detecting and characterizing heart failure that are based upon novel non-invasive means, involving biological indicators and signatures found in biological samples that are easy to obtain, store, and analyse. SUMMARY OF THE INVENTION

[0010] The present invention relates to the novel understanding of the diagnostic value of circulating nucleosomes, histones and histone complexes as biomarkers of HF, in general or with respect to specific subtypes, in particular those defined by left ventricular ejection fraction (EF). These findings allow defining methods for determining HF subtypes or related HF risk in subjects by performing liquid biopsies that are analysed using imaging technologies, in particular flow cytometry, permitting determination of the size and composition of circulating, subcellular biological complexes. This approach may provide clinicians with relevant data that can integrate those generated using other technologies for refining diagnosis that are based upon the detection of one or more additional biochemical, metabolic, genetic, or physiological criteria within biological fluids, such as plasma, isolated from HF patients and / or individuals presenting a HF risk that is defined by ejection fraction. The technologies that can be further combined for assessing HF subtypes may involve, for instance, the analysis of sequences and epigenetic features found in cfDNA, such as those obtained by NGS (Next-Generation Sequencing) and bioinformatics. Thus, canonical histones and their isoforms, variants, and subsequent combinations, as detected within circulating histone complexes, can be exploited as circulating biomarkers that, alone or in association with sequence or epigenetic information within cell-free DNA, can be clustered according to clinical parameters and / or information defining other biological entities relating to HF subtypes, for instance allowing the in vitro determination of the cellular and / or tissue provenance of circulating histone complexes.

[0011] In one embodiment, the present invention relates to in vitro use of an extracellular, circulating histone canonical form, isoform, and / or variant, (such as those associated with circulating nucleosomes or other extracellular chromatin fractions found in serum or plasma) for diagnosing HF and determining HF subtypes as defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF) in a subject. In particular, the canonical form, isoforms, or variants of H2A, alone or in combination with other histones (as identified within dimer / multimer complexes or as mono / oligonucleosomes^ can be used as a biomarker within plasma for differentiating HFpEF patients from HFrEF patients and / or healthy individuals and thus allowing HFpEF assessment using a biological fluid in a more precise manner. In a further embodiment, the present invention relates to the use of a binding agent that specifically binds a histone canonical form, isoform, and / or variant (in particular the canonical form or a variant of H2A, alone or in combination with other histones) for detecting, isolating and / or purifying circulating nucleosomes or other extracellular chromatin fractions (as extracellular, circulating complexes including dimer / multimer histones or as mono / oligonucleosomes) that are associated with subtypes as defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF), when compared to those detected in biological fluids obtained from patients affected by other HF subtypes or healthy individuals.

[0012] In another aspect, the present invention relates to an in vitro method for detecting or diagnosing a Heart Failure subtype (in a subject, the method comprising detecting or measuring at least one circulating histone and / or histone complex in a body fluid sample of said subject, identifying the HF status of said subject based on the level of the circulating histone(s) and / or histone complex(es) detected or measured in said body fluid sample, wherein the HF subtype is defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF). In an embodiment, the in vitro method comprises detecting or measuring at least one circulating histone (being its canonical form, isoform, and / or variant) or at least two different circulating histones (being any of their canonical form, isoform, and / orvariants). In anotherembodiment, the in vitro method comprises detecting or measuring at least one circulating histone complex (including dimer / multimer histones or mono / oligonucleosomes), at least two different circulating histone complexes, or at least one different circulating histone and at least one circulating histone complex. In an embodiment, the histone and / or histone complex is an extracellular, circulating histone and / or histone complex, or the histone and / or histone complex is associated with an extracellular, circulating nucleosome. In another embodiment, the extracellular, circulating nucleosome is a mononucleosome or an oligonucleosome, that may also contain DNA. In an embodiment, the circulating histone and / or the histone complex is or comprises a canonical histone, a histone variant, or a histone isoform. In an embodiment, the histone is histone H2A, macroH2Al, H2B, H3, or H4, and / or the histone complex comprises at least an H2A-H2B heterodimer or at least an H3-H4 heterodimer. In another embodiment, the histone isoform or variant is an isoform or variant of histone H2A. In an embodiment, the variant or isoform of histone H2A is selected from macroH2Al.l, macroH2A1.2, macroH2A2 and H2AZ.

[0013] In another embodiment, the body fluid sample is a blood, serum, or plasma sample. In an embodiment, the body fluid sample is a plasma sample. In another embodiment, the histone and / or histone complex is detected using antibodies specific for the histone and / or histone complex. In another embodiment, the circulating histone and / or histone complex is detected or measured using flow cytometry, such as advanced imaging flow cytometry ImageStream-X or other technology allowing detecting and measuring circulating biological objects within a subcellular size range. In an embodiment, the heart failure is selected from Heart Failure with reduced ejection fraction (HFrEF, <40%), Heart Failure with mid-range reduced ejection fraction (HFmrEF, 41-49%), and Heart Failure with preserved ejection fraction (HFpEF, >50%). In an embodiment, an elevated level of H2A is positively correlated with increased ejection fraction (EF). In an embodiment, an elevated level of H2A in the body fluid sample is indicative of Heart Failure with preserved ejection fraction (HFpEF, >50%) in the subject.

[0014] In another aspect, the present invention relates to a diagnostic method for diagnosing a HF subtype that is defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF) in a subject, the method comprising detecting or measuring at least one circulating histone and / or histones complex in a body fluid sample of said subject, and identifying the HF status of said subject based on the level of the circulating histone(s) and / or histone complex(es) level detected or measured in said body fluid sample.

[0015] In another main embodiment, the invention provides a method for detecting / diagnosing HF subtypes that is defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF in an animal or a human subject comprising the steps of (i) detecting or measuring a histone canonical form, isoform, and / or variant (in particular the canonical form or variants of H2A, alone or in combination with other histones) in a biological fluid (such as plasma) obtained from a subject; and (ii) using the nucleosome-associated histone isoform level detected to identify the HF status of the subject (such as HFpEF when compared to other HF subtypes or health individuals). In another embodiment, the invention provides a method for assessing the suitability (or the effects) of a treatment that is standard-of-care or under development for one or more HF subtypes (such as HFpEF when compared to other HF subtypes or healthy individuals) comprising the steps of: (i) detecting or measuring a histone canonical form, isoform, and / or variant (in particular the canonical form or variants of H2A, alone or in combination with other histones) in a biological fluid (such as plasma) obtained from a subject; and (ii) using the nucleosome-associated histone isoform level detected to identify the HF status of the subject (such as HFpEF when compared to other HF subtypes or healthy individuals).

[0016] In a further embodiment, the invention provides an immunoassay method comprising the steps of (i) contacting a biological sample with a binding agent that specifically binds a histone canonical form, isoform, and / or variant (in particular those of H2A, alone or in combination with those of other histones); (ii) contacting the sample with a second binding agent that binds to an epitope of a nucleosome (such as DNA or another histone); (iii) detecting and / or quantifying the binding of said first and / or second binding agent to said epitope; and (iv) using such binding as to identify an HF subtype as defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF).

[0017] In a further embodiment, the present invention relates to a kit comprising a binding agent that specifically binds a histone canonical form, isoform, and / or variant (in particular the canonical form or a variant of H2A, alone or in combination with other histones) for detecting, isolating and / or purifying circulating nucleosomes (as extracellular complexes including dimer / multimer histones or as mono / oligonucleosomes) that are associated with a HF subtype that is defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF) from a biological fluid.

[0018] In some preferred embodiments, the methods of the invention may further comprise additional steps that involve the use of technologies for simultaneously or sequentially identifying, the DNA associated to circulating histone complexes, and / or other circulating molecular entities that may allow improving or confirming the assessment of HF subtypes as defined by ejection fraction (in particular HFrEF, HFrmEF, and HFpEF, such as other nucleic acids (DNA or RNA), proteins, lipids, sugars, or other antigens or epitopes known to be relevant for defining HF subtypes, when compared to other HF subtypes or healthy individuals. These other circulating molecular entities can be identified and quantified using other technologies (such as immunoassays, DNA / protein sequencing, proteomics, or mass spectrometry). These methods may also be combined with one or more clinically validated tests for evaluating HF status, such as measuring a left ventricular ejection fraction (i.e. EF measurement) of the subject's heart by using an echocardiogram, cardiac catheterization, magnetic resonance imaging, computerized tomography, and / or a nuclear medicine scan, in order to establish the most appropriate diagnosis and / or drug treatment.

[0019] Further embodiments, such as uses, methods, and kits according to the present invention, are disclosed in the Detailed Description and in the Examples herein. BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1: Detection and categorization of histone-positive entities in blood fractions from the plasma of a healthy volunteer, using a fluorescent antibody. Depicted are exemplary images for Circulating Histone Complexes (CHICO; small CHICO with a size comprised within 0-20 p.m2; medium CHICO with a size comprised within 20 and 40 p.m2; large CHICO with a size beyond 40 p.m2 such as cells) that are Histone 2A (H2A)-positive when observing the sample treated with a fluorescent anti-H2A antibody using fluorescence or bright-field image acquisition (A). The normalized frequencies for the three CHICO types within such an exemplary blood sample are analyzed on the basis of the size of the H2A-positive fluorescent area, as measured in square micrometres (p.m2; B).

[0021] Figure 2: CHICO signatures within plasma obtained from subjects previously diagnosed with HFpEF (diagonally hatched boxes, n=25) or HFrEF (vertically hatched boxes, n=25) when compared to 20 healthy subjects (white boxes, n=30) in plasma. Analysis of relative histone abundance (upon log and quantile transformation) within CHICO is performed at the level of the specific histone (A) or at the level of specific H3 / H4-containing complexes (B), using optical density (defined as Arbitrary Units; AU). Statistically significant differences between groups are indicated with one star (p-value<0.01), two stars (p-value<0.001), or three stars (p-value<0.0001). DETAILED DESCRIPTION

[0022] Diagnostic Targets

[0023] The methods, uses, and kits of the invention are intended to be applied to extracellular circulating histones and / or histone complexes (CHICO, collectively), as identified with appropriate binding agents and technologies, in particular as extracellular biological entities found within body fluids (in particular blood, sera or plasma) defined on the basis of the presence of histones and of a subcellular size. The literature and sequence databases disclose many details about histone sequences and their medical or biological features in humans, not only with respect to main members of this protein family (Hl, H2A, H2B, H3, H4), but also for a large number of other histone subtypes that defined on the basis of sequence and / or post-translational modifications (Kirkiz E et al., 2023). The detection of specific histone canonical forms, variants and isoforms within CHICO, such as those described in the Examples, may be extended in parallel to multiple histones and / or to combinations of histones that are combined and co-localized within CHICO. For instance, the analysis may combine the detection and measurement of CHICO that are positive for at least 2, 3, 4 or more histones (such as H3 and H4; H2A and H2B; H2A and macroH2Al, in general or a specific isoform; H2A, H2B, and one or more among macroH2Al, H3 and H4).

[0024] The determination of histone components within CHICO may be associated to simultaneous evaluation of other CHICO criteria. The first main one is the size, as determined by the detection technology, which preferably below the size of circulating human cells, in particular below 50p.m2, 40 p.m2 or 30 p.m2. The analysis may be limited to specific ranges (for instance between 10 and 40p.m2; 20 and 40p.m2; 0 and 20p.m2; or 0 and 40p,m2) that may be relevant to establish CHICO subgroups that combine distinctive size and histone composition and that may be present different distribution or abundance among different HF subtypes as determined by ejection fraction or other criteria.

[0025] Diagnostic Products

[0026] The analysis of CHICO composition is preferably based on the use of products that allows the identification of histone sequence and / or post-translational modifications within biological fluids. The most commonly used tools for performing such analysis are natural, recombinant, or synthetic antibodies (or artificial molecules that contain histone-binding protein sequences) that are raised and selected for binding one or more of the canonical histone isoforms, variants, and post-translationally modified forms. The Examples provides details for only a few of all histone-specific antibodies that are commercially available from many different companies (such as Abeam, Merck Millipore, Cell Signaling, Santa Cruz Biotech, Sigma-Aldrich) or public repositories and institutions.

[0027] Aside from histone specificity, the choice of the antibody should be made also on the basis of other criteria such as the compatibility and correct data interpretation when using the selected technology for CHICO analysis (such as advanced flow cytometry technologies), when combining the analysis with other antibodies that may be used for evaluating CHICO composition (being or not histone-specific), and / or when choosing detection approach (using directly labelled antibodies or, as in Examples, amplifying the signal by using an unlabelled anti-histone antibody and a secondary, host-specific labelled antibody). The antibody-specific signal may be measured using a radioactive, enzymatic, isotopic, fluorescent, or other type of labelling that is compatible with the selected technology for CHICO analysis and, if needed, that allow obtaining distinctive signals when two or more anti-histone antibodies that are simultaneously applied to the biological sample that contains CHICO.

[0028] Diagnostic Technologies

[0029] CHICO analysis may be pursued using pure, partially purified, or fractionated biological fluids, preferably after eliminating the cellular elements within the biological fluid. Preferably the fluid is plasma or sera, depending on whether obtained from blood samples in which clotting has been prevented (using an anti-coagulant) or after eliminating the clotted fraction. The separation of the CHICO-containing liquid fraction may also comprise a centrifugation, an affinity binding protocol, and / or any other technical step that allows eliminating cells or other entities under or above a given size, followed or not by an appropriate protocol for sample archiving and maintenance if analysis is not pursued immediately after its preparation.

[0030] The label detection, size measurement, and other CHICO features are determined in the biological fluid preferably by flow cytometry, in particular using those advanced technologies that allows sample acquisition and data analysis using very small volumes of sample (below 500p.l, 200p.l, 100p.l, or 50p.l ) and combined measurement of size and labels that are detected in a given micrometer or wavelength range. Such technologies have been recently reviewed (Dimiatridis S et al., 2024). These Imaging flow cytometry technologies include Imagestream, FlowSight and other commercial platforms having the speed, throughput, and other features that are required for analysing panels of CHICO-containing samples, and that may be also associated to software for data and image analysis involving statistical analysis, machine learning, and artificial intelligence tools.

[0031] Diagnostic Readout

[0032] The detection and measurement of CHICO may be combined with methods to anticipate, reinforce or refine the prognosis and / or diagnosis of heart failure subtypes related to ejection fraction that are reported in the literature, in particular for HFpEF (Albani S et al., 2024), such as: measurement of biomarkers (such as BNP,NT-proBNP, troponins, or sST2), echocardiography (ECG), score-based algorithms, cardiac magnetic resonance (CMR), cardiac computed tomography (CCT), cardiopulmonary exercise testing (CPET), nuclear medicine (such as single photon emission computed tomography imaging). The comparison of histone components within CHICO may be pursued using panels of samples from different groups of subjects that are used as control or representative of a given HF subtype, in particular as determined on the basis of ejection fraction, prior drug treatment, drug response, age, sex, body mass index, co-morbidities and / or any other clinically relevant criteria for HF that is established by medical community or health authorities.

[0033] The abundance of CHICO-based biomarker signatures (based upon the presence of one or more histone canonical form, isoform, or variant) having different composition should be evaluated in panels of subjects that can be compared and used for evaluating any statistically relevant difference by applying the appropriate tests and algorithms for confirming any under- or over-representation of one or more CHICO subpopulations among the panels. This qualitative and quantitative analysis may lead, as shown in the Examples, to defining p-values that define how any CHICO-based readout may be associated to HF patients with respect to general population and / or to a specific HF subtype.

[0034] This analysis may be also completed or extended by analysing the genetic information that is associated to CHICO (such as sequence and / or epigenetic modifications of residual DNA molecules) found circulating in the biological fluid (such as from DNA or RNA of human or non-human origin), expressed in specific cells or tissues (for instance those present in the heart), or at the genomic level within a subject or a population.

[0035] Therapeutic Decisions Consequent to the Diagnostic Readout

[0036] Such CHICO-based differences, together with the level of statistical confidence based upon the p-values (below 0.05, 0.01, 0.001, 0.0001 or even lower), may then be analysed with respect to the criteria for establishing each panel of samples, or any other criteria that has been assessed in the entire population or study, so that some diagnostic, prognostic, or medical conclusion can be made and consequent decision can be taken. This analysis may lead to the start (or interruption) of a HF-specific medical intervention in the subject that has been object of the CHICO analysis or in other subjects that may share other HF-related parameters. This decision may involve starting, continuing, monitoring, reducing, or interrupting a drug treatment (HF-specific or related to a co-morbidity such as hypertension, type 2 diabetes mellitus, obesity, and renal insufficiency), a device-based therapy (such as remote pulmonary artery pressure monitoring, interatrial shunt device, implantable hemodynamic monitoring by remote pulmonary artery pressure, Interatrial shunt device, HF pacemaker), or HF-relevant lifestyle interventions.

[0037] Other features of the invention will also become apparent in the Examples, including the biological samples, the technologies, and the comparative assays which provide the required experimental support, without limiting its scope. EXAMPLES

[0038] Example 1: Analysis of specific circulating histones and / or histone complexes for discriminating HF patients that present different ejection fraction profiles

[0039] Materials &methods

[0040] Protocol for detecting and measuring circulating histones in human plasma samples. The protocol for detection of circulating histones and histone complexes using ImageStream-X technology has been previously described (Buzova D et al., 2022). Some changes have been included compared to the prior publication since some of the primary antibodies were no longer available from manufacturers. Thus, different primary antibodies were used in this protocol, while maintaining the same staining conditions. Additionally, a lower amount of plasma (25 pl instead of 50 pl) in combination with a lower amount of primary antibody (0.5 pg instead of 1 pg) was used for determining histone presence within CHICO, thus maintaining the same ratio between the amount of primary antibody and plasma volume (1:50). Lower volumes of sample and antibody reagents could be used to measure the level of histones and histones complexes, which is of utmost importance in case of limited sample volumes being available, depending on the chosen technology.

[0041] The presence of the following human histones within CHICO has been determined in each group of plasma samples: H2A, H2B, H3, H4, macroH2Al.l, and macroH2A1.2. The primary antibodies that were used for detecting each human histone are listed in Table 1.

[0042] TABLE 1 Histone Antibody name (host species) Antibody provider (catalog no.) H2A Anti-H2A antibody - ChIP Grade (Rabbit) Abeam (abl8255) macroH2Al.l MacroH2Al.l antibody D5F6N (Rabbit) CST(12455S) macroH2A1.2 MacroH2A1.2 antibody #4827 (Rabbit) CST (4827S) H2B Purified anti-Histone H2B antibody (Rat) BioLegend (606302) H3 IHC-plus™ Polyclonal H3 antibody (Sheep) LSBio (LS-B6334-125) H4 Anti-H4 antibody - ChIP Grade (Mouse) Abeam (ab31830)

[0043] The fluorescently labelled, host species-specific secondary antibodies that were used for detecting the binding of each primary antibody to CHICO are listed in Table 2.

[0044] TABLE 2 Antibody name Antibody provider (catalog no.) Goat Anti-Rabbit IgG H&L - Alexa Fluor® 488 Abeam (abl50077) Donkey Anti-Sheep IgG H&L - Alexa Fluor® 555 Abeam (abl50178) Goat Anti-Rat IgG H&L - AlexaFluor® 594 Abeam (abl50160) Goat Anti-Mouse IgG H&L - Alexa Fluor® 647 Abeam (abl50115)

[0045] Preparation and storage of human plasma samples before use for histone detection. A total of 30 healthy sex / age matched individuals (mean age: 62 ± 9.9), 25 patients diagnosed with HFrEF (mean age: 69.7 ± 10.7) and 22 HFpEF patients (mean age: 72.1 ± 10), were recruited. Blood was collected in K2 EDTA-coated collection tubes and mixed by inverting the tubes three times. The blood was then centrifuged at 3,000 g for 20 min at 4°C. Following the centrifugation, the separated plasma fraction was collected, aliquoted (0.5 microliter per each aliquot), and stored at -80°C.

[0046] Data analysis. The software IDEAS was used to analyse the images that were obtained using ImageStream-X (without any subsequent modifications). The comparison of the CHICO signatures between two groups of samples was performed in Python by a Kruskal-Wallis test, followed by a post-hoc analysis. Since histones H2B, H3, and H4 are detected in all three antibody sets, the relative abundance of individual histones and histone complexes was log-transformed, scaled, and averaged to mitigate potential batch effects. The box plot in Figure 2 was generated through Matplotlib (version 3.8.1). The p-value for each data comparison is shown in Table 3.

[0047] TABLE 3 Histone-based criteria for defining CHICO p-Value Control vs. HFrEF Control vs. HFpEF HFrEF vs. HFpEF H2A 0.218 <0.01 <0.0001 H2B Undetectable H3 <0.0001 0.0889 H4 <0.0001 <0.001 0.1312 macroH2Al.l 0.0370 0.262 0.2615 macroH2A1.2 Not signi leant in the Kruskal-Wallis test H3 / H4 <0.001 0.6313 H2A / H2B / H3 / H4 <0.0001 0.4799 macroH2Al.l / H2B / H3 / H4 <0.0001 0.6090 macroH2A1.2 / H2B / H3 / H4 <0.0001 0.1120

[0048] Spearman correlation analysis between the levels of specific histones and / or histone complexes that are detected as CHICO and EF data was used to establish correlations with clinical HF criteria. The p-value for each data comparison is shown in Table 4.

[0049] TABLE 4 Histone / histone complex p-Value Correlation H2A 0.0003 Positive H3 0.1160 Not significant H4 0.1989 Not significant macroH2Al.l 0.3338 Not significant macroH2A1.2 0.0524 Not significant H3 / H4 0.4515 Not significant H2A / H2B / H3 / H4 0.3245 Not significant macroH2Al.l / H2B / H3 / H4 0.8645 Not significant macroH2A1.2 / H2B / H3 / H4 0.0782 Not significant

[0050] Results

[0051] The composition of extracellular circulating histones and / or histone complexes (CHICO, collectively) can be identified using Imagestream, a multi-channel imaging methodology that allows detecting the histone complexes within small amounts (such as 20-200 microliters) of blood samples and fractions (Dimiatridis S et al., 2024). This technology combines the speed, sensitivity, and phenotyping abilities of multicolor flow cytometry with the functional insights and size assessment of microscopy, so that CHICO can be observed and measured in a plasma sample using one or more fluorescently-labelled histone-specific antibodies in appropriate combinations, as recently published (Buzova D et al., 2022).

[0052] The diagnostic role of CHICO determination in HF was established using an imaging approach based on histone profiles in plasma to detect and distinguish healthy individuals and patients with specific HF subtypes as previously assigned by clinicians, in particular HFpEF or HFrEF. Blood was collected prior to any therapeutic intervention for generating comparable panels of HF subtype-specific plasma samples wherein the presence of individual histones, histone dimers, and nucleosomes (isolated or aggregated) can be characterized. Thus, specific histones were initially selected to analyse CHICO presence in human plasma by imaging with a flow cytometry method based on ImageStream-X technology: H2A (UniProt AC B2R5B3), selected isoforms of the macroH2Al variant (UniProt AC 075367) including those already identified as being clinically relevant such as macroH2Al.l (MacroH2Al isoform 1, binding poly-ADP-ribose), and macroH2A1.2 (MacroH2Al isoform 2, not binding poly-ADP-ribose), together with other major histones such as H2B, H3, and H4 (Kirkiz E et al., 2023). It should be noted that a splicing switch from macroH2A1.2 to macroH2Al.l expression is reported in association with cell differentiation.

[0053] As an initial validation of the assay, the plasma of a healthy volunteer was tested with an anti-histone H2A antibody coupled to Alexa-Fluor588 (green dye) in order to establish the size range and the frequency of fluorescently labelled biological entities in such plasma samples. Interestingly, the cell-size population accounts for <1% of the overall H2A-labelled object count, while the large majority of fluorescently labelled objects are extracellular, smaller, biological entities of variable size comprising H2A and possibly other histones (Figure 1). Thus, this approach may be tested to establish a standardized profiling method to compare plasma from HF patients that have previously been assigned to a given HF subtype according to presently applied clinical standard criteria, taking into account the fluorescent signal that can be assigned to CHICO having specific, subcellular size range.

[0054] The validation of this analytical method can be pursued in two exemplary groups of HF patients previously defined by standard clinical criteria as belonging to either HFpEF or HFrEF subtypes, extending detection to other histones within CHICO. When the presence of specific histones within CHICO is analysed separately, the level of each of them is generally lower in healthy individuals when compared to both groups of HF patients, but the difference is more statistically relevant for H3 and H4. Interestingly, the difference in H2A is statistically relevant for the HFpEF subtype when compared to the samples from both the healthy population and the group of HFrEF patients, suggesting that higher levels of CHICO containing this histone isoform may be used to anticipate with more precision (or at least confirm) the assignment of a HF patient to the HFpEF subtype (Figure 2A). The statistical relevance of the difference in the amount of specific combinations of histone variants or isoforms within CHICO may be evaluated, for instance when CHICO are analysed on the basis of the simultaneous presence of at least H3 and H4. In this case, CHICO are again more frequently identified in both groups of HF patients, but the difference is even more statistically relevant when the presence of any of the selected H2A isoforms is also tested (Figure 2B). Moreover, the findings that are based upon the composition of circulating histone isoforms and / or complexes comprising them can be compared with the ejection fraction (EF) data and other specific clinical criteria that are measured in the different groups. H2A was positively correlated to EF (p-value=0.0003).

[0055] Thus, an approach that allows distinguishing the size and composition of circulating histone isoforms and / or complexes comprising them that are detectable in human plasma allows discriminating HF subtypes (both HFpEF and HFrEF or between them), using a rapid and non-invasive ImageStream-X imaging technology-based liquid biopsy. These findings may be exploited to screen HF patients requiring a proper, more precise assignment to a given subtype (for instance any of those based on ejection fraction) as well as to stratify accordingly HF patients before and after treatment with a given drug regimen or dosage, in particular for anticipating safety and efficacy of the HF treatment that standard HF clinical criteria would suggest. This method may also be combined with methods for characterizing other features of CHICO in the plasma of HF patients to be assigned to a given subgroup, including the increased or decreased presence of post-translationally modified histones (Liu R et al., 2023), a specific cell or tissue origin, as potentially determined from co-identified cfDNA (Oberhofer et al., 2022, Qi T et al., 2023), altered protein-protein and protein-DNA interactions, nucleosome occupancy or position changes, activation / suppression of gene expression, and other properties that may have diagnostic and / or therapeutic relevance for a better medical management in HF patients according to the actual HF subtype.

[0056] Example 2: Combining the analysis of circulating histone isoforms and / or complexes from the plasma of HF patients with other clinical or in vitro HF criteria

[0057] The findings described in Example 1 are based on the specific detection and qualitative / quantitative characterization of CHICO, circulating histone isoforms and / or complexes found in human plasma. This approach can be integrated in methods for HF prevention, diagnosis and treatment that involve using appropriate binding tools for specific histone variant and / or isoforms to test plasma samples from HF patients. This assay may be combined with other means for evaluating the status of such patients, either in vitro, such as assays of biomarkers that are clinically, genetically, or biochemically defined, using biological samples, such as liquid or solid biopsies; or in clinical tests for evaluating heart function (for instance electrocardiograms) or other HF-specific phenotypes related to the ejection fraction (Albani S et al., 2024). Moreover, CHICO may include nuclear DNA that can be identified using labelled anti-DNA antibodies that co-localize with the labelled anti-histones antibodies. These DNA molecules may be quantified, and their sequence or epigenetic modifications determined, forthose CHICO in which the two signals are actually co-localized. Recent approaches aiming at mapping nucleosome occupancy suggested that it is possible to identify the tissue of origin of circulating free DNA (Qi T et al. 2023; Oberhofer et al., 2022). A systematic approach may be developed for elucidating the tissue or cell type of origin of cfDNA bound to histone complexes in healthy individuals or in patients assigned to any specific HF subtype and potentially establishing relationship with HF causes and symptoms.

[0058] The features of CHICO can be determined regarding any mechanisms of their release into blood, not only as a response to apoptotic signals (Wang Y et al., 2024). For instance, histones and histone complexes have been found interacting with Neutrophil ExtracellularTraps (NETs), networks of extracellularfibres composed of neutrophil genomic DNA and core histones and other antimicrobial factors, which capture and degrade invading microorganisms. Indeed, increased release of NETs can lead to a unique form of immune cell death termed 'NETosis' which causes histone release. The acquisition of HF plasma samples and associated clinical phenotyping may be followed by imaging flow microscopy technology to assess CHICO composition and amount. These data may be further combined to NGS-based or epigenetic analysis of circulating cell free DNA. These consolidated datasets may be evaluated using a hierarchical clustering analysis that applies unsupervised, probabilistic machine learning, and / or an analysis of the cell / tissue of origin of the liquid biopsies. This combination of epigenetic, genetic, and cardiovascular markers may allow obtaining a more robust, not invasive, and consistent diagnosis of HF subtypes with respect to ejection fraction, and potentially an improved medical management of HF patients using standard-of-care or experimental drugs and therapeutic protocols.

[0059] This assessment may lead to defining the risk of HF and potential treatments, including stratifying HF patients that are selected for clinical trials and elaborating the conclusions about the different endpoints. For example, the different criteria and data may be elaborated using clinical, prognostic, genetic, and other phenotyping, benchmarking data from electronic health record sources by techniques such as machine learning (ML; Banerjee A et al., 2023; Tian P et al., 2023), or Generative Topographic Mapping (GTM) that allows clustering, data stratification and visualisation for a hierarchical cluster analysis of the data from cross-sectional studies. Similar techniques can be used to identify and interpret existing and novel phenotypes among HF subtypes by integrating circulating biochemical and epigenetic biomarkers clustered according to their clinical parameters.

[0060] Moreover, using commercial kits, circulating cell-free DNA may be extracted, quantified from plasma and used for the preparation of libraries specific for blood samples from age- and sex-matched healthy volunteers with good or poor cardiovascular health (as defined by the American Heart Association or in other internationally clinical standards), and from patients assigned to specific HF subtypes such as HFrEF, HFmrEF, or HFpEF. The library DNA profiles may be sequenced, and such sequences annotated against the human genome according to implemented pipelines (Zhang W et al., 2021; Liorni N et al., 2023), to identify genome-wide loci specifically enriched in the HF subtypes. For each HF patient in the cohort, demographic data, self- and physician-reported cardiovascular-related symptoms, medical history, comorbidities, medications, lifestyle, dietary patterns, anthropometric measurements, blood pressure, ECG, and / or psychological assessment can be retrieved and compared. A panel of laboratory tests (BNP, cTnT-hs, HbAlc, hs-CRP, GDF-15, full blood count, electrolytes and / or lipid profiles) may be combined using ML and non- invasive liquid biopsy for assessing status in subjects affected by a HF subtype, in particular if associated to the ejection fraction that measured. REFERENCES Albani S et al., 2024. Hellenic Jour Card. 75:60-73. Banerjee A et al., 2023. Lancet Digit Health. 5: e370-e9. Bragazzi N et al., 2021. Eur J Prev Cardiol. 28:1682-90. Buzova D et al., 2022.Hepatol Commun.6: 3311-3323. Chen Z et al., 2023.MedComm. 4: e329. Church M et al., 2021. Int J Mol Sci. 22:10274. Dimiatridis S et al., 2024. Methods Protoc. 7: 28. Everitt M et al., 2021. ACS Sens. 6: 3006-3012. Formiga F et al., 2024. Heart Failure Reviews. 29:179-189. Garcia-Gimenez J et al., 2023. J Transl Med. 21: 344. Golla M and Shams P, 2024. NCBI Bookshelf. NBK599960. Hamdani N et al., 2021. Ham Eur Heart J. 42: 1940-1958. Jasinska-Piadlo A and Campbell P, 2023. Heart. 109: 874-83. Javed N et al., 2024. Jour Int Med Res. 52: 03000605241254330. Kirkiz E et al., 2023. Hemasphere. 7: e927. Liorni N et al., 2023. Epigenomics. 15: 863-77. Liu R et al., 2023. MedComm. 4: e292. Mansisidor A and Risca V. 2022. Nucleus. 13: 236-276. Marsman G et al., 2016. Cell Death Dis. 7: e2518. McAnena P et al., 2017. Cancers; 9: 5. McDonagh T et al., 2021. Eur Heart J. 42: 3599-726. Oberhofer et al., 2022. Diagnostics. 12:1834. Qi T et al., 2023. Int J Mol Sci. 24: 1503. Rabkin S and Wong C, 2023. Review Biomedicines. 11: 2815. Salzano A et al., 2021.Eur J Prev Cardiol 28: e28-e31. Santulli S et al., 2022. Eur Heart Jour - Cardiovasc Pharmacoth. 8: e23-e24. Savarese G et al., 2022. Nat Rev Cardiol. 19:100-16. Savarese G et al., 2023. Cardiovasc Res. 118: 3272-87. Shahriari S and Ravi Selvaganapathy P, 2024. Gels. 10:186. Simmonds S et al., 2020. Cells. 9: 242. Sinictyn P et al., 2023. Nat Biotechnol. 4: 1776-1786. Storci G et al., 2018. Semin Immunol. 40: 6-16. Su F et al., 2024. Biomedicines.12:1385. Tian P et al., 2023. J Am Heart Assoc. 12: e029124. Tsoneva D et al. 2023, J Clin Transl Hepatol. 11:1520-41. Tulanov O and Tamkovich S, 2022. Int J Mol Sci. 23: 7224. Von Haehling S et al., 2024. Clin Res Cardiol, doi: 10.1007 / s00392-024-02396-4. Wang Y et al., 2024. Biomolecules. 14: 416. Wei S et al., 2023, Eur Heart J Qual Care Clin Outcomes. 9: 662-672. Zhang W et al., 2021. Bioinformatics. 37: 4251-2.

Claims

1. An in vitro method for detecting or diagnosing a Heart Failure (HF) subtype defined by left ventricular ejection fraction (EF) in a subject, the method comprising:a) detecting or measuring at least one extracellular circulating histone and / or histone complex in a body fluid sample of said subject;b) identifying the HF status of said subject based on the level of the extracellular circulating histone and / or histone complex detected or measured in said body fluid sample.

2. The in vitro method of claim 1, wherein the in vitro method comprises detecting or measuring at least one circulating histone or at least two different circulating histones.

3. The in vitro method of claim 1, wherein the in vitro method comprises detecting or measuring at least one circulating histone complex, at least two different circulating histone complexes, or at least one different circulating histone and at least circulating histone complex.

4. The in vitro method of claim 1 or 2, wherein the histone and / or histone complex is associated with an extracellular nucleosome, preferably a mononucleosome or an oligonucleosome.

5. The in vitro method of claim 4, wherein the extracellular nucleosome contains DNA.

6. The in vitro method of any one of claims 1 to 5, wherein the circulating histoneand / or histone complex is or comprises a canonical histone, a histone variant, or a histone isoform.

7. The in vitro method of any one of claims 1 to 5, wherein the circulating histone is histone H2A, macroH2Al, H2B, H3, or H4.

8. The in vitro method of claim 6, wherein the circulating histone is a variant or an isoform of histone H2A or macroH2Al, preferably selected from macroH2Al.l, macroH2A1.2, macroH2A2 and H2AZ.

9. The in vitro method of claim 8, wherein H2B, H3, and / or H4 are further detected or measured, preferably at least H2A and H2B, or at least H3 and H4.

10. The in vitro method of any one of claims 1 to 9, wherein the body fluid sample is a blood, serum, or plasma sample.

11. The in vitro method of claim 10, wherein the body fluid sample is a plasma sample.

12. The in vitro method of any one of claims 1 to 11, wherein the circulating histoneand / or histone complex is detected using an antibody specific for a canonical sequence, a sequence variant, or an isoform of histone H2A, H2B, H3, and / or H4.

13. The in vitro method of any one of claims 1 to 12, wherein the circulating histone and / or histone complex is detected or measured using imaging flow cytometry.

14. The in vitro method of any one of claims 1 to 13, wherein the heart failure defined by left ventricular ejection fraction is selected from Heart Failure with reduced ejection fraction (HFrEF), Heart Failure with mid-range reduced ejection fraction (HFmrEF), and Heart Failure with preserved ejection fraction (HFpEF).

15. The in vitro method of claim 14, wherein an elevated level of H2A is positively correlated with increased ejection fraction (EF).

16. The in vitro method of claim 15, wherein an elevated level of H2A in the body fluid sample is indicative of Heart Failure with preserved ejection fraction (HFpEF) in the subject.

17. A diagnostic method for diagnosing a Heart Failure (HF) subtype defined by left ventricular ejection fraction (EF) in a subject, the method comprising:a) detecting or measuring at least one extracellular circulating histone and / or histone complex in a body fluid sample of said subject;b)identifying the HF status of said subject based on the level of the extracellular circulating histone and / or histone complex detected or measured in said body fluid sample.A

Images