Patent Application
20220170943
The present invention relates to an assessment test for liver disease severity. More specifically, this invention relates to a blood or plasma-based panel to assess chronic liver disease severity.
Coagulopathy and inflammation-driven vascular derangements are common in chronic liver disease (CLD). Hepatocytes produce many clotting and fibrinolytic factors and in CLD, biosynthesis of these factors is altered leading to thrombotic complications such as portal vein thrombosis (PVT). PVT is a major cause of morbidity and mortality in CLD. While some progress has been made recently with regard to the approaches to treat PVT, the ability to predict the onset of PVT and/or response to PVT management is lacking. Portal vein flow velocity is a useful parameter but there are no adequate markers and/or scores to predict PVT in CLD. Preventive anticoagulation is risky in CLD, and it is desirable to be able to identify patients for whom the benefit of thromboprophylaxis outweighs the risk of bleeding. Because systemic aberrations in hemostatic, inflammatory, and vascular systems are frequently observed in CLD, a blood panel that captures them may not only be a viable alternative to CLD staging in a point-of-care setting, but also be particularly useful in predicting PVT onset and/or response to PVT management.
The Child-Pugh score (CP) is a widely used tool to assess prognosis in patients with chronic liver disease (CLD) and cirrhosis. The score considers five factors, three of which assess the synthetic function of the liver (i.e., total bilirubin level, serum albumin, and international normalized ratio, or INR) and two of which are based on clinical assessment (i.e., degree of ascites and degree of hepatic encephalopathy).
The CP score is calculated as follows: Factor 1—Encephalopathy: None (1 point), Grade 1: Altered mood/confusion (2 points), Grade 2: Inappropriate behavior, impending stupor, somnolence (2 points), Grade 3: Markedly confused, stuporous but arousable (3 points), Grade 4: Comatose/unresponsive (3 points). Factor 2—Ascites: Absent (1 point), Slight (2 points), Moderate (3 points). Factor 3—Bilirubin: <2 mg/dL (1 point), 2-3 mg/dL (2 points), >3 mg/dL (3 points). Factor 4—Albumin: >3.5 g/dL (1 point), 2.8-3.5 g/dL (2 points), <2.8 g/dL (3 points). Factor 5—Prothrombin time/International normalized ratio (PT/INR): Less than 4 seconds above control/INR <1.7 (1 point), 4-6 seconds above control/INR 1.7-2.3 (2 points), More than 6 seconds above control/INR >2.3 (3 points). The points for each of the five factors are summated to attain a CP score.
For Child Pugh scores, Class A (“Child-Pugh-A”) is assigned for Child-Pugh assessments with 5 to 6 points. This is considered the least severe level of liver disease. The one- to five-year survival rate is 95%. Class B (“Child-Pugh-B”) is assigned for assessments with 7 to 9 points. This is considered moderately severe liver disease, with a one- to five-year survival rate of 75%. Class C (“Child-Pugh-C”) is assigned for assessments with 10 to 15 points. This is considered the most severe level of liver disease. The one- to five-year survival rate is 50%.
Critics of the Child-Pugh score have noted its reliance on clinical assessment, which may result in inconsistency in scoring. In particular, the usefulness of PT/INR has been questioned. Hepatocytes produce a variety of clotting and fibrinolytic factors. In chronic liver disease, biosynthesis of these factors is often altered leading to hemostatic aberrations.
We have discovered a novel blood or plasma-based panel to assess CLD severity. This panel comprises clotting (co)factors, i.e. fVIII, fV, protein S, and protein C; a marker of fibrinolysis (D-dimer), and markers of endothelial activation, i.e. soluble P-selectin and alternatively spliced Tissue Factor (asTF). Of these seven proteins, asTF is a recently discovered molecule. asTF may contribute to prothrombotic states and endothelial dysfunction due to its ability to activate integrin subsets on endothelial cells. We devised a seven-component scoring system that included asTF. Our scoring system was able to predict CLD severity correlating to the Child-Pugh score (CP) with high accuracy. The present invention assesses CLD severity without the use of PT/INR and/or such subjective parameters as the extent of ascites and hepatic encephalopathy.
In one embodiment of the present invention, a method for assessing the presence or severity of liver disease in a subject and treating the subject is disclosed. The method involves obtaining a blood or plasma sample from a subject, analyzing the sample and obtaining data concerning the activities or levels of the following seven proteins: protein C, factor V, factor VIII, protein S, D-dimer, sP-selectin, and asTF. A score is assigned to each of the seven protein activities and/or levels as follows: a) a protein C activity >65% is assigned a score of 0, a protein C activity of 35-65% is assigned a score of 1, and a protein C activity <35% is assigned a score of 2; b) a factor V activity >80% is assigned a score of 0, a factor V activity of 55-80% is assigned a score of 1, and a factor V activity <55% is assigned a score of 2; c) a factor VIII activity <140% is assigned a score of 0, a factor VIII activity of 140-200% is assigned a score of 1, and a factor VIII activity >200% is assigned a score of 2; d) a protein S activity >50% is assigned a score of 0, and a protein S activity <50% is assigned a score of 2; e) a D-dimer level <0.5 ng/ml is assigned a score of 0, a D-dimer level of 0.5-1 ng/ml is assigned a score of 1, and a D-dimer level >1 ng/ml is assigned a score of 2; f) a sP-selectin level >100 pg/mL is assigned a score of 0, a sP-selectin level of 50-100 pg/mL is assigned a score of 1, and a sP-selectin level <50 pg/mL is assigned a score of 2; and g) an asTF level <0.3 pg/mL is assigned a score of 0, an asTF level of 0.3-0.5 pg/mL is assigned a score of 1, and an asTF level >0.5 pg/mL is assigned a score of 2. The assigned scores of the seven proteins are summated to obtain a cumulative assessment value. The method next determines the presence or severity of liver disease in the subject based on the cumulative assessment value. Finally, a subject having been identified with liver disease is administered a treatment, a therapeutic agent, or both based on the identified severity of the liver disease. In another embodiment, the method further involves determining the presence or severity of liver disease in the subject based on the cumulative assessment value as follows: a) a cumulative assessment value of 0-1 is normal; b) a cumulative assessment value of 2-3 is the least severe level of liver disease, comparable to Child-Pugh-A; c) a cumulative assessment value of 4-7 is a moderate level of liver disease, comparable to Child-Pugh-B; and d) a cumulative assessment value of 8 or greater is the most severe level of liver disease, comparable to Child-Pugh-C.
In another embodiment of the present invention, a method for assessing the presence or severity of liver disease in a subject and treating the subject is disclosed. The method involves: obtaining a blood or plasma sample from a subject; analyzing the sample and obtaining data concerning the level of alternatively spliced Tissue Factor (asTF); incorporating the asTF level as an element in assigning a severity of liver disease score; determining the presence or severity of liver disease in the subject based on the severity of liver disease score; and administering to a subject having liver disease a treatment, a therapeutic agent, or both based on the identified severity of the liver disease. In one embodiment, the method further involves obtaining data from the blood or plasma sample concerning the activities and/or levels of the following proteins: protein C, factor V, factor VIII, protein S, D-dimer, sP-selectin; and incorporating the protein activities and/or levels as elements in assigning a severity of liver disease score. In another embodiment, the method further involves obtaining data from the blood or plasma sample concerning the activities and/or levels of the following proteins: factor V, factor VIII, protein S, D-dimer, sP-selectin; and incorporating said protein activities and/or levels as elements in assigning a severity of liver disease score. In yet another embodiment, the method further involves obtaining data from the blood or plasma sample concerning the activities and/or levels of the following proteins: protein C, factor V, protein S, D-dimer, sP-selectin; and incorporating said protein activities and/or levels as elements in assigning a severity of liver disease score. In still another embodiment, the method further involves obtaining data from the blood or plasma sample concerning the activities and/or levels of the following proteins: protein C, factor V, factor VIII, D-dimer, sP-selectin; and incorporating said protein activities and/or levels as elements in assigning a severity of liver disease score.
In one embodiment of the present invention, an assessment tool to determine the severity of liver disease is disclosed. The assessment tool comprises assigning a score to each of seven protein activities and/or levels as follows: a) a protein C activity >65% is assigned a score of 0, a protein C activity of 35-65% is assigned a score of 1, and a protein C activity <35% is assigned a score of 2; b) a factor V activity >80% is assigned a score of 0, a factor V activity of 55-80% is assigned a score of 1, and a factor V activity <55% is assigned a score of 2; c) a factor VIII activity <140% is assigned a score of 0, a factor VIII activity of 140-200% is assigned a score of 1, and a factor VIII activity >200% is assigned a score of 2; d) a protein S activity >50% is assigned a score of 0, and a protein S activity <50% is assigned a score of 2; e) a D-dimer level <0.5 ng/ml is assigned a score of 0, a D-dimer level of 0.5-1 ng/ml is assigned a score of 1, and a D-dimer level >1 ng/ml is assigned a score of 2; f) a sP-selectin level >100 pg/mL is assigned a score of 0, a sP-selectin level of 50-100 pg/mL is assigned a score of 1, and a sP-selectin level <50 pg/mL is assigned a score of 2; and g) an asTF level <0.3 pg/mL is assigned a score of 0, an asTF level of 0.3-0.5 pg/mL is assigned a score of 1, and an asTF level >0.5 pg/mL is assigned a score of 2. The assigned scores of the seven proteins are summated to obtain a cumulative assessment value.
In another embodiment of the present invention, a severity of liver disease score based on the assessment tool is determined as follows: a) a cumulative assessment value of 0-1 is normal; b) a cumulative assessment value of 2-3 is the least severe level of liver disease, comparable to Child-Pugh-A; c) a cumulative assessment value of 4-7 is a moderate level of liver disease, comparable to Child-Pugh-B; and d) a cumulative assessment value of 8 or greater is the most severe level of liver disease, comparable to Child-Pugh-C.
The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein.
While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
As used herein, the term “liver disease” means a disorder that causes the liver to malfunction or cease functioning all together.
The term “chronic liver disease” as used herein denotes a disorder of the liver that involves progressive destruction and regeneration of the liver parenchyma leading to fibrosis and cirrhosis. Chronic liver disease causes can be any condition that results in the gradual degradation and renewal of the tissue cells with a body's liver.
The term “comparable,” as used herein, means of the same approximate value.
The terms “treat,” “treatment,” and “treating,” as used herein, refer to a method of alleviating or abrogating a disease, disorder, and/or symptoms thereof in a subject.
As used herein, a “subject” refers to a mammal. Optionally, a subject is a human or non-human primate. Optionally, the subject is selected from the group consisting of mouse, rat, rabbit, monkey, pig, and human. In a specific embodiment, the subject is a human.
One of the leading methods for diagnosing the severity of liver disease, Child-Pugh, involves clinical assessments and subjective parameters such as the extent of ascites and hepatic encephalopathy. We have discovered a method of diagnosing the severity of liver disease that is simpler and less subjective. For patients as well as physicians, non-subjective (e.g. all-labs based) scores are more reliable and “portable” between health care providers because they cannot be influenced by such subjective elements of physical examination as “degree of ascites” and/or mental status. This comprises a major advantage for the new system that we have discovered.
Also, our system is less prone to subjective adjustments by practitioners. For example, even though the CP scale is split into A, B, and C, there are sub-sections in each CP group that liver specialists often note and use for tracking individual patient's disease dynamics longer term. Since our new scoring system does not require clinical assessment beyond laboratory testing, we contend that it is more objective and reliable. In addition, our scoring system appears to be particularly reflective of the current/future hemostatic & vascular statuses of the patient. Its seven components are proteins participating in clot formation (or a result of clot breakdown: d-dimer) as well as markers of perturbed vasculature (asTF, soluble p-selectin).
We explored the patterns of plasma alterations relative to CP scores to establish a plasma-based panel that can help evaluate the extent of CLD severity without the use of PT/INR and/or such subjective parameters as the extent of ascites and hepatic encephalopathy.
Our novel panel comprises a combination of clotting (co)factors (fVIII, fV, protein S, and protein C); a marker of fibrinolysis (D-dimer), and markers of endothelial activation, i.e. soluble P-selectin and alternatively spliced Tissue Factor (asTF). We devised a seven-component scoring system that was able to predict CLD severity with a high correlation to previously assigned CP scores. In fact, when the seven parameters are used together, the method exhibited a 97.2% correlation to previously assigned CP scores. We also note that reducing the panel to six factors (by removing either protein C or factor VIII) still showed a correlation of over 95%.
Of the seven proteins in our panel, asTF is a recently discovered molecule. asTF may contribute to prothrombotic states and endothelial dysfunction due to its ability to activate integrin subsets on endothelial cells. We found that asTF activates track CLD severity, after studying asTF plasma levels in healthy subjects, patients suffering from stage F0-3 liver fibrosis, liver cirrhosis, as well as hepatocellular carcinoma (HCC). asTF activities were found to increase with worsening CP scores. In fact, we found that asTF activities are higher in subjects previously assigned a CP-C score vs those previously assigned a CP-B or A score.
Once the severity of liver disease is determined using our method, the subject can receive treatment. The treatment will vary, based on the level of liver disease severity. For example, treatment for the least severe level of liver disease may include a weight loss program comprising, for example, a regimen of dieting and exercise. Treatment for the most severe level of liver disease may be a transplant.
Additionally, subjects may be administered one or more therapeutic agents for treating liver disease. Such therapeutic agents can include, for example, metformin (e.g., Glucophage XR, Bristol Myers Squibb, Princeton, N.J.), ursodiol, and ppar-gamma agonists (e.g., pioglitizaone).
An effective amount of a therapeutic agent may include a dosage sufficient to provide a medically desirable result in a subject having a particular disease or condition. The effective amount will vary with the particular disease or condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of any concurrent therapy, the specific route of administration, and the like factors within the knowledge and expertise of the health practitioner. It will be recognized that when the therapeutic agent is used in acute circumstances, it can be used to prevent one or more medically undesirable results that typically flow from such adverse events. It is expected that dosages will range depending on the method of administration. In the event that a response in a subject is insufficient at the initial dosages applied, higher dosages (or effectively higher dosages by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple dosages per day are contemplated to achieve appropriate systemic levels of the therapeutic agent.
A study was conducted in CLD patients suffering from cirrhosis due to untreated hepatitis C virus (HCV) and/or excessive alcohol consumption and patients were assessed on a CP scale: CP-A, n=12; CP-B, n=19; CP-C, n=11. Healthy control subjects (n=30) were age & sex matched. The following was measured: pro-coagulant phospholipid, fXa, D-dimer, fVIII, sP-selectin, thrombomodulin, protein C, protein S, asTF, free TFPI, and TFPI/protein S ratio. The activities and/or levels of fVIII, D-dimer, and asTF increased with CLD severity while protein C, fV, protein S, and sP-selectin activities and/or levels decreased (Table 2). In combination, these seven parameters were able to predict CP scores with high accuracy. We devised a scoring system which assigns a numeric value (0, 1, or 2) for each parameter's range (Table 1). A cumulative score of <2 is normal, 2-3 CP-A, 4-7 CP-B, and ≥8 CP-C (Table 3). The system was 97.2% accurate at predicting the CP score: of the 72 plasma samples analyzed, there was 1 false positive (a healthy subject scored as a CP-A), and 1 CP-C scored as a CP-B. Omission of any of the seven components of the panel markedly lowered the scoring system's accuracy. Tables 1-3 are below:
Regarding Table 2, as CLD severity increased, activities and/or levels of fVIII, D-dimer and asTF increased while the activities and/or levels of protein C, fV, protein S, and sP-selectin decreased; the activities and/or levels of these seven proteins correlated to previously assigned CP scores with high accuracy. Our scoring system, which assigns a numeric value for each parameter (Table 1), exhibited 97.2% correlation to previously assigned CP scores. The correlation diminished if we removed any of the seven parameters. Table 4 shows the reduction in the degree of correlation to previously assigned CP scores when a given component is excluded from the panel.
As stated above, when the seven parameters are used together, the method exhibited a 97.2% correlation to previously assigned CP scores. Removing either the protein C or factor VIII parameter gave a 95.77 correlation. Removing the protein S parameter gave a 94.36 correlation.
A novel element in this panel is alternatively spliced Tissue Factor (asTF), a soluble, secreted form of TF. Structures of the major pro-coagulant full-length TF (flTF) and asTF are shown in
We analyzed asTF plasma levels in healthy subjects, patients suffering from stage F0-3 liver fibrosis, liver cirrhosis, as well as hepatocellular carcinoma (HCC). asTF plasma levels were measured using custom sandwich enzyme-linked immunosorbent assay (ELISA) prototypes. Values were expressed as median with interquartile range (IQR). The lowest median plasma asTF concentration (94 pg/ml, IQR: 33-275) was found in the healthy control group. Significant differences between asTF levels in the plasma of healthy subjects and those in grade F0-1 fibrosis patients (p<0.001), grade F2-3 fibrosis patients (p=0.019), cirrhosis patients (p=0.004), and HCC patients (p<0.001) were found using a Wilcoxon rank-sum test. asTF levels were found to increase with worsening CP scores (
In another study, plasma was isolated from citrated whole blood. All CLD patients (n=42) were previously assigned a CP score; healthy controls (n=30) were age and sex matched. Consent was obtained. D-dimer, fV, fVIII, sP-selectin, protein C, protein S, and asTF were measured. Thrombin generation tests were performed. MVs of various origin were studied. Specified cutoffs for each parameter were derived using clinical ranges and the data generated in this study to compile a CLD severity score. Immortalized human liver sinusoidal endothelial cell line TMNK-1 was assessed for the expression of TF and MV release.
MVs are released in increased quantities by activated endothelial cells. To evaluate whether plasma asTF levels positively associate with the levels of endothelium-derived MVs, we measured the levels of CD31+/CD41− MVs in all CLD plasma samples by FACS. The levels of CD31+/CD41− MVs increased with disease severity (p<0.0001, data not shown). Strikingly, there was a strong and highly significant positive correlation between the levels of asTF and CD31+/CD41− MVs (r=0.63; p=7.81×10-6) (see
Given the strong positive correlation between plasma levels of asTF and CD31+/CD41− MVs in patients with CLD, we sought to determine whether cytokine stimulation of an immortalized human liver sinusoidal endothelium cell line (TMNK1) can mimic these observations in an in vitro setting.
In one embodiment, the method of the present invention is a blood and/or plasma panel with utility in assessing CLD severity in a point-of-care setting. In another embodiment, the method is useful in predicting PVT onset and/or response to PVT management. The scoring system of the present invention exhibits a 97.2% correlation to previously assigned CP scores and does not require clinical assessment beyond laboratory testing. In addition, we have shown that asTF levels positively correlate with those of CD31+/CD41− MVs and both increase with CLD severity.
All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” and/or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
The present application is a continuation of PCT Application No. PCT/US2019/045092, filed on Aug. 5, 2019, which claims the benefit of U.S. Provisional Application Ser. No. 62/714,161, filed Aug. 3, 2018, which application is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/45092 | 8/5/2019 | WO | 00 |
