WO2020130111A1 - Method for evaluating possibility for recovery of cardiac function in patients with cardiac insufficiency - Google Patents

Abstract

The purpose of this invention is to provide a method for evaluating the possibility for recovery of cardiac function in patients with cardiac insufficiency. Another purpose of this invention are to provide: a method for identifying patients requiring non-drug treatment from among patients with cardiac insufficiency; and a method for identifying patients predicted to have a good effect from drug treatment from among patients with cardiac insufficiency. According to this invention, a method for evaluating the possibility for recovery of cardiac function in patients with cardiac insufficiency is provided, said method including a step for measuring the level of DNA damage in cardiomyocytes. According to this invention, a method for identifying patients requiring non-drug treatment from among patients with cardiac insufficiency and a method for identifying patients predicted to have a good effect from drug treatment from among patients with cardiac insufficiency are provided, said methods including a step of measuring the level of DNA damage to cardiomyocytes.

Description

Evaluation method of heart function recovery possibility in patients with heart failure Reference to related applications

This application enjoys the benefit of the priority of prior US Patent Application No. 62/781,927 (filing date: December 19, 2018), the entire disclosure content of which is incorporated herein by reference. To be a part.

The present invention relates to a method for evaluating the possibility of recovering cardiac function in a heart failure patient using the degree of DNA damage in cardiomyocytes as an index.

Heart failure affects an estimated 38 million patients worldwide and is a leading cause of morbidity and mortality despite advances in cardiovascular therapy over the last few decades. Dilated cardiomyopathy (DCM) is a common cause of heart failure. DCM is diagnosed by left ventricular dilation and systolic dysfunction, but without myocardial dysfunction, such as pressure overload and coronary artery disease. Common therapies such as beta blockers, renin-angiotensin-aldosterone inhibitors, and cardiac resynchronization therapy are characterized by reduced left ventricular volume and improved contractile function in some patients with DCM. Leading to (LVRR), many clinical trials of DCM report decreasing mortality with increased left ventricular ejection fraction (LVEF) and decreased left ventricular end-diastolic and left-systolic volume. (Non-patent documents 1 to 3).

Since LVRR occurs only in about 40% of DCM patients, it is important to identify patients with a possibility of LVRR at an early stage when determining a treatment policy (Non-patent Documents 1 and 2). Many attempts have been made to identify LVRR predictors in patients with DCM, such as hemodynamic parameters (blood pressure, etc.), echocardiographic parameters (left ventricular end diastolic diameter, etc.), and cardiac magnetic resonance gadolinium. Interstitial fibrosis evaluated by late augmentation has been reported as a useful predictor of LVRR (Non-patent Documents 4 to 6). However, general tests such as blood pressure measurement and echocardiography are not strong predictors of LVRR, and late gadolinium enhancement evaluation by cardiac magnetic resonance has the problem of false positives and should be performed in patients with renal dysfunction. I can't. Therefore, there remains a lack of accurate methods for predicting cardiac function recovery in patients with DCM.

MerloMetetal,JAmCollCardiol2011;57:1468-76. MatsumuraYetetal, AmJJCardiol 2013;111:106-10. MerloMetetal,JAmHeartAssoc2015;4:e001504. McNamaraDMet etal,JAmColl Cardiol2011;58:1112-8. Kubanek Met et al,J Am Coll Cardiol 2013;61:54-63. Broch Ket et al, Am J Cardiol 2015;116:952-9.

The present invention aims to provide a method for evaluating the possibility of recovery of cardiac function in patients with heart failure. The present invention also provides a method for identifying a patient in need of non-drug treatment from a heart failure patient, a method for identifying a patient in a heart failure patient who is predicted to have a better effect on drug treatment, and a method for treating a heart failure patient. With the goal.

The inventors of the present invention have conducted intensive research focusing on the DNA damage of cardiomyocytes. As a result, in the biopsy specimens of patients with heart failure who did not recover the cardiac function, the degree of the DNA damage of the cardiomyocytes was the cardiac function. It was found to be significantly higher for patients who recovered The present inventors have also found that the possibility of recovering cardiac function in heart failure patients can be evaluated by using the degree of DNA damage in cardiomyocytes as an index. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A method for evaluating the possibility of recovering cardiac function in a heart failure patient, which comprises the step of measuring the degree of DNA damage in cardiomyocytes.
[2] The evaluation method according to [1] above, wherein the degree of DNA damage in cardiomyocytes correlates with the possibility of recovery of cardiac function in patients with heart failure.
[3] The evaluation method according to the above [1] or [2], wherein the degree of DNA damage is measured based on a DNA damage marker.
[4] The evaluation method according to [3] above, wherein the degree of DNA damage is indicated by the ratio of the number of DNA damage marker positive nuclei in cardiomyocytes to the total number of nuclei.
[5] The evaluation method according to the above [3] or [4], wherein the DNA damage marker is detected by immunological analysis.
[6] The evaluation method according to any one of [3] to [5] above, wherein the presence of a DNA damage marker in cardiomyocytes indicates a low possibility of recovery of cardiac function.
[7] The evaluation method according to any one of [1] to [6] above, wherein the degree of DNA damage is measured using a myocardial biopsy sample of a heart failure patient.
[8] The evaluation method according to any of [1] to [7] above, wherein the heart failure patient is a cardiomyopathy patient.
[9] The evaluation method according to [8] above, wherein the cardiomyopathy patient is a dilated cardiomyopathy patient.
[10] The evaluation method according to any one of [1] to [9] above, which is used for determining a treatment policy for patients with heart failure.
[11] A method for identifying a patient in need of non-drug treatment from a heart failure patient, comprising the step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient.
[12] The method according to claim 11, wherein the non-drug treatment is surgical treatment or regenerative medicine.
[13] The method according to claim 12, wherein the surgical treatment is heart transplantation, implantation of an assisted artificial heart, or device treatment.
[14] A method of identifying a patient from a heart failure patient who is predicted to have a better effect on drug treatment, the method comprising a step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient.
[15] A method for treating a heart failure patient, which comprises performing the method according to any one of [11] to [13] above to identify a patient in need of non-drug treatment from the heart failure patient; Carrying out non-drug treatment against said method.

According to the present invention, the possibility of recovery of cardiac function of a heart failure patient can be evaluated by measuring the degree of DNA damage in cardiomyocytes of the heart failure patient. This is advantageous in that it is possible to early determine a treatment policy such as heart transplantation or implantation of an auxiliary artificial heart for a heart failure patient who is evaluated to have a low possibility of recovering cardiac function.

FIG. 1 shows PAR or γ-H2A.P in LVRR-positive or LVRR-negative patients. Examples of distribution of fluorescence intensity of nuclei stained with X are shown below. FIG. 2 shows an example of images of PAR staining in biopsy specimens of LVRR-negative patients and LVRR-positive patients. 2A-D show raw images of staining by PAR, and FIGS. 2E and F show the same images after automatic determination by the hybrid cell number program. Wheat germ agglutinin (WGA) labels the cell membrane and 4,6-diamidino-2-phenylindole (DAPI) labels the nucleus. Scale bar=50 μm. FIG. 3 shows γ-H2A. in biopsy specimens of LVRR negative and LVRR positive patients. An example of an X-stained image is shown. 3A-D show γ-H2A. Raw images of staining with X are shown, and FIGS. 3E and F show the same images after automatic determination by the hybrid cell number program. Wheat germ agglutinin (WGA) labels the cell membrane and 4,6-diamidino-2-phenylindole (DAPI) labels the nucleus. Scale bar=50 μm. FIG. 4 shows PAR and γ-H2A. Images of immunostaining for X and types of PAR stained cells are shown. FIG. 4A shows PAR and γ-H2A. X is a double-stained image, and arrows indicate PAR and γ-H2A. Costaining of X is shown. FIG. 4B is an image of double staining of PAR and vimentin (fibroblast marker), and arrows indicate PAR-positive fibroblasts. FIG. 4C shows an image of double staining of PAR and CD31 (endothelial cell marker). Scale bar=10 μm. FIG. 4D shows a comparison of the average percentage of PAR-positive cardiomyocytes and non-cardiomyocytes. ^*** indicates p<0.001. FIG. 5 shows the PAR-positive nuclei of the LVRR-negative group and the LVRR-positive group and γ-H2A. A comparison of the proportion of X-positive nuclei is shown. FIG. 5A shows a comparison of the number of counted nuclei of each specimen of the LVRR negative group and the LVRR positive group. 5B and C show PAR positive nuclei or γ-H2A.N in LVRR negative and LVRR positive groups. A comparison of the proportions of X-positive nuclei is shown. ^*** with p<0.001, n. s. Indicates that there is no significant difference. FIG. 6 shows a comparison of the percentage of PAR-positive nuclei in the LVRR-negative group and the LVRR-positive group in familial and non-familial DCM patients, respectively. FIG. 7: Occurrence of events corresponding to composite endpoints (death, assisted artificial heart implantation and heart transplantation) comparing the LVRR-positive and LVRR-negative patient groups on the long-term prognostic impact of LVRR in DCM patients. Kaplan-Meier curves for FIG. 8 shows% PAR nuclei (left) and% γ-H2A. The ROC curve which evaluated the usefulness of X nucleus (right) is shown, respectively.

Detailed explanation of the invention

The subject of the evaluation method of the present invention is a heart failure patient, preferably a cardiomyopathy patient, more preferably a dilated cardiomyopathy patient. Here, "heart failure" refers to some kind of cardiac dysfunction, that is, as a result of organic and/or functional abnormality in the heart resulting in failure of compensatory mechanism of heart pump function, resulting in dyspnea, malaise and edema. Along with this, it means a clinical syndrome in which exercise tolerance decreases (Acute/chronic heart failure medical care guideline (2017 revised version)). In addition, "cardiomyopathy" is classified into four basic pathologies in addition to hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and restrictive cardiomyopathy among cardiomyopathy accompanied by cardiac dysfunction. It means unclassifiable cardiomyopathy, and "dilated cardiomyopathy" means a group of diseases characterized by diffuse contraction disorder of the left ventricle and left ventricular enlargement (Cardiomyopathy Practice Guideline (2018 revised version)).

“Recovery of cardiac function” in the present invention means recovery of left ventricular function, and is meant to include left ventricular reverse remodeling (LVRR, Left Ventricular Reverse Remodeling). Here, in LVRR, the left ventricular ejection fraction increased by 10% or more and exceeded 35%, and the left ventricular end diastolic diameter decreased by 10% or more 12 months after the start of appropriate treatment in accordance with the medical practice guidelines. Defined as companion.

In the present invention, it was confirmed that the degree of DNA damage of cardiomyocytes in myocardial biopsy specimens of heart failure patients whose heart function is not recovered is significantly higher than that of patients whose heart function is recovered (Example 1). And 2). This indicates a correlation (negative correlation) between the degree of DNA damage in cardiomyocytes and the possibility of recovering cardiac function in patients with heart failure. That is, in the evaluation method of the present invention, the degree of DNA damage in cardiomyocytes that exceeds (or exceeds the reference value) indicates that the possibility of recovery of cardiac function is low, and myocardium that falls below the reference value (or below the reference value). The degree of cellular DNA damage can be defined as indicating a high likelihood of recovery of cardiac function. The reference value can be set in advance as described later.

In the present invention, “DNA damage” means single-strand break and/or double-strand break generated in nuclear DNA. The measurement of the degree of DNA damage is not particularly limited as long as it is a method that can quantify the degree of DNA damage. The degree of DNA damage can be measured, for example, by detecting a DNA damage marker. DNA damage markers are known and include, for example, poly(ADP-ribose) (PAR), γ-H2A. X,8-hydroxy-2'-deoxyguanosine (8-OHdG) can be the detection target, and only one kind may be detected or two or more kinds may be detected in combination.

When measuring the degree of DNA damage based on a DNA damage marker, the presence of a DNA damage marker in cardiomyocytes (particularly the nucleus of cardiomyocytes) can be used as an index. That is, in one embodiment of the present invention, the presence of a DNA damage marker in cardiomyocytes (particularly the nucleus of cardiomyocytes) indicates that the possibility of recovery of cardiac function in heart failure patients is low, and DNA in cardiomyocytes (particularly the nucleus of cardiomyocytes) is shown. The absence of damage markers indicates a high likelihood of heart function recovery in patients with heart failure. The presence of a DNA damage marker in cardiomyocytes (particularly the nucleus of cardiomyocytes) can be quantified, and the degree of DNA damage can also be quantitatively evaluated. For example, based on the number of DNA damage marker-positive cardiomyocytes (particularly the nucleus of the cardiomyocyte) within a predetermined range as in Examples described later, or the total number of cardiomyocytes (particularly the nucleus of the whole cardiomyocyte) within the predetermined range. The presence of a DNA damage marker in cardiomyocytes can be quantified based on the ratio of the number of DNA damage marker-positive cardiomyocytes (particularly myocardial fat nuclei) to the number, and thus the degree of DNA damage in cardiomyocytes can be quantified. Can be converted.

In the evaluation method of the present invention, the ratio of the number of DNA damage marker-positive cardiomyocytes (particularly the nucleus of the cardiomyocyte) measured in the heart failure patient to be evaluated to the number of all cardiomyocytes (particularly the nucleus of all cardiomyocytes) is predetermined. If it is larger than the reference value (or is larger than the reference value), it is possible to evaluate or judge that the possibility of recovery of cardiac function is low. In the evaluation method of the present invention, the ratio of the number of DNA damage marker-positive cardiomyocytes (particularly the nucleus of the cardiomyocyte) measured in the heart failure patient to be evaluated to the total number of the cardiomyocytes (particularly the nucleus of the whole cardiomyocyte) is preset. If it is smaller than the defined reference value (or is less than or equal to the reference value), it is possible to evaluate or judge that the possibility of recovery of cardiac function is high. That is, the evaluation method of the present invention may further include a step of comparing the degree of DNA damage measured for the cardiomyocytes to be evaluated with a predetermined reference value. Here, when the DNA damage marker is PAR, the reference value can be, for example, 5.47%, and the DNA damage marker is γ-H2A. When it is X, it can be set to 6.3%, for example.

In the evaluation method of the present invention, the reference value is a group in which heart function is recovered in a heart failure patient whose cardiac damage is measured in advance (heart function recovery group) and a group in which heart function is not recovered (heart function non-recovery). It can be determined based on the average value of the degree of DNA damage in the cardiac function recovery group and the average value of the degree of DNA damage in the non-cardiac function recovery group. That is, the evaluation method of the present invention may include a step of preparing the reference value in advance. In the evaluation method of the present invention, the reference value can be, for example, the average value of the degree of DNA damage in the non-cardiac function recovery group.

As a method for measuring the degree of DNA damage based on the DNA damage marker, detection of the DNA damage marker by immunological analysis can be mentioned. Examples of such techniques include ELISA (eg, direct method, indirect method, sandwich method, competitive method), immunoassays such as Western blotting, immunohistochemical staining, and the like. For example, DNA used in Examples described later. The extent of DNA damage can be measured by immunohistochemical staining using a damage marker.

In the evaluation method of the present invention, the degree of DNA damage can be measured for a biopsy sample of a heart failure patient (particularly myocardial biopsy sample). A biopsy sample of a heart failure patient can be collected from the right ventricle, the left ventricle, or the like by using biopsy forceps at the time of cardiac catheterization. In the treatment of heart failure patients, it is usual that a catheterization of the heart is carried out depending on the severity, but since the evaluation method of the present invention can use a biopsy sample that can be collected by this catheterization, It is advantageous in that no additional invasion is required for heart failure patients.

In the evaluation method of the present invention, in addition to the measurement of the degree of DNA damage, measurement of other clinical variables can be performed in combination. Clinical variables to be combined include age, body mass index (BMI), systolic blood pressure, heart failure morbidity, NYHA classification (heart failure severity), B-type natriuretic peptide (BNP), echo indexes such as left ventricle diameter, and the like. ..

The evaluation of the present invention can also be performed by setting a cutoff value by statistical analysis. Specifically, the reference value can be determined based on a cut-off value obtained by a statistical analysis such as a receiver operating characteristic curve (Receiver Operating Characteristic curve, ROC) analysis. For example, the cutoff value may be set using ROC curve analysis, as shown in Example 3 below. In the ROC curve analysis, a cutoff value having a desired sensitivity and specificity can be selected, and a point at which the Youden index (sensitivity-(1-specificity)) becomes maximum can be used as the cutoff value.

According to the evaluation method of the present invention, it is possible to predict the possibility of recovery of cardiac function (particularly LVRR) in a heart failure patient, and thus it is possible to predict the prognosis of the heart failure patient. Among the patients with heart failure, those with a low possibility of recovery of cardiac function (particularly LVRR) have low therapeutic responsiveness to drug treatment, and thus were evaluated as having a low possibility of recovery of cardiac function (particularly LVRR) by the evaluation method of the present invention. Non-drug treatment can be administered to the patient. In the present invention, non-drug treatment is used to include surgical treatment and regenerative medicine. Non-limiting examples of surgical treatments include heart transplantation, implantation of an assisted artificial heart, device treatment (ICD, Implantable Cardioverter Defibrillator), cardiac resynchronization therapy (CRT, Cardiac Resynchronization Therapy). The treatment used). Non-limiting examples of regenerative medicine include treatment using a cell processed product defined in Article 2 of the Act on ensuring safety of regenerative medicine. On the other hand, patients evaluated to have a high possibility of recovery of cardiac function (particularly LVRR) by the evaluation method of the present invention are expected to have high therapeutic responsiveness to drug treatment, and thus have a better effect on drug treatment. Patients that are expected to be treated can be evaluated, and drug treatment can be selected and performed for such patients. In the present invention, drug treatment means treatment by administration of a therapeutic agent for heart failure such as angiotensin converting enzyme inhibitor (ACE inhibitor), β blocker, angiotensin II receptor blocker, antimineralocorticoid, diuretic, SGLT2 inhibitor. To do. Thus, the evaluation method of the present invention can be used by a doctor to predict treatment response of a heart failure patient, prognostic diagnosis of a heart failure patient, and further be used to determine a treatment policy of a heart failure patient. It is advantageous in that it enables medical treatment (individualized medical treatment and precision medical treatment) that provides appropriate treatment.

According to another aspect of the present invention, there is provided a method for identifying a patient in need of non-drug treatment from a heart failure patient, the method comprising the step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient. It The method for identifying a patient in need of non-drug treatment of the present invention can be carried out according to the evaluation method of the present invention. That is, the degree of DNA damage of cardiomyocytes in patients with heart failure can be measured according to the evaluation method of the present invention, and the patients evaluated or judged to have a low possibility of recovering cardiac function can be identified as patients in need of non-drug treatment. .. The method of identifying a patient in need of non-drug treatment from the heart failure patients of the present invention can be used as an auxiliary in determining the treatment policy of a heart failure patient by a doctor.

According to another aspect of the present invention, there is also provided a method for identifying a patient from a heart failure patient who is predicted to have a better effect on drug treatment, the method comprising the step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient. A method comprising is provided. The method of identifying a patient predicted to have a favorable effect by the drug treatment of the present invention can be carried out according to the evaluation method of the present invention. That is, according to the evaluation method of the present invention, the degree of DNA damage of cardiomyocytes in a heart failure patient is measured, and a patient who is evaluated or judged to have a high possibility of recovering cardiac function is a patient who is predicted to have a good effect by drug treatment. Can be specified. The method of identifying a patient predicted to have a better effect on drug treatment from the heart failure patients according to the present invention can be used as an aide in determining a treatment policy for a heart failure patient by a doctor.

According to still another aspect of the present invention, a method of identifying a patient in need of non-drug treatment of the present invention is performed to identify a patient in need of non-drug treatment, and the non-drug treatment is performed on the patient. A method for treating a patient with heart failure is provided. Among the treatment methods of the present invention, the method of identifying a patient in need of the non-drug treatment of the present invention can be carried out according to the evaluation method of the present invention as described above.

The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

Example 1: DNA damage in cardiomyocytes of patients with dilated cardiomyopathy In Example 1, biopsy specimens of patients with dilated cardiomyopathy were used to evaluate the degree of DNA damage in cardiomyocytes.

(1) Method a Biopsy specimens Of the 82 patients with heart failure who were diagnosed with dilated cardiomyopathy (DCM) at the University of Tokyo Hospital from 2009 to 2016 and were admitted to hospital, 24 cases had already undergone therapeutic intervention. The removed 58 patients' myocardial biopsy specimens were used. DCM diagnosis was based on various modalities including coronary angiography, echocardiography, and myocardial biopsy, according to current guidelines.

Patients with DCM are optimal for heart failure, including administration of ACE inhibitors or angiotensin II receptor blockers, antimineralocorticoid, and beta blocker dose escalation immediately after being diagnosed with DCM on myocardial biopsy. Treatment started. The endpoint is a composite endpoint defined as the combined outcome of death, implantation of a ventricular assist device, and heart transplantation. LVRR negative for patients with endpoint events 12 months before the start of treatment above. They were divided into groups. The clinical background at the time of biopsy of the LVRR positive group (sometimes referred to as “LVRR(+)” herein) and the LVRR negative group (sometimes referred to as “LVRR(−)” herein) is shown in the table below. It was as shown in 1.

B. Immunohistochemistry Using formalin-fixed paraffin-embedded biopsy specimens from DCM patients, PAR (poly(ADP-ribose)) and γ-H2A. X was measured. 4 μm sections were cut from paraffin blocks and placed on slides. After deparaffinization and rehydration, the antigen was unmaximized by boiling the slides for 20 minutes with Dako S1699 antigen retrieval solution (Agilent) using a MI-33 microwave processor (Toya Medical Instruments). Slides were blocked with 5% normal goat serum for 60 minutes at room temperature, followed by overnight incubation with anti-PAR polymer antibody (ab14459, 1:100, Abcam). After washing with phosphate buffered saline, the samples were stained with anti-mouse IgG-Alexa647 (1:300, Thermo Fisher Scientific) for 1 hour at room temperature. Cell membranes and nuclei were treated with wheat germ agglutinin (WGA)-Alexa488 (1:200, Thermo Fisher Scientific) and 4',6-diamidino-2-phenylindole (DAPI) (1:1,000, Dojindo Laboratories). Counterstained. Other antibodies and dyes used here were γ-H2A. X (ab81299, 1:200, Abcam), γ-H2A. X (MA1-2022, 1:200, Thermo Fisher Scientific), WGA-Alexa350 (1:200, Thermo Fisher Scientific), Vimentin (ab92547, 1:200, Abcam), PECAM1 (HPA004690, 1:100, Sigma-Aldrich). ), and anti-mouse IgG-Alexa488 and 594 (1:300, Thermo Fisher Scientific). For each patient, two sections from the remaining biopsy specimens, one for PAR staining and the other for γ-H2A. Used for X staining. Images were acquired with an inverted fluorescence microscope (BZ-X700, Keyence Corporation) equipped with a 20x objective, covering most of the biopsy specimen within one field of view. Raw image data was analyzed using BZ-X analyzer software (Keyence Corporation) to quantify the fluorescence intensity of PAR signal associated with DAPI in each nucleus. Then, a threshold for detecting PAR positive nuclei was set based on the fluorescence intensity histogram, and it was confirmed that each PAR positive region recognized by the software showed high PAR signal intensity in each section. FIG. 1 shows PAR or γ-H2A.P in LVRR-positive or LVRR-negative patients. An example of distribution of fluorescence intensity of each nucleus stained with X is shown. The software automatically calculated the percentage of PAR positive nuclei (% PAR nuclei) ([PAR stained nuclei]/[all nuclei stained with DAPI]). All raw image data were analyzed using the same algorithm. γ-H2A. The same analysis was performed with X immunostaining. To analyze the type of PAR-positive cells, all image data of PAR-stained cells were evaluated and it was determined whether each stained cell was a cardiomyocyte or a non-cardiomyocyte based on the difference in morphology. Non-cardiomyocytes are very small compared to typical large mature cardiomyocytes, their nuclei are near the cell membrane and are detected by WGA staining.

C. Statistical analysis Continuous variables were expressed as mean±SD, and categorical variables were expressed as counts and percentages. For variables with skewed distributions, the median (interquartile range [IQR]) was reported and compared using the Wilcoxon rank sum test. Statistical significance between the two groups was determined by an unpaired, two-tailed Student's t-test.

All analyzes were performed using SAS software version 9.4 (SAS Institute, Inc) and p-values of p<0.05 were considered significant in all tests.

(2) Results FIGS. 2A to 2D are the raw images of immunofluorescence staining of PAR using the myocardial biopsy specimens of the LVRR-negative patient and the LVRR-positive patient, respectively, and the PAR and DAPI (nuclear DNA) stained areas are It was confirmed that they match. 2E and F are the same images after automated evaluation by the hybrid cell counting program, with PAR-positive nuclei represented in white.

FIGS. 3A to 3D show γ-H2A. using myocardial biopsy specimens of LVRR-negative and LVRR-positive patients, respectively. X is a raw image of γ-H2A. It was confirmed that the stained locations of X and DAPI (nuclear DNA) were the same. 3E and F are the same images after automated evaluation by the hybrid cell counting program, with PAR positive nuclei represented in white.

FIG. 4A shows PAR and γ-H2A. It is a raw image of double staining of X, confirming that it was co-stained in the nucleus. Figures 4B and C show raw images of PAR and vimentin (fibroblast marker) and PAR and CD31 (vascular endothelial cell marker) double stain, respectively. Vimentin-positive cells were co-stained with PAR, whereas CD-positive cells were not co-stained with PAR, confirming that all positive non-cardiomyocytes belong to cardiac fibroblasts. As shown in FIG. 4D, the average percentage of cardiomyocytes and non-cardiomyocytes in PAR-positive cells of all biopsy specimens of 58 patients (1068 cells) was 94.5% and 5.5%, respectively.

Further, as shown in FIG. 5, the average number of nuclei analyzed in the PAR-stained specimens of the LVRR-negative group and the LVRR-positive group was 887±41 and 903±69, respectively (p=0.832) ( FIG. 5A). The ratio of nuclei stained by PAR was LVRR positive group (3.7% [IQR: 0.6% to 3.9%]) and LVRR negative group (16.3% [IQR: 6.3% to 19.3]. %]) (p<0.001) (FIG. 5B). γ-H2A. The proportion of X-stained nuclei was also LVRR positive group (3.5% [IQR: 1.2% to 6.4%]) and LVRR negative group (11.7% [IQR: 6.0% to 14.%]. 6%]) (p<0.001) (FIG. 5C).

Furthermore, as shown in FIG. 6, in both familial and non-familial DCM patients, LVRR-negative patients showed high% PAR nuclei.

From these results, it was confirmed that the degree of DNA damage in LVRR-negative patients was significantly higher than that in LVRR-positive patients.

Example 2: Treatment course and LVRR in patients with dilated cardiomyopathy
In Example 2, a statistical analysis was performed on the progress and LVRR of patients with dilated cardiomyopathy.

(1) Method A univariate screen of all patient parameters at baseline was performed first and Student's t-test was used for continuous variables. Fisher's exact test was used for categorical variables and mid-p values were calculated. The Kaplan-Meier method and log-rank test were used to assess the impact of LVRR on endpoints. An inverse probability weighted Cox proportional hazards regression model using robust standard errors was used to calculate the% PAR kernels and% γ-H2A. The effect of X nuclei was estimated. Weight of each subject was calculated using a generalized propensity score, but included the following variables in the model to adjust for baseline confounding factors: age, BMI, family history, duration of heart failure, New York Heart Association. (NYHA) functional class, systolic blood pressure, B-type natriuretic peptide (BNP), left ventricular end diastolic diameter, severe mitral regurgitation (grade III or IV). % PAR nuclei to LVRR relationship, and% γ-H2A. The relationship between X nuclei and LVRR was also examined using the propensity scoring method of logistic regression modeling. Statistical analysis other than the above was performed according to the method described in Example 1(1)C.

(2) Results The median survey period was 1386 (IQR:667-2032) days. During the LVRR assessment period, 25 of 58 patients (43.1%) achieved LVRR after multimodality treatment including renin-angiotensin-aldosterone inhibitors, beta-blockers, and cardiac resynchronization therapy. .. Treatment of neurohormonal drugs with angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, antimineralcorticoids and beta-blockers is tailored to most patients at target doses (95%, 78%, 100%, respectively) It was Twelve months after the initiation of optimal drug therapy, there was no significant difference in dose rate between patients with LVRR and LVRR for the following drugs: Angiotensin-converting enzyme inhibitor (68.0 patients with LVRR). %, patients without LVRR 72.7%, p=0.775), angiotensin II receptor blocker (28.0% with LVRR, 21.2% without LVRR, p=0.758), antimineralocorticoid (72.0% of patients with LVRR, 81.8% of patients without LVRR, p=0.527), β-blocker (carvedilol equivalent daily dose of 22.7 mg of patients with LVRR, 19.5 mg of patients without LVRR) All patients were administered 100%, p=0.281).

Fig. 7 shows the survival curves of the subjects classified by the presence or absence of LVRR. Patients with LVRR had a significantly better prognosis than patients without LVRR (log-rank test, p<0.001). During the follow-up period, 17 patients reached the composite endpoint: 8 heart transplants (13.8%), 16 LVAD (left ventricular assist device) implants (27.6%). ), 1 death (1.7%).

Age, BMI, family history, duration of heart failure, NYHA functional class, systolic blood pressure, B-type natriuretic peptide, left ventricular end diastolic diameter, and severe mitral regurgitation (grade) as confounding factors for adjustment in propensity score analysis. III or IV) was used. As shown in Table 2, by composite endpoint propensity score analysis, adjusted for other major clinical factors,% PAR nuclei (for every 10% increase, hazard ratio: 1.36; 95% confidence interval [CI]: 1.02 to 1.81; p=0.035) was found to be a significant and independent prognostic factor. Other major prognostic factors predicting poor outcome with univariate Cox analysis were age, high NYHA functional class, low blood pressure, and low BMI. In the current cohort study, only a small number of patients underwent cardiac magnetic resonance (27.6%) and cardiopulmonary exercise (51.7%), so the model for propensity score analysis included the extent of late gadolinium enhancement and peak oxygen consumption. The quantity could not be included.

In addition, as shown in Table 3, as a significant and independent predictor of LVRR by propensity score analysis,% PAR nucleus (for each 1% increase, odds ratio: 0.87; 95% CI: 0.79 to 0.95). ; P=0.003) was identified. γ-H2A. A similar analysis using the data from the X staining showed that% γ-H2A. It was revealed that X nuclei can also independently predict LVRR. From the results of Table 3, it was confirmed that the possibility of LVRR in heart failure patients can be evaluated using the degree of DNA damage in cardiomyocytes as an index.

Example 3: LVRR prediction of patients with dilated cardiomyopathy In Example 3, the prediction of LVRR of patients with dilated cardiomyopathy was performed using %PAR nuclei and %γ-H2A. Receiver operating characteristic (ROC) analysis was performed using X nuclei.

(1) Method In the ROC analysis, the point at which the Youden index (sensitivity-(1-specificity)) is the maximum was determined as the cutoff value by the cut point analysis. Area under the ROC curve (AUC) was calculated using logistic regression.

(2) Results The results are as shown in FIG. Regarding %PAR nuclei, when the cutoff value is set to 5.74%, the sensitivity is 77.8% (95% CI: 57.7% to 91.4%), and the specificity is 87.1% (95% CI: 70.2% to 96.4%), and the result was that LVRR could be predicted by AUC 0.879. % ΓH2A. Regarding the X nucleus, when the cutoff value is set to 6.3%, the sensitivity is 69.6% (95% CI: 47.1% to 86.8%) and the specificity is 75.9% (95% CI: 56). 0.5% to 89.7%), and it was the result that LVRR can be predicted by AUC 0.880.

Claims (15)

A method for evaluating the possibility of recovery of cardiac function in a heart failure patient, which comprises a step of measuring the degree of DNA damage in cardiomyocytes. The evaluation method according to claim 1, wherein the degree of DNA damage in cardiomyocytes correlates with the possibility of recovering cardiac function in patients with heart failure. The evaluation method according to claim 1 or 2, wherein the degree of DNA damage is measured based on a DNA damage marker. The evaluation method according to claim 3, wherein the degree of DNA damage is indicated by the ratio of the number of DNA damage marker positive nuclei in cardiomyocytes to the total number of nuclei. The evaluation method according to claim 3 or 4, wherein the DNA damage marker is detected by immunological analysis. The evaluation method according to any one of claims 3 to 5, wherein the presence of a DNA damage marker in cardiomyocytes indicates a low possibility of recovery of cardiac function. The evaluation method according to any one of claims 1 to 6, wherein the degree of DNA damage is measured using a myocardial biopsy sample of a heart failure patient. The evaluation method according to any one of claims 1 to 7, wherein the heart failure patient is a cardiomyopathy patient. The evaluation method according to claim 8, wherein the cardiomyopathy patient is a dilated cardiomyopathy patient. The evaluation method according to any one of claims 1 to 9, which is used for determining a treatment policy for a heart failure patient. A method for identifying a patient in need of non-drug treatment from a heart failure patient, comprising a step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient. The method according to claim 11, wherein the non-drug treatment is surgical treatment or regenerative medicine. 13. The method of claim 12, wherein the surgical treatment is a heart transplant, an implantable artificial heart surgery or a device treatment. A method of identifying a patient from a heart failure patient who is predicted to have a favorable effect on drug treatment, the method comprising the step of measuring the degree of DNA damage of cardiomyocytes in the heart failure patient. A method for treating a patient with heart failure, which comprises performing the method according to any one of claims 11 to 13 to identify a patient in need of non-drug treatment from the patient with heart failure, and a non-drug for the patient. Administering the treatment.

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