A marker combination and analysis system for primary aldosteronism diagnosis or prognosis evaluation

Abstract

This invention relates to a biomarker combination and analysis system for the diagnosis or prognostic assessment of primary aldosteronism. It includes one or more of the following: CD56 positivity distribution range, CD56 positivity staining depth, CD56 staining localization, CYP11B1 positivity distribution range, CYP11B1 positivity staining depth, CYP11B1 staining localization, CYP11B2 positivity distribution range, CYP11B2 positivity staining depth, and CYP11B2 staining localization. The comprehensive scoring algorithm of this invention enables standardized interpretation of the test results, providing reliable clinical evidence for the pathological diagnosis of primary aldosteronism, the differential classification of non-functional adenomas, and prognostic assessment.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a biomarker combination and analysis system for the diagnosis or prognostic assessment of primary aldosteronism. Background Technology

[0002] The detection rate of adrenal incidentaloma (AI) in the general population is 4%-7%, of which about 1%-2% are aldosterone-producing adenoma (APA), while non-functional adenoma (NFA) accounts for more than 70%.

[0003] Primary aldosteronism (PA) is a systemic disorder characterized by hypertension, increased aldosterone secretion, and decreased plasma renin activity. It is caused by the autonomous secretion of excessive aldosterone by the adrenal cortex and accounts for 5%–15% of hypertension cases, with an even higher proportion (17%–23%) in refractory hypertension. Excessive aldosterone secretion leads to sodium retention, increased potassium excretion, and blood volume expansion, resulting in elevated blood pressure and damage to target organs (such as the heart, kidneys, and blood vessels).

[0004] The causes of PA include APA (adrenal cortical adenoma): accounting for 35% of primary aldosteronism, mostly unilateral adrenal cortical adenoma, usually <2 cm in diameter, pathologically characterized by a golden-yellow cut surface and an intact capsule; idiopathic hyperaldosteronism (IHA): accounting for 65%, manifesting as bilateral adrenal zona glomerulosa hyperplasia, possibly related to increased angiotensin II (AT-II) sensitivity or genetic factors; familial hyperaldosteronism... FH (adrenocorticoid-mediated hyperplasia), accounting for about 5% of PA, is further divided into FH-I (glucocorticoid-inhibitory type), caused by a chimeric gene (CYP11B1 / CYP11B2), which has an onset in adolescence and is caused by exogenous glucocorticoids that can inhibit aldosterone secretion, and FH-II (glucocorticoid-non-inhibitory type), which is related to gene mutations (such as KCNJ5, CACNA1D) and requires surgical or drug treatment; other rare types include adrenocortical carcinoma, ectopic aldosterone-secreting tumors, and primary adrenal hyperplasia.

[0005] According to the "Expert Consensus on the Diagnosis and Treatment of Primary Aldosteronism," the diagnostic process includes three stages: screening, confirmation, and subtyping. We routinely screen patients with refractory hypertension (those whose blood pressure is not controlled despite using three antihypertensive drugs), hypertension complicated by spontaneous or diuretic-induced hypokalemia, early-onset hypertension (<40 years old) or a family history of the condition, or adrenal incidental tumors. Screening is performed using the plasma aldosterone / renin ratio (ARR): an ARR > 3.7 (aldosterone units: ng / dL, renin units: ng / mL / h) suggests possible primary aldosteronism. For patients with positive screening results, confirmatory tests are performed: saline loading test: after intravenous infusion of 2L of normal saline, aldosterone > 10 ng / dL supports the diagnosis; captopril suppression test: aldosterone is not suppressed after oral captopril (> 12 ng / dL).

[0006] Adrenal imaging examinations (such as adrenal computed tomography / magnetic resonance imaging, CT / MRI) are simple and non-invasive, and can directly visualize adrenal lesions, but they cannot prove the function of adrenal tumors. Therefore, adrenal venous sampling (AVS) is currently considered the gold standard for classifying adrenal aldosteronism (PA). AVS has a sensitivity of 95% and a specificity of 100% for detecting unilateral aldosterone excess. However, AVS is an invasive examination, not very easy to perform, and exposes patients to radiation. Even for a confirmed diagnosis of APA, there is controversy regarding whether to remove only the tumor or simultaneously remove the surrounding adrenal tissue that appears normal on imaging. This is because some patients do experience recurrence of ipsilateral functional abnormalities after simple resection of APA.

[0007] Adrenal non-adrenal adenomas (NAFAs) are benign adrenal tumors that do not secrete hormones or secrete insufficient amounts. Some adenomas may gradually enlarge and compress surrounding organs (such as the kidneys and blood vessels), causing lower back pain or abdominal discomfort. However, the surgical cure rate for APA patients can reach 90%. If misdiagnosed as NFA and treated conservatively, it may lead to persistent hyperaldosteronism and cardiovascular damage. Therefore, accurate differentiation between APA and NFA is crucial for optimizing treatment strategies.

[0008] In recent years, significant progress has been made in the study of molecular markers for adrenocortical tumors. Among them, the combined analysis of CD56 (neural cell adhesion molecule), CYP11B1 (11β-hydroxylase, a key enzyme in cortisol synthesis) and CYP11B2 (aldosterone synthase) has provided new insights into the differentiation between APA and NFA.

[0009] CYP11B2, a core biomarker for aldosterone synthesis, is specifically expressed in the zona glomerulosa of the adrenal gland, catalyzing the final step in aldosterone synthesis. CYP11B2 is strongly positive in aldosterone-producing arthritis (APA), but almost absent in non-aldosterone-producing arthritis (NFA). In 2022, the World Health Organization proposed the HISTALDO international consensus, which states that CYP11B2 immunohistochemistry can classify PA pathology into six subtypes (such as solitary APA and aldosterone-producing micronodules), significantly improving the functional relevance of pathological diagnosis.

[0010] The 11β-hydroxylase encoded by CYP11B1 is the dominant cortisol synthase and is highly expressed in the zona fasciculata. In adrenal adenoma (APA), 11β-hydroxylase catalyzes the production of 18-deoxycorticosterone, a precursor of aldosterone, and is involved in the growth and enlargement of the adrenal adenoma. Differentiation between APA and cortisol adenoma requires simultaneous analysis of the expression ratio of CYP11B1 and CYP11B2. For example, case studies show that the CYP11B2 / CYP11B1 ratio is >5 in APA, while it is <0.2 in cortisol adenoma.

[0011] CD56 is a novel biomarker for neuroendocrine differentiation, a cell surface glycoprotein belonging to the immunoglobulin superfamily. It is expressed in various cell types, including natural killer (NK) cells, T cells, dendritic cells, and monocytes, and plays important roles in cell adhesion, signal transduction, and cell migration. High expression of CD56 in APA (aplastic angioplasty) may be associated with abnormal electrical signal transduction caused by tumor cell depolarization and ion channel mutations (such as KCNJ5 and CACNA1D), suggesting the potential link between APA and pheochromocytoma cell proliferation.

[0012] Most importantly, conventional pathological examination can generally only determine whether an adrenal adenoma originates from the cortex, but cannot distinguish its function, such as whether it is an aldosterone-secreting adenoma or a cortisol adenoma. Furthermore, conventional pathological examination cannot differentiate between adrenal adenomas (APNs) and adrenal motility tumors (APMs). The combined application of the aforementioned molecular markers provides a new direction for overcoming the bottlenecks of traditional pathological diagnosis. For example, co-expression of CD56 and CYP11B2 can label the neuroendocrine characteristics and aldosterone-secreting activity of APAs, while CYP11B1 and CYP11B2 can effectively distinguish mixed hormone-secreting tumors. By integrating multiplex immunohistochemistry and gene detection technologies, it is hoped that a dual-track diagnostic system of preoperative non-invasive subtyping and postoperative pathological verification can be established in the future, thereby optimizing treatment decisions and reducing the risk of recurrence.

[0013] Since Jerome Conn first systematically described PA in 1955, inventors have continuously deepened their research on the molecular mechanisms and subtyping diagnosis of PA, but the following core challenges still exist in clinical practice: 1) Differentiation of functional adenomas in adrenal adenomas (AIs) is difficult. Approximately 5%-10% of AIs are acute adrenal adenomas (APA), but the majority (around 75%) are non-functional adenomas (NFAs). Computed tomography / magnetic resonance imaging (CT / MRI) has a diagnostic sensitivity of only 52%-81% for APAs and cannot distinguish between functional and non-functional lesions. For example, 20% of patients with unilateral adenomas on imaging are confirmed to have bilateral lesions by adrenal vein sampling (AVS). 2) Pathological diagnosis has certain limitations. Traditional hematoxylin-eosin (HE) staining can only determine the cortical origin of the tumor and cannot differentiate between APA and NFA or other functional adenomas, thus failing to provide an accurate postoperative pathological diagnosis. Furthermore, a standardized system for postoperative pathological diagnosis is lacking. Currently, there is no standardized scoring standard for CYP11B2 immunohistochemical staining, and the scoring standard lacks a quantitative unified standard. Different centers have significantly different thresholds for positive cells (e.g., >10% vs. >30%), affecting the comparability of results. Existing studies mostly analyze CD56, CYP11B1, and CYP11B2 independently, without clarifying the weighting ratio of the combined diagnosis of the three.

[0014] 3) Biomarker expression heterogeneity and false-negative risk. CYP11B2 expression in APA is focally distributed, with APA tissue showing uniform positive staining. False negatives in other cases may occur due to sampling bias (e.g., only non-secreting areas are detected). Although CD56 is highly expressed in APA, its association mechanism with CYP11B2 or other driver gene mutations is unclear, and multicenter cohort validation data are lacking. Some APA cases may also have co-existing cortisol secretion abnormalities (subclinical Cushing's syndrome), leading to elevated CYP11B1 expression. Summary of the Invention

[0015] Purpose of the invention To overcome the above shortcomings, the present invention aims to provide a biomarker combination and analysis system for the diagnosis or prognostic assessment of primary aldosteronism. This invention utilizes the positive distribution range, positive staining depth, and staining location of three immunohistochemical biomarkers—CD56, CYP11B1, and CYP11B2—as a biomarker combination for the diagnosis or prognostic assessment of primary aldosteronism. This invention constructs a disease prediction model based on the positive distribution range, positive staining depth, and staining location of these three immunohistochemical biomarkers, and establishes a comprehensive scoring algorithm to facilitate the interpretation of test results, providing clinical evidence for the diagnosis or prognostic assessment of primary aldosteronism.

[0016] Solution To achieve the above objectives, in a first aspect, the present invention provides a combination of biomarkers for the diagnosis or prognostic assessment of primary aldosteronism, comprising one or more of the following: CD56 positive distribution range, CD56 positive staining depth, CD56 staining localization, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining localization, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining localization. The CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the breadth of CD56 expression distribution at the tissue level. CD56 positive staining depth refers to the degree of staining of CD56 positive sites in an adrenal tissue section stained with immunohistochemistry. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CD56 antigen. CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. The CYP11B1 positive staining depth refers to the degree of staining of the CYP11B1 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B1 antigen. CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B2 refers to the percentage of the area of ​​the CYP11B2 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. The CYP11B2 positive staining depth refers to the degree of staining of the CYP11B2 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B2 antigen. CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm.

[0017] The quantitative assignment rules for each indicator are as follows: Positive distribution range: The value is directly assigned as the percentage of the area of ​​the positive region to the effective observation area (0~100); Positive staining depth: A semi-quantitative grading system of 0-3 points was used: 0 points was no color development (positive areas showed no specific brownish-yellow staining, consistent with the background color), 1 point was light yellow (weak positive, positive areas were light yellow with weak staining), 2 points was brownish-yellow (moderate positive, positive areas were distinctly brownish-yellow with moderate staining intensity), and 3 points was brownish-brown / dark brown (strong positive, positive areas were dark brownish-brown with high staining intensity and a clear contrast with the background). Staining localization: An ordered classification system was used for localization: 0 points for localization of the cell membrane alone, 2 points for localization of the cytoplasm alone, and 3 points for localization of both the cell membrane and cytoplasm.

[0018] Secondly, the present invention provides a combination of markers for distinguishing nonfunctional adenomas from primary aldosteronism, including one or more of the following: CD56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining location, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining location. Among them, the CD56 positive distribution range refers to the percentage of the area of ​​CD56 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CD56 at the tissue level; CD56 positive staining depth refers to the degree of staining of CD56 positive sites in an adrenal tissue section stained with immunohistochemistry. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CD56 antigen. CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. The CYP11B1 positive staining depth refers to the degree of staining of the CYP11B1 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B1 antigen. CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B2 refers to the percentage of the area of ​​the CYP11B2 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. The CYP11B2 positive staining depth refers to the degree of staining of the CYP11B2 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B2 antigen. CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm.

[0019] The quantitative assignment rules for each indicator are as follows: Positive distribution range: The value is directly assigned as the percentage of the area of ​​the positive region to the effective observation area (0~100); Positive staining depth: A semi-quantitative grading system of 0-3 points was used: 0 points was no color development (positive areas showed no specific brownish-yellow staining, consistent with the background color), 1 point was light yellow (weak positive, positive areas were light yellow with weak staining), 2 points was brownish-yellow (moderate positive, positive areas were distinctly brownish-yellow with moderate staining intensity), and 3 points was brownish-brown / dark brown (strong positive, positive areas were dark brownish-brown with high staining intensity and a clear contrast with the background). Staining localization: An ordered classification system was used for localization: 0 points for localization of the cell membrane alone, 2 points for localization of the cytoplasm alone, and 3 points for localization of both the cell membrane and cytoplasm.

[0020] Thirdly, the present invention provides a predictive system for the diagnosis or prognostic assessment of primary aldosteronism, comprising a data analysis module. The data analysis module is used to collect the assigned values ​​of biomarkers of the target subject to be predicted, using these biomarker values ​​as input features, and further used to calculate a predictive value for whether the target subject has primary aldosteronism based on the biomarker values. The input feature is the assignment of a combination of markers described in the first or second aspect; The CD56 positive distribution range refers to the percentage of the CD56 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CD56 at the tissue level; optionally, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. CD56 positive staining depth refers to the intensity of CD56 positive staining in an adrenal tissue section stained with immunohistochemistry. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CD56 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. Alternatively, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. The CYP11B1 positive staining depth refers to the intensity of CYP11B1 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B1 antigen expression. Optionally, the gradient is assigned a semi-quantitative grading of 0-3 points: 0 points for consistency with the background color, 1 point for light yellow (weakly distinguishable from the background), 2 points for brownish-yellow (moderately distinguishable from the background), and 3 points for brownish-black / dark brown (strongly distinguishable from the background). CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The CYP11B2 positive distribution range refers to the percentage of the area of ​​the CYP11B2 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. Alternatively, the percentage of the positive region in the effective observation area (0~100) can be directly used for assignment. The CYP11B2 positive staining depth refers to the intensity of CYP11B2 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B2 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections. This includes three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points.

[0021] Fourthly, the present invention provides a predictive system for distinguishing between non-functional adenomas and primary aldosteronism, comprising a data analysis module. The data analysis module is used to collect the values ​​of biomarkers of the target subject to be predicted, using the biomarker values ​​as input features, and to calculate the predicted probability value of the target subject having primary aldosteronism based on the biomarker values; wherein, The input feature is the assignment of a combination of markers described in the first or second aspect; in, The CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in an immunohistochemically stained adrenal tissue section, which is used to characterize the breadth of CD56 expression and distribution at the tissue level; optionally, the percentage of the positive area to the effective observation area (0~100) can be directly used for assignment. CD56 positive staining depth refers to the intensity of CD56 positive staining in an adrenal tissue section stained with immunohistochemistry. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CD56 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. Alternatively, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. The CYP11B1 positive staining depth refers to the intensity of CYP11B1 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B1 antigen expression. Optionally, the gradient is assigned a semi-quantitative grading of 0-3 points: 0 points for consistency with the background color, 1 point for light yellow (weakly distinguishable from the background), 2 points for brownish-yellow (moderately distinguishable from the background), and 3 points for brownish-black / dark brown (strongly distinguishable from the background). CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The CYP11B2 positive distribution range refers to the percentage of the area of ​​the CYP11B2 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. Alternatively, the percentage of the positive region in the effective observation area (0~100) can be directly used for assignment. The CYP11B2 positive staining depth refers to the intensity of CYP11B2 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B2 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections. This includes three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points.

[0022] It should be noted that cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm are mutually exclusive categories, not parallel indicators, and there is no "weighting" issue. Cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm are three mutually exclusive phenotypic states of the intracellular distribution of the same marker (such as CD56). For any given sample, the staining localization result can only be one of the three; two or all three will not appear simultaneously.

[0023] The quantitative assignment rules for each indicator are as follows: In the third and fourth aspects mentioned above, the assigned values ​​of the target object marker combination are used as input features to calculate the predicted value of whether the target object has primary aldosteronism. The model prediction formula is as follows: glm(formula=data$Group~-0.1521900 CD56 positive distribution range -0.0008638 CD56 positive staining depth +0.1592934 CD56 staining localization -0.7659794 CYP11B1 positive distribution range +0.5342818 CYP11B1 positive staining depth +2.2903715 CYP11B1 staining localization +0.7705341 CYP11B2 positive distribution range +0.0190591 CYP11B2 positive staining depth -0.2857227 CYP11B2_staining localization, family=binomial, data=data); in, The CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in an immunohistochemically stained adrenal tissue section, which is used to characterize the breadth of CD56 expression and distribution at the tissue level; optionally, the percentage of the positive area to the effective observation area (0~100) can be directly used for assignment. CD56 positive staining depth refers to the intensity of CD56 positive staining in an adrenal tissue section stained with immunohistochemistry. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CD56 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. Alternatively, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. The CYP11B1 positive staining depth refers to the intensity of CYP11B1 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B1 antigen expression. Optionally, the gradient is assigned a semi-quantitative grading of 0-3 points: 0 points for consistency with the background color, 1 point for light yellow (weakly distinguishable from the background), 2 points for brownish-yellow (moderately distinguishable from the background), and 3 points for brownish-black / dark brown (strongly distinguishable from the background). CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The CYP11B2 positive distribution range refers to the percentage of the area of ​​the CYP11B2 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. Alternatively, the percentage of the positive region in the effective observation area (0~100) can be directly used for assignment. The CYP11B2 positive staining depth refers to the intensity of CYP11B2 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B2 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections. This includes three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points.

[0024] It should be noted that cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm are mutually exclusive categories, not parallel indicators, and there is no "weighting" issue. Cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm are three mutually exclusive phenotypic states of the intracellular distribution of the same marker (such as CD56). For any given sample, the staining localization result can only be one of the three; two or all three will not appear simultaneously.

[0025] The quantitative assignment rules for each indicator are as follows: Positive distribution range: The value is directly assigned as the percentage of the area of ​​the positive region to the effective observation area (0~100); Positive staining depth: A semi-quantitative grading system of 0-3 points was used: 0 points was no color development (positive areas showed no specific brownish-yellow staining, consistent with the background color), 1 point was light yellow (weak positive, positive areas were light yellow with weak staining), 2 points was brownish-yellow (moderate positive, positive areas were distinctly brownish-yellow with moderate staining intensity), and 3 points was brownish-brown / dark brown (strong positive, positive areas were dark brownish-brown with high staining intensity and a clear contrast with the background). Staining localization: An ordered classification system was used for localization: 0 points for localization of the cell membrane alone, 2 points for localization of the cytoplasm alone, and 3 points for localization of both the cell membrane and cytoplasm.

[0026] Among them, when the predicted value is greater than 0.615, it is considered to be primary aldosteronism, otherwise it is adrenal nonfunctioning adenoma. Optionally, the target population includes individuals with incidental adrenal tumors, individuals suspected of having primary aldosteronism, or individuals requiring pathological classification assessment after adrenal adenoma surgery.

[0027] The third and fourth aspects mentioned above include an analysis module for processing the following combinations of markers: CD56 positive distribution range, CD56 positive staining depth, CD56 staining localization, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining localization, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining localization. The processing method of the analysis module is as follows: (1) Receive the immunohistochemical staining image data of the adrenal tissue section to be tested; (2) Delineate the effective area: delineate the effective observation area of ​​adrenal tumor tissue in the image, and exclude necrotic tissue, stromal tissue and normal adrenal tissue area; (3) Preprocess the image, including color standardization, background correction and area labeling; (4) Identify and quantify the positive distribution range, positive staining depth and staining location of CD56, CYP11B1 and CYP11B2 in the effective observation area; (5) Substitute the assigned value into the regression prediction model to calculate the predicted probability value; (6) Output the predicted probability value and the corresponding judgment result.

[0028] Fifthly, a detection system is provided for distinguishing between nonfunctional adenomas and primary aldosteronism, optionally including a reagent for detecting combinations of the markers of the first or second aspect, and outputting the detection results.

[0029] Optionally, it also includes an image processing system for identifying and performing the following analyses or scores: the percentage of positive distribution ranges of CD56, CYP11B1, and CD56; the staining depth of positive CD56, CYP11B1, and CYP11B2 and assigning a value based on the difference from the background color; and the staining localization of CD56, CYP11B1, and CYP11B2 and assigning a value based on the localization result.

[0030] Optionally, the percentage of positive distribution range of each marker and the percentage of the area of ​​the positive region to the effective observation area (0~100) are assigned values; And / or, the positive staining depth of each marker is assigned a gradient value based on the difference between light yellow, brownish yellow, brownish brown to dark brown; optionally, the gradient value is assigned in a semi-quantitative grade of 0 to 3 points. And / or, the method for assigning values ​​to the staining localization of each marker is as follows: based on staining localization, simple cell membrane localization is assigned 0 points, simple cytoplasm localization is assigned 2 points, and joint localization of cell membrane and cytoplasm is assigned 3 points.

[0031] Beneficial effects This invention fills a gap in the availability of biomarker combinations for the diagnosis or prognosis of essential aldosteronism: It proposes nine biomarker combinations that could potentially serve as a tool for the diagnosis or prognosis of essential aldosteronism. Extensive clinical diagnostic analysis demonstrates that the combined diagnostic value of the positive distribution range, staining depth, and staining localization of the three immunohistochemical biomarkers CD56, CYP11B1, and CYP11B2 can be considered as biomarkers for essential aldosteronism.

[0032] Another major innovation of this invention is its concise result analysis system: Instead of analyzing results from a single indicator (which has low diagnostic efficacy), this invention imports the screened biomarker detection results into a self-developed comprehensive scoring system. This system comprehensively scores the positive distribution range, positive staining depth, and staining location of the three immunohistochemical markers CD56, CYP11B1, and CYP11B2, overcoming the problem of low diagnostic efficacy associated with single indicators. It avoids the confusion caused by inconsistent individual results in clinical interpretation, and the comprehensive interpretation makes the results more reliable and referential, facilitating clinical promotion and application. Attached Figure Description

[0033] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0034] Figure 1 This is a heatmap showing the correlation analysis between various immunohistochemical indicators in this invention.

[0035] Figure 2 This is the ROC curve of the three-marker combined diagnostic model of the present invention. Detailed Implementation

[0036] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0037] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0038] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0039] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0040] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0041] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0042] This invention provides a combination of indicators for detecting various tissue markers in patients with essential aldosteronism: CD56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining location, CYP11B2 positive distribution range, CYP11B2 staining depth, CYP11B2 staining location.

[0043] In this invention, the definitions of each marker index are as follows: 1. Positive distribution range (_positive distribution range): refers to the percentage of the area of ​​the corresponding antigen positive staining region in the effective observation area of ​​the target tumor tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of the antigen at the tissue level.

[0044] 2. Positive staining depth (_positive staining depth): refers to the degree of staining of positive staining sites in an adrenal tissue section subjected to immunohistochemical staining. It can be determined by semi-quantitative grading and usually presents a gradient difference from light yellow, brownish-yellow, brownish-black to dark brown, which is used to characterize the expression abundance of the corresponding antigen.

[0045] 3. Staining localization (_staining localization): refers to the substructural distribution of positive staining signals within cells in an immunohistochemically stained section of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm.

[0046] The quantitative assignment rules for each indicator are as follows: Positive distribution range: The value is directly assigned as the percentage of the area of ​​the positive region to the effective observation area (0~100); Positive staining depth: A semi-quantitative grading system of 0-3 points was used: 0 points for no staining, 1 point for light yellow (weak positive), 2 points for brownish-yellow (moderate positive), and 3 points for brownish-black / dark brown (strong positive). Staining localization: Classification assignment was used: 0 points for localization of cell membrane alone, 2 points for localization of cytoplasm alone, and 3 points for localization of both cell membrane and cytoplasm.

[0047] Example 1: Real Sample Detection One hundred clinically diagnosed cases of primary aldosteronism and one hundred control cases of non-functional adrenal adenoma were selected from the postoperative adrenal tissue samples. The expression of CD56, CYP11B1, and CYP11B2 was detected by immunohistochemistry. The indicators were assigned values ​​according to the above interpretation rules and then substituted into the prediction model of the present invention to calculate the prediction probability value.

[0048] Inclusion criteria: (1) Age 18-75 years old; (2) The postoperative pathology confirmed it as an adrenocortical adenoma; (3) The adrenal tissue specimens after surgery were fixed in 4% neutral formaldehyde and embedded in paraffin. The specimen quality met the requirements for immunohistochemical detection.

[0049] Exclusion criteria: (1) Adrenocortical carcinoma, pheochromocytoma, metastatic tumors and other non-cortical adenoma lesions; (2) Comorbid systemic diseases such as severe liver and kidney dysfunction, malignant tumors, and autoimmune diseases; (3) Tissue specimens with poor fixation, necrosis area >30%, or failed immunohistochemical staining, or results that are uninterpretable; All specimens were subjected to the same immunohistochemical staining protocol, performed by the same team of researchers on the same experimental platform, to ensure the consistency and comparability of the test results. (1) Paraffin sections were 4 μm thick, baked at 60 °C for 2 h, dewaxed with xylene, and hydrated with graded ethanol. (2) High-pressure antigen retrieval for 2 min using citrate buffer (pH 6.0); (3) Incubation with 3% H2O2 at room temperature for 10 min blocks endogenous peroxidase; (4) Add primary antibodies: CD56 (clone number 123C3, working concentration 1:200), CYP11B1 (polyclonal antibody, working concentration 1:100), CYP11B2 (clone number 41-17B, working concentration 1:100), and incubate overnight at 4°C; (5) EnVision two-step color development, DAB color developer for 3~5 min, hematoxylin counterstaining, dehydration and clearing, and neutral resin mounting; (6) For each batch of staining, a positive control (adrenal tissue sections with known positive staining) and a negative control (PBS instead of primary antibody) are set to ensure that the staining quality is stable and controllable.

[0050] 4. Indicator Interpretation Two independent pathologists interpreted the slides using a double-blind method, quantifying the positive distribution range, positive staining depth, and staining location of CD56, CYP11B1, and CYP11B2 according to the aforementioned interpretation rules.

[0051] 5. Test Results The nine indicators of 200 samples were assigned values ​​and substituted into the binary logistic regression prediction model of this invention to calculate the predicted probability value P for each sample. A diagnostic threshold of 0.615 was used for judgment, and the results are as follows: Primary aldosteronism case group (100 cases): 88 cases had a predictive probability value P>0.615 and were judged as positive, with a concordance rate of 88.0%; 12 cases had a P≤0.615 and were judged as negative (false negative), with a false negative rate of 12.0%.

[0052] In the control group of non-functional adrenal adenomas (100 cases): 85 cases with a predicted probability value P≤0.615 were judged as NFA negative, with a concordance rate of 85.0%; 15 cases with P>0.615 were judged as positive (false positive), with a false positive rate of 15.0%.

[0053] Overall diagnostic performance: The biomarker combination and prediction model of this invention achieved an overall diagnostic accuracy of 86.5%, sensitivity of 88.0%, and specificity of 85.0% in 200 real samples, consistent with the results of the modeling set, thus verifying the diagnostic efficacy and stability of this invention.

[0054] Experimental Example 1: Establish models and software for diagnosing or prognostically assessing nonfunctional and essential aldosteronism: This invention is based on a multivariate logistic regression algorithm, which calculates binary classification probabilities to establish a scoring model for primary aldosteronism.

[0055] First, prepare the data matrix to be analyzed (immunohistochemical detection of CD56, CYP11B1, and CYP11B2 expression in patients with non-functional adenomas and patients with primary aldosteronism, respectively, and assign values ​​to the indicators according to the above interpretation rules). Use the ggcorrplot and pheatmap tools in R language, and use the cor() function to calculate the correlation coefficient (i.e., the correlation between each pair of indicators). Then, use the corrplot package for visualization (e.g., ...). Figure 1 ), intergroup correlation analysis.

[0056] Secondly, the data were preprocessed and their integrity was checked. Nine immunohistochemical indicators (positive distribution range, positive staining depth, and staining location of CD56, CYP11B1, and CYP11B2) were used as input variables, with primary aldosteronism and nonfunctional adenoma as dependent variables, and clinically confirmed primary aldosteronism / nonfunctional adenoma as dependent variables. Data with a difference between predicted and true values ​​greater than 0.55 in the control group were removed as outliers. Disease grouping was used as the stratification variable, and stratified random sampling was used to divide the remaining samples into a training set and a test set in a 7:3 ratio. The purpose of stratified sampling is to ensure that the proportion of non-functional adenomas to primary aldosteronism in the training and test sets remains consistent with the original population, avoiding sampling bias that could lead to an imbalance in the proportion of a particular subgroup in either the training or test set. The specific division is as follows: the training set comprises approximately 70% of the remaining samples, and the test set comprises approximately 30%. Based on the original population proportions, the training set contains approximately 52 cases of non-functional adenomas and approximately 88 cases of primary aldosteronism, while the test set contains approximately 28 cases of non-functional adenomas and approximately 32 cases of primary aldosteronism. Subsequently, a multivariate logistic regression model is constructed and trained based on the training set. The model parameters (regression coefficients) are estimated entirely from the training set data. After training, the trained model is independently applied to the test set to calculate the predicted probability of each sample in the test set belonging to the primary aldosteronism group. The test set data is not involved in model training or parameter tuning throughout the process to ensure the objectivity and accuracy of the model performance evaluation. Subsequently, the model performance was evaluated based on different performance metrics, and the AUC value of the model, i.e. the area under the ROC curve, was tested. An AUC value between 0.5 and 1 indicates that the predictive model performs better than random guessing; the closer the AUC is to 1, the better. The model's prediction result for each sample is a probability value. An optimal critical point is obtained from the ROC curve as the cutoff value to be found. The cutoff value found indicates that the model has at least one threshold that can accurately separate the data above and below the threshold.

[0057] 1. Indicator Screening Using the positive distribution range, staining depth, and staining location of CD56 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~-0.06952) CD56 positive distribution range -0.54621 CD56 positive staining depth +1.57651 CD56 staining localization, family=binomial, data=data) Using the positive distribution range, staining depth, and staining location of CYP11B1 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~-0.1542) CYP11B1 positive distribution range +0.2469 CYP11B1 positive staining depth +17.9452 CYP11B1_staining localization, family=binomial, data=data).

[0058] Using the positive distribution range, staining depth, and staining location of CYP11B2 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~0.3859) CYP11B2 positive distribution range -0.6982 CYP11B2 positive staining depth +1.1739 CYP11B2_staining localization, family=binomial, data=data) Using the positive distribution range, staining depth, and staining location of CD56 and CYP11B1 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~0.02923) CD56 positive distribution range -1.02965 CD56 positive staining depth +0.95944 CD56 staining localization -0.17617 CYP11B1 positive distribution range +0.19598 CYP11B1 positive staining depth +1.77827 CYP11B1_staining localization,family=binomial,data=data)) Using the positive distribution range, staining depth, and staining location of CD56 and CYP11B2 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~0.1076) CD56 positive distribution range -0.8831 CD56 positive staining depth +0.4218 CD56 staining localization +0.6426 CYP11B2 positive distribution range -1.4211 CYP11B2 positive staining depth +0.9107 CYP11B2_staining localization, family=binomial, data=data) Using the positive distribution range, positive staining depth, and staining location of the target object (CYP11B1+CYP11B2) as input features, the predicted value for whether the target object has primary aldosteronism is calculated. The model prediction formula is: glm(formula=data$Group~-0.5145) CYP11B1 positive distribution range +0.5685 CYP11B1 positive staining depth +2.8566 CYP11B1 staining localization +0.6733 CYP11B2 positive distribution range -1.4341 CYP11B2 positive staining depth +1.1323 CYP11B2_staining localization, family=binomial, data=data) Using the positive distribution range, positive staining depth, and staining location of CD56, CYP11B1, and CYP11B2 in the target subject as input features, the predicted value for whether the target subject has primary aldosteronism is calculated. The model prediction formula is as follows: glm(formula=data$Group~-0.1521900 CD56 positive distribution range -0.0008638 CD56 positive staining depth +0.1592934 CD56 staining localization -0.7659794 CYP11B1 positive distribution range +0.5342818 CYP11B1 positive staining depth +2.2903715 CYP11B1 staining localization +0.7705341 CYP11B2 positive distribution range +0.0190591 CYP11B2 positive staining depth -0.2857227 CYP11B2_staining localization, family=binomial, data=data) Based on the above combination of indicators, ROC curves were plotted, and the results are shown in Table 2.

[0059] Table 2. AUC, sensitivity, and specificity of different indicator combinations

[0060] The results showed that the AUC predicted by the three indicators per unit point for CD56 (positive distribution range, positive staining depth, staining location), CYP11B1 (positive distribution range, positive staining depth, staining location), and CYP11B2 (positive distribution range, positive staining depth, staining location) was all below 0.842.

[0061] The combined AUC of the six indicators for CD56 (positive distribution range, positive staining depth, staining location) + CYP11B1 (positive distribution range, positive staining depth, staining location), CD56 (positive distribution range, positive staining depth, staining location) + CYP11B2 (positive distribution range, positive staining depth, staining location), and CYP11B1 (positive distribution range, positive staining depth, staining location) + CYP11B2 (positive distribution range, positive staining depth, staining location) for the two loci were all below 0.842.

[0062] Simultaneous selection of 9 indicators from 3 loci (CD56 (positive distribution range, positive staining depth, staining location), CYP11B1 (positive distribution range, positive staining depth, staining location), and CYP11B (positive distribution range, positive staining depth, staining location)) for combined diagnosis is necessary for better diagnostic performance.

[0063] Results Analysis Analysis of tissue tests on 278 subjects (101 patients with non-functional adenomas and 177 patients with primary aldosteronism) showed that the analytical system scoring method described in this invention has high predictive value for the diseases.

[0064] The formula for the multivariate logistic regression model obtained by this invention is as follows: glm(formula=data$Group~-0.1521900 CD56 positive distribution range -0.0008638 CD56 positive staining depth +0.1592934 CD56 staining localization -0.7659794 CYP11B1 positive distribution range +0.5342818 CYP11B1 positive staining depth +2.2903715 CYP11B1 staining localization +0.7705341 CYP11B2 positive distribution range +0.0190591 CYP11B2 positive staining depth -0.2857227 CYP11B2_staining localization, family=binomial, data=data) Among them, CD56_positive distribution range, CD56_positive staining depth, CD56_staining location, CYP11B1_positive distribution range, CYP11B1_positive staining depth, CYP11B2_positive staining depth, CYP11B2_staining location, and CYP11B2_positive distribution range are the values ​​of each indicator in the tissue.

[0065] A value >0.615 indicates primary aldosteronism; otherwise, it indicates a nonfunctional adenoma.

[0066] Genotype values ​​at each locus (D56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 positive distribution range, CYP11B2 positive staining depth, CYP11B2 staining location, CYP11B2 positive distribution range) were used as input variables, with the patient status of primary aldosteronism as the dependent variable and the predicted probability of having primary aldosteronism as the output. Logistic multivariate regression analysis was performed to obtain a model for predicting primary aldosteronism, and the data distribution and model performance were calculated. The AUC value of the model was tested, and the ROC curve results are shown below. Figure 2 .

[0067] The results of modeling and validation using nearly 278 samples show that the detection and scoring method of this invention, with an optimal threshold of 0.615 (specificity of 0.785 and sensitivity of 0.772), can effectively (AUC value of 0.842) identify patients with primary aldosteronism.

[0068] Independent external verification To further verify the reliability of the model, an additional 20 independent samples (10 patients with primary aldosteronism and 10 patients with non-functional adrenal adenomas) that did not participate in model training were selected as an external validation set. These samples were then input into the prediction model of this invention for blind validation. The results are as follows: Of the 10 samples of primary aldosteronism, 9 had a predictive probability value > 0.615 and were therefore considered positive, with a concordance rate of 90%. Of the 10 samples of non-functional adrenal adenoma, 8 had a predicted probability value ≤0.615 and were judged as negative, with a concordance rate of 80%. The overall accuracy of the external validation set was 85%, consistent with the results of the modeling set, proving that the model of this invention has good stability and generalization ability.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of biomarkers for the diagnosis or prognostic assessment of primary aldosteronism, characterized in that, This includes one or more of the following: CD56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining location, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining location. Among them, the CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in the test adrenal tissue section after immunohistochemical staining, which is used to characterize the expression and distribution breadth of CD56 at the tissue level; CD56 positive staining depth refers to the degree of staining of CD56 positive sites in an adrenal tissue section stained with immunohistochemistry. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CD56 antigen. CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. The CYP11B1 positive staining depth refers to the degree of staining of the CYP11B1 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B1 antigen. CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B2 refers to the percentage of the area of ​​the CYP11B2 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. The CYP11B2 positive staining depth refers to the degree of staining of the CYP11B2 positive staining sites in the immunohistochemically stained adrenal tissue sections. It is determined by semi-quantitative grading and is assigned gradient values ​​based on the differences in light yellow, brownish-yellow, brownish-black to dark brown to characterize the expression abundance of CYP11B2 antigen. CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm.

2. A combination of biomarkers for differentiating nonfunctional adenomas from primary aldosteronism, characterized in that, This includes one or more of the following: CD56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining location, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining location. Among them, the CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in the test adrenal tissue section after immunohistochemical staining, which is used to characterize the expression and distribution breadth of CD56 at the tissue level; CD56 positive staining depth refers to the degree of staining of CD56 positive sites in an adrenal tissue section stained with immunohistochemistry. It can be determined by semi-quantitative grading and usually shows a gradient difference from light yellow, brownish-yellow, brownish-black to dark brown, which is used to characterize the expression abundance of CD56 antigen. CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained sections of adrenal tissue, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. The CYP11B1 positive staining depth refers to the degree of staining of the CYP11B1 positive staining sites in the immunohistochemically stained adrenal tissue sections. It can be determined by semi-quantitative grading and usually presents a gradient difference from light yellow, brownish-yellow, brownish-black to dark brown, which is used to characterize the expression abundance of CYP11B1 antigen. CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. The positive distribution range of CYP11B2 refers to the percentage of the area of ​​the CYP11B2 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section, which is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. The CYP11B2 positive staining depth refers to the degree of staining of the CYP11B2 positive staining sites in the immunohistochemically stained adrenal tissue sections. It can be determined by semi-quantitative grading and usually presents a gradient difference from light yellow, brownish-yellow, brownish-black to dark brown, which is used to characterize the expression abundance of CYP11B2 antigen. CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm.

3. A predictive system for the diagnosis or prognostic assessment of primary aldosteronism, characterized in that, The system includes a data analysis module, which collects and assigns values ​​to the detection results of biomarkers of the target object to be predicted. These biomarkers are used as input features, and the predicted probability of the target object suffering from primary aldosteronism is calculated based on the biomarker values. The input feature is the assignment of the marker combination as described in claim 1 or 2; wherein, The CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in an immunohistochemically stained adrenal tissue section, which is used to characterize the breadth of CD56 expression and distribution at the tissue level; optionally, the percentage of the positive area to the effective observation area (0~100) can be directly used for assignment. CD56 positive staining depth refers to the intensity of CD56 positive staining in an adrenal tissue section stained with immunohistochemistry. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CD56 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. Alternatively, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. The CYP11B1 positive staining depth refers to the intensity of CYP11B1 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B1 antigen expression. Optionally, the gradient is assigned a semi-quantitative grading of 0-3 points: 0 points for consistency with the background color, 1 point for light yellow (weakly distinguishable from the background), 2 points for brownish-yellow (moderately distinguishable from the background), and 3 points for brownish-black / dark brown (strongly distinguishable from the background). CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The CYP11B2 positive distribution range refers to the percentage of the area of ​​the CYP11B2 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. Alternatively, the percentage of the positive region in the effective observation area (0~100) can be directly used for assignment. The CYP11B2 positive staining depth refers to the intensity of CYP11B2 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B2 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections. This includes three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points.

4. A predictive system for distinguishing between non-functional adenomas and primary aldosteronism, characterized in that, The system includes a data analysis module, which collects biomarker values ​​for the target object to be predicted, uses these biomarker values ​​as input features, and calculates the predicted probability value of the target object having primary aldosteronism based on the biomarker values; wherein, The input feature is the assignment of the marker combination as described in claim 1 or 2; wherein, The CD56 positive distribution range refers to the percentage of the effective observation area of ​​the target tissue in an immunohistochemically stained adrenal tissue section, which is used to characterize the breadth of CD56 expression and distribution at the tissue level; optionally, the percentage of the positive area to the effective observation area (0~100) can be directly used for assignment. CD56 positive staining depth refers to the intensity of CD56 positive staining in an adrenal tissue section stained with immunohistochemistry. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CD56 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CD56 staining localization refers to the substructural distribution of CD56 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The positive distribution range of CYP11B1 refers to the percentage of the area of ​​the CYP11B1 antigen-positive stained area in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B1 at the tissue level. Alternatively, the percentage of the positive area in the effective observation area (0~100) can be directly used for assignment. The CYP11B1 positive staining depth refers to the intensity of CYP11B1 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B1 antigen expression. Optionally, the gradient is assigned a semi-quantitative grading of 0-3 points: 0 points for consistency with the background color, 1 point for light yellow (weakly distinguishable from the background), 2 points for brownish-yellow (moderately distinguishable from the background), and 3 points for brownish-black / dark brown (strongly distinguishable from the background). CYP11B1 staining localization refers to the substructural distribution of CYP11B1 positive staining signals within cells in immunohistochemically stained adrenal tissue sections, including three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points. The CYP11B2 positive distribution range refers to the percentage of the area of ​​the CYP11B2 antigen positive staining region in the effective observation area of ​​the target tissue in the immunohistochemically stained adrenal tissue section. It is used to characterize the expression and distribution breadth of CYP11B2 at the tissue level. Alternatively, the percentage of the positive region in the effective observation area (0~100) can be directly used for assignment. The CYP11B2 positive staining depth refers to the intensity of CYP11B2 positive staining in immunohistochemically stained adrenal tissue sections. It is determined through semi-quantitative grading, using a gradient of light yellow, brownish-yellow, brownish-black to dark brown to characterize the abundance of CYP11B2 antigen expression. Optionally, the gradient is assigned a semi-quantitative score of 0-3: 0 points for consistency with the background color, 1 point for light yellow (weakly distinct from the background), 2 points for brownish-yellow (moderately distinct from the background), and 3 points for brownish-black / dark brown (strongly distinct from the background). CYP11B2 staining localization refers to the substructural distribution of CYP11B2 positive staining signals within cells in immunohistochemically stained adrenal tissue sections. This includes three categories: cell membrane localization, cytoplasmic localization, and co-localization of cell membrane and cytoplasm. Optionally, simple cell membrane localization is assigned 0 points, simple cytoplasmic localization is assigned 2 points, and co-localization of cell membrane and cytoplasm is assigned 3 points.

5. The prediction system according to claim 3 or 4, characterized in that, Using the assigned values ​​of the target object's biomarkers as input features, the model calculates the predicted value for whether the target object has primary aldosteronism. The prediction formula is as follows: glm(formula=data$Group~-0.1521900 CD56 positive distribution range -0.0008638 CD56 positive staining depth +0.1592934 CD56 staining localization -0.7659794 CYP11B1 positive distribution range +0.5342818 CYP11B1 positive staining depth +2.2903715 CYP11B1 staining localization +0.7705341 CYP11B2 positive distribution range +0.0190591 CYP11B2 positive staining depth -0.2857227 CYP11B2_staining localization, family=binomial, data=data); Specifically, CD56_positive distribution range, CYP11B1_positive distribution range, and CYP11B2_positive distribution range are assigned values ​​for the positive distribution ranges of CD56, CYP11B1, and CYP11B2, respectively; CD56_positive staining depth, CYP11B1_positive staining depth, and CYP11B2_positive staining depth are assigned values ​​for the positive staining depths of CD56, CYP11B1, and CYP11B2, respectively; and CD56_staining location, CYP11B1_staining location, and CYP11B2_staining location are assigned values ​​for the staining locations of CD56, CYP11B1, and CYP11B2, respectively. Among them, when the predicted value is greater than 0.615, the patient is considered to have primary aldosteronism; otherwise, the patient is considered to have a non-functional adenoma.

6. The prediction system according to any one of claims 3 to 5, characterized in that, The target population includes individuals with incidental adrenal tumors, individuals suspected of having primary aldosteronism, or individuals who require pathological classification evaluation after adrenal adenoma surgery.

7. The prediction system according to any one of claims 3 to 6, characterized in that, It includes an analysis module for processing the following combinations of biomarkers: CD56 positive distribution range, CD56 positive staining depth, CD56 staining location, CYP11B1 positive distribution range, CYP11B1 positive staining depth, CYP11B1 staining location, CYP11B2 positive distribution range, CYP11B2 positive staining depth, and CYP11B2 staining location. The processing method of the analysis module is as follows: (1) Receive the immunohistochemical staining image data of the adrenal tissue section to be tested; (2) Delineate the effective area: delineate the effective observation area of ​​adrenal tumor tissue in the image, and exclude necrotic tissue, stromal tissue and normal adrenal tissue area; (3) Preprocess the image, including color standardization, background correction and area labeling; (4) Identify and quantify the positive distribution range, positive staining depth and staining location of CD56, CYP11B1 and CYP11B2 in the effective observation area; (5) Substitute the assigned value into the regression prediction model to calculate the predicted probability value; (6) Output the predicted probability value and the corresponding judgment result.

8. A detection system for differentiating non-functional adenomas from primary aldosteronism, characterized in that, It includes reagents for detecting the combination of markers as described in claim 1 or 2, and outputs detection results.

9. The detection system according to claim 8, characterized in that, It also includes an image processing system for identifying and performing the following analyses or scores: the percentage of positive distribution range of CD56, CYP11B1, and CD56; the staining depth of positive CD56, CYP11B1, and CYP11B2 and assigning a value based on the difference from the background color; and the staining localization of CD56, CYP11B1, and CYP11B2 and assigning a value based on the localization result.

10. The detection system according to claim 9, characterized in that, The percentage of positive distribution range for each marker is assigned a value (0~100) representing the percentage of the area of ​​the positive region relative to the effective observation area; And / or, the positive staining depth of each marker is assigned a gradient value based on the difference between light yellow, brownish yellow, brownish brown to dark brown; optionally, the gradient value is assigned in a semi-quantitative grade of 0 to 3 points. And / or, the method for assigning values ​​to the staining localization of each marker is as follows: based on staining localization, simple cell membrane localization is assigned 0 points, simple cytoplasm localization is assigned 2 points, and joint localization of cell membrane and cytoplasm is assigned 3 points.

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