Cancer detection method and kit based on alpha-1 antitrypsin biomarker

Abstract

Disclosed is a method for diagnosing a cancer health status, or a change in cancer health status, or for diagnosing a risk of cancer change or presence in a patient, comprising determining in a plasma sample from the patient one or more biomarker values corresponding to a complex structure containing alpha-1 antitrypsin (A1AT), and based on the biomarker values, determining the patient as suffering or not suffering from cancer, or having or not having a change in cancer health status, or having or not having a risk of cancer, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

Description

[0001] Cross-referencing This non-provisional application claims priority to U.S. Provisional Application No. 63 / 597,490, filed November 9, 2023, pursuant to Section 119(e) of the U.S. Patent Act, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to biomarkers and the use of biomarkers based on a complex structure containing α-1 antitrypsin (A1AT) for the detection and monitoring of cancer. Background Technology

[0003] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for approximately 80% of cases. HCC is more prevalent in men, with a male-to-fetal ratio estimated to be between 2:1 and 4:1. To date, alpha-fetoprotein (AFP) is the only widely used cancer biomarker for HCC screening. AFP has an overall sensitivity of approximately 70% for HCC across all stages. However, it is not the optimal choice for detecting small, early-stage HCC tumors.

[0004] Ovarian cancer (OC) is the third most common gynecological cancer, with a poor prognosis and the highest mortality rate. Ovarian cancer is often called a silent killer because it presents with a variety of symptoms that typically only appear when the disease has reached an incurable stage. Currently, CA125 and human epididymal protein 4 (HE4) are the only two FDA-approved biomarkers for monitoring ovarian cancer treatment and detecting disease recurrence. While HE4 has limited efficacy, CA125 has a sensitivity of 55% for stage I and II ovarian cancer.

[0005] Breast cancer (BC) is the second most common cancer among women, accounting for approximately 2.5% of deaths. Despite a high cure rate for the localized disease, only 20% of BC cases are diagnosed at an early stage. International research reports that the biomarkers CA15-3 and CA27.29 can be used for BC screening, with sensitivities of 30%–57% and 55%–62%, respectively. In the Taiwanese population, the sensitivity of these two markers is quite low, at 5.5% and 6.4%, respectively.

[0006] Overall, there remains an urgent need for biomarkers with more comprehensive performance in HCC, BC, and OC screening. Protein complexes are true structures with biological functions. In traditional Western ink dot analysis, these protein complexes are difficult to quantify accurately because proteins of different sizes exhibit differences in electrotransfer. Summary of the Invention

[0007] The inventors of this case unexpectedly discovered that different types of α-1 antitrypsin (A1AT) complex structures (or multimer structures) can be detected and quantified in human plasma samples, and their content and ratio can indicate the risk of cancers including hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0008] In one aspect, the present invention provides a method for diagnosing cancer health status in a patient, comprising: determining one or more biomarker values ​​in a plasma sample from the patient corresponding to a complex structure containing α-1 antitrypsin (A1AT); and determining, based on the biomarker values, whether the patient has or does not have cancer, or has or does not have a change in cancer health status, or has or does not have a risk of developing cancer, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0009] In another aspect, the present invention provides a method for diagnosing changes in cancer health status in a patient, comprising: determining one or more biomarker values ​​in a plasma sample from the patient corresponding to a complex structure containing α-1 antitrypsin (A1AT); and determining, based on the biomarker values, whether the patient has or does not have cancer, or has or does not have changes in cancer health status, or has or does not have a risk of developing cancer, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0010] In another aspect, the present invention provides a method for diagnosing changes in or risk of cancer in a patient, comprising: detecting one or more biomarker values ​​in a plasma sample from the patient corresponding to a complex structure containing α-1 antitrypsin (A1AT); and determining, based on the biomarker values, whether the patient has or does not have cancer, or has or does not have changes in cancer health status, or has or does not have a risk of cancer, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0011] In another aspect, the present invention provides the use of a complex structure containing α-1 antitrypsin (A1AT) as a biomarker for a cancer selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC) and breast cancer (BC).

[0012] In another aspect, the present invention provides a capture reagent targeting α-1 antitrypsin (A1AT) or a complex containing A1AT for (in vitro) diagnosis of cancer health status in patients, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC). Specifically, this use includes using the capture reagent containing A1AT or a complex containing A1AT to determine one or more biomarker values ​​corresponding to the complex containing A1AT.

[0013] Also provided is a kit for carrying out the methods described herein, comprising a capture reagent for AlAT or a composite structure containing AlAT, and instructions for carrying out the method.

[0014] In some specific embodiments, the values ​​of one or more biomarkers are determined by performing an in vitro analysis. This in vitro analysis may be an immunoassay, including but not limited to Western blotting and capillary electrophoresis.

[0015] In some specific embodiments, determining the biomarker value includes performing an in vitro analysis, wherein the in vitro analysis comprises a capture reagent targeting AlAT or a complex containing AlAT.

[0016] In some specific embodiments, the capture reagent is an antibody.

[0017] In some embodiments, the one or more biomarker values ​​are determined by performing capillary electrophoresis under non-reducing conditions. According to some preferred embodiments, the one or more biomarker values ​​include IP58, IP130, IP180, or combinations thereof. According to some preferred embodiments, the determination is based on a ratio of the biomarker values, the ratio being selected from IP... 130 / IP 58 IP 180 / IP 58 and (IP) 130 +IP 180 ) / IP 58 The group formed by them.

[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and not intended to limit the invention. Attached Figure Description

[0019] The foregoing overview and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. Preferred embodiments are shown in the drawings to illustrate the invention.

[0020] In the diagram: Figure 1This shows the results of Western ink dot analysis of plasma A1AT species in three healthy male subjects and three hepatocellular carcinoma patients under reducing and non-reducing conditions. The numbers on the left indicate the molecular weight marker (kDa).

[0021] Figure 2 This display shows the migration patterns of plasma A1AT species in capillary electrophoresis from 9 healthy subjects (male), 28 healthy subjects (female), 53 patients with hepatocellular carcinoma (male), 9 patients with hepatocellular carcinoma (female), 51 patients with ovarian cancer, and 161 patients with breast cancer. All patients had cancer severity no greater than stage 2. The numbers on the left represent the percentage of signal relative to the ~60-kDa peak, while the numbers at the bottom indicate the migration location of the protein corresponding to its molecular weight. The pair of numbers at the top of the shaded box (containing P58, P130, or P180) indicates the boundary of the mass range used for summing the signal; for example, region P58 primarily represents A1AT monomers. The immunoblots on the right show how the peaks in the capillary western dot analysis correspond to the structures resolved in SDS-PAGE.

[0022] Figure 3 Box plots show statistical analyses of A1AT levels in patients with hepatocellular carcinoma (male), hepatocellular carcinoma (female), ovarian cancer, and breast cancer. Statistical analyses were performed on the three A1AT multimer indices. Each point in the plot represents the index value for a single individual subject, and the mean value for each group is also shown.

[0023] Figure 4 This graph displays receiver operating characteristic (ROC) curves for three A1AT indicators that differentiate healthy individuals from patients with hepatocellular carcinoma (male), hepatocellular carcinoma (female), ovarian cancer, and breast cancer. Sensitivity is shown on the vertical axis, and (1 – specificity) is shown on the horizontal axis for the critical value of each A1AT indicator. Each point in the graph corresponds to the point with the maximum value (sensitivity + specificity). Specific critical values ​​are marked as points, and the corresponding performance is represented by dashed lines aligned with the axes.

[0024] Figure 5 This presentation summarizes the results of two indicators for all healthy subjects (HS) and patients with hepatocellular carcinoma (HCC) in this specific embodiment. Dark gray shading indicates values ​​that are more than twice the threshold, while light gray shading indicates values ​​that are between one and two times the threshold.

[0025] Figure 6 This presentation summarizes the results of two indicators for all female healthy subjects (HS) and ovarian cancer (OC) patients in this specific embodiment. Dark gray shading indicates values ​​more than twice the threshold, while light gray shading indicates values ​​between one and two times the threshold.

[0026] Figure 7This presentation summarizes the results of two indicators for all female healthy subjects (HS) and breast cancer (BC) patients in this specific embodiment. Dark gray shading indicates values ​​more than twice the threshold, while light gray shading indicates values ​​between one and two times the threshold. Detailed Implementation

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] The singular forms “a,” “an,” and “the” used herein include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to “a sample” includes multiple such samples and their equivalents known to those skilled in the art.

[0029] As used in this article, the term "biomarker" refers to a measurable characteristic, whether internal or external, of an organism that indicates the presence of a specific physiological state or disease. Biomarkers serve as indicators for assessing physiological processes, disease progression, drug response, or treatment efficacy. They may include molecular, cellular, tissue, physiological indicators, or imaging features, and their changes are often closely related to the occurrence, progression, and treatment response of diseases. Biomarkers have important applications in clinical diagnosis, prediction, monitoring, and treatment, helping to improve the accuracy of early disease detection, diagnosis, prognostic assessment, and the evaluation of the effectiveness and safety of treatment regimens.

[0030] As used in this article, the term "cancer" refers to a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. These cells can invade and destroy surrounding healthy tissue and can also metastasize to distant parts of the body. Cancer can arise from almost any type of cell in the body and can develop in a variety of organs and tissues. It is usually caused by gene mutations or other factors that disrupt the normal regulation of cell growth and division.

[0031] The biomarker values ​​of the biomarkers described herein can be determined using any of a variety of known analytical methods. In some specific embodiments, biomarker values ​​can be determined by performing in vitro analyses such as immunoassays. In one specific embodiment, the determination of biomarker values ​​involves the use of capture reagents. Biomarker values ​​can also refer to ratios calculated based on two or more biomarker values, such as IP130 / IP58, IP180 / IP58, and (IP130+IP180) / IP58.

[0032] As used in this article, “capture agent” or “capture reagent” refers to a molecule capable of specifically binding to a biomarker. Capture reagents include, but are not limited to, aptamers, antibodies, antigens, adnectins, ankyrins, other antibody mimics and other protein scaffolds, autoantibodies, chimeras, small molecules, F(ab')2 fragments, single-chain antibody fragments, Fv fragments, single-chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, affibodies, nanobodies, imprinted polymers, Avimers, peptide mimics, hormone receptors, cytokine receptors and synthetic receptors, as well as modifications and fragments of these substances.

[0033] α-1 antitrypsin (A1AT protein) in humans is... SERPINA1 The gene encodes a protein belonging to the serine protease inhibitor (serpin) superfamily. According to the BioGPS database, this protein is expressed in various human tissues, including the lung, small intestine, bone marrow, and liver. The link between A1AT deficiency and an increased risk of liver disease has been well documented (Teckman JH, Jain A (2014) Advances in alpha-1-antitrypsin deficiency liver disease. Curr Gastroenterol Rep 16(1):367). In patients expressing the defective A1AT protein, A1AT mutants tend to accumulate and polymerize in hepatocytes. It is speculated that these polymers are formed by non-covalent bonds and can dissociate into monomers even in the absence of reducing agents (Lomas DA, Evans DL, Finch JT, Carrell RW. (1992) The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature. 357(6379):605). In contrast, our findings concern A1AT entities of unique sizes that are observed only under non-reducing conditions and disappear upon thiol-mediated reduction (Figure 1). Therefore, these cyclic A1AT polymers may be assembled via disulfide bonds. Furthermore, while our findings reveal an association between A1AT complex formation and cancer, there have been no previous reports on how these A1AT polymers are associated with any disease or health condition.

[0034] The terms “A1AT multimer,” “A1AT-containing complex,” and “A1AT complex” used herein are used interchangeably to refer to a protein complex containing at least one α-1 antitrypsin (A1AT) subunit, wherein the at least one A1AT subunit may be linked to one or more partners (proteins or peptides other than A1AT).

[0035] In one aspect, the present invention provides a method for diagnosing a patient’s cancer health status, or a change in cancer health status, or for diagnosing a change in cancer or the risk of cancer in a patient, comprising determining one or more biomarker values ​​corresponding to a complex structure containing A1AT in a plasma sample from the patient, and determining the patient as having or not having cancer, or having or not having a change in cancer health status, or having or not having a risk of cancer based on the biomarker values, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0036] The one or more biomarker values ​​may or may not include biomarker values ​​corresponding to the A1AT monomer.

[0037] In another aspect, the present invention provides the use of the aforementioned composite structure containing A1AT as a biomarker for cancers selected from the group consisting of HCC, OC and BC.

[0038] The following is a simplified Chinese translation prepared for you, using patent terms such as "disclosed" and "described" as requested: The present invention also provides a capture reagent targeting A1AT or a complex containing A1AT for (in vitro) diagnosis of cancer health status in patients, wherein the cancer is selected from the group consisting of HCC, OC, and BC. The use may include using the capture reagent targeting A1AT or a complex containing A1AT to determine one or more biomarker values ​​corresponding to the complex containing A1AT.

[0039] In another aspect, the present invention provides a kit for carrying out the methods described herein, comprising a capture reagent for AlAT or a composite structure containing AlAT, and instructions for carrying out the methods.

[0040] In another aspect, the present invention provides the use of the described capture reagent for A1AT or a composite structure containing A1AT in the preparation of kits for carrying out the methods described herein.

[0041] According to the present invention, a patient may be determined to have or not have HCC, OC, or BC, or have or not have changes in health status of HCC, OC, or BC, or have or not be at risk of having HCC, OC, or BC, based on a higher biomarker value corresponding to a complex structure containing A1AT, or a lower biomarker value corresponding to a complex structure containing A1AT.

[0042] As used herein, “higher (biomarker) value” or “lower (biomarker) value” can refer to a value that is higher or lower than a reference level. For example, a lower value can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than a reference level; and a higher value can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than a reference level. In some specific embodiments, the reference level can be a standard (or cutoff) value in normal individuals or a control group. For example, the standard or cutoff value can be set based on the mean or median level obtained from a group of normal subjects. In some specific embodiments, the subject group can be a normal population (without cancer, or without HCC, OC, or BC). Furthermore, the cutoff value can be further set based on the sensitivity and / or specificity required to detect or diagnose HCC, OC, or BC.

[0043] According to certain specific embodiments of the present invention, conventional SDS-PAGE and Western ink dot analysis under non-reducing conditions can be used to resolve four substances or groups of human plasma containing A1AT proteins, including: (i) a 56-kDa monomeric substance, (ii) a 62-kDa substance (migrating slightly slower than the A1AT monomer), (iii) a 150-kDa group (with two protein bands at approximately 135 and approximately 160 kDa), and (iv) a 260-kDa group (containing three polypeptides with molecular weights of approximately 225, 265, and 295 kDa, respectively).

[0044] According to the present invention, the human plasma A1AT complex structure can also be resolved by capillary gel electrophoresis into three main peaks, including P58, P130 and P180, which correspond to the 56 / 62-kDa substance, the 150-kDa group and the 260-kDa group, respectively.

[0045] Biomarker values ​​indicate the concentration of a biomarker in a sample, or a ratio of biomarker concentrations. The biomarker values ​​of this invention can be the signal intensity or normalized signal intensity (denoted as IP58, IP130, and IP180, respectively) of any one of peaks P58, P130, and P180, or a ratio of signal intensities. Signal intensity can be measured as the area under the peak. In some specific embodiments, signal intensity is measured as the area under the peak in immunoassays.

[0046] In some specific embodiments, the one or more biomarker values ​​are determined by performing capillary electrophoresis under non-reducing conditions. Specifically, biomarker signals are detected by performing capillary electrophoresis and immunoassay, and the one or more biomarker values ​​are then determined based on the detected biomarker signals. According to some preferred embodiments, the one or more biomarker values ​​include IP58, IP130, IP180, or combinations thereof. According to some preferred embodiments, the values ​​are based on a selection from… , and The ratio of the biomarker values ​​of the group to which the patient is classified as having or not having HCC, OC, or BC, or having or not having changes in health status of HCC, OC, or BC, or having or not having the risk of developing HCC, OC, or BC.

[0047] The following is a simplified Chinese translation of the examples section prepared for you: The present invention is further illustrated by the following embodiments, which are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] Example 1. Materials and Methods 1.1 Clinical Samples Plasma samples from patients with hepatocellular carcinoma (HCC), breast cancer (BC), and ovarian cancer (OC) were provided by Dr. Ming-Chih Ho, Dr. Wen-Hung Kuo, and Dr. Pao-Ling Tung of the National Taiwan University Hospital. Before centrifuging at 3000 RPM for 15 minutes at 4°C in a benchtop centrifuge, the blood samples were treated with 0.5M EDTA and a protease inhibitor. The supernatant was collected as the plasma fraction and stored at -80°C until use.

[0049] 1.2 Analysis of Western Ink Spots In each well, 0.3 μL of plasma sample was mixed with a sample dye containing SDS (0.04 M Tris-HCl pH 6.8, 1 M glycerol, 0.05 M SDS with bromophenol blue), and 0.3 μL of β-mercaptoethanol was added or omitted for reduction or non-reduction analysis. After heating for 5 min, the mixed sample was loaded into the wells of a Tris-based polyacrylamide gel with a 4% stacking gel / 12% separating gel. After SDS-PAGE, the gel was incubated in electrophoresis solution (0.025 M Tris, 0.2 M glycine, 3 mM SDS), and proteins were transferred to a nitrocellulose membrane using 0.02 M 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS) buffer (pH 11) containing 10% methanol. After electrotransfer, the membrane was immunoblotted with the specified antibody. Blocking was performed using 1% BSA in TBST buffer (0.02 M Tris, 0.14 M NaCl, 0.1% Tween 20, pH 7.6), followed by overnight incubation at 4°C with anti-A1AT (Abcam, ab207303, rabbit-derived, 1:5,000 dilution). Incubation was then performed at room temperature for 1 hour with an anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (Jackson, donkey-derived, 711-035-152, 1:10,000 dilution). The membrane was washed three times with TBST after each antibody incubation. Immediately after coating with chemiluminescence assay reagents, the signal from the membrane was acquired using a LAS-4000 (Fujifilm, Japan).

[0050] 1.3 Automated capillary electrophoresis immunoassay Unless otherwise specified, reagents and equipment were purchased from BioTechne, Inc., USA. Plasma samples were diluted 1:200, and 5X Fluorescent Master Mix was added to each sample. The samples were heated in a water bath for 30 minutes. In the sample plate, 4 μL of each sample and electrophoresis buffer for separating proteins in the 12 to 230 kDa range were loaded. Additionally, 1% bovine serum albumin (or antibody diluent, Bioovas, AA0530-0250), primary and secondary antibody solutions, chemiluminescence reagents, and wash buffer were used according to the manufacturer's instructions. For biotinylated SimpleWestern molecular weight standards, antibody diluent and streptavidin-HRP (Genetex, GTX27403) were used instead of primary and secondary antibody solutions. The primary antibody was anti-A1AT (Abcam, ab207303, rabbit-derived, 1:2,000 dilution), and the secondary antibody was an anti-rabbit HRP conjugate (Jackson, donkey-derived, 711-035-152). After centrifugation at 1000 g for 5 minutes at room temperature, the plate and capillary tube were loaded into a SimpleWestern™ system (BioTechne, USA) running Compass software (version 6.1.0). The mass range was set to the standard 12 to 230 kDa protocol, and the separation time was set to the default 25 minutes. The chemiluminescence signal was correlated with the apparent molecular weight (MW) using fluorescently labeled protein standards.

[0051] 1.4 Migration Map Analysis Data from the SimpleWestern system was analyzed using Microsoft Excel 2021 and its Visual Basic for Applications (VBA) software package. Raw data obtained from Compass for SW was converted to text format and then to charts, where the x-axis represents molecular weight and the y-axis represents the intensity of the immunoassay signal. To quantify peaks within a specific mass range, a self-developed peak detection and integration program was used, which was further validated manually. Subsequently, the area within the specific mass range was used to construct a labeling index.

[0052] The following is a simplified Chinese translation of the "2. Results" section, which continues the writing style and terminology of the aforementioned patent specification: 2. Results 2.1 In the plasma of patients diagnosed with hepatocellular carcinoma (HCC), ovarian cancer (OC), or breast cancer (BC), the levels of 62-kDa, 135-160 kDa, and 225-295 kDa polymeric A1AT were increased relative to the 56-kDa monomeric form. Plasma samples from three healthy subjects and three HCC patients underwent routine SDS-PAGE and Western ink dot analysis under non-reducing conditions to compare disulfide bond-mediated A1AT conjugates between the two groups. In addition to the 56-kDa monomer, three groups of A1AT conjugates were identified based on molecular weight. The first group, a 62-kDa conjugate, migrated slightly slower than the A1AT monomer. The second group, a 150-kDa conjugate, exhibited two protein bands at 135 and 160 kDa. The third group, a 260-kDa conjugate, contained three peptides with molecular weights of 225, 265, and 295 kDa. Western ink dot analysis showed increased A1AT levels in all three groups, which were recorded by density analysis. For healthy subjects, the signal values ​​for the 150-kDa and 260-kDa groups were 0.21–0.30 and 0.26–0.38, respectively. In contrast, the values ​​increased to 0.26–0.36 and 0.34–0.43 in HCC patients (data not shown). Similar findings were also observed in A1AT material in ovarian cancer (OC) or breast cancer (BC) (data not shown). All three groups of multimeric structures disappeared in reduced SDS-PAGE ( Figure 1 Therefore, it is hypothesized that disulfide bonds are the linking mechanism between the AlAT subunits and their partners. Since many factors can interfere with accurate quantification based on conventional Western ink dot analysis, we decided to employ a different approach to evaluate the structures of these AlAT polymers.

[0053] 2.2 Peaks P130 and P180 in capillary electrophoresis correspond to the 150-kDa and 260-kDa groups in SDS-PAGE analysis. To better quantify changes in A1AT polymeric structure in cancer patients, we utilized an automated capillary electrophoresis immunoassay. This platform allowed us to observe these A1AT polymeric structures in the migration profiles of test plasma samples under non-reducing conditions. In the profiles of healthy subjects, three distinct peaks were observed, including P58, P130, and P180. The overall pattern differences observed between men and women were minimal. Due to the limited resolution of capillary electrophoresis, the 62-kDa substance may have been integrated into the P58 peak of the monomeric A1AT and therefore could not be measured by this analysis. Two other groups of A1AT substances were resolved to peaks P130 and P180. Based on their migration positions, the former corresponds to the 150-kDa group, and the latter to the 260-kDa group. To quantify these increases, we established three indicators: , and IP58, IP130, and IP180 represent the peak areas of the corresponding substances. Three healthy control subjects... and The values ​​were 0.02–0.04 and 0.05–0.11, respectively, while those for HCC patients were 0.04–0.11 and 0.13–0.31 (data not shown). These findings are generally consistent with the results of density analysis (Figure 1), strongly suggesting that the described automated system can be used for quantitative analysis of these two groups of polymeric structures.

[0054] 2.3 For most patients with HCC, BC, and OC, both peaks P130 and P180 showed a significant increase compared to peak P58. Automated capillary electrophoresis immunoassay was performed on samples from 9 male and 28 female healthy controls. We found that gender had almost no effect on the migration profiles of the A1AT multimer structure (Figure 2). Plasma samples from 53 male HCC patients, 9 female HCC patients, 51 OC patients, and 161 BC patients (stages 0-2) were analyzed using the SimpleWestern system. These results showed a significant increase in the types of proteins corresponding to P130 and P180 in the patient groups (Figure 2). For BC patients, the change in P180 appeared to be more significant than in other cancer groups, but the increase in P130 was not as consistent as in the other two cancers (Figure 2).

[0055] for The mean value for this indicator in healthy subjects was approximately 0.04. In contrast, the mean values ​​for male HCC, female HCC, OC, and BC patients were 0.08, 0.12, 0.08, and 0.13, respectively. Regarding... The mean value for the index was approximately 0.1 in the healthy control group, while the values ​​in patients with HCC, OC, and BC ranged from 0.2 to 0.4 (Figure 3). These results are consistent with the increased peak heights of P130 and P180 observed in the migration plot (Figure 2). To account for the total changes in P130 and P180 polymers, we introduced […]. The values ​​ranged from 0.15 to 0.16 in healthy controls, increasing to 0.3 to 0.35 in HCC patients, 0.33 in the OC group, and 0.5 in the BC group (Figure 3). These all indicate that the peak P130 and P180 were significantly increased in most HCC, OC, and BC patients. and The statistical parameters of the indicators showed a very high degree of similarity, consistent with the observation that the increase in peak P180 was much higher than that of peak P130 in most migration maps. Considering the distribution of these values ​​across quartiles (Figure 3), cutoffs can be established to distinguish patients from healthy controls.

[0056] 2.4 The A1AT multimer index based on peak P130 and P180 showed excellent performance in screening patients with HCC, OC, and BC. We used receiver operating characteristic (ROC) curves to assess the potential of these A1AT indices in distinguishing between patients and healthy subjects. The curves for HCC and OC patients are usually closer to the randomization line, but for BC patients they are closer to the ideal classifier. The ROC curves of the indicator showed a clearer separation from the random classification line, with a sensitivity ranging from 77% to 100% at a critical value of 0.11 to 0.14. This differentiation was most pronounced in the BC patient group. The sensitivity of the indicator is approximately 66% to 70% when the cutoff value is between 0.14 and 0.20. Although this indicator still performs best in BC patients, it is not superior to... How much better are the indicators (Figure 4)?

[0057] We then explored the potential of the A1AT index in cancer patient screening. First, based on our ROC curve analysis (Figure 4), we selected... and To test their sensitivity in detecting cancer patients. Secondly, in clinical practice, a single test typically only has one standard for different types of cancer. Taking these factors into consideration, we tentatively decide to use A1AT. The critical value is set to 0.13, while The threshold value is set to 0.19. Overall, using A1AT... and The indicators could identify 82%, 80%, and 94% of patients with HCC, OC, and BC, respectively (Figures 5 to 7). These cancer patient groups showed better performance than... More patients had a value twice the threshold than This is consistent with the separation results in ROC analysis. Furthermore, compared to HCC or OC patients, more BC patients showed higher [specific abnormalities / conditions]. In conclusion, our results demonstrate that these indicators have excellent sensitivity and specificity in detecting cancer patients.

[0058] While this specification contains numerous details, these details should not be construed as limiting the scope of the invention or the content for which protection may be claimed, but rather as descriptions of features specific to particular embodiments or examples of the invention. Some features described in the context of individual embodiments or examples in this specification may also be implemented in combination in a single embodiment.

Claims

1. A method for diagnosing the cancer health status of a patient, characterized in that, include: Determine the value of one or more biomarkers corresponding to the complex structure containing α-1 antitrypsin (A1AT) in the plasma sample from the patient; as well as Based on the aforementioned biomarker values, the patient is determined to have or not have cancer, or to have or not have a cancer-related health status change, or to have or not have a risk of developing cancer. The cancers mentioned therein are selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

2. The method of claim 1, wherein determining the biomarker value comprises performing an in vitro analysis, wherein the in vitro analysis comprises a capture reagent targeting AlAT or a complex containing AlAT.

3. The method of claim 2, wherein the capture reagent is an antibody.

4. The method of claim 2, wherein the in vitro analysis is performed under non-reducing conditions using capillary electrophoresis.

5. The method of claim 2, wherein the one or more biomarker values ​​include IP58, IP130, IP180, or a combination thereof.

6. The method of claim 1, wherein the determination is based on a ratio of the biomarker values, the ratio being selected from... , and The group formed by them.

7. A method for diagnosing changes in or the risk of cancer in a patient's body, characterized in that, include: Determine the value of one or more biomarkers corresponding to the complex structure containing α-1 antitrypsin (A1AT) in the plasma sample from the patient; as well as Based on the aforementioned biomarker values, the patient is determined to have or not have cancer, or to have or not have a cancer-related health status change, or to have or not have a risk of developing cancer. The cancers mentioned therein are selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

8. The method of claim 7, wherein determining the biomarker value comprises performing an in vitro analysis, wherein the in vitro analysis comprises a capture reagent targeting AlAT or a complex containing AlAT.

9. The method of claim 8, wherein the capture reagent is an antibody.

10. The method of claim 8, wherein the in vitro analysis is performed under non-reducing conditions using capillary electrophoresis.

11. The method of claim 7, wherein the one or more biomarker values ​​are based on IP58, IP130, IP180, or a combination thereof.

12. The method of claim 7, wherein the determination is based on a ratio of the biomarker values, the ratio being selected from... , and The group formed by them.

13. A kit for implementing a method for diagnosing the health status of a patient with cancer, characterized in that, The method includes a capture agent targeting A1AT or a composite structure containing A1AT, and instructions for carrying out the method, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

14. The kit of claim 13, wherein the method comprises using the capture reagent to determine one or more biomarker values ​​corresponding to the complex structure containing AlAT, and determining the patient as having or not having cancer, or having or not having a cancer-related health status change, or having or not having a risk of developing cancer based on the biomarker values.

15. The kit as claimed in claim 13 or 14, wherein the capture agent is an antibody.

16. The kit of claim 14, wherein determining the biomarker value comprises performing an in vitro analysis using the capture reagent.

17. The kit of claim 16, wherein the in vitro analysis is performed under non-reducing conditions using capillary electrophoresis.

18. The kit of claim 14, wherein the one or more biomarker values ​​include IP58, IP130, IP180, or a combination thereof.

19. The kit of claim 14, wherein the determination is based on a ratio of the biomarker values, the ratio being selected from... , and The group formed by them.

20. A capture agent targeting α-1 antitrypsin (A1AT) or a complex containing A1AT, for diagnosing cancer health status in a patient, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

21. The capture reagent for diagnosis as claimed in claim 20, wherein the use includes using the capture reagent to detect one or more biomarker values ​​corresponding to a complex structure containing AlAT, and determining the patient based on the biomarker values ​​whether the patient has or does not have cancer, or has or does not have a cancer-related health status change, or has or does not have a risk of developing cancer.

22. The capture reagent for diagnosis as claimed in claim 20 or 21, wherein the capture reagent is an antibody.

23. The capture reagent for diagnosis as claimed in claim 21 or 22, wherein the one or more biomarker values ​​are based on IP58, IP130, IP180, or a combination thereof.

24. The capture reagent for diagnosis as described in claim 21 or 22, wherein the determination is based on a ratio of the biomarker values ​​selected from... , and The group formed by them.

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