Cancer Detection Method and Kit Based on Lipoprotein A-II Biomarker

Abstract

Discloses a method for diagnosing cancer health status, or changes in cancer health status, or for diagnosing changes in cancer or the risk of cancer, comprising determining the value of one or more biomarkers corresponding to a complex structure containing lipoprotein A-II (ApoA2) in a plasma sample from the patient, and determining the patient as having or not having cancer, or having or not having changes in cancer health status, or having or not having the 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).

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 a biomarker, and the use of a biomarker based on a complex structure containing lipoprotein A-II (ApoA2) 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, causing death in approximately 2.5% of cases. Despite a high cure rate for the 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. For the local 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 unexpectedly discovered that different types of lipoprotein A-II (ApoA2) 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 lipoprotein A-II (ApoA2); 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 lipoprotein A-II (ApoA2); 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 HCC, OC and BC.

[0010] In another aspect, the present invention provides a method for diagnosing changes in or risk of cancer in a patient, comprising: determining one or more biomarker values ​​in a plasma sample from the patient corresponding to a complex structure containing lipoprotein A-II (ApoA2); 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 HCC, OC and BC.

[0011] In another aspect, the present invention provides the use of a complex structure containing lipoprotein A-II (ApoA2) 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 lipoprotein A-II (ApoA2) or a complex structure containing ApoA2 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).

[0013] Specifically, this use includes using a capture reagent of ApoA2 or an ApoA2-containing complex to determine one or more biomarker values ​​corresponding to the ApoA2-containing complex. A kit for carrying out the methods described herein is also provided, comprising a capture reagent for ApoA2 or an ApoA2-containing complex, and instructions for carrying out the methods.

[0014] In some embodiments, one or more biomarker values ​​are determined by performing an in vitro analysis. This in vitro analysis may be an immunoassay, including but not limited to Western blot analysis and capillary electrophoresis. In some embodiments, determining the biomarker value includes performing an in vitro analysis comprising a capture reagent targeting ApoA2 or a complex containing ApoA2. In some embodiments, the capture reagent is an antibody. In some embodiments, one or more biomarker values ​​are determined by performing capillary electrophoresis under non-reducing conditions.

[0015] According to some preferred embodiments, one or more biomarker values ​​include IP20, IP60, IP150, or combinations thereof. According to some preferred embodiments, the determination is based on a ratio of the biomarker values, which is selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

[0016] 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

[0017] 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.

[0018] In the diagram: Figure 1 shows the results of Western ink dot analysis of plasma ApoA2 species in three healthy male subjects and three hepatocellular carcinoma patients under reducing and non-reducing conditions. The numbers on the left represent molecular weight markers (kDa).

[0019] Figure 2 shows the migration patterns of plasma ApoA2 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 ~20-kDa peak, while the numbers at the bottom represent the migration location of the protein corresponding to its molecular weight. The pair of numbers at the top of the shaded box (including P20, P60, or P150) indicates the boundary of the mass range used for summing the signal; for example, region P20 represents the ApoA2 dimer. The immunoblots on the right show how the peaks in the capillary western dot analysis correspond to the structures resolved in SDS-PAGE.

[0020] Figure 3 shows a box plot illustrating the statistical analysis of ApoA2 levels in patients with hepatocellular carcinoma (male), hepatocellular carcinoma (female), ovarian cancer, and breast cancer. Statistical analysis was performed on the three ApoA2 multimer indicators. Each point in the figure represents the indicator value for a single subject, and the mean value for each group is shown.

[0021] Figure 4 shows the receiver operating characteristic (ROC) curves for three ApoA2 indicators that distinguish healthy individuals from patients with hepatocellular carcinoma (male), hepatocellular carcinoma (female), ovarian cancer, and breast cancer. Sensitivity for each ApoA2 indicator's cutoff value is shown on the vertical axis, and (1 – specificity) is shown on the horizontal axis. Each point in the figure corresponds to the point with the maximum (sensitivity + specificity). Specific cutoff values ​​are marked as points, and the corresponding performance is represented by dashed lines aligned with the axes.

[0022] Figure 5 shows a summary of the results for both indicators for all healthy subjects (HS) and hepatocellular carcinoma (HCC) patients in this specific embodiment. Dark gray shading represents values ​​more than twice the threshold, while light gray shading represents values ​​between 1 and 2 times the threshold.

[0023] Figure 6 summarizes the results of the two ApoA2 indicators for all female healthy subjects (HS) and ovarian cancer (OC) patients in this study. Dark gray shading represents values ​​more than twice the cutoff value, while light gray shading represents values ​​between one and two times the cutoff value.

[0024] Figure 7 shows a summary of the ApoA2 results for all female healthy subjects (HS) and breast cancer (BC) patients in this study. Dark gray shading indicates values ​​more than twice the cutoff value, while light gray shading indicates values ​​between 1 and 2 times the cutoff value. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] As used in this article, "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 can 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 changes in these biomarkers are often closely related to the occurrence, progression, and treatment response of diseases.

[0028] 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.

[0029] The biomarker values ​​of the biomarkers described herein can be determined using any of a variety of known analytical methods. In some specific embodiments, the biomarker values ​​can be determined by performing in vitro analyses (e.g., immunoassays). In one specific embodiment, the determination of the biomarker value involves the use of a capture reagent. A biomarker value can also refer to a ratio calculated based on two or more biomarker values, such as IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

[0030] As used herein, “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.

[0031] Lipoprotein A-II (ApoA2), encoded by the APOA2 gene, is the second most abundant lipoprotein found in high-density lipoprotein (HDL) particles. While one report has suggested ApoA2 as a potential serum biomarker for cancer screening, it is noteworthy that, based on its original data, the distribution of ApoA2 levels in ovarian cancer (OC) patients significantly overlaps with the upper half of the distribution in healthy controls (Timms et al. (2014), Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies, Proteomics Clin Appl. 8(11-12):982-93). Based on these results, demonstrating a reliable criterion for identifying OC patients solely based on ApoA2 levels presents a significant challenge. ApoA2 has been identified in plasma in monomeric, homodimeric, and ApoA2-ApoD heterodimeric forms. Notably, these two dimers are presumably formed via disulfide bonds. Recent studies have shown that the homodimer is the most common form (Kobayashi et al. (2018), Serum apolipoprotein A2 isoforms in autoimmune pancreatitis, BiochemBiophys Res Commun. 497(3):903-907; Wilkins et al. (2021), Spectrum of apolipoprotein AI and apolipoprotein AII proteoforms and their associations with indices of cardiometabolic health: The CARDIA study. J Am Heart Assoc. 2021 Sep 7;10(17):e019890), which is largely consistent with our findings (see Figure 1). The terms “ApoA2 multimer,” “complex containing ApoA2,” and “ApoA2 complex” are used interchangeably herein and refer to a protein complex containing at least one lipoprotein A-II (ApoA2) subunit, wherein the at least one ApoA2 subunit may be linked to one or more partners (proteins or peptides other than ApoA2).

[0032] In one aspect, the present invention provides a method for diagnosing a patient’s cancer health status, or changes in cancer health status, or for diagnosing changes in cancer or the risk of cancer in a patient, comprising: determining, in a plasma sample from the patient, the value of one or more biomarkers corresponding to a complex structure containing lipoprotein A-II (ApoA2); and determining, based on the biomarker value, 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 the risk of cancer, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), ovarian cancer (OC), and breast cancer (BC).

[0033] The one or more biomarker values ​​may or may not include biomarker values ​​corresponding to the ApoA2 dimer.

[0034] On the other hand, the present invention provides a composite structure containing ApoA2 for use as a biomarker for cancers selected from the group consisting of HCC, OC and BC.

[0035] This invention also provides a capture reagent targeting ApoA2 or a complex containing ApoA2 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 ApoA2 or a complex containing ApoA2 to determine one or more biomarker values ​​corresponding to the complex containing ApoA2.

[0036] In another aspect, the present invention provides a kit for carrying out the methods described herein, comprising a capture reagent for ApoA2 or a composite structure containing ApoA2, and instructions for carrying out the method.

[0037] In another aspect, the present invention provides the use of a capture agent targeting ApoA2 or a composite structure containing ApoA2 in the preparation of a kit for carrying out the methods described herein.

[0038] According to the present invention, based on higher biomarker values ​​corresponding to complex structures containing ApoA2, or lower biomarker values ​​corresponding to complex structures containing ApoA2, a patient can 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 have the risk of HCC, OC, or BC.

[0039] As used herein, a higher (biomarker) value or a 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 threshold) in normal individuals or a control group. For example, the standard or threshold can be set based on the mean or median level obtained from a cohort of normal subjects. In some specific embodiments, the cohort of subjects can be a normal population (without cancer, or without HCC, OC, or BC). Furthermore, the threshold can be further set based on the required sensitivity and / or specificity for detecting or diagnosing HCC, OC, or BC.

[0040] According to certain specific embodiments of the present invention, under non-reducing conditions, conventional SDS-PAGE and Western ink dot analysis can be used to resolve three species or groups of proteins containing ApoA2 in human plasma, including: (i) 11-kDa dimer species, (ii) 60-kDa group with two protein bands at approximately 59 and 62 kDa, and (iii) 330-kDa group with two protein bands at approximately 325 and 345 kDa.

[0041] According to the present invention, the human plasma ApoA2 complex structure can also be resolved by capillary gel electrophoresis into three main peaks, including P20, P60 and P150, which correspond to the 11-kDa class, the 60-kDa group and the 330-kDa group, respectively.

[0042] Biomarker values ​​indicate the concentration of a biomarker in a sample or the ratio of biomarker concentrations in a sample. The biomarker values ​​of this invention can be the signal intensity or normalized signal intensity (denoted as IP20, IP60, and IP150, respectively) of any one of peaks P20, P60, and P150, or the ratio of said 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 immunoassay.

[0043] 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 IP20, IP60, IP150, or combinations thereof. According to some preferred embodiments, based on the ratio of the biomarker values ​​selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20, the patient is determined to have or not have HCC, OC, or BC, or have or not have a change in health status of HCC, OC, or BC, or have or not have a risk of HCC, OC, or BC.

[0044] The present invention is further illustrated by the following embodiments, which are provided for illustrative purposes and not for limiting purposes.

[0045] Example 1. Materials and Methods 1.1 Clinical Samples Preoperative HCC, BC, and OC plasma samples were provided by Dr. Ming-Chih Ho, Dr. Wen-Hong Kuo, and Dr. Pao-Ling Torng. Blood samples were treated with 0.5M EDTA and a protease inhibitor before centrifugation at 3000 RPM for 15 minutes at 4°C. The supernatant was collected as the plasma fraction and stored at -80°C until use.

[0046] 1.2 Analysis of Western Ink Spots For each well, 0.3 μL of plasma sample was mixed with SDS-PAGE sample dye consisting of 0.04 M Tris-HCl pH 6.8, 1 M glycerol, 0.05 M SDS, and bromophenol blue, with 0.3 μL of β-mercaptoethanol added or omitted for reducing or non-reducing analysis. After incubation at 95°C for 5 min, the sample mixture was loaded into the wells of a Tris-based polyacrylamide gel with a 4% stacking gel and a 12% separating gel. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane in CAPS buffer (0.02 M 3-(cyclohexylamino)-1-propanesulfonic acid, containing 10% methanol, pH 11). The ink dots were blocked with 1% BSA in TBST buffer (0.02 M Tris, 0.14 M NaCl, 0.1% Tween 20, pH 7.6), incubated overnight at 4°C with anti-ApoA2 (Cusabio, CSB-RA001915A0HU, rabbit-derived, dilution 1:2,000), and then incubated at room temperature for 1 hour with anti-rabbit HRP conjugate (Jackson, donkey-derived, 711-035-152, dilution 1:10,000), followed by washing with TBST. A chemiluminescent signal was generated using the horseradish peroxidase (HRP)-conjugated secondary antibody and detected using a LAS-4000 (Fujifilm, Japan).

[0047] 1.3 Automated capillary electrophoresis immunoassay Unless otherwise specified, reagents were purchased from BioTechne, USA. Plasma samples were diluted 1:200 and 5X Fluorescent Master Mix was added to each sample. The samples were incubated in a 37°C water bath for 30 minutes. Four microliters of each sample were loaded into the top row wells of a plate pre-filled with proprietary electrophoresis buffer designed to separate proteins in the 12 to 230 kDa range. The other rows of the plate were filled with 1% bovine serum albumin (Bionovas, AA0530-0250; antibody diluent). Primary and secondary antibody solutions, chemiluminescent reagents, and wash buffers were used according to the manufacturer's instructions. For biotinylated SimpleWestern molecular weight standards, these rows were filled with antibody diluent and streptavidin-HRP (Genetex, GTX27403), instead of primary and secondary antibody solutions. Anti-ApoA2 (Cusabio, CSB-RA001915A0HU, rabbit-derived, dilution 1:2,000) was used as the primary antibody; anti-rabbit HRP conjugate (Jackson, donkey-derived, 711-035-152, dilution 1:10,000) was used as the secondary antibody. Plates were centrifuged at 1000 g for 5 minutes at room temperature. The plates and capillaries were then loaded into a SimpleWestern™ system (BioTechne USA) and analyzed using the standard 12 to 230 kDa separation range protocol introduced in the accompanying Compass software version 6.1.0. The separation time was set to the default 25 minutes. The Compass software reported the data as chemiluminescent signal relative to apparent molecular weight (MW). Apparent molecular weight (MW) is determined by aligning the molecular weight marker peak with the capillary position and compensating for variability in intercapillary migration using the signal from fluorescently labeled protein standards in the 5X stock solution.

[0048] 1.4 Migration Map Analysis Data analysis collected via capillary electrophoresis was performed using Microsoft Excel 2021 Visual Basic for Applications (VBA). Raw data obtained from Compass for Switch software was converted to text format and processed into charts, where the x-axis represents molecular weight and the y-axis represents the intensity of the immunoassay signal. To identify specific mass ranges, an internal peak detection and integration procedure was employed, which was further validated manually. These identified ranges were then used to sum the specified peak areas. The sensitivity and specificity of different cutoff values ​​were characterized using receiver operating characteristic (ROC) curve analysis.

[0049] The following is a simplified Chinese translation prepared for you, referencing the language style, format, and terminology of TYG0011CN and removing paragraph numbers: 2. Results 2.1 Compared with the 11-kDa dimer, HCC patients showed a significant increase in the number of circulating ApoA2 multimers with sizes of approximately ~60 kDa and ~330 kDa. To investigate the significance of changes in ApoA2 polymeric structure among the three patient groups, we performed Western ink dot analysis after conventional SDS-PAGE under non-reducing conditions. In healthy controls, in addition to the 11-kDa dimer ApoA2, many other ApoA2-containing variants of varying sizes were observed. Since most of these variants disappeared upon the addition of thiol reagents, they are likely conjugated products linked by disulfide bonds. Analysis of samples from HCC patients showed the presence of most of these ApoA2 variants, with two groups of protein bands showing significant increases. The ~60-kDa group contained 59- and 62-kDa variants; optical density analysis showed a signal intensity of 30–87 × 10⁻⁶ in healthy subjects. 3 AU, while the signal in HCC patients is 41 ~ 180 × 10 3 AU. The ~330-kDa group consisted of 325- and 345-kDa bands, with intensities ranging from 9.5 to 15 × 10⁻⁶ in healthy controls. 3 AU, in HCC patients, is 3.8 ~ 40 × 10 3 AU (Data not displayed).

[0050] 2.2 In female subjects, using the dimer P20 as a standard, the peaks P60 and P150 resolved by capillary electrophoresis showed a significant increase. To quantitatively determine changes in the ApoA2 multimer structure in cancer patients, we attempted to analyze plasma samples under non-reducing conditions using an automated system. This platform resolved the ApoA2 multimer structure into multiple peaks in a migration map. In the maps of healthy subjects, at least eight peaks were clearly identifiable, a complexity largely consistent with patterns observed using SDS-PAGE. The dimer was detected as P20 in this system due to its highest signal. We were impressed that female subjects appeared to have higher signals in peaks P60 and P150. To validate this sex-dependent expression, we used the same analysis to record three parameters in nine male and 28 female healthy controls: IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20. For male subjects, these values ​​averaged approximately 0.07, 0.04, and 0.12, while for female controls, the averages were 0.09, 0.12, and 0.22, respectively (Figure 3). All three indicators did indeed have high values ​​in the female healthy subjects.

[0051] Previous reports have not shown significant differences in ApoA2 levels between male and female subjects. In a recent report on plasma lipoprotein profiling, ApoA2 concentrations in both male and female subjects were within the range of 25–50 mg / dL, with no significant difference. Due to limited knowledge of disulfide-mediated ApoA2 conjugates, there are no reports of sex-dependent changes observed in the two ApoA2 conjugates as revealed by capillary electrophoresis.

[0052] 2.3 The peaks P60 and P150 corresponding to the ~60 kDa and ~330 kDa categories observed by SDS-PAGE showed a significant increase in all three cancer patient groups. For the three patients examined above, their IP60 / IP20 and IP150 / IP20 ratios were approximately 0.06–0.23 and 0.08–0.56, respectively, while the values ​​for the three healthy controls were 0.07–0.09 and 0.03–0.06 (data not shown). This increases the likelihood that P60 and P150 represent the increased 60 kDa and 330 kDa classes in the three HCC patients (Figure 1). Considering gender, we investigated the increases in both sex groups of patients with stage 1 or 2 HCC. For male patients, there were indeed significant increases in IP60 / IP20 and IP150 / IP20 (Figure 2). Compared to 0.07, 0.04, and 0.12 in male subjects, the average values ​​of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20 increased to 0.19, 0.20, and 0.39, respectively. Although the number of female HCC patients was limited, the average values ​​of the three indicators in these patients were 0.24, 0.39, and 0.53, respectively, significantly higher than the 0.09, 0.12, and 0.21 in female controls (Figure 3). These findings suggest that peak P60 and P150 (likely corresponding to the 60 kDa and 330 kDa classes observed in SDS-PAGE) are significantly increased in both male and female HCC patients.

[0053] We then used the same analysis to determine the values ​​of three indicators in 51 patients with stage 1–2 ovarian cancer and 161 patients with stage 0–2 breast cancer. In the case of ovarian cancer patients, the mean values ​​of the multimer indicators IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20 were approximately 0.24, 0.27, and 0.45, respectively (Figure 3). For breast cancer patients, the mean values ​​of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20 were 0.22, 0.33, and 0.55, respectively (Figure 3). All three indicators based on IP60 and IP150 were significantly increased in all three cancer patient groups and could be used to distinguish these cancer patients from healthy controls.

[0054] 2.4 All three ApoA2 multimer markers demonstrated good performance in distinguishing HCC, OC, and BC patients from healthy controls. Since quartile analysis revealed good differentiation between healthy controls and cancer patients (Figure 3), we decided to use ROC curves to investigate whether critical values ​​for these three indicators could be found to distinguish patients from healthy controls. Overall, all these curves were closer to the perfect classifier point than the random classifier line. As expected, the critical values ​​for these three indicators differed significantly between the two sexes. The critical values ​​for female subjects were sometimes twice as high as those for males. For different cancers, these indicators performed better in identifying male HCC and breast cancer patients. IP150 / IP20 appeared to perform better than IP60 / IP20, and (IP60+IP150) / IP20 improved the discriminative power of IP60 / IP20 (Figure 4).

[0055] We then explored the ability of ApoA2 indicators in cancer patient screening. Several factors needed to be considered. First, based on our ROC curve analysis (Figure 4), we selected IP150 / IP20 and (IP60+IP150) / IP20 to test their sensitivity in detecting cancer patients. Second, in clinical practice, only one set of criteria can be used for a single test. Therefore, the cutoff values ​​for each indicator should be the same when screening for different cancers. Third, gender has a significant impact on ApoA2 multimer expression. Taking these factors into account, we set the cutoff value for ApoA2 IP150 / IP20 at 0.07 for men and 0.18 for women, while the cutoff value for (IP60+IP150) / IP20 was set at 0.14 for men and 0.29 for women. Using these criteria, we were able to identify 99%, 84%, and 99% of patients with HCC, OC, and BC, respectively (Figures 5 to 7). Furthermore, higher expression was observed in HCC and BC patients, consistent with ROC analysis results. For example, compared to OC patients, more HCC and BC patients had values ​​more than twice the cutoff value (Figures 5, 6, and 7). Moreover, higher expression was indeed associated with later stages. For example, more stage 1 and stage 2 BC patients had values ​​more than twice the standard value compared to stage 0 patients (Figure 7).

[0056] While this specification contains numerous details, these details should not be construed as limiting the scope of the invention or what can 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 cancer-related health status in a patient, comprising: Determine the value of one or more biomarkers corresponding to the complex structure containing lipoprotein A-II (ApoA2) in the plasma sample from the patient; as well as Based on the biomarker values, the patient is determined to have or not have cancer, or to have or not have changes in cancer-related health status, or to have or not have a risk of developing cancer. The cancer in question is selected from a 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 includes a capture reagent targeting ApoA2 or a complex containing ApoA2.

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 capillary electrophoresis under non-reducing conditions.

5. The method of claim 2, wherein the one or more biomarker values ​​include IP20, IP60, IP150, or a combination thereof.

6. The method of claim 1, wherein the determination is based on a ratio of the biomarker values ​​selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

7. A method for diagnosing changes in or risk of cancer in a patient, comprising: Determine the value of one or more biomarkers corresponding to the complex structure containing lipoprotein A-II (ApoA2) in the plasma sample from the patient; as well as Based on the biomarker values, the patient is determined to have or not have cancer, or to have or not have changes in cancer-related health status, or to have or not have a risk of developing cancer. The cancer in question is selected from a 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 ApoA2 or a complex containing ApoA2.

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 capillary electrophoresis under non-reducing conditions.

11. The method of claim 8, wherein the one or more biomarker values ​​are based on IP20, IP60, IP150, or a combination thereof.

12. The method of claim 7, wherein the determination is based on a ratio of the biomarker values ​​selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

13. A kit for performing a method of diagnosing cancerous health status in a patient, comprising a capture reagent targeting ApoA2 or a complex containing ApoA2, and instructions for performing 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 a complex structure containing ApoA2, 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.

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

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

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

18. The kit of claim 16, wherein the one or more biomarker values ​​include IP20, IP60, IP150, or a combination thereof.

19. The kit of claim 14, wherein the determination is based on a ratio of the biomarker values ​​selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

20. A capture agent targeting lipoprotein A-II (ApoA2) or a complex containing ApoA2 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. A capture reagent for ApoA2 or a complex containing ApoA2 as claimed in claim 20, wherein the use includes using the capture reagent to detect one or more biomarker values ​​corresponding to the complex containing ApoA2, 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 agent for ApoA2 or a complex containing ApoA2 as claimed in claim 20 or 21, wherein the capture agent is an antibody.

23. The capture agent for ApoA2 or a composite structure containing ApoA2 as claimed in claim 21 or 22, wherein the one or more biomarker values ​​are based on IP20, IP60, IP150 or a combination thereof.

24. A capture agent for ApoA2 or a composite structure containing ApoA2 as claimed in claim 21 or 22, wherein the determination is based on a ratio of the biomarker values ​​selected from the group consisting of IP60 / IP20, IP150 / IP20, and (IP60+IP150) / IP20.

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