Methods for the management of prostate cancer based on PSA glycosylation

Abstract

The present invention relates to a method for assessing whether a patient has aggressive prostate cancer by determining the level of a specific glycoform attached to the prostate specific antigen (PSA) protein in a biological fluid sample from the subject and comparing the determined level or concentration to a standard. The method is particularly useful for assessing subjects having 2-10 ng / ml of total PSA in the subject's serum.

Description

[Technical field]

[0001] The present invention relates to methods for the management of prostate cancer based on PSA glycosylation. [Background technology]

[0002] Worldwide, more than 1.4 million patients were diagnosed with prostate cancer (PCa) in 2020 (Sung et al., CA Cancer J. Clin. 71: 209-249, 2021). Serum prostate-specific antigen (PSA) concentrations are used as an early detection and prediction method for PCa, but this test has low sensitivity, specificity, and poor predictive value (Wolf et al., Ca-Cancer J. Clin. 60: 70-98, 2010). Early studies have shown that glycosylation of PSA may provide a promising lead for more specific PCa diagnosis. Recently, the Center for Proteomics and Metabolomics (CPM) at the Leiden University Medical Center established a high-performance PSA glycomics assay (PGA) that allows detailed glycosylation analysis, including discrimination of α2,6- and α2,3-sialylated isomers (Figure 1; Kammeijer et al., Anal. Chem. 90:4414-4421, 2018). These isomers have also been suggested to be potential biomarkers for aggressive types of cancer (Schultz et al., Cancer Metastasis Rev. 31:501-518, 2012). PGA involves a PSA affinity purification step followed by tryptic digestion and mass spectrometry (MS) analysis by capillary electrophoresis-electrospray ionization (CE-ESI) (Kammeijer et al., Anal. Chem. 90:4414-4421, 2018).

[0003] The use of PSA as a biomarker has significant drawbacks. First, PSA is not a PCa-specific biomarker because its fluid levels (generally blood) do not efficiently distinguish PCa from other prostate diseases, such as benign prostatic hyperplasia (BPH) or prostatitis (Hudson et al., J Urol. 151:1291-1291, 1989). Furthermore, serum levels of PSA are influenced by several factors, such as age, comorbidities, ejaculation, catheterization, and medication (Hatekeyama et al., Int J Clin Oncol, 22:214-221, 2017). PSA discrimination between PCa and benign prostate disease is particularly inefficient in the so-called "gray area" in the range of 2-10 ng / ml (Barry, MJ, N Engl J Med. 360(13):1351-4, 2009), and in distinguishing between indolent and aggressive PCa (Lamy et al., Eur Urol Focus. 4(6):790-803, 2018).

[0004] Screening for PCa with PSA has led to a reduction in advanced disease and disease-specific mortality. However, the trade-off is overdiagnosis of cases that, if left untreated, would not have caused clinical consequences during a person's lifetime. Overdiagnosis, in turn, has led to overtreatment with significant risks as side effects from biopsy or negative outcomes from treatment (Loeb et al., Eur Urol, 65(6):1046-1055, 2014). According to www.uspreventiveservicestaskforce.org (Final Recommendation Statement: Screening for Prostate Cancer and Final Evidence Review: Screening for Prostate Cancer. US Preventive Services Task Force. May 2018. www.uspreventiveservicestaskforce.org), 20-50% of men who test positive have an indolent, non-threatening form of PCa. These data clearly indicate a major problem of overdiagnosis and the need for improved and more specific tools for PCa diagnosis and assessment of cancer aggressiveness.

[0005] A single N-linked glycosylation site (N 69) is dominated by a highly heterogeneous group of glycans, resulting in various so-called glycoforms with the same protein backbone. Previous studies speculated that detailed analysis of PSA glycoforms could provide more efficient PCa diagnosis (Vermassen et al. (supra); Jankovic et al., Clin. Biochem. 38:58-65, 2005; Peracaula et al., Glycobiology. 13:457-470, 2003); Saldova et al. Glycobiology. 21:195-205, 2011). Follow-up studies on molecular features of PSA glycosylation (e.g., antennary modifications and core fucosylation) also suggested improved PCa diagnosis by combining conventional PSA testing with specific N-glycan features (Kyselova et al. J. Proteome Res. 6:1822-1832, 2007; Vermassen et al. Electrophoresis. 35:1017-1024, 2014; Yoneyama et al., Biochem. Biophys. Res. Commun. 448:390-396, 2014), but the size of the cohorts tested was limited, and there is still a high need to identify new glycol structures. Furthermore, there is a need to identify biomarkers for assessing PCa aggressiveness.

[0006] Detection of PSA glycoforms is currently performed using lectin or mass spectrometry-based methods (Llop et al., Theranostics. 6(8):1190-204, 2016; Kuzmanov et al., BMC Med. 11:31, 2013). A limitation of lectin application is their reactivity only towards sugar residues and not towards the protein backbone. Furthermore, the specificity of lectins to discriminate between related glycan structures is low and their reactivity is mostly based on binding activity rather than affinity. On the other hand, mass spectrometry-based methods require more purification steps, especially for complex matrices (e.g. serum or plasma). Summary of the Invention

[0007] The present inventors have demonstrated that prostate-specific antigen (PSA), secreted from prostate cells into biological fluids such as blood and urine, is variously glycosylated, and have surprisingly found that in subjects with aggressive PCa, the levels of certain PSA glycoforms are increased, while the levels of other PSA glycoforms are decreased. The present invention relies on these changes in the levels of one or more specific PSA glycoform(s) to detect whether a subject has or is likely to have aggressive PCa. Thus, the method of the present invention can be used to determine whether a subject who has or is suspected of having prostate disease (which may be any form of PCa or a non-cancerous prostate disorder such as prostatitis or BPH) is likely to have indolent disease or aggressive PCa. This characterization can then be used to inform subsequent therapeutic or analytical interventions. For example, individuals determined to be likely to have aggressive PCa can be selected for biopsy and / or therapeutic treatment. Individuals determined to be unlikely to have aggressive PCa can be selected for "watchful waiting", i.e. regular monitoring but no (direct) intervention.

[0008] According to a first aspect of the present invention there is provided a method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) determining a level of at least one monoantennary PSA glycoform and a level of at least one diantennary PSA glycoform in a biological fluid sample from a subject; (b) calculating an aggressiveness score for the subject using the level determined in (a); and (c) comparing the subject's aggressiveness score in (b) with a reference value for that aggressiveness score; (d) using the comparison in (c) to help determine whether the subject has aggressive prostate cancer. A method is provided, comprising:

[0009] According to a second aspect of the invention there is provided a method for determining whether a subject has aggressive prostate cancer comprising the steps of: (i) isolating the PSA protein from other proteins in a biological fluid sample from a subject; (ii) treating the isolated PSA protein with a protease; (iii) Two or more Ns in the sample 69 determining the level of each of the glycopeptide forms, 69 At least one of the glycopeptide forms is a monoantennary glycoform, 69 determining a level of each of two or more N69 glycopeptide forms in the sample, wherein at least one of the glycopeptide forms is a diantennary glycoform; (iv) Each N in (iii) 69 determining an aggressiveness score for the subject using the level of glycopeptide form; (v) comparing the aggressiveness score in (iv) with a reference value for that aggressiveness score; (vi) determining whether the subject has aggressive PCa based on the comparison in step (v); A method is provided, comprising:

[0010] In certain embodiments, the level determined in step (iii) is a normalized level. In certain embodiments, the PSA protein is isolated from a biological fluid sample derived from the subject using a binding protein (such as an antibody) specific for the PSA protein.

[0011] In certain embodiments, the level of a PSA glycoform is determined by normalizing the amount of a PSA glycoform (e.g., N 69 glycopeptide).

[0012] In certain embodiments, the level of a PSA glycoform is measured using a normalized signal for all PSA glycoforms measured (e.g., all glycoforms shown in Table 1A). 69 The signal corresponds to a normalized signal (e.g., area under the curve) that correlates with the amount of glycopeptide (glycopeptide) in the sample. In certain embodiments, the PSA protein is isolated / separated from other components in the biological fluid sample, such as other proteins, using a binding protein (such as an antibody) specific for the PSA protein.

[0013] It will be appreciated that the isolated PSA protein may contain a number of different proteoglycan species (PSA glycoforms). Proteases serve to digest the PSA protein into smaller peptide forms so that the various proteoglycan species can be attached to the smaller peptides. Suitable proteases include N 69 Another suitable protease is trypsin, which produces a K dipeptide proteoglycan population. 69 KSVILLGR (SEQ ID NO:3) is an Arg-C (clostripain) producing proteoglycan population.

[0014] In certain embodiments, the aggressiveness score is determined with respect to the first aspect of the invention.In certain embodiments, a baseline aggressiveness score is determined with respect to the first aspect of the invention.

[0015] The first and second aspects of the present invention comprise the measurement of the level of at least two PSA glycoforms.However, as can be seen from Table 6, the levels of three PSA glycoforms (compounds 21, 2 and 9) can individually better distinguish aggressive PCa from indolent PCa and BPH than free PSA (higher area under the curve (AUC) in receiver operating characteristic (ROC) analysis).Therefore, the levels of each of these three PSA glycoforms can be used individually in a method to help determine whether a subject has aggressive PCa.

[0016] Thus, according to a third aspect of the present invention there is provided a method of assisting in determining whether a subject has aggressive prostate cancer, comprising the steps of: (a) H4N5F1S in a biological fluid sample from a subject 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 determining the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform in (a) with a reference value for that PSA glycoform, or comparing the aggressiveness score taking into account the level of that PSA glycoform in (a) with a reference value for that aggressiveness score; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer.

[0017] According to a fourth aspect of the present invention there is provided a computer implemented method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) receiving a value for a level of a first monoantennary PSA glycoform in a biological fluid sample of a subject; (b) receiving a value for the level of the first diantennary PSA glycoform in the subject's biological fluid sample; (c) calculating an aggressiveness score for the subject based on the levels received in (a) and (b); (d) comparing the subject's aggressiveness score in (c) with a reference value for said aggressiveness score; (e) using the comparison in (d) to help determine whether the subject has an aggressive form of prostate cancer; A computer-implemented method is provided, comprising:

[0018] In certain embodiments, the first monoantennary PSA glycoform in step (a) is selected from compounds 2, 3, 4, or 5 (as shown in Tables 1A and B).

[0019] In certain embodiments, the first diantennary PSA glycoform in step (b) is selected from compounds 7, 9, 10, 11, 13, 16, 18, 21, 24, or 25 (as shown in Tables 1A and B).

[0020] In certain embodiments, the aggressiveness score of step (c) is calculated from a combination of the levels received in steps (a) and (b). In certain embodiments, the aggressiveness score of step (c) is calculated from importing the levels received in steps (a) and (b) into a formula (e.g., a weighted calculation matrix, a ratio, etc.).

[0021] The values ​​received in (a) and (b) may be obtained from the PSA glycoforms determined in the first aspect of the invention. Where appropriate, embodiments applicable to the first aspect of the invention may be applied to this fourth aspect of the invention.

[0022] According to a fifth aspect of the present invention there is provided a computer implemented method for assisting in determining whether a subject has an aggressive form of PCa, comprising: (a) H4N5F1S in biological fluid samples of interest 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 receiving a value for the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform received in (a) with a reference value for that PSA glycoform, or comparing an aggressiveness score taking into account the level of the PSA glycoform received in (a) with a reference value for that aggressiveness score; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer; A method is provided, comprising:

[0023] In certain embodiments of the fifth aspect of the invention, there is provided a computer-implemented method for assisting in determining whether a subject has aggressive prostate cancer, comprising: (a) H4N5F1S in biological fluid samples of interest 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 receiving a value for the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of a PSA glycoform received in (a) with a reference value for that PSA glycoform; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer.

[0024] The value received in (a) may be obtained from the PSA glycoforms determined in the third aspect of the invention. Where appropriate, embodiments applicable to the third aspect of the invention may be applied to this fifth aspect of the invention.

[0025] Suitably, the monoantennary PSA glycoform for use in the methods according to the first, second, fourth and fifth aspects of the invention is selected from one or more of compounds 2, 3, 4 and 5 identified in Tables 1A and B.

[0026] Suitably, the diantennary PSA glycoform for use in the methods according to the first, second, fourth and fifth aspects of the invention is selected from one or more of compounds 7, 9, 10, 11, 13, 16, 18, 21, 24 and 25 identified in Tables 1A and B.

[0027] The methods of the first, second, third, fourth and fifth aspects of the invention can be used to help determine whether a subject has aggressive PCa, or to help determine whether a subject does not have aggressive PCa, such as indolent prostate disease. Suitably, the methods of the first, second, third, fourth or fifth aspects of the invention can be used to help determine whether a subject has indolent prostate disease, such as BPH.

[0028] According to a sixth aspect of the present invention, there is provided a kit (or use of a kit) for use in or with a method according to the first, second, third, fourth or fifth aspect of the present invention, comprising a prostate cancer aggressiveness score or scoring system (e.g., a determination system having a PSA glycoform threshold indicative of aggressive PCa). Optionally, the kit also comprises a PSA protein binding agent, e.g., a monoclonal antibody. Such a PSA protein binding agent may be used to isolate or purify PSA protein. In an embodiment, the kit may comprise a protease. The protease may be used to generate glycopeptides of PSA that can be measured by mass spectrometry. In an embodiment, the kit may be a kit for mass spectrometry detection of PSA glycoforms. [Brief description of the drawings]

[0029] [Figure 1] Figure 1 is a schematic diagram of the PSA glycomic assay. Step 1 involves sample collection of a urine sample. Step 2 isolates PSA from the urine. Step 3 prepares the sample for analysis by reducing and alkylating the PSA followed by in-solution digestion (e.g., using trypsin). After sample preparation, the sample can be analyzed by capillary electrophoresis (step 4.A) followed by mass spectrometry for detection (step 4.B). After analysis, data is collected and further processed (step 5). [Diagram 2] AUC ROC of urinary Comp.21 and serum fPSA% for discrimination of aggressive (Gleason ≥ 7) vs indolent PCa and BPH. [Diagram 3] AUC ROC (Comp.3+7) and serum fPSA% of the best urinary 2-compound combination for discrimination of aggressive vs indolent PCa and BPH. [Figure 4] AUC ROC of the best urinary 3-compound combination (Comp. 4+7+monoantennary glycan) and serum fPSA% for discrimination of aggressive vs indolent PCa and BPH. [Diagram 5]AUC ROC (Comp.2+9+13+22) and serum fPSA% of the best urinary 4-compound combination for discrimination of aggressive vs indolent PCa and BPH. [Figure 6] Major glycoforms with the same group trait (up- or down-regulated). Structural glycosylation traits (e.g., sialylation, fucosylation, and branching) are annotated in the left column. A description of the structure is provided in the right column under "Glycan Structure." A "1" indicates the presence of a particular trait for a given glycan, and a "0" indicates the absence of a particular trait for a given glycoform. [Figure 7] AUC ROC of best ratios between upregulated and downregulated urinary PSA glycostructures and serum fPSA% for discrimination of aggressive vs. indolent PCa and BPH. Figures 8-18 show exemplary structures of various PSA glycoforms (compounds) of the present invention attached to a peptide (PEP). The figure on the right shows the SNFG (Symbol Nomenclature for Glycans) system (Ajit Varki et al., Symbol Nomenclature for Graphical Representations of Glycans, Glycobiology, Volume 25, Issue 12, December 2015, Pages 1323-1324, https: / / doi.org / 10.1093 / glycob / cwv091 and Sriram Neelamegham et a.al, The SNFG Discussion Group, Updates to the Symbol Nomenclature for Glycans guidelines, Glycobiology, Volume 29, Issue 9, September 2019, Pages 620-624, https: / / doi.org / 10.1093 / glycob / cwz045). The linkage types of sialic acid (2,3 or 2,6) are shown in Table 1A above. [Figure 8] The structure of exemplary compound 2 is shown. [Figure 9] The structure of exemplary compound 3 is shown. [Figure 10] The structure of exemplary compound 4 is shown. [Figure 11] The structure of exemplary compound 5 is shown. [Figure 12] The structure of exemplary compound 7 is shown. [Figure 13] The structure of exemplary compound 9 is shown. [Figure 14] The structure of exemplary compound 13 is shown. [Figure 15] The structure of exemplary compound 18 is shown. [Figure 16] The structure of exemplary compound 21 is shown. [Figure 17] The structure of exemplary compound 22 is shown. [Figure 18] The structure of exemplary compound 24 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although similar or equivalent methods and materials described herein can be used in the practice or testing of this invention, suitable methods and materials are described below.In case of conflict, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0031] The disclosed methods may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing specific embodiments by way of example only, and is not intended to limit the methods claimed.

[0032] The methods of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of those in the art. Exemplary techniques are described in "Molecular Cloning: A Laboratory Manual", 2 nd edition (Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989); "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., Current Protocols of Molecular Biology, John Wiley and Sons (1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., Birhauser, Boston, 1994).

[0033] It should be understood that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0034] In describing the present invention, the following terms will be used and are intended to be defined as indicated below.

[0035] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. As used herein, the term "species," such as "PSA-glycoform species," may be singular or plural, depending on the context.

[0036] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0037] The term "and / or" should be understood to mean either or both of the alternatives.

[0038] As used herein, unless otherwise specified, it is to be understood that the term "about" is used synonymously with the term "approximately." Illustratively, unless otherwise specified, the use of the term "about" when used in conjunction with a stated numerical value or range indicates something greater or less than the stated value or range, up to within ±15% of the stated value or range, ±10% of the stated value, ±5% of the stated value, or for convenience, ±2% of the stated value. Such values ​​are therefore encompassed within the scope of the claims reciting the term "about" or "approximately."

[0039] The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of stated integers or steps or groups of integers or steps, but not the exclusion of any other integers or steps or groups of integers or steps.

[0040] The term "analyte" refers to the molecule (eg, a PSA glycoform or a protein) being detected and measured.

[0041] As used herein, the term "level" or "amount" encompasses the absolute level or amount of a biomarker (e.g., a PSA glycoform) referred to herein, the normalized amount, the relative amount or concentration of the biomarker (e.g., the normalized signal (e.g., area under the curve) of the biomarker (e.g., a PSA glycoform) relative to the total signal of all PSA glycoforms integrated or measured (e.g., if five PSA glycoforms are measured, the level may be the amount or area under the curve of PSA glycoform 1 divided by the equivalent scale of PSA glycoforms 1+2+3+4+5; see also Table 1A), as well as any value or parameter that correlates thereto or can be derived therefrom. Such values ​​or parameters may include integrated area values ​​or intensity signals from any particular physical or chemical property obtained from the biomarker by direct measurement, e.g., mass spectral intensity values. Additionally, all values ​​or parameters obtained by indirect measurement as specified elsewhere herein are encompassed. It should be understood that values ​​correlating to the levels of the aforementioned PSA glycoforms or parameters can also be obtained by any standard mathematical operation.

[0042] In certain embodiments, the level of a PSA glycoform is determined by normalizing the amount of a PSA glycoform (e.g., N 69 glycopeptide).

[0043] In certain embodiments, the level of a PSA glycoform is measured using a normalized signal for all PSA glycoforms measured (e.g., all glycoforms shown in Table 1A). 69 The corresponding normalized signal (e.g., area under the curve) correlates with the amount of a glycopeptide.

[0044] The level of PSA glycoforms may be used directly to compare with a reference value, or may be combined with the level of one or more other PSA glycoforms, for example, as an aggressiveness score based on the ratio or percentage of the level of one or more other PSA glycoforms, for example, the level of one or more other PSA glycoforms also holds biochemical significance for the clinical condition of interest. For example, the level of monoantennary glycoforms may be combined with the level of diantennary glycan species to obtain a ratio level. Alternatively, the levels of two or more PSA glycoforms may be imported into an equation to obtain a score that is used to generate or can be used to generate a PCa aggressiveness score (herein, aggressiveness score).

[0045] As used herein, the term "assess" refers to assessing or determining whether a subject has a particular condition, such as aggressive PCa.Accordingly, as used herein, assessment includes determining or identifying whether a subject may have aggressive PCa and / or whether a subject may have indolent prostate disease.

[0046] As will be understood by those skilled in the art, the assessment made according to the present invention is preferable, but may not be correct for 100% of the subjects usually examined. This term typically requires that the statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley&Sons, New York 1983. Typically, the assumed confidence interval is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0047] The term aggressiveness score as used herein refers to a score, e.g., a numerical score, that correlates with the degree of aggressiveness of PCa in a sample. It is typically determined from any mathematical combination of the determined levels, optionally including other biomarkers and / or clinical information. Suitably, this score can distinguish aggressive PCa from indolent prostate disease, etc., by comparing with a reference value for that aggressiveness score that indicates some degree of aggressiveness. The reference aggressiveness score can be a threshold value such that any test score that falls into one or the other of its values ​​(depending on how the scoring is calibrated, e.g., whether a high score or a low score means more aggressive) can identify or help identify subjects who are likely to have aggressive PCa or not. The aggressiveness score is determined by considering the combined levels of the determined PSA glycoforms. As used herein, "by considering the combined levels of the determined PSA glycoforms" means that the determined levels of each PSA glycoform (as the case may be, from the test subject, control sample or historical control sample) are used in a formula that includes the determined levels of each PSA glycoform to obtain a score that may be an aggressiveness score. As an example, this may be an aggregation / sum of the individual levels (so that, for example, aggressiveness score = level of PSA glycoform 1 + level of PSA glycoform 2, such that a level of x for PSA glycoform 1 and a level of y for PSA glycoform 2 in the subject's biological "test" sample gives a combined level of x + y). Alternatively, this may be, for example, a ratio of PSA glycoform 1 / PSA glycoform 2 (=x / y in the above example), or, for example, a ratio of PSA glycoform 2 / PSA glycoform 1 (=y / x in the above example). Alternatively, this can be some other formula that includes the levels of each PSA glycoform determined (for example, if the equation is Aggressiveness Score = 2.5 times PSA glycoform 1 + 1.5 times PSA glycoform 2, then using the x and y values ​​as above would result in an aggressiveness score of 2.5x + 1.5y for the test subject).The appropriate formula for use in determining the aggressiveness score using any particular combination of PSA glycoforms can be determined using standard statistical analysis on appropriate reference samples.

[0048] For use in the method of the present invention, the level or aggressiveness score of one or more PSA glycoforms in a subject sample (test sample) is compared with a respective reference value that can distinguish subjects with aggressive PCa from subjects without aggressive PCa. The reference value may be based on the same calculation method of the level or aggressiveness score of PSA glycoforms as the determined level or aggressiveness score. Such a reference value may be referred to as the reference value for aggressiveness score, and as described herein, such a reference value for aggressiveness score may be determined by comparing the measured levels of PSA glycoforms found in individuals with aggressive PCa and / or individuals without aggressive PCa.

[0049] For example, the level of a measured PSA glycoform in a subject's test sample, alone or when combined with the levels of one or more other PSA glycoform(s) in a suitable formula to give an aggressiveness score, can be compared to values ​​known to be associated with aggressive PCa and / or values ​​known to be associated with individuals not having aggressive PCa. Typically, the levels in the test sample are directly or indirectly correlated with PCa aggressiveness status, and the marker levels are used, for example, to determine whether an individual is likely to have aggressive PCa.

[0050] In the context of the present invention, an aggressiveness score can be calculated in that the individual parameters (including or consisting of the determined or received PSA glycoform levels) are mathematically combined. The levels can be used as is or can be mathematically transformed (e.g., by logarithmic transformation, such as log2 or log10 transformation) to determine the score. The score can take into account one or more other factors other than the level of PSA glycoform(s), including, but not limited to, the presence or level of one or more other biomarkers in the sample and / or one or more clinical parameters of the subject (e.g., tumor histology, smoking status, disease stage and / or age).

[0051] In certain embodiments of the invention, the aggressiveness score may be obtained by a weighted calculation of the levels of the biomarker molecule(s) (e.g., PSA glycoform(s)) in the sample. This means that a marker may be weighted differently than other markers. For example, if the score takes into account the level of a PSA glycoform (e.g., monoantennary compound 2) and another PSA glycoform (e.g., diantennary compound 9) in the sample, the score may be calculated by the following formula:

[0052] Score = a * [compound 2] + b * [compound 9], where a and b represent weighting factors. Preferably, the weighting factor or factors (in the above example, a and b) are obtained by analyzing a reference population (e.g., any reference population defined in relation to the reference values ​​below). In embodiments, the weighting factor may be obtained by a machine learning approach applied to a training data set obtained from samples of a reference population defined herein in relation to the reference values ​​and / or control samples.

[0053] Those skilled in the art will recognize that the aggressiveness score and corresponding reference values ​​for the score may be optimized based on a reference population (eg, any of those defined in conjunction with determining the cutoffs below).

[0054] In an embodiment of the invention, the aggressiveness score may comprise or consist of the ratio of the level of at least one diantennary PSA glycoform from a subject sample to at least one monoantennary PSA glycoform, or vice versa.

[0055] In embodiments where more than one monoantennary or diantennary PSA glycoform(s) are determined, a sum of all respective monoantennary or diantennary levels can be generated and used as a ratio value. The ratio is the sum of the levels of one category of PSA glycoforms to the sum of the other category of PSA glycoforms, e.g., the category of PSA glycoforms includes monoantennary and diantennary forms. For example, the ratio can be all measured diantennary species to all measured monoantennary species, e.g., (A+B) / C (where A and B are the levels of different diantennary species and C is the level of a single monoantennary species), or (A+B) / (C+D) (where D is the second monoantennary species). Similarly, if the ratio is all measured monoantennary species to all measured diantennary species, it can be C / (A+B) or (C+D) / (A+B). Such a ratio can then be used to determine whether the PCa is an aggressive PCa or not.

[0056] Thus, in embodiments in which more than one monoantennary or diantennary PSA glycoform is determined, the sum of the respective monoantennary or diantennary levels is calculated and used as the ratio value.

[0057] In one embodiment, the aggressiveness score may be a binary score, and the corresponding reference value may also be binary. "Binary" means that the score includes two values, for example, the first value is the determined or received level or value derived therefrom of one or more diantennary PSA glycoforms, and the second value is the level or value derived therefrom of one or more monoantennary PSA glycoforms. The "value derived therefrom" may be, for example, a value obtained by mathematical operation. The "value derived therefrom" is preferably directly proportional to the respective level. The value of the binary reference value may be obtained as described below for each individual PSA glycoform.

[0058] Comparing the binary score to the binary cut-off score means comparing the first value of the determined binary score to the first value of the reference value of the binary score, and comparing the second value of the determined binary score to the second value of the reference value of the binary score. In embodiments, the aggressiveness score is a binary score comprising a first value corresponding to the level of at least one monoantennary PSA glycoform and a second value corresponding to the level of at least one diantennary PSA glycoform, and the reference value for the aggressiveness score comprises a reference value for the first value and a reference value for the second value. In these embodiments, if the level of at least one monoantennary PSA glycoform (i.e., the first value of the binary score) is decreased compared to the reference value for the first value, and the level of at least one diantennary PSA glycoform (i.e., the second value of the binary score) is increased compared to the reference value for the second value, the subject is determined to have aggressive PCa. Vice versa, if only one or neither of the first and second values ​​of such binary score meets the aggressiveness criteria, the subject is determined to have non-aggressive prostate disease.

[0059] As used herein, the term "comparison" has its usual meaning, e.g., the act of examining whether they are similar or different. In the context of the present invention, the term "comparison" includes comparing the level of PSA glycoforms in a sample from a subject with a reference level of PSA glycoforms, or, for example, comparing a calculated aggressiveness score (e.g., calculated based on a mathematical combination of the levels of various PSA glycoforms measured, e.g., using weighted calculations, calculation of aggregate (e.g., sum) levels, or construction of ratios) with a reference value for such aggressiveness score. As used herein, comparison usually refers to the comparison of corresponding parameters, values, or scores, e.g., absolute amounts are compared with absolute reference amounts, concentrations are compared with reference concentrations, or intensity signals obtained from biomarkers in a sample are compared with the same type of intensity signals obtained from a reference sample, or matched values ​​(e.g., aggressiveness scores) in which values ​​of levels of multiple biomarkers (e.g., PSA glycoforms) are entered into a formula are compared with reference values ​​for matched levels from the same biomarkers (e.g., obtained from a reference control sample or reference cohort). As described herein, the reference value can also be obtained from a plurality of control or reference samples, for example, a reference cohort with known prostate (cancer) disease status (e.g., Gleason score). The comparison can be performed manually or computer-assisted. Thus, the comparison can be performed by a computing device. The measured or detected levels of the biomarkers in the sample from the subject and the reference level values ​​can be compared, for example, with each other, and the comparison can be performed automatically by a computer program that executes an algorithm for comparison. The computer program that performs the evaluation provides the desired assessment in a suitable output format. In a computer-assisted comparison, the measured level values ​​can be compared by the computer program with values ​​corresponding to suitable standards stored in a database. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired assessment in a suitable output format.In a computer-assisted comparison, the measured level values ​​may be compared by a computer program with values ​​corresponding to suitable standards stored in a database, which may further evaluate the comparison results, i.e. automatically provide the desired rating in a suitable output format.

[0060] As used herein, the term "reference value" refers to a known (e.g., predetermined) value against which a test value can be compared, and thus is a value that distinguishes between two states (e.g., yes, no; disease / health; aggressive / non-aggressive PCa). There are essentially two options for determining the reference value: 1) a control sample (which can be an internal or external sample or a pool of samples); 2) multiple samples of a reference population with a known disease state (e.g., aggressive PCa and / or indolent prostate disease).

[0061] Suitably, the reference value can be determined from one or more control samples that are analyzed and determined in substantially the same manner as the test sample of interest, and the information of the control sample of interest is compared with that of the test sample of interest.In one embodiment, the control sample is obtained from a control subject that does not have aggressive PCa.In another embodiment, the control sample is obtained from a subject that has an indolent prostate disease, such as indolent (slowly growing) PCa or BPH.In another embodiment, the control sample is obtained from a control subject that has aggressive PCa.

[0062] The control sample can be evaluated substantially simultaneously with the test sample, and therefore can be used as a direct contemporaneous comparison, or can be from an existing sample. Thus, the reference value can be determined together (contemporaneously) with the reference value of the test sample, or can be pre-determined. Pre-determined reference values ​​are preferred. Thereby, the values ​​from one or more control samples provide a standard that allows the evaluation of information obtained from the sample of interest (test sample). In certain embodiments, the subject sample is the same sample type as the sample type of the subject of interest. For example, if the sample of interest is urine, the control sample can be urine. The control sample can be derived from a body fluid obtained from a healthy individual, particularly urine, serum or plasma for non-invasive testing, thereby providing a standard of health or a defined disease state of the tissue, organ or individual. Alternatively, the control sample can be obtained from at least one individual with aggressive PCa from the same species, for example, an individual with cancer with a Gleason score of ≧7. Alternatively, the control sample may be obtained from at least one individual from the same species who does not have aggressive PCa, or may be a sample obtained from at least one individual from the same species who has a non-cancerous prostate disorder, such as BPH or prostatitis. In certain embodiments, the control sample may be derived from an individual with benign PCa with a Gleason score <7. The individual may be of the same age, or of the same health state or condition, as the subject from whom the test sample is obtained. The difference between the state of the control sample and the state of the test sample of interest may indicate the presence of disease or the risk of developing disease, or the presence or further progression of such disease or disorder. The control sample may be derived from an abnormal or diseased tissue, organ, body fluid, or individual (e.g., one with indolent prostate disease), thereby providing a standard for the disease state of the tissue, organ, or individual. The difference between the state of the abnormal reference sample and the state of the sample of interest may indicate PCa aggressiveness. The reference sample may also be derived from the same tissue, organ, body fluid, or individual as the sample of interest, but taken at an earlier time point. A difference between the status of the previously taken reference sample and the status of the sample of interest may indicate disease progression, i.e., improvement or worsening of disease over time.

[0063] The control sample can be an internal or external control sample. An internal control sample is used, i.e., in the test sample and one or more other sample(s) taken from the same subject, the marker level(s) is evaluated to determine whether there is a change in the level(s) of the marker(s). For an external control sample, the level of the marker in the sample from the individual is compared with the level of the marker in an individual known to suffer from or known to be at risk of a given condition, or an individual known to be free of a given condition (i.e., "normal individual").

[0064] It will be understood by those skilled in the art that such external control samples can be obtained from a single individual or from an age-matched reference population. Typically, samples from 100 well-characterized individuals from a suitable reference population are used to set a "reference value". However, the reference population can also be selected to consist of, for example, 20, 30, 50, 200, 500 or 1000 individuals.

[0065] Therefore, alternatively, the reference value can be determined based on samples obtained from a reference population, for example, samples of a reference population with known prostate disease and PCa aggressiveness status. Those skilled in the art can establish a reference value for the level of PSA glycoforms or a reference value for aggressiveness score based on a representative reference population. Using a reference population, a reference value can be determined such that the level or score value set as the reference value divides the reference population into aggressive PCa and indolent prostate disease with a certain sensitivity and specificity. The reference value can be selected differently depending on whether higher sensitivity is desired at the expense of lower specificity, or vice versa. For example, the level or score that separates aggressive PCa from non-aggressive PCa can be determined using commonly known statistical methods so that the desired sensitivity and specificity are defined. The reference value used for comparison with the test sample can be a value calculated as the average or median value of multiple (e.g., 2 or more, 5 or more, 10 or more, groups, etc.) control samples. Alternatively, the control sample can be a sample from more than one (e.g., two or more, five or more, ten or more, a group, etc.) (i.e., a mix) individuals who are not afflicted with PCa (or have BPH).

[0066] The reference value is typically determined from a statistical assessment of the readouts / levels of biomarkers from multiple (e.g., >49, >99, >249, >499, >999) subject samples (e.g., from a reference population) with or without the particular condition (e.g., aggressive PCa) to be determined. The reference value may be an aggressiveness score value value derived from the level of a single biomarker (i.e., an individual PSA glycoform) or the individual levels of a combination of biomarkers (i.e., two or more PSA glycoforms as described herein), which may be applied to an algorithm or equation to provide a reference value that can be used when measuring and depending on that particular combination of biomarkers (e.g., a combination of PSA glycoforms). Typically, the reference value achieves a certain statistical significance threshold, for example, a quantile in the case or control distribution. For example, to determine a reference value from a particular control cohort (BPH or indolent PCa), a certain percentile may be determined from the measurements of this control cohort (e.g., 90%, 95%, 95.7 or 97%). For example, if the 90th percentile is used, 90% of the control samples will have values ​​below this cutoff, thereby reducing the number of false positive predictions.

[0067] In one example, the reference value is the average or median level of one or more PSA glycoforms(s) taken from a group or population of individuals with aggressive PCa from the same species. In one example, the reference value is the average or median value obtained from a group or population of individuals not affected by PCa. For example, the reference value can be calculated as the average or median value obtained from a group or population of individuals with aggressive PCa and / or indolent prostate disease (e.g., indolent PCa, and / or BPH, and / or prostatitis, etc.). The individual or population of individuals can be of the same age, or of the same health status or condition, as the subject from whom the test sample is obtained.

[0068] In certain embodiments, the reference population for establishing the reference value (e.g., for PSA glycoform level or aggressiveness score) is derived from healthy individuals. In another embodiment, the reference population may include healthy individuals and individuals with non-aggressive and aggressive PCa. In another embodiment, the reference population may include individuals with non-aggressive (BPH and / or indolent) and aggressive PCa. In another embodiment, the reference population may include individuals with indolent prostate disease. In another embodiment, the reference population may be the one used in the examples. Preferably, the reference value in each of these embodiments is a predetermined reference value established from historical control samples, e.g., derived from a reference population.

[0069] The reference value may also be a reference value for an aggressiveness score (when an aggressiveness score is determined) generated from measurements of the levels of two or more PSA glycoforms, again from a group or population of individuals with known PCa status. Such levels may be input into a formula to generate a score (e.g., aggressiveness score). This formula may be a ratio or aggregate (e.g., sum) or any other formula in which the measured levels of each PSA glycoform are used in the formula, and the output provides a score / value that distinguishes whether an individual falls into a particular category (e.g., likely to have aggressive PCa, likely not to have aggressive PCa).

[0070] As used herein, an individual or subject who is "not having PCa" is an individual or subject who has histologically normal-looking prostate tissue. Methods for histologically testing prostate tissue and identifying whether an individual has histologically normal-looking prostate tissue are well known in the art (see, for example, Litwin MS and Tan HJ., The Diagnosis and Treatment of Prostate Cancer: A Review. JAMA. 2017 Jun 27; 317 (24): 2532-254). Thus, in this context, a control sample obtained from an individual who does not have PCa refers to a biological fluid sample (e.g., blood or urine sample, as appropriate) obtained from an individual of the same species, the individual having histologically normal-looking prostate tissue. Examples of individuals who do not have PCa include individuals who have BPH, prostatitis and / or prostatic hyperplasia.

[0071] Benign prostatic hyperplasia (BPH; also known as benign prostatic hyperplasia) is a common medical condition in men over the age of 50. Although BPH can affect how urine passes, it is not cancer and is a condition that does not pose an increased risk of developing PCa.

[0072] Thus, as used herein, an individual / subject / patient with "benign prostatic hyperplasia" or "BPH" is an individual / subject / patient with an enlarged prostate with histologically normal-appearing prostate tissue. Methods for histologically testing prostate tissue and identifying whether an individual has BPH are well known in the art (see, for example, Chughtai et al. Benign prostatic hyperplasia. Nat Rev Dis Primers. 2016 May 5; 2: 16031). Thus, in this context, a control sample obtained from an individual with BPH refers to a biological fluid sample (e.g., blood or urine sample, as appropriate) obtained from an individual of the same species, the individual having an enlarged prostate with histologically normal-appearing prostate tissue.

[0073] Prostatitis is the name given to a group of symptoms thought to be caused by infection or inflammation of the prostate. Prostatitis is not a form of PCa. It is a common condition that can affect men of any age, but is most common in young and middle-aged men, typically aged 30-50 years. Prostatitis can cause a wide range of symptoms, which vary from person to person. Common symptoms include problems and pain with urination, or discomfort around the testicles, back passage, or lower abdomen. There are four types of prostatitis: chronic pelvic pain syndrome (CPPS), acute bacterial prostatitis, chronic bacterial prostatitis, and asymptomatic prostatitis.

[0074] Thus, a control sample obtained from an "individual with prostatitis" refers to a biological fluid sample (e.g., a blood or urine sample, as appropriate) obtained from an individual of the same species, where the individual has been diagnosed with one of the above forms of prostatitis.

[0075] Prostatic hyperplasia is an increase in the size of the prostate gland that is not due to cancer. The medical term for prostatic hyperplasia is benign prostatic enlargement (BPE). BPE is also known as benign prostatic hyperplasia (BPH), which is explained in more detail above.

[0076] The term "measurement" or "measurement" or "determination" in the context of analysis preferably includes qualitative, semi-quantitative or quantitative measurements. The term "determine" is also used in the context of making a decision regarding the subject's condition, for example, identifying whether or not the subject has aggressive PCa. In this context, the term "identify" may be used.

[0077] As will be understood by those skilled in the art, the determination made according to the present invention, although preferred, may not be correct for 100% of the subjects examined. This term typically requires that the statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley&Sons, New York 1983. Typically, the assumed confidence interval is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05. Thus, as used herein, when determining whether a subject has aggressive PCa, the determining / determination can be for the probability that someone has aggressive PCa.

[0078] The terms "cancer" and "cancerous" refer to or describe a physiological condition typically characterized by uncontrolled cell growth. Examples of cancer include cancer of the genitourinary tract, such as PCa. As used herein, the term "prostate cancer (PCa)" refers to all stages and all forms of cancer that arise from the tissues of the prostate gland.

[0079] As used herein, the term "prostate disease" refers to any and all diseases or disorders of the prostate, such as PCa (whether slow-growing / indolent or aggressive), prostatitis (including bacterial prostatitis - acute or chronic bacterial infection and non-bacterial prostatitis), prostatic inflammation, also known as chronic pelvic pain syndrome (CPPS) and BPH.

[0080] As used herein, "gray zone", "PSA grey zone", "grey area" or "PSA grey area" as used herein refers to a range of 2-10 ng / ml of total PSA in a subject's serum. As described elsewhere herein, the PSA test is a non-conclusive measure and subjects with total serum PSA levels in the range of 2-10 ng / ml may or may not have PCa. The method of the present invention is particularly suitable for subjects whose serum PSA levels are in this "grey zone". Suitably, the method of the present invention is performed on a sample from a subject whose serum PSA levels are in this "grey zone".

[0081] Methods for diagnosing and staging PCa are well known in the art. For example, according to the tumor, node, metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC), AJCC Cancer Staging Manual (7th ed., 2010), the various stages of PCa are defined as follows: Tumor: T1: clinically inapparent tumor not palpable or visible on imaging, T1a: paraneoplastic histological findings in 5% or less of the resected tissue, T1b: paraneoplastic histological findings in more than 5% of the resected tissue, T1c: tumor identified by needle biopsy, T1d: tumor identified by needle biopsy, T1e: tumor identified by needle biopsy, T1f: tumor identified by needle biopsy, T1g: tumor identified by needle biopsy, T1h: tumor identified by needle biopsy, T1i: tumor identified by needle biopsy, T1j: tumor identified by needle biopsy, T1m ... Tumor;T2: tumor confined to the prostate, T2a: tumor involves less than 1 / 2 of one lobe, T2b: tumor involves more than half of one lobe but not both lobes, T2c: tumor involves both lobes;T3: tumor extends through prostatic capsule, T3a: extracapsular extension (unilateral or bilateral), T3b: tumor invades seminal vesicle(s);T4: tumor is fixed or invades adjacent structures other than seminal vesicles (bladder neck, external sphincter, rectum, levator, or pelvic wall). Nodes:N0: no regional lymph node metastasis;N1: metastasis in regional lymph nodes. Metastases:M0: no distant metastasis;M1: distant metastasis.

[0082] The Gleason grading system is also commonly used to help assess the prognosis of men with PCa. Along with other parameters, it is incorporated into a strategy of PCa staging that helps predict prognosis and guide treatment. A Gleason "score" or "grade" is given to PCa based on its microscopic appearance. Tumors with a low Gleason score (e.g., a Gleason score of 6 or less) typically grow slowly enough that they may not pose a significant threat to the patient during survival. These patients are monitored over time ("watchful waiting" or "active surveillance"). Cancers with a high Gleason score are more aggressive and have a worse prognosis, and these patients are generally treated with surgery (e.g., radical prostatectomy) and sometimes therapy (e.g., radiation, hormones, ultrasound, chemotherapy, immunotherapy). The Gleason score (or sum) includes the grades of the two most common tumor patterns. These patterns are called Gleason patterns 1 to 5, with pattern 1 being the most well-differentiated. Most have a mixture of patterns. To obtain the Gleason score or grade, the predominant pattern is added to the second most common pattern to obtain a number between 2 and 10. Gleason grades include: G1: well differentiated (mild anaplasia) (Gleason 2-4); G2: moderately differentiated (moderate anaplasia) (Gleason 5-6); G3-4: poorly differentiated / undifferentiated (marked anaplasia) (Gleason 7-10).

[0083] As used herein, "indolent prostate disease" refers to subjects / patients with a Gleason score of 6 or less.

[0084] As used herein, "aggressive prostate cancer" or "aggressive PCa" refers to a subject / patient with a Gleason score > 7. Such a score can be, for example, 3+4.

[0085] The methods described herein can be used for samples from patients with prostate disease or patients with increased risk of developing PCa. In this context, the phrase "increased risk" indicates that a subject has a higher level of risk (or likelihood) of experiencing a particular clinical outcome. A subject may be at increased risk of developing PCa if there is a family history of the disease. A subject may be at increased risk of developing PCa if they have genetic mutations that predispose to the development of PCa, such as BRCA1 and BRCA2 and HPC1, androgen receptor mutations and TMPRSS2:ERG gene fusions.

[0086] A biomarker is a characteristic that is objectively measured and evaluated as an indicator of the biological state of a system (e.g., normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention). A biomarker can be any type of molecule present in a living organism, such as a nucleic acid (DNA, mRNA, miRNA, rRNA, etc.), a protein, a polypeptide, a peptide, or an immunologically detectable fragment thereof (cell surface receptors, cytosolic proteins, etc.), a metabolite or hormone (blood glucose, insulin, estrogen, etc.), or a molecule characteristic of a particular modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA). Suitably, a biomarker is differentially present in a sample taken from a subject with a disease or disease state compared to a subject without the disease or disease state. A biomarker is differentially present if the mean or median levels of the biomarker in different groups are calculated to be statistically significant. Common tests for statistical significance include, among others, t-test (e.g., Student's t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, receiver operating characteristic (ROC curve), precision and odds ratio.Biomarkers, alone or in combination, provide a measure of the relative risk that a subject belongs to one phenotypic state or another phenotypic state.Therefore, they are useful as markers of disease (diagnosis), therapeutic efficacy of drugs and drug toxicity.

[0087] Typically, the biomarker referred to herein is the N-linked glycan attached to the PSA protein via the nitrogen atom to asparagine. A glycan is a carbohydrate or sugar-based unit composed of monosaccharides linked by glycosidic bonds. The PSA glycoforms herein are biomarkers.

[0088] The terms "disease" and "disorder" are used interchangeably herein and refer to abnormal conditions, particularly abnormal medical conditions such as illness or injury in which a tissue, organ or individual / subject can no longer perform its function efficiently. Typically, but not necessarily, a disease is associated with certain symptoms or signs that indicate the presence of such a disease. Thus, the presence of such symptoms or signs may indicate a tissue, organ or individual suffering from a disease. Changes in these symptoms or signs may indicate the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a "worsening" or "improvement" of the disease. A "worsening" of a disease is characterized by a decrease in the ability of a tissue, organ or organism to perform its function efficiently, whereas an "improvement" of a disease is typically characterized by an increase in the ability of a tissue, organ or individual to perform its function efficiently. A tissue, organ or individual that is "at risk of developing" a disease is in a healthy state but exhibits the potential for the disease to become manifest. Typically, the risk of developing a disease is associated with early or weak signs or signs of such a disease. In such cases, the onset of the disease may still be prevented by treatment. Examples of diseases include, but are not limited to, PCa and prostatitis.

[0089] "Glycan" or "carbohydrate" refers to a polymer of monosaccharide residues or sugar moieties found attached to proteins such as glycoproteins, proteoglycans, and lipids. Glycans can be linear or branched. Glycans can be found covalently attached to non-sugar moieties such as lipids or proteins. Attachment to proteins typically occurs through nitrogen (N-linked) or oxygen atoms (O-linked). Covalent conjugates that include glycans are referred to as, for example, glycosylated polypeptides, glycoproteins, glycopeptides, peptidoglycans, proteoglycans, glycolipids, and lipopolysaccharides. Glycans also exist in free form (i.e., separated from and not associated with other moieties).

[0090] As used herein, the terms "PSA glycoform", "PSA glycoform species", "PSA glycopeptide", and "PSA glycan" are used interchangeably and, when singular, refer to a particular type of glycan structure attached to the PSA protein or peptides thereof. When plural, it refers to multiple PSA glycoforms / glycoform species. The term "glycosylation marker" or "glycan biomarker" may also be used to indicate a particular glycan species bound to PSA whose levels are altered, either enhanced or decreased, in a particular disease state (e.g., indolent prostate disease or aggressive PCa), and thus can be used, including measured, in the methods of the present invention.

[0091] Prostate specific antigen (PSA) has a single site where N-linked glycosylation occurs, namely amino acid residue 69 (numbering according to Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 1991). Using proteases, the PSA protein can be cleaved into smaller peptides, ideally with only one unique peptide having an asparagine at position 69 (so that all the different glycoforms are on the same peptide length), referred to herein as glycopeptides or N-glycosylation.69 Glycopeptide-containing N 69 The cleavage of PSA by trypsin is called N 69 Cleavage of PSA by Arg-C (clostripain) releases the N dipeptide. 69 KSVILLGR peptide (SEQ ID NO: 3). 69 Any glycan attached to the glycopeptide (N 69 Glycopeptides, e.g. N 69 K glycopeptide or N 69 Since the PSA-glycan moiety is presented as a glycopeptide (KSVILLGR glycopeptide), the level of each PSA-glycan moiety detected according to the method of the present invention is 69 It can be determined by relative quantification of the levels of K-glycoforms (PSA-glycan species or moieties).

[0092] As used herein, the term "N69 glycopeptide" or "N69 containing glycopeptide" refers to any peptide (e.g., 20 amino acids or less) derived from PSA (e.g., by protease digestion) that includes a residue corresponding to N69 of PSA and has an N-glycan (e.g., a specific sugar chain) attached to N69.

[0093] The glycan / carbohydrate / glycoform species or moieties of the present invention are described with reference to the nomenclature commonly used to describe oligosaccharides (see, for example, Glycobiology 25:1323-1324, 2015 and Glycobiology 29:620-624, 2019, as well as the legend below Table 1A herein). A review of carbohydrate chemistry using this nomenclature can be found in Hubbard, SC, and Ivatt, RJ, Ann. Rev. Biochem. 50:555-583, 1981. Examples of PSA glycoforms that can be used in the present invention are shown in Table 1.

[0094] The term "glycosylation" refers to the attachment of a sugar moiety (e.g., a polysaccharide) to a protein. Glycosylation is a form of co-translational and post-translational modification. Suitably, a polysaccharide consists of two or more sugars linked together via glycosidic bonds. Most glycosylation in mammalian cells is N- or O-linked to proteins. Typically, polysaccharides are attached via the OH group of serine or threonine (O-linked to produce an O-glycosylated polypeptide) or via the amide group of asparagine (NH 2 ) (N-linked to produce an N-glycosylated polypeptide). The glycosylation of PSA is N-linked.

[0095] The terms "glycosylated polypeptide," "glycoprotein," "glycosylated peptide," and "glycopeptide," used interchangeably within this application, refer to a protein or polypeptide or peptide having at least two amino acids, at least one of which has a glycan or polysaccharide covalently attached thereto. In reference to the glycosylated peptide of PSA, in one embodiment, it is 69 K glycopeptide.

[0096] The term "glycoform" refers to a protein, polypeptide or peptide having a particular type and distribution of attached polysaccharides, i.e., two identical peptides / polypeptides are the same glycoform if they contain glycans with the same number, kind and sequence of monosaccharides. On the other hand, two identical peptides (identical with respect to amino acid sequence) are different glycoforms if they contain glycans with different numbers, kinds and sequences of monosaccharides. In the context of glycoforms of PSA, this appropriately refers to the N-glycan moieties attached to a particular glycan moiety. 69 K glycopeptide. A glycoform is also a glycosylated polypeptide.

[0097] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies compounds by their mass. Typically, samples are analyzed by generating gas phase ions from the sample, which are then separated and detected according to their mass-to-charge ratio (m / z). MS techniques generally include (1) ionizing compounds to form charged compounds, (2) calculating the mass-to-charge ratio (m / z), and (3) determining the molecular weight of the charged compounds. Compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized and the ions are then introduced into a mass spectrometry instrument where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z").

[0098] Methods for generating gas phase ions from a sample include electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption ionization (SELDI) and chemical ionization. Separation of ions by m / z ratio can be achieved with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers, magnetic sector mass analyzers, 3D and linear ion traps (IT), Fourier transform ion cyclotron resonance (FT-ICR) analyzers, and combinations thereof (e.g., quadrupole-time-of-flight analyzers, or Q-TOF analyzers). Prior to ionization, the sample may be subjected to one or more dimensions of chromatographic or electrophoretic separation, such as one or more dimensions of liquid or size exclusion chromatography.

[0099] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to an ionization technique that uses a laser energy absorbing matrix to produce ions from large molecules with minimal fragmentation. The MALDI method is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, and third, the analyte molecules are protonated (H) in a hot plume of ablated gas. + addition) or deprotonation (H + The particles are then ionized by being removed from the atmosphere (removed from the atmosphere). They can then be accelerated into any mass spectrometer that is used to analyze them.

[0100] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a high voltage is applied to a liquid to produce an aerosol of very small droplets of a solution in a solvent vapor. This mist of droplets flows through an evaporation chamber. Based on current understanding, as the droplets get smaller, the electric surface charge density increases until the natural repulsion between similar charges causes ions as well as neutral molecules to be ejected. ESI differs from other ionization methods in that it can produce multiply charged ions, effectively expanding the mass range of the analyzer. Heated ESI is similar, but includes a heat source to heat the sample within the capillary tube. Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS).

[0101] Prostate-specific antigen (PSA) (also known as kallikrein-related peptidase 3, APS, prostate-specific antigen (PSA), hK3, KLK2A1, P-30 antigen, gamma-seminoprotein, kallikrein-3, semenogelase, seminin NP_001025218.1 (EC 3.4.21.77), NP_001025219.1 (EC 3.4.21.77), NP_001639.1 (EC 3.4.21.77)) is a glycoprotein enzyme encoded by the KLK3 gene. KLK3 is a member of the kallikrein-related peptidase family and is secreted by epithelial cells of the prostate gland. PSA mRNA is translated as an inactive 261 amino acid preproPSA precursor. PreproPSA has a preregion (signal polypeptide) and 24 additional residues that make up the propolypeptide. Release of the propolypeptide results in the enzymatically active 237-amino acid mature extracellular form. PSA is organ specific and is thus produced by epithelial cells in BPH, primary and metastatic PCa tissues.

[0102] PSA is concentrated in prostate tissue and serum PSA levels are usually very low. For example, destruction of normal prostate structure by prostate disease, inflammation or trauma releases large amounts of PSA into the circulation (e.g. blood). PSA measurement is used to detect potential problems in the prostate and to track the progress of PCa therapy. However, PSA is not a specific marker for PCa since its levels increase due to other conditions including BPH and prostatitis, and PSA levels are also known to be affected by factors such as trauma, drug therapy, urological manipulations and urinary inflammation. PSA is present in small amounts in the serum of men with healthy prostates, but is often elevated in subjects with PCa and other prostate disorders. It has been demonstrated that 40-45% of the variability in PSA levels in the general population is due to genetic factors (Gudmundsson et al. Sci Transl Med. 2010 December 15;2(62):62ra92. doi:10.1126 / scitranslmed.3001513).

[0103] As used herein, the terms "sample" or "biological fluid sample", "test sample", and variations thereof, encompass biological samples obtained from a patient or subject, which may include blood, plasma, serum, urine, prostatic fluid (such as prostatic secretion sample), semen, saliva, or sputum. For purposes described herein, a sample is or includes a biological fluid (also referred to herein as bodily fluid) sample. Suitably, the sample is a urine sample, such as a digital rectal examination (DRE) urine sample. Suitably, the sample is the subject, or is obtained from or derived from the subject. A DRE urine sample is one that is collected immediately after a DRE. By way of example, this may include performing a DRE test, applying some pressure to the left and right lobes of the prostate before urination, and collecting the first 50 mL (approximately) of the first urine stream of urine. This urine is referred to as a DRE urine.

[0104] As used herein, "providing," "obtaining," or "obtaining" in the context of a sample can be any means of coming into possession of the sample by "direct" or "indirect" means. Obtaining a sample directly means performing a process to obtain the sample (e.g., performing a physical method such as extraction). Obtaining a sample indirectly refers to receiving a sample from another entity or source (e.g., a third-party laboratory that directly obtained the sample). In certain embodiments, the methods of the invention are performed on an indirectly obtained sample. In certain embodiments, the methods of the invention include the additional step of obtaining the sample directly from the subject.

[0105] The methods provided herein can include providing a biological fluid sample (e.g., a blood sample or a urine sample) from a subject.

[0106] Generally, the methods described herein are ex vivo methods performed using a sample already obtained from a subject (i.e., the sample is provided for the method and steps performed to obtain the sample from the subject are not included as part of the method).

[0107] For the avoidance of doubt, the term "ex vivo" has its ordinary meaning in the art and refers to a method carried out in or on a sample obtained from a subject in an artificial environment outside the subject's body from which the sample was obtained. In certain embodiments, the subject's biological fluid sample has previously been isolated / collected from the subject and sample isolation does not form part of the method of the invention. In another embodiment, sample isolation may be a step of the method of the invention.

[0108] As used herein, the term "subject" refers to an animal, more preferably a mammal, such as a human, chimpanzee, rhesus monkey, dog, cow, horse, cat, rat, or mouse, provided that they also have a prostate. The subject is preferably a mammal, most preferably a human. Suitably, the subject is male, such as a male human.

[0109] A subject may be referred to as a patient herein. The terms "subject", "individual" and "patient" are used interchangeably herein. A subject may be symptomatic (e.g., the subject exhibits symptoms associated with PCa or prostate disease) or the subject may be asymptomatic (e.g., the subject does not exhibit symptoms associated with PCa or prostate disease).

[0110] As used herein, "watchful waiting" or "active surveillance" refers to a strategy to manage patients with prostate disease. PCa is often slow growing and may not cause any problems or symptoms. Many treatments and diagnostic procedures for PCa can cause side effects. For some men, these side effects can be long-term and can have a significant impact on their lives. Therefore, watchful waiting is a way to monitor PCa that does not cause symptoms or problems over time and avoid treatment unless symptoms appear. Many men on watchful waiting never need treatment for PCa.

[0111] According to a first aspect of the present invention, (a) determining a level of at least one monoantennary PSA glycoform and a level of at least one diantennary PSA glycoform in a biological fluid sample from a subject; (b) calculating an aggressiveness score for the subject based on the level determined in (a); and (c) comparing the subject's aggressiveness score in (b) with a reference value for that aggressiveness score; (d) using the comparison in (c) to help determine whether the subject has an aggressive form of prostate cancer.

[0112] This method can be used for various purposes, such as to determine or help determine whether a subject has aggressive PCa, or to determine or help determine whether a subject has indolent prostate disease.

[0113] Suitably, a method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) determining a level of at least one monoantennary PSA glycoform and a level of at least one diantennary PSA glycoform in a biological fluid sample from a subject; (b) calculating an aggressiveness score for the subject based on the level determined in (a); and (c) comparing the subject's aggressiveness score in (b) with a reference value for that aggressiveness score; (d) using the comparison in (c) to help determine whether the subject has an aggressive form of prostate cancer.

[0114] Suitably, the comparison and / or calculated score in (b) determines whether the subject has an aggressive form of prostate cancer.

[0115] In certain embodiments, the baseline aggressiveness score is determined, or has been determined, by considering the combined levels of the determined PSA glycoforms.

[0116] In certain embodiments, a subject's aggressiveness score is determined from or takes into account the individual levels of PSA glycoforms.

[0117] In certain embodiments, a subject's aggressiveness score is determined by considering the combined levels of two or more determined PSA glycoforms, at least one of which is a monoantennary PSA glycoform and at least one of which is a diantennary PSA glycoform.

[0118] In certain embodiments, a subject's aggressiveness score is determined by considering the combined levels of two or more determined PSA glycoforms that have a shared structural feature (e.g., two or more monoantennary or two or more diantennary PSA glycoforms).

[0119] In certain embodiments, a subject's aggressiveness score may include or be determined by constructing a ratio between the determined level of one PSA glycoform and the level of another PSA glycoform (e.g., the ratio of monoantennary PSA glycoform to diantennary PSA glycoform, or vice versa).

[0120] In a variation of this first aspect of the invention there is provided a method of aiding in determining whether a subject has an aggressive form of prostate cancer, comprising the steps of: (a) determining a level of at least one monoantennary PSA glycoform and a level of at least one diantennary PSA glycoform in a biological fluid sample from a subject; (b) calculating a score for assessing whether the subject has aggressive PCa by comparing the level of each PSA glycoform with a control or reference value for that PSA glycoform and / or taking into account the level of each PSA glycoform; and / or calculating a score for assessing whether the subject has aggressive PCa by comparing the value of the combined level of each measured PSA glycoform with a control or reference value for that combined PSA glycoform and / or taking into account the combined level of each measured PSA glycoform. (c) using the comparison and / or calculated score in (b) to help determine whether the subject has an aggressive form of prostate cancer.

[0121] In certain embodiments, subject refers to an animal, more preferably a mammal, such as a human, chimpanzee, rhesus monkey, dog, cow, horse, cat, rat or mouse, provided that they also have a prostate.Subject is preferably a mammal, most preferably a human.Suitably, subject is male, such as a male human.

[0122] In certain embodiments, if the subject is not determined to have an aggressive form of PCa, they are determined to have or likely to have an indolent form of prostate disease.

[0123] In certain embodiments, the at least one monoantennary PSA glycoform and the at least one diantennary PSA glycoform are selected from the monoantennary PSA glycoforms identified in Tables 1A and B as compounds 2, 3, 4 or 5, and the diantennary PSA glycoforms identified as compounds 7, 9, 10, 11, 13, 16, 18, 21, 24 and 25.

[0124] In other specific embodiments, the at least one monoantennary PSA glycoform and the at least one diantennary PSA glycoform are selected from the monoantennary PSA glycoforms identified in Tables 1A and B as compounds 2, 3 and 4 and the diantennary PSA glycoforms identified as compounds 7, 9, 18, 11, 13, 24.

[0125] In yet other specific embodiments, the at least one monoantennary PSA glycoform and the at least one diantennary PSA glycoform are selected from the monoantennary PSA glycoforms identified in Tables 1A and B as 2, 3, 4 and 5 and the diantennary PSA glycoforms identified as compounds 7, 9, 24 and 18.

[0126] In certain embodiments, at least one monoantennary PSA glycoform is H4N3S 2,6 1(compound 2);H4N3F1S 2,6 1(compound 3);H4N3F1S 2,3 1 (compound 4); and H3N4F1S 2,6 1 (Compound 5).

[0127] In certain embodiments, at least one diantennary PSA glycoform is H4N4F1S 2,6 1(compound 7);H5N4F1S 2,6 1(Compound 9);H5N4F1S 2,3 1(Compound 10);H4N5F1S 2,6 1 (compound 11); H5N4S 2,3 1S 2,6 1(Compound 13);H4N5S 2,3 1S 2,6 1(Compound 16);H4N5F1S 2,3 1S 2,6 1 (compound 18); H4N5S 2,3 1(SO 3 )1(compound 21);H4N5F1S 2,6 1(SO 3 ) 1 (compound 24); and H4N5F1S 2,61(SO 3 ) 1 (Compound 25).

[0128] In certain embodiments, at least one diantennary PSA glycoform is a diantennary PSA glycoform that includes alpha 2,6-linked sialylation. Such diantennary and alpha 2,6-linked sialylated PSA glycoforms may be selected from the group consisting of compounds 18, 7, 9, 11, 13, 16, 18, 21 and 25 in Tables 1A and B.

[0129] In certain embodiments, at least one diantennary PSA glycoform is a diantennary PSA glycoform comprising alpha 2,6-linked sialylated and fucosylated. Such a diantennary, alpha 2,6-linked sialylated and fucosylated PSA glycoform may be selected from the group consisting of compounds 18, 7, 9, 11, 21 and 25 of Tables 1A and B. In embodiments, such a diantennary, alpha 2,6-linked sialylated and fucosylated PSA glycoform may be selected from the group consisting of compounds 18, 7 and 9 of Tables 1A and B.

[0130] In certain embodiments, at least one diantennary PSA glycoform is a diantennary PSA glycoform comprising alpha 2,3-linked sialylation. Such diantennary and alpha 2,3-linked sialylated PSA glycoforms may be selected from the group consisting of compounds 10, 13, 16, 18, 21, 24 and 25 of Tables 1A and B. In certain embodiments, at least one diantennary PSA glycoform is a diantennary PSA glycoform comprising alpha 2,6-linked sialylation and alpha 2,3-linked sialylation. Such diantennary alpha 2,6-linked sialylated and alpha 2,3-linked sialylated PSA glycoforms may be selected from the group consisting of compounds 13, 16 and 21 of Tables 1A and B.

[0131] In certain embodiments, the methods of the present invention can determine whether a subject has aggressive PCa if the level of at least one monoantennary PSA glycoform is decreased and the level of at least one diantennary PSA glycoform is increased in the subject's sample when compared to a suitable control or reference level or value.

[0132] It will be appreciated that different test samples are likely to contain different amounts of PSA, and therefore the absolute levels of any PSA-glycan species detected in the sample will need to be normalized. Normalization is a standard approach in diagnostic testing. The measured levels of PSA-glycan species can be normalized to the amount of PSA in the original sample. Alternatively, normalization can be incorporated into the test assay by adding a known amount of a reference analyte or marker to the sample being analyzed. For example, the examples herein determined PSA-glycan levels by reference to a known amount of a specific peptide that was added / spiked to the sample by protease digestion (e.g., with trypsin or Arg-C (clostripain)). Thus, in certain embodiments, a known amount of a reference PSA peptide derivative (spiked peptide) may be spiked to normalize the levels of the sample being analyzed by MS. Suitably, the spiked peptide may differ from the native PSA peptide (released during protease digestion) by a single amino acid, and thus be classified as a PSA peptide derivative, and MS quantification of a known amount of the PSA peptide derivative, together with quantification of the endogenous peptide, allows normalization of the level of any and each measured PSA-glycan species in the test / case sample. In one embodiment, the spiked peptide may comprise the sequence disclosed in SEQ ID NO: 2. In another embodiment, the spiked peptide may be labeled, for example radioactively labeled. Various techniques for normalization are known to those skilled in the art.

[0133] In certain embodiments of the present invention, the detected level of each PSA-glycan species is normalized. In certain embodiments, a normalized level of one or each PSA-glycan species is determined and used to determine an aggressiveness score.

[0134] If a ratio or other formula is used that includes levels of multiple PSA glycoforms, normalization may not be required.

[0135] In certain embodiments of the invention, the reference value for the aggressiveness score is determined from at least one reference population including aggressive PCa subjects and / or subjects not having aggressive PCa, such as patients with prostatitis, BPH or indolent PCa.

[0136] In certain embodiments of the present invention, if the level of at least one monoantennary PSA glycoform is decreased and the level of at least one diantennary PSA glycoform is increased compared to the control or reference value, the subject is determined to have aggressive PCa. Otherwise, the subject is determined to not have aggressive PCa or to have indolent prostate disease.

[0137] In certain embodiments of the invention, an aggressiveness score for a subject is calculated from the ratio of the levels of each measured PSA glycoform determined from the samples.

[0138] In certain embodiments of the invention, the method comprises calculating a score for assessing whether the subject has aggressive PCa by considering the combined level of each determined PSA glycoform.

[0139] In certain embodiments of the invention, the method comprises calculating a score (aggression score) for assessing whether a subject has aggressive PCa by taking into account the individual levels of at least one monoantennary PSA glycoform and at least one diantennary PSA glycoform.

[0140] In a particular embodiment, comparison of the individual levels of at least one monoantennary PSA glycoform and at least one diantennary PSA glycoform with appropriate reference levels is used to help determine whether a subject has an aggressive form of PCa.

[0141] In one particular embodiment, comparing the combined level of at least one monoantennary PSA glycoform and at least one diantennary PSA glycoform compared to a reference level of the combined PSA glycoforms is used to help determine whether a subject has an aggressive form of PCa.

[0142] In one particular embodiment, the aggressiveness score is used to help determine whether a subject has an aggressive form of PCa.

[0143] In one particular embodiment, the score in step (b) comprises or is generated from the ratio of at least one monoantennary PSA glycoform to at least one diantennary PSA glycoform, or vice versa.

[0144] In certain embodiments, a ratio value comprising the ratio of (i) to (ii), or vice versa, is prepared, where (i) is the normalized level of at least one monoantennary PSA glycoform in the sample and (ii) is the normalized level of at least one diantennary PSA glycoform in the sample, and optionally the ratio value is used to calculate a risk score for assessing whether the subject has aggressive PCa, and the ratio value or risk score is then compared to a control or reference value to determine whether the subject has aggressive PCa.

[0145] The method may be applied using measurements from one or more monoantennary species and one or more diantennary species in a test sample.

[0146] In certain embodiments, the levels of all measured monoantennary species are summed (added together) and the levels of all measured diantennary species are summed and one sum is divided by the other to obtain a ratio or "aggression score." It will be understood that if only one monoantennary species or one diantennary species in the category is measured, the level of that species is considered the sum level. In this manner, the method can be used using one monoantennary species and two or more diantennary species, or one diantennary species and two or more monoantennary species.

[0147] In certain embodiments of the first aspect of the invention, at least three PSA glycoforms are determined.

[0148] In certain embodiments of the first aspect of the invention, at least four PSA glycoforms are determined.

[0149] In certain embodiments of the first aspect of the invention, at least five PSA glycoforms are determined.

[0150] In certain embodiments of the first aspect of the invention, at least six PSA glycoforms are determined.

[0151] The inventors have discovered that the levels of all monoantennary PSA glycoforms (e.g., compounds 2, 3, 4, and 5) measured in biological fluid samples (e.g., in urine) are decreased in subjects with aggressive PCa, whereas the levels of most of the diantennary PSA glycoforms tested, including, for example, compounds 7, 9, 18, 24 (see Tables 1A and B or Table 6), are increased in subjects with aggressive PCa.

[0152] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention include PSA glycoforms selected from any one of the combinations shown in Table 7 having an AUC (1 x CV) of at least 66.0, preferably having an AUC (1 x CV) of at least 68.0.

[0153] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention are (i) H4N3F1S 2,6 1 and H4N4F1S 2,6 1; (ii) H4N3F1S 2,6 1 and H5N4F1S 2,6 1; (iii) H4N3S 2,6 1 and H4N5S 2,3 1(SO3)1; (iv) H4N3S 2,6 1 and H5N4F1S2,31S 2,6 1; (v) H4N3S 2,6 1 and H4N4F1S 2,6 1; (vi)H4N3F1S 2,6 1 and H5N4F1S2,31S 2,6 1; (vii) H4N3S 2,6 1 and H4N5F1S 2,3 1S 2,6 1; (viii) H4N3S 2,6 1 and H4N5F1S 2,6 1; and (ix)H3N4F1S 2,6 1 and H4N5F1S2,31S 2,6 1.

[0154] As can be seen from Table 1B, H4N3S 2,6 1. H4N3F1S 2,6 1. H3N4F1S 2,6 1 (underlined directly above) are the monoantennary compounds 2, 3 and 5.

[0155] In certain embodiments of the first aspect of the invention, the levels of at least two PSA glycoforms, at least one monoantennary PSA-glycan species and at least one diantennary PSA-glycan species, are determined or measured. In certain embodiments of the first aspect of the invention, the levels of only two PSA glycoforms, one monoantennary PSA glycan species and one diantennary PSA glycan species, are determined or measured. In certain embodiments, the two PSA glycoforms measured are those identified in any one of groups (i)-(ix) immediately above or groups (a)-(i) immediately below.

[0156] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention are (a)H4N4F1S 2,6 1 and a combination of monoantennary glycoforms, (b) H4N3S 2,6 1 and a combination of monoantennary glycoforms, (c)H4N3S 2,6 1 and a2.3. A combination of sialylated glycoforms; (d) H4N3S 2,6 1 and a2.6. A combination of sialylated glycoforms; (e)H5N4F1S 2,3 1S 2,6 1 and a combination of monoantennary glycoforms, (f) H5N4F1S 2,3 1 and a combination of monoantennary glycoforms, (g) a2.3. Combinations of sialylated glycoforms and combinations of monoantennary glycoforms; (h)H4N5F1S 2,3 1S 2,6 1 and a combination of monoantennary glycoforms, and (i) H4N3F1S 2,3 1 and a combination of monoantennary glycoforms.

[0157] In certain embodiments, in each of (a)-(i) above, the combination of glycan structure classes (e.g., combinations of monoantennary glycoforms) includes at least two members, e.g., 2, 3, 4 or more members, from the same class of glycans, e.g., as shown in Tables 1A and 1B.

[0158] In certain other embodiments, in each of (a)-(i) above, the combination of a class of glycan structures (e.g., a combination of monoantennary glycoforms) includes all members of that class, as shown in Tables 1A and B.

[0159] Monoantennary PSA glycoforms suitable for use in the methods of the invention are selected from compounds 2, 3, 4 and 5 identified in Tables 1A and B. Diantennary PSA glycoforms suitable for use in the methods of the invention are selected from one or more of compounds 7, 9, 10, 11, 13, 16, 18, 21, 24 and 25 identified in Tables 1A and B.

[0160] In certain embodiments of the first aspect of the invention, the levels of three, four, five or more PSA glycoforms are determined or measured.

[0161] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention include PSA glycoforms selected from any one of the combinations shown in Table 8 having an AUC (1 x CV) of at least 70.0, preferably having an AUC (1 x CV) of at least 72.0.

[0162] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention include PSA glycoforms selected from any one of the combinations shown in Table 9 having an AUC (1 x CV) of at least 70.0, preferably having an AUC (1 x CV) of at least 72.0.

[0163] In certain embodiments, the PSA glycoforms used or determined in the methods of the invention comprise a combination of PSA glycoforms selected from any one of the combinations shown in Table 10 having an AUC (1 x CV) of at least 73.0, preferably having an AUC (1 x CV) of at least 72.5.

[0164] In certain embodiments, the combination of PSA glycoforms used is a combination of monoantennary PSA glycoforms selected from at least two of compounds 2, 3, 4, and 5, and a combination of diantennary PSA glycoforms selected from at least two of compounds 7, 9, 18, and 24.

[0165] In certain embodiments, the combination of PSA glycoforms used is selected from the following: (a) Compounds 2, 3, and 5 (as monoantennary species) and compounds 24, 7, and 9 (as diantennary species); (b) Compounds 2 and 3 (as monoantennary species) and compounds 7 and 9 (as diantennary species); (c) Compounds 3 and 5 (as monoantennary species) and compounds 24, 7 and 9 (as diantennary species); (d) Compounds 2, 3 and 5 (as monoantennary species) and compounds 7 and 9 (as diantennary species); (e) Compounds 2 and 3 (as monoantennary species) and compound 7 (as diantennary species), and (c) Compounds 3 and 5 (as monoantennary species) and compounds 24 and 9 (as diantennary species).

[0166] In certain embodiments, when the method uses the measurement of the level of two PSA-glycan species, the PSA-glycan species measured / used are one PSA-glycan species that has a higher level (+ direction) in the sample of the subject with aggressive PCa compared to the sample of the subject with indolent prostate disease, and one PSA-glycan species that has a lower level (- direction) in the sample of the subject with aggressive PCa compared to the sample of the subject with indolent prostate disease.Suitable combinations of two PSA-glycan species for use in the method of the present invention are any of those shown in Table 7.

[0167] In certain embodiments, when the method uses measurements of the levels of three PSA glycoforms, the PSA glycoforms measured / used consist of two PSA glycoforms (PSA-glycan species) whose levels are increased (+ direction) in samples from subjects with aggressive PCa, and one PSA-glycan species (PSA glycoform) whose levels are decreased (- direction) in samples from subjects with aggressive PCa. Suitable combinations of three PSA glycoforms for use in the methods of the present invention are any of those shown in Table 8.

[0168] In certain embodiments, when the method uses measurements of the levels of four PSA glycoforms, the PSA glycoforms measured / used consist of three PSA-glycan species whose levels are increased in samples from subjects with aggressive PCa (+ direction) and one PSA glycoform whose levels are decreased in samples from subjects with aggressive PCa (- direction). Suitable combinations of the four PSA glycoforms for use in the methods of the present invention are any of those shown in Table 9.

[0169] In certain embodiments of any aspect of the invention, the aggressiveness score is calculated by or includes (i) a weighted calculation, or (ii) a ratio of the levels of PSA glycoform(s).

[0170] In certain embodiments, calculation is performed based on or includes the ratio of the levels of PSA glycoform species, and uses the levels of at least one PSA glycoform known to increase in subjects with aggressive PCa and at least one PSA glycoform known to decrease in subjects with aggressive PCa.Suitably, the PSA glycoform known to decrease in subjects with aggressive PCa is monoantennary PSA glycoform.Suitably, the PSA glycoform known to increase in subjects with aggressive PCa is diantennary PSA glycoform.

[0171] In certain embodiments, the method of the present invention uses a combination of PSA glycoforms of one glycan class and a combination of PSA glycoforms of another glycan class. Suitably, one glycan class comprises a monoantennary PSA glycoform species. Suitably, one glycan class comprises a diantennary PSA glycoform species or a subset thereof. The subset means that the diantennary species also has one or more specific types of glycosylation (e.g., sialylation, fucosylation, etc.).

[0172] In certain embodiments, the aggressiveness score calculation comprises or consists of forming a ratio of the total level of a combination of PSA glycoform species from one PSA glycoform class to the total level of a combination of PSA glycoform species from different PSA glycoform classes. In certain embodiments, one of the classes of PSA glycoform species is the monoantennary class of PSA glycoform species. In certain embodiments, one of the classes of PSA glycoform species is the diantennary class of PSA glycoform species.

[0173] In certain embodiments, the aggressiveness score is calculated by the ratio of the total level of the combination of monoantennary PSA glycoform species to the total level of the combination of diantennary PSA glycoform species. Specific examples of combinations are provided in Table 10 herein below.

[0174] In certain embodiments, the aggressiveness score is calculated by the ratio of the sum of the levels of the combination of mono-PSA glycoform species to the sum of the levels of the combination of diantennary PSA glycoform species with fucosylation.

[0175] In certain embodiments, the aggressiveness score is calculated by the ratio of the sum of the levels of the combination of mono PSA glycoform species to the sum of the levels of the combination of diantennary PSA glycoform species with α2,3-sialylation.

[0176] In certain embodiments, the aggressiveness score is calculated by the ratio of the sum of the levels of the combination of mono PSA glycoform species to the sum of the levels of the combination of diantennary PSA glycoform species with α2,6-sialylation.

[0177] The inventors found that determining aggressiveness based on the ratio of PSA glycoform species levels was particularly effective and, in fact, resulted in improved performance in assessing aggressiveness (see Table 10).

[0178] One advantage of calculating the aggressiveness score from the ratio of PSA glycoform species levels is that no normalization of the levels is required (as normalization is eliminated by ratio formation).

[0179] The method of the present invention can be used on any suitable subject. Such a subject may not have had any prior prostate evaluation. More typically, such a subject may have had a digital rectal examination (DRE) and / or a PSA test. The method of the present invention is particularly suitable for analyzing samples from subjects with free PSA serum levels in the gray zone (2-10 ng / mL). The method of the present invention is particularly suitable for analyzing samples from subjects with an enlarged prostate (benign prostatic hyperplasia).

[0180] Thus, in certain embodiments, the subject has benign prostatic hyperplasia.

[0181] Suitably, prostatic hyperplasia has been previously determined, for example from a DRE or imaging.

[0182] In certain embodiments, the subject is pre-selected based on having benign prostatic hyperplasia.

[0183] In certain embodiments, the subject is pre-selected based on total serum PSA level.

[0184] In certain embodiments, the subject has a total PSA serum level of 2-10 ng / mL.

[0185] In certain embodiments, the subject is preselected based on having a total PSA serum level between 2 and 10 ng / mL.

[0186] In certain embodiments, subjects are preselected based on their level of percent free serum PSA.

[0187] In certain embodiments, the subject is pre-selected based on having a free serum PSA level below 25%, for example between about 0% and 20%.

[0188] In certain embodiments, the subject has a free serum PSA level below 25%, for example about 0% to 20%.

[0189] The method of the present invention is based on the measurement of PSA-glycan species levels in a sample of a subject.In certain embodiments, the sample is a biological fluid sample, such as a body fluid of a subject.In certain embodiments, the biological fluid sample is selected from blood, serum, plasma, semen, prostatic fluid and urine.

[0190] In one embodiment, the sample is a urine sample, such as a digital rectal exam (DRE) urine sample.

[0191] In certain embodiments, the PSA protein in the sample is treated to separate the PSA protein from other components in the sample.

[0192] In certain embodiments, the PSA protein in the sample is isolated or purified from the sample.This ensures that any glycan species detected thereafter is a glycan species related to PSA in the sample.In certain embodiments, the PSA protein in the sample is isolated or purified from the sample before determining the level of PSA-glycan species.

[0193] In certain embodiments, the PSA protein in the subject's biological fluid sample is separated from other components in the sample. In certain embodiments, the PSA protein in the subject's biological fluid sample is separated from other components in the sample before determining the level of PSA-glycan species.

[0194] As used herein, the terms "isolated," "isolated," and "purified" can be used interchangeably.

[0195] PSA can be separated from the sample using various conventional purification methods such as affinity chromatography. Most suitably, PSA is isolated or purified from the sample by using a PSA binding partner, such as an antibody, that can capture the protein, for example, the PSA binding partner / antibody can be bound to a solid surface such as a microtiter plate or beads, and once PSA binds to the binding partner, the sample can be washed away, leaving the PSA protein bound to the immobilized PSA binding partner / antibody. The separated / purified sample PSA can then be dissociated from the binding partner.

[0196] In certain embodiments, a biological fluid sample is processed to isolate PSA protein from the sample.

[0197] In certain embodiments, PSA in a sample is isolated from the sample by use of a capture binding partner, such as an anti-PSA antibody. Suitably, the capture binding partner is attached to a solid surface, such as a bead.

[0198] The specific glycan species attached to the PSA need to be detected and quantified. This can be done using a variety of techniques. Most suitably, detection and quantification is performed by mass spectrometry followed by a means of separating the various analytes, such as by using capillary electrophoresis. To facilitate detection by mass spectrometry, it is convenient to release the N-glycans from the PSA and either analyze the N-glycans separately, or digest the PSA (with a suitable protease, such as trypsin) to create smaller molecules (with intact glycan species) that are more suitable for MS analysis and analyze the N-glycopeptides.

[0199] Thus, in certain embodiments, the PSA in the sample or isolated from the sample is treated to release glycan structures from the PSA.

[0200] PSA is N-glycosylated at the asparagine residue at position 69 of the PSA protein (Kabat numbering). This position is conveniently located adjacent to a trypsin cleavage site. Thus, the glycan structure(s) attached to PSA can be released as glycopeptide species (different glycoforms) by treating PSA with a suitable protease, such as trypsin or Arg-C (clostripain).

[0201] In certain embodiments, the PSA protein, once separated from the sample, is treated with a protease to obtain N 69 Glycopeptide forms are generated.

[0202] In certain embodiments, the PSA protein, once isolated from the sample, is treated with trypsin to obtain N 69 Generate the K dipeptide glycoform.

[0203] In certain embodiments, the PSA protein, once isolated from the sample, is treated with Arg-C (clostripain) to obtain N 69 It produces the KSVILLGR glycopeptide form, the sequence of which is identified herein as SEQ ID NO:3.

[0204] Glycan species or fragments thereof can be released from the purified PSA or N69 glycopeptide produced as described above by treatment with a glycosidase, e.g., a glycosidase selected from the group consisting of PNGase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3 and endoglycosidase H.

[0205] Thus, in certain embodiments, isolated / purified PSA or N 69 The glycopeptide(s) are treated with glycosidases to produce the PSA protein or N 69 Glycopeptides (e.g., N 69 K or N 69 Releases glycoforms from the KSVILLGR glycopeptide.

[0206] In certain embodiments, the level of each measured PSA glycoform is determined by mass spectrometry, such as CE-ESI-MS or LC-ESI-MS( / MS), or MALDI-TOF MS.

[0207] CE-ESI-MS techniques suitable for use in the method of the present invention are described in Kammeiger et al., supra.

[0208] In certain embodiments, a particular PSA glycoform is 69 It is detected as the K dipeptide glycoform.

[0209] In certain embodiments, a particular PSA glycoform is 69 Detected as KSVILLGR glycopeptide.

[0210] The method of the present invention provides useful information about the state of prostate disease in subjects.The ability to more accurately determine whether a subject is likely to have aggressive PCa or indolent prostate disease provides the attending physician and the subject with additional options for further diagnosis or treatment.For example, the subject that is determined to have indolent prostate disease can be managed by careful follow-up.The subject that is determined to have or is likely to have aggressive PCa can be identified or selected for biopsy test and / or PCa treatment (for example, surgery, radiation therapy, chemotherapy, androgen therapy, etc.).

[0211] Thus, in certain embodiments, the determination by the methods of the invention is used to provide a treatment recommendation to the subject and / or to determine whether a prostate biopsy is necessary.

[0212] It will be appreciated that the method may be performed remotely from the subject or his / her physician, and may in fact be performed off-shore, with the results being communicated.

[0213] In certain embodiments, the results of the methods of the invention (e.g., determining whether a subject has aggressive PCa) are provided to the subject or a third party, such as their physician, laboratory, or health center.

[0214] In certain embodiments, in the methods of the invention, the aggressiveness score is derived from an algorithm into which the level of PSA glycoforms is input.

[0215] In certain embodiments, the algorithm has been generated by machine learning trained on samples from subjects with aggressive PCa as well as subjects with indolent PCa and / or BPH and / or healthy subjects.

[0216] The method of the present invention may comprise analyzing a sample from a subject to determine the level of at least one PSA glycoform, and then interpreting the data to determine whether the subject has or is likely to have aggressive PCa.However, the level(s) of one or more PSA glycoform(s) have already been established, but no question or analysis or data has been performed, so the subject's status may not be determined.The data of the level of at least one PSA glycoform can be inputted into a computer, for example, via a computer program, to perform analysis.Suitably, the computer comprises software having an algorithm and a reference value that can result in the determination of the subject's status.

[0217] According to a second aspect of the invention there is provided a method for determining whether a subject has aggressive prostate cancer comprising the steps of: (i) isolating the PSA protein from other proteins in a biological fluid sample from a subject; (ii) treating the isolated PSA protein with a protease; (iii) Two or more Ns in the sample 69 determining the level of each of the glycopeptide forms, 69 At least one of the glycopeptide forms is a monoantennary glycoform, 69 determining a level of each of two or more N69 glycopeptide forms in the sample, wherein at least one of the glycopeptide forms is a diantennary glycoform; (iv) Each N in (iii) 69 determining an aggressiveness score for the subject using the level of glycopeptide form; (v) comparing the aggressiveness score in (iv) with a reference value for that aggressiveness score; (vi) determining whether the subject has aggressive PCa based on the comparison in step (v); A method is provided, comprising:

[0218] This second aspect of the invention is a subset of the first aspect of the invention, but involves treating a sample from a subject to isolate PSA, cleavage with a protease (such as trypsin or Arg-C (clostripain)), and isoforms of N-acetylglucosamine (NAc), N-acetylglucosamine (NAc), and ... 69 The levels of glycopeptides are determined and assessed. Thus, the embodiments described for the first aspect of the invention also apply to this second aspect of the invention, unless it is clear from the context that they do not apply.

[0219] In certain embodiments, the protease is a N-containing glycopeptide. 69 In certain embodiments, the protease is trypsin or Arg-C (clostripain).

[0220] In certain embodiments, the first monoantennary PSA glycoform in step (a) is selected from compounds 2, 3, 4, or 5 (as shown in Tables 1A and B).

[0221] In certain embodiments, the first diantennary PSA glycoform in step (b) is selected from compounds 7, 9, 10, 11, 13, 16, 18, 21, 24, or 25 (as shown in Tables 1A and B).

[0222] The first, second and fourth aspects of the present invention include measuring the levels of at least two PSA glycoforms. However, as can be seen from Table 2, the levels of three PSA glycoforms (compounds 21, 2 and 9) when analyzed individually can distinguish aggressive PCa from indolent PCa and BPH with higher specificity (higher AUC) than free PSA. Therefore, these PSA glycoforms can be used individually in a method to help determine whether a subject has aggressive PCa.

[0223] Thus, according to a third aspect of the present invention there is provided a method of assisting in determining whether a subject has aggressive prostate cancer, comprising the steps of: (a) H4N5F1S in a biological fluid sample from a subject 2,3 1S2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 determining the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform in (a) with a reference value for that PSA glycoform, or comparing the aggressiveness score taking into account the level of that PSA glycoform in (a) with a reference value for that aggressiveness score; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer; A method is provided, comprising:

[0224] In certain embodiments, the reference value is a value that correlates with aggressive PCa. In certain embodiments, the reference value is a value that correlates with non-aggressive PCa, such as BPH.

[0225] Where appropriate, embodiments applicable to the first aspect of the invention may be applied to this third aspect of the invention.

[0226] In certain embodiments of the third aspect of the invention, the method comprises: 2,3 1S 2,6 1 (Compound 21). In certain embodiments, the H4N5F1S 2,3 1S 2,6 1 (compound 21) is the only PSA glycoform measured. The levels of this PSA glycoform are increased in subjects with aggressive PCa compared to subjects with indolent prostate disease. Thus, in certain embodiments, H4N5F1S 2,3 1S 2,6 If the level of H4N5F1S is increased relative to the baseline, the subject is determined to have an aggressive form of PCa. 2,3 1S 2,6 If the level of 1 is not increased relative to the baseline value, the subject is determined to not have aggressive PCa and / or to have indolent prostate disease.

[0227] In a particular embodiment of the third aspect of the invention, the method comprises: 2,6 1 (Compound 2). In certain embodiments, the H4N3S 2,6 1 (compound 2) is the only PSA glycoform measured, and levels of this PSA glycoform are decreased in subjects with aggressive PCa compared to subjects with indolent prostate disease.

[0228] Thus, in certain embodiments, H4N3S 2,6 If the level of H4N3S is decreased relative to the baseline, the subject is determined to have an aggressive form of PCa. 2,6 If the level of 1 is not decreased relative to the baseline value, the subject is determined to not have aggressive PCa and / or to have indolent prostate disease.

[0229] In a particular embodiment of the third aspect of the invention, the method comprises: 2,6 1 (Compound 9). In certain embodiments, the H5N4F1S 2,6 1 (compound 9) is the only PSA glycoform measured, and levels of this PSA glycoform are increased in subjects with aggressive PCa compared to subjects with indolent prostate disease.

[0230] Thus, in certain embodiments, H5N4F1S 2,6 If the level of 1 is increased relative to the baseline value, the subject is determined to have an aggressive form of PCa.

[0231] In another embodiment, H5N4F1S 2,6 If the level of 1 is not increased relative to the baseline value, the subject is determined to not have aggressive PCa and / or to have indolent prostate disease.

[0232] In certain embodiments of the present invention, the detected level of each PSA-glycan is normalized. In certain embodiments, the normalized level of one or each PSA-glycan is determined and / or compared to a control or reference value.

[0233] In certain embodiments of the invention, the reference value is determined from at least one reference population including aggressive PCa subjects and subjects not having aggressive PCa, such as patients with prostatitis, BPH or indolent PCa.

[0234] In certain embodiments of the invention, the method comprises calculating a score for assessing whether a subject has aggressive PCa by taking into account the individual levels of at least one PSA glycoform.

[0235] In one particular embodiment, the score calculated by considering the levels of the individual PSA glycoforms is used to help determine whether a subject has an aggressive form of PCa.

[0236] In certain embodiments, the subject has benign prostatic hyperplasia. Suitably, benign prostatic hyperplasia is previously determined, for example, from a DRE or imaging.

[0237] In certain embodiments, the subject is pre-selected based on total serum PSA level.

[0238] In certain embodiments, the subject has a total PSA serum level of about 2-10 ng / mL.

[0239] In certain embodiments, the subject is preselected based on having a total PSA serum level of about 2-10 ng / mL.

[0240] In certain embodiments, subjects are preselected based on their level of percent free serum PSA.

[0241] In certain embodiments, the subject is pre-selected based on having a free serum PSA level below 25%, for example between about 0% and 20%.

[0242] In certain embodiments, the subject has a free serum PSA level below 25%, for example about 0% to 20%.

[0243] In certain embodiments, the sample is any bodily fluid that contains PSA. In certain embodiments, the sample is a biological fluid sample. In certain embodiments, the biological fluid sample is a bodily fluid. In certain embodiments, the sample is selected from blood, serum, plasma, semen, prostatic fluid and urine.

[0244] In one embodiment, the sample is a urine sample, such as a digital rectal exam (DRE) urine sample.

[0245] In certain embodiments, the PSA protein in the sample is isolated or purified from the sample, thereby ensuring that any glycan species subsequently detected are glycan species associated with PSA in the sample.

[0246] In certain embodiments, the PSA protein in a biological fluid sample of a subject is separated from other components in the sample.

[0247] PSA can be separated from the sample using various conventional purification methods such as affinity chromatography. Most suitably, PSA is isolated or purified from the sample by using a PSA binding partner, such as an antibody, that can capture the protein, for example, the PSA binding partner / antibody can be bound to a solid surface such as a microtiter plate or beads, and once PSA binds to the binding partner, the sample can be washed away, leaving the PSA protein bound to the immobilized PSA binding partner / antibody. The separated / purified sample PSA can then be dissociated from the binding partner.

[0248] In certain embodiments, a biological fluid sample is processed to isolate PSA protein from the sample.

[0249] In certain embodiments, PSA in a sample is isolated from the sample by use of a capture binding partner, such as an anti-PSA antibody. Suitably, the capture binding partner is attached to a solid surface, such as a bead.

[0250] There is a need to detect and quantitate the specific glycans attached to PSA, which can be accomplished using a variety of techniques.

[0251] Most suitably, detection and quantification is accomplished by means of separation of the various analytes, such as using capillary electrophoresis, followed by mass spectrometry.

[0252] To facilitate detection by mass spectrometry, it is advantageous to release the glycans from PSA or to process the PSA-glycans to generate smaller molecules (with intact glycan species) that are more amenable to MS analysis.

[0253] Thus, in certain embodiments, the PSA in the sample or isolated from the sample is treated to release glycan structures from the PSA.

[0254] In certain embodiments, the PSA protein, once isolated from the sample, is treated with trypsin to obtain N 69 K dipeptide glycopeptides (glycoforms) are produced.

[0255] In certain embodiments, the PSA protein, once isolated from the sample, is treated with Arg-C (clostripain) to isolate N-glycopeptides containing 69 is produced.

[0256] In certain embodiments, isolated / purified PSA or glycopeptides (e.g., N-containing glycopeptide forms) are 69 ) is incubated with glycosidase to release glycoforms from the PSA protein or glycopeptide.

[0257] In certain embodiments, the level of each measured PSA glycoform is determined by mass spectrometry, such as CE-ESI-MS or LC-ESI-MS( / MS), or MALDI-TOF MS.

[0258] In certain embodiments, a particular PSA glycoform is 69 K dipeptide glycoform or its N 69 Detected as the KSVILLGR glycopeptide form.

[0259] The method of the present invention provides useful information about the state of prostate disease in subjects.The ability to more accurately determine whether a subject is likely to have aggressive PCa or indolent prostate disease provides the attending physician and the subject with additional options for further diagnosis or treatment.For example, the subject that is determined to have indolent prostate disease can be managed by careful follow-up.The subject that is determined to have or is likely to have aggressive PCa can be identified or selected for biopsy test and / or PCa treatment (for example, surgery, radiation therapy, chemotherapy, androgen therapy, etc.).

[0260] Thus, in certain embodiments, the determination by the methods of the invention is used to provide a treatment recommendation to the subject and / or to determine whether a prostate biopsy is necessary.

[0261] In certain embodiments, in the methods of the invention, an algorithm is applied to the levels of PSA glycoforms to determine whether a subject has an aggressive form of PCa.

[0262] In certain embodiments, the algorithm has been generated by machine learning trained on samples from subjects with aggressive PCa as well as subjects with indolent PCa and / or BPH and / or healthy subjects.

[0263] The method of the present invention may comprise analyzing a sample from a subject to determine the level of at least one PSA glycoform, and then interpreting the data to determine whether the subject has or is likely to have aggressive PCa.However, the subject's status may not be determined because the level(s) of one or more PSA glycoforms have already been established, but no question or analysis or data has been performed.The data of the level of at least one PSA glycoform can be input into a computer to perform analysis.Suitably, the computer comprises software having an algorithm and a reference value that can result in the determination of the subject's status.

[0264] Computer-implemented methods According to a fourth aspect of the present invention there is provided a computer implemented method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) receiving a value for a level of a first monoantennary PSA glycoform in a biological fluid sample of a subject; (b) receiving a value for the level of the first diantennary PSA glycoform in the subject's biological fluid sample; (c) calculating an aggressiveness score for the subject based on the levels received in (a) and (b); (d) comparing the subject's aggressiveness score in (c) with a reference value for that aggressiveness score; (e) using the comparison in (d) to help determine whether the subject has an aggressive form of prostate cancer.

[0265] In certain embodiments, the first monoantennary PSA glycoform in step (a) is selected from compounds 2, 3, 4, or 5 (as shown in Tables 1A and B).

[0266] In certain embodiments, the first diantennary PSA glycoform in step (b) is selected from compounds 7, 9, 10, 11, 13, 16, 18, 21, 24, or 25 (as shown in Tables 1A and B).

[0267] In certain embodiments, the PSA glycoforms used or determined in the fourth aspect of the invention comprise PSA glycoforms selected from any one of the combinations shown in Table 8 having an AUC(1×CV) of at least 70.0, preferably having an AUC(1×CV) of at least 72.0.

[0268] In certain embodiments, the PSA glycoforms used or determined in the fourth aspect include PSA glycoforms selected from any one of the combinations shown in Table 9 having an AUC(1×CV) of at least 70.0, preferably having an AUC(1×CV) of at least 72.0.

[0269] In certain embodiments, the PSA glycoforms used or determined in this aspect of the invention comprise a combination of PSA glycoforms selected from any one of the combinations shown in Table 10 having an AUC (1 x CV) of at least 73.0, preferably having an AUC (1 x CV) of at least 72.5.

[0270] In certain embodiments, the combination of PSA glycoforms used is a combination of monoantennary PSA glycoforms selected from at least two of compounds 2, 3, 4, and 5, and a combination of diantennary PSA glycoforms selected from at least two of compounds 7, 9, 18, and 24. In certain embodiments, the combination of PSA glycoforms used is selected from the following: (a) Compounds 2, 3 and 5 (as monoantennary species) and compounds 24, 7 and 9 as diantennary species. (b) Compounds 2 and 3 (as monoantennary species) and compounds 7 and 9 as diantennary species. (c) Compounds 3 and 5 (as monoantennary species) and compounds 24, 7, and 9 as diantennary species. (d) Compounds 2, 3, and 5 (as monoantennary species) and compounds 7 and 9 as diantennary species. (e) Compounds 2 and 3 (as monoantennary species) and compound 7 as a diantennary species. (f) Compounds 3 and 5 (as monoantennary species) and compounds 24 and 9 as diantennary species.

[0271] Where appropriate, embodiments applicable to the first aspect of the invention may be applied to this fourth aspect of the invention.

[0272] According to a fifth aspect of the present invention there is provided a computer implemented method for assisting in determining whether a subject has aggressive prostate PCa, comprising: (a) H4N5F1S in biological fluid samples of interest 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 receiving a value for the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform received in (a) with a reference value for that PSA glycoform, or comparing an aggressiveness score taking into account the level of the PSA glycoform received in (a) with a reference value for that aggressiveness score; and assisting in determining whether the

[0273] The value received in (a) may be obtained from the PSA glycoforms determined in the third aspect of the invention.

[0274] According to a variation of this fifth aspect of the invention there is provided a computer-implemented method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) H4N5F1S in biological fluid samples of interest 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 receiving a value for the level of a PSA glycoform selected from the group consisting of: (b) calculating an aggressiveness score for the subject taking into account the values ​​received in (a); and (c) comparing the aggressiveness score in (b) with a baseline aggressiveness score; (d) using the comparison in (c) to help determine whether the subject has an aggressive form of prostate cancer.

[0275] Where appropriate, embodiments applicable to the third aspect of the invention may be applied to this fifth aspect of the invention.

[0276] As used herein, the term "computer-implemented" means that the method is typically carried out in an automated manner on a data processing unit contained in a computer or similar data processing device. The data processing unit shall receive values ​​of the levels of biomarkers (i.e., the levels of each PSA glycoform). Such values ​​may be amounts, relative amounts, or any other calculated values ​​reflecting amounts as described in detail elsewhere herein. Thus, it should be understood that the above-mentioned method does not require the determination of the amount of the biomarker, but rather uses values ​​for the amount that are already predetermined.

[0277] The present invention also contemplates in principle a computer program, a computer program product, or a computer readable storage medium tangibly embodied with said computer program, the computer program comprising instructions which, when executed on a data processing device or computer, perform the inventive method as specified above. - a computer or computer network comprising at least one processor, the processor being adapted to execute a method according to one of the aspects described herein, - a computer-loadable data structure adapted to carry out a method according to one of the aspects described herein while said data structure is executed on a computer, - a computer script, the computer script being adapted to carry out a method according to one of the aspects described herein while the program is being run on a computer, a computer program comprising program means for carrying out a method according to one of the aspects described herein while said computer program is being run on a computer or on a computer network, a computer program comprising program means according to any preceding embodiment, the program means being stored on a computer readable storage medium; - a storage medium on which a data structure is stored and adapted to perform a method according to one of the aspects described herein after the data structure has been loaded into a primary and / or working storage device of a computer or a computer network, a computer program product having program code means in which, or capable of being stored on a storage medium, the program code means for performing a method according to one of the aspects described in this specification when the program code means is executed on a computer or a computer network, a typically encrypted data stream signal containing data of the parameters defined elsewhere in this specification, and - a typically encrypted data stream signal containing the assessment provided by the method of the present invention.

[0278] The methods of the first, second, third, fourth and fifth aspects of the invention can be used to determine or help determine whether a subject has aggressive PCa, or to determine or help determine whether a subject does not have aggressive PCa, such as indolent prostate disease. Suitably, the methods of the first, second, third, fourth or fifth aspects of the invention can be used to determine or help determine whether a subject has an indolent disease, such as BPH.

[0279] "Aiding in determining" means that other factors may or may not be used in addition to making the decision, and thus the measure that is "aiding" is only part of the decision. As used herein, if the outcome can be determined directly, for example from the aggressiveness score, this can be considered an aid in determining.

[0280] Kit of parts According to a sixth aspect of the invention there is provided a kit (or use of a kit) for use in a method of the invention (including any one of the first, second, third, fourth or fifth aspects of the invention), the kit comprising a scoring system having a PSA glycoform threshold or ratio indicative of aggressive PCa.

[0281] Optionally, the kit also contains instructions for use.

[0282] Optionally, the kit also includes a PSA binding partner, such as an antibody, particularly a monoclonal antibody, optionally wherein the PSA binding partner is immobilized on a solid surface, such as a bead.

[0283] Such PSA binding partners may be for isolating or purifying PSA protein. In embodiments, the kit may include a protease such as trypsin or Arg C. Trypsin or Arg-C (clostripain) may be used to generate glycopeptides of PSA that can be measured by mass spectrometry. In embodiments, the kit may be a kit for mass spectrometry detection of PSA glycoforms.

[0284] Further aspects and embodiments of the invention According to a seventh aspect of the invention there is provided a method for determining whether a patient has aggressive prostate cancer or indolent prostate disease comprising the steps of: (a) determining the level of one or more PSA glycoform(s) in a urine sample from a patient; (b) calculating an aggressiveness score for the subject taking into account the level(s) of (a); and (c) comparing the aggressiveness score of (b) with a reference value for that aggressiveness score; (d) determining whether the patient has aggressive prostate cancer or indolent prostate disease based on the comparison in step (b).

[0285] In certain embodiments, the one or more PSA glycoform(s) identified in Tables 1A and B are selected from one or more of compounds 2, 3, 4, 5, 7, 9, 10, 11, 13, 16, 18, 21, 24 and 25. Most suitably, the PSA glycoform is H4N5F1S 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 1. Where appropriate, embodiments applicable to the third aspect of the invention may be applied to this seventh aspect of the invention.

[0286] According to an eighth aspect of the present invention there is provided a method of identifying whether a subject is likely to have aggressive prostate cancer or indolent prostate disease, comprising the steps of: (a) determining or receiving a level of (i) one or more monoantennary PSA glycoform(s) and (ii) one or more diantennary PSA glycoform(s) in a biological fluid sample from a subject; (b) calculating an aggressiveness score using the levels detected in (a); (c) comparing the aggressiveness score determined in step (b) with a reference value for said aggressiveness score; (d) determining whether the subject has aggressive prostate cancer or indolent prostate disease based on the comparison in step (c).

[0287] In certain embodiments, the level in step (a) is determined from a biological fluid sample of the subject.

[0288] In certain embodiments, the one or more PSA glycoforms identified in Tables 1A and B are selected from one or more of compounds 2, 3, 4, 5, 7, 9, 10, 11, 13, 16, 18, 21, 24, and 25.

[0289] In certain embodiments, the PSA glycoforms used or determined in the seventh or eighth aspect of the invention comprise PSA glycoforms selected from any one of the combinations shown in Table 8 having an AUC(1×CV) of at least 70.0, preferably having an AUC(1×CV) of at least 72.0.

[0290] In certain embodiments, the PSA glycoforms used or determined in the seventh or eighth aspect of the invention comprise PSA glycoforms selected from any one of the combinations shown in Table 9 having an AUC(1×CV) of at least 70.0, preferably having an AUC(1×CV) of at least 72.0.

[0291] In certain embodiments, the PSA glycoforms used or determined in the seventh or eighth aspect of the invention comprise a combination of PSA glycoforms selected from any one of the combinations shown in Table 10 having an AUC(1×CV) of at least 73.0, preferably having an AUC(1×CV) of at least 72.5.

[0292] In certain embodiments, the combination of PSA glycoforms used is a combination of monoantennary PSA glycoforms selected from at least two of compounds 2, 3, 4, and 5, and a combination of diantennary PSA glycoforms selected from at least two of compounds 7, 9, 18, and 24.

[0293] In certain embodiments, the combination of PSA glycoforms used is selected from the following: (a) Compounds 2, 3 and 5 (as monoantennary species) and compounds 24, 7 and 9 as diantennary species. (b) Compounds 2 and 3 (as monoantennary species) and compounds 7 and 9 as diantennary species. (c) Compounds 3 and 5 (as monoantennary species) and compounds 24, 7, and 9 as diantennary species. (d) Compounds 2, 3, and 5 (as monoantennary species) and compounds 7 and 9 as diantennary species. (e) Compounds 2 and 3 (as monoantennary species) and compound 7 as a diantennary species. (f) Compounds 3 and 5 (as monoantennary species) and compounds 24 and 9 as diantennary species.

[0294] Where appropriate, embodiments applicable to the first aspect of the invention may be applied to this eighth aspect of the invention.

[0295] [Table 1-1] [Table 1-2] [Table 1-3]

[0296] [Table 2]

[0297] The present invention also provides, inter alia, the following embodiments: 1. A method for assisting in determining whether a subject has an aggressive form of prostate cancer, comprising: (a) determining a level of at least one monoantennary PSA glycoform and a level of at least one diantennary PSA glycoform in a biological fluid sample from the subject; (b) calculating an aggressiveness score for the subject based on the level determined in (a); and (c) comparing the subject's aggressiveness score in (b) with a reference value for the aggressiveness score; (d) using the comparison in (c) to help determine whether the subject has an aggressive form of prostate cancer.

[0298] 2. A method for determining whether a subject has an aggressive form of prostate cancer, comprising: (i) isolating the PSA protein from other proteins in a biological fluid sample from said subject; (ii) treating the isolated PSA protein with a protease; (iii) two or more N 69 determining the level of each of the glycopeptide forms, 69 At least one of the glycopeptide forms is a monoantennary glycoform, 69 determining a level of each of two or more N69 glycopeptide forms in the sample, wherein at least one of the glycopeptide forms is a diantennary glycoform; (iv) Each N in (iii) 69 determining an aggressiveness score for the subject using the level of glycopeptide form; (v) comparing the aggressiveness score in (iv) with a reference value for that aggressiveness score; (vi) determining whether the subject has aggressive PCa based on the comparison in step (v); A method comprising:

[0299] 3. The method of embodiment 2, wherein said protease is selected from trypsin and Arg-C (clostripain).

[0300] 4. The method of embodiment 1, 2 or 3, wherein if the subject is not determined to have an aggressive form of PCa, the subject is determined to have an indolent form of prostate disease.

[0301] 5. The at least one monoantennary PSA glycoform is H4N3S2,6 1;H4N3F1S 2,6 1;H4N3F1S 2,3 1; and H3N4F1S 2,6 The method according to any one of embodiments 1 to 4, wherein the method is selected from the group consisting of: 1.

[0302] 6. The at least one diantennary PSA glycoform is H4N4F1S 2,6 1;H5N4F1S 2,6 1;H5N4F1S 2,3 1;H4N5F1S 2,6 1;H5N4S 2,3 1S 2,6 1;H4N5S 2,3 1S 2,6 1;H4N5F1S 2,3 1S 2,6 1;H4N5S 2,3 1(SO 3 )1; and H4N5F1S 2,6 1(SO 3 6. The method according to any one of the preceding claims, wherein the compound is selected from the group consisting of:

[0303] 7. The method of any one of the preceding embodiments, wherein the levels of three, four, five or more PSA glycoforms are determined.

[0304] 8. The method of any one of the preceding embodiments, wherein said level of each determined PSA glycoform is normalized.

[0305] 9. The method of any one of the preceding embodiments, wherein the aggressiveness score comprises or consists of a ratio of the level of the at least one diantennary PSA glycoform from the subject sample to the at least one monoantennary PSA glycoform, or vice versa.

[0306] 10. The method of any one of the preceding embodiments, wherein the aggressiveness score is a binary score comprising a first value corresponding to the level of the at least one monoantennary PSA glycoform and a second value corresponding to the level of the at least one diantennary PSA glycoform, and the reference value for the aggressiveness score comprises a reference value for the first value and a reference value for the second value, and the subject is determined to have an aggressive form of PCa if the level of the at least one monoantennary PSA glycoform is decreased compared to the reference value for the first value and the level of the at least one diantennary PSA glycoform is increased compared to the reference value for the second value.

[0307] 11. The PSA glycoform(s) used or determined in the methods of the present invention are (i) H4N3F1S 2,6 1 and H4N4F1S 2,6 1. (ii) H4N3F1S 2,6 1 and H5N4F1S 2,6 1. (iii) H4N3S 2,6 1 and H4N5S 2,3 1(SO 3 )1, (iv) H4N3S 2,6 1 and H5N4F1S2,31S 2,6 1. (v) H4N3S 2,6 1 and H4N4F1S 2,6 1. (vi)H4N3F1S 2,6 1 and H5N4F1S 2,3 1S 2,6 1. (vii) H4N3S2,61 and H4N5F1S 2,3 1S 2,6 1. (vii) H4N3S 2,6 1 and H4N5F1S 2,6 1. (ix)H3N4F1S 2,6 1 and H4N5F1S2,31S2,6 1 2. The method of any one of the preceding embodiments, comprising a PSA glycoform(s) selected from the group consisting of:

[0308] 12. The PSA glycoform used or determined in the method of the present invention is a.H4N4F1S 2,6 1 and a combination of monoantennary glycoforms, b.H4N3S 2,6 1 and a combination of monoantennary glycoforms, c.H4N3S 2,6 1 and a2.3. A combination of sialylated glycoforms; d.H4N3S 2,6 1 and a2.6. A combination of sialylated glycoforms; e.H5N4F1S 2,3 1S 2,6 1 and a combination of monoantennary glycoforms, f.H5N4F1S 2,3 1 and a combination of monoantennary glycoforms, g.a2.3. combinations of sialylated glycoforms and combinations of monoantennary glycoforms; h.H4N5F1S 2,3 1S 2,6 1 and a combination of monoantennary glycoforms, and i.H4N3F1S 2,3 1 and combination of monoantennary glycoforms The method of any one of embodiments 1 to 10, comprising a PSA glycoform selected from the group consisting of:

[0309] 13. A method for assisting in determining whether a subject has aggressive prostate cancer, comprising: (a) H4N5F1S in a biological fluid sample from a subject 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 determining the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform in (a) with a reference value for that PSA glycoform, or comparing the aggressiveness score taking into account the level of that PSA glycoform in (a) with a reference value for that aggressiveness score; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer.

[0310] 14. The method of any one of the preceding embodiments, wherein the subject is a mammal, such as a male human.

[0311] 15. The method of any one of the preceding embodiments, wherein said biological fluid sample is selected from blood, serum, plasma, semen, prostatic fluid and urine.

[0312] 16. The method of embodiment 15, wherein the sample is a urine sample, optionally a digital rectal exam (DRE) urine sample.

[0313] 17. The method of any one of the preceding embodiments, wherein the subject is preselected based on having benign prostatic hyperplasia.

[0314] 18. The method of any one of the preceding embodiments, wherein the subject is preselected based on having benign prostatic hyperplasia.

[0315] 19. The method of embodiment 18, wherein the level of total serum PSA in the subject is about 2-10 ng / ml.

[0316] 20. The method of any one of the preceding embodiments, wherein PSA protein in a biological fluid sample of said subject is isolated from said sample.

[0317] 21. The method of embodiment 20, wherein the PSA is isolated from the sample by use of a capture binding partner, such as an anti-PSA antibody.

[0318] 22. The isolated PSA protein is treated with a protease, such as trypsin or Arg-C (clostripain), to obtain N-glycopeptide-containing peptides. 69 22. The method of embodiment 20 or 21, wherein the method produces

[0319] 23. The isolated PSA or N-glycopeptide-containing 69 is treated with glycosidase to produce PSA protein or N 69 23. The method of any one of embodiments 20 to 22, wherein the glycoform is released from the K dipeptide.

[0320] 24. The method of any one of the preceding embodiments, wherein said level of each measured PSA glycoform is determined by mass spectrometry, such as CE-ESI-MS or LC-ESI-MS( / MS) or MALDI-TOF MS.

[0321] 25. The method of any of the preceding embodiments, wherein the determining is used to provide a treatment recommendation to the patient and / or to determine whether a prostate biopsy is necessary.

[0322] 26. The method according to any one of embodiments 1 to 12 and 14 to 25, wherein the reference value for the aggressiveness score is determined from samples of at least one reference population comprising aggressive PCa subjects and subjects not having aggressive PCa, e.g. patients with prostatitis, BPH or indolent PCa, and optionally the disease state is known.

[0323] 27. The method of any one of claims 13 to 25, wherein the reference values ​​for the PSA glycoforms are determined from samples of at least one reference population comprising aggressive PCa subjects and subjects not having aggressive PCa, such as patients with prostatitis, BPH or indolent PCa, optionally with known disease status.

[0324] 28. A kit for use in the method according to any one of embodiments 1 to 27, comprising a scoring system having PSA glycoform reference values ​​or ratios indicative of aggressive PCa, and optionally instructions for use.

[0325] 29. The kit of embodiment 28, further comprising a PSA binding partner, such as a monoclonal antibody, optionally wherein said PSA binding partner is immobilized on a solid surface, such as a bead.

[0326] 30. Use of a kit in the method according to any one of embodiments 1 to 27, comprising a scoring system having a PSA glycoform reference value or ratio indicative of aggressive PCa, and, optionally, instructions for use.

[0327] 31. The use according to embodiment 30, further comprising a PSA binding partner, such as a monoclonal antibody, optionally wherein said PSA binding partner is immobilized on a solid surface, such as a bead.

[0328] 32. A computer-implemented method for assisting in determining whether a subject has aggressive prostate cancer, comprising: (a) receiving a value for the level of a first monoantennary PSA glycoform in a biological fluid sample of the subject; (b) receiving a value for the level of a first diantennary PSA glycoform in the biological fluid sample of the subject; (c) calculating an aggressiveness score for the subject based on the levels received in (a) and (b); (d) comparing the subject's aggressiveness score in (c) with a reference value for that aggressiveness score; (e) using the comparison in (d) to help determine whether the subject has an aggressive form of prostate cancer.

[0329] 33. A computer-implemented method for assisting in determining whether a subject has aggressive prostate cancer, comprising: (a) H4N5F1S in biological fluid samples of interest 2,3 1S 2,6 1;H4N3S 2,6 1; and H5N4F1S 2,6 receiving a value for the level of a PSA glycoform selected from the group consisting of: (b) comparing the value of the level of the PSA glycoform received in (a) with a reference value for that PSA glycoform, or comparing an aggressiveness score taking into account the level of the PSA glycoform received in (a) with a reference value for that aggressiveness score; (c) using the comparison in (b) to help determine whether the subject has an aggressive form of prostate cancer.

[0330] Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference. Aspects and embodiments of the present invention will now be described with reference to the following examples and accompanying drawings. EXAMPLES

[0331] Example 1 1.1 Abbreviations: HAc Acetic acid BGE Background electrolyte: CE-ESI-MS Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry Using a Sheathless Porous Sprayer Prostate-specific antigen (PSA) BPH Benign Prostatic Hyperplasia LE Major electrolytes AmAc Ammonium Acetate PCa Prostate Cancer RT room temperature ABC Ammonium Bicarbonate FA Formic Acid PGA PSA Glycomic Assay FUP Female Urine Pool DTT Dithiothreitol IAA Iodoacetamide Serum fPSA% Free PSA as a percentage of total PSA in serum

[0332] 1.2 Materials 1.2.1 Clinical samples A total of 110 digital rectal examination (DRE) urine samples from 106 patients with elevated "gray zone" PSA concentrations in serum (2-10 ng / ml total PSA) were analyzed with an optimized PSA glycomic assay (PGA). The panel included quadruplicates for technical robustness assessment.

[0333] DRE urine samples obtained from a prospective multicenter study were analyzed. The sample panel consisted of urine from 33 benign controls (benign prostatic hyperplasia; BPH) and 73 PCa patients. Prostate cancer (PCa) samples were further divided into indolent PCa (Gleason score ≤ 6, n = 29) and aggressive PCa (Gleason ≥ 7, n = 44). The majority of PCa patients had total PSA serum values ​​between 2 and 10 ng / ml and therefore represented a gray zone population (see Tables 2 and 3 for demographic and clinical information of the sample panel). 1 mL of DRE urine was used for the PGA approach.

[0334] All samples were diluted to 100 ng / mL PSA before the capture procedure unless the concentration was below this threshold (based on the total PSA Elecsys assay - Roche Diagnostics GmbH, Mannheim, Gemany). Dilutions were performed using a female urine pool (FUP) created by pooling urine samples from healthy female volunteers.

[0335] 1.2.2 Positive Control A PSA-protein standard from human semen (obtained from Lee BioSolutions-St. Louis, MO) was used as a positive control. A positive control sample was generated by spiking 100 ng of PSA-protein standard into FUP.

[0336] 1.2.3 Negative Control As a negative control, FUP from urine samples obtained from healthy female volunteers was used.

[0337] 1.2.4 System Suitability Samples To test whether the analytical performance was as expected before analyzing the DRE urine samples, a tryptic digest of the PSA-protein standard was used as a system suitability sample. For this purpose, several aliquots containing 50 ng of the PSA-protein standard and an internal peptide standard were prepared (see Table 4), followed by reduction, alkylation and tryptic digestion of the proteins. After digestion, the samples were pooled and redistributed into aliquots of equal volume (for protocol, see Kammeiger et al. Anal. Chem. 90, 4414-4421, 2018).

[0338] [Table 3]

[0339] [Table 4]

[0340] 1.3 Method 110 patient samples were randomly divided into 6 batches, each of which contained one positive and one negative control.

[0341] 1.3.1 Anti-PSA beads IgG-M36 Biotinylated monoclonal IgG clone M36 anti-PSA antibody was obtained from Roche Diagnostics GmbH (Mannheim, Germany). 6 mg (3 mg / mL) of antibody solution was added to 600 μL of washed streptavidin beads (ThermoFischer) and incubated overnight at 4 °C with continuous mixing. The next day, the beads were washed with 1 × PBS to remove unbound antibody. Non-specifically bound antibody was removed by incubating the beads in 100 mM formic acid (FA) for 5 min. The pH was readjusted to 7 and the beads were incubated in 0.02% sodium azide (NaN 3) at 4° C. Such beads are used immediately without pre-washing.

[0342] 1.3.2 PSA acquisition Samples were thawed, mixed by hand, and spun at 500g for 1 minute to pellet and remove precipitate. The supernatant was collected and used for further analysis, and samples were placed on ice. Diluted to a concentration of 100ng / mL based on urinary PSA concentration and added to 1mL with FUP. If concentration was less than 100ng / mL, the full sample was used.

[0343] If sufficient sample remained, an additional 20 ng of each urine sample was taken to generate a pooled sample. For all experimental steps, samples and reagents were kept on ice to keep the PSA as stable as possible before the start of the capture procedure.

[0344] A total of 250 uL of 5x PBS and 2 μL of 50% antibody bead suspension (IgG-M36) were added to the samples and capture was carried out overnight at 4 °C. The beads were then washed once with PBS (pH 7-8) and twice with ammonium bicarbonate (ABC) (pH 8). Prior to elution, internal standards (Table 4) were added to the sample wells. PSA was eluted with 100 mM FA (pH 2-3). After elution, the samples were dried.

[0345] [Table 5]

[0346] 1.3.3 Digestion Samples were digested as described by Kammeijer et al. (supra). Briefly, the dried eluate of the captured PSA glycoprotein sample was reconstituted in 25 mM ammonium bicarbonate (ABC) buffer. Reduction was performed with dithiothreitol (DTT) at a final concentration of 2 mM for 30 min at 60°C. Samples were cooled to room temperature (RT) and sulfide alkylation was performed with iodoacetamide (IAA) at RT in the dark for 30 min (final concentration 6 mM). Additional DTT was added to ensure the alkylation process was stopped (final concentration 6 mM). 0.15 ug porcine trypsin was added for overnight digestion at 37°C.

[0347] 1.3.4 Equipment All capillary electrophoresis (CE) experiments were performed on a CESI8000 system (SCIEX Separations, Framingham, MA) with either a bare fused capillary, a dynamic coated neutral capillary or a static coated neutral capillary (all 30 μm i.d. and 150 μm o.d.) of 91 cm length. Prior to analysis, the capillaries were rinsed extensively with background electrolyte (BGE) consisting of 0.1 M NaOH (2.5 min), 0.1 M HCl (2.5 min), water (4 min) and 10% acetic acid (HAc) (v / v, pH 2.3).

[0348] For analysis, an online preconcentration step was used (e.g., transient isotachophoresis (t-ITP)). Prior to injection, ammonium acetate (AmAc) was added to the samples (400 mM). AmAc acted as the main electrolyte. For all experiments, the samples were injected at 25 psi for 24 s (corresponding to approximately 13.5% of the capillary volume), followed by a BGE postplug by applying 0.5 psi for 25 s (corresponding to approximately 0.3% of the capillary volume). Separation was performed by applying a voltage of 20 kV.

[0349] The CE system was coupled to a UHR-QqTOF maXis Impact HD MS (Bruker Daltonics) via a sheathless CE-ESI-MS interface from SCIEX. A capillary voltage of -1000 to -1300 V was applied to ensure stable electrospray. All experiments were performed in positive ionization mode. The flow rate of drying gas (nitrogen) was 1.5 L / min and the temperature was 150 °C. MS data were acquired between m / z 200 and 2200 with a spectral acquisition rate of 1 Hz.

[0350] 1.3.5 Data Analysis CE-ESI-MS data were analyzed with DataAnalysis (Bruker Daltonics). Prior to data analysis, all MS spectra were calibrated using the sodium adduct detected at the beginning of the electropherogram. Data were manually screened for endogenous peptides (Table 4) and spiked internal standard I (synthetic peptide with one amino acid substitution from the endogenous peptide, see Table 4). Extracted ion electropherograms (EIE smoothed with a Gaussian fit) were acquired for the first three isotopes of doubly and triply charged analytes using a width of m / z ± 0.05 units.

[0351] 1.4 Results 1.4.1 System Suitability The peak areas observed for the endogenous peptides of the system suitability standards and the spiked internal standard I (total CE-MS intensity of MS1) revealed that the system was working well throughout the entire measurement of all samples (data not shown).

[0352] 1.4.2 Clinical evaluation of PSA glycans Measurement of 110 clinical samples resulted in the identification of 26 N-glycopeptides (areas expressed as relative peak areas [%] normalized to all identified glycoforms and summed to 100%).

[0353] The individual glycoform regions were grouped based on the same glycosylation trait, e.g., fucosylation, mono-sialylation, di-sialylation, α2,3-sialylation, α2,6-sialylation or sulfation (shown in Table 1B and identified as "Total" in Table 5), and used in univariate and multivariate biostatistical analyses to identify structures (biomarkers) or compositions of several structures (biomarker panels) that provide the best discrimination between aggressive PCa and indolent PCa and BPH. See Table 4 for a list of individual and combined glycan biomarkers.

[0354] Table 1B indicates what category / glycosylation characteristics a particular glycoform has (e.g., whether it is monoantennary, diantennary, fucosylated, a2,3-sialylated, etc.). When a compound ID is marked as Total (Yes) in Table 4 above, it means that each member of that class (e.g., in Table 1B) was measured and summed (e.g., compare sulfation = compounds 23, 24, 25 and 26).

[0355] The clinical outcomes of urinary glycoforms or glycosylation categories were compared with the efficacy of serum free PSA% (Elecsys serum free PSA% = free PSA / total PSA) in the same cohort.

[0356] Statistical analysis was performed as follows.

[0357] Univariate analysis was based on: (1) p-value from Wilcoxon test, (2) false discovery rate (FDR) of 30% and (3) biomarker performance as indicated by area under receiver operating characteristic (AUC ROC) curves.

[0358] Multivariate analyses included (1) ROC analysis for all linear combinations in 2-, 3-, and 4-marker panels (1× cross-validation) and (2) selection frequencies of combinations from 2× cross-validation (inner loop: feature selection, outer loop: performance estimates, splitting training and test sets in 100 outer loops).

[0359] Moreover, as estimated from the multivariate analysis, the best combinations are mainly in the direction + / - (increased compound and decreased compound) or - / + (decreased compound and increased compound). Therefore, the ratio between the best upregulated compound groups (Comp.2, 3, 4, 5) and the downregulated compound groups (Comp.7, 9, 18, 24; Figure 6) was calculated, and their clinical outcomes were estimated in the study population.

[0360] [Table 6]

[0361] 1.4.3 Univariate analysis results Univariate analysis of all 26 distinct glycoforms and 10 combined glycosylation features revealed good clinical performance (discrimination ability) of PSA glycoforms with at least 3 glycoforms. The AUC ROC for discrimination of aggressive vs. indolent PCa and BPH was higher compared to serum fPSA%.

[0362] In particular, compound (Comp.) 21 performed better as serum fPSA% with AUC61.8, with AUC64.1, Comp.2 with AUC63.0 and Comp.9 with AUC62.0. See Table 6 for detailed structures of compounds and Figure 2 for comparison of AUC ROC of Comp.21 against serum free PSA%.

[0363] [Table 7]

[0364] 1.4.4 Multivariate analysis results In multivariate analyses combining two, three, or four compounds or groups of compounds, all glycoform combinations perform better in discriminating aggressive vs indolent PCa and BPH compared to serum fPSA% (Tables 7-9 and Figures 3-5). Addition of further compounds to the combination panel steadily increased panel performance from AUC 69.8 for two structures (Comp.3+7), AUC 73.0 for three structures (Comp.4+7+monantennary); and AUC 75.1 for four structure combinations (Comp.2+9+13+22).

[0365] [Table 8]

[0366] [Table 9]

[0367] [Table 10]

[0368] 1.4.5 Results - Ratio analysis The combination of the two selected markers was most frequent in the + / - or - / + combination, and the coefficients of the multivariate model (logistic regression with log-transformed markers was used) had comparable absolute values, so identity was confirmed.

number

[0369] FIG. 6 shows the strongest up- and down-regulated traits.

[0370] Biostatistical analysis demonstrated a significant benefit on clinical outcome through multivariate combination by including artificially combined traits (upregulated, downregulated) and calculation of their ratios (Table 10 and Figure 7).

[0371] [Table 11]

[0372] Interestingly, all the major downregulated structures, Comp. 2, 3, 4, 5, represent complex monoantennary N-glycans, whereas the upregulated groups, Comp. 7, 9, 18 and 24, specifically contain complex diantennary, sialylated and fucosylated N-glycans. It may therefore be concluded that the ratio between monoantennary and diantennary PSA glycoforms is a novel marker (signature) for improved discrimination of aggressive PCa from its indolent and benign controls.

[0373] 1.5 Conclusion CE-MS-based glycoanalysis of urinary PSA glycoforms revealed several novel structures and structure combinations with superior clinical performance for discriminating between aggressive and indolent prostate cancer compared to serum fPSA%. In the univariate setting: Three glycoforms with greater AUC than serum fPSA% In a multivariate two-marker setting: Approximately 6% improvement over the univariate model In a multivariate 3-marker setting: Approximately 10% improvement over univariate models In a multivariate 3-marker setting: Approximately 12% improvement over the univariate model, approximately 5-10% improvement over the Beckman Prostate Health Index (PHI) (serum) Significant benefit on clinical outcome from multivariate combination of artificially combined traits (up-regulators, down-regulators) and ratios, approximately 10% improvement over univariate models, approximately 5-10% improvement on Beckmann PHI (serum).

Claims

1. A method to assist in determining whether or not a subject has invasive prostate cancer, (a) Determining the level of at least one monoantenna PSA glycoform and at least one diantenna PSA glycoform in the biofluid sample from the subject, (b) Calculate an invasiveness score for the subject based on the level determined in (a), (c)(b) Compare the invasiveness score of the subject with the reference value for the invasiveness score, (d)(c) The comparison described above is used to assist in determining whether the subject has aggressive prostate cancer, Methods that include...

2. Step (a) is, (i) Isolating PSA protein from a biofluid sample from the subject, (ii) Treating the isolated PSA protein with a protease, such as trypsin or Arg-C (clostrypine), (iii) Two or more N containing glycopeptide forms in the sample ６９ The determination of each level of the N including glycopeptide form ６９ At least one of the is a monoantenna glycoform, and the N includes a glycopeptide form. ６９ Two or more N2s in the sample comprising glycopeptide forms, at least one of which is a dianthenic glycoform. ６９ To determine each level, The method according to claim 1, including the method described in claim 1.

3. The aforementioned at least one mono-antenna PSA Glycoform is H4N3S ２，６ 1. H4N3F1S ２，６ 1. H4N3F1S ２，３ 1, and H3N4F1S ２，６ The method according to claim 1, selected from the group consisting of 1.

4. The at least one diantenna PSA glycoform is H4N4F1S ２，６ 1, H5N4F1S ２，６ 1, H5N4F1S ２，３ 1, H4N5F1S ２，６ 1, H5N4S ２，３ 1S ２，６ 1, H4N5S ２，３ 1S ２，６ 1, H4N5F1S ２，３ 1S ２，６ 1, H4N5S ２，３ 1(SO ３ )1, and H4N5F1S ２，６ 1(SO ３ )1, the method according to claim 1, selected from the group consisting of.

5. The method according to claim 1, wherein the invasiveness score includes or comprises the ratio of the level of the at least one diantenna PSA glycoform from the target sample to the level of the at least one monoantenna PSA glycoform, or the reverse ratio.

6. The PSA glycoform used or determined in the method of the present invention is (i) H4N3F1S ２，６ 1 and H4N4F1S ２，６ 1. (ii) H4N3F1S ２，６ 1 and H5N4F1S ２，６ 1. (iii) H4N3S ２，６ 1 and H4N5S ２，３ 1 (SO ３ ) 1. (iv) H4N3S ２，６ 1 and H5N4F1S ２，３ 1S ２，６ 1. (v) H4N3S ２，６ 1 and H4N4F1S ２，６ 1. (vi) H4N3F1S ２，６ 1 and H5N4F1S2,31S ２，６ 1. (vii) H4N3S2, 61 and H4N5F1S ２，３ 1S ２，６ 1. (viii) H4N3S ２，６ 1 and H4N5F1S ２，６ 1. and (ix) H3N4F1S ２，６ 1 and H4N5F1S2,31S ２，６ 1 The method according to claim 1, comprising a PSA glycoform selected from the group consisting of the following.

7. The PSA glycoform used or determined in the method of the present invention is a. H4N4F1S ２，６ 1 and a combination of monoantenna glycoform, b. H4N3S ２，６ 1 and a combination of monoantenna glycoform, c. H4N3S ２，６ 1 and a2.

3. Combinations of sialylated glycoforms, d. H4N3S ２，６ 1 and a2.

6. Combinations of sialylated glycoforms, e. H5N4F1S ２，３ 1S ２，６ 1 and a combination of monoantenna glycoform, f. H5N4F1S ２，３ 1 and a combination of monoantenna glycoform, g. a2.

3. Combinations of sialylated glycoforms and combinations of monoantennae glycoforms, h. H4N5F1S ２，３ 1S ２，６ 1 and the combination of monoantenna glycoform, and i. H4N3F1S ２，３ 1 and a combination of monoantenna glycoform The method according to claim 1, comprising a PSA glycoform selected from the group consisting of the following.

8. The method according to claim 1, wherein the subject is a mammal, for example, a male human, and / or the biofluid sample is selected from blood, serum, plasma, semen, prostatic fluid, and urine, for example, a digital rectal examination (DRE) urine sample.

9. The method according to claim 1, wherein the subject is pre-selected based on having benign prostatic hyperplasia or based on the level of total serum PSA, and optionally the level of total serum PSA in the subject is between approximately 2 and 10 ng / ml.

10. The method according to claim 1, wherein the PSA protein in the target biological fluid sample is optionally isolated from the sample by the use of a capture and binding partner, such as an anti-PSA antibody.

11. The isolated PSA protein was treated with protease to obtain a glycopeptide containing N ６９ The method according to claim 10, which produces the following.

12. The method according to claim 1, wherein the level of each measured PSA glycoform is determined by mass spectrometry, for example, CE-ESI-MS, LC-ESI-MS ( / MS), or MALDI-TOF MS.

13. The method according to claim 1, wherein the reference value for the invasiveness score is determined from a sample of at least one reference population, including subjects with invasive PCa and subjects without invasive PCa, such as patients with prostatitis, BPH, or slowly progressive PCa, and optionally the disease state is known.

14. A kit for use in a method according to any one of claims 1 to 13, comprising: a scoring system having a PSA glycoform reference value or ratio indicating invasive PCa; and optionally, (i) instructions for use, and / or (ii) a PSA-binding partner, such as a monoclonal antibody, wherein the PSA-binding partner is optionally immobilized on a solid surface such as a bead.

15. A computer implementation method for assisting in determining whether or not a subject has invasive prostate cancer, (a) A step of receiving a value for the level of the first mono-antenna PSA glycoform in the target biological fluid sample, (b) A step of receiving a value for the level of the first dianthentic PSA glycoform in the target biological fluid sample, (c) A step of calculating an invasiveness score for the object based on the levels received in (a) and (b), (d) A step of comparing the invasiveness score of the target in (c) with a reference value for the invasiveness score, (e) A step of assisting in determining whether the subject has aggressive prostate cancer using the comparison in (d), Computer implementation methods, including those mentioned above.

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