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    • 专利摘要:
    In vitro method for the diagnosis of prostate cancer. The invention relates to a method for the in vitro differential diagnosis of sensitive and specific prostate cancer and which comprises partially purifying the prostate specific antigen from a fluid sample isolated from a subject and then determining the percentage of sialic acid α -2, 3 of the prostate-specific antigen and/or the ratio (ratio) of internal fucosylation of the prostate-specific antigen. The invention relates in particular to a highly sensitive and specific method which makes it possible to distinguish samples from patients with high-risk prostate cancer, from samples from patients with benign prostate diseases or disorders and from low or intermediate risk prostate cancer patients. . The method is also useful for the prognosis of this cancer. It also refers to kits for carrying out said diagnostic methods. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2615538A1
    申请号:ES201531608
    申请日:2015-11-06
    公开日:2017-06-07
    发明作者:Rosa PERACAULA MIRÓ；Rafael De Llorens Duran；Esther LLOP ESCORIHUELA；Sílvia BARRABÉS VERA；Josep COMET BATLLE；Manel RAMÍREZ MALAGÓN；Rosa Núria ALEIXANDRE CERAROLS；Montserrat FERRER BATALLÉ；Radka FAHEY；Pauline Mary RUDD
    申请人:Universitat de Girona；Fundacio Institut dInvestigacio Biomedica de Girona Dr Josep Trueta Idibgi；
    IPC主号:
    专利说明:

    DESCRIPTION
    Table 1: Sample characteristics In vitro method for the diagnosis of prostate cancer.

    The present invention is related to medicine, and particularly to the diagnosis of cancer. Specifically it refers to a method of diagnosis of prostate cancer.

    STATE OF THE TECHNIQUE

    Prostate cancer (PCa) has the highest incidence among carcinomas and is the second leading cause of cancer death in men in Western countries. The diagnosis of PCa is currently based on digital rectal examination (DRE) and serum levels of prostate specific antigen (PSA). In men who could develop CaP, PSA levels are elevated for years, even before cancer is diagnosed. Actually, PSA is a good prognostic marker of the risk of suffering from a clinically relevant long-term prostate cancer. The wide availability of trials to detect PSA revolutionized the screening of CaP and resulted in a 29% decrease in the risk of deaths from CaP. However, the ubiquitous application of the PSA test has also resulted in an excess of false positives that have undergone treatment (surgery, radio and chemotherapy) when it was not necessary, while unnecessary biopsies have been performed and aggressive therapies for cancer listed as 20 low risk. Considering that the clinical approach should be redirected towards patients who are more likely to have clinically significant tumors and, therefore, will benefit from the treatment, more sensitive and specific tests should be developed and implemented.
    image 1

    PSA (prostate-specific antigen) is a serine protease glycoprotein secreted by the 25 epithelial cells of the prostate. It is a member of the human kallikrein family. Its protein sequence in humans is 237 amino acids, in its active form without the signal peptide or the propeptide, and is accessible in the UniProtKB database, edition of September 16, 2015, under accession number P07288, whose isoform 1 it is version 2 of the sequence dated July 1, 1989. The detection of serum PSA levels is currently used for the detection and monitoring of prostate pathologies, because their levels rise mainly as a result of Destruction of the basement membrane of prostate cells affected by the pathology. Virtually all epithelial cells of the prostate, whether normal, hyperplastic or cancerous, synthesize PSA. Neoplastic cells produce lower and variable PSA levels compared to benign epithelial cells, although both conditions cause total elevation of PSA in the blood. Therefore, serum PSA values do not allow to specifically differentiate between changes in these levels caused by cancer (CaP), benign prostatic hyperplasia (BPH), prostatitis, or manipulations (cycling, DRE, catheterization) of the prostate, since all these situations can also cause an increase in serum PSA concentrations. Serum PSA levels are therefore not very specific and cause a high rate of false positives, especially when serum PSA levels are in the so-called "gray zone" (4.0 to 10.0 ng / ml). ), so several authors have extended this concentration range of 2-20 ng / ml or 1-27 ng / mL. To this we must add that in high-risk cancers, serum levels of total PSA may be low. Therefore, there is also no cut-off point (or reference threshold control value) of serum PSA 45 to distinguish between aggressive and indolent cancers. To improve the specificity of the tests in which serum PSA levels are measured, the measurement of the ratio of free PSA (fPSA) / total PSA, which is lower in prostate cancer, is applied in clinical practice, although it continues to give false positives. Free PSA (abbreviated fPSA) refers to free glycoprotein, not bound to whey proteins. Total PSA (abbreviated tPSA) includes free serum glycoprotein and also serum-bound protein. Other alternatives to improve the specificity and sensitivity of the assays are PSA density, PSA velocity, and the different molecular isoforms of PSA. Unfortunately none of these approaches substantially improves the ability to
    distinguish between BPH and PCa. Non-PSA markers are Prostate Cancer Antigen 3 (PCA3) and the Prostate Health Index (PHI), the latter recently approved by the FDA and Europe for the diagnosis of PCa. Sophisticated imaging systems are also available that have improved the diagnosis of prostate cancer.
     5
    Taking advantage of the fact that the oncogenic process leads to an important alteration of the cell glycosylation pattern, there are also scientific works in the literature that propose the analysis of the glycosylation pattern of PSA, which refers to the type and proportion of modifications of the monosaccharide molecules of the polypeptide chain, as a useful element for the diagnosis of PCa. As indicated above, PSA is a glycoprotein and contains 8% of its weight in carbohydrates, in a single chain of N-oligosaccharide linked to asparagine 45 (Asn-45).
    Some of these modifications with monosaccharides correspond to internal fucosylation (core fucosylation), which is the binding of fucose with an α-1,6 bond to the β-N-acetylglucosamine in the nucleus of N-glycans (or N -oligosaccharide). In eukaryotes, N-glycans derive from the transfer onto an Asn consensus (Asn-X-Ser / Thr) of an initial oligosaccharide of 15 14 monosaccharides containing 3 glucose units, 9 mannose units and 2 N-acetylglucosamine units , which is subsequently processed in the secretory pathway (endoplasmic reticulum and Golgi apparatus). All the generated glycans contain a nucleus that derives from this initial oligosaccharide, and which is formed by the 5 internal monosaccharides, which are the two units of N-acetylglucosamine and three hands. When talking about internal fucosylation, it should be understood as the presence of this monosaccharide (fucose) linked to the first N-acetylglucosamine in the N-oligosaccharide chain of PSA. When the internal fucosylation of an oligosaccharide molecule bound to a protein (for example PSA) is determined, it refers to the level of fucosylation or internal fucose level, or the proportion of protein molecules in a sample (for example PSA) that contain internal fucose in said oligosaccharide molecules linked to the polypeptide chain.

    Another type of glycosylation that can be found in the oligosaccharide chain of PSA is sialylation. Sialic acid is the generic term used for derivatives of neuraminic acid (monosaccharide with a 9-carbon structure) that are N- or O-substituted. Of the two anomeric forms, the alpha (α) anomer is the form that is linked to glycans. The union of sialic acid to one of the monosaccharides of the N-glycans can be of the α2.6 link type between units, α2.3 or α2.8, 2 corresponding to the number of carbon in the structure of sialic acid.
     35
    Previous studies have analyzed and compared the glycosylation pattern of purified PSA seminal fluid from healthy donors with PSA produced by prostate tumor cells, from a human prostate tumor cell line and from sera from patients with cancer. prostate. An example of this follows from the document Tabarés et al. "Different glycan structures in prostate-specific antigen from prostate cancer will be in relation to seminal plasma 40 PSA", Glycobiology - 2006, vol. No. 16 (2), pp .: 132-145. These studies allowed us to conclude that compared to seminal plasma PSA, the internal fucose content (α1.6 fucose) of the serum PSA N-glycans of patients with PCa was significantly lower. In addition, the degree of sialylation of PSA, evaluated by changes in the proportion of PSA isoforms separated by two-dimensional electrophoresis (2-DE), indicated a decrease in sialylation (sialic acid that is part of the N-oligosaccharide chain of PSA) in PSA of patients with PCa, compared with the same monosaccharide levels in seminal plasma. Other authors also observed this decrease in PSA sialylation of patients with PCa compared to that of PSA in patients with BPH, (Sarrats et al., “Differential percentage of serum prostate-specific antigen subforms suggest a new way to improve prostate cancer 50 diagnosis ”, Prostate - 2010 vol. No. 70 (1), pp.:1-9). These results suggested, therefore, a possible way to discriminate between BPH and PCa focused on the determination of glycosylation changes in serum PSA. In addition to two-dimensional electrophoresis, for the analysis of these
    Glycosylation patterns were carried out in immunobsorption tests of carbohydrates and capture using lectins, such as Sambucus nigra lectin (SNA), Maackia amurensis lectin (MAA) and Aleuria aurantia lectin (AAL). In addition to characterizing glycosylation patterns, the objective of Tabarés et al., And Sarrats et al., Was to avoid having to purify PSA from patient sera, which requires large amounts of serum and also translates into long processes. and expensive.

    Also by analyzing sialic acid bound to serum PSA, other authors besides Tabarés et al. (supra) have proposed and analyzed the levels of sialic acid bound to PSA through the use of lectins. Thus, the document Ohyama et al. "Carbohydrate structure and 10 differential binding of prostate-specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy", Glycobiology - 2004, vol. No. 14 (8), pp .: 671-679, describes a trial between patients with PCa (n = 15) and patients with BPH (n = 15), where it is concluded that the levels of α2,3-sialic acid bound A free PSA are significantly higher in CaP. However, there are no sensitivity and specificity values. fifteen

    Similarly, patent application WO2010011357 proposes a two-phase analytical platform that combines a high density lectin microarray with an immunoassay with an antibody and fluorescent reagents to determine the glycosylation pattern in clinical samples with PSA (in particular α2 acid , 3-sialic and α2,6-sialic bound to PSA). twenty

    Along these same lines, which aims to determine the glycosylation pattern in serum PSA of patients, the document by Meany et al., "Glycoproteomics for Prostate Cancer Detection: Changes in Serum PSA Glycosylation Patterns", J Proteome Res - 2009, vol. No. 8 (2), pp .: 613-619. Meany et al., Compared the sialylation pattern of free and total serum PSA of 25 patients with PCa (n = 26) and controls without cancer (n = 26). To do this, they developed five immunosorbent assays with lectins. The authors proposed that the α2,6-sialic acid assay linked to total serum PSA improved the detection of prostate cancer compared to the percentage of free serum PSA in the so-called gray zone. If this analysis was carried out with the SNA lectin, the ROC curves could even achieve area values under the curve (AUC) of 0.71. 30

    Another document focused on the comparison of the glycosylation pattern of PSA, in this case of the internal fucosa linked in α1,6 bond (core fucosa), among patients with PCa of different degrees of aggressiveness or risk, is the document by Li et al ., "Serum Fucosylated Prostate-specific Antigen (PSA) Improves the Differentiation of Agressive from Non-aggressive Prostate Cancers", 35 Theranostics - 2015, vol.no. 5 (3), pp .: 267-276. Li et al. Analyzed PSA fucosylation levels of different patients by immunoabsorption with the AAL lectin. They concluded that the "internal fucose / total serum PSA" ratio allowed a better prediction (AUC = 0.7762) than the value of total serum PSA levels between tumors with a Gleason score of 6 and a score greater than 6 (more aggressive). The Gleason score allows classifying the different 40 degrees of tumor according to the risk associated with them in terms of survival.

    Despite all these attempts, there is still a need to develop a test and / or parameters that allow the diagnosis of PCa in fluid samples, particularly in serum, and that not only distinguish cancer from other prostate diseases with greater specificity. and 45 sensitivity, but also to be able to distinguish aggressive or high-risk PCa, which need to apply as soon as possible the available therapies, of indolent PCs (or low-risk or intermediate risk), to which it is recommended to follow-up to control their evolution ("wait and watch" or "active surveillance"). There is also a lack of means or kits that allow these serum tests to be practiced in a simple and routinely applicable way in the clinic. fifty

    EXPLANATION OF THE INVENTION

    The inventors propose the use of a method of purification and analysis of the serum PSA and some parameters or relationships between levels or proportions of different types of glycosylation of the PSA, specifically its sialylation and the internal fucose, which make it possible to distinguish patients with aggressive CaP 5 from indolent CaP patients and patients without cancer and other prostate diseases, especially patients with BPH. The diagnostic method also includes the determination of a parameter (the percentage of PSA that comprises sialic acid α2,3 or levels of α2,3-sialic acid) that correlates very well with the classification of prostate tumors according to the score of Gleason, which is one of the gradation systems used to classify patients with PCa among high-risk, medium-risk or low-risk cancers, based on patient biopsies.

    Thus, in a first aspect the invention relates to a method for in vitro diagnosis of high-risk prostate cancer in a fluid sample isolated from a subject. This method includes the determination of the PSA glycosylation pattern of the sample, which is defined by the presence, quantity and proportion of internal fucosylation (understanding proportion as the fucosylated protein ratio with respect to the one that is not) and / or by the presence and percentage of sialic acid α2,3, and where the method comprises:
     twenty
    (A) Determine the amount of PSA of the sample comprising internal fucose and calculate a parameter called the internal fucosylation ratio (abbreviated RFI) of the sample, such as the ratio between the amount of PSA comprising internal fucose (abbreviated fucose-PSA) with respect to the amount of free PSA in the sample (fPSA), according to formula (II)
     25
    RFI = (fucose-PSA) / fPSA (II);

    I

    (B) Separate the PSA from the sample into two fractions B and UB, by interacting with a solid phase comprising specific sialic acid lectins, where the UB fraction comprises PSA comprising α2.3 sialic acid and does not comprise PSA with α2.6 sialic acid (abbreviated α2,6-PSA), and where fraction B comprises PSA comprising α2.6 sialic acid (abbreviated α2,6-PSA); determine in each of these fractions UB and B the amount of PSA; and calculate the percentage of PSA comprising sialic acid α2.3 35 (abbreviated% α2,3 – PSA), calculating the ratio between the amount of PSA of the UB fraction (UB-PSA) with respect to the sum of the amount of PSA of fractions UB (UB-PSA) and B (B-
    PSA) according to formula (Ia)

    % α2,3 – PSA = [(UB-PSA) / ((UB-PSA) + (B-PSA))] x 100 (Ia); 40


    where:

    if the percentage of PSA comprising α2.3 sialic acid (abbreviated% α2,3-PSA) is greater than a reference control value, the subject is diagnosed with high-risk prostate cancer; and / or if the internal fucosylation (RFI) ratio in the isolated sample is less than a reference control value, the subject is diagnosed of high-risk prostate cancer, where high-risk prostate cancer is the one that corresponds with a Gleason grade score equal to or greater than 8, or Gleason 7 with metastasis. fifty

    Prostate cancers are also classified according to their aggressiveness as aggressive and indolent cancers also based on (or based on) the Gleason score. In the sense
    of the present invention, high-risk cancers would correspond to those considered aggressive according to this other classification, while indolent ones would include intermediate and low-risk prostate cancers.

    The inventors have observed that with the analysis of these parameters defined by formulas 5 (Ia) and (II) very high specificity and sensitivity values are obtained in the discrimination between high risk prostate cancer with respect to this same type of cancer but low or intermediate risk. It also allows differentiation from other benign prostate diseases (BPH). This makes it possible to make a more accurate diagnosis and proceed quickly to treatment when high-risk cancer is detected. In addition, these parameters 10 related to the glycosylation pattern of PSA are calculated from data that comes from fluid samples of a subject, avoiding biopsies, as will be seen later.

    Although for the determination of% α2,3-PSA both the amount of free PSA (fPSA) and total PSA (tPSA) can be analyzed, in a particular embodiment, optionally in combination with any aspect or embodiment of the invention, proceed by calculating the percentage of free PSA comprising α2.3 sialic acid (abbreviated% α2,3 – PSA), as the ratio between the amount of free PSA of the UB fraction (UB-fPSA) with respect to the sum of the amount of Free PSA of the UB (UB-fPSA) and B (B-fPSA) fractions according to formula (Ib), therefore using fPSA-specific antibodies:

    % α2,3 – PSA = [(UB-fPSA) / ((UB-fPSA) + (B-fPSA))] x 100 (Ib).

    The first aspect of the invention relates, more particularly, to a method for in vitro diagnosis of high-risk prostate cancer in a fluid sample isolated from a subject. Method 25 consists in determining the glycosylation pattern of the PSA in the sample, and this pattern is defined by the presence, quantity and proportion, with respect to the entire PSA, of internal fucosylation and / or by the presence and percentage of PSA that It contains sialic acid α2,3, according to the following steps:
     30
    (a) mixing the isolated fluid sample with a solution of ethanolamine at a final molar concentration of 0.5 to 2.5 M ethanolamine;
    (b) immunoprecipitate the solution obtained in step (a) with a prostate specific antigen specific antibody (PSA) and elute the immunoprecipitated PSA to recover the PSA from the sample;
    (c) in the solution of step (b): 35

    (c.1) Separate the PSA into two fractions B and UB, by interaction with a solid phase comprising sialic acid specific lectins, where the UB fraction comprises PSA comprising α2.3 sialic acid and does not comprise PSA with sialic acid α2,6 (abbreviated α2,6-PSA), and where fraction B comprises PSA comprising sialic acid α2,6 40 (abbreviated α2,6-PSA);
    (c.2) determine in each of these UB and B fractions the amount of PSA, more particularly the amount of free PSA (fPSA); Y
    (c.3) calculate the percentage of PSA comprising sialic acid α2,3 (abbreviated% α2,3 – PSA), calculating the ratio between the amount of PSA of the UB fraction (UB-PSA) with respect to the sum of the amount of PSA of the fractions UB (UB-PSA) and B (B-PSA) according to formula (Ia)

    % α2,3 – PSA = [(UB-PSA) / ((UB-PSA) + (B-PSA))] x 100 (Ia);
     fifty
    I

    (d) In the solution of step (b) determine the amount of free PSA (fPSA) and the amount of PSA comprising internal fucose; and calculate a parameter called internal fucosylation ratio (abbreviated RFI) of the sample as the ratio between the amount of PSA comprising internal fucose (abbreviated fucose-PSA) with respect to the amount of free PSA in the sample (fPSA), according to the formula (II) 5

    RFI = (fucose-PSA) / fPSA (II);

    where:
     10
    if the% α2,3-PSA is greater than a reference control value, the subject is diagnosed with high-risk prostate cancer; and / or if the RFI in the isolated sample is less than a reference control value, the subject is diagnosed with high-risk prostate cancer, where high-risk prostate cancer is one that corresponds to a Gleason grade score. equal to or greater than 8, or Gleason 7 with metastases. fifteen

    When reference is made to PSA in this description, it refers in particular to human PSA whose active protein sequence (without signal peptide or propeptide) consists of 237 amino acids and is accessible in the UniProtKB database, September 16, 2015 edition, under accession number P07288, whose isoform 1 is version 2 of the sequence dated July 1, 1989. 20

    Steps (a) and (b) of the particular diagnostic method allow the PSA to be purified very efficiently from the fluid sample isolated from the subject, separating it from other proteins that could interfere with the calculation of the proposed parameters. As will be seen in the examples, the combination of the mixture with an ethanolamine solution at the expected final concentrations and the immunoprecipitation of tPSA with an antibody, allows an amount to be recovered from a small volume (750 microliters of fluid sample) an amount of free PSA (fPSA) optimal for subsequent analyzes of different types of glycosylation. That is, it is placed in the compromise solution to recover the maximum of PSA and isolate it from whey proteins that could then interfere in glycosylation analyzes, for example because they are also glycosylated. If the determination of percentages and ratios of the different types of glycosylation mentioned above is added to these purification steps, it is possible to determine values that, when compared with reference values, give rise to high specificity and sensitivity. In addition, the statistical analysis confirms high positive and negative predictive values and also values of the area under the curve (AUC) in an ROC curve (acronym 35 of Receiver Operating Characteristic, or Receiver Operating Characteristic) with values close to 1. This means that the probability of randomly choosing a positive value (sick with aggressive prostate cancer) is greater than the probability of obtaining a negative value (sick with BPH or another benign prostate disease or indolent prostate cancer, the latter including cancers of prostate of low or intermediate risk). In other words, the probability that the diagnosis made to a patient with aggressive PCa is correct is greater than that which would be made to a healthy person or benign disease chosen at random. Sensitivity is the concept that indicates that the method is capable of detecting patients with aggressive PCa despite minimal changes in the levels of the markers regarding a healthy control or with another disease. Specificity is the concept that indicates that we unequivocally classify well among a cohort of patients, those who have aggressive PCa of those who do not.

    As can be seen from the examples, carried out with a cohort of patients with an n of 65, in particular the detection and quantification of the percentage of PSA comprising 50 α-2.3 sialic acid (% α2,3-PSA) allows separate patients with high-risk PCa from patients with BPH or low-risk prostate cancer, with a sensitivity of 85.7% and a specificity of 95.3%, using a cut-off point or reference control value as a PSA percentage
    with sialic acid α2.3 of 30.05%, and reaching a high yield (AUC of 0.97). According to the inventors' knowledge, these values have not yet been achieved. In addition, the percentage of serum PSA bound to sialic acid α2,3 (% α2,3-PSA) correlated positively with the Gleason system score, a milestone not achieved with the markers currently used in the clinic, such as tPSA or% levels fPSA in serum. This correlation between the percentage of 5 serum PSA bound to or comprising α2.3 sialic acid and the Gleason score can improve the classification of the type of CaP. All this means that the use of this biomarker in serum can be routinely monitored in patients with serum PSA values indicative of prostate pathology, to identify patients with high-risk PCa. In the case of a serum marker, it is a non-invasive method, with the advantage of avoiding unnecessary and uncomfortable biopsies for the patient.

    In the present description the PSA containing (or synonymously comprising) in its oligosaccharide chain α2,3 sialic acid can also be referred to as α2,3 sialic acid in the specific prostate antigen (abbreviated as α2,3-PSA). When the description refers to 15 percentage of sialic acid α2,3 in the prostate specific antigen (abbreviated% α2,3-PSA), it should be understood as the percentage of PSA of the isolated sample that contains exclusively α2,3 sialic acid in its oligosaccharide chain and does not contain α2.6 sialic acid.

    In a particular embodiment, when the diagnostic method includes the steps of (a) and (b) of purification, this is characterized in that the molar concentration of ethanolamine in the solution of step (a) is 1 to 1.5 molar ( M), said concentration with respect to the total volume of ethanolamine solution plus fluid sample isolated from the patient.

    When concentration ranges are indicated herein, it should be understood that these include the extreme values of the range, and in particular the range of 0.5 to 2.5 M includes 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 , 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 M.

    In another particular embodiment, optionally in combination with any previous or subsequent embodiment, the method is carried out at a temperature of 20 ° C to 40 ° C. This range includes the 30 temperature values 20 ºC, 25 ºC, 30 ºC, 35 ºC and 40 ºC.

    The method according to the invention is also characterized in that step (a), according to the particular method described above, is carried out for a period of 20 hours to 75 hours, more particularly 40 to 75 hours, and even more in particular from 70 to 75 hours and at a final 35 molar concentration of ethanolamine from 1 to 1.5 molar, more particularly 1 molar.

    In another particular embodiment, optionally in combination with any previous or subsequent embodiment and in the case where steps (a) and (b) of purification of the sample are applicable, after step (a) of mixing the isolated fluid sample With a solution of ethanolamine and subsequent incubation, the pH is adjusted between 7 and 8, particularly 7.8, before proceeding with step (b) of immunoprecipitation.

    In another particular embodiment, the elution of the PSA in step (b), if any, is performed by three successive incubations with a high saline buffer, such as the Gentle Elution Buffer 45 (Pierce ™); and optionally the sample is desalted by filtration, in particular using a filter of the type Amicon Ultra-0.5, 3K of Millipore.

    In another particular embodiment of the method according to the first aspect of the invention, the fluid sample isolated from the subject is selected from the group consisting of whole blood, serum, plasma and urine. In another even more particular embodiment, the isolated sample is serum.

    Precisely, one of the advantages of the method according to the invention is that it is carried out in a sample that is easily obtained in clinical routine, thus avoiding biopsies that are annoying and not without problems for the patient and are more expensive for the healthcare system . This also reduces the number of interventions using diagnostic imaging techniques (MRI) that are also more expensive than the test in blood samples (serum). 5

    In another particular embodiment, step (b) of immunoprecipitation with a specific antibody against the specific prostate antigen is carried out with a monoclonal or polyclonal antibody and selected from the group consisting of a total anti-PSA antibody and a free anti-PSA antibody . More particularly with a total anti-PSA antibody. The expert will understand that in the case of an immunoprecipitation these antibodies are attached to a support (such as beads, beads or beads) that can be captured by different methods, such as by a magnetic field if the support is sensitive to it.

    The method according to the invention comprises any technique for determining the presence and quantity of sialic acid α2,3 bound to the specific prostate antigen (abbreviated here as indicated above α2,3-PSA). In a particular embodiment, the method according to the invention, optionally in combination with any embodiment explained herein, is characterized in that the amount of α2,3-PSA and then the% α2,3-PSA is determined by affinity chromatography with a lectin sialic acid specific α2,6. twenty

    In a still more particular embodiment, the method according to the invention, optionally in combination with any embodiment explained herein, is characterized in that the amount of α2,3-PSA and then the% α2,3-PSA is determined by affinity chromatography in a column with a solid phase containing the lectin of Sambucus nigra (SNA) and proceeding according to the following steps:

    (i) the PSA of the sample, optionally obtained after steps (a) of mixing with ethanolamine and (b) of immunoprecipitation and elution, is incubated with the SNA lectin in a chromatography column for a period of 15 hours to 20 hours , more particularly from 16 to 18 hours, and at a temperature of 2 ° C to 7 ° C, more particularly from 2 ° C to 4 ° C;

    (ii) the column is centrifuged and eluted at least twice for 3-7 minutes, in particular 5 minutes, with a volume of 200-700 µl, in particular 500 µl, of a buffer solution (LB), to obtain the fraction not bound to column (UB) and comprising α2,3-PSA; 35

    (iii) the PSA fraction bound to column (B), comprising α2,6-PSA, is eluted according to the following two sub-steps:
    - incubating the lectin twice with a buffer solution at pH between 7 and 8 comprising lactose at a temperature of 20 to 30 ° C (corresponding to room temperature) to obtain a first fraction of eluate; and
    - incubating the lectin with an acid solution, particularly with 0.1M to 1M acetic acid, more particularly with 0.2M acetic acid, between 2.5 and 3 minutes, in particular 2.7 minutes, where said acid solution contains lactose in LB , and at a temperature of 20 to 30 ° C (rt) to obtain a second fraction of eluate, which is neutralized with a solution of pH = 9;

    (iv) the PSA, either fPSA or tPSA, is determined from each of the stages (ii) and (iii) and the percentage of prostate specific antigen comprising the sialic acid α-2,3 (%) is calculated α-2,3-PSA), as the ratio between the PSA of the UB fraction of step (ii) with respect to the sum of 50 PSA of the UB and B fractions of steps (ii) and (iii), of according to formula (Ia), or (Ib) if it is a question of comparing amounts of fPSA, described above.

    In a particular embodiment, the centrifugation of step (ii) is carried out at a speed between 1500-2000 revolutions per minute (rpm), in particular 2000 rpm, for a time of 2 to 5 minutes (min).

    In another particular embodiment, the buffer solution comprises HEPES (4- (2-hydroxy ethyl) -1-5 piperazinetanesulfonic acid) and a mixture of salts such as sodium and calcium chloride. When this buffer solution contains lactose, it is in particular at a concentration of 0.5 to 0.8 M, in particular 0.5 M. The buffer solution also contains bovine serum albumin (BSA) in a weight percentage of 0.1% to 1.5%. Incubation with this buffer solution is carried out for 20 to 25 minutes, in particular for 20 minutes. 10

    Similarly, the method according to the invention comprises any technique for determining the level of internal fucosylation and with it the proportion of free PSA fucosylated in the sample (internal fucosylation ratio, RFI), which includes determining by any methodology the amount of internal fucosylation of the PSA in the subject's sample and the amount or levels of fPSA in this same sample.

    In a particular embodiment, the method according to the first aspect of the invention is characterized in that the determination of the level of internal fucosylation (or amount of PSA comprising internal fucose) and then of the internal fucosylation ratio, is carried out by means of a Enzyme-linked lectin assay (ELLA) with the biotin-conjugated Pholiota squarrosa lectin (PhoSL), according to the following steps:

    (i) the PSA is incubated, optionally obtained after steps (a) of mixing with ethanolamine and (b) of immunoprecipitation and elution, with a specific antibody against the free prostate specific antigen for a time of 1 to 3 hours, in particular 2 hours;

    (ii) after stage (i) the biotin-conjugated Pholiota squarrosa (PhoSL) lectin is added for a time of 1 to 3 hours, in particular for a time of 2 hours;
     30
    (iii) a solution of streptavidin conjugated to horseradish peroxidase is added after step (ii) for a period of 1 to 1.5 hours, more particularly 1 hour, period after which the substrate of the enzyme conjugated peroxidase is added to a fluorescence emitting molecule and the emitted fluorescence is determined, which is proportional to the amount of free PSA in the sample comprising internal fucose; and 35

    (iv) the fPSA of the fluid sample isolated from the subject, or of the optionally obtained solution obtained after steps (a) of mixing with ethanolamine and (b) of immunoprecipitation and elution; and the internal fucosylation ratio is calculated, as the ratio between the amount of PSA comprising internal fucose (or what is the same, internal fucose level of the PSA of the sample) determined in (iii) with respect to the amount of PSA free of the sample or in particular with respect to the solution optionally obtained after steps (a) of mixing with ethanolamine and (b) of immunoprecipitation and elution, all in accordance with the formula (II) cited above.

    To transfer the fluorescence values emitted in step (iii) with the internal fucosylation of free PSA, a standard curve made from standard PSA with 77% of PSA molecules with internal fucosylation is used in a particular embodiment. Then, the internal fucosylation ratio of the sample refers to the amount of PSA with internal fucose compared to the standard PSA, which contains 77% of PSA molecules with internal fucosylation. Rigorously, the internal fucosylation (RFI) ratio of the sample corresponds to the ratio between the internal fucose level of the PSA in the sample, calculated in relation to the internal fucose level of the standard PSA, relative to the amount of free PSA of the sample, in particular the PSA free of the solution obtained after steps (a) of mixing with ethanolamine and (b) of immunoprecipitation and
    elution This ratio of internal fucosylation is also referred to in this description as the proportion of free PSA fucosylated.

    In an even more particular embodiment, steps (iv) of any of the methods for determining% α2,3-PSA and internal fucosylation ratio (RFI) are carried out with a fPSA specific antibody, which can be monoclonal or polyclonal and selected from commercially available ones, such as the reference monoclonal antibody M-30 from Roche Diagnostics.

    In another particular embodiment of the method according to the invention, this is characterized in that the reference control value of the internal fucosylation ratio is 0.86; and the reference control value of the percentage of PSA with sialic acid α-2,3 is 30%. This means that a value of the internal fucosylation ratio, determined in the isolated sample of the subject, less than 0.86 is indicative of aggressive or high irrigation CaP according to the Gleason score. On the other hand, if the PSA value (measured as free or total PSA) eluted in the UB fraction of the SNA chromatography (or of the corresponding lectin) is equal to or greater than 30% of all eluted PSA (UB + B) means that the PSA in the sample has a content equal to or greater than 30% α2.3 sialic acid bound to PSA and is therefore indicative of aggressive prostate cancer.

    "Reference control value" means a value with which the proportion of fucosylated PSA 20 is compared (which is the same as internal fucosylation ratio) and / or the% α2,3-PSA determined in the isolated sample according to the invention for the diagnosis of high-risk PCa. It is a value of each one of these parameters that allows to distinguish with a high degree of probability a subject with high-risk PCa from the rest of individuals who do not suffer from this cancer or who suffer from low-risk PCa, and even individuals suffering from diseases benign of the prostate. The reference value is a value, also called the cut-off point or threshold value, which allows us to rule out with a high sensitivity that the diagnosed patient suffers high-risk PCa. This threshold value represents an internal fucosylation ratio or a percentage of α2,3-PSA below or above which, depending on each case, the subject's sample is associated with the disease (high-risk or aggressive CaP) or not. 30

    As indicated above, the method according to the invention makes it possible to differentially diagnose aggressive or high-risk prostate cancer, intermediate and low-risk prostate cancer, and benign prostate diseases. In a particular embodiment, benign prostate diseases are selected from the group consisting of benign prostatic hyperplasia, prostatitis, and manipulations of the prostate. Prostate manipulations should be understood as all those situations that involve an increase in serum PSA and selected from the practice of cycling, catheterization and digital rectal exams (DRE).
     40
    The parameters indicated above (% α-2,3-PSA and the internal fucosylation ratio) give significant information not only on the diagnosis of prostate cancer, but also on the prognosis of it, correlating with the established aggressive or non-aggressive CaP classification of according to the Gleason system score, where a Gleason grade score equal to or greater than 8 means high-risk cancer. These patients after undergoing only 45 radical prostatectomy have a probability of relapse-free survival at 50 years of between 50 and 80% (Chang et al., "High-risk prostate cancer-Classification and therapy", Nat .Clin Rev. Onco -2014, vol. No. 11, pp .: 308-323) The parameters also correlate with the Tumor Node Metastasis (TNM) staging, another method of classifying the aggressiveness of this type of cancer. fifty

    The Gleason gradation system for prostate cancer prognosis is described in Humphrey, et al. “Gleason grading and prognostic factors in carcinoma of the prostate”, Modern
    Pathology -2004, vol. no. 17, pp .: 292-306. Gleason's gradation system is based on the histological pattern of carcinoma: its glandular pattern and its degree of differentiation. The Gleason score values between 2-4 correspond to a highly differentiated pattern (anaplasia); between 5 and 6 moderately differentiated and between 7 and 10 poorly differentiated. While values of 6 and lower have been associated with a good prognosis or 5 indolent or low-risk cancers, values above 8 are indicative of aggressive or high-risk cancer. The value of 7 is in an intermediate stage of aggressiveness between 5-6 and 8-10.

    The invention relates, in a second aspect, to a method for deciding or recommending a medical regimen for prostate cancer, said medical regimen selected from pharmacological treatment and surgical intervention, which comprises carrying out the diagnostic method according to any of the embodiments described above; and where, if the subject is diagnosed with aggressive or high-risk prostate cancer the recommended medical regimen for this type of prostate cancer is recommended or decided, and if the subject is not diagnosed with aggressive prostate cancer but is diagnosed with a Benign prostate disease or cancer of 15 indolent prostate is recommended or decides the medical regimen for this other disease.

    The invention also relates, according to another aspect, to the use of:
    (a) the internal fucosylation ratio, which results from determining in a fluid sample isolated from a subject the ratio between the amount of PSA comprising internal fucose and the amount of free PSA 20 in the sample; I
    (b) the percentage of prostate specific antigen comprising α2.3 sialic acid, and that results from calculating the PSA ratio of a fraction not bound to lectin (in particular to a specific lectin of α2.6 sialic acid, more in particular SNA lectin) with respect to the sum of the PSA fractions bound and not bound to said lectin, as independent biomarkers of high-risk prostate cancer, where high-risk prostate cancer is one that corresponds to a score Gleason grade equal to or greater than 8, or grade 7 with metastasis.

    According to the information available to the inventors, this is the first time that the relationship of these parameters, RFI and% α2,3-PSA calculated as described above, with high-risk prostate cancer has been described. . In a particular embodiment, for the determination of these parameters the isolated fluid sample of the subject where the amount of PSA comprising internal fucose and / or the amount of PSA of the fractions bound and not bound to lectin is determined, is a sample that previously it has been mixed with a solution of ethanolamine at a final molar concentration of 0.5 M to 2.5 M, in particular 1 M, and then immunoprecipitated with a specific antibody against the specific prostate antigen.

    In a particular embodiment, the use of these biomarkers is for the differential diagnosis of high-risk prostate cancer, of low intermediate risk prostate cancer; and / or 40 benign diseases of the prostate. The determination of these biomarkers is carried out, in a particular embodiment in a fluid sample isolated from the subject that is selected from the group consisting of whole blood, serum, plasma and urine. More particularly, it is carried out in serum.
     Four. Five
    In practice, the method according to the invention is carried out in kits comprising means for determining in an isolated fluid sample of a subject the amount of PSA comprising α-2,3 sialic acid and / or the amount of PSA comprising internal fucosylation. These kits are characterized because they include:
    (a) a specific antibody against the free specific prostate antigen; and 50
    (b) a solid phase for affinity chromatographic column comprising a specific lectin against sialic acid and, optionally, solutions for elution of the sample loaded into said
    column as means for detecting and quantifying the amount of prostate specific antigen comprising sialic acid α-2,3; I
    (c) a specific lectin against fucose, said lectin conjugated with biotin; optionally a free specific prostate antigen specific antibody that is immobilized in a well plate; and optionally, a solution of streptavidin conjugated to horseradish peroxidase and a substrate of the enzyme peroxidase conjugated to a fluorescent emitting molecule, all as a means to detect and quantify the specific prostate antigen comprising internal fucosylation.

    In a more particular embodiment, the kit comprises a specific lectin against fucose, said biotin-conjugated lectin; and the free specific prostate antigen specific antibody that is immobilized in a well plate, and optionally, a solution of streptavidin conjugated to horseradish peroxidase and a substrate of the enzyme peroxidase conjugated to a fluorescence emitting molecule. This particular embodiment of the kit is advantageous for the user, who already has the immobilized free prostate specific antigen specific antibody. Thus, the method for carrying out the analysis of the amount of PSA comprising internal fucosylation is simplified, since the antibody also has not been previously immobilized in the kit.

    In a particular embodiment of these kits, the sialic acid specific lectin is the 20 Sambucus nigra agglutinin (SNA) and the internal fucose specific lectin is the Pholiota squarrosa lectin (PhoSL).

    In another even more particular embodiment, optionally in combination with any previous or subsequent embodiment described herein, the solid phase comprising Sambucus 25 nigra lectin consists of 4% cross-linked agarose beads with a molecular exclusion size limit of 2x107 daltons ( Gives). At this solid phase the lectin covalently binds.

    In another particular embodiment the kits are characterized in that they further comprise:
    (a) a solution of ethanolamine to be mixed with the subject's sample and obtain a final concentration of ethanolamine from 0.5 to 2.5 M; Y
    (b) a specific prostate antigen specific antibody bound to a support for immunoprecipitation.

    In the present invention, "support for immunoprecipitation" should be understood as any solid material, generally beads (beads) where specific antibodies or fragments thereof are covalently bound against proteins that want to be isolated from a complex mixture. These supports are then separated from the complex mixture either by sedimentation means or by application of external forces (magnetic or electric fields) to which said supports are sensitive. 40

    These kits according to the invention are used for the diagnosis of prostate cancer, and in particular for the diagnosis of high-risk prostate cancer, and / or for the differential diagnosis of high-risk prostate cancer and benign diseases of the prostate.
     Four. Five
    In fact, the invention also relates to a method for purifying PSA in the fluid sample isolated from a subject, specifically in a sample volume of 0.25 to 1.5 mL, in particular from 0.75 to 1.5 mL, wherein said purified PSA is also in conditions to be analyzed to detect and quantify its glycosylation at the level of sialic acid and internal fucosylation.
     fifty
    This purification method comprises the steps of:
    (a) mixing the isolated fluid sample with a solution of ethanolamine at a molar concentration of 0.5 to 2.5 M in the final mixture, in particular of 1 M;
    (b) immunoprecipitate the solution obtained in step (a) with a prostate specific antigen specific antibody and elute the immunoprecipitated PSA to recover the PSA from the sample.

    In a particular embodiment, this elution of the PSA is performed by three successive incubations with a high saline buffer, specifically Gentle Elution Buffer (Pierce ™). 5 After elution of the PSA the sample is desalted by filtration, in particular using Amicon Ultra-0.5, 3K Millipore.

    In a particular embodiment of the purification method, the specific antibody against the prostate specific antigen of step (b) is selected from the group consisting of a total anti-PSA antibody and a free anti-PSA antibody. More particularly with a total anti-PSA antibody. This antibody, in turn, can be monoclonal or polyclonal.

    Throughout the description and the claims the word "comprises" and "contains" should be understood as synonymous and their variants are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "comprises" or "contains" includes the case "consists of". For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations 20 of particular and preferred embodiments indicated herein.

    BRIEF DESCRIPTION OF THE DRAWINGS

    FIG. 1 (A) Representation of the PSA's internal fucosylation relationship with respect to serum levels of total PSA (tPSA) (panel A) or depending on the pathology (panel B). Serum samples from patients with benign prostatic hyperplasia (BPH) are indicated with white circles and serum samples from high-risk CaP (CaP HR) with black circles. The dotted line indicates the cut-off value to discriminate the two pathologies. Significance was set at p <0.05. In panel (C) the ROC curves for the internal fucosylation ratio (RFI) are compared with a solid line, for the tPSA with a dotted line; and for the fPSA% with a discontinuous line in segments. The diagonal line is the reference line of the ROCs. The internal fucosylation ratio gave the highest value AUC (AUC = 0.94). The cut-off value of 0.86 gave the highest sensitivity (90%) and specificity (95%).
     35
    FIG. 2 (A) Representation of the percentage of PSA comprising α2,3-sialic acid (% α2,3-SA) with respect to serum PSA concentration (tPSA) (panel A) or with respect to pathology (panel B). BPH serum samples are indicated with white circles, low risk CaP (CaP LR) with a cross, intermediate risk CaP (CaP IR) with a black triangle and high risk CaP (CaP HR) with a black circle The dotted line indicates the cut-off value to discriminate the high-risk CaP 40 from the other three pathologies. Significance was set at p <0.05. In panel (C) the ROC curves for% α2,3-SA, tPSA and% fPSA are compared. The% α2,3-SA gave the highest value of AUC (AUC = 0.97) and the cut-off value 30.05 showed the highest sensitivity (85.7%) and specificity (95.3%).
     Four. Five
    FIG. 3 is a graph showing the correlation between the percentage of PSA comprising α2.3 sialic acid in PSA (% α2,3-SA) of serum samples from patients with PCa with their Gleason score (PG). Low-risk CaP serum samples are indicated by a cross, intermediate risk CaP with a black triangle and high-risk CaP with a black circle. The mean of% α2,3-SA for each Gleason score is indicated with a horizontal line. fifty

    FIG. 4 shows the chromatograms (HPLC) of the N-glycan profiles after digestion with sialidases (NAN1, specific sialic acid α2,3; and ABS, which digests all
    sialic acids α2,6 and α2,3) for the unbound UB and bound B fractions. The profile of the chromatograms from samples not digested with these sialidases is also shown. The profiles are standardized with respect to a dextran hydrolyzate (glucose units; GU). The structures of the N-glycans are abbreviated according to: all N-glycans have two N-acetylglucosamines (GlcNac) and three hands in the nucleus; F at the beginning of the abbreviation means a nucleus with fucose (internal fucose); A represents in number of antennas; G represents galactose linked by β1-4 bond in the antenna; S represents sialic acid bound to galactose. In the peaks with more than one N-glycan structure, the main one is highlighted. The legend corresponds to, UB PSA of SNA column is the profile of N-glycans in the unbound fraction leaving the SNA lectin column; UB PSA of SNA + NAN1 column is the profile of N-glycans in the unbound fraction leaving the SNA lectin column subsequently digested with the NAN1 enzyme; UB PSA of SNA + ABS column is the profile of N-glycans in the unbound fraction that exits the SNA lectin column subsequently digested with the ABS enzyme; B PSA of SNA column is the profile of N-glycans in the bound fraction leaving the SNA lectin column; B PSA of SNA + NAN1 column is the profile of N-glycans in the bound fraction leaving the SNA lectin column subsequently digested with the NAN1 enzyme; and B PSA of SNA + ABS column is the profile of N-glycans in the bound fraction that leaves the SNA lectin column subsequently digested with the ABS enzyme.

    EXAMPLES 20

    1. Clinical characteristics of the study population

    The serum samples analyzed (73 in total) came from 44 patients with PCa, including 22 high-risk, 11 medium-risk and 11 low-risk CaP samples, and 29 samples of benign prostatic hyperplasia (BPH). The 44 patients with PCa were classified as described in Table 1 according to the TNM * system of the International Union Against Cancer (UICC). Patients with low-risk CaP 11 had Gleason scores of 6 and tPSA levels below 10 ng / ml. Of the 11 patients with intermediate risk PCa, 8 had a Gleason score of 7, 1 presented a focal Gleason score of 8 and 2 patients with 30 intermediate risk PCa were Gleason 6, one with PSA levels above 10 ng / mL and the other presented extracapsular extension (T3a in the TNM * classification). Of the 22 patients with high-risk prostate cancer, 17 were patients with Gleason scores equal to or greater than 8, three had a Gleason score of 7 with metastases. The other two high-risk metastases, although data on the Gleason score of the primary tumor were not available. The age and average and range of tPSA and fPSA levels of the different groups (BPH, high risk PCa, intermediate risk PCa and low risk PCA) are shown in Table 1.

    (*) The TNM system is a widely used system for tumor staging and has been accepted by the "Union for International Cancer Control (UICC)", and by the "American Joint Committee on Cancer, AJCC".





































    2. Sample preparation

    To release the PSA complexed with α1-anti-chymotrypsin (ACT), serum samples (750 µl) were first treated with ethanolamine (200 µl 5M, Sigma-Aldrich, Germany), which gave a concentration of 1M ethanolamine to the mixture of serum plus ethanolamine. The pH was subsequently adjusted to 10.3 with 5M hydrochloric acid and the mixtures were incubated 72 hours at 25 ° C. The samples were then neutralized to pH 7.8 with 5M hydrochloric acid.

    In order to obtain maximum performance without compromising protein integrity, different concentrations of ethanolamine and different incubation times were previously evaluated in a serum of a patient with BPH (PSA was determined therein). The percentage cleavage of the PSA-ACT complex was dependent on the concentration of ethanolamine. Increasing the molar concentration of ethanolamine from 0.1 to 2.5 increased the percentage of fPSA from 32.9% to 57.7%, respectively. However, the results showed that the higher the concentration of ethanolamine, the lower the recovery of tPSA (see Table 2). Therefore, in order to preserve the integrity of the PSA to be analyzed, different incubation times and temperatures were tested with 1 M ethanolamine.

    Table 2
     Ethanolamine concentration (M)  Incubation time (h) Temperature ºC% fPSA after treatment with ethanolamine% tPSA after treatment with ethanolamine
     0.1 0.5 1 2 2.5  24 25 32.88 48.10 51.63 54.93 57.74 87.5 89.2 85.5 77.3 73.1
     one  24 48 72 24 48 72 25 37 50.87 60.06 60.00 64.78 71.32 67.10 77.0 78.7 81.1 66.8 61.9 53.6
     twenty
    The best conditions to release PSA were 1 M ethanolamine for 72 hours at 25 ° C. Using this protocol, the percentage of fPSA increased from 19% (untreated serum) to 60% (treated ethanolamine serum). The fPSA values from the 73 serum samples of the study after ethanolamine treatment ranged from 50-80% of the tPSA values.
     25
    3. Immunopurification of tPSA samples

    After PSA release from the ACT, serum tPSA was isolated using the PSA Access Hybritech test kit (Beckman Coulter, Brea, CA, USA). In summary, a suspension of 100 µl of paramagnetic particles (beads) coated with anti-30 PSA mouse monoclonal antibody (PSA Access Hybritech assay, Beckman Coulter, Brea, CA, USA) was washed with 500 µl of buffer incubation (50 mM Tris, 150 mM NaCl, pH 7.4, 0.1% Tween-20, 1% BSA) using magnetic separation. The beads were then incubated with 1 ml of treated and neutralized serum (containing 750 µl of original serum) for 1 hour at room temperature (RT) with stirring. Then, the beads were washed with 500 µl of wash buffer (35 mM Tris, 150 mM NaCl, pH 7.4, 1% Triton X-100) using magnetic separation and the immunoadsorbed PSA eluted in three steps using 100 µl Gentle Ag / Ab elution buffer, pH
    6.6 (Thermo Scientific, Rockford, IL, USA) for 30 minutes each. The final elution volume was 300 µl. Samples were desalted and concentrated to a final volume of 40 µl using an Amicon Ultra-0.5 3K Centrifugal Filter Devices (Millipore, Cork, IRL) filter device. The Amicon filters had previously been pre-treated with 5% Brij-35 (Sigma-Aldrich, Germany) and were used for desalting the sample according to the manufacturer's instructions.

    4. Determination of the internal fucosylation ratio (RFI)

    The immunoprecipitated samples as discussed in the previous section were subjected to a second immunopurification stage by means of an enzyme-linked lectin assay (ELLA; from Enzyme linked lectin assay).

    To do this, M-30 mouse monoclonal antibodies against fPSA from Roche Diagnostics were diluted to 8 µg / ml in phosphate buffered saline (PBS) and bound to black Maxisorp 15 microplates (FluoroNunc / LumiNunc Maxisorp Surface) overnight at room temperature (rt; which means from 20 to 30 ° C). The plates were washed 3 times with T-saline (0.9% NaCl solution, 0.05% v / v Tween-20) and incubated for 1 hour at room temperature with blocking solution (2% w / v PVP in PBS). After washing with T-saline solution, samples obtained after immunoprecipitation with anti-tPSA antibody (total PSA IP) were added in a final volume of 100 µl in PBS - 0.1% Tween-20 (PBST) and They were allowed to incubate for 2 h at room temperature. After washing the plates with saline, the biotinylated PhoSL lectin diluted to 1 µg / ml in lectin solution (150 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2) was added and allowed to incubate for 2 It has room temperature. The plates were then washed with T-saline and streptavidin conjugated to horseradish peroxidase 25 (GE Healthcare-, United Kingdom) [diluted 1: 5000 in PBS] and allowed to incubate for 1 h at room temperature. The plates were washed with PBS and detection was performed with 100 µl / well of the QuantaRed Enhanced Chemifluorescent HRP Substrate radish peroxidase substrate (Thermo Scientific, USA). The reaction was stopped with 10 µl / well of QuantaRed Stop Solution (Thermo Scientific, USA) and the fluorescence was read at 585 nm after excitation at 530 nm in an automated microplate reader (BIO-TEK , USA). Wells containing only PBST buffer were used as negative controls (white) and wells with female sera (without endogenous PSA but processed in parallel with the samples) were used as controls to normalize the background signal.
     35
    This assay was used to analyze the PSA content containing internal fucose (internal fucosylation levels) of a serum cohort consisting of 20 samples of BPH (tPSA from 7.82 to 18.20 ng / ml) and 20 cancer samples of High-risk prostate (CaP) (12 with a tPSA from 8.72 to 19.53 ng / ml and 8 with a tPSA of more than 20 ng / ml). For this, the sera were treated as indicated above with ethanolamine, and immunoadsorbed (immunoprecipitated). 40 They were then analyzed with the ELLA assay described above. For each sample, two duplicate dilutions were made that could be interpolated in a calibration curve performed with commercial standard PSA. The calibration curve ranged from 0.8 to 13 ng of standard PSA per well.
     Four. Five
    The standard PSA corresponds to a PSA with a characterized glycosylation pattern, which is added to a woman's serum pool (therefore, without endogenous PSA). The standard PSA that is N-glycosylated contains 77% of its N-glycans with internal fucosylation.

    To perform the corresponding replications, 1.5 ml of serum from each sample with 50 tPSA values above 8 ng / mL were required. Only samples with a coefficient of variation (CV) below 15% were used. The inter-assay CV of ELLA was determined with a sample of
    High-risk CaP of 12.4 ng / ml of tPSA in five independent trials and was found to be below 10%.

    For each sample, the amount of PSA containing internal fucose (obtained from the ELLA test) was divided by the fPSA value of the same immunoprecipitated sample, using the ELECSYS® platform, and the resulting ratio was called internal fucosylation ratio. (RFI). The ELECSYS® platform on the Modular Analytics E170 (Roche Diagnostics) device contains the biotinylated anti-free PSA monoclonal antibody (anti-fPSA) called M-30 to measure fPSA and the biotinylated monoclonal antibody called M-36 anti total PSA (anti-PSA). tPSA).
     10
    The internal fucosylation ratio of the samples was plotted against the concentration of serum tPSA in ng / mL (FIG. 1 (A)) and against the group by pathologies (BPH and high-risk PCa) (FIG. 1 (B )). As shown in FIG. 1, the BPH group showed a significantly higher internal fucosylation ratio (0.72 to 1.56) than patients with high-risk PCa (0.53-1.00) (significance established at p <0.05). An ROC curve of the ratio of internal fucosylation was performed and compared with the ROC curve of tPSA and the percentage of fPSA. The ROC trial conducted with the 40 serum samples from patients showed that patients with high-risk PCa were discriminated against those with BPH with a 90% sensitivity and with a specificity of 95%, being the cut-off point of the parameter called internal fucosylation ratio (RFI) = 0.86 with an area under the curve (AUC) of 0.94 (Figure 1C). twenty

    The low amounts of serum PSA (2-20 ng / ml), which represent only between 0.0000025 and 0.000025% by weight of the total whey protein, and the fact that other whey proteins are glycoproteins that include fucose in the nucleus of its N-glycans, makes developing an assay to specifically detect fucosylated PSA represent a major challenge. Generally, the detection of this monosaccharide is carried out by lectins. The method must include a step of immunopurification of PSA to eliminate most of the glycosylated serum proteins, in particular those that may contain fucose in oligosaccharide chains, such as immunoglobulins, in order for the signal generated by the fucose to be Recognized by lectin can be attributed to the PSA molecule and not to others. 30

    Therefore, in order to fine-tune the method of the invention, it was first checked whether other proteins other than PSA containing fucose in the nucleus of their N-glycans were recovered in this immunopurification process. For this purpose, the standard PSA was used with a glycosylation pattern characterized by enriching a serum pool of women (therefore, without endogenous PSA) (control serum). The standard PSA that is N-glycosylated contains 77% of its N-glycans with internal fucosylation. This standard PSA was added in control sera and was immunopurified using antibodies against tPSA (anti-tPSA) covalently bound to magnetic beads (total PSA IP) and also using biotinylated monoclonal antibody against fPSA M-30 (free PSA IP), the latter bound to streptavidin coated beads. 40

    The immunoadsorbed PSA in the two previous immunoprecipitations (with anti-tPSA or anti-fPSA) was eluted with a high salt strength buffer, desalted and concentrated using centricon type filters (Merk-Millipore). Quantification of proteins and immunoadsorbed / immunoprecipitated PSA indicated that 99% of whey proteins had been eliminated and that PSA was recovered in percentages of about 40% by weight. The PSA of these two immunoprecipitations was evaluated in Western blot and with lectins that recognize the fucose to visualize yes with the immunoprecipitations there were other proteins that had been recovered and that presented internal fucosylation. The data is not shown but, bands of higher molecular weight proteins than PSA appeared. fifty

    That is why, in order to eliminate them, it was decided to carry out a double immunoprecipitation. Thus, after immunoprecipitating the tPSA, he underwent a
    immunoprecipitation with anti-fPSA antibody. It was found in a Western blot that a band of PSA was detected with the PhoSL and only other glycoproteins with internal fucosylation were detected. That is why the PhoSL lectin was chosen to more specifically determine the amount of PSA containing internal fucose (also referred to herein as fucosylation levels or fucose levels). 5

    5. Determination of the percentage of serum PSA containing α2,3 sialic acid.

    Lectin columns (one per isolated sample to be analyzed) were prepared by loading 250 µl of agarose containing elder bark lectin (SNA; Sambucus nigra agglutinin) (Vector 10 Laboratories, INC.) In spin-X centrifuge tubes (0.22 microns cellulose acetate) from Corning, Costar (NY, USA). The agarose was in the form of 4% cross-linked beads with a molecular exclusion size limit of 2x107 daltons (Da). The lectin columns were vigorously washed with 4 ml of lectin buffer (LB) [10 mM Hepes (pH 7.6), 150 mM NaCl, 1 mM CaCl2]. The PSA of the serum samples (0.75 ml per sample) was purified using the immunoprecipitation protocol (section 3) described above. The samples were then diluted in LB to a final volume of 405 µl and incubated with SNA lectin for 16 hours at 4 ° C. The columns were centrifuged 2 minutes at 2,000 rpm at 4 ° C and washed twice (5 min each) with 500 µl of LB containing 1% bovine serum albumin (BSA) (UB; it is the unbound fraction and it contains PSA with only α2.3 sialic acid in the oligosaccharide chain as described below). Then, the bound fraction was eluted first by incubating the lectin twice with 500 µl of a 0.5 M lactose solution in 1% BSA in LB, for 20 min at room temperature (fraction B1). A second elution (B2) was performed with 0.5 M lactose in 1% BSA in LB, and 0.2 M acetic acid. The bound fractions (B, containing PSA with α2.6 sialic acid) were collected by centrifugation at 2,000 rpm for 2 minutes. In the acid elution, before centrifugation, 150 µl of 1M Tris pH 9.0 was added to the collecting tube in order to neutralize the sample. Total PSA (tPSA) and free PSA (fPSA) loaded in the column and eluted in each fraction were quantified by the ELECSYS trial.

    The percentage of PSA comprising α2.3 sialic acid (% α2.3 – PSA) in each isolated serum sample 30 was calculated as the ratio between the amount of fPSA of the unbound fraction (UB-fPSA) in the lectin relative to of the sum of the amounts of fPSA of the unbound (UB-fPSA) and bound (B-fPSA) fractions, according to the following formula (Ib):

    % α2,3 – PSA = [(UB-fPSA) / ((UB-fPSA) + (B-fPSA))] x 100 (Ib) 35

    With this methodology, the percentage of PSA comprising α2.3 sialic acid in serum samples from a cohort of subjects consisting of 22 samples of BPH (tPSA from 5.53 to 14.71 ng / mL), 11 prostate cancer samples was analyzed (CaP) of low risk (tPSA from 5.10 to 9.75 ng / mL), 11 samples of prostate cancer (CaP) of intermediate or moderate risk (tPSA from 40 5.44 to 17.63 ng / mL) and 21 cancer samples of High-risk prostate (CaP) (13 with a tPSA from 4.14 to 19.53 ng / mL and 8 with a tPSA over 20 ng / mL). The quantification of fPSA of the unbound and bound fractions of each sample once subjected to chromatography allowed to calculate the relative percentages of PSA in the unbound fraction (sialylated PSA-α2.3) and in the bound fractions (PSA α2.6 -sialylated). The inter-assay variation of the method was determined using a CaP serum (tPSA of 31.89 ng / mL) in 10 independent experiments, and was found to be below 5%.

    In FIG. 2 (A) shows the percentage of unbound PSA of the samples, which corresponds to the percentage of PSA comprising α2,3 sialic acid (or α2,3-sialylated PSA), based on the total PSA concentration (tPSA) in the serum of each sample. In FIG. 2 (B) this same parameter is shown for each of the four groups or types of samples / patients tested.

    From this FIG. 2 (A) and (B) it follows that there is a significant increase in PSA comprising α2.3 sialic acid in the group of patients with high-risk PCa (HRP 26.5 to 61.4%) compared to the other three groups: Intermediate risk PC (IR CaP; 16.9-35.5%); Low risk CaP (CaP LR; 12.5 to 29.9%); BPH (13.4-33.5%), with a significance of p <0.05). No significant differences were found between the percentage of α2,3-sialylated PSA in BPH and the 5 patients with low and moderate risk PCa.

    In addition, ROC curves of the percentage of PSA containing α2.3 sialic acid (% α2.3 – PSA) were plotted and compared with the ROC curves of tPSA and the percentage of fPSA (FIG. 2 (C)) . The ROC trial for% α2,3 – PSA performed with the 65 serum samples from 10 patients showed that patients with high-risk PCa discriminated very well from patients with BPH and from patients with intermediate-risk prostate cancers or low. The sensitivity was 85.7% and the specificity was 95.3% if the% α2,3 – PSA of the samples was analyzed for a reference or cut-off control value of 30%. The area value under the curve (AUC = 0.968) was calculated as an indication of the accuracy of the prediction of parameter 15% α2,3 – PSA.

    In addition, the% α2,3 – PSA correlated significantly (p <0.01) with the tumor Gleason score (correlation coefficient of 0.54) (FIG. 3). With this, this relationship entails the advantage that can help doctors improve the prognostic evaluation by using a parameter measured in serum, thus reducing the number of prostate biopsies.

    The methodology for determining α2.3 sialic acid levels in PSA (or PSA comprising α2.3 sialic acid) was therefore based on a solid phase chromatography comprising lectin, in particular using agarose columns with SNA . LaSNA binds to glycoconjugates comprising α2.6 sialic acid.

    As stated before, the UB fraction comprised PSA with α2.3 sialic acid because previously the inventors carried out a specificity test of the chromatography column (or what is the same as the solid phase with lectin). The specificity of the SNA chromatography was validated using first the standard PSA dissolved in female serum. First, the SNA-agarose / serum ratio was established to avoid saturation of the lectin column, which was 10 µl of serum in 500 µl of agarose-SNA. To increase the amount of serum and to avoid column saturation due to other sialylated serum proteins, the serum sample containing standard PSA was first immunoadsorbed using 35 monoclonal antibodies against total PSA (anti-tPSA) covalently bound to magnetic beads such as described above, which allowed loading 0.75 ml of initial serum. After optimizing the protocol, the selectivity of the SNA lectin column for α2.6 sialic acid was evaluated using this standard PSA treated. The standard PSA was chromatographed and the retained fraction was eluted using 0.5 M lactose. A second fraction was eluted with 0.5 M 40 lactose in 0.2 M acetic acid in 0.001% BSA. The quantification of the fPSA of the UB fraction and of the two joined fractions Bs was performed using the ELECSYS platform (detailed above).

    Next, the relative percentages of fPSA in the unbound fraction (PSA α2,3-sialylated) and in the bound fractions (PSA-α2,6 sialylated) were calculated. Therefore, it could be determined that 45% of the standard PSA contained N-glycans-α2,3 sialylated according to previous published data (Tabares et al, supra). The PSA of the UB and B fractions was isolated using polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue. Densitometry analysis of the gel bands revealed 25.7% PSA in the unbound fraction (UB) vs 75.4% in bound fractions (B1 + B2) that was consistent with the percentage of sialic acid α2.3 / α2.6 50 sialic acid obtained in previous analyzes (Tabarés et al., supra).

    The cleavage of the protein band was performed and the release of the N-glycans from the PSA was carried out with the enzyme PNGase. In summary, the gel fractions were washed and treated with PNGase F to release the N-glycans from the PSA. These N-glycans were fluorescently labeled with 2-aminobenzamide (2AB) by reductive amination using the Ludger Tag 2AB kit. The excess of 2AB was removed by ascending chromatography on Whatman 3MM paper in acetonitrile. The 2AB-labeled glycans 5 were digested in 10 µl of 50 mM sodium acetate buffer, pH 5.5 for 18 hours at 37 ° C with the Arthrobacter ureafaciens sialidase (ABS) (EC 3.2.1.18), 0.5 U / ml or the sialidase of Streptococcus pneumonia (NAN1) (EC 3.2.1.23) specific for sialic acid α2.3, 1.7 U / ml. The enzymes were then separated (filtration by Millipore EZ filters) and the N-glycans were analyzed by hydrophilic interaction liquid chromatography (HILIC). These N-glycans derivatized with 2AB were separated by ultra-liquid liquid chromatography and with fluorescence detection (Waters Acquiti UPLC device). The column for separation consisted of a 2.1 x 150 mm BEH glycan column, 1.7 µm of BEH particles (Solvent A: 50 mM formic acid adjusted to pH 4.4 with ammonia solution; Solvent B: acetonitrile; Tª column 30 ° C; The chromatographic method was n 30 min with a linear gradient of 70-53% acenonitrile at 0.56 15 mL / min. The injection volume was 20 µl of sample in 60% v / v acetonitrile; Fluorescence detection: 330 nm excitation and emission at 420 nm). Retention times were transferred to glucose units (GU) with the help of a standard made with hydrolyzed dextran.

    The sequencing of the N-glycans released from PSA showed that the unbound fraction (UB) 20 contained mainly monosialylated glycans and a few disialylated glycans that were digested to the corresponding neutral structures with NAN1, specific treatment with α2,3-sialidase. Therefore, the free fraction contained only PSA with α2.3 sialic acid that was not retained in the SNA column. Both bound fractions (B = B1 + B2) contained N-glycans with α2.6 sialic acid. FIG. 4 shows the retention profile of the different UB and B samples of 25 standard PSA treated with PNGase and with the N-glycates digested by NAN1 or ABS. The profiles from the UB fraction show how initially the different types of oligosaccharides (N-glycans) comprised sialic acid α2,3, which disappeared once digested with NAN1 and also with ABS (which digests any form of sialic acid α2,6 or α2,3). On the other hand, the retention profiles of the bound fractions (Bs) show that they mostly comprised 30 PSA with α2.6 sialic acid.

    In all the tests carried out and described in this description, the statistical analysis was carried out as described below:
     35
    The patients were divided into four groups; BPH, low risk CaP, intermediate risk CaP and high risk CaP. All statistical analyzes were performed on patients within the range of tPSA between 4.14 and 109.7 ng / mL.

    The means of comparison and the correlation analysis of the variables were performed using the IBM SPSS Statistics 19 program for Windows. Data with a coefficient of variation between CV trials> 15% were not included in the statistical analyzes. The variables were analyzed for normality and homocedasticity using the Kolmogorov-Smirnov and Levene tests. When these criteria were met (study of sialic acid α2.3% (SA)), the differences between the groups were analyzed by ANOVA with the post-hoc 45 Bonferroni test. On the contrary, for the non-normally distributed groups (fucosylation study) the Mann-Whitney U test was performed. ROC curves were generated with the SPSS software. For the α2,3-SA content, the upper criterion value corresponding to the highest of the average sensitivity and specificity was calculated with the statistical software R (version 3.1.2). Bivariate regression (Pearson's correlation) was performed to assess the diagnostic value of α2,3-SA content. In 50 all statistical analyzes, the significant level was set at 5%. The results are shown as the means of the values (95% confidence interval, CI).

    REFERENCES CITED

    - Tabarés et al. "Different glycan structures in prostate-specific antigen from prostate cancer will be in relation to seminal plasma PSA", Glycobiology - 2006, vol. No. 16 (2), pp .: 132-145.
    - Sarrats et al., "Differential percentage of serum prostate-specific antigen subforms 5 suggests a new way to improve prostate cancer diagnosis." Prostate - 2010 vol. No. 70 (1), pp.:1-9.
    - Meany et al., "Glycoproteomics for Prostate Cancer Detection: Changes in Serum PSA Glycosylation Patterns", J Proteome Res - 2009, vol. No. 8 (2), pp .: 613-619.
    - Li et al., "Serum Fucosylated Prostate-specific Antigen (PSA) Improves the Differentiation 10 of Agressive from Non-aggressive Prostate Cancers", Theranostics - 2015, vol.no. 5 (3), pp .: 267-276.
    - Ohyama et al. "Carbohydrate structure and differentiak binding of prostate-specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy", Glycobiology - 2004, vol. No. 14 (8), pp .: 671-679. fifteen
    - Chang et al., "High-risk prostate cancer-Classification and therapy", Nat.Rev.Clin. Onco -2014, vol. no. 11, pp .: 308-323.
    - Humphrey, et al. "Gleason grading and prognostic factors in carcinoma of the prostate", Modern Pathology -2004, vol. no. 17, pp .: 292-306.
    权利要求:
    Claims (21)
    [1]

    1.- Method for in vitro diagnosis of high-risk prostate cancer in a fluid sample isolated from a subject, characterized in that it comprises determining the glycosylation pattern of the specific prostate antigen of the sample, said pattern defined by the presence and proportion 5 of internal fucose and / or by the presence and percentage of specific prostate antigen comprising α2,3 sialic acid, and where the method comprises:
    (A) Determine the amount of prostate specific antigen of the sample comprising internal fucose and calculate a parameter called the internal fucosylation ratio of the sample, such as the ratio between the amount of specific prostate antigen comprising internal fucose with respect to the amount of specific prostate antigen free of the sample, according to formula (II)
    RFI = (fucose-PSA) / fPSA (II), 15
    where RFI is the ratio of internal fucosylation, fucose-PSA is the amount of prostate specific antigen of the sample comprising internal fucose and fPSA is the amount of specific prostate antigen free of the sample;
     twenty
    I
    (B) Separate the specific prostate antigen from the sample into two fractions B and UB, by interacting with a solid phase comprising specific lectins against sialic acid, where the UB fraction comprises prostate specific antigen with sialic acid 25 α2,3 and not comprises prostate specific antigen with α2.6 sialic acid, and where fraction B comprises prostate specific antigen with α2.6 sialic acid; determine in each of these UB and B fractions the amount of prostate specific antigen; and calculate the percentage of prostate specific antigen comprising α2.3 sialic acid, calculating the ratio between the amount of prostate specific antigen of the UB 30 fraction (UB-PSA) with respect to the sum of the amount of prostate specific antigen of the fractions UB (UB-PSA) and B (B-PSA) according to formula (I)
    % α2,3 – PSA = [(UB-PSA) / ((UB-PSA) + (B-PSA))] x 100 (Ia);
     35
    where:
    if the percentage of specific prostate antigen comprising α2.3 sialic acid is greater than a reference control value, the subject is diagnosed with high-risk prostate cancer; and / or 40 if the internal fucosylation ratio in the isolated sample is less than a reference control value, the subject is diagnosed with high-risk prostate cancer, where high-risk prostate cancer is one that corresponds to a Gleason grade score equal to or greater than 8 or Gleason grade 7 with metastasis.
     Four. Five
    [2]
    2. - Method according to claim 1, characterized in that it comprises determining the glycosylation pattern of the specific prostate antigen of the sample, said pattern defined by the presence and proportion of internal fucosylation and / or by the presence and percentage of α2,3 sialic acid , according to the following stages:
     fifty
    (a) mixing the isolated fluid sample with a solution of ethanolamine at a final molar concentration of ethanolamine from 0.5 to 2.5 M;
    (b) immunoprecipitate the solution obtained in step (a) with a specific prostate antigen specific antibody and elute the prostate specific immunoprecipitated antigen to recover the specific prostate antigen from the sample; Y
    (c) in the solution of step (b):
     5
    (c.1) Separate the specific prostate antigen into two fractions B and UB, by interacting with a solid phase comprising specific lectins against sialic acid, where the UB fraction comprises prostate specific antigen with α2.3 sialic acid and does not comprise antigen prostate specific with α2.6 sialic acid, and where fraction B comprises prostate specific antigen with α2.6 sialic acid; 10
    (c.2) determine in each of these UB and B fractions the amount of prostate specific antigen; Y
    (c.3) calculate the percentage of specific prostate antigen comprising α2.3 sialic acid, calculating the ratio between the amount of prostate specific antigen of the UB fraction (UB-PSA) with respect to the sum of the amount of specific prostate antigen of the 15 fractions UB (UB-PSA) and B (B-PSA) according to formula (Ia)
    % α2,3 – PSA = [(UB-PSA) / ((UB-PSA) + (B-PSA))] x 100 (Ia);
    I 20
    (d) In the solution of step (b) determine the amount of free specific prostate antigen and determine the amount of specific prostate antigen comprising internal fucose; and calculate a parameter called the internal fucosylation ratio of the sample as the ratio between the amount of specific prostate antigen comprising internal fucose (fucose-PSA) with respect to the amount of specific free prostate antigen in the sample according to formula (II)
    RFI = (fucose-PSA) / fPSA (II);
    where: 30
    if the percentage of specific prostate antigen comprising α2.3 sialic acid according to formula (Ia) is greater than a reference control value, the subject is diagnosed with high-risk prostate cancer; and / or if the internal fucosylation ratio according to formula (II) in the isolated sample is less than a reference control value, the subject is diagnosed with high-risk prostate cancer, where high-risk prostate cancer is one that corresponds to a Gleason grade score equal to or greater than 8 or Gleason 7 grade with metastasis.

    [3]
    3. Method according to claim 2, characterized in that the molar concentration of ethanolamine in the solution of step (a) is 1 to 1.5 molar. 40

    [4]
    4. Method according to any one of claims 2-3, characterized in that step (a) is carried out at a temperature of 20 ° C to 40 ° C.

    [5]
    5. Method according to any one of claims 2-4, characterized in that step (a) is carried out for a period of time from 20 hours to 75 hours.

    [6]
    Method according to any one of claims 1-5, characterized in that the isolated fluid sample from the subject is selected from the group consisting of whole blood, serum, plasma and urine. fifty

    [7]
    7. Method according to claim 6, characterized in that the isolated sample is serum.

    [8]
    Method according to any one of claims 1-7, characterized in that the percentage of specific prostate antigen comprising α-2,3 sialic acid is determined by affinity chromatography on a column with a solid phase comprising Sambucus nigra lectin ( SNA) and proceed according to the following stages:
     5
    (i) the sample-specific prostate antigen is incubated with the Sambucus nigra lectin on a chromatography column for a period of 15 hours to 20 hours and at a temperature of 2 ° C to 7 ° C, said Sambucus nigra lectin with the capacity of retaining the specific prostate antigen comprising α-2,6 sialic acid;
     10
    (ii) the column is centrifuged and eluted at least twice for 3-7 minutes, with a volume of 200-700 µl of a buffer solution, to obtain the fraction not bound to the column (UB) and comprising the antigen prostate specific comprising α2,3 sialic acid;
    (iii) the specific prostate antigen fraction bound to column (B), comprising α2.6 sialic acid, is eluted according to the following two sub-stages:
    - incubating the lectin twice with a buffer solution comprising lactose at a temperature of 20 ° C to 30 ° C and at a pH of 7 to 7.5 to obtain a first fraction of eluate; and
    - incubating the lectin with an acid solution at a pH of 2.5 to 3 comprising lactose at a temperature of 20 to 30 ° C to obtain a second fraction of eluate and neutralize it at a pH of 7 to 8;
    (iv) the specific prostate antigen of each of stages (ii) and (iii) is determined and the percentage of specific prostate antigen comprising α-2,3 sialic acid is calculated, as the ratio between the specific prostate antigen of the UB fraction of stage (ii) with respect to the sum of the prostate specific antigen of the UB and B fractions of stages (ii) and (iii), in accordance with formula (Ia) above.

    [9]
    9. Method according to any one of claims 1-7, characterized in that the determination of the internal fucosylation ratio is carried out by means of an enzyme-linked lectin (ELLA) assay with Pholiota squarrosa (PhoSL) conjugate conjugated with biotin, according to the following stages:
    (i) the prostate specific antigen of the isolated fluid sample is incubated with a free prostate specific antigen specific antibody 35 for a time of 1 to 3 hours;
    (ii) after stage (i) the biotin-conjugated Pholiota squarrosa (PhoSL) lectin is added for a time of 1 to 3 hours;
     40
    (iii) a solution of streptavidin conjugated to horseradish peroxidase is added after step (ii) for a period of 1 to 1.5 hours, period after which the substrate of the conjugated peroxidase enzyme is added to a fluorescence emitting molecule and the emitted fluorescence is determined, which is proportional to the amount of prostate specific antigen comprising internal fucose; Y
     Four. Five
    (iv) the specific prostate antigen free of the sample is determined and the internal fucosylation ratio is calculated according to the formula (II) above.

    [10]
    10. Method according to any one of claims 1-9, characterized in that the reference control value of the internal fucosylation ratio is 0.86 and the reference control value of the 50 percent specific prostate antigen comprising α-2,3 sialic acid It is 30%.

    [11]
    11. Method according to any of claims 1-10, characterized in that it is for the differential diagnosis of high-risk prostate cancer of intermediate and low-risk prostate cancer and benign diseases of the prostate.

    [12]
    12. Method according to claim 11, characterized in that the benign diseases of the prostate are selected from the group consisting of benign prostatic hyperplasia, prostatitis and manipulations of the prostate.

    [13]
    13. Method for deciding or recommending a medical regimen for high-risk prostate cancer, said medical regimen selected from pharmacological treatment and surgical intervention, which comprises carrying out the diagnostic method according to any one of claims 1-12; and where, if the subject is diagnosed with high-risk prostate cancer, the recommended medical regimen for prostate cancer is recommended or decided, and if the subject is not diagnosed with high-risk prostate cancer but is diagnosed with a benign disease Prostate or low or moderate risk prostate cancer is recommended or decided by the medical regime for this other disease.

    [14]
    14. Use of (a) the internal fucosylation ratio, which results from determining in a fluid sample isolated from a subject the ratio between the amount of specific prostate antigen comprising internal fucose and the amount of specific prostate free antigen in the sample; and / or (b) of the 20 percent specific prostate antigen comprising α-2,3 sialic acid, and which results from calculating the ratio between the specific prostate antigen of a fraction not bound to specific lectin against sialic acid with respect to the sum of the specific prostate antigen fractions bound and not bound to said lectin, as independent biomarkers of high-risk prostate cancer, where high-risk prostate cancer is one that corresponds to 25 with a Gleason grade score equal to or greater than 8th grade Gleason 7 with metastasis.

    [15]
    15. Use according to claim 14, characterized in that the isolated fluid sample where the amount of specific prostate antigen comprising internal fucose and / or the amount of prostate specific antigen of the fractions bound and not bound to lectin is determined, is a sample which has previously been mixed with a solution of ethanolamine, this ethanolamine at a final molar concentration of 0.5 M to 2.5 M, and then immunoprecipitated with a specific prostate antigen specific antibody.

    [16]
    16. Use according to any one of claims 14-15, wherein the isolated fluid sample from the subject is selected from the group consisting of whole blood, serum, plasma and urine.

    [17]
    17. Use according to claim 16, wherein the isolated fluid sample from the subject is serum.

    [18]
    18. Use according to any one of claims 14-17, wherein the biomarker is for differential diagnosis of high-risk prostate cancer and low or intermediate risk prostate cancer and / or benign prostate diseases.

    [19]
    19. Kit comprising means for determining in an isolated fluid sample of a subject the amount of specific prostate antigen comprising α-2,3 sialic acid and / or the amount of specific prostate antigen comprising internal fucose, characterized in that it comprises:
    (a) a free prostate specific antigen specific antibody; Y
    (b) a solid phase for affinity chromatographic column comprising a specific lectin against sialic acid and, optionally, solutions for elution of the sample loaded in said column, as means for detecting and quantifying the specific amount of prostate antigen comprising acid sialic α-2,3; I
    (c) a specific lectin against fucose and conjugated with biotin; optionally a free specific prostate antigen specific antibody, said antibody immobilized in a well plate; and optionally, a solution of streptavidin conjugated to horseradish peroxidase and a substrate of the enzyme peroxidase conjugated to a fluorescent emitting molecule, as means for detecting and quantifying the specific prostate antigen comprising internal fucosylation.

    [20]
    20. Kit according to claim 19, wherein the specific lectin against sialic acid is the agglutinin of Sambucus nigra (SNA) and the specific lectin against internal fucose is the lectin of Pholiota squarrosa (PhoSL). 10

    [21]
    21. Kit according to any one of claims 19-20, characterized in that it further comprises:
    (a) a solution of ethanolamine to be mixed with the subject's sample and obtain a final concentration of ethanolamine from 0.5 to 2.5 M; and 15
    (b) a specific prostate antigen specific antibody bound to a support for immunoprecipitation.
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    WO2017077162A1|2017-05-11|
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2010011357A2|2008-07-25|2010-01-28|The Johns Hopkins University|Detection of prostate cancer using psa glycosylation patterns|KR20180103919A|2016-01-27|2018-09-19|후지필름 와코 준야쿠 가부시키가이샤|How to judge prostate cancer|
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