Glycan structures of haptoglobin as biomarkers for hepatocellular carcinoma

Abstract

The present invention relates to an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject. The method may include determining the amount of one or more N-glycan structures bound to haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from a subject, and comparing the amount of one or more glycan structures to a reference amount of one or more glycan structures, where a change in the amount of one or more glycan structures in a patient sample relative to the reference amount of one or more glycan structures indicates HCC. Furthermore, the present invention relates to the use of one or more glycan structures bound to haptoglobin of a haptoglobin-derived glycopeptide in combination with AFP and / or PIVKA-II in the detection of HCC.

Description

[Technical field]

[0001] The present invention relates to an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject. The method may include determining the amount of one or more N-glycan structures bound to haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from a subject, and comparing the amount of one or more glycan structures to a reference amount of one or more glycan structures, where a change in the amount of one or more glycan structures in a patient sample relative to the reference amount of one or more glycan structures indicates HCC. Furthermore, the present invention relates to the use of one or more glycan structures bound to haptoglobin of a haptoglobin-derived glycopeptide in combination with AFP and / or PIVKA-II in the detection of HCC. [Background technology]

[0002] Liver cancer is the seventh most common cancer and the second leading cause of cancer death worldwide. In 2012, the incidence and mortality rates were 10.1 and 9.5 per 100,000, respectively.

[0003] Hepatocellular carcinoma (HCC) is the predominant histological type of primary liver cancer occurring worldwide, accounting for 70% to 85% of the total burden. Underlying liver disease, such as hepatic fibrosis and cirrhosis, is known to be the main risk factor for the development of HCC. HCC can be treated by resection, liver transplantation, or local radiofrequency ablation in patients diagnosed at an early stage.

[0004] If this malignancy is diagnosed early, the 5-year survival rate of HCC patients can be as high as 70%. However, the 5-year survival rate of HCC patients decreases significantly the later the disease is diagnosed, dropping to only 15% if HCC is diagnosed at a later stage of the disease (Tsuchiya N, Sawada Y, Endo I, et al. Biomarkers for the early diagnosis of hepatocellular carcinoma. World J Gastroenterol. 2015; 21(37): 10573-83; Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: A Cancer Journal for Clinicians. 2013; 63(1): 11-30). Due to the absence of symptoms, more than 60% of HCC patients are diagnosed at a late stage when metastasis has already occurred (Altekruse SF, McGlynn KA, Reichman ME. Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2009;27(9):1485-91). Therefore, the development of a non-invasive early HCC diagnostic test is essential to reduce the mortality and increase the efficiency / success rate of HCC treatment.

[0005] The most common methods for the diagnosis of HCC are ultrasound detection, imaging techniques such as computed axial tomography (CAT scan) or magnetic resonance imaging (MRI), and serological biomarkers. However, ultrasound detection has poor prognostic value because it requires a tumor mass of at least 2 cm, and imaging techniques have low sensitivity per lesion and high cost. Meanwhile, in recent years, much interest has been paid to the discovery of new blood biomarkers that can be used in surveillance programs for early detection of HCC in high-risk patients (Yang JD.Detect or not to detect very early stage hepatocellular carcinoma? The western perspective.Clin Mol Hepatol.2019;25(4):335-43).

[0006] Some current medical guidelines recommend monitoring high-risk patients every 6 months using ultrasound or other imaging modalities. However, limitations of imaging approaches include low sensitivity for early-stage tumors, operator dependency, and poor quality in patients with obesity or nonalcoholic steatohepatitis. The adjunctive use of the tumor biomarker AFP may improve detection rates but has limited sensitivity for detecting small tumors.

[0007] Haptoglobin (Hp) is an acute-phase glycoprotein with four N-glycosylation sites. Like many other inflammatory markers and many other tumor-related biomarkers, it has been occasionally mentioned that Hp may be a marker candidate in the field of HCC (Tai CS, Lin YR, Teng TH, Lin PY, Tu SJ, Chou CH, Huang YR, Huang WC, Weng SL, Huang HD, Chen YL, Chen WL. Haptoglobin expression correlates with tumor differentiation and five-year overall survival rate in hepatocellular carcinoma. PLoS ONE 2017;12(2):e0171269). Aberrant glycosylation of serum haptoglobin, especially on glycosite Asn241, has been recently shown to correlate with the development of gastric cancer (Jeong S, Kim U, Oh MJ, Nam J, Park SH, Choi YJ, Lee DH, Kim J, An HJ. Detection of Aberrant Glycosylation of Serum Haptoglobin for Gastric Cancer Diagnosis Using a Middle-Up-Down Glycoproteome Platform. J. Pers. Med. 2021;11:575). Specific glycosylation changes of haptoglobin have also been reported for other types of cancer, such as liver, breast, lung, prostate, pancreas, and colon cancer (Oh MJ, Lee SH, Kim U, An HJ. In-depth investigation of altered glycosylation in human haptoglobin associated cancer by mass spectrometry. Mass Spec Rev. 2021;1-23).

[0008] Recently, Ramachandran P. et al. (2022) described serum glycoprotein markers in nonalcoholic steatohepatitis and hepatocellular carcinoma. The study also included mass spectrometry-based glycan assessment at amino acid residues 184, 207, 211, and 241 of haptoglobin (Ramachandran, Gege Xu, Hector H. Huang, Rachel Rice, Bo Zhou, Klaus Lindpaintner, and Daniel Serie Journal of Proteome Research 2022 21(4), 1083-1094. DOI:10.1021 / acs.jproteome.1c00965).

[0009] Alpha-fetoprotein (AFP) is the most established blood biomarker for HCC. However, even AFP shows suboptimal sensitivity and specificity for early detection of HCC. In addition, false elevation of AFP levels has been reported in patients with chronic hepatitis or cirrhosis without HCC.

[0010] AFP is a glycoprotein and various glycosylated forms of AFP have been described. Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the glycan structures of glycoproteins, it is possible to separate and enrich one or more marker glycoprotein fractions with specific glycan structures. For AFP, lectins derived from lentil lectin-A (LCA) are widely used. The lentil lectin (LCA) reactive fraction of alpha-fetoprotein (AFP-L3) is specifically increased in HCC patients. Many attempts have been made to specifically measure AFP-L3, for example by affinity electrophoresis with LCA, lectin-based ELISA, or by antibodies that specifically bind to the L3 form of AFP.

[0011] Rapid / sensitive qualitative / quantitative analysis using mass spectrometry is also used for glycoprotein analysis. Multiple reaction monitoring mass spectrometry (MRM) facilitates quantification of peptides generated from protein hydrolysis and is highly reliable. Parallel reaction monitoring (PRM) method uses a mass spectrometer equipped with a trap and a time-of-flight mass spectrometer, which results in the product ion spectrum of peptides, allowing simultaneous quantification and qualification of peptides. This method can also analyze trace amounts of glycoproteins that show low signals with high reproducibility and good sensitivity.

[0012] Methods for analyzing specific glycans using a mass spectrometer include methods based on the analysis of one or more glycans separated from a glycoprotein, and methods based on the analysis of peptides bound to glycans, i.e., glycopeptides.

[0013] Glycans attached to the exact same amino acid position of a protein can have various structures and exhibit heterogeneity. It is also known that the sugar structure can differ depending on the amino acid position at which the glycan is located.

[0014] Based on MRM-MS analysis, Kim et al. demonstrated that deglycopeptides of alpha-fetoprotein are better able to distinguish cancer status between normal subjects and hepatocellular carcinoma patients than non-glycopeptides (Kim H, Kim K, Jin J, Park J, Yu SJ, Yoon JH, Kim Y. Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Form in Hepatocellular Carcinoma. PLOS One, 2014, 9: e110366). In EP 3 415 918, various glycopeptides of AFP-L3 have been analyzed by mass spectrometry and used, for example, to compare samples obtained from HCC patients with samples obtained from subjects with cirrhosis or hepatitis.

[0015] Protein induced by vitamin K absent / antagonist-II (PIVKA-II), also known as des-gamma-carboxy-prothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and is used alone or in combination with AFP as a surrogate HCC biomarker. Prothrombin has 10 potential gamma-carboxylation sites, and various forms of PIVKA-II with different levels of carboxylation exist in the blood circulation. Different assays of PIVKA-II may detect different sets of PIVKA-II-forms, and the specificity / sensitivity of PIVKA-II may vary depending on the assay used, limiting its usefulness in detecting early HCC.

[0016] To date, international guidelines lack consensus on the use of AFP-L3 and PIVKA-II or alternative serum biomarkers for the monitoring and diagnosis of at-risk patients. However, many recent publications clearly point out that a score including the input variables sex, age, AFP and descarboxyprothrombin (DCP=PIVKA-II) (=GALAD score) or sex, age, AFP, AFP-L3 and descarboxyprothrombin (DCP=PIVKA-II) (=GALAD score) can and does improve the outcome of HCC screening efforts.

[0017] Nonetheless, there is a great need for further improved in vitro methods to aid in the detection of HCC.

[0018] Surprisingly, novel glycan structures on haptoglobin have been discovered that have high diagnostic value, for example, in the early detection of HCC. The combination of these glycan structures with established reference protein biomarkers (e.g., AFP, PIVKA-II) further improves their clinical utility. Summary of the Invention

[0019] The present inventors have found that analysis of one or more glycan structures attached to the β-chain of haptoglobin can overcome some of the problems of current in vitro diagnostic methods aiding in the detection of HCC.

[0020] In particular, the inventors have identified a glycan structure at the N184 position of the beta chain of haptoglobin (i.e., at the amino acid asparagine at position 184 of SEQ ID NO: 1) that is a good biomarker for detecting HCC, and particularly for detecting early stage HCC.

[0021] In a first aspect, the present invention relates to an in vitro method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, comprising the steps of: a) determining the amount of one or more glycan structures at position N184 of haptoglobin (i.e. the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from said subject; and b) comparing the amount of said one or more glycan structures detected in a) with a reference amount of said one or more glycan structures, wherein a change in the amount of said one or more glycan structures in said patient sample relative to the reference amount of said one or more glycan structures is indicative of HCC.

[0022] In certain embodiments, the one or more glycan structures attached to N184 is the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0023] In a second aspect, there is provided an in vitro method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e. the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from said subject; and b) comparing the amount of said N-glycan structure determined in (i)a) with a reference amount of said N-glycan structure, wherein the N184 amount of haptoglobin in the sample from said subject is compared to a reference amount of said N-glycan structure. or (ii) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin determined in a) and comparing the determined score for detecting HCC (e.g., early stage HCC) to a reference value of said score that is indicative of HCC (e.g., early stage HCC).

[0024] In a third aspect, the present invention relates to an isolated glycopeptide having a peptide portion and an N-glycan portion, wherein the peptide portion comprises or consists of the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO:2), and the N-glycan portion is HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and the N-glycan portion is linked to N at position 6 of SEQ ID NO:2. In an embodiment, the methionine at position 1 of SEQ ID NO:2 is optionally oxidized.

[0025] In certain embodiments of the third aspect of the present disclosure, the compound of formula 1: [ka] (Formula 1) (wherein GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). The lines represent covalent bonds. The glycan in the glycopeptide is covalently attached as an N-glycan via the GlcNAc of the indicated glycan to the side chain of an asparagine corresponding to N184 of haptoglobin (i.e., N184 of the β-chain of haptoglobin in SEQ ID NO:1). Provided herein is a glycopeptide of the formula:

[0026] A peptide contained in this glycopeptide has the amino acid sequence shown in SEQ ID NO: 2. Another pictorial representation of a glycopeptide having the same structure as shown in Formula 1 above is shown in the right panel of FIG.

[0027] In a fourth aspect, the present invention relates to the use of the glycopeptide of the third aspect (eg, the glycopeptide of formula 1, see above) in the detection of HCC.

[0028] According to a fifth aspect of the present disclosure, there is provided a method for detecting a glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1), comprising the steps of: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) with GluC and trypsin; and c) detecting glycopeptides comprising position N184 of haptoglobin having the glycan structure produced in b) attached thereto, thereby detecting the glycan structure at position N184.

[0029] In a sixth aspect, the present disclosure relates to an (in vitro) method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from said subject; b) determining the amount of an HCC biomarker other than the N-glycan structure at position N184 of a) (e.g., the amount of AFP and / or the amount of PIVKA) in a sample obtained from said subject; and c) combining the amounts determined in a) and b) to a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of said composite value, wherein a change in the composite value is indicative of HCC (e.g., early stage HCC).

[0030] In a seventh aspect, there is provided a computer-implemented method for aiding in detection of HCC (e.g., early stage HCC) in a subject, comprising: a) receiving data comprising the amount of a glycan structure at the N184 position of haptoglobin (i.e., the beta chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from the subject; and b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at N184 position of haptoglobin, wherein a change in the amount of the N-glycan structure in the patient sample relative to the reference amount of the one or more glycan structures indicates HCC; or (ii) taking into account the amount of the glycan structure at N184 position of haptoglobin received in a), calculating a score for detecting HCC (e.g., early stage HCC) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of the score indicating HCC (e.g., early stage HCC).

[0031] In an eighth aspect, there is provided a computer-implemented method for aiding in detection of HCC (e.g., early stage HCC) in a subject, comprising: a) receiving data comprising the amount of one or more glycan structures (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2)) at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from the subject; b) receiving data comprising the amount of PIVKA-II and / or AFP in a sample obtained from the subject; c) calculating a score taking into account the quantities determined in a) and b); and d) comparing the score calculated in c) with a reference value of said score indicative of HCC (eg, early stage HCC).

[0032] There is also provided herein a computer program product comprising instructions which, when executed by a computer, cause the computer to carry out the computer-implemented method according to the seventh or eighth aspect.

[0033] Further provided is a computer readable medium comprising instructions which, when executed by a computer, cause the computer to perform a computer implemented method according to the seventh or eighth aspect.

[0034] There is also provided a data processing system comprising a receiving unit configured to receive data as defined in the seventh or eighth aspect, a processing unit configured to perform the calculation and / or comparison steps and / or any determination / evaluation steps of the seventh or eighth aspect, and optionally an output unit configured to output a result of said evaluation. [Brief description of the drawings]

[0035] [Figure 1] Schematic diagram showing the various forms of haptoglobin. The three types of haptoglobin contain either two α1 chains (1-1), an α1 chain and one α2 chain (2-1) or two α2 chains (2-2) of haptoglobin (always together with a β chain). [Diagram 2]Structure of formula 1 (compound 41): HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at N-184 position. On the left is the glycan structure drawn using monosaccharide abbreviations according to the Symbol Nomenclature for Glycans (SNFG) system (PMID 26543186, Glycobiology 25:1323-1324, 2015), with connections shown as black lines. On the right is a symbolic representation of the glycan structure, with monosaccharide symbols according to SNFG. [Diagram 3] Box blot of various glycopeptides at N184 position of haptoglobin beta chain (SEQ ID NO: 1) indicative of HCC. Site-specific glycan analysis revealed that a significant number of glycopeptides were elevated or decreased in HCC. Hp glycopeptide abundance was compared among controls, early HCC cohorts, and late HCC cohorts. Statistical significance between early HCC (ESH) and late HCC (LSH) in controls (C) was determined by Wilcoxen (Mann-Whitney U) test. [Figure 4] Overview of upregulated glycopeptides on glycosylation site N-184. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG) system (PMID 26543186, Glycobiology 25:1323-1324, 2015). Only the top 5 glycopeptides distinguishing early and late HCC versus controls are shown. [Diagram 5] Receiver operating curve (ROC) for the compound HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at N-184 position showing diagnostic performance for detection of early and late stage HCC. Specificity and sensitivity values ​​are for a sensitivity and specificity of 0.9 cutoff, respectively. [Figure 6]Overview of up- / downregulated glycopeptides at all haptoglobin glycosylation sites (N-184, N-207, N-211 and N-241). Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) system (PMID 26543186, Glycobiology 25:1323-1324, 2015). On the left side, Hp glycopeptides that distinguish early HCC cases from controls are listed. The middle column shows glycopeptides present in both early and late HCC, while the right column shows glycopeptides that distinguish late HCC versus controls. Only glycopeptides with AUC >70% are shown. The direction of expression change (decreased or increased versus control) is indicated as up (regulated) or down (regulated). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention Below, elements of the invention are described that, although listed with specific aspects and embodiments, it is understood that they can be combined in any way and in any number to create additional aspects and embodiments.

[0037] In a first aspect, the present invention relates to an in vitro method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, comprising the steps of: a) determining the amount of one or more glycan structures at position N184 of haptoglobin (i.e. the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from said subject; and b) comparing the amount of said one or more glycan structures determined in a) with a reference amount of said glycan structures, wherein a change in the amount of said one or more glycan structures in said patient sample relative to the reference amount of said one or more glycan structures is indicative of HCC.

[0038] In an embodiment of a first aspect of the present invention, there is provided an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, comprising: a) determining the amount of a glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from the subject; and b) comparing the amount of the glycan structure determined in a) with a reference amount of the glycan structure, wherein a change in the amount of the glycan structure in the patient sample relative to the reference amount of the glycan structure is indicative of HCC.

[0039] The method of the first aspect of the present invention is based on the finding that the glycosylation pattern at position 184 of the β-chain of haptoglobin can be used to detect HCC (e.g., early stage HCC). As demonstrated in the accompanying examples, the glycan structures at this position in samples from patients with HCC (e.g., early stage HCC) show characteristic patterns, for example, compared to reference samples and compared to samples from patients with cirrhosis.

[0040] In an embodiment of the first aspect of the present invention, the determined amount or amounts of one or more glycan structures at N184 position of haptoglobin do not have to be compared with the reference amount of each of the above glycan structures, but instead, a score for determining HCC (e.g., early stage HCC) can be calculated taking into account the determined amount or amounts of one or more glycan structures at N184 position of haptoglobin. The score can be compared with the respective reference value of the score that determines the presence of HCC (e.g., early stage HCC).

[0041] Therefore, the present specification provides an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, comprising: a) determining the amount of glycan structure at N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from the subject; b) determining the score for detecting HCC (e.g., early HCC) taking into account the amount of the glycan structure determined in a); and c) comparing the score for detecting HCC (e.g., early HCC) determined with a reference value indicating HCC (e.g., early HCC). Based on this comparison, the presence or absence of HCC (e.g., early HCC) can be determined.

[0042] In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) determined by a method according to the present disclosure is HexNAc(5)Hex(6)Fuc(1)NeuAc(2);HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4). Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3) , HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(3 ) and HexNAc(6)Hex(7)NeuAc(1). In embodiments, the glycan structure at N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) determined in a method according to the present disclosure is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In embodiments, the glycan structure at N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) determined in a method according to the present disclosure is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0043] In an embodiment, an increase in the amount of the determined glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) relative to a reference amount may be indicative of HCC (e.g., early stage HCC).

[0044] In embodiments in which the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin is determined, an increase in the amount of this glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) relative to a reference amount may be indicative of HCC (e.g., early stage HCC).

[0045] In a particularly preferred embodiment, the determined glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0046] In an embodiment of the method according to the first aspect, the method further comprises determining the amount of PIVKA-II and / or the amount of AFP in the sample or another sample from the same subject, and the score for detecting HCC takes into account the determined amount of PIVKA-II and / or the determined amount of AFP.As demonstrated in the accompanying examples, the amount of glycan structure at N184 position of haptoglobin in combination with the amount of AFP and / or PIVKA-II enhances the diagnostic utility of HCC (e.g., early stage HCC).

[0047] In a second aspect, the disclosure provides an in vitro method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from said subject; and b) comparing the amount of said N-glycan structure determined in (i)a) to a reference amount of said N-glycan structure, or (ii) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position 184 of haptoglobin determined in a) and comparing the determined score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). b) Based on the comparison in i) or ii), the presence or absence of HCC (e.g., early stage HCC) can be determined.

[0048] The method of the second aspect is based on the finding that an increased amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position 184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) was found in samples obtained from subjects suffering from HCC (particularly early stage HCC) compared to control samples representing chronic liver diseases including HBV, HCV and liver cirrhosis. As shown by the attached examples, the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) is a very promising marker for HCC (e.g., early stage HCC) and shows the highest AUC in the detection of HCC (early stage HCC and late stage HCC combined) of all identified N-glycan structures (including all glycan structures at position N184 but also including other glycosylation sites). The structure of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) can be as shown in Figure 2. Thus, in embodiments, the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) can have a glycan structure as shown in the glycan portion of the glycopeptide in Figure 2 or the glycan structure of formula 2 referred to elsewhere herein.

[0049] What is said herein in relation to the method according to the first aspect of the invention applies mutatis mutandis.

[0050] In an embodiment of the second aspect of the present invention, there is provided an in vitro method for aiding in the detection of HCC (e.g. early stage HCC) in a subject, the method comprising the steps of: a) determining the amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e. the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from said subject; and b) comparing the amount of said N-glycan structure determined in a) with a reference amount of said N-glycan structure, wherein a change in the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin in the subject's sample relative to the reference amount of said N-glycan structure is indicative of HCC (e.g. early stage HCC).

[0051] When a reference amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) is selected to be representative of the amount of said N-glycan structure in healthy subjects and / or subjects suffering from a non-cancerous chronic liver disease (e.g., selected from the group consisting of HBV, HCV and cirrhosis), an increased amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) compared to the reference amount is indicative of HCC (e.g., early stage HCC).

[0052] Thus, in embodiments, an increase in the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) is indicative of HCC (e.g., early stage HCC).

[0053] In an embodiment, a second aspect of the present invention relates to an in vitro method for assisting in the detection of HCC (e.g., early stage HCC) in a subject, comprising: a) determining the amount of N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from said subject; b) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin determined in a); and c) comparing the determined score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). Then, the comparison in c) can be used to determine the presence or absence of HCC (e.g., early stage HCC).

[0054] In embodiments, the score may be configured such that a higher value of the score indicates an increased risk of HCC. In these embodiments, the value of the score will increase with increasing levels of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin.

[0055] Determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e. the β chain of haptoglobin having the sequence shown in SEQ ID NO:1) in a sample means that a measurement reflecting the absolute or relative amount of haptoglobin glycosylated with each glycan structure at position N184 of haptoglobin in the sample is determined.

[0056] Disclosed herein below are embodiments for determining the amount of a glycan structure, such as the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2), at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1).

[0057] In embodiments, the level of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) can be determined by determining the level of a glycopeptide comprising N184 and the glycan structure, e.g., by mass spectrometry. In embodiments, a glycopeptide having the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), used to determine the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), comprises a peptide sequence of MVSHHNLTTGATLINE (SEQ ID NO:2), where the N at position 6 of SEQ ID NO:2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO:1). In certain embodiments, the peptide portion of the glycopeptide may consist of MVSHHNLTTGATLINE (SEQ ID NO:2). Thus, the amount of glycopeptide of formula 1 (see elsewhere herein) can be determined to determine the amount of the glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin. Depending on the method used for sample preparation and determination, the methionine at position 1 of SEQ ID NO:2 can be oxidized when the level of the glycopeptide is measured.

[0058] In an embodiment, a glycopeptide having the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1), detected to determine the amount of the N-glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin, has an m / z of 1118,2098, a charge of 4, and a glycopeptide mass of 4469,8163 Da.

[0059] In other words, a glycopeptide can be analyzed to determine the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1), said glycopeptide can comprise the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO:2), where the N at position 6 of SEQ ID NO:2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO:1), and the glycan structure is HexNAc(5)Hex(6)Fuc(1)NeuAc(2). In certain embodiments, the peptide portion of the glycopeptide can consist of MVSHHNLTTGATLINE (SEQ ID NO:2). Thus, a glycopeptide of formula 1 (see elsewhere herein) can be analyzed to determine the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin.

[0060] In an embodiment, to determine the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1), a glycopeptide having the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO:2) may be analyzed, where N at position 6 of SEQ ID NO:2 corresponds to N at position 184 of the β-chain of haptoglobin (SEQ ID NO:1), and the glycan is HexNAc(5)Hex(6)Fuc(1)NeuAc(2). This glycopeptide may be the glycopeptide shown in Formula 1 (see elsewhere herein) or in FIG. 2. It has a characteristic m / z value and is well defined in this alternative method. For example, a glycopeptide for determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin may have an m / z of 1118,2098, a charge of 4, and a glycopeptide mass of 4469,8163 Da. In embodiments, the glycopeptide may comprise the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO:2) as the peptide moiety and HexNAc(5)Hex(6)Fuc(1)NeuAc(2) as the glycan structure attached to the N at position 6 of SEQ ID NO:2, where the N at position 6 of SEQ ID NO:2 corresponds to the N at position 184 of the β-strand of the haptoglobin (SEQ ID NO:1) sequence, and the glycopeptide may have an m / z of 1118,2098927.9544, a charge of 54, and a glycopeptide mass of 4469,81634636.8124 Da.

[0061] To reduce the effects of sample handling and differences in the absolute amount of haptoglobin between individuals, determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e. the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) may mean determining the amount relative to the amount of a second analyte or group of analytes. Such a second analyte may be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide or glycopeptide may be exogenously added to the sample before the determination step or may be a peptide or glycopeptide of haptoglobin contained in the sample. The added peptide may be labelled with a heavy isotope.

[0062] In an embodiment of the method according to the second aspect, the method further comprises determining the amount of PIVKA-II and / or the amount of AFP in the sample or another sample from the same subject, and the score for detecting HCC takes into account the determined amount of PIVKA-II and / or the determined amount of AFP.As demonstrated in the accompanying examples, the amount of glycan structure at N184 position of haptoglobin in combination with at least the amount of AFP increases the diagnostic utility of HCC (e.g., early stage HCC).

[0063] In a third aspect, the present invention relates to an isolated glycopeptide having a peptide portion and an N-glycan portion, wherein the peptide portion comprises or consists of the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO:2), and the N-glycan portion is HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and the N-glycan portion is linked to N at position 6 of SEQ ID NO:2. In an embodiment, the methionine at position 1 of SEQ ID NO:2 is optionally oxidized.

[0064] In a preferred embodiment, the glycopeptide has Formula 1: [ka] (wherein GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). The lines represent covalent bonds. The glycans in the glycopeptide are covalently attached as N-glycans via the GlcNac of the indicated glycan to the side chain of an asparagine corresponding to N184 of haptoglobin (i.e., N184 of the β-chain of haptoglobin in SEQ ID NO: 1).

[0065] The isolated glycopeptide of formula 1 is shown only in a diagrammatic alternative representation in FIG. 2 (right panel). The right panel of Figure 2 is a representation according to 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).

[0066] As shown throughout this disclosure, the glycopeptide of formula 1 is very promising for the detection of HCC (e.g., early stage HCC). It has been found herein to be a reliable biomarker for detecting early and late stage HCC.

[0067] Thus, in a fourth aspect, the present disclosure relates to the use of a glycopeptide according to the fifth aspect of the present disclosure (ie, of formula 1) in detecting HCC (eg, early stage HCC).

[0068] The method embodiments according to the first and second aspects apply mutatis mutandis to this use.

[0069] According to a fifth aspect of the present disclosure, there is provided a method for detecting a glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1), comprising the steps of: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) with GluC and trypsin; and c) detecting glycopeptides comprising position N184 of haptoglobin having attached thereto the glycan structure produced in b), thereby detecting the glycan structure at position N184.

[0070] In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2);HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc (5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2), HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0071] The accompanying examples show that glycopeptides containing an N-glycan modification at the asparagine corresponding to position N184 of haptoglobin can be reliably determined with good sensitivity after purification and digestion followed by a suitable detection method, preferably LC-MS. In particular, all glycopeptides specifically mentioned herein (e.g., the glycopeptides of the fifth aspect of the invention) could be reliably determined using such a method.

[0072] The accompanying examples also show that, in particular, glycopeptides according to the fourth aspect of the disclosure can be reliably determined with good sensitivity after purification and digestion and after carrying out a suitable detection method, preferably LC-MS.Accordingly, in an embodiment, there is provided a method for detecting the glycan structure of a glycopeptide according to the fourth aspect (e.g., a glycopeptide of formula 1), comprising the steps of: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) with GluC and trypsin; and c) detecting the glycopeptide obtained in b), thereby detecting the glycopeptide according to the fourth aspect (e.g., a glycopeptide of formula 1).

[0073] In clinical routine, the diagnosis of HCC, especially early stage HCC, is very difficult, and many cases of HCC are diagnosed too late and can no longer be cured. Ultrasound is frequently used to "look for" the presence of liver cancer. However, the results obtained by ultrasound vary from clinician to clinician, depend on the quality of the ultrasound equipment used, and are influenced by factors such as weight / fat. Nevertheless, late stages of HCC are often first found by ultrasound.

[0074] It is envisioned that the glycan structure disclosed in the present invention significantly improves the detection of HCC, particularly the detection of early HCC. In one embodiment, the method disclosed for detecting HCC is a method for detecting early HCC (i.e., early HCC).

[0075] The glycan structures of the present invention can be combined with other biomarkers (eg, AFP) for detecting HCC.

[0076] In a sixth aspect, the present disclosure relates to an (in vitro) method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from said subject; b) determining the amount of an HCC biomarker other than the N-glycan structure at position N184 of a) in a sample obtained from said subject; and c) combining the amounts determined in a) and b) into a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of said composite value, wherein a change in the composite value is indicative of HCC (e.g., early stage HCC).

[0077] The embodiments of the aspects described above apply mutatis mutandis.

[0078] An HCC biomarker is a biomarker that is indicative of HCC (e.g., early stage HCC). In a specific example, the HCC biomarker can be AFP or PIVKA-II.

[0079] In an embodiment of a sixth aspect of the present invention, the present disclosure provides an (in vitro) method for assisting in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from the subject; b) determining the amount of AFP and / or the amount of PIVKA-II in a sample obtained from the subject; and c) combining the amounts determined in a) and b) to obtain a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of the composite value, wherein a change in the composite value indicates HCC (e.g., early stage HCC).

[0080] In an embodiment of the method of the sixth aspect, the present disclosure provides an (in vitro) method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from said subject; b) determining the amount of AFP in the sample obtained from said subject; and c) combining the amounts determined in a) and b) into a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of said composite value, wherein a change in the composite value is indicative of HCC (e.g., early stage HCC).

[0081] In an embodiment of the method of the sixth aspect, the present disclosure provides an (in vitro) method for assisting in the detection of HCC (e.g., early stage HCC) in a subject, comprising: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from the subject; b) determining the amount of PIVKA-II in the sample obtained from the subject; and c) combining the amounts determined in a) and b) to obtain a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of the composite value, wherein a change in the composite value indicates HCC (e.g., early stage HCC).

[0082] In an embodiment of the method of the sixth aspect, the present disclosure provides an (in vitro) method for assisting in the detection of HCC (e.g., early stage HCC) in a subject, comprising: a) determining the amount of an N-glycan structure at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) in a sample obtained from the subject; b) determining the amount of PIVKA-II and the amount of AFP in a sample obtained from the subject; and c) combining the amounts determined in a) and b) to obtain a composite value (e.g., a score for detecting HCC, e.g., early stage HCC) and comparing the composite value with a reference value of the composite value, wherein a change in the composite value indicates HCC (e.g., early stage HCC).

[0083] In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2);HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc (5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2), HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0084] What has been said elsewhere in this specification (e.g., in other aspects and in the following context of this specification) regarding the determination of the amount of each glycan structure applies mutatis mutandis. Thus, the amount of glycan structure can be determined by determining the amount of glycopeptide (e.g., by mass spectrometry).

[0085] The sample used for determining one or more of a) and b) may be the same sample obtained from the subject or may be different samples.Preferably, the samples are obtained from the subject at the same time.In certain embodiments, the sample for determining one or more of a) is the same as the sample for determining one or more of b).

[0086] "Composite value" may refer to, in embodiments, determining a score indicative of HCC (e.g., early stage HCC), said score taking into account the amounts determined in a) and b). The score may further take into account additional biomarkers. Alternatively and additionally, the score may take into account the subject's clinical data (e.g., age, sex, smoking status, cancer history, family cancer history, and the presence of existing liver disease).

[0087] Exemplary, but non-limiting, methods for calculating the composite value or score are disclosed herein below.

[0088] Methods for determining the amount of AFP and PIVKA-II are well known in the art. For example, microchip capillary electrophoresis and liquid-phase binding assay using uTASWako i30 autoanalyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions. AFP can also be detected, for example, with Elecsys® AFP (material number: 044817981190). PIVKA-II can be detected, for example, with Elecsys® PIVKA-II (material number: 08333602190).

[0089] In the method according to the sixth aspect, the N-glycan structure is preferably HexNAc(5)Hex(6)Fuc(1)NeuAc(2), even more preferably a glycan comprised in a glycopeptide of formula 1 (see above). In further embodiments of these aspects and embodiments, the amount of HexNAc(4)Hex(5)NeuAc(2) at position N184 or HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e. the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) may also be determined, the ratio of the amounts calculated and such ratio used to calculate a composite value.

[0090] In a seventh aspect, there is provided a computer-implemented method for aiding in detection of HCC (e.g., early stage HCC) in a subject, comprising: a) receiving data comprising the amount of a glycan structure at the N184 position of haptoglobin (i.e., the beta chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in a sample obtained from the subject; and b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at N184 position of haptoglobin, wherein a change in the amount of the N-glycan structure in the patient sample relative to the reference amount of the one or more glycan structures indicates HCC; or (ii) taking into account the amount of the glycan structure at N184 position of haptoglobin received in a), calculating a score for detecting HCC (e.g., early stage HCC) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of the score indicating HCC (e.g., early stage HCC).

[0091] The embodiments of the aspects described above apply mutatis mutandis.

[0092] In an embodiment, the method may include assisting in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0093] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0094] In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2);HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc (5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In embodiments, the glycan structure at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(5)Hex(6)Fuc(1)NeuAc(2), HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0095] In an eighth aspect, there is provided a computer-implemented method for assisting in detection of hepatocellular carcinoma (HCC) in a subject, comprising: a) receiving data comprising the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from the subject; b) comparing the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) received in i)a) with a reference amount of said glycan structure, wherein an increase in the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin in the subject's sample relative to the reference amount of said glycan structure is indicative of HCC; or (ii) calculating a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin received in a) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with said reference value of score indicative of HCC (e.g., early stage HCC).

[0096] The embodiments of the aspects described above apply mutatis mutandis.

[0097] In an embodiment, the method may include assisting in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0098] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0099] Where there are two or more steps of receiving data in the computer-implemented methods disclosed herein, one of ordinary skill in the art will readily appreciate that this includes both (i) embodiments in which one single data set is received that includes all data received in two or more steps, and (ii) embodiments in which two or more separate data sets are received. Any of the above computer-implemented methods may optionally include a step of outputting whether the subject has or is suspected of having HCC (e.g., early stage HCC) and / or whether further clinical testing for HCC (e.g., early stage HCC) is required based on the comparison step. The output may be via a display.

[0100] Also disclosed herein is a computer program product comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to the seventh or eighth aspect.

[0101] Further provided is a computer readable medium comprising instructions which, when executed by a computer, cause the computer to perform a computer implemented method according to the seventh or eighth aspect.

[0102] There is also provided a data processing system comprising a receiving unit configured to receive data as defined in the seventh or eighth aspect, a processing unit configured to perform the calculation and / or comparison steps and / or any determination / evaluation steps of the seventh or eighth aspect, and optionally an output unit configured to output a result of said evaluation.

[0103] The above embodiment relates to the glycan structure at N184 of the β-chain of haptoglobin. As is evident from the accompanying examples, several other glycan structures at N207, N211 or N241 positions have also been identified herein that are useful for detecting HCC.

[0104] The respective glycan structures at N207 of haptoglobin are HexNAc(6)Hex(7)Fuc(1)NeuAc(4), HexNAc(4)Hex(5)NeuAc(2), HexNAc(5)Hex(5)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0105] Furthermore, the respective glycan structures at the N207 position are shown in Tables 2 and 3.

[0106] The respective glycan structures at N211 of haptoglobin are HexNAc(6)Hex(7)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0107] Furthermore, the respective glycan structures at the N211 position are shown in Tables 2 and 3.

[0108] The respective glycan structures at N241 of haptoglobin are HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(4)Hex(5)NeuAc(2), HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and HexNAc(4)Hex(5)Fuc(1)NeuAc(1).

[0109] Furthermore, the respective glycan structures at the N241 position are shown in Tables 2 and 3.

[0110] Accordingly, all aspects and embodiments described herein for N184 of haptoglobin are also disclosed mutatis mutandis for N207, N211 and N241 of haptoglobin. Furthermore, all aspects and embodiments disclosed herein for a particular glycan structure at position N184 are also disclosed mutatis mutandis for any of the above-mentioned glycan structures at positions N207, N211 and N241.

[0111] To detect the amount of glycan structure at the N207 position, NLFL N A glycopeptide having a peptide portion that comprises or consists of HSE (SEQ ID NO:3) may be used, where N at position 5 corresponds to N207.

[0112] To detect the amount of a glycan structure at position N211, a glycopeptide having a peptide portion comprising or consisting of NATAK (SEQ ID NO: 4) may be used, where N at position 1 corresponds to N211.

[0113] To detect the amount of a glycan structure at position N241, a glycopeptide having a peptide portion comprising or consisting of VVLHPNYSQVD (SEQ ID NO: 5) may be used, where N at position 6 corresponds to position N241.

[0114] 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0115] The following definitions and embodiments apply throughout this disclosure and in particular to all aspects and embodiments of the present invention.

[0116] 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.

[0117] It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps.

[0118] Thus, the use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0119] The term "and / or" should be understood to mean either or both of the alternatives.

[0120] As used herein, unless otherwise specified, the term "about" should be understood to be 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 something less than the stated value or range, and indicates within ±15% of the stated value or range, ±10% of the stated value, ±5% of the stated value, or ±2% of the stated value for convenience. Such values ​​are therefore encompassed within the scope of the claims reciting the term "about" or "approximately."

[0121] As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, a protein, a carbohydrate structure, or a glycolipid, a metabolite, an mRNA, an miRNA, a protein, a DNA (cDNA or genomic DNA), a DNA copy number, or an epigenetic change, such as an increase, decrease, or change in DNA methylation (e.g., cytosine methylation, or CpG methylation, non-CpG methylation); a histone modification (e.g., (de)acetylation, (de)methylation, (de)phosphorylation, ubiquitination, sumoylation, ADP-ribosylation); a change in nucleosome arrangement, the expression or presence of which can be detected in or on a mammalian tissue or cell by standard methods (or the methods disclosed herein), which may be predictive, diagnostic, and / or prognostic of an individual's health or disease. Thus, hereinafter, the more general term "marker" may also be used when describing the more general terms and definitions. The term marker also includes the glycan structure or glycan, or glycopeptide, analyzed in this disclosure.

[0122] The term "in vitro method" is used to indicate that the method is performed outside of an organism, preferably on a body fluid, isolated tissue, organ or cell. In vitro methods are sometimes also called ex vivo methods.

[0123] Hepatocellular carcinoma (HCC) is the predominant histological type of primary liver cancer occurring worldwide, accounting for 70%-85% of the total burden. Underlying liver disease, such as hepatic fibrosis and cirrhosis, is known to be the main risk factor for the development of HCC. HCC can be treated by resection, liver transplantation, or local radiofrequency ablation in patients diagnosed at an early stage. If this malignancy is diagnosed early, the 5-year survival rate of HCC patients can be as high as 70%. However, the 5-year survival rate of HCC patients decreases significantly the later the disease is diagnosed, dropping to only 15% if HCC is diagnosed at a late stage of the disease (Tsuchiya N, Sawada Y, Endo I, et al. Biomarkers for the early diagnosis of hepatocellular carcinoma. World J Gastroenterol. 2015;21(37):10573-83; Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: A Cancer Journal for Clinicians. 2013;63(1):11-30).

[0124] Haptoglobin is an acute phase protein. It is synthesized in the liver and secreted into plasma, and has a rather complex biochemistry and diverse biological functions. As shown in Figure 1, haptoglobin can form dimers (or even polymers) through disulfide bonds. The basic form of haptoglobin is a dimer. The α-chain of human haptoglobin shows genetic polymorphisms (α1 or α2 chains, respectively) resulting in three types of haptoglobin, 1-1, 2-1 and 2-2 (see Figure 1). The concentration of haptoglobin in human plasma is usually in the range of 0.3-3 mg / ml. Haptoglobin binds to free hemoglobin, thereby preventing oxidative stress. It also plays a role in regulating the immune response through binding to both resting and activated CD4+ and CD8+ T cells.

[0125] The haptoglobin beta chain does not exhibit genetic polymorphism. As used herein, the haptoglobin beta chain preferably has the sequence shown in SEQ ID NO: 1. Haptoglobin can undergo secondary modifications, for example in the form of glycosylation. The haptoglobin beta chain has four N-glycosylation sites (asparagine (N)) at amino acid positions 183, 207, 211 and 241, respectively.

[0126] As used herein, and unless otherwise stated, the phrase "position N184 of haptoglobin" refers to position N184 of the β chain of haptoglobin (eg having the sequence shown in SEQ ID NO:1).

[0127] As will be understood by those skilled in the art who are familiar with methods to aid in the detection of HCC (e.g., early stage HCC), the assessment of HCC performed according to the present invention is preferably correct for 100% of the subjects examined, but may not usually be correct for 100% of the subjects examined (as with essentially all diagnostic methods). The term typically requires that a statistically significant portion of the subjects can be accurately evaluated. Whether a portion is statistically significant can be determined by the skilled artisan without further difficulty 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 intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values ​​are typically 0.2, 0.1, 0.05.

[0128] The term "indicative of HCC" is used to explain that an increase in the level or amount of a determined marker (e.g., glycan structure or glycopeptide), and optionally its combination with other biomarkers or variables, although very informative, is not about an infallible diagnosis, but rather indicates that the subject has a high probability of having HCC. Not all (100%) of HCC patients have the amount of a marker above the reference level, and not all healthy individuals have the level of a marker below the reference level or cut-off level. As the skilled artisan will understand, in many diseases, a biochemical marker does not have 100% specificity and at the same time 100% sensitivity. Rather, the presence of an analyzed marker or a combination of markers including this marker, for example, at a given specificity level or a given sensitivity level, indicates a certain probability that the individual whose sample was analyzed has a particular clinical condition, for example, has HCC. The skilled artisan will be fully familiar with the mathematical / statistical methods used to calculate specificity, sensitivity, positive predictive value, negative predictive value, reference value, or total error. Any of these parameters can be calculated and used to obtain an indication of the presence or absence of HCC.

[0129] The term "aiding in the detection of hepatocellular carcinoma (HCC)" is used to indicate that the method according to the present invention helps / assists a medical professional, such as a physician, in assessing whether an individual has HCC or is at risk of developing HCC. As will be understood, to detect or exclude the presence of HCC, a physician can use several alternative methods (e.g., ultrasound, radiography, MRT, or CT), which can be combined with in vitro biomarker data, such as glycan structure data. The final diagnosis of HCC is usually made by tissue biopsy or tissue sample after surgery. The term "aiding in the detection of HCC" includes the method being used as the sole diagnostic utility or as one of multiple diagnostic utilities.

[0130] In an embodiment of the present invention, "aiding in the detection of hepatocellular carcinoma (HCC)" can be "aiding in the detection of early-stage HCC." As demonstrated in the accompanying examples, a particular glycan at position 184 of the β-chain of haptoglobin exhibits particularly good performance in detecting early-stage HCC.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0132] As used herein, 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.

[0133] In the above aspects and embodiments, a particular marker value, e.g., the amount of a glycan structure or the amount of a glycopeptide, may be combined (e.g., as a score) with amounts determined for one or more other biomarkers. Such combinations are performed using standard mathematical / statistical techniques.

[0134] One convenient goal for quantifying the diagnostic accuracy of a laboratory test is to express its performance in a single number. The most common global measure is the area under the curve (AUC) of the ROC plot. The area under the ROC curve is a measure of the probability that the captured measurements allow for correct identification of a condition (or discrimination between one condition and the other). Values ​​typically range from 1.0 (perfect separation of the test values ​​of the two groups) to 0.5 (no obvious distribution difference between the two groups of test values). The area does not depend only on a particular part of the plot, such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. It is a quantitative descriptive expression of how close the ROC plot is to the perfect one (area = 1.0). In the context of the present invention, the two different conditions can be whether the patient has HCC or not.

[0135] In the present invention, the terms "HCC", "early stage HCC" and "late stage HCC" are used.

[0136] As used herein, "early stage HCC", "early stage HCC" or "early stage HCC" refers to patients classified as stage 0 and A according to the Barcelona Clinical Liver Cancer (BCLC) classification (Llovet JM, Bru C, Bruix J, Semin Liver Dis. 1999;19(3):329-38).

[0137] As used herein, "late stage HCC", "late stage HCC" or "late stage HCC" refers to patients classified as stages B, C and D according to the Barcelona Clinical Liver Cancer (BCLC) classification (Llovet JM, Bru C, Bruix J, Semin Liver Dis. 1999;19(3):329-38).

[0138] "HCC" refers to any form of HCC, including early and late stage HCC.

[0139] The BCLC classification has been recommended as the standard system for the management of HCC by the American Association for the Study of Liver Disease, the American Gastroenterology Association, the European Association for the Study of Liver, and the European Organization for the Research and Treatment of Cancer. According to the BCLC staging, patients are assigned to five categories (0, A, B, C, and D). BCLC stage 0 (defined as very early disease) includes patients with well-preserved liver function diagnosed with one asymptomatic nodule less than 2 cm without vascular invasion or satellites (Child-Pugh A; Cholongitas E, Papatheodoridis GV, Vangeli M, Terreni N, Patch D, Burroughs AK. Systematic review: The model for end-stage liver disease--should it replace Child-Pugh's classification for assessing prognosis in cirrhosis?. 2005; Alimentary Pharmacology&Therapeutics. 22(11-12):1079-89.). BCLC stage A (defined as early disease) includes patients with Child-Pugh A or B status diagnosed with one nodule of any size or up to three nodules less than 3 cm. In the context of this disclosure, BCLC stages 0 and A were defined as the early HCC group. BCLC stage B (defined as intermediate stage disease) corresponds to patients with Child-Pugh grade A or B status diagnosed with multiple nodules without vascular invasion or extrahepatic metastases. Patients with Child-Pugh grade A or B, vascular invasion or extrahepatic metastases and cancer-related symptoms (PS1-2) are classified as having BCLC C disease (defined as advanced stage disease).Finally, patients with Child-Pugh grade C at any tumor stage and cancer-related symptoms (PS>2) were classified as belonging to BCLC D disease (defined as end-stage disease). In the context of this disclosure, patients with BCLC stages B, C, and D were defined as the late-stage HCC group.

[0140] The term "subject" or "individual" as used herein refers to a single human being. A subject may be healthy or a patient, for example, a patient with cirrhosis, at risk of developing HCC, experiencing or having experienced one or more signs, symptoms, or other indicators of HCC. Subjects are intended to include any subject involved in a clinical research trial that does not show any clinical signs of disease, or subjects involved in epidemiological studies, or subjects whose samples serve as controls. In some embodiments, a subject may be known to be at risk of developing HCC, for example, due to chronic alcohol consumption, hepatitis B and / or hepatitis C infection, non-alcoholic fatty liver disease, Wilson's disease, hereditary hemochromatosis, alpha 1-antitrypsin deficiency, primary biliary cirrhosis, autoimmune hepatitis, and other risk factors.

[0141] In one embodiment according to the present disclosure, the subject from which the sample to be investigated is obtained is a healthy subject, screened for (the presence of) HCC as part of routine oncology surveillance.

[0142] In one embodiment according to the present disclosure, the subject from whom the sample to be investigated was obtained is a subject at risk for developing HCC and is screened for the (presence of) HCC as part of routine oncology surveillance.

[0143] A subject may be at risk for developing HCC if the subject is known to be suffering from chronic liver disease, viral or non-viral hepatitis and / or cirrhosis.

[0144] In one embodiment according to the present disclosure, the subject from whom the sample to be investigated was obtained has chronic liver disease, viral or non-viral hepatitis, cirrhosis, and is subject to differential diagnosis of the presence or absence of HCC.

[0145] The terms "sample", "subject sample", "patient sample" or "individual sample" as used herein refer to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the present invention, the sample, patient sample or sample obtained from an individual can preferably be any type of bodily fluid. Bodily fluid samples include blood, serum, plasma, urine, saliva and synovial fluid. Preferred sample types are whole blood, serum or plasma. In one embodiment, the sample type is serum or plasma. In one embodiment, the sample type is plasma. In one embodiment, the sample type is serum. As will be appreciated by those skilled in the art, the sample is used for the analysis of markers of interest in vitro. The patient sample is discarded after the analysis. The patient sample is used only for the in vitro methods of the present invention, and the contents of the patient sample are not returned to the patient's body.

[0146] The term "determining" the amount of an N-glycan structure or the amount of a glycopeptide as used herein refers to measuring the amount or level of said N-glycan structure or said glycopeptide. The level or amount of an N-glycan structure or glycopeptide in a sample is determined, for example, by using any suitable method known in the art or by using the methods described herein.

[0147] In the context of the present invention, determining the amount of a glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) or any other glycan structure mentioned herein) at the N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) in a sample means that a measurement is determined that reflects the absolute or relative amount of haptoglobin glycosylated with the respective glycan structure at the N184 position of haptoglobin in the sample. In an embodiment, determining the amount of a glycan structure can be determining the presence of said glycan structure and quantifying the amount / level of said glycan structure. In an embodiment, the amount of a glycan structure at the N184 position corresponds to the amount of a glycopeptide that comprises said glycan structure at N184. Such a glycopeptide can be generated by hydrolyzing the haptoglobin in the sample (e.g., using a protease such as the proteases described herein below).

[0148] In an embodiment of the present disclosure, determining the amount of one or more glycan structures (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2)) at position N184 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence shown in SEQ ID NO:1) may be determining the amount relative to a second analyte (e.g., a control analyte). This setting makes it possible to reduce the effects of sample handling and differences in the absolute amount of haptoglobin between individuals. The second analyte may be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide may be added to the sample before the determination step or may be a peptide or glycopeptide of haptoglobin contained in the sample. The added peptide may be labeled with a heavy isotope.

[0149] In the present disclosure, determining the amount of a glycan structure at the N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) in a sample obtained from the subject may include generating one or more glycopeptides from the haptoglobin contained in the sample, the glycopeptides comprising the N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) and, if present, a glycan structure attached thereto, and determining the amount of one or more glycopeptides comprising the glycan of interest, for example, by mass spectrometry. The amount of each glycan structure at the N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) may correspond to or be derived from the amount of each glycopeptide comprising the respective glycan structure at the N184 position.

[0150] In an exemplary embodiment, determining the amount of a glycan structure at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) may include purifying haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) from a sample obtained from a subject, hydrolyzing the purified haptoglobin such that a glycopeptide comprising the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) and a respective glycan attached thereto is produced, and determining the amount of one or more glycopeptides having a glycan structure at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1). The amount of each glycan structure at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) may correspond to or be derived from the amount of each glycopeptide comprising the respective glycan structure at the N184 position.

[0151] In the present disclosure, determining the amount of a glycan structure at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) may include, for example, purification of haptoglobin from a sample, digestion of the purified haptoglobin with GluC and trypsin, and MS analysis to determine the amount of each glycopeptide having one or more respective glycan structures at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO: 1). The amount of each glycan structure at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO: 1) may correspond to or be derived from the determined amount of the corresponding glycopeptide comprising each glycan structure at the N184 position.

[0152] As will be appreciated by those skilled in the art, other methods can be used to determine the glycan structure at N184 of haptoglobin. Such analysis only requires that this glycan structure is or was attached to N184 of the haptoglobin β chain. Non-limiting examples of such methods are disclosed in Oh MJ, Lee SH, Kim U, An HJ. Mass Spectrom Rev. 2021. Similar methods can be used to detect glycan structures at positions N207, N211 and / or N241 of the haptoglobin β chain.

[0153] In embodiments, one or more glycopeptides comprising N184 of haptoglobin used to determine the amount of glycan structures may comprise the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO: 2), where N at position 6 of SEQ ID NO: 2 corresponds to N at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1) to which the respective glycan to be detected is attached. In certain embodiments, the peptide portion of one or more glycopeptides may consist of MVSHHNLTTGATLINE (SEQ ID NO: 2). In embodiments, the glycopeptide may be oxidized, particularly on methionine. The skilled artisan will understand that such oxidation may occur depending on the sample handling and the method used to detect such glycopeptide.

[0154] In an embodiment, the glycan structure whose amount is being determined is part of a glycopeptide, said glycopeptide comprising the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO:2), wherein N at position 6 of SEQ ID NO:2 corresponds to N at position 184 of the β chain of haptoglobin (SEQ ID NO:1).

[0155] In the context of the present invention, the level or amount of the N-glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) of the haptoglobin beta chain or the glycopeptide (e.g., formula 1) in the sample is determined. For this purpose, any suitable method known in the art may be used. In an embodiment of the method according to the present disclosure, the step of determining one or more N-glycan structures (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) of the haptoglobin beta chain or the corresponding glycopeptide (e.g., formula 1) comprises that the haptoglobin (comprising at least the beta chain) is purified before such determination is performed. Preferably, the purification of the haptoglobin is achieved using an antibody that binds to the haptoglobin. In particular, a monoclonal antibody that specifically binds to the haptoglobin may be used. Such monoclonal antibodies or antigen-binding fragments thereof can be biotinylated and used in combination with streptavidin-coated magnetic beads (SA beads). Non-limiting examples of such antibodies are commercially available, and monoclonal antibodies detecting human haptoglobin have been applied to various immunological methods in various publications. Non-limiting examples are Abcam, No. AB13429, clone HG-36; Abnova, No. MAB12976, clone 2F4; Acris Antibodies, No. UM500010, clone UMAB10; Novus Biologicals, No. NBP2-03008, clone OTI4H5; OriGene, No. TA00399, clone OTI2B8; Thermo Fisher Scientific, No. HYB 170-06-02, clone 9G10. In certain embodiments of the methods according to the present disclosure, determining one or more N-glycan structures (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) of the β chain of haptoglobin or the corresponding glycopeptide (e.g., Formula 1) comprises purifying the haptoglobin (including at least the β chain) using a biotinylated anti-haptoglobin monoclonal antibody in combination with streptavidin-coated beads (SA beads).In such a purification step, the sample may be incubated (simultaneously or sequentially) with biotinylated antibody and (e.g. magnetic or magnetizable) SA beads under conditions appropriate for antigen / antibody and biotin / streptavidin binding. The beads (with haptoglobin attached) are then separated (e.g. by using magnetic forces) from other components contained in the sample.

[0156] In an embodiment of the disclosure, the step of determining the amount of one (or more) N-glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at N184) of haptoglobin comprises chemically or biochemically / enzymatically cleaving / hydrolyzing the haptoglobin into peptides / glycopeptides. Various methods for hydrolysis of polypeptides by chemicals are known and can be used. In an embodiment, the haptoglobin is enzymatically cleaved into peptides / glycopeptides.

[0157] In an embodiment of the disclosure, the step of determining the amount of one (or more) N-glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) of haptoglobin comprises enzymatically cleaving haptoglobin into peptides / glycopeptides using the enzymes GluC and trypsin. GluC cleaves the protein chain C-terminal to the amino acid glutamic acid, while trypsin cleaves the protein chain C-terminal to the amino acids arginine and lysine. Both enzymes cleave with high specificity.

[0158] As shown above, one (or more) N-glycans (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) of haptoglobin or glycopeptides containing N184 (e.g., glycopeptide of formula 1) can be measured by mass spectrometry (MS). Various MS methods are known. Discovery and quantification of glycopeptides can be performed by high-resolution data-dependent mass spectrometry (Mol Cell Proteomics. 2014 Jan;13(1):329-338). The mass spectrometer selects the most intense peptides entering the mass spectrometer at a given time, then sequentially selects the most intense species for gas-phase fragmentation, and then analyzes the resulting fragments. The fragments are analyzed computationally by comparing the resulting fragment spectra with predicted spectra (obtained from a database), bearing sequence information of the peptide and glycan structures. Rarely, manual inspection of isotopic patterns is required to distinguish similar structures.

[0159] Highly sensitive determination of glycopeptides can also be performed by triple quadrupole mass spectrometry in single reaction monitoring mode (SRM) (Mol Cell Proteomics. 2013 Apr;12(4):1005-1016.). In this technique, the mass spectrometer selects only the target peptide. This peptide is fragmented in the gas phase, and unique and representative fragments are determined and quantified.

[0160] MS analysis of glycan structures or glycopeptides requires a significant number of handling steps. These handling steps may result in considerable variability, especially with respect to the measurement of (absolute) levels or amounts. To compensate for differences in sample handling, samples are usually spiked with an internal standard (usually the analyte of interest but in an isotopically labeled form). Sample handling affects the absolute values, but is unlikely to affect the ratio (glycan structure / glycopeptide) based on the same method of MS analysis. This ratio is not an absolute value, but rather a relative measure, and is therefore independent of differences in sample handling. It may therefore be advantageous to determine the amount of a glycan structure or glycopeptide relative to another glycan structure or glycopeptide, respectively. In an embodiment, this ratio may be the ratio between increased and decreased glycan structures at the same position of the polypeptide. Specific but non-limiting examples of such ratios are described elsewhere herein.

[0161] The term "amount" or "level" of an analyte (e.g., glycan or glycopeptide) in a sample as used herein refers to any absolute measure that corresponds to the amount or concentration of said analyte in the sample or is proportional to the absolute amount or concentration of said analyte in the sample, or any relative measure, i.e., a measure that expresses the amount or concentration relative to a reference amount or concentration, respectively, of said analyte. The reference amount or concentration may be the amount of an internal control (e.g., a standard glycopeptide, another glycopeptide or peptide detected in the sample) measured simultaneously with the analyte of interest using the same method.

[0162] In embodiments, the amount of a glycan structure at position N184 of haptoglobin in a sample can be the amount of a glycopeptide that contains the determined glycan structure at N184 from the sample. In these embodiments, the method can include generating a glycopeptide from haptoglobin (e.g., by protease digestion as described elsewhere). In embodiments, the peptide portion of the glycopeptide can be as described above.

[0163] In embodiments, the amount of glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal, respectively, of a non-glycosylated peptide (e.g., from haptoglobin). In embodiments, the amount of glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal, respectively, of all glycopeptides (e.g., haptoglobin) detected.

[0164] The term "change in amount" refers to the fact that the amount is outside the reference range, ie, either above a certain reference amount or below a certain reference amount. The term "elevation" or "increase" in the level or amount of a marker refers to an amount or level of such marker in the sample being investigated that is higher (i.e., does not contain) compared to the amount or level of such marker in a reference or control sample.

[0165] The term "reduction" in the level or amount of a marker refers to the amount or level of such marker in the sample being investigated being lower than (i.e., not containing) the amount or level of such marker in a reference or control sample.

[0166] As used herein, the term "score" taking into account the amount of a glycan structure or glycopeptide or "score for detecting HCC" refers to a score (e.g., value) obtained by combining the amount of a glycan structure or glycopeptide of the present invention with at least one further parameter, such as, for example, the amount of a second glycan structure, the amount of one or more other biomarkers, or a clinical parameter. Illustrative but non-limiting examples of other biomarkers are AFP, PIVKA-II, and AFP-L3. Illustrative examples of clinical parameters are age, sex, smoking status, ultrasound data, liver disease history, cancer history, and family cancer history. The score should be configured such that it can indicate HCC (e.g., early HCC).

[0167] "Taking into account" as used in the context of a score includes embodiments in which only one or more of the quantities and parameters specifically recited are taken into account, as well as embodiments in which other, unrecited parameters are taken into account.

[0168] Determining or calculating a score (e.g., a score for detecting HCC) can be accomplished in many different ways. As referred to herein, combining certain biomarker data or other information into a composite value can also be referred to as determining or calculating a score.

[0169] Determining or calculating the score includes any mathematical synthesis of the amount of glycan structures or glycopeptides mentioned herein and further parameters (see above). Thus, the score can be calculated in such a way that the individual parameters (including or consisting of the determined or received amount of glycans attached to N184 of haptoglobin or each glycopeptide) are mathematically synthesized. The level 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 may take into account one or more other factors other than the level of the PSA-glycoform species, such as, 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, stage of the disease and / or age).

[0170] In certain embodiments of the present invention, the score of the detection of HCC can be obtained by or can include a weighting calculation. This means that biomarkers (such as the amount of glycan structure at N184 position of haptoglobin) can be given different weights. For example, if the score takes into account the amount of a first glycan structure at N184 position of haptoglobin and the amount of another glycan structure at N184 position of haptoglobin in a sample, the score can be calculated by the following formula:

[0171] Score = a * [amount of first glycan structure] + b * [amount of second glycan structure], where a and b represent weighting coefficients. Preferably, the weighting coefficients or factors (a and b in the above example) are obtained by analyzing a control sample from a reference population (e.g., any reference population defined in the context of the reference values ​​below), etc. In an embodiment, the weighting coefficients or factors can be obtained by a machine learning method applied to a training data set obtained from a sample of a reference population defined herein.

[0172] Those skilled in the art will recognize that the score and the corresponding reference value of the score may be optimized based on a reference population (e.g., any of those defined herein or disclosed in the accompanying examples).

[0173] In an embodiment, the score may be a binary score and the corresponding reference value may be binary. "Binary" means that the score includes two values, for example, a first value is the determined or received level of one or more di-antenna PSA-glycoforms or a value derived therefrom, and a second value is the level of one or more mono-antenna PSA-glycoforms or a value derived therefrom. The "value derived therefrom" may be, for example, a value obtained by a 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.

[0174] Comparing the binary score to the reference value of 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. If a binary score is constructed from the amount of a first glycan structure at the N184 position of haptoglobin and the amount of a glycan structure at the N184 position of haptoglobin, HCC can only be detected if both amounts are indicative of HCC relative to their reference values ​​forming part of the binary score.

[0175] In aspects and embodiments using two or more markers (or even more general input parameters that may also include clinical parameters such as age or sex), they may be combined by a suitable algorithm (e.g., logistic regression) derived from a multivariate analysis of the available data, preferably in a full search for feature selection. This combination / calculation results in a multivariate score. Other methods, for example selected from DA (i.e., linear, quadratic, regularized discriminant analysis), Kernel Methods (i.e., SVM), Nonparametric Methods (i.e., k-nearest neighbor classifiers), PLS (Partial Least Squares), Tree-Based Methods (i.e., logistic regression, CART, random forest methods, boosting methods), may also be used to combine the biomarkers / input values ​​into a score.

[0176] The performance of the results of the applied mathematical / statistical methods used according to the present disclosure can be best described by their receiver operating characteristics (ROC). The ROC curve corresponds to both the sensitivity and specificity of the test. Thus, the sensitivity and specificity values ​​for a given biomarker or combination of biomarkers are indicators of the performance of the test. For example, if a combination of biomarkers has a sensitivity value of 80%, 80 out of 100 diseased patients will be correctly identified, or if it has a specificity value of 80%, 80 out of 100 patients without the disease will be correctly analyzed as negative for the disease.

[0177] As before, the ROC curve can be used to assess the performance of the aforementioned logistic regression model in discriminating between cases and controls.

[0178] The present disclosure, particularly in the context of the methods disclosed herein, refers to comparing the amount or level of a glycan structure, glycopeptide or score with a reference amount / level or score of said glycan structure, glycopeptide or score. It should be understood that such a comparison as used herein generally refers to a comparison of corresponding parameters, amounts, values ​​or scores, e.g., absolute amounts are compared to absolute reference amounts, while concentrations are compared to reference concentrations, or intensity signals obtained for glycan structures in a sample are compared to the same type of intensity signal obtained from a reference sample. The score is typically compared to a reference value of such a particular score that is indicative of HCC (e.g., early stage HCC). The comparison can be performed by a suitable device, e.g., a computer. The measured or detected level / amount value of the glycan structure in a sample from an individual or patient and the reference level / amount can, for example, be compared with each other, and the comparison can be performed automatically by a computer program that executes an algorithm for the comparison. The computer program that performs the evaluation provides the desired evaluation in a suitable output format. In a computer-assisted comparison, the determined amount value may be compared by the computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the results of the comparison, i.e. automatically provide the desired evaluation in a suitable output format. For computer-assisted comparison, the determined quantity values ​​may be compared by the computer program with values ​​corresponding to suitable references stored in a database. The computer program may further evaluate the results of the comparison, i.e. automatically provide the desired evaluation in a suitable output format.

[0179] The terms "glycan structure" or "glycan" are used interchangeably. In the present disclosure, the glycan investigated is an N-glycan, and thus the terms glycan and N-glycan are used interchangeably herein. The glycan or glycan structure is composed of various types of carbohydrates. The glycan structure can be, for example, linked to an amino acid, for example, the amino acid asparagine. In this case, the glycan linked to the asparagine at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1) is analyzed. The glycan structure or glycan of the present disclosure serves as a biomarker or marker for HCC (e.g., early stage HCC).

[0180] The term "glycopeptide" is used to refer to a peptide or peptide fragment of a larger polypeptide that comprises an amino acid to which a glycan is covalently attached. In the present disclosure, the glycopeptide that is preferably analyzed is a peptide sequence derived from the β-chain of haptoglobin and contains the amino acid asparagine (N) at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1). Preferred embodiments in which the peptide portion of the glycopeptide is preferably detected or quantified are disclosed elsewhere herein.

[0181] Glycans are typically represented herein as sum formulas. In the present specification, we represent glycan compositions as follows: HexNAc(x)Hex(x)Fuc(x)NeuAc(x). The numbers in parentheses represent the number of monomers in the glycan. This nomenclature is well known to those skilled in the art of glycan biology. HexNAc can be GlcNAc or GalNAc (in embodiments, GlcNAc), and Hex can be Glu or Gal (in embodiments, Gal). HexNAc means N-acetylhexosamine, GlcNAc means N-acetylglucosamine, GalNAc means N-acetylgalactosamine, Hex means hexose, Man means mannose, Glu means glucose, Gal means galactose, Fuc means fucose, and NeuAc or Neu5Ac means N-acetyl-neuraminic acid (sialic acid).

[0182] Preferred glycans are represented by a specific formula or using a schematic diagram according to 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). An exemplary glycan is the glycan of formula 2:

[0183] In an embodiment of the invention, the glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) has the formula 2: [ka] (Formula 2) may have the following structure:

[0184] As used in formulae herein (e.g., Formula 1 and Formula 2), GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetyl-neuraminic acid (or sialic acid). The lines represent the covalent bond between monohalides. GlcNac below is the monosaccharide that attaches the glycan to a peptide or protein (e.g., N184 in SEQ ID NO: 1) when it is part of a glycopeptide or glycoprotein, respectively. The brackets used in the context of NeuAc (or Neu5Ac, used synonymously) indicate that NeuAc can be attached to any of the three galactose residues.

[0185] As used herein, the term "reference amount" (or "reference level") for an analyte (e.g., a glycan structure or a glycopeptide) refers to an independently established, predetermined amount of said analyte. Those skilled in the art will understand that the reference amount is predetermined and set, for example, to meet routine requirements with respect to specificity and / or sensitivity for the purpose of detecting HCC (e.g., early stage HCC). Thus, the reference amount can be selected such that it is indicative of HCC (e.g., early stage HCC). The requirements for detecting HCC can vary, for example, from one regulatory agency to another. For example, the sensitivity or specificity of an assay may have to be set at a certain limit, for example, 80%, 90%, 95%, or 98%, respectively. These requirements may also be defined in terms of positive predictive value or negative predictive value. For example, for any requirement selected, respectively, with respect to the level of sensitivity or specificity, the reference range (when an assessed or reduced value indicates an abnormal state) or the reference level or cut-off level (when an assessed or reduced value indicates an abnormal state) can be determined by the skilled artisan. Similarly, the reference ratio can be determined according to the same principle.

[0186] As used herein, the term "reference value" in the context of, for example, a reference value of a score, refers to a predetermined value that is established independently for each parameter (e.g., score). Those skilled in the art will understand that the reference value is predetermined and set to meet routine requirements, for example, for the purpose of detecting HCC (e.g., early stage HCC) in terms of specificity and / or sensitivity. Thus, the reference value can be selected such that it indicates HCC (e.g., early stage HCC). What has been said above regarding the reference amount applies mutatis mutandis.

[0187] The reference amount or value of the score is typically determined in one reference sample or a reasonable number of reference samples. A reference sample is also called a control sample. Generally, the reference sample is obtained from an individual or a group of individuals known to suffer from or at risk of a given condition, or from an individual or a group of individuals known to be free of a given condition, i.e., "normal" or "healthy" individuals. Usually, the marker level of the sample is directly or indirectly correlated with a diagnosis, and the marker level is used to determine, for example, whether an individual is at risk of HCC. Depending on the intended diagnostic application, a suitable reference sample is selected, in which the control or reference value of the marker is set. It will be understood by the skilled artisan that such a reference sample in one embodiment is obtained from a reference population that is age-matched and free of confounding diseases. As will be clear to the skilled artisan, the absolute marker value established in a reference sample or set of reference samples (e.g., forming a reference population) will vary depending on the assay used. Preferably, samples from 100 well-characterized individuals from a suitable reference population are used to establish the reference amount or value. A preferred reference population may also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a reference population that is frequently used to establish a control or reference amount or value. In one embodiment, the reference level or value is determined in a reference sample obtained from a healthy individual.

[0188] For the detection of HCC in clinical routine, it is of utmost importance to obtain an indication that HCC may develop or exist in patients with cirrhosis or other HCC risk factors.Therefore, in an embodiment, the reference level is determined in a reference sample obtained from a patient with cirrhosis.

[0189] In embodiments, reference populations from which control or reference samples may be obtained include samples obtained from control subjects (e.g., healthy individuals or subjects with liver disease who are at high risk of developing HCC) and subjects suffering from HCC (e.g., early stage HCC).

[0190] As will be appreciated by those skilled in the art, any method or use for detecting or aiding in the detection of HCC may include a step of detecting or aiding in the detection of HCC, which aid in detection or detection is typically achieved based on a comparison step of such a method.

[0191] Alpha-fetoprotein is a glycoprotein, and various glycosylated forms of AFP have been reported. Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the glycan structures of glycoproteins, it is possible to separate and enrich one or more marker glycoprotein fractions with specific glycan structures. For AFP, lectins derived from lentil lectin-A (LCA) are widely used. The lentil lectin (LCA) reactive fraction of alpha-fetoprotein (AFP-L3) is specifically increased in HCC patients. Many attempts have been made to specifically measure AFP-L3, for example, by affinity electrophoresis using LCA, lectin-based ELISA, or by antibodies that specifically bind to the L3 form of AFP.

[0192] Protein induced by Vitamin K Absence / Antagonist-II (PIVKA-II), also known as des-gamma-carboxy-prothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and is used alone or in combination with AFP as a surrogate HCC biomarker. Prothrombin has 10 potential gamma-carboxylation sites, and various forms of PIVKA-II with different levels of carboxylation exist in the blood circulation. Different assays of PIVKA-II may detect different sets of PIVKA-II-forms, and the specificity / sensitivity of PIVKA-II may vary depending on the assay used, limiting its usefulness in detecting early HCC. As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding properties.

[0193] As used herein, the terms "specific binding" or "specifically bind" refer to a binding reaction in which the binding pair molecules exhibit binding to one another under conditions in which they do not significantly bind other molecules. The terms "specific binding" or "specifically binds" when referring to an antibody or protein or peptide as a binding agent means that the binding agent binds to a corresponding target molecule. -7 K below M D The term "specific binding" or "specifically binding" preferably refers to a binding reaction in which the target molecule is bound to the target molecule at a specific binding site. -8 M or less, or even more preferably 10 -9 K below M D The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the binding agent specific for the target molecule. Preferably, the level of binding to molecules other than the target molecule results in a binding affinity that is no more than 10% of the affinity for the target molecule, more preferably no more than 5%. In other words, preferably, the K D is at most 10-fold, more preferably at most 20-fold, lower than that of binding to a non-target protein.

[0194] The disclosed methods and uses provide valuable information regarding the presence of HCC in a subject. In certain embodiments, detection by the disclosed methods and uses is used to provide treatment recommendations to the subject.

[0195] Of course, the disclosed methods and uses can be performed remotely from the subject or his / her doctor, and can even be performed overseas and the results returned. In certain embodiments, the results of the methods of the present invention (e.g., determining whether the subject has HCC) are provided to a third party, such as the subject or his / her doctor, a laboratory, or a health center.

[0196] The elements of the present invention will now be described. Although these elements are listed as aspects having specific embodiments, it will be understood that they may be combined in any manner and in any number to create additional aspects and embodiments. In particular, an embodiment disclosed in the context of one aspect applies mutatis mutandis to other aspects. The various described examples and preferred embodiments should not be construed to limit the invention to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0197] All references mentioned in this specification are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0198] The following examples and figures are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Also provided are descriptions of the amino acid sequences disclosed herein.

[0199] Array Description SEQ ID NO:1 shows the amino acid sequence of human haptoglobin, with N184 printed in bold and underlined. MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT EGDGVYTLND KKQWINKAVG DKLPECEADD GCPKPPEIAH GYVEHSVRYQ CKNYYKLRTE GDGVYTLNNE KQWINKAVGD KLPECEAVCG KPKNPANPVQ RILGGHLDAK GSFPWQAKMV SHH N LTTGAT LINEQWLLTT AKNLFLNHSE NATAKDIAPT LTLYVGKKQL VEIEKVVLHP NYSQVDIGLI KLKQKVSVNE RVMPICLPSK DYAEVGRVGY VSGWGRNANF KFTDHLKYVM LPVADQDQCI RHYEGSTVPE KKTPKSPVGV QPILNEHTFC AGMSKYQEDT CYGDAGSAFA VHDLEEDTWY ATGILSFDKS CAVAEYGVYV KVTSIQDWVQ KTIAEN (Sequence listing; Uniprot P00738, version 221).

[0200] SEQ ID NO:2 shows the amino acid sequence of a peptide portion contained in an exemplary glycopeptide for detecting a glycan structure at position N184 (e.g., a glycopeptide of formula 1), which corresponds to amino acid positions 179 to 194 of SEQ ID NO:1, with the Asn residue corresponding to N184 of SEQ ID NO:1 printed in bold and underlined. MVSHH N LTGATLINE

[0201] SEQ ID NO:3 shows the amino acid sequence of a peptide contained in an exemplary glycopeptide for detecting a glycan structure at position N207, with the Asn residue corresponding to N207 in SEQ ID NO:3 printed in bold and underlined. NLFL N HSE

[0202] SEQ ID NO: 4 shows the amino acid sequence of a peptide contained in an exemplary glycopeptide for detecting a glycan structure at position N211, with the Asn residue corresponding to N211 in SEQ ID NO: 4 printed in bold and underlined. N ATAK

[0203] SEQ ID NO:5 shows the amino acid sequence of a peptide contained in an exemplary glycopeptide for detecting a glycan structure at position N241, with the Asn residue corresponding to N241 in SEQ ID NO:5 printed in bold and underlined. VVLHP N YSQVD EXAMPLES

[0204] Example 1 1.1 Experimental setup EDTA-plasma samples were obtained from 57 controls representing chronic liver diseases such as HBV, HCV and cirrhosis, and HCC patients, including 33 with early stage and 32 with late stage HCC (see Table 1 for demographic information).

[0205] Staging of the samples was based on the Barcelona Liver Cancer (BCLC, Llovet JM, Bru C, Bruix J, Semin Liver Dis. 1999;19(3):329-38) approach, with BCLC stages 0 and A classified as early HCC and stages B-D as late HCC. After plasma preparation, samples used in this analysis were stored at -80°C until analyzed as described below. Repeated freezing and thawing of samples was avoided. [Table 1] Table 1: Summary of demographic variables grouped according to clinical status.

[0206] 1.2 Hp immunoprecipitation from plasma In the first step, the immunocapture beads were prepared. 10 mg of streptavidin (SA)-coated latex beads were co-incubated with 150 μg of biotin (Bi)-labeled F(ab')2 fragments of mouse monoclonal antibody against Hpβ chain MAK<haptoglobin>M-1.1.13-F(ab')2-Bi (Roche Diagnostics GmbH, Mannheim, Germany) for 1 h at room temperature on a rotator. The co-incubation buffer used was phosphate-buffered saline (PBS buffer: 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH: 7.4). After coating the F(ab')2 fragments on the beads, the resulting coated beads were washed three times with PBS. 50 microliters of each plasma sample was incubated with 5 μl of 20 mM DTT (dithiothreitol) for 1 h at 37 °C. The samples were then treated with 10 μl of 50 mM IAM (iodoacetamide) for 30 min at room temperature. Afterwards, the samples were diluted with 2 ml of PBS and incubated with MAK<haptoglobin>M-1.1.13-F(ab')2-Bi-SA-beads (antibody-bound beads from the previous step). After 2 h of incubation at room temperature on a rotator, unbound proteins were washed away using two washing steps with PBST (PBS buffer + 0.1% Tween 20®) and two washing steps with PBS buffer. Hp bound to the antibody-coated beads was eluted from the beads by incubating the beads in 500 μl of glycine buffer (0.2 M glycine, pH 2.6) in two steps. The two-step elution facilitated the recovery. To adapt the pH, 200 μl of NaOH (1 N) was added to the eluate after each elution step.

[0207] 1.3 FASP digestion The two eluted Hp fractions from the previous step were combined and loaded into Nanosep Centrifugal Devices equipped with an Omega™ Membrane-10K (PALL, USA) filter (cut-off: 10 kDa) and the samples were centrifuged at 10,000 g for 20 min. In this step, molecules, e.g., proteins with molecular weights of 10,000 or more, remain on the filter while small molecules, e.g., salts, pass through the filter and are removed. Then, 75 ng of heavy haptoglobin (recombinant Hp with isotope-labeled heavy chains Lys and Arg) protein was added to each sample / filter as an internal standard. Then, 50 μl of denaturing buffer (1 mg / ml PPS (3-[3-(1,1-bisheptyloxyethyl)pyridin-1-yl]propane-1-sulfonate) in 50 mM ammonium bicarbonate) and 5 μl of DTT (10 mM) were added to the samples and the samples were incubated at 50° C. for 30 min. In the next step, 5 μl of IAM (55 mM) was added to the samples and they were incubated at 37° C. for 30 min in the dark. The samples were centrifuged at 10,000 g for 20 min to wash out the buffer and 100 μl of ABC (ammonium bicarbonate) were added to the samples. The filters were washed once with 50 μl ABC buffer (50 mM). Proteins remaining on the filters were then first digested by adding 6 μg trypsin in 50 μl ABC buffer for 3 h at 37° C. Trypsin digestion was stopped by incubating the filters at 95° C. for 10 min. For the second digestion, 10 μg GluC was added to the samples. For this second digestion, the samples were incubated overnight at 25° C. The digested samples were eluted from the membrane by centrifugation at 10,000 g for 20 min.

[0208] 1.4 LC-MS / MS analysis Twenty microliters of peptides obtained by the enzymatic digestion described in 1.3 were injected into an LC-MS / MS system (HF-X mass spectrometer (Thermo Fisher Scientific, Germany) coupled to a Vanquish (Thermo Fisher Scientific, Germany) UHPLC system). Peptides were separated by reversed-phase chromatography on a C18 column (Waters, XSelect CSH C18 column, 130 Å, 3.5 μm, 2.1 mm × 150 mm) at 50 °C. The LC flow rate was 320 μl / min, and the gradient was set as follows: 0% to 30% B (0–30 min), 30% to 80% B (30–31 min), 80% B (31–36 min), 80% to 0% B (36–37 min), and 0% B (37–42 min) (eluents A and B were HO containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively). The separated peptides were ionized by electrospray ionization (ESI) source and analyzed in positive ion mode and data-dependent acquisition. Full scan MS spectra were acquired in the m / z range of 300-2000 with a resolution of 60,000, 10e6 autogain control (AGC), and 50ms injection time. The top five most intense peaks from this scan, the survey scan, were selected for fragmentation by higher energy collisional dissociation (HCD) with a normalized collision energy of 28%, a resolution of 15000, 1e5 AGC, and 150ms injection time.

[0209] 1.5 AFP and PIVKA-II assays AFP and PIVKA-II were measured using microchip capillary electrophoresis and liquid-phase binding assays with a uTASWako i30 automated analyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions.

[0210] 1.6 Data Analysis Raw files obtained from the mass spectrometer were processed by the Byonic (Proteome Quantitative, CA, USA) search engine integrated in Proteome Discoverer 2.2 (Thermo Fisher Scientific). The dataset was searched against the Uniprot haptoglobin protein sequence (P00738). For glycopeptide identification, the Byonic curated database was used. Byonic / Proteome Discoverer was configured as follows: Mass tolerance was set to 10 ppm for MS1 and 20 ppm for MS2. GluC and trypsin were set as proteases, with two missed cleavages allowed. Carbamidomethylation of cysteine ​​was set as fixed modification, while methionine oxidation and glycosylation on asparagine were designated as variable modifications. Results were filtered with a false discovery rate (FDR) of 1% and a confidence threshold of Byonic score >100. Glycopeptide compositions with significant differences between cohort groups and AUC >0.7 were manually checked. In these cases, we confirmed the retention time, charge state, glycan oxonium ion and isotopic pattern by comparing it with the predicted isotopic pattern of the proposed glycopeptide. In this paper, we represent the glycan composition as HexNAc(x)Hex(x)Fuc(x)NeuAc(x), where the numbers in brackets represent the number of each monomer present in the glycan structure. HexNAc means N-acetylhexosamine, Hex means hexose, Fuc means fucose, and NeuAc or Neu5Ac means N-acetyl-neuraminic acid (sialic acid).The schematic diagram is based on the SNFG (Symbol Nomenclature for Glycans) system (PMID 26543186, Glycobiology) of NCBI (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). 25:1323-1324, 2015). The peak areas of glycopeptide XICs were automatically integrated by Proteome Discoverer and used as relative quantification values.

[0211] 1.7 Statistical analysis To correct for possible handling, digestion or MS measurement variability, the abundance of glycopeptides in the samples was normalized to the abundance of the top three peptides spiked with heavy Hp. Missing values ​​for a particular glycopeptide (i.e., glycopeptides below the detection limit) were replaced by the minimum abundance value of that glycopeptide in the data set. The significance of the differences in glycopeptides between clinical groups was examined by calculating p-values ​​using the Wilcoxen (Mann-Whitney U) test. To correct for multiple testing, the Benjamini-Hochberg correction with a 20% FDR control was used. To evaluate the diagnostic value of glycopeptides, receiver operating characteristic (ROC) curves were constructed and area under the curve (AUC) values ​​were calculated by the DeLong method (Elisabeth R. DeLong, David M. DeLong and Daniel L. Clarke-Pearson (1988), Biometrics 44, 837-845) using R software (version 3.5.2) available at https: / / www.R-project.org.

[0212] 2.Results 2.1 Glycan performance for HCC diagnosis Global analysis of glycan characteristics (e.g., fucosylation) provides insight into the main changes in the glycosylation pattern of Hp during HCC progression. However, the types and abundance of glycoforms differ at different Hp glycosites. Therefore, we investigated whether specific analysis of Hp glycosylation sites and glycoforms could be indicative of HCC. We compared site-specific glycopeptide levels between controls, early HCC, and late HCC. Our observations revealed specific glycopeptides that were significantly upregulated in early HCC compared to controls with an AUC of 0.70 or higher and could be clearly assigned to specific glycan structures (Figure 6). This set of glycopeptides includes highly branched and fucosylated glycopeptides (compounds 126, 131, or 140) and highly mannosylated glycopeptides (compounds 58, 60, 24, and 27) and sialylated glycopeptide (compound 126).

[0213] Besides the early HCC-specific biomarkers, we found 14 other glycopeptides that were significantly highly expressed in both early and late HCC and provided an AUC of 0.70 or higher in distinguishing early HCC patients from cirrhosis controls (Figure 6). Among these, compound 41 showed the best AUC. These glycopeptides were more expressed as HCC progressed from early to late. The top 5 Hp glycopeptides that were significantly highly expressed in both early and late HCC are listed in Figure 4.

[0214] In addition to the upregulated glycopeptides in HCC, we identified two glycopeptides that were significantly downregulated in early and late HCC compared to cirrhosis located at the N207 glycosylation site (Figure 6). One of the glycoforms is a biantennary glycan with two sialic acids (HexNAc(4)Hex(5)NeuAc(2)), one of the most abundant glycans. The other glycoform is a biantennary glycan with one sialic acid (HexNAc(5)Hex(5)NeuAc(1)). These downregulated glycopeptides can be used to build a ratio with upregulated glycopeptides (e.g., compound 126 or compound 41) to further improve robustness by serving as an internal control. In total, we identified 29 glycan structures that distinguished early and late HCC from controls with an AUC of more than 70% (Figure 6).

[0215] By far the best glycopeptide for detecting early and late HCC was compound 41 on N184 (see Figures 4 and 5). Thus, among all glycolytic sites analyzed herein, haptoglobin glycosylation at position N184 appears to be the most suitable for detecting all stages of HCC. Figures 4 and 6 show the data for all altered glycopeptides using an AUC of 0.70 or greater as a cutoff.

[0216] The results, glycan structures, peptide sequences and m / z values ​​of the detected glycopeptides are summarized in Tables 2 and 3 below.

[0217] These results consistently revealed that glycopeptide analysis of Hp, especially at position N184 (but also at positions N207, N211 and N241), could provide glycobiomed- ic markers with better clinical value than established biomarkers for the diagnosis of early HCC. [Table 2] Table 2: Glycan structures, peptide sequences and m / z values ​​of detected glycopeptides are significantly different between early and late HCC compared to controls. The AUC[%] and direction of expression change for each group tested are shown. *Methionine residues may be oxidized. [Table 3] Table 3: Glycan structures, peptide sequences and m / z values ​​of detected glycopeptides are significantly different between early and late HCC compared to controls. The AUC[%] and direction of expression change for each group tested are shown. *Methionine residues may be oxidized.

[0218] 2.2 Top Glycans for Detection of HCC (Early or Early and Late) The results indicate that especially Hp glycopeptide compound 41 (i.e., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at N184 position) is likely to have clinical utility in detecting HCC, especially early stage HCC. These are evident from the area under the curve (AUC) values ​​shown for each of these glycopeptides. It is noted that for this glycopeptide, the calculated AUC exceeds the AUC value of AFP, and the glycopeptide improves the performance of each individual protein biomarker and their combination. Other Hp glycopeptide compounds with good diagnostic potential for early stage HCC are shown in Table 2 and Figure 6.

[0219] In addition, several glycans were identified that had good performance in identifying early and late HCC cases. In particular, compound 41 has high potential clinical utility. This Hp glycopeptide has the best AUC for early HCC and also shows a significant AUC for late HCC. Other Hp glycopeptide compounds with good diagnostic potential for early and late HCC are shown in Table 2 and Figures 4 and 6.

[0220] 2.3 Marker Combinations To analyze the added value of compound 41 to PIVKA-II and / or AFP, logistic regression models consisting of compound 41, PIVKA-II and / or AFP were constructed. In these models, logarithmic transformation was applied to the markers to reduce the skewness of the marker distribution. For all compounds, these multivariate models were constructed and their performance in the form of combined AUC was compared. The combinations of markers are summarized in Table 4. Other models other than logistic regression (or logistic regression models with interaction terms between variables) could not significantly improve the performance of the aforementioned logistic regression models. [Table 4] Table 4: Combining glycopeptide compound 41 improves the clinical value of AFP. The AUC value of AFP for early diagnosis of HCC in our cohort is 83%. Combining these two biomarkers improves the AUC to 87%, and the combination of glycopeptide with AFP and PIVKA-II improves the AUC to 93%.

Claims

1. An in vitro method to support the detection of hepatocellular carcinoma (HCC) in a subject, a) A step of determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin (i.e., the β chain of haptoglobin having the sequence shown in Sequence ID No. 1) in the sample obtained from the subject, b) A step of comparing the amount of the N-glycan structure determined in (i) a) with a reference amount of the N-glycan structure, wherein an increase in the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position 184 of haptoglobin in the sample with respect to the reference amount of the N-glycan structure indicates HCC, or (ii) A step of determining a score for detecting HCC, taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin determined in (ii) a), and comparing the determined score for detecting HCC with a reference value of the score indicating HCC. In vitro methods, including

2. An isolated glycopeptide having a peptide portion and an N-glycan portion, wherein the peptide portion contains or consists of the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO: 2), the N-glycan portion is HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and the N-glycan portion is bonded to the N at position 6 of SEQ ID NO:

2.

3. Formula 1 【Chemistry 1】 (Formula 1) The glycopeptide according to claim 2, having the structure shown.

4. Use of the glycopeptide according to claim 2, in an embodiment, to support the detection of early HCC.

5. The method according to claim 1, further comprising determining the amount of AFP in the aforementioned sample or another sample from the same subject, the score for detecting HCC taking into account the amount of AFP for which the score was determined.

6. The method according to claim 1 or 5, wherein the haptoglobin is hydrolyzed to a glycopeptide, the glycopeptide comprising the glycopeptide described in claim 2 or 3, and the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of the haptoglobin is determined by determining the amount of the glycopeptide described in claim 2 or 3, or corresponds to the amount of the glycopeptide described in claim 2 or 3.

7. The method according to claim 6, comprising purifying the haptoglobin from the sample before the haptoglobin is hydrolyzed.

8. The method according to claim 1, wherein the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin is determined by mass spectrometry.

9. A method for detecting and / or quantifying the glycopeptide described in claim 2 or 3, a) A step of purifying haptoglobin from the sample to be analyzed, b) A step of digesting the haptoglobin obtained in step a) with GluC and trypsin, A step of detecting the glycopeptide according to claim 2 or 3 by detecting the glycopeptide obtained in c) b), Methods that include...

10. A computer implementation method for supporting the detection of hepatocellular carcinoma (HCC) in a subject, a) Receiving data including the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) in the sample obtained from the subject, and b) Comparing the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) received in i) a) with a reference amount of the glycan structure, wherein an increase in the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin in the sample with respect to this reference amount of glycan structure indicates HCC, or (ii) Calculating a score for detecting HCC taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at the N184 position of haptoglobin received in a), and comparing the calculated score for detecting HCC with a reference value of the score indicating HCC, and To assist in determining whether the subject has HCC based on the comparison of c) and b), Computer implementation methods, including those mentioned above.

11. The computer implementation method according to claim 10, further comprising receiving data including the amount of AFP in the sample or another sample from the same subject, wherein the calculation of the score for detecting HCC in c) further takes into account the determined amount of AFP.

12. A computer program product, wherein when the program is executed by a computer, it includes an instruction that causes the computer to execute the computer implementation method described in claim 10 or 11.

13. The method according to any one of claims 1, 5, 8, 10, and 11, or the use according to claim 4, wherein the HCC is early HCC.

14. The method according to any one of claims 1, 5, 8, 10, and 11, wherein the sample is a blood sample, particularly serum or plasma.

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