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, comprising determining the amount of one or more glycan structures at position N207 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. Glycan structures and glycopeptides comprising said glycan structures are also disclosed, both of which have great utility in the detection of HCC. Furthermore, the present invention relates to the use of one or more glycan structures at position N207, or glycopeptides comprising N207 of haptoglobin in combination with AFP and / or PIVKA in the detection of HCC.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an in vitro method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, comprising determining the amount of one or more glycan structures at position N207 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, and comparing the amount of the one or more glycan structures to a reference amount of the one or more glycan structures, wherein an altered amount of the one or more glycan structures in the patient sample relative to the reference amount of the one or more glycan structures is indicative of HCC.Furthermore, the present invention relates to the use of one or more glycan structures at position N207 or a glycopeptide comprising N207 of haptoglobin in combination with AFP and / or PIVKA-II in the detection of HCC. [Background technology]

[0002] 2. Background of the Invention 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 leading histological type among primary liver cancers 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.

[0004] If this malignancy is diagnosed at an early stage, 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 lack 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 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 requires a tumor mass of at least 2 cm, and imaging techniques have poor prognostic value due to their low sensitivity per lesion and high cost. Meanwhile, in recent years, much focus has been placed on discovering 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 techniques include low sensitivity for early-stage tumors, operator dependence, 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 shown in recent years 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, including liver, breast, lung, prostate, pancreatic, 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] Ramachandran P. et al. (2022) recently described serum glycoprotein markers in nonalcoholic steatohepatitis and hepatocellular carcinoma. The study also included mass spectrometry-based glycan assessment at amino acid residue positions 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. It has also been reported that AFP levels may be falsely elevated 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 marker glycoprotein fraction(s) with specific glycan structures. For AFP, lectins derived from lentil agglutinin-A (LCA) are widely used. The lentil agglutinin (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 / high-sensitivity qualitative / quantitative analysis using mass spectrometers 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) technology can obtain product ion spectra of peptides using a mass spectrometer equipped with a trap and a time-of-flight mass spectrometer, allowing quantitative and qualitative analysis of peptides to be performed simultaneously. This method can also analyze trace amounts of glycoproteins that show low signals with good reproducibility and sensitivity.

[0012] Methods for analyzing specific glycans using mass spectrometry include methods based on the analysis of glycan(s) separated from glycoproteins and methods based on the analysis of peptides to which glycans are attached, i.e., glycopeptides(s).

[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 vary depending on the amino acid position at which the glycan is located.

[0014] Based on MRM-MS analysis, Kim and coworkers demonstrated that deglycopeptides of alpha-fetoprotein are better able to distinguish cancer status between normal subjects and hepatocellular carcinoma patients than nonglycopeptides (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 3415918, 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 individually or in combination with AFP as an alternative HCC biomarker. Prothrombin has 10 potential gamma-carboxylation sites, and various forms of PIVKA-II with different levels of undercarboxylation exist in the circulation. Different assays for PIVKA-II may detect different sets of PIVKA-II-forms, and the specificity / sensitivity of PIVKA-II may vary depending on the assay used and its limited usefulness in detecting early-stage 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 will 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] Summary of the Invention The inventors have found that analysis of the glycan structure(s) at position 207 of the beta chain of haptoglobin (i.e., at amino acid position 207 of SEQ ID NO:1) may overcome some of the problems of current in vitro diagnostic methods aiding in the detection of HCC.

[0020] 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 N207 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 an altered 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.

[0021] In certain embodiments, the one or more glycan structures attached to N207 is the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0022] In a second embodiment, 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-strand 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 N-glycan structure determined in (i)a) to a reference amount of the N-glycan structure, wherein the amount of haptoglobin in the subject's sample relative to the reference amount of the N-glycan structure is or (ii) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin determined in a) and comparing the determined score for detecting HCC (e.g., early stage HCC) to a reference value for said score that is indicative of HCC (e.g., early stage HCC).

[0023] In a third 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 the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of said haptoglobin in said sample obtained from said subject; c) determining a score for detecting HCC (e.g., early stage HCC) that takes into account or consists of the ratio of the two glycan structures determined in a) and b), respectively, by dividing a) by b) or vice versa; and d) comparing the score determined in c) to a reference score value indicative of HCC (e.g., early stage HCC). Such a comparison can then be used to determine the presence (or absence) of HCC.

[0024] In a fourth 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 the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of said haptoglobin in said sample obtained from said subject; c) determining a score for detecting HCC (e.g., early stage HCC) that takes into account or consists of the ratio of the two glycan structures determined in a) and b), respectively, by dividing a) by b) or vice versa; and d) comparing the score determined in c) to a reference score value indicative of HCC (e.g., early stage HCC). Such a comparison can then be used to determine the presence (or absence) of HCC.

[0025] In a fifth 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 NLFLNHSE (SEQ ID NO:2), and the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and the N-glycan portion is linked to the N at position 5 of SEQ ID NO:2.

[0026] In a preferred embodiment of the fifth aspect, provided herein is a glycopeptide of formula 1: [ka] where GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, 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 through the indicated GlcNac of the glycan to the side chain of an asparagine that corresponds to N207 of haptoglobin (i.e., N207 of the β-chain of haptoglobin in SEQ ID NO:1).

[0027] A peptide contained in this glycopeptide has the amino acid sequence shown in SEQ ID NO: 2. An alternative graphical representation of a glycopeptide having the same structure as shown in Formula 1 above is shown in the right panel of FIG.

[0028] In a sixth aspect, the present invention relates to the use of the glycopeptide of formula 1 (see above) in the detection of HCC.

[0029] According to a seventh aspect of the present disclosure, there is provided a method for detecting a glycan structure at position N207 of haptoglobin (i.e., the beta 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 a glycopeptide comprising position N207 of haptoglobin having attached thereto the glycan structure produced in b), thereby detecting the glycan structure at position N207.

[0030] In an eighth 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 N207 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 an HCC biomarker other than the N-glycan structure at position N207 at a) (e.g., the amount of AFP and / or the amount of PIVKA) in a sample obtained from the subject; c) combining the amounts determined in a) and b) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC) and comparing the binding value with a reference value for the binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0031] In a ninth aspect, the present disclosure relates to a clinical workflow for screening HCC (e.g., early stage HCC) in a subject, comprising: a) determining the amount of one or more N-glycan structures at position N207 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 determining the amount of PIVKA-II in a sample obtained from the subject; c) combining the amounts of markers determined in a) and b), and optionally d) further using the results of an ultrasound (US) investigation; an elevated score for the markers determined in a) and b), and / or optionally a positive US result, indicates HCC (e.g., early stage HCC).

[0032] In a tenth aspect, there is provided herein a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of a glycan structure at position N207 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) i) comparing the amount of the glycan structure received in a) with a reference amount of said N-glycan structure at position N207 of haptoglobin, wherein an altered amount of the N-glycan structure in said patient sample relative to the reference amount of said one or more glycan structures is indicative of HCC, or (ii) calculating a score for detecting HCC (e.g., early stage HCC) taking into account the amount of said glycan structure at position N207 of haptoglobin received in a) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). Includes.

[0033] In an eleventh aspect, there is provided a computer-implemented method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, the method comprising: a) receiving data comprising the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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) i) comparing the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) received in a) with a reference amount of said glycan structure, wherein an increase in HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in the subject 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin received in a) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). Includes.

[0034] In a twelfth aspect, the present invention provides a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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) receiving data comprising the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of the haptoglobin in the sample obtained from the subject; c) calculating a score that includes or consists of the ratio of the amounts of the two glycan structures of a) and b) (or vice versa); and d) comparing the score calculated in c) with a reference value for said score indicative of HCC (e.g., early stage HCC). Includes.

[0035] In a thirteenth aspect, there is provided herein a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of one or more glycan structures (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4)) at position N207 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 amounts determined in a) and b); and d) comparing the score calculated in c) with a reference value for said score indicative of HCC (e.g., early stage HCC). Includes.

[0036] The present specification also provides a computer program product, the computer program product including instructions that, when executed by a computer, cause the computer to execute the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0037] Further provided is a computer readable medium comprising instructions that, when executed by a computer, cause the computer to perform a computer-implemented method according to any one of the tenth to thirteenth aspects.

[0038] There is also provided a data processing system comprising: a receiving unit configured to receive data as defined in any one of the tenth to thirteenth aspects; a processing unit configured to perform the calculation and / or comparison steps and / or any decision / evaluation steps in any one of the tenth to thirteenth aspects; and optionally an output unit configured to output a result of the evaluation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] 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.

[0040] 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 N207 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 an altered 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.

[0041] In an embodiment of a first aspect of the present invention, there is provided an in vitro method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, comprising: a) determining the amount of a glycan structure at position N207 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 an altered amount of the glycan structure in the patient sample relative to the reference amount of the glycan structure is indicative of HCC.

[0042] The method of the first aspect of the present invention is based on the finding that the glycosylation pattern at position 207 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 HCC (e.g., early stage HCC) patients show a characteristic pattern, for example, compared to reference samples and compared to samples from cirrhosis patients.

[0043] In an embodiment of the first aspect of the present invention, the determined amount(s) of one or more glycan structures at position N207 of haptoglobin may not be compared to a reference amount of each of the glycan structures, but instead, a score for determining HCC (e.g., early stage HCC) may be calculated that takes into account the determined amount(s) of one or more glycan structures at position N207 of haptoglobin. The score may be compared to a respective reference value for the score that determines the presence of HCC (e.g., early stage HCC).

[0044] Thus, an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject is provided herein, the method comprising: a) determining the amount of glycan structure at position N207 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; and b) determining a 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 determined for detecting HCC (e.g., early HCC) 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.

[0045] In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) determined by a method according to the present disclosure is selected from the group consisting of 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). These glycan structures correspond to compound IDs 126, 150, 172, 131, 138 and 140 listed in Tables 2 and 3, respectively. In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1).

[0046] In embodiments in which any one of the glycan structures HexNAc(6)Hex(7)Fuc(1)NeuAc(4); 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) at position N207 of haptoglobin is determined, an increased amount of this glycan structure at position N207 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. In embodiments in which the glycan structures HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin are determined, a decreased amount of each glycan structure is indicative of HCC (e.g., early stage HCC).

[0047] In a particularly preferred embodiment, the determined glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0048] 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 the glycan structure at position N207 of haptoglobin in combination with AFP and / or PIVKA-II increases the diagnostic utility of HCC (e.g., early stage HCC).

[0049] 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-strand 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 N-glycan structure determined in (i)a) to a reference amount of the N-glycan structure, or (ii) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin determined in a) and comparing the determined score for detecting HCC (e.g., early stage HCC) to a reference value for 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.

[0050] The method of the second embodiment is based on the finding that an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 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) relative to control samples representing chronic liver diseases including HBV, HCV and cirrhosis. As shown by the attached examples, the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO: 1) represents a very promising marker for HCC (e.g., early stage HCC), showing the highest AUC in the detection of early stage HCC among all identified N-glycan structures (including all glycan structures at position N207, but also including other glycosylation sites). The structure of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) can be as shown in Figure 2. Thus, in embodiments, the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence set forth 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.

[0051] What is said herein in relation to the method according to the first aspect of the invention applies mutatis mutandis.

[0052] 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, comprising the steps of: a) determining the amount of the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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; and b) comparing the amount of the N-glycan structure determined in a) to a reference amount of the N-glycan structure, wherein an altered amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin in the subject's sample relative to the reference amount of the N-glycan structure is indicative of HCC (e.g. early stage HCC).

[0053] When a reference amount of the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) relative to the reference amount is indicative of HCC (e.g., early stage HCC).

[0054] Thus, in embodiments, an increase in the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) is indicative of HCC (e.g., early stage HCC).

[0055] In an embodiment, a second aspect of the present invention relates to 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 N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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) determining a score for detecting HCC (e.g., early stage HCC) taking into account the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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). The comparison in c) may then be used to determine the presence or absence of HCC (e.g., early stage HCC).

[0056] In embodiments, the score may be configured such that a higher score value indicates an increased risk of HCC. In these embodiments, the score value will increase with increasing levels of the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin.

[0057] Determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. the β chain of haptoglobin having the sequence shown in SEQ ID NO:1) in a sample means that a measure reflecting the absolute or relative amount of haptoglobin glycosylated with the respective glycan structure at position N207 of haptoglobin in the sample is determined.

[0058] Disclosed herein below are embodiments for determining the amount of a glycan structure such as the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1).

[0059] In embodiments, the level of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) can be determined by determining the level of a glycopeptide comprising N207 and the glycan structure, e.g., by mass spectrometry.

[0060] In an embodiment, the glycopeptide having the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is a glycopeptide having the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), N HSE (SEQ ID NO:2), where the middle N (bold and underlined; position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the beta chain of haptoglobin (SEQ ID NO:1). N HSE (SEQ ID NO:2). Thus, the level of Formula 1 (see elsewhere herein) is determined to determine the level of the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin.

[0061] In an embodiment, a glycopeptide having the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin, has an m / z of 927.9544, a charge of 5 and a glycopeptide mass of 4636.8124 Da.

[0062] In other words, a glycopeptide may be analyzed to determine the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1), the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), where the central N (i.e., the N at position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan structure is HexNAc(6)Hex(7)Fuc(1)NeuAc(4). In certain embodiments, the peptide portion of the glycopeptide is NLFL N HSE (SEQ ID NO: 2). Thus, the glycopeptide of formula 1 (see elsewhere herein) can be analyzed to determine the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin.

[0063] In an embodiment, to determine the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO:2) may be analyzed, where the central N corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4). This glycopeptide may be the glycopeptide shown in Formula 1 (see elsewhere herein) or FIG. 2, which 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin may have an m / z of 927.9544, a charge of 5 and a glycopeptide mass of 4636.8124 Da.

[0064] In embodiments, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as a peptide moiety and HexNAc(6)Hex(7)Fuc(1)NeuAc(4) as a glycan structure attached to the central N (i.e., the N at position 5) of SEQ ID NO:2, which corresponds to the N at position 207 of the beta strand of the haptoglobin (SEQ ID NO:1) sequence, and may have an m / z of 927.9544, in particular with a charge of 5 and a glycopeptide mass of 4636.8124 Da.

[0065] 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. the beta chain of haptoglobin having the sequence shown in SEQ ID NO:1) can mean determining the amount relative to the amount of a second analyte or group of analytes. Such a second analyte can be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide or glycopeptide can be spiked exogenously into the sample before the determination step or can be a peptide or glycopeptide of haptoglobin contained in the sample. The spiked peptide can be labeled with a heavy isotope.

[0066] 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 the glycan structure at position N207 of haptoglobin in combination with AFP and / or PIVKA-II increases the diagnostic utility of HCC (e.g., early stage HCC).

[0067] According to a third 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 the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of said haptoglobin in said sample obtained from said subject; c) determining a score for detecting HCC (e.g., early stage HCC) that takes into account or consists of the ratio of the amounts of the two glycan structures determined in a) and b), respectively, by dividing a) by b) or vice versa; and d) comparing the score determined in c) to a reference score value indicative of HCC (e.g., early stage HCC). Such a comparison can then be used to determine the presence (or absence) of HCC.

[0068] What is said herein in relation to the methods according to the first and second aspects of the invention applies mutatis mutandis.

[0069] As shown in Figures 4-6, the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin has been identified as a potential marker of HCC (e.g., early stage HCC) (see compound ID 150). The amount of HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin was found to be lower in samples obtained from subjects with HCC (e.g., early stage HCC) compared to the amount of this glycan structure in samples from control subjects.

[0070] Constructing a ratio between the amount of a first glycan structure that is more abundant in HCC (e.g., early stage HCC) subjects than in control subjects and the amount of a second glycan structure that is less abundant in HCC (e.g., early stage HCC) subjects than in control subjects (or the inverse ratio) can increase diagnostic performance.

[0071] In an embodiment of the method according to the third aspect of the present invention, an in vitro method for aiding in the detection of HCC (e.g., early stage HCC) in a subject is provided, comprising the steps of: a) determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin in the sample obtained from the subject; c) determining a ratio of the amounts of the two glycan structures determined in a) and b), respectively, by dividing a) by b); and d) comparing the ratio determined in c) to a reference ratio, wherein an increased ratio calculated for the sample of the subject relative to the reference ratio of the N-glycan structure is indicative of HCC (e.g., early stage HCC).

[0072] Determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. the β chain of haptoglobin having the sequence shown in SEQ ID NO:1) in a sample means that a measure reflecting the absolute or relative amount of haptoglobin glycosylated with the respective glycan structure at position N207 of haptoglobin in the sample is determined.

[0073] The embodiments for determining the amount of a glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) described elsewhere herein apply mutatis mutandis to determining the amount of HexNAc(4)Hex(5)NeuAc(2).

[0074] Thus, determining the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) includes generating a glycopeptide from haptoglobin contained in a sample, said glycopeptide comprising the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), and The amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) can be derived from or corresponds to the amount of glycopeptides comprising the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1).

[0075] In an embodiment, for determining the amount of HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), a glycopeptide having the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 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 NLFL N HSE (SEQ ID NO:2), where the middle N (bold and underlined) corresponds to the N at position 207 of the beta chain of haptoglobin (SEQ ID NO:1). N HSE (SEQ ID NO:2).

[0076] In an embodiment, a glycopeptide having the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 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(4)Hex(5)NeuAc(2) at position N207 of haptoglobin, has an m / z of 1060.0883, a charge of 3 and a glycopeptide mass of 3178.2503.

[0077] In an embodiment, the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin is determined by determining the amount of a glycopeptide of formula 5. [ka]

[0078] In other words, a glycopeptide may be analyzed to determine the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1), the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), where the central N (i.e., the N at position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan structure is HexNAc(4)Hex(5)NeuAc(2). In certain embodiments, the peptide portion of the glycopeptide is NLFL N HSE (SEQ ID NO:2).

[0079] In an embodiment, to determine the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO:2) can be analyzed, where the central N corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan is HexNAc(4)Hex(5)NeuAc(2). This glycopeptide may be a glycopeptide as shown in formula 5 (see above), which 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(4)Hex(5)NeuAc(2) at position N207 of haptoglobin may have an m / z of 1060.0883, a charge of 3, and a glycopeptide mass of 3178.2503.

[0080] In embodiments, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as a peptide moiety and HexNAc(4)Hex(5)NeuAc(2) as a glycan structure attached to the central N (i.e., the N at position 5) of SEQ ID NO:2, which corresponds to the N at position 207 of the beta strand of the haptoglobin (SEQ ID NO:1) sequence, and may have an m / z of 1060.0883, a charge of 3, and a glycopeptide mass of 3178.2503.

[0081] In an embodiment of the method according to the third 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 the glycan structure at position N207 of haptoglobin in combination with AFP and / or PIVKA-II increases the diagnostic utility of HCC (e.g., early stage HCC).

[0082] In a fourth 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 the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of said haptoglobin in said sample obtained from said subject; c) determining a score for detecting HCC (e.g., early stage HCC) that takes into account or consists of the ratio of the amounts of the two glycan structures determined in a) and b), respectively, by dividing a) by b) or vice versa; and d) comparing the score determined in c) to a reference score value indicative of HCC (e.g., early stage HCC). Such a comparison can then be used to determine the presence (or absence) of HCC.

[0083] What is said herein in relation to the methods according to the first, second and third aspects of the invention applies mutatis mutandis.

[0084] As shown in Figures 4-6, the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin has been identified as a potential marker of HCC (e.g., early stage HCC) (see compound ID 172). The amount of HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin was found to be lower in samples obtained from subjects with HCC (e.g., early stage HCC) compared to the amount of this glycan structure in samples from control subjects.

[0085] Constructing a ratio between the amount of a first glycan structure that is more abundant in HCC (e.g., early stage HCC) subjects than in control subjects and the amount of a second glycan structure that is less abundant in HCC (e.g., early stage HCC) subjects than in control subjects (or the inverse ratio) can increase diagnostic performance.

[0086] In an embodiment of a fourth aspect of the present disclosure, an in vitro method for aiding in the detection of HCC (e.g., early stage HCC) in a subject is provided, comprising: a) determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in a sample obtained from the subject; b) determining the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin in the sample obtained from the subject; c) determining a ratio of the amounts of the two glycan structures determined in a) and b), respectively, by dividing a) by b); and d) comparing the ratio determined in c) to a reference ratio, wherein an increased ratio calculated for the sample of the subject relative to the reference ratio of the N-glycan structure is indicative of HCC (e.g., early stage HCC).

[0087] The embodiments for determining the amount of a glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) described elsewhere herein apply mutatis mutandis to determining the amount of HexNAc(5)Hex(5)NeuAc(1).

[0088] Thus, determining the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) comprises generating a glycopeptide from haptoglobin contained in a sample, said glycopeptide comprising the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), and The amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) can be derived from or corresponds to the amount of glycopeptides comprising the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1).

[0089] In an embodiment, for determining the amount of HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1), a glycopeptide having the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 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 NLFL N HSE (SEQ ID NO:2), where the middle N (bold and underlined) corresponds to the N at position 207 of the beta chain of haptoglobin (SEQ ID NO:1). N HSE (SEQ ID NO:2).

[0090] In an embodiment, the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin is determined by determining the amount of a glycopeptide of formula 6. [ka]

[0091] In an embodiment, a glycopeptide having the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 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 N-glycan HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin, has an m / z of 982.0654, a charge of 3 and a glycopeptide mass of 2116.7564 Da.

[0092] In other words, a glycopeptide may be analyzed to determine the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence set forth in SEQ ID NO:1), the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), where the central N (i.e., the N at position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan structure is HexNAc(5)Hex(5)NeuAc(1). In certain embodiments, the peptide portion of the glycopeptide is NLFL N HSE (SEQ ID NO:2).

[0093] In an embodiment, to determine the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO:2) can be analyzed, where the central N corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO:1), and the glycan is HexNAc(5)Hex(5)NeuAc(1). This glycopeptide may be a glycopeptide as shown in formula 6 (see above), which 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(5)NeuAc(1) at position N207 of haptoglobin may have an m / z of 982.0654, a charge of 3, and a glycopeptide mass of 2116.7564 Da.

[0094] In embodiments, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as a peptide moiety and HexNAc(5)Hex(5)NeuAc(1) as a glycan structure attached to the central N (i.e., the N at position 5) of SEQ ID NO:2, which corresponds to the N at position 207 of the beta strand of the haptoglobin (SEQ ID NO:1) sequence, and may have an m / z of 982.0654, a charge of 3, and a glycopeptide mass of 2116.7564 Da.

[0095] In an embodiment of the method according to the fourth 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 the glycan structure at position N207 of haptoglobin in combination with AFP and / or PIVKA-II increases the diagnostic utility of HCC (e.g., early stage HCC).

[0096] In a fifth 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 NLFLNHSE (SEQ ID NO:2), and the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and the N-glycan portion is linked to the N at position 5 of SEQ ID NO:2.

[0097] In a preferred embodiment, the glycopeptide is a glycopeptide of formula 1: [ka] where GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, 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 through the indicated GlcNac of the glycan to the side chain of an asparagine corresponding to N207 of haptoglobin (i.e., N207 of the β-chain of haptoglobin in SEQ ID NO:1).

[0098] The isolated glycopeptide of formula 1 is shown in merely a graphical 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).

[0099] As demonstrated throughout this disclosure, the glycopeptides of Formula 1 show great promise for the detection of HCC (eg, early stage HCC).

[0100] Thus, in a sixth 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).

[0101] The method embodiments according to the first, second, third and fourth aspects apply to this use mutatis mutandis.

[0102] According to a seventh aspect of the present disclosure, there is provided a method for detecting a glycan structure at position N207 of haptoglobin (i.e., the beta 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 a glycopeptide comprising position N207 of haptoglobin having attached thereto the glycan structure produced in b), thereby detecting the glycan structure at position N207.

[0103] In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of 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). These glycan structures correspond to compound IDs 126, 150, 172, 131, 138 and 140 listed in Tables 2 and 3, respectively. In embodiments, the glycan structure at position N207 of haptoglobin is selected from the group consisting of: HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1). In a particularly preferred embodiment, the glycan structure at position N207 of haptoglobin is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0104] In the appended examples it is shown that glycopeptides containing an N-glycan modification at the asparagine corresponding to position N207 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.

[0105] The accompanying examples also show that, in particular, glycopeptides according to the fifth aspect of the present disclosure can be reliably determined with good sensitivity after purification and digestion, and after carrying out a suitable detection method, preferably LC-MS.Thus, in an embodiment, a method for detecting a glycan structure (e.g., glycopeptide of formula 1) according to the fifth aspect is provided, the method comprising 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) by detecting the glycopeptide according to the fifth aspect (e.g., glycopeptide of formula 1).

[0106] 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 body weight / fat. Nevertheless, later stages of HCC are often first spotted by ultrasound.

[0107] Previous biomarkers have not been routinely used in the detection of HCC. Some treatment guidelines indicate that the use of alpha-fetoprotein (AFP) is useful. However, this rather old biomarker is not very reliable, and very often tests negative for patients with early stages of HCC. The same is true for the biomarker PIVKA-II (=descarboxyprothrombin). However, both AFP and PIVKA-II tend to be frequently elevated in late stage HCC. The opposite is true for the glycan structures and glycopeptides disclosed and claimed in the present invention.

[0108] It is envisioned that the glycan structure disclosed in the present invention significantly improves the detection of HCC, particularly the detection of early stage HCC. In one embodiment, the method disclosed for detecting HCC is a method for detecting early stage HCC (i.e., early stage HCC).

[0109] In embodiments, the glycan structures of the present invention may also be combined with other biomarkers, such as biomarkers that reliably detect late-stage HCC. Such combinations are highly useful for detecting HCC at any stage (and early HCC), as confirmed by the combined AUC values ​​shown in the accompanying examples.

[0110] Thus, in an eighth 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 N207 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 N207 at a) in a sample obtained from said subject; c) combining the amounts determined in a) and b) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC) and comparing the binding value with a reference value for said binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0111] The embodiments of the aspects described above apply mutatis mutandis.

[0112] An HCC biomarker is a biomarker that indicates HCC (e.g., early stage HCC). In a specific example, the HCC biomarker can be AFP or PIVKA-II.

[0113] In an embodiment of an eighth 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, comprising the steps of: a) determining the amount of an N-glycan structure at position N207 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; c) combining the amounts determined in a) and b) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC)) and comparing the binding value with a reference value for the binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0114] In an embodiment of the method of the eighth 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 an N-glycan structure at position N207 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 in the sample obtained from the subject; c) combining the amounts determined in a) and b) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC) and comparing the binding value with a reference value for the binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0115] In an embodiment of the method of the eighth 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 an N-glycan structure at position N207 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) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC) and comparing the binding value with a reference value for the binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0116] In an embodiment of the method of the eighth 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 an N-glycan structure at position N207 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) into a binding value (e.g., a score for detecting HCC (e.g., early stage HCC) and comparing the binding value with a reference value for the binding value, wherein an altered binding value is indicative of HCC (e.g., early stage HCC).

[0117] In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of 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). These glycan structures correspond to compound IDs 126, 150, 172, 131, 138 and 140 listed in Tables 2 and 3, respectively. In the context of the eighth aspect of the invention, the N-glycan structure at position N207 of haptoglobin may be selected from the group consisting of: HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1). In a particularly preferred embodiment according to the eighth aspect of the invention, the glycan structure at position N207 of haptoglobin is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0118] 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., via mass spectrometry).

[0119] The sample used for the determination(s) of a) and b) may be the same sample or may be different samples obtained from the subject. Preferably, the samples are obtained from the subject at the same time.

[0120] "Combined value" may refer to, in an embodiment, determining a score indicative of HCC (e.g., early stage HCC), which score takes into account the amounts determined in a) and b). The score can further take into account additional biomarkers. Alternatively and additionally, the score can further take into account subject clinical data (e.g., age, sex, smoking status, cancer history, family cancer history and the presence of existing liver disease).

[0121] Exemplary, but non-limiting, methods for calculating binding values ​​or scores are disclosed herein below.

[0122] 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 on 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).

[0123] In the method according to the eighth aspect, the N-glycan structure is preferably HexNAc(6)Hex(7)Fuc(1)NeuAc(4), 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) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e. the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1) may also be determined; the ratio of the amounts may be calculated as described elsewhere herein and the binding value may be calculated using such ratio.

[0124] In an ideal clinical situation, an ultrasound examination of the liver is performed or the corresponding results from such an examination (previously performed) are received and, in parallel, a blood sample obtained from the subject is tested for the markers described herein above.

[0125] Thus, in a ninth aspect, the present disclosure relates to a clinical workflow for screening HCC (e.g., early stage HCC) in a subject, comprising the steps of: a) determining the amount of one or more N-glycan structures at position N207 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 determining the amount of PIVKA-II in a sample obtained from the subject; c) combining the amounts of the markers determined in a) and b); and d) further using the results of an ultrasound (US) investigation; wherein a positive US result and / or an elevated score for the markers determined in a) and b) is indicative of HCC (e.g., early stage HCC).

[0126] Embodiments described herein with respect to any of the preceding aspects of the invention apply mutatis mutandis to the ninth aspect of the invention.

[0127] In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of 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). These glycan structures correspond to compound IDs 126, 150, 172, 131, 138 and 140 listed in Tables 2 and 3, respectively. In embodiments, the N-glycan structure at position N207 of haptoglobin may be selected from the group consisting of: HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1). In a particularly preferred embodiment according to the ninth aspect of the invention, the glycan structure at position N207 of haptoglobin is HexNAc(6)Hex(7)Fuc(1)NeuAc(4). What has been said elsewhere herein (e.g. in the context of other aspects) regarding the detection of glycan structures as described in this paragraph applies mutatis mutandis.

[0128] In the method according to the ninth aspect, the N-glycan structure is preferably HexNAc(6)Hex(7)Fuc(1)NeuAc(4), 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) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e. the β-strand of haptoglobin having the sequence shown in SEQ ID NO:1) may also be determined; the ratio of the amounts may be calculated as described elsewhere herein and the binding value may be calculated using such ratio.

[0129] In an embodiment, the workflow may be for monitoring patients at risk of HCC (e.g., early stage HCC).In other words, the subject may be known to be at risk of developing HCC through, for example, 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In a tenth aspect, there is provided herein a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of a glycan structure at position N207 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) i) comparing the amount of the glycan structure received in a) with a reference amount of said N-glycan structure at position N207 of haptoglobin, wherein an altered amount of the N-glycan structure in said patient sample relative to the reference amount of said one or more glycan structures is indicative of HCC, or (ii) calculating a score for detecting HCC (e.g., early stage HCC) taking into account the amount of said glycan structure at position N207 of haptoglobin received in a) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). Includes.

[0135] The embodiments of the aspects described above apply mutatis mutandis.

[0136] In an embodiment, the method may include aiding in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0137] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0138] In embodiments, the glycan structure at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence set forth in SEQ ID NO:1) is selected from the group consisting of 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). These glycan structures correspond to compound IDs 126, 150, 172, 131, 138 and 140 listed in Tables 2 and 3, respectively. In embodiments, the glycan structure is selected from the group consisting of HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1).

[0139] In certain preferred embodiments, the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0140] In an eleventh aspect, there is provided a computer-implemented method for aiding in the detection of hepatocellular carcinoma (HCC) in a subject, the method comprising: c) receiving data comprising the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 d) comparing the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) received in i)a) with a reference amount of said glycan structure, wherein an increase in HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in the subject 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin received in a) and comparing the calculated score for detecting HCC (e.g., early stage HCC) with a reference value of said score indicative of HCC (e.g., early stage HCC). Includes.

[0141] The embodiments of the aspects described above apply mutatis mutandis.

[0142] In an embodiment, the method may include aiding in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0143] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0144] In a twelfth aspect, the present invention provides a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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) receiving data comprising the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of the haptoglobin in the sample obtained from the subject; c) calculating a score that includes or consists of the ratio of the amounts of the two glycan structures of a) and b) (or vice versa); and d) comparing the score calculated in c) with a reference value for said score indicative of HCC (e.g., early stage HCC). Includes.

[0145] The embodiments of the aspects described above apply mutatis mutandis.

[0146] In an embodiment, the method may include aiding in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0147] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0148] In a thirteenth aspect, there is provided herein a computer-implemented method for aiding in the detection of HCC (e.g., early stage HCC) in a subject, the method comprising: a) receiving data comprising the amount of one or more glycan structures (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4)) at position N207 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 amounts determined in a) and b); and d) comparing the score calculated in c) with a reference value for said score indicative of HCC (e.g., early stage HCC). Includes.

[0149] The embodiments of the aspects described above apply mutatis mutandis.

[0150] In an embodiment, the method may include aiding in the detection of HCC (eg, early stage HCC) based on i) or ii) of b).

[0151] In embodiments, the method may include outputting (eg, via a display) whether the subject is afflicted with HCC (eg, early stage HCC).

[0152] In embodiments, the glycan structure is selected from the group consisting of HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); and HexNAc(5)Hex(5)NeuAc(1).

[0153] In certain preferred embodiments, the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0154] 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) one single data set that includes all data received in the two or more steps, and (ii) embodiments in which two or more separate data sets are received.

[0155] 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 necessary based on the comparison step. The output may be via a display.

[0156] This specification also discloses a computer program product, the computer program product including instructions that, when executed by a computer, cause the computer to execute the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0157] Further provided is a computer readable medium comprising instructions that, when executed by a computer, cause the computer to perform a computer-implemented method according to any one of the tenth to thirteenth aspects.

[0158] There is also provided a data processing system comprising: a receiving unit configured to receive data as defined in any one of the tenth to thirteenth aspects; a processing unit configured to perform the calculation and / or comparison steps and / or any decision / evaluation steps in any one of the tenth to thirteenth aspects; and optionally an output unit configured to output a result of the evaluation.

[0159] The above embodiment relates to the glycan structure at position N207 of the β-chain of haptoglobin. As is evident from the accompanying examples, also identified herein are some other glycan structures at positions N184, N211 or N241 that are useful for detecting HCC. These glycan structures are shown in FIG. 6, and further information on them and on exemplary glycopeptides for determining the level of such glycan structures is provided in Tables 2 and 3.

[0160] The respective glycan structures at position N184 of haptoglobin are as follows: HexNAc(2)Hex(8), HexNAc(2)Hex(9), (HexNAc(3)Hex(4)NeuAc(1), HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(5)Hex(6)Fuc(1)NeuAc(3), HexNAc(6)Hex(7)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2), HexNAc(5). Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5) Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(2)NeuAc(2), HexNAc(2)Hex(12), HexNAc(6)Hex(7) and HexNAc(4)Hex(5)Fuc(3)NeuAc(1).

[0161] The respective glycan structures at position N211 of haptoglobin are as follows: HexNAc(6)Hex(7)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2) and HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0162] The respective glycan structures at position N241 of haptoglobin are as follows: 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).

[0163] Thus, all aspects and embodiments described herein for N207 of haptoglobin are disclosed mutatis mutandis for N184, 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 glycan structures (and / or the glycan structures referred to in Figure 6 in conjunction with Table 2) at positions N184, N211 and N241.

[0164] To detect the amount of glycan structure at position N184, MVSHH N A glycopeptide having a peptide portion comprising or consisting of LTTGATLINE (SEQ ID NO:3) may be used, where N at position 6 corresponds to N184.

[0165] 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.

[0166] To detect the amount of a glycan structure at position N241, a glycopeptide may be used having a peptide portion comprising or consisting of VVLHPNYSQVD (SEQ ID NO: 5), where N at position 6 corresponds to N241.

[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of this invention, suitable methods and materials are described below.In case of conflict, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0168] The following definitions and embodiments apply throughout this disclosure and in particular to all aspects and embodiments of the present invention.

[0169] 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.

[0170] 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.

[0171] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0172] The term "and / or" should be understood to mean either or both of the alternatives.

[0173] As used herein, unless otherwise indicated, the term "about" is understood to be used synonymously with the term "approximately." Illustratively, unless otherwise indicated, the use of the term "about" when used in conjunction with a stated numerical value or range indicates something greater or less than the stated value or range, up to within ±15% of the stated value, ±10% of the stated value, ±5% of the stated value, or, for convenience, ±2% of the stated value. Such values ​​are therefore encompassed within the scope of the claims reciting the term "about" or "approximately."

[0174] 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, whose expression or presence in or on a mammalian tissue or cell can be detected by standard methods (or the methods disclosed herein) and can be predictive, diagnostic and / or prognostic of an individual's health or disease. Thus, hereinafter, the more general term "marker" may also be used at times while discussing the more general terms and definitions. The term marker also includes the glycan structure or glycan, or glycopeptide, analyzed in this disclosure.

[0175] 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.

[0176] Hepatocellular carcinoma (HCC) is the major histologic type among primary liver cancers 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 ablation with radiofrequency for patients diagnosed at an early stage. If this malignancy is diagnosed at an early stage, 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).

[0177] 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 exhibits 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 usually ranges from 0.3 to 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.

[0178] The haptoglobin beta chain does not show genetic polymorphism. As used herein, the haptoglobin beta chain preferably has the sequence shown in SEQ ID NO: 1. Haptoglobin can undergo secondary modification, 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.

[0179] As used herein, and unless otherwise stated, the phrase "at position N207 of haptoglobin" refers to position N207 of the β chain of haptoglobin (eg, having the sequence shown in SEQ ID NO:1).

[0180] 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, although preferably, may not usually be correct for 100% of the subjects examined (essentially for all diagnostic methods). This term typically requires that a statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be determined by those skilled in the art 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.

[0181] The term "indicative of HCC" is used to explain that an increased level or amount of the determined marker (e.g., glycan structure or glycopeptide), and its combination, optionally with other biomarkers or variables, is very informative, but does not diagnose without error, but rather indicates that the subject has a high probability of having HCC. Not all (100%) of HCC patients have the amount of the marker above the reference level, and not all healthy individuals have the level of the 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 analyzed marker or a combination of markers including this marker gives a certain probability that the individual whose sample was analyzed has a certain clinical condition, for example, has HCC, for example, at a given specificity level or a given sensitivity level. 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.

[0182] 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, including, for example, a physician, in assessing whether an individual has HCC or is at risk of developing HCC. As will be understood, several alternative methods (e.g., ultrasound, radiography, MRT, or CT) can be used by a physician to detect or exclude the presence of HCC, and can be combined with in vitro biomarker data, such as glycan structure data. The final diagnosis of HCC is usually made from tissue biopsy or tissue samples 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.

[0183] In an embodiment of the present invention, "aiding in the detection of hepatocellular carcinoma (HCC)" may be "aiding in the detection of early-stage HCC." As demonstrated in the accompanying examples, a particular glycan at position 207 of the β-chain of haptoglobin exhibits particularly good performance in detecting early-stage HCC.

[0184] 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.

[0185] 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.

[0186] In the above aspects and embodiments, a particular marker value, such as the amount of a glycan structure or the amount of a glycopeptide, may be combined (e.g., into a score) with amounts determined for one or more other biomarkers. Such combinations are performed using standard mathematical / statistical techniques.

[0187] 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 a perceived measurement allows for correct identification of a condition (or differentiation of one condition from the other). Values ​​typically range between 1.0 (perfect separation of the two groups of test values) and 0.5 (no obvious distribution difference between the two groups of test values). The area does not depend only on a specific 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 a 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.

[0188] In the present invention, the terms "HCC", "early HCC" and "late HCC" are used.

[0189] As used herein, "early stage HCC," "early stage HCC," or "early stage HCC" refers to patients classified as stages 0 and A according to the Barcelona Clinic Liver Cancer (BCLC) classification (Llovet JM, Bru C, Bruix J, Semin Liver Dis. 1999;19(3):329-38).

[0190] 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 Clinic Liver Cancer (BCLC) classification (Llovet JM, Bru C, Bruix J, Semin Liver Dis. 1999;19(3):329-38).

[0191] "HCC" refers to any form of HCC, including early and late stage HCC.

[0192] The BCLC classification is recommended as the standard system for managing 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 classification, patients are assigned to five categories (0, A, B, C and D): BCLC stage 0 (defined as very early stage disease) includes patients with well-preserved liver function diagnosed with one asymptomatic nodule smaller 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 stage 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 metastasis. Patients with Child-Pugh grade A or B, vascular invasion or extrahepatic metastasis and cancer-related symptoms (PS 1-2) 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) are classified as belonging to BCLC D disease (defined as late-stage disease). In the context of this disclosure, patients with BCLC stages B, C and D were defined as the late-stage HCC group.

[0193] The term "subject" or "individual" as used herein refers to a single person. 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. Intended to be included as a subject is any subject involved in a clinical research trial that does not show any clinical signs of disease, or a subject involved in an epidemiological study, or a subject whose sample can serve as a control. In an embodiment, the subject may be known to be at risk of developing HCC, for example, through 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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. The 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 understood 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 material of the patient sample is not returned to the patient's body.

[0199] As used herein, the term "determining" the amount of an N-glycan structure or the amount of a glycopeptide 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.

[0200] In the context of the present invention, determining the amount of a glycan structure (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) or any of the other glycan structures mentioned herein) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence shown in SEQ ID NO:1) in a sample means that a measure is determined that reflects the absolute or relative amount of haptoglobin glycosylated with the respective glycan structure at position N207 of the haptoglobin in the sample. In an embodiment, determining the amount of a glycan structure can be measuring the presence of the glycan structure and quantifying the amount / level of the glycan structure. In an embodiment, the amount of a glycan structure at position N207 corresponds to the amount of a glycopeptide comprising the glycan structure at N207. Such glycopeptides can be generated by hydrolyzing haptoglobin in a sample (e.g., using a protease such as the proteases described below).

[0201] In an embodiment of the present disclosure, determining the amount of the glycan structure (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4)) at position N207 of haptoglobin (i.e., the beta chain of haptoglobin having the sequence shown in SEQ ID NO:1) can be a relative amount 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 can be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide can be spiked into the sample before the determination step or can be a peptide or glycopeptide of haptoglobin contained in the sample. The spiked peptide can be labeled with a heavy isotope.

[0202] In the present disclosure, determining the amount of a glycan structure at position N207 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 glycopeptide(s) from haptoglobin contained in the sample, the glycopeptide(s) comprising position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence set forth in SEQ ID NO:1) and a glycan structure attached thereto (if present), and determining, for example, via mass spectrometry, the amount(s) of the glycopeptide comprising the glycan of interest. The amount of each glycan structure at position N207 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 position N207.

[0203] In an exemplary embodiment, determining the amount of a glycan structure at position N207 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 position N207 of haptoglobin (i.e., the β chain of haptoglobin having the sequence set forth in SEQ ID NO:1) and respective glycans attached thereto are generated, and determining the amount(s) of glycopeptides having a glycan structure at position N207 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 position N207 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 position N207.

[0204] In the present disclosure, determining the amount of a glycan structure at position N207 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 a respective one or more glycan structures at position N207 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 position N207 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 the respective glycan structure at position N207.

[0205] As those skilled in the art will appreciate, other methods can be used to determine the glycan structure at N207 of haptoglobin. Such analysis only requires that this glycan structure is or was attached to N207 (and N184, 211 and 241) of the haptoglobin beta chain. An exemplary, but non-limiting method is described by Oh and coworkers (Mass Spectrom Rev. 2021).

[0206] In an embodiment, the glycopeptide(s) comprising position N207 of haptoglobin used to determine the amount of glycan structure has the peptide sequence NLFL N HSE (SEQ ID NO:2), where the middle N (bold and underlined; position 5) corresponds to the N at position 207 of the beta chain of haptoglobin (SEQ ID NO:1) to which the respective glycan to be detected is attached. In certain embodiments, the peptide portion of the glycopeptide(s) is / are NLFL N HSE (SEQ ID NO:2).

[0207] In embodiments, the glycan structure being quantified is part of a glycopeptide, the glycopeptide having the peptide sequence NLFL NIt contains an HSE (SEQ ID NO:2) and the middle N (bold and underlined) corresponds to the N at position 207 of the β chain of haptoglobin (SEQ ID NO:1).

[0208] In the context of the present invention, the level or amount of an N-glycan structure (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or a glycopeptide (e.g., Formula 1) at position N207 of the β-chain of haptoglobin in a sample is determined. Any suitable method known in the art may be used for this purpose. In an embodiment of the method according to the present disclosure, the step of determining one or more N-glycan structures (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or a corresponding glycopeptide (e.g., Formula 1) at position N207 of the β-chain of haptoglobin includes that the haptoglobin (comprising at least the β-chain) is purified before such determination is performed. Preferably, the purification of haptoglobin is achieved via an antibody that binds to haptoglobin. In particular, a monoclonal antibody that specifically binds to haptoglobin can 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, #AB13429, clone HG-36; Abnova, #MAB12976, clone 2F4; Acris Antibodies, #UM500010, clone UMAB10; Novus Biologicals, #NBP2-03008, clone OTI4H5; OriGene, #TA00399, clone OTI2B8; Thermo Fisher Scientific, #HYB 170-06-02, clone 9G10.In a particular embodiment of the method according to the present disclosure, the step of determining one or more N-glycan structures at position N207 of the β-chain of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or the corresponding glycopeptide (e.g., Formula 1) comprises purifying the haptoglobin (including at least the β-chain) via a biotinylated anti-haptoglobin monoclonal antibody in combination with streptavidin-coated beads (SA-beads). In such a purification step, the sample is incubated with a biotinylated antibody and (simultaneously or sequentially) with (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.

[0209] In an embodiment of the present disclosure, the step of determining the amount of (or one or more) N-glycan structures at position N207 of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) comprises chemically or biochemically / enzymatically hydrolyzing the haptoglobin to cleave it 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.

[0210] In an embodiment of the disclosure, the step of determining the amount of N-glycan structure (or structures) at position N207 of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) 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, and trypsin cleaves the protein chain C-terminal to the amino acids arginine and lysine. Both enzymes cleave with high specificity.

[0211] As shown above, the N-glycan at position 207 (or one or more) of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or glycopeptides containing N207 (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, after which the resulting fragments are analyzed. The fragments are analyzed computer-assisted 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.

[0212] 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.

[0213] MS analysis of glycan structures or glycopeptides requires a significant number of handling steps. These handling steps can cause considerable variation, 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 of (glycan structure / glycopeptide) based on the same method of MS analysis. This ratio is not absolute, 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 embodiments, the ratio may be between an increase and a decrease of a glycan structure at the same position of a polypeptide. Specific but non-limiting examples of such ratios are described elsewhere herein.

[0214] As used herein, the term "amount" or "level" of an analyte (e.g., glycan or glycopeptide) in a sample refers to any absolute measure that corresponds to the amount or concentration of the analyte in the sample or is proportional to the absolute amount or concentration of the analyte in the sample; or any relative measure, i.e., a measure that expresses the amount or concentration of the analyte relative to a reference amount or concentration, respectively. 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.

[0215] In embodiments, the amount of a glycan structure at position N207 of haptoglobin in a sample can be the amount of a glycopeptide that includes the glycan structure at N207 determined from the sample. In these embodiments, the method can include generating a glycopeptide from haptoglobin (e.g., via protease digestion as described elsewhere). In embodiments, the peptide portion of the glycopeptide can be as described above.

[0216] In embodiments, the amount of glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal of the non-glycosylated peptide (e.g., from haptoglobin), respectively. In embodiments, the amount of glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal of all glycopeptides (e.g., haptoglobin) detected, respectively.

[0217] The term "altered 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.

[0218] The term "elevated" or "increased" 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 include) than the amount or level of such marker in a reference or control sample. The term "decreased" level or amount of a marker refers to an amount or level of such marker in the sample being investigated that is lower (i.e., does not include) than the amount or level of such marker in a reference or control sample.

[0219] As used herein, the term "score" or "score for detecting HCC" considering the amount of glycan structure or glycopeptide refers to a score (e.g., value) obtained by combining the amount of the 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 clinical parameters. 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, etc. The score should be configured to be able to indicate HCC (e.g., early HCC).

[0220] "Taking into account" as used in the context of a score includes embodiments in which only the specifically recited quantity(s) and parameters are taken into account, as well as embodiments in which other, unrecited parameters are taken into account.

[0221] 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 combined value can also be referred to as determining or calculating a score.

[0222] Determining or calculating the score includes any mathematical combination of the amount of glycan structures or glycopeptides mentioned herein with additional parameters (see above). Thus, the score can be calculated in that the individual parameters (including or consisting of the determined or received amount of glycans attached to N207 of haptoglobin or each glycopeptide) are mathematically combined. 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 can take into account one or more other factors other than the level of PSA-glycoform species, including, but not limited to, the presence or level of one or more other biomarkers in the sample and / or one or more clinical parameters of the subject (e.g., tumor histology, smoking status, stage of disease, and / or age).

[0223] In certain embodiments of the present invention, the score of detection of HCC may be obtained by or may include weighting calculation.This means that biomarkers (such as the amount of glycan structure at position N207 of haptoglobin) can be given different weights.For example, if the score takes into account the amount of a first glycan structure at position N207 of haptoglobin in a sample and the amount of another glycan structure at position N207 of haptoglobin, the score can be calculated by the following formula:

[0224] Score = a * [amount of first glycan structure] + b * [amount of second glycan structure], where a and b represent weighting factors. Preferably, the weighting factors or coefficients (in the above examples a and b) are obtained by analyzing control samples from a reference population (e.g., any reference population defined in the context of the reference values ​​below), etc. In embodiments, the weighting factors or coefficients can be obtained by machine learning techniques applied to a training data set obtained from samples of the reference population defined herein.

[0225] One of skill in the art will recognize that the scores and corresponding reference values ​​of the scores may be optimized based on a reference population (e.g., any of those defined herein or disclosed in the accompanying examples).

[0226] In an embodiment, the score may be a binary score and the corresponding reference value may be binary. "Binary" means that the score contains two values, for example, the first value is the determined or received level or value derived therefrom of one or more biantennary PSA-glycoforms, and the second value is the level or value derived therefrom of one or more monoantennary PSA-glycoforms. The "value derived therefrom" may be, for example, a value obtained by mathematical operation. The "value derived therefrom" is preferably directly proportional to the respective levels. The value of the binary reference value may be obtained as described below for each individual PSA-glycoform.

[0227] Comparing the two-component score to a reference value for a two-component cut-off score means comparing a first value of the determined two-component score to a first value of the reference value of the two-component score and comparing a second value of the determined two-component score to a second value of the reference value of the two-component score. When a two-component score is constructed from the amount of a first glycan structure at position N207 of haptoglobin and the amount of a glycan structure at position N207 of haptoglobin, HCC can only be detected if both amounts are indicative of HCC relative to their reference values ​​forming part of the two-component score.

[0228] In aspects and embodiments using two or more markers (or even more general input parameters, which may also include clinical parameters such as age or sex), they can be combined by a suitable algorithm (e.g., logistic regression) derived from multivariate analysis of available data, preferably in an exhaustive search for feature selection. This combination / calculation produces a multivariate score. Other methods, such as selected from DA (i.e., linear, quadratic, regularized discriminant analysis), kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbors), PLS (partial least squares), tree methods (i.e., logistic regression, CART, random forest, boosting), can also be used to combine biomarkers / input values ​​into a score.

[0229] 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 addresses 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 tested negative for the disease.

[0230] As above, the ROC curve can be used to assess the performance of the aforementioned logistic regression model in discriminating between patients and controls.

[0231] 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 for 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, whereas 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 for such a particular score that indicates HCC (e.g., early stage HCC). The comparison may be performed by a suitable device, e.g., a computer. The measured or detected level / amount value and the reference level / amount of a glycan structure in a sample from an individual or patient 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 above evaluation provides the desired evaluation in a suitable output format. For 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 assessment 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 standards stored in a database. The computer program may further evaluate the results of the comparison, i.e. automatically provide the desired assessment in a suitable output format.

[0232] The terms "glycan structure" or "glycan" are used interchangeably. In the present disclosure, the glycan investigated is N-glycan, so the terms glycan and N-glycan are used interchangeably herein. Glycans or glycan structures are composed of various types of carbohydrates. Glycan structures can be, for example, linked to amino acids, such as the amino acid asparagine. In this case, the glycan linked to asparagine at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1) is analyzed. The glycan structures or glycans of the present disclosure serve as biomarkers or markers for HCC (e.g., early HCC).

[0233] The term "glycopeptide" is used to refer to a peptide or peptide fragment of a larger polypeptide that includes an amino acid to which a glycan is covalently attached. In the present disclosure, a glycopeptide that is preferably analyzed is a peptide sequence derived from the beta chain of haptoglobin and includes the amino acid asparagine (N) at position 207 of the beta chain of haptoglobin (SEQ ID NO: 1). Preferred embodiments of peptide portions of glycopeptides that are preferably detected or quantified are disclosed elsewhere herein.

[0234] Glycans are typically referred to herein by their sum formula. In this context, we refer to 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 of skill in the art working in the field 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).

[0235] Preferred glycans are shown with 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,). Such preferred glycans are shown, for example, in FIG. 4 for the glycan structure at N207 of haptoglobin.

[0236] In an embodiment of the invention, the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) may have the formula 2. [ka]

[0237] In a preferred embodiment of the invention, the glycan HexNAc(4)Hex(5)NeuAc(2) may have the formula 3. [ka]

[0238] In a preferred embodiment of the invention, the glycan HexNAc(5)Hex(5)NeuAc(1) may have the formula 4: [ka]

[0239] In a preferred embodiment of the invention, the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) can have formula 2, the glycan HexNAc(4)Hex(5)NeuAc(2) can have formula 3, and the glycan HexNAc(5)Hex(5)NeuAc(1) can have formula 4.

[0240] As used in any one of formulas 2-4 and any other formulas herein, GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). The lines represent covalent bonds between monohalides. The dashed lines indicate through which monosaccharide the glycan is attached to the peptide or protein when it is part of a glycopeptide or glycoprotein, respectively (e.g., N207 in SEQ ID NO:1). The dashed lines used in the context of NeuAc (or Neu5Ac, used synonymously) indicate that NeuAc may be attached to either sugar.

[0241] As used herein, the term "reference amount" (or "reference level") for an analyte (e.g., a glycan structure or 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 to meet routine requirements for specificity and / or sensitivity for the purpose of detecting HCC (e.g., early stage HCC), for example. Thus, the reference amount may be selected to indicate HCC (e.g., early stage HCC). Requirements for detecting HCC may 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 for the level of sensitivity or specificity, respectively, the reference range (when evaluated or reduced values ​​indicate an abnormal state) or the reference level or cutoff level (when evaluated or reduced values ​​indicate an abnormal state) can be determined by the skilled artisan. Similarly, the reference ratio can be determined according to the same principle.

[0242] 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., a score). Those skilled in the art will understand that the reference value is predetermined and set to meet the routine requirements for, for example, specificity and / or sensitivity for the purpose of detecting HCC (e.g., early stage HCC). Thus, the reference value can be selected to indicate HCC (e.g., early stage HCC). What has been said above regarding the reference amount applies mutatis mutandis.

[0243] The reference amount or value of the score is typically determined in a 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 be affected by or known to be at risk for a given condition; or 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, for example, to determine whether an individual is at risk for HCC. Depending on the intended diagnostic application, a suitable reference sample is selected, in which a control value or a 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 apparent to the skilled artisan, the absolute marker value established in a reference sample or set of reference samples (e.g., forming a reference population) depends 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. Also preferred reference populations may 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 from a healthy individual.

[0244] For the detection of HCC in clinical routine, it is most important 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 from a patient with cirrhosis.

[0245] 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 at increased risk of developing HCC) and subjects suffering from HCC (e.g., early stage HCC).

[0246] As will be understood 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. This aid in detection or detection is typically achieved based on the comparison step of such a method.

[0247] Alpha-fetoprotein 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 marker glycoprotein fraction(s) with specific glycan structures. For AFP, lectins derived from lentil agglutinin-A (LCA) are widely used. The lentil agglutinin (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.

[0248] 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 individually or in combination with AFP as an alternative HCC biomarker. Prothrombin has 10 potential gamma-carboxylation sites, and various forms of PIVKA-II with different levels of undercarboxylation exist in the 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 and its limited usefulness in detecting early-stage 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.

[0249] As used herein, the terms "specific binding" or "specifically binds" refer to a binding reaction in which the molecules of the binding pair exhibit binding to one another under conditions in which they do not significantly bind to another molecule.

[0250] 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 is -7 K below M D The term "specific binding" or "specifically binding" refers to a binding reaction that binds to a corresponding target molecule at a specific concentration, preferably at least 10 -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 least 10-fold lower, more preferably at least 20-fold lower than binding to non-target proteins.

[0251] 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 a subject.

[0252] It will be understood that the disclosed method and use can be performed remotely from the subject or his / her doctor, and can actually be performed overseas, and the results are communicated. In certain embodiments, the results of the method 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.

[0253] 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. This description 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.

[0254] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein.

[0255] 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 described without departing from the spirit of the invention. Also provided are descriptions of the amino acid sequences disclosed herein. Array Description

[0256] SEQ ID NO: 1 shows the amino acid sequence of human haptoglobin, with N207 printed in bold and underlined. MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT EGDGVYTLND KKQWINKAVG DKLPECEADD GCPKPPEIAH GYVEHSVRYQ CKNYYKLRTE GDGVYTLNNE KQWINKAVGD KLPECEAVCG KPKNPANPVQ RILGGHLDAK GSFPWQAKMV SHHNLTTGAT LINEQWLLTT AKNLFL N HSE 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).

[0257] SEQ ID NO:2 shows the amino acid sequence of a peptide portion contained in an exemplary glycopeptide (e.g., the glycopeptides of formulas 1, 5 and 6) for detecting a glycan structure at position N207. The sequence corresponds to amino acid positions 203 to 210 of SEQ ID NO:1, with the Asn residue corresponding to N207 of SEQ ID NO:1 printed in bold and underlined. NLFL N HSE

[0258] SEQ ID NO: 3 shows the amino acid sequence of a peptide contained in an exemplary glycopeptide for detecting a glycan structure at position N184. The Asn residue corresponding to N184 in SEQ ID NO: 1 is printed in bold and underlined. MVSHH N LTGATLINE

[0259] 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. The Asn residue corresponding to N211 in SEQ ID NO: 1 is printed in bold and underlined. N ATAK

[0260] 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. The Asn residue corresponding to N241 in SEQ ID NO: 1 is printed in bold and underlined. VVLHP N YSQVD [Brief description of the drawings]

[0261] [Figure 1] Schematic diagram representing the various forms of haptoglobin: The three types of haptoglobin contain (always together with a β chain) either twice the α1 chain of haptoglobin (1-1); an α1- and one α2-chain (2-1) or two α2-chains (2-2). [Diagram 2] Exemplary structure of formula 1 (compound 126): HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N-207. Left: Glycan structure drawn using monosaccharide abbreviations according to the SNFG (Symbol Nomenclature for Glycans) system (PMID 26543186, Glycobiology 25:1323-1324, 2015). Connections are shown as black lines. Right: Symbolic representation of glycan structure: monosaccharide symbols follow SNFG. [Diagram 3]Box blot of various glycopeptides at position N207 of the β-chain of haptoglobin (SEQ ID NO: 1) indicative of HCC: Site-specific glycan analysis revealed a significant number of glycopeptides elevated or decreased in HCC. The amount of Hp glycopeptides was compared between control, early and late stage cohorts. Compared to controls, some glycopeptides were specifically upregulated in early stage HCC (compound 126), some were upregulated in early as well as late stage HCC (compound 140 and compound 131), and some were downregulated in early and late stage HCC (compound 172). Statistically significant differences between control (C), early stage HCC (ESH) and late stage HCC (LSH) were determined by Wilcoxen (Mann-Whitney U) test. See Figure 4 for glycopeptide structures. [Figure 4] Overview of up / downregulated glycopeptides on glycosylation site N-207. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) system (PMID 26543186, Glycobiology 25:1323-1324, 2015). On the left, Hp glycopeptides are listed separately between early stage HCC cases and controls. In the right column, glycopeptides with differences in early and late stage HCC vs. controls are shown, whereas no glycans on glycosylation site N-207 were found that differed only between late stage HCC and controls. Only glycopeptides with AUC>70% are shown. The direction of expression change (decreased or increased) is indicated by up(regulated) or down(regulated). [Figure 5-1] Receiver Operator Curve (ROC): Several Hp glycopeptides at site N-207 are likely to have clinical utility in the detection of early HCC. These are evident from the ROC shown and the area under the curve (AUC) values ​​given. Specificity and sensitivity values ​​are for a sensitivity and specificity of 0.9 cutoff, respectively. [Figure 5-2]Receiver Operator Curve (ROC): Several Hp glycopeptides at site N-207 are likely to have clinical utility in the detection of early HCC. These are evident from the ROC shown and the area under the curve (AUC) values ​​given. Specificity and sensitivity values ​​are for a sensitivity and specificity of 0.9 cutoff, respectively. [Figure 6] Overview of up / downregulated glycopeptides on 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, Hp glycopeptides are listed with distinction between early HCC cases and controls. In the middle column, glycopeptides present in both early and late stage HCC are shown, while in the right column, glycopeptides with differences in late stage HCC vs. controls are shown. Only glycopeptides with AUC>70% are shown. The direction of expression change (decrease or increase relative to control) is indicated by up(regulated) or down(regulated). EXAMPLES

[0262] Example 1 1.1 Research composition EDTA-plasma samples were obtained from 57 controls representing chronic liver disease including HBV, HCV, and cirrhosis, and HCC patients, including 33 with early stage and 32 with late stage HCC (see Table 1 for demographic information).

[0263] 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) procedure, 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 analysis as described below. Repeated freezing and thawing of samples was avoided. [Table 1]

[0264] 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 of the F(ab')2 fragments on the beads, the resulting coated beads were washed three times with PBS. 50 microliters of each plasma sample were 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 steps of washing with PBST (PBS buffer + 0.1% Tween 20®) and two steps of 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 recovery. To neutralize the pH, 200 μl of NaOH (1 N) was added to the eluate after each elution step.

[0265] 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, US) 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 a molecular weight of 10.000 or more, are retained 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-sulfonato) 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. Next, step 5 μl of IAM (55 mM) was added to the samples and they were incubated at 37° C. in the dark for 30 min. The samples were centrifuged at 10.000 g for 20 min to wash out the buffer and washed once with 100 μl of ABC (ammonium bicarbonate, 50 mM) buffer. The proteins retained on the filter were then first digested by adding 6 μg of trypsin in 50 μl of 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 of GluC was added to the sample. For this second digestion, the sample was incubated overnight at 25° C. The digested sample was eluted from the membrane by centrifugation at 10.000 g for 20 min.

[0266] 1.4 LC-MS / MS analysis Twenty microliters of peptides obtained by enzymatic digestion as 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 flow rate of LC 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 method. Full scan MS spectra were acquired in the m / z range of 300-2000 with a resolution of 60000, 10e6 automatic gain control (AGC), and 50ms injection time. The top five most intense peaks from this scan, i.e., survey scan, were selected for fragmentation by higher energy collision dissociation (HCD) with a normalized collision energy of 28%, a resolution of 15000, 1e5 AGC, and 150ms injection time.

[0267] 1.5 AFP and PIVKA-II assays AFP and PIVKA-II were measured using microchip capillary electrophoresis and liquid-phase binding assays on a uTASWako i30 automated analyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions.

[0268] 1.6 Data Analysis Raw files obtained from the mass spectrometer were processed by the Byonic (Protein Metrics, CA, US) 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, allowing two missed cleavages. Carbamidomethylation of cysteine ​​was set as fixed modification, while methionine oxidation and glycosylation on asparagine were specified as variable modifications. Results were filtered with a false discovery rate (FDR) of 1% and a confidence threshold of Byonic score >100. Glycopeptide composition with significant differences between cohort groups and AUC >0.7 was manually checked. In these cases, we confirmed the retention time, charge state, glycan oxonium ion, and isotopic pattern compared to the predicted isotopic pattern of the proposed glycopeptide. In this text, we refer to the glycan composition as follows: 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).

[0269] The schematic diagrams presented herein are based on the SNFG (Symbol Nomenclature for Glycans) system (PMID) detailed in 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). Glycopeptide XIC peak areas were automatically integrated by Proteome Discoverer and used as relative quantification values.

[0270] 1.7 Statistical analysis To correct for possible handling, digestion or MS measurement variations, the abundance of glycopeptides in the samples was normalized to the abundance of the top three peptides of the spike-in 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 dataset. The significance of the differences in glycopeptides between clinical groups was tested by calculating p-values ​​using the Wilcoxen (Mann-Whitney U) test. To correct for multiple testing, the Benjamini-Hochberg correction was used with an FDR control of 20%. 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.

[0271] 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 type and abundance of glycoforms differ at different Hp glycosites. Therefore, we investigated whether Hp glycosylation site and glycoform-specific analysis can be indicative of HCC. We compared the levels of site-specific glycopeptides between control, early and late stage HCC. Our observations revealed specific glycopeptides that were significantly upregulated in early stages of HCC compared to controls with AUCs of 0.70 or higher and could be unambiguously assigned to specific glycan structures (Figure 6). This set of glycopeptides includes highly branched (see Figure 4, compounds 126 or 140), fucosylated (see compounds 126, 131 or 140 in Figure 4) and highly mannosylated (see compounds 58, 60, 24 and 27 in Figure 6), and sialylated (see compound 126 in Figures 4 and 6) glycopeptides. Furthermore, as early-stage HCC-specific biomarkers, we discovered 14 other glycopeptides that were significantly highly expressed in both early- and late-stage HCC and provided an AUC of 0.70 or greater in discriminating early-stage HCC patients and CLD controls (Figure 6).

[0272] In addition to the upregulated glycopeptides in HCC, we observed two glycopeptides that were significantly downregulated in early and late stage HCC compared to CLD located at the N207 glycosylation site (Figures 4 and 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) to further improve robustness by serving as an internal control. In total, we identified 29 glycan structures that distinguished controls from early and late stage HCC with an AUC of over 70% (Figure 6).

[0273] By far the best glycopeptide for detecting early stage HCC was compound 126 on N207 (see Figure 6). Thus, among all glycosylation sites analyzed herein, haptoglobin glycosylation at position N207 appears to be the most suitable for detecting early stage HCC. Figure 6 shows the data for all altered glycopeptides using an AUC of 0.70 or greater as a cutoff.

[0274] The results, glycan structures, peptide sequences and m / z values ​​of the detected glycopeptides are summarized in Tables 2 and 3 below.

[0275] These results consistently revealed that glycopeptide analysis of Hp, especially at position N207 (but also at positions N184, N211, and N241), could provide a glycobiom- marker with better clinical value than established biomarkers for the diagnosis of early-stage HCC. [Table 2] [Table 3-1] [Table 3-2]

[0276] 2.2 Top glycans for detection of (early stage) HCC The results indicate that in particular Hp glycopeptide compound 126 (i.e., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) has high potential for clinical utility in the detection of HCC, especially early stage HCC. These are evident from the receiver operator curves (ROC) shown in FIG. 5 and the area under the curve (AUC) values ​​given for each of these glycopeptides. It is noted that for this glycopeptide, the calculated AUC exceeded the AUC values ​​for AFP and PIVKA-II, respectively, and their combination.

[0277] 2.3 Marker Combinations To analyze the added value of compound 126 to PIVKA-II and / or AFP, logistic regression models consisting of compound 126, PIVKA-II and / or AFP were constructed. In these models, logarithmic transformation was applied to the markers to reduce 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 results for compound 126 are summarized in Table 4 below. 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]

Claims

1. An in vitro method to assist in the detection of hepatocellular carcinoma (HCC) in a subject, a) A step of determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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, and 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 the increase in HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 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(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 those mentioned above.

2. An in vitro method to assist in the detection of hepatocellular carcinoma (HCC) in a subject, a) A step of determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 determining the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of the haptoglobin (i.e., the β chain of haptoglobin having the sequence shown in Sequence ID No. 1) in the sample obtained from the subject, c) A step of determining a score for detecting HCC by taking into account or consisting of the ratio of the amounts of the two glycan structures determined in a) and b) by dividing a) by b) or vice versa, d) A step of comparing the score determined in c) with a reference score value indicating HCC (e.g., early HCC). In vitro methods, including those mentioned above.

3. An isolated glycopeptide having a peptide portion and an N-glycan portion, wherein the peptide portion contains or consists of the amino acid sequence NLFLNHSE (SEQ ID NO: 2), the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and the N-glycan portion is bonded to the N at position 5 of SEQ ID NO:

2.

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

5. Use of the glycopeptide according to claim 3 or 4 to assist in the detection of HCC, or, in an embodiment, early HCC.

6. The method according to claim 2, wherein the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) is determined by determining the amount of a glycopeptide containing haptoglobin N207 and the glycan structure bound thereto, and in an embodiment, the peptide portion of the glycopeptide has the amino acid sequence of SEQ ID NO:

2.

7. The method according to any one of claims 1, 2, or 6, further comprising determining the amount of PIVKA-II and / or AFP in the sample or another sample from the same subject, wherein the score for detecting HCC takes into account the determined amount of PIVKA-II and / or the determined amount of AFP.

8. The method according to any one of claims 1, 2, and 6, wherein the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin is determined by determining the amount of the glycopeptide described in claim 3, or corresponds to the amount of the glycopeptide described in claim 3.

9. A method for detecting and / or quantifying a glycopeptide according to claim 3 or 4, a) A step to purify 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 3 or 4 by detecting the glycopeptide obtained in c) or b). Methods that include...

10. A clinical workflow for screening hepatocellular carcinoma (HCC) in a target population, a) A step of determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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, and b) A step of determining the amount of AFP in the sample obtained from the subject and / or determining the amount of PIVKA-II, c) A step of combining the amounts determined in a) and b) to obtain a combined value, and comparing the combined value with a reference value for the combined value, d) A clinical workflow further comprising the step of using the results of an ultrasound examination, wherein the HCC-positive ultrasound result and / or the bound value changed relative to the reference value indicates HCC.

11. A computer implementation method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, a) A step of receiving data including the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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, and b) A step of comparing the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) received in i)a) with a reference amount of the glycan structure, wherein the increase in HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of the haptoglobin in the sample with respect to the reference amount of the glycan structure indicates HCC, or (ii) A step of calculating a score for detecting HCC taking into account the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of the haptoglobin received in a), and comparing the calculated score for detecting HCC with a reference value of the score indicating HCC, and c) A step that assists in detecting whether the subject has HCC based on the comparison in b). Computer implementation methods, including those mentioned above.

12. A computer implementation method for assisting in the detection of HCC in a target, a) A step of receiving data including the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 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 receiving data including the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of the haptoglobin in the sample obtained from the subject, c) A step of calculating a score that includes or consists of the ratio of the amounts of two glycan structures in a) and b) (or vice versa), d) A step of comparing the score calculated in c) with a reference value of the score indicating HCC. Computer implementation methods, including those mentioned above.

13. The computer implementation method according to claim 11 or 12, further comprising receiving data including the amount of PIVKA-II and / or 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 PIVKA-II and / or the determined amount of AFP.

14. 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 11.

15. The method according to any one of claims 1, 2, 6, and 10 to 13, the use according to claim 5, or the clinical workflow according to claim 10, wherein the HCC is early HCC.

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