Glycan structure of haptoglobin as biomarker of hepatocellular carcinoma

Abstract

The present invention relates to an in vitro method for aiding 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. Of the beta-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject. The invention further discloses a glycan structure and a glycopeptide containing the glycan structure, and the glycan structure and the glycopeptide play an important role in HCC detection. Furthermore, the present invention relates to the use of one or more glycan structures at position N207 or a glycopeptide comprising haptoglobin N207 in combination with AFP and / or PIVKA in the detection of HCC.

Description

Technical Field

[0001] The present invention relates to in vitro methods for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, the methods comprising determining the amount of one or more glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject, and comparing the amount of the one or more glycan structures with a reference amount of the one or more glycan structures, wherein a change in the amount of the one or more glycan structures in the patient sample relative to the reference amount of the one or more glycan structures indicates HCC. In addition, the present invention relates to the use of a combination of one or more glycan structures at position N207 or a glycopeptide comprising N207 of haptoglobin with AFP and / or PIVKA-II in the detection of HCC. Background Art

[0002] Liver cancer is the seventh most common cancer and the second leading cause of cancer death worldwide, with an incidence and mortality rate of 10.1 and 9.5 per 100,000 population, respectively, in 2012.

[0003] Hepatocellular carcinoma (HCC) is the main histological type of primary liver cancer occurring worldwide, accounting for 70% to 85% of the total burden. It is well known that underlying liver diseases such as liver fibrosis and cirrhosis are the main risk factors for the development of HCC. For patients diagnosed at an early stage, HCC can be treated by resection, liver transplantation or radiofrequency ablation.

[0004] If this malignancy is diagnosed early, the 5-year survival rate of HCC patients may be as high as 70%. However, the 5-year survival rate of HCC patients decreases significantly as the disease is diagnosed later, and if HCC is diagnosed in the late stage of the disease, the 5-year survival rate of patients drops to only 15% (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 an advanced 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: the 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 crucial to reducing mortality and improving the efficiency / success rate of HCC treatment.

[0005] The most common methods for diagnosing HCC are ultrasound detection, imaging techniques such as computerized axial tomography (CAT scan) or magnetic resonance imaging (MRI), and serological biomarkers. However, the prognostic value of ultrasound detection is poor because it requires a tumor mass of at least 2 cm, and imaging techniques have poor sensitivity to each lesion and high cost. On the other hand, in recent years, people have devoted a lot of energy to discovering new blood biomarkers that can be used for early detection monitoring programs 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] Several current medical guidelines recommend monitoring high-risk patients every 6 months using ultrasound or other imaging modalities. However, limitations of imaging methods include poor sensitivity for early-stage tumors, operator dependence, and lower quality for patients with obesity or nonalcoholic steatohepatitis. 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 like many other tumor-related biomarkers, Hp is occasionally mentioned as a possible marker candidate in the field of HCC (Tai CS, Lin YR, Teng TH, Lin PY, Tu SJ, Chou CH, Huang YR, Huang WC, Weng S-L, 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 at the sugar site Asn241, has recently been shown to be associated 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 cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, 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 for 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] α-fetoprotein (AFP) is the best blood biomarker for HCC. However, even AFP shows suboptimal sensitivity and specificity for early detection of HCC. In addition, there are reports that AFP levels may be falsely elevated in patients with chronic hepatitis or cirrhosis but without HCC.

[0010] AFP is a glycoprotein, and various glycosylation forms of AFP have been described. Lectins can be used for the analysis of glycoproteins. By using the selective binding ability of lectins to the sugar chain structure of glycoproteins, it is possible to separate and concentrate marker glycoprotein parts with specific sugar chain structures. In the case of AFP, lectins derived from lentil lectin-A (LCA) have been widely used. The lentil lectin (LCA) reactive α-fetoprotein part (AFP-L3) is particularly increased in patients with HCC. Many attempts have been made to specifically measure AFP-L3, such as by affinity electrophoresis using LCA, lectin-based ELISA, or by antibodies that specifically bind to the L3 form of AFP.

[0011] High-speed / high-sensitivity qualitative / quantitative analysis using a mass spectrometer is also used for the analysis of glycoproteins. Multiple reaction monitoring mass spectrometry (MRM) helps to quantify peptides produced by protein hydrolysis and is very reliable. Parallel reaction monitoring (PRM) technology uses a mass spectrometer equipped with a trap and a time-of-flight mass analyzer so that the product ion spectrum of the peptide can be obtained, thereby allowing quantitative and qualitative analysis of the peptide at the same time. This method can also analyze trace glycoproteins that exhibit low signals, with high reproducibility and good sensitivity.

[0012] Methods for analyzing specific sugar chains using a mass spectrometer include methods based on analysis of sugar chains separated from glycoproteins and methods based on analysis of sugar chain-binding peptides (ie, glycopeptides).

[0013] Sugar chains bound to the same amino acid position of a protein may have various structures and show heterogeneity. It is well known that sugar structures may differ depending on the amino acid position at which the sugar chain is located.

[0014] Based on MRM-MS analysis, Kim and colleagues demonstrated that deglycopeptides of α-fetoprotein can better distinguish the cancer status between normal subjects and liver cancer patients than non-glycopeptides (Kim H, Kim K, Jin J, Park J, Yu SJ, Yoon JH, Kim Y. Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Formin Hepatocellular Carcinoma. PLOS One, 2014, 9: e110366). In EP3415918, various glycopeptides of AFP-L3 have been analyzed by mass spectrometry and used, for example, to compare samples obtained from patients with HCC with samples obtained from subjects with cirrhosis or hepatitis.

[0015] Vitamin K deficiency / antagonist-II-induced protein (PIVKA-II), also known as des-γ-carboxyprothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and can be used as a surrogate HCC biomarker alone or in combination with AFP. Prothrombin has 10 potential γ-carboxylation sites, and various forms of PIVKA-II with varying levels of undercarboxylation exist in the circulation. Different assays for PIVKA-II can detect different groups of PIVKA-II forms, and the specificity / sensitivity of PIVKA-II may vary depending on the assay used and has limited utility in detecting early HCC.

[0016] To date, international guidelines lack consensus on the use of AFP-L3 and PIVKA-II or alternative serum biomarkers for monitoring and diagnosing high-risk patients. However, many recent publications clearly indicate that including the input variables sex, age, AFP and decarboxy prothrombin (DCP = PIVKA-II) (= GAAD-score) or a score for sex, age, AFP, AFP-L3 and decarboxy prothrombin (DCP = PIVKA-II) (= GALAD-score) can and will improve the results of HCC screening efforts.

[0017] Nevertheless, there is still a great need for further improvement of in vitro methods that aid in the detection of HCC.

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

[0019] The present inventors have discovered that analysis of the glycan structure at position 207 of the haptoglobin β-chain (ie, amino acid position 207 of SEQ ID NO: 1) can overcome some of the problems of current in vitro diagnostic methods to aid in the detection of HCC.

[0020] In a first aspect, the present invention relates to an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, the method comprising the following steps: a) determining the amount of one or more glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject, and b) comparing the amount of the one or more glycan structures detected in a) with a reference amount of the one or more glycan structures, wherein a change in the amount of the one or more glycan structures in the patient sample relative to the reference amount of the one or more glycan structures indicates HCC.

[0021] In a specific embodiment, the one or more glycan structures attached to N207 are the glycan structures HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0022] In a second aspect, the present invention provides an in vitro method for assisting in detecting HCC (eg, early HCC) in a subject, the method comprising the following steps: a) determining the haptoglobin (i.e., having SEQ ID NO:1), and b) (i) comparing the amount of the N-glycan structure determined in a) with a reference amount of the N-glycan structure, wherein a change in the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin in the sample of the subject relative to the reference amount of such N-glycan structure indicates HCC (e.g., early HCC); or (ii) determining a score for detecting HCC (e.g., early 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 HCC) with a reference value for the score indicating HCC (e.g., early HCC).

[0023] In a third aspect, the present disclosure relates to an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) 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 given 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 the haptoglobin in the sample obtained from the subject, c) determining a score for detecting HCC (e.g., early HCC) by dividing a) by b) or vice versa, the score taking into account or consisting of the ratio of the two glycan structures determined in a) and b), respectively, and d) comparing the score determined in c) with a reference score value indicative of HCC (e.g., early HCC). The 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 assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) 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 given in SEQ ID NO: 1) 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 the haptoglobin in the sample obtained from the subject, c) determining a score for detecting HCC (e.g., early HCC) by dividing a) by b) or vice versa, the score taking into account or consisting of the ratio of the two glycan structures determined in a) and b), respectively, and d) comparing the score determined in c) with a reference score value indicative of HCC (e.g., early HCC). The 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 wherein the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and wherein the N-glycan portion is attached to the N in position 5 of SEQ ID NO:2.

[0026] In a preferred embodiment of the fifth aspect, provided herein is a glycopeptide of Formula 1:

[0027]

[0028] Wherein GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). Lines represent covalent bonds. The glycans in the glycopeptide are covalently attached as N-glycans to the side chain of asparagine corresponding to N207 of haptoglobin (i.e., N207 of the β-chain of haptoglobin of SEQ ID NO: 1) via the GlcNac of the glycan shown.

[0029] The glycopeptide comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 2. An alternative graphical representation of a glycopeptide having the same structure as that represented by Formula 1 above is as follows Figure 2 As shown in the figure on the right.

[0030] In a sixth aspect, the present invention relates to the use of the glycopeptide of formula 1 (see above) in the detection of HCC.

[0031] According to the seventh aspect of the present disclosure, a method for detecting the glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) is provided, the method comprising the following steps: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) by GluC and trypsin; and c) detecting the glycopeptide produced in b) comprising N207 of haptoglobin and the glycan structure attached thereto, thereby detecting the glycan structure at position N207.

[0032] In an eighth aspect, the present disclosure relates to an (in vitro) method for assisting in the detection of HCC (e.g., early-stage HCC) in a subject, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject, b) determining the amount of an HCC biomarker (e.g., the amount of AFP and / or the amount of PIVKA) in the sample obtained from the subject other than the amount of the N-glycan structure at position N207 in a), c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC, e.g., early-stage HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early-stage HCC).

[0033] In a ninth aspect, the present disclosure relates to a clinical workflow for screening a subject for HCC (e.g., early HCC), the clinical workflow comprising the following steps: a) determining the amount of one or more N-glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 the sample obtained from the subject; c) combining the amounts of markers determined in a) and b); and optionally d) additionally using the results of an ultrasound (US) examination, wherein an elevated score of the markers determined in a) and b) and / or optionally a positive US result indicates HCC (e.g., early HCC).

[0034] In a tenth aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0035] a) receiving data comprising the amount of a glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject; and

[0036] b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at position N207 of haptoglobin, wherein a change in the amount of the N-glycan structure in the patient sample relative to the reference amount of the one or more glycan structures is indicative of HCC; or (ii) calculating a score for detecting HCC (e.g., early HCC) taking into account the amount of the glycan structure at position N207 of haptoglobin received in a), and comparing the calculated score for detecting HCC (e.g., early HCC) with a reference value for the score indicating HCC (e.g., early HCC).

[0037] In an eleventh aspect, a computer-implemented method for aiding in detecting hepatocellular carcinoma (HCC) in a subject is provided, the method comprising the steps of:

[0038] 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. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject, and

[0039] b) i) comparing the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) received in a) to a reference amount of the glycan structure, wherein an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in the sample from the subject relative to the reference amount of the glycan structure is indicative of HCC, or (ii)

[0040] Considering the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin received in a), a score for detecting HCC (e.g., early HCC) is calculated, and the calculated score for detecting HCC (e.g., early HCC) is compared with a reference value for the score indicating HCC (e.g., early HCC).

[0041] In a twelfth aspect, the present invention provides a computer-implemented method for assisting in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising the following steps:

[0042] 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. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject,

[0043] 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,

[0044] c) calculating the ratio of the amounts of the two glycan structures comprising a) and b) (or vice versa) or the fraction consisting thereof, and

[0045] d) comparing the score calculated in c) with a reference value for the score indicative of HCC (eg, early stage HCC).

[0046] In a thirteenth aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0047] 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. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject

[0048] b) receiving data comprising the amount of PIVKA-II and / or AFP in a sample obtained from the subject,

[0049] c) calculating the fraction taking into account the quantities determined in a) and b); and

[0050] d) comparing the score calculated in c) with a reference value for the score indicative of HCC (eg, early stage HCC).

[0051] There is also provided herein a computer program product comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0052] Furthermore, a computer-readable medium is provided, comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0053] A data processing system is also provided, comprising: a receiving unit configured to receive data defined in any one of aspects ten to thirteen; and a processing unit configured to perform the calculation and / or comparison steps and / or any determination / assessment steps in any one of aspects ten to thirteen; and optionally an output unit configured to output an assessment result. DETAILED DESCRIPTION

[0054] Elements of the present invention will be described below. These elements are listed with specific aspects and embodiments, however, it should be understood that they can be combined in any manner and in any number to form additional aspects and embodiments.

[0055] In a first aspect, the present invention relates to an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, the method comprising the following steps: a) determining the amount of one or more glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject, and b) comparing the amount of the one or more glycan structures determined in a) with a reference amount of the glycan structures, wherein a change in the amount of the one or more glycan structures in the patient sample relative to the reference amount of the one or more glycan structures indicates HCC.

[0056] In an embodiment of the first aspect of the present invention, an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject is provided, the method comprising the following steps: a) determining the amount of a glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) in a sample obtained from the subject, and b) comparing the amount of the glycan structure determined in a) with a reference amount of the glycan structure, wherein a change in the amount of the glycan structure in the patient sample relative to the reference amount of the glycan structure indicates HCC.

[0057] The method of the first aspect of the invention is based on the discovery that the glycosylation pattern at position 207 of the β-chain of haptoglobin can be used to detect HCC (e.g., early HCC). As demonstrated in the accompanying examples, the glycan structure at this position in samples from patients with HCC (e.g., early HCC) shows a characteristic pattern, for example compared to a reference sample, and compared to a sample from a patient with cirrhosis.

[0058] In an embodiment of the first aspect of the present invention, the determined amount of one or more glycan structures at position N207 of haptoglobin may not be individually compared with a reference amount of the corresponding glycan structure, but a score for determining HCC (e.g., early HCC) may be calculated taking into account the determined amount of one or more glycan structures at position N207 of haptoglobin. The score may be compared with a corresponding reference value for the score, which is decisive for the presence of HCC (e.g., early HCC).

[0059] Therefore, the present invention provides an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject, the method comprising the following steps: a) determining the amount of a glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given 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 determined score for detecting HCC (e.g., early HCC) with a reference value indicating HCC (e.g., early HCC). Based on the comparison, the presence or absence of HCC (e.g., early HCC) can be determined.

[0060] In an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) determined in 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 an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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).

[0061] In embodiments where 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 increase in the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) relative to a reference amount indicates HCC (e.g., early HCC). In embodiments where the glycan structure HexNAc(4)Hex(5)NeuAc(2) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin is determined, a decrease in the amount of the corresponding glycan structure indicates HCC (e.g., early HCC).

[0062] In a particularly preferred embodiment, the determined glycan structure at position N207 of haptoglobin (ie of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0063] 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 wherein 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, combining the amount of the glycan structure at position N207 of haptoglobin with the amount of AFP and / or PIVKA-II increases the diagnostic utility for HCC (e.g., early HCC).

[0064] In a second aspect, the present invention relates to an in vitro method for assisting in detecting HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) determining the haptoglobin (i.e., having SEQ ID NO:1), and b) (i) comparing the amount of the N-glycan structure determined in a) with a reference amount of the N-glycan structure, wherein a change (e.g., an increase in the amount) of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin in the sample of the subject relative to the reference amount of such N-glycan structure indicates HCC (e.g., early HCC); or (ii) determining a score for detecting HCC (e.g., early 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 HCC) with a reference value for the score indicating HCC (e.g., early HCC). Based on the comparison in b) i) or ii), the presence or absence of HCC (eg, early stage HCC) can be determined.

[0065] The method of the second aspect is based on the discovery that an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) is found in samples obtained from subjects suffering from HCC (e.g., early HCC) relative to control samples representing chronic liver diseases (including HBV, HCV, and cirrhosis). As shown in the accompanying examples, the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) represents a very promising marker for HCC (e.g., early HCC), showing the highest AUC when detecting early HCC of all identified N-glycan structures (including all glycan structures at position N207 as well as other glycosylation sites). The structure of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) can be shown as Figure 2 Thus, in an embodiment, 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 given in SEQ ID NO: 1) may have the following structure: Figure 2 The glycan structure shown in the glycan portion of the glycopeptide in , or the glycan structure of Formula 2 referred to elsewhere herein.

[0066] What has been said herein with respect to the method according to the first aspect of the invention also applies mutatis mutandis.

[0067] In an embodiment of the second aspect of the present invention, an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: 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 given 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) with a reference amount of the N-glycan structure, wherein a change in the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin in the sample of the subject relative to the reference amount of the N-glycan structure indicates HCC (e.g., early HCC).

[0068] If a reference amount of the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) is selected so that it represents the amount of this N-glycan structure in healthy subjects and / or subjects suffering from non-cancerous chronic liver diseases (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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) relative to the reference amount indicates HCC (e.g., early HCC).

[0069] Thus, in embodiments, an increase in the N-glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) is indicative of HCC (eg, early stage HCC).

[0070] In an embodiment, the second aspect of the present invention relates to an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: 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 given 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 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 HCC) with a reference value for the score indicating HCC (e.g., early HCC). The comparison in c) can then be used to determine the presence or absence of HCC (e.g., early HCC).

[0071] In embodiments, the score may be configured such that higher values ​​of the score indicate 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.

[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 given in SEQ ID NO:1) in a sample means determining a measure of the absolute amount or relative amount of haptoglobin that reflects the glycosylation of the corresponding glycan structure at position N207 of haptoglobin in the sample.

[0073] The following discloses an example 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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1).

[0074] In an embodiment, the level of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) can be determined by determining the level of a glycopeptide comprising N207 and the glycan structure, for example, by mass spectrometry.

[0075] 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 given in SEQ ID NO: 1) for determining 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 given in SEQ ID NO: 1) comprises (SEQ ID NO: 2), wherein the middle N (bold and underlined; in position 5 of SEQ ID NO: 2) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1). In a specific embodiment, the peptide portion of the glycopeptide can be composed of (SEQ ID NO: 2). Therefore, the level of Formula 1 (see elsewhere herein) was determined to determine the level of the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin.

[0076] In the embodiments, the glycopeptide having the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) detected for determining the amount of 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.

[0077] In other words, to determine the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), a glycopeptide may be analyzed, wherein the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), wherein the middle N (i.e., the N in position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO:1), and wherein the glycan structure is HexNAc(6)Hex(7)Fuc(1)NeuAc(4). In a specific embodiment, the peptide portion of the glycopeptide may consist of (SEQ ID NO: 2). Therefore, to determine the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin, the glycopeptide of formula 1 (see elsewhere herein) can be analyzed.

[0078] 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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO: 2) wherein the middle N corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1) and wherein the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4) can be analyzed. The glycopeptide can be as in Formula 1 (see elsewhere herein) or Figure 2 . It has a characteristic m / z value and this substitution is also well defined. For example, a glycopeptide used to determine 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.

[0079] In an embodiment, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as the peptide portion and HexNAc(6)Hex(7)Fuc(1)NeuAc(4) as the glycan structure attached to the middle N (i.e., the N in position 5) of SEQ ID NO:2, wherein the middle N corresponds to the N at position 207 of the β-chain of the haptoglobin (SEQ ID NO:1) sequence and may have an m / z of 927.9544, in particular a charge of 5, and a glycopeptide mass of 4636.8124 Da.

[0080] In order to reduce the influence of sample processing and the difference in the absolute amount of haptoglobin between individuals, determining 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 given in SEQ ID NO:1) may mean determining the relative amount with respect to the amount of a second analyte or group of analytes. This second analyte may be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide or glycopeptide may be externally incorporated into the sample prior to the determination step, or may be a peptide or glycopeptide of haptoglobin contained in the sample. The spiked peptide may be labeled with a heavy isotope.

[0081] 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 wherein 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, combining the amount of the glycan structure at position N207 of haptoglobin with the amount of AFP and / or PIVKA-II increases the diagnostic utility for HCC (e.g., early HCC).

[0082] According to a third aspect, the present disclosure relates to an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) 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 given 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 the haptoglobin in the sample obtained from the subject, c) determining a score for detecting HCC (e.g., early HCC) by dividing a) by b) or vice versa, the score taking into account or consisting of the ratio of the amounts of the two glycan structures determined in a) and b), respectively, and d) comparing the score determined in c) with a reference score value indicative of HCC (e.g., early HCC). The comparison can then be used to determine the presence (or absence) of HCC.

[0083] What has been said herein with respect to the methods according to the first and second aspects of the invention applies mutatis mutandis.

[0084] The glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin has been identified as a potential marker for HCC (e.g., early HCC), such as Figures 4 to 6(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) relative to the amount of this glycan structure in samples from control subjects.

[0085] Constructing a ratio of the amount of a first sugar structure that is shown in a higher amount in HCC (e.g., early HCC) subjects than in control subjects to the amount of a second sugar structure that is shown in a lower amount in HCC (e.g., early HCC) subjects than in control subjects (or vice versa) may improve diagnostic performance.

[0086] In an embodiment of the method according to the third aspect of the present invention, an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: a) 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 given 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 the 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) with a reference ratio, wherein an increase in the ratio calculated for the sample of the subject relative to the reference ratio of the N-glycan structure indicates HCC (e.g., early HCC).

[0087] 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 given in SEQ ID NO:1) in a sample means determining a measure of the absolute amount or relative amount of haptoglobin that reflects the glycosylation of the corresponding glycan structure at position N207 of haptoglobin in the sample.

[0088] The examples described elsewhere herein for determining the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) are also applicable, with appropriate modifications, to determining the amount of HexNAc(4)Hex(5)NeuAc(2).

[0089] Therefore, determining the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) may include generating a glycopeptide from the haptoglobin contained in the sample, the glycopeptide comprising the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), and determining the amount of the glycopeptide, for example, by mass spectrometry. The amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) can be derived from or correspond to the amount of a glycopeptide comprising the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1).

[0090] In an embodiment, the 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 given in SEQ ID NO: 1) 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 given in SEQ ID NO: 1) comprises (SEQ ID NO: 2), wherein the middle N (bold and underlined) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1). In a specific embodiment, the peptide portion of the glycopeptide can be composed of (SEQ ID NO:2).

[0091] In the embodiments, the 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 given in SEQ ID NO:1) detected for determining the amount of 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.

[0092] In the examples, the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin is determined by determining the amount of the glycopeptide of formula 5.

[0093]

[0094] In other words, in order to determine the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), a glycopeptide may be analyzed, wherein the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), wherein the middle N (i.e., the N in position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO:1), and wherein the glycan structure is HexNAc(4)Hex(5)NeuAc(2). In a specific embodiment, the peptide portion of the glycopeptide may consist of (SEQ ID NO:2).

[0095] In an embodiment, in order to determine the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO: 2), wherein the middle N corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1), and wherein the glycan is HexNAc(4)Hex(5)NeuAc(2) can be analyzed. The glycopeptide can be a glycopeptide as shown in Formula 5 (see above). It has a characteristic m / z value, and this substitution is also well defined. For example, the glycopeptide used to determine the amount of the glycan structure HexNAc(4)Hex(5)NeuAc(2) at position N207 of haptoglobin can have an m / z of 1060.0883, a charge of 3, and a glycopeptide mass of 3178.2503.

[0096] In an embodiment, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as the peptide portion and HexNAc(4)Hex(5)NeuAc(2) as the glycan structure attached to the middle N (i.e., the N in position 5) of SEQ ID NO:2, wherein the middle N corresponds to the N at position 207 of the β-chain 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.

[0097] 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 wherein 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, combining the amount of the glycan structure at position N207 of haptoglobin with the amount of AFP and / or PIVKA-II increases the diagnostic utility for HCC (e.g., early HCC).

[0098] In a fourth aspect, the present disclosure relates to an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) 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 given in SEQ ID NO: 1) 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 the haptoglobin in the sample obtained from the subject, c) determining a score for detecting HCC (e.g., early HCC) by dividing a) by b) or vice versa, the score taking into account or consisting of the ratio of the amounts of the two glycan structures determined in a) and b), respectively, and d) comparing the score determined in c) with a reference score value indicative of HCC (e.g., early HCC). The comparison can then be used to determine the presence (or absence) of HCC.

[0099] What has been said herein with respect to the methods according to the first, second and third aspects of the invention also applies mutatis mutandis.

[0100] The glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin has been identified as a potential marker for HCC (e.g., early HCC), such as Figures 4 to 6 (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) relative to the amount of this glycan structure in samples from control subjects.

[0101] Constructing a ratio of the amount of a first sugar structure that is shown in a higher amount in HCC (e.g., early HCC) subjects than in control subjects to the amount of a second sugar structure that is shown in a lower amount in HCC (e.g., early HCC) subjects than in control subjects (or vice versa) may improve diagnostic performance.

[0102] In an embodiment of the fourth aspect of the present disclosure, an in vitro method for assisting in the detection of HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: 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 the 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) with a reference ratio, wherein an increase in the ratio calculated for the sample of the subject relative to the reference ratio of the N-glycan structure indicates HCC (e.g., early HCC).

[0103] The examples described elsewhere herein for determining the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) are also applicable, mutatis mutandis, to determining the amount of HexNAc(5)Hex(5)NeuAc(1).

[0104] Therefore, determining the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) may include generating a glycopeptide from the haptoglobin contained in the sample, the glycopeptide comprising the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), and determining the amount of the glycopeptide, for example, by mass spectrometry. The amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) can be derived from or correspond to the amount of a glycopeptide comprising the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1).

[0105] In an embodiment, the 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 given in SEQ ID NO: 1) 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 given in SEQ ID NO: 1) comprises (SEQ ID NO: 2), wherein the middle N (bold and underlined) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1). In a specific embodiment, the peptide portion of the glycopeptide can be composed of (SEQ ID NO:2).

[0106] In the examples, the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin is determined by determining the amount of the glycopeptide of Formula 6.

[0107]

[0108] In the embodiments, the glycopeptide having the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) detected for determining 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.

[0109] In other words, in order to determine the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), 1) a glycopeptide may be analyzed, wherein the glycopeptide may comprise the peptide sequence NLFLNHSE (SEQ ID NO:2), wherein the middle N (i.e., the N in position 5 of SEQ ID NO:2) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO:1), and wherein the glycan structure is HexNAc(5)Hex(5)NeuAc(1). In a specific embodiment, the peptide portion of the glycopeptide may consist of (SEQ ID NO:2).

[0110] In an embodiment, in order to determine the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), a glycopeptide having the amino acid sequence NLFLNHSE (SEQ ID NO:2) wherein the middle N corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO:1) and wherein the glycan is HexNAc(5)Hex(5)NeuAc(1) can be analyzed. The glycopeptide can be a glycopeptide as shown in Formula 6 (see above). It has a characteristic m / z value and this substitution is also well defined. For example, a glycopeptide used to determine the amount of the glycan structure HexNAc(5)Hex(5)NeuAc(1) at position N207 of haptoglobin can have an m / z of 982.0654, a charge of 3 and a glycopeptide mass of 2116.7564 Da.

[0111] In an embodiment, the glycopeptide may comprise the amino acid sequence NLFLNHSE (SEQ ID NO:2) as the peptide portion and HexNAc(5)Hex(5)NeuAc(1) as the glycan structure attached to the middle N (i.e., the N in position 5) of SEQ ID NO:2, wherein the middle N corresponds to the N at position 207 of the β-chain 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.

[0112] 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 wherein 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, combining the amount of the glycan structure at position N207 of haptoglobin with the amount of AFP and / or PIVKA-II increases the diagnostic utility for HCC (e.g., early HCC).

[0113] 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 wherein the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and wherein the N-glycan portion is attached to the N in position 5 of SEQ ID NO:2.

[0114] In a preferred embodiment, the glycopeptide is a glycopeptide of Formula 1:

[0115]

[0116] Where GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). Lines represent covalent bonds. The glycans in the glycopeptide are covalently attached as N-glycans to the side chain of asparagine corresponding to N207 of haptoglobin via the GlcNac of the glycan shown. (i.e., N207 of the β-chain of haptoglobin of SEQ ID NO: 1).

[0117] Figure 2 (Right panel) shows the isolated glycopeptide of Formula 1 only in a graphical alternative representation. Figure 2 The right figure is a representation based on 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 al., The SNFG Discussion Group, Updates to the Symbol Nomenclature for Glycansguidelines, Glycobiology, Volume 29, Issue 9, September 2019, Pages 620-624, https: / / doi.org / 10.1093 / glycob / cwz045).

[0118] As shown throughout this disclosure, the glycopeptide of Formula 1 holds great promise for detecting HCC, such as early-stage HCC.

[0119] Therefore, in a sixth aspect, the present disclosure relates to use of the glycopeptide according to the fifth aspect of the present disclosure (ie, the glycopeptide of Formula 1) in detecting HCC (eg, early-stage HCC).

[0120] The embodiments of the methods according to the first, second, third and fourth aspects are equally applicable to this purpose with appropriate modifications.

[0121] According to the seventh aspect of the present disclosure, a method for detecting the glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) is provided, the method comprising the following steps: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) by GluC and trypsin; and c) detecting the glycopeptide produced in b) comprising N207 of haptoglobin and the glycan structure attached thereto, thereby detecting the glycan structure at position N207.

[0122] In an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 an embodiment, 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).

[0123] In the attached examples, it is shown that after purification and digestion and subjecting to an appropriate detection method (preferably LC-MS), glycopeptides comprising an N-glycan modification at the asparagine corresponding to position N207 of haptoglobin can be reliably determined with good sensitivity. In particular, all glycopeptides specifically mentioned herein (e.g., glycopeptides according to the fifth aspect of the invention) can be reliably determined using this method.

[0124] The attached examples also specifically show that after purification and digestion and subjecting to an appropriate detection method (preferably LC-MS), the glycopeptide according to the fifth aspect of the present disclosure can be reliably determined with good sensitivity. Therefore, in an embodiment, a method for detecting a glycan structure according to the fifth aspect (e.g., a glycopeptide of Formula 1) is provided, the method comprising the following steps: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) by GluC and trypsin, and c) detecting the glycopeptide obtained in b), thereby detecting (e.g., a glycopeptide of Formula 1) according to the fifth aspect.

[0125] In clinical routine, the diagnosis of HCC, especially early-stage HCC, is very challenging, and many cases of HCC are diagnosed too late to be cured. Ultrasound is often 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 affected by factors such as weight / fatness. Despite this, advanced HCC is often first discovered by ultrasound.

[0126] To date, biomarkers have not been routinely used for HCC detection. In some treatment guidelines, the use of alpha-fetoprotein (AFP) is considered useful. However, this rather old biomarker is not very reliable and the test results are often negative for patients with early HCC. The same is true for the biomarker PIVKA-II (= decarboxylated prothrombin). However, both AFP and PIVKA-II tend to be elevated in advanced HCC. The opposite applies to glycan structures and to the glycopeptides disclosed and claimed in the present invention.

[0127] It is expected that the disclosed glycan structures of the present invention will significantly improve the detection of HCC, especially the detection of early stage HCC.In one embodiment, the method for detecting HCC disclosed herein is a method for detecting HCC at an early stage (ie, early stage HCC).

[0128] In an embodiment, the glycan structure of the present invention can also be combined with other biomarkers, such as with biomarkers that also reliably detect advanced HCC. Such combinations are very useful for detecting HCC (and early HCC) at any stage, as demonstrated by the combined AUC values ​​shown in the attached examples.

[0129] Therefore, in an eighth aspect, the present disclosure relates to an (in vitro) method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) in a sample obtained from the subject, b) determining the amount of an HCC biomarker in the sample obtained from the subject other than the amount of the N-glycan structure at position N207 in a), c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC, e.g., early HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early HCC).

[0130] The embodiments of the aforementioned aspects are equally applicable with appropriate modifications.

[0131] HCC biomarkers are biomarkers indicative of HCC (eg, early-stage HCC). In a specific example, the HCC biomarker may be AFP or PIVKA-II.

[0132] In an embodiment of the eighth aspect of the present invention, there is provided an (in vitro) method for assisting in the detection of HCC (e.g., early HCC) in a subject, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given 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 the sample obtained from the subject, c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC, e.g., early HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early HCC).

[0133] In an embodiment of the method of the eighth aspect, an (in vitro) method for assisting in the detection of HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given 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 combined value (e.g., a score for detecting HCC, e.g., early HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early HCC).

[0134] In an embodiment of the method of the eighth aspect, an (in vitro) method for assisting in the detection of HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given 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, c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC, e.g., early HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early HCC).

[0135] In an embodiment of the method of the eighth aspect, an (in vitro) method for assisting in detecting HCC (e.g., early HCC) in a subject is provided, the method comprising the following steps: a) determining the amount of the N-glycan structure at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given 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 the sample obtained from the subject, c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC, e.g., early HCC), and comparing the combined value with a reference value for the combined value, wherein a change in the combined value indicates HCC (e.g., early HCC).

[0136] In an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 present 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 present invention, the glycan structure at position N207 of haptoglobin is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0137] What has been described elsewhere herein (e.g. in the context of other aspects and below) with respect to determining the amount of individual glycan structures also applies mutatis mutandis. Thus, the amount of glycan structures can be determined by determining the amount of glycopeptides (e.g. by mass spectrometry).

[0138] The samples used for determination in a) and b) may be the same or may be different samples obtained from the subject. Preferably, the samples are obtained from the subject at the same time.

[0139] In an embodiment, a "combined value" may mean determining a score indicative of HCC (e.g., early HCC) that takes into account the amounts determined in a) and b). The score may further take into account other biomarkers. The score may alternatively and additionally further take into account clinical data of the subject (e.g., age, sex, smoking status, cancer history, family cancer history, and the presence of pre-existing liver disease).

[0140] An exemplary but non-limiting manner for calculating a combined value or score is disclosed below.

[0141] 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 assays were performed on an uTASWako i30 autoanalyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions. For example, AFP can also be measured using AFP (material number: 044817981190) can be used for detection. For example, PPIVKA-II can be used PIVKA-II (material number: 08333602190).

[0142] In the method according to the eighth aspect, the N-glycan structure is preferably HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and even more preferably is a glycan contained in the 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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) can also be determined; the ratio of the amounts can be calculated as described elsewhere herein and the combined value can be calculated using this ratio.

[0143] In an ideal clinical setting, an ultrasound examination of the liver would be performed, or corresponding results from such an examination (previously performed) would be received, and a parallel blood sample obtained from the subject would be examined for the markers discussed above.

[0144] Therefore, in a ninth aspect, the present disclosure relates to a clinical workflow for screening a subject for HCC (e.g., early HCC), the clinical workflow comprising the following steps: a) determining the amount of one or more N-glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 the sample obtained from the subject; c) combining the amounts of the markers determined in a) and b); and d) additionally using the results of an ultrasound (US) examination, wherein a positive US result and / or an increased score of the markers determined in a) and b) indicates HCC (e.g., early HCC).

[0145] The embodiments described herein with respect to any of the preceding aspects of the present invention are equally applicable to the ninth aspect of the present invention with appropriate modifications.

[0146] In an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 an embodiment, the N-glycan structure at position N207 of haptoglobin can 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 present invention, the glycan structure at position N207 of haptoglobin is HexNAc(6)Hex(7)Fuc(1)NeuAc(4). The contents described elsewhere herein (e.g. in the context of other aspects) regarding the detection of the glycan structure mentioned in this paragraph are equally applicable with appropriate modifications.

[0147] In the method according to the ninth aspect, the N-glycan structure is preferably HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and even more preferably is a glycan contained in the 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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) can also be determined; the ratio of the amounts can be calculated as described elsewhere herein and the combined value can be calculated using this ratio.

[0148] In an embodiment, the workflow can be used to monitor patients at risk for HCC (e.g., early HCC). In other words, the subject may be known to be at risk for 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.

[0149] In one embodiment according to the present disclosure, the subject from which the sample to be studied has been obtained is a healthy subject and the subject is screened for (the presence of) HCC as part of routine oncology monitoring.

[0150] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained is a subject at risk of developing HCC and is screened for (the presence of) HCC as part of routine oncology monitoring.

[0151] If the subject is known to suffer from chronic liver disease, viral or non-viral hepatitis, and / or cirrhosis, the subject may be at risk for developing HCC.

[0152] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained suffers from chronic liver disease, viral or non-viral hepatitis, liver cirrhosis, and is differentially diagnosed due to the presence or absence of HCC.

[0153] In a tenth aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0154] a) receiving data comprising the amount of a glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject; and

[0155] b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at position N207 of haptoglobin, wherein a change in the amount of the N-glycan structure in the patient sample relative to the reference amount of the one or more glycan structures is indicative of HCC; or (ii) calculating a score for detecting HCC (e.g., early HCC) taking into account the amount of the glycan structure at position N207 of haptoglobin received in a), and comparing the calculated score for detecting HCC (e.g., early HCC) with a reference value for the score indicating HCC (e.g., early HCC).

[0156] The embodiments of the aforementioned aspects are equally applicable with appropriate modifications.

[0157] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0158] In an embodiment, a method may include outputting (eg, via a display) whether the subject suffers from HCC (eg, early-stage HCC).

[0159] In an embodiment, the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given 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 an embodiment, 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).

[0160] In a particularly preferred embodiment, the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0161] In an eleventh aspect, a computer-implemented method for aiding in detecting hepatocellular carcinoma (HCC) in a subject is provided, the method comprising the steps of:

[0162] c) receiving a sample containing haptoglobin (i.e., having SEQ ID

[0163] Data on the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of the β-chain of haptoglobin of the sequence given in NO:1, and

[0164] d) i) comparing the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) received in a) to a reference amount of the glycan structure, wherein an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in the sample from the subject relative to the reference amount of the glycan structure is indicative of HCC, or (ii)

[0165] Considering the amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin received in a), a score for detecting HCC (e.g., early HCC) is calculated, and the calculated score for detecting HCC (e.g., early HCC) is compared with a reference value for the score indicating HCC (e.g., early HCC).

[0166] The embodiments of the aforementioned aspects are equally applicable with appropriate modifications.

[0167] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0168] In an embodiment, a method may include outputting (eg, via a display) whether the subject suffers from HCC (eg, early-stage HCC).

[0169] In a twelfth aspect, the present invention provides a computer-implemented method for assisting in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising the following steps:

[0170] a) receiving a sample containing haptoglobin (i.e., having SEQ ID

[0171] Data on the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of the β-chain of haptoglobin of the sequence given in NO:1,

[0172] 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,

[0173] c) calculating the ratio of the amounts of the two glycan structures comprising a) and b) (or vice versa) or the fraction consisting thereof, and

[0174] d) comparing the score calculated in c) with a reference value for the score indicative of HCC (eg, early stage HCC).

[0175] The embodiments of the aforementioned aspects are equally applicable with appropriate modifications.

[0176] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0177] In an embodiment, a method may include outputting (eg, via a display) whether the subject suffers from HCC (eg, early-stage HCC).

[0178] In a thirteenth aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0179] 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. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject

[0180] b) receiving data comprising the amount of PIVKA-II and / or AFP in a sample obtained from the subject,

[0181] c) calculating the fraction taking into account the quantities determined in a) and b); and

[0182] d) comparing the score calculated in c) with a reference value for the score indicative of HCC (eg, early stage HCC).

[0183] The embodiments of the aforementioned aspects are equally applicable with appropriate modifications.

[0184] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0185] In an embodiment, a method may include outputting (eg, via a display) whether the subject suffers from HCC (eg, early-stage HCC).

[0186] In an embodiment, 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).

[0187] In a particularly preferred embodiment, the glycan is HexNAc(6)Hex(7)Fuc(1)NeuAc(4).

[0188] If there are two or more steps of receiving data in the computer-implemented method disclosed herein, those skilled in the art will readily understand that this includes (i) embodiments in which a single data set includes all of the received data in the two or more steps, and (ii) embodiments in which two or more separate data sets are received.

[0189] Any of the above computer-implemented methods may optionally include the following steps: Based on the comparison step, whether the subject suffers from or is suspected of suffering from HCC (eg, early HCC) and / or whether further clinical examination of HCC (eg, early HCC) is required may be outputted via a display.

[0190] A computer program product is also disclosed herein, comprising instructions, which, when the program is executed by a computer, cause the computer to perform the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0191] Furthermore, a computer-readable medium is provided, comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to any one of the tenth to thirteenth aspects.

[0192] A data processing system is also provided, comprising: a receiving unit configured to receive data defined in any one of aspects ten to thirteen; and a processing unit configured to perform the calculation and / or comparison steps and / or any determination / assessment steps in any one of aspects ten to thirteen; and optionally an output unit configured to output an assessment result.

[0193] The above aspects relate to the glycan structure at position N207 of the β-chain of haptoglobin. As is apparent from the attached examples, many other glycan structures at positions N184, N211 or N241 that can be used to detect HCC are also identified herein. These glycan structures are shown in Figure 6 and further information about them and about exemplary glycopeptides used to determine the levels of such glycan structures is provided in Tables 2 and 3.

[0194] The corresponding glycan structures at position N184 of haptoglobin are: 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), HexNA c(5)Hex(6)Fuc(2)NeuAc(2), HexNAc(2)Hex(12), HexNAc(6)Hex(7) and HexNAc(4)Hex(5)Fuc(3)NeuAc(1).

[0195] The corresponding glycan structures at position N211 of haptoglobin are: HexNAc(6)Hex(7)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2) and HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0196] The corresponding glycan structures at position N241 of haptoglobin are: HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(4)Hex(5)NeuAc(2), HexNAc(5)Hex(6)Fuc(1)NeuAc(2) and HexNAc(4)Hex(5)Fuc(1)NeuAc(1).

[0197] Therefore, all aspects and embodiments as described herein with respect to N207 of haptoglobin are disclosed with appropriate modifications with respect to N184, N211 and N241 of haptoglobin. In addition, all aspects and embodiments disclosed herein with respect to the specific glycan structure at position N184 are also disclosed with appropriate modifications with respect to the above glycan structures (and / or Figure 6 and any of the glycan structures mentioned in Table 2).

[0198] To detect the amount of glycan structure at position N184, a PCR product containing MVSHH NA glycopeptide of a peptide portion of LTTGATLINE (SEQ ID NO: 3) or consisting thereof, wherein the N in position 6 corresponds to N184.

[0199] To detect the amount of glycan structure at position N211, a glycopeptide having a peptide portion comprising or consisting of NATAK (SEQ ID NO: 4) can be used, wherein the N in position 1 corresponds to N211.

[0200] To detect the amount of the glycan structure at position N241, a glycopeptide having a peptide portion comprising or consisting of VVLHPNYSQVD (SEQ ID NO: 5) can be used, wherein the N in position 6 corresponds to N241.

[0201] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although in the practice or testing of the present invention, methods and materials similar or equivalent to those described herein may be used, suitable methods and materials are described below. In the event of ambiguity, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0202] The following definitions and examples apply to this disclosure as a whole and in particular to all aspects and embodiments of the invention.

[0203] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise.

[0204] 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.

[0205] The use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

[0206] The term "and / or" should be understood to mean either or both of the alternatives.

[0207] As used herein and unless otherwise specified, it should be understood that the term "about" is used synonymously with the term "approximately". Illustratively and unless otherwise specified, the term "about" when used in conjunction with a stated value or range means slightly more or slightly less than the stated value or range, within ±15% of the stated value or range, within ±10% of the stated value or range, within ±5% of the stated value or range, or conveniently within ±2% of the stated value or range. Therefore, these values ​​are encompassed within the scope of the claims that reference the term "about" or "approximately".

[0208] As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, a protein, a carbohydrate structure or glycolipid, a metabolite, an mRNA, a miRNA, a protein, a DNA (cDNA or genomic DNA), a DNA copy number, or an epigenetic change, for example, increased, decreased, or altered DNA methylation (e.g., cytosine methylation or CpG methylation, non-CpG methylation); histone modification (e.g., (de)acetylation, (de)methylation, (de)phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation); altered nucleosome positioning, the expression or presence of a biomarker in or on a mammalian tissue or cell can be detected by standard methods (or methods disclosed herein), and the biomarker may be predictive, diagnostic, and / or prognostic for the health or disease of an individual. Therefore, in the following, sometimes when discussing more general terms and definitions, the more general term "marker" will also be used. The term marker also includes a glycan structure or glycan, or a glycopeptide as analyzed in the present disclosure.

[0209] The term "in vitro method" is used to indicate that the method is performed outside a living organism, and preferably is performed on body fluids, isolated tissues, organs or cells. In vitro methods may also be referred to as ex vivo methods.

[0210] Hepatocellular carcinoma (HCC) is the main histological type of primary liver cancer occurring worldwide, accounting for 70% to 85% of the total burden. It is well known that underlying liver diseases such as liver fibrosis and cirrhosis are major risk factors for the development of HCC. For patients diagnosed at an early stage, HCC can be treated by resection, liver transplantation, or radiofrequency local ablation. If this malignancy is diagnosed early, the 5-year survival rate of HCC patients may be as high as 70%. However, the 5-year survival rate of HCC patients decreases significantly as the disease is diagnosed later, and if HCC is diagnosed in the late stage of the disease, the patient's 5-year survival rate drops to only 15% (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).

[0211] Haptoglobin is an acute phase protein. It is synthesized in the liver and secreted into the plasma and has a fairly complex biochemistry and multiple biological functions. Figure 1 As shown in , 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 polymorphism (α1-chain or α2-chain, respectively), resulting in three types of haptoglobin 1-1, 2-1 and 2-2 (see Figure 1 ). The concentration of haptoglobin in human plasma is usually in the range of 0.3mg / ml to 3mg / ml. Haptoglobin binds to free hemoglobin, thereby protecting against oxidative stress. It also plays a role in regulating immune responses by binding to resting and activated CD4+ and CD8+ T cells.

[0212] The β-chain of haptoglobin does not show genetic polymorphism. As used herein, the β-chain of haptoglobin preferably has a sequence as given in SEQ ID NO: 1. Haptoglobin can be modified secondary, for example in the form of glycosylation. The β-chain of haptoglobin has four N-glycosylation sites (asparagine (N)) at amino acid positions 183, 207, 211 and 241, respectively.

[0213] As used herein and if not mentioned otherwise, the expression "at position N207 of haptoglobin" refers to position N207 of the β-chain of haptoglobin (eg having the sequence given in SEQ ID NO: 1).

[0214] As will be understood by those skilled in the art who are familiar with the method of assisting detection of HCC (e.g., early HCC), the assessment of HCC performed according to the present invention is preferred, but may not usually be 100% correct research subjects (basically for each diagnostic method). The term generally requires that a statistically significant portion of subjects can be correctly assessed. Those skilled in the art can use various well-known statistical evaluation tools (e.g., determining confidence intervals, determining p values, Student's t test, Mann-Whitney test, etc.) to effortlessly determine whether a part is statistically significant. Detailed information can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The confidence intervals generally envisioned are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p value is generally 0.2, 0.1, 0.05.

[0215] The term "indicative of HCC" is used to illustrate that the level or amount of the marker (e.g., of a glycan structure or glycopeptide) determined is increased, and optionally its combination with other biomarkers or variables is very valuable, but it cannot be diagnosed without error but indicates that the subject is likely to suffer from HCC. Not all (100%) patients with the disease HCC have marker amounts above the reference level, and not all healthy individuals have marker levels below the reference level or cut-off level. As those skilled in the art will appreciate, in many diseases, no biochemical marker has 100% specificity and 100% sensitivity at the same time. On the contrary, the marker analyzed or the marker combination comprising the marker gives a certain probability, for example, at a given level of specificity or a given level of sensitivity, the individual whose sample has been analyzed has a certain clinical state, for example, suffering from HCC. Those skilled in the art are fully familiar with mathematical / statistical methods for calculating 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.

[0216] The term "aided detection of hepatocellular carcinoma (HCC)" is used to indicate that the method according to the present invention will help / assist medical professionals including, for example, doctors in assessing whether an individual suffers from HCC or is at risk of developing HCC. It should be understood that doctors can use several alternative methods (e.g., ultrasound, radiography, MRT or CT) and combine with in vitro biomarker data (e.g., glycan structure data) to detect or exclude the presence of HCC. The final diagnosis of HCC is usually performed by tissue biopsy or postoperative tissue samples. The term "aided detection of HCC" includes that the method is used as the only diagnostic tool or as one of multiple diagnostic tools.

[0217] In an embodiment of the present invention, “assisting the detection of hepatocellular carcinoma HCC” may be “assisting the detection of early HCC.” As demonstrated in the accompanying examples, certain glycans at position 207 of the β-chain of haptoglobin exhibit particularly good performance in detecting early HCC.

[0218] Unless defined otherwise, all scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0219] 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.

[0220] In the aspects and embodiments set forth above, certain marker values, such as the amount of glycan structures or the amount of glycopeptides, can be combined (eg, combined into a score) with the amounts determined for one or more other biomarkers. Such combination is performed using standard mathematical / statistical methods.

[0221] A convenient goal of quantifying the diagnostic accuracy of a laboratory test is to express its performance with a single number. The most common global metric is the area under the curve (AUC) of the ROC graph. The area under the ROC curve is a measure of the probability that a perceptual measurement allows a condition (or the distinction between one condition and another) to be correctly identified. Values ​​are typically between 1.0 (perfect separation of the two sets of test values) and 0.5 (no obvious distribution difference between the two sets of test values). The area depends not only on a specific part of the graph, such as, for example, the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire graph. This is a quantitative descriptive expression of the proximity of the ROC graph to the perfect graph (area = 1.0). In the context of the present invention, two different conditions can be that the patient suffers from HCC or does not suffer from HCC.

[0222] The present invention uses the terms "HCC", "early HCC" and "advanced HCC".

[0223] As used herein, "early HCC", "early stage HCC" or "HCC of early stage" refers to patients classified as stage 0 and stage A according to the Barcelona Clinic Liver Cancer (BCLC) classification (Llovet JM, Brú C, Bruix J, Semin Liver Dis. 1999; 19(3): 329-38).

[0224] As used herein, "late HCC", "late stage HCC" or "HCC of late stage" refers to patients classified as stage B, C and D according to the Barcelona Clinic Liver Cancer (BCLC) classification (Llovet JM, Brú C, Bruix J, Semin Liver Dis. 1999; 19(3): 329-38).

[0225] "HCC" refers to any form of HCC, including early-stage and advanced-stage HCC.

[0226] The BCLC classification is recognized by the American Association for the Study of Liver Disease, the American Gastroenterology Association, the European Association for the Study of the Liver, and the European Organization for the Research and Treatment of Cancer as the standard system for the management of HCC. According to the BCLC stage, patients are divided into five categories (0, A, B, C, and D): BCLC stage 0 (defined as very early disease) includes patients who present with well-preserved liver function (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.), diagnosed with one asymptomatic nodule measuring less than 2 cm without vascular invasion or satellites. BCLC stage A (defined as early disease) includes patients with Child-Pugh A or B status, diagnosed with one nodule of any size or up to three nodules measuring <3 cm. In the context of the present disclosure, BCLC 0 and A stages are defined as early HCC groups. BCLC B stages (defined as mid-stage disease) correspond to patients with Child-Pugh A or B status, diagnosed as multiple nodules, no vascular invasion or extrahepatic metastasis. Patients with Child-Pugh A or B, vascular invasion or extrahepatic metastasis, and cancer-related symptoms (PS 1-2) are classified as suffering from BCLC C disease (defined as advanced disease). Finally, patients with Child-Pugh C, any tumor stage, and cancer-related symptoms (PS>2) are classified as belonging to BCLC D disease (defined as terminal disease). In the context of the present disclosure, patients with BCLC B, C, and D stages are defined as advanced HCC groups.

[0227] As used herein, the term "subject" or "individual" refers to a single person. The subject may be healthy or a patient, for example, suffering from cirrhosis, at risk of developing HCC, experiencing or having experienced one or more signs, symptoms or other indicators of HCC. It is intended to be included as a subject any subject who participates in a clinical research trial but does not show any clinical signs of the disease, or a subject who participates in an epidemiological study, or a subject whose sample can be used as a control. In an embodiment, it may be known that the subject has a risk of developing HCC, for example, through long-term drinking, hepatitis B and / or hepatitis C infection, non-alcoholic fatty liver disease, Wilson's disease, hereditary hemochromatosis, α1-antitrypsin deficiency, primary biliary cirrhosis, autoimmune hepatitis and other risk factors.

[0228] In one embodiment according to the present disclosure, the subject from which the sample to be studied has been obtained is a healthy subject and the subject is screened for (the presence of) HCC as part of routine oncology monitoring.

[0229] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained is a subject at risk of developing HCC and is screened for (the presence of) HCC as part of routine oncology monitoring.

[0230] If the subject is known to suffer from chronic liver disease, viral or non-viral hepatitis, and / or cirrhosis, the subject may be at risk for developing HCC.

[0231] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained suffers from chronic liver disease, viral or non-viral hepatitis, liver cirrhosis, and is differentially diagnosed due to the presence or absence of HCC.

[0232] As used herein, the terms "sample", "subject's sample" or "patient sample" 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 preferably can be any type of body fluid. Body fluid samples include blood, serum, plasma, urine, saliva and synovial fluid. Preferred sample types are whole blood, serum or plasma. In one embodiment, the sample type is serum or plasma. In one embodiment, the sample type is plasma. In one embodiment, the sample type is serum. As will be appreciated by those skilled in the art, the sample is used for in vitro analysis of markers of interest. The patient sample is discarded after analysis. The patient sample is used only for the in vitro methods of the present invention, and no material from the patient sample is transferred back into the patient's body.

[0233] 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 the N-glycan structure or the glycopeptide. The level or amount of an N-glycan structure or glycopeptide in a sample is determined by using any suitable method, for example, as known in the art or by using a method described herein.

[0234] In the context of aspects 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., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample means determining a measure of the absolute amount or relative amount of haptoglobin that reflects glycosylation of the corresponding glycan structure at position N207 of haptoglobin in the sample. In embodiments, determining the amount of a glycan structure may be measuring the presence of the glycan structure and quantifying the amount / level of the glycan structure. In embodiments, the amount of a glycan structure at position N207 corresponds to the amount of a glycopeptide comprising the glycan structure at N207. Such a glycopeptide may be produced by hydrolyzing (e.g., using a protease, such as a protease as described below) haptoglobin in a sample.

[0235] In an embodiment of the present disclosure, determining the amount of a glycan structure (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4)) at position N207 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) can be used to determine the relative amount relative to a second analyte (e.g., a control analyte). This arrangement can reduce the impact of sample processing and the difference in the absolute amount of haptoglobin between individuals. The second analyte can be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide can be incorporated into the sample prior to the determination step, or it can be a peptide or glycopeptide of haptoglobin contained in the sample. The spiked peptide can be labeled with a heavy isotope.

[0236] In the present disclosure, determining the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject may include generating a glycopeptide from the haptoglobin contained in the sample, the glycopeptide comprising position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) and, if present, a glycan structure attached thereto, and determining the amount of the glycopeptide comprising the glycan of interest, for example, by mass spectrometry. The amount of the corresponding glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may correspond to or be derived from the amount of the corresponding glycopeptide comprising the corresponding glycan structure at position N207.

[0237] In an exemplary embodiment, determining the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may include purifying haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) from a sample obtained from a subject, hydrolyzing the purified haptoglobin to generate a glycopeptide comprising position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) and the corresponding glycan attached thereto, and determining the amount of the glycopeptide comprising the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1). The amount of the corresponding glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may correspond to or be derived from the amount of the corresponding glycopeptide comprising the corresponding glycan structure at position N207.

[0238] In the present disclosure, determining the amount of the glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may, for example, comprise purifying haptoglobin from a sample, digesting the purified haptoglobin with GluC and trypsin, and determining the amount of the corresponding glycopeptide having the corresponding one or more glycan structures at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) by MS analysis. The amount of the corresponding glycan structure at position N207 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may correspond to or be derived from the determined amount of the corresponding glycopeptide comprising the corresponding glycan structure at position N207.

[0239] As will be appreciated by those skilled in the art, other methods can be used to determine the glycan structure at N207 of haptoglobin. In this analysis, it is only necessary to ensure that the glycan structure is or has been attached to N207 (as well as N184, 211, and 241) of the β-chain of haptoglobin. An exemplary but non-limiting method is described by Oh et al. (Mass Spectrom Rev. 2021)

[0240] In an embodiment, a glycopeptide comprising position N207 of haptoglobin used to determine the amount of glycan structures may comprise a peptide sequence (SEQ ID NO: 2), wherein the middle N (bold and underlined; in position 5) corresponds to the N at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1), to which the corresponding glycan to be detected is attached. In a specific embodiment, the peptide portion of the glycopeptide can be composed of (SEQ ID NO:2).

[0241] In embodiments, the glycan structure whose amount is determined is part of a glycopeptide, wherein the glycopeptide comprises the peptide sequence (SEQ ID NO: 2), and wherein the middle N (bold and underlined) corresponds to the N at position 207 of the β-strand of haptoglobin (SEQ ID NO: 1).

[0242] 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., of Formula 1) at position N207 of the β-chain of haptoglobin in a sample is determined. For this purpose, any suitable method known in the art may be used. In an embodiment of the method according to the present disclosure, the step of determining one or more N-glycan structures (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or a corresponding glycopeptide (e.g., of Formula 1) at position N207 of the β-chain of haptoglobin includes purifying haptoglobin (at least comprising the β-chain) prior to making such a determination. Preferably, purification of haptoglobin is achieved by an antibody that binds to haptoglobin. In particular, a monoclonal antibody that specifically binds to haptoglobin may be used. Such a monoclonal antibody or an antigen-binding fragment thereof may 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 that detect human haptoglobin have been used in different publications in different immunological methods. Non-limiting examples are: Abcam, #AB13429, clone HG-36; Abnova, #MAB12976, clone 2F4; AcrisAntibodies, #UM500010, clone UMAB10; Novus Biologicals, #NBP2-03008, clone OTI4H5; OriGene, #TA00399, clone OTI2B8; Thermo Fisher Scientific, #HYB 170-06-02, clone 9G10. In a specific 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., of Formula 1) comprises purifying haptoglobin (at least comprising the β-chain) by a combination of a biotinylated anti-haptoglobin monoclonal antibody and streptavidin-coated beads (SA-beads). In such a purification step, the sample is incubated with the biotinylated antibody and (simultaneously or sequentially) with (e.g., magnetic or magnetizable) SA-beads under conditions suitable for antigen / antibody and biotin / streptavidin binding. The beads (with attached haptoglobin) are then separated from other components contained in the sample (e.g., by using magnetic forces).

[0243] In an embodiment of the present disclosure, the step of determining the (or one or more) N-glycan structure at position N207 of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) comprises chemical hydrolysis or biochemical / enzymatic cleavage of haptoglobin into peptides / glycopeptides. Various methods for hydrolyzing polypeptides by chemicals are known and can be used. In an embodiment, haptoglobin is enzymatically cleaved into peptides / glycopeptides.

[0244] In an embodiment of the present disclosure, the step of determining the (or one or more) N-glycan structure at position N207 of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) comprises enzymatically cleaving the haptoglobin into peptides / glycopeptides using the enzymes GluC and trypsin. GluC cleaves the C-terminus of the protein chain into the amino acid glutamic acid, while trypsin cleaves the C-terminus of the protein chain into the amino acids arginine and lysine. Both enzymes cleave with high specificity.

[0245] As described above, the (or one or more) N-glycans at position 207 of haptoglobin (e.g., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) or glycopeptides comprising N207 (e.g., the glycopeptide of Formula 1) can be measured by mass spectrometry (MS). Various MS methods are known. The 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 peptide entering the mass spectrometer at a given time, and then sequentially selects the most intense species for gas phase fragmentation, and subsequently analyzes the obtained fragments. These fragments carry sequence information of the peptide and glycan structure, and computer-assisted analysis is performed by comparing the obtained fragment spectra with predicted spectra (derived from a database). In rare cases, manual inspection of isotope patterns is required to distinguish similar structures.

[0246] Highly sensitive identification 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. The peptide is fragmented in the gas phase, and a unique and representative fragment is identified and quantified.

[0247] For the MS analysis of glycan structures or glycopeptides, quite a few processing steps are required. These processing steps may cause considerable changes, especially when measuring (absolute) levels or amounts. In order to compensate for the differences in sample treatment, samples are usually mixed with internal standards (usually the analyte of interest, but in isotope-labeled form). Sample treatment will affect the absolute value, but is unlikely to affect the (glycan structure / glycopeptide) ratio based on the same MS analysis method. This ratio is not absolute, but a relative measure, and is therefore unrelated to the differences in sample treatment. Therefore, it may be advantageous to determine the amount of a glycan structure or a glycopeptide relative to another glycan structure or glycopeptide. In an embodiment, the ratio can be the ratio between the increase and decrease of the glycan structure at the same position of a polypeptide. Specific but non-limiting examples of such ratios are described elsewhere herein.

[0248] As used herein, the term "amount" or "level" of an analyte (e.g., a glycan or glycopeptide) in a sample relates to any absolute measure, which corresponds to or is proportional to the absolute amount or concentration of the analyte in the sample; or any relative measure, i.e., a measure expressing 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 a sample) measured simultaneously with the analyte of interest using the same method.

[0249] In embodiments, the amount of the glycan structure at position N207 of haptoglobin in the sample can be the amount of a glycopeptide comprising the glycan structure at N207 determined from the sample. In these embodiments, the method can include generating a glycopeptide from haptoglobin (e.g., by protease digestion as described elsewhere). In embodiments, the peptide portion of the glycopeptide can be as described above.

[0250] In embodiments, the amount of a glycopeptide can be the amount or raw signal of a glycopeptide divided by the amount or raw signal of a non-glycosylated peptide (e.g., from haptoglobin), respectively. In embodiments, the amount of a glycopeptide can be the amount or raw signal of a glycopeptide divided by the amount or raw signal of all detected glycopeptides (e.g., of haptoglobin), respectively.

[0251] The term "amount changes" refers to the fact that the amount is out of a reference range, that is, is higher than a certain reference amount or is lower than a certain reference amount.

[0252] The term "increase" or "increase" in the level of a marker refers to a higher amount or level of this marker in the sample under investigation compared to the amount or level of this marker in a reference or control sample (i.e., excluding it). The term "decrease" in the level or amount of a marker refers to a lower amount or level of this marker in the sample under investigation than the amount or level of this marker in a reference or control sample (i.e., excluding it).

[0253] As used herein, the term "score" or "score for detecting HCC" taking into account the amount of a glycan structure or glycopeptide relates to a score (e.g., value) obtained by combining the amount of a glycan structure or glycopeptide of the present invention with at least one additional parameter, such as, for example, the amount of a second glycan structure, the amount of one or more other biomarkers, or a clinical parameter. Exemplary but non-limiting examples of other biomarkers are AFP, PIVKA-II, and AFP-L3. Exemplary examples of clinical parameters are age, sex, smoking status, ultrasound data, history of liver disease, history of cancer, and family history of cancer, etc. The score is configured so that it can indicate HCC (e.g., early HCC).

[0254] As used in the context of scores, "take into account" includes embodiments that take into account only the specifically listed quantities and parameters as well as embodiments that take into account other non-listed parameters.

[0255] Determining or calculating a score (eg, a score for detecting HCC) can be accomplished in many different ways. As mentioned herein, combining certain biomarker data or other information into a combined value can also be referred to as determining or calculating a score.

[0256] Determining or calculating a score includes any mathematical combination of the amount of a glycan structure or glycopeptide mentioned herein and another parameter (see above). Thus, a score can be calculated in which the various parameters (including or consisting of the determined or received amount of a glycan or corresponding glycopeptide attached to N207 of haptoglobin) are mathematically combined. These levels can be used as is or can be mathematically transformed (e.g., by a logarithmic transformation, such as a log2 or log10 transformation) to determine the score. In addition to the level of PSA glycoform species, the score may also take into account one or more other factors, including but not limited to the presence or level of one or more other biomarkers in the sample and one or more clinical parameters of the subject (e.g., tumor histology, smoking status, disease stage and / or age).

[0257] In certain embodiments of the present invention, the score for detecting HCC may be obtained by weighted calculation or may include weighted calculation. This means that biomarkers (e.g., the amount of the glycan structure at position N207 of haptoglobin) may be assigned different weights. For example, when the score considers the amount of the first glycan structure at position N207 of haptoglobin in the sample and the amount of another glycan structure at position N207 of haptoglobin, the score may be calculated by the following equation:

[0258] Score = a*[amount of the first glycan structure] + b*[amount of the second glycan structure], where a and b represent weight factors. Preferably, the weight factors or coefficients (a and b in the above examples) have been obtained by analyzing control samples such as from a reference population (e.g., any reference population as defined in the context of reference values ​​below). In an embodiment, the weight factors or coefficients can be obtained by a machine learning method applied to a training data set obtained from samples of a reference population as defined herein.

[0259] One skilled in the art will appreciate that the score and the corresponding reference value for the score may be optimized based on a reference population (eg, any population as defined herein or as disclosed in the accompanying Examples).

[0260] 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 level of one or more biantennary PSA-glycoforms determined or received or a value derived therefrom, and the second value is the level of one or more monoantennary PSA-glycoforms or a value derived therefrom. The "value derived therefrom" may, for example, be a value obtained by mathematical operation. The "value derived therefrom" is preferably proportional to the corresponding level. For each PSA-glycoform, the value of the binary reference value may be obtained as described below.

[0261] Comparing the binary score to a reference value for the binary cutoff score means comparing a first value of the determined binary score to a first value of the reference value of the binary score, and comparing a second value of the determined binary score to a second value of the reference value of the binary score. If a binary score is constructed based on the amount of the first glycan structure at position N207 of haptoglobin and the amount of the glycan structure at position N207 of haptoglobin, HCC can be detected only if both amounts are indicative of HCC relative to the reference values ​​of the two amounts constituting part of the binary score.

[0262] In aspects and embodiments, two or more markers (or even more general input parameters, which may also include clinical parameters such as age or gender) are used, which may be combined by an appropriate algorithm (e.g., logistic regression) derived from a multivariate analysis of the available data, preferably an exhaustive search for feature selection. In such a combination / calculation, a multivariate score is calculated. Other methods may also be used, such as selected from DA (i.e., linear, quadratic, regularized discriminant analysis), kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbor classification), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forests, Boosting), to combine biomarkers / input values ​​into scores.

[0263] The performance of the results of the applied mathematical / statistical methods used in accordance with the present disclosure can be best described by their receiver operating characteristics (ROC). The ROC curve addresses the sensitivity and specificity of the test. Therefore, the sensitivity and specificity values ​​for a given biomarker or combination of biomarkers are an indication of the performance of the test. For example, if the sensitivity value of a biomarker combination is 80%, then 80 out of 100 patients with the disease will be correctly identified, or if its specificity value is 80%, then 80 out of 100 patients without the disease will be accurately tested as negative for the disease.

[0264] In the same manner as previously described, ROC curves can be used to assess the performance of the aforementioned logistic regression model in discriminating between patients and controls.

[0265] 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 fraction with a reference amount / level or fraction of the glycan structure, glycopeptide or fraction. It should be understood that this comparison as used herein generally refers to the comparison of corresponding parameters, amounts, values ​​or fractions, for example, comparing an absolute amount with an absolute reference amount, and comparing a concentration with a reference concentration, or comparing an intensity signal obtained for a glycan structure in a sample with an intensity signal of the same type obtained from a reference sample. The score is usually compared with a reference value of this specific score indicating HCC (e.g., early HCC). The comparison can be performed by an appropriate device, for example, by a computer. For example, the value of the measured or detected level / amount of a glycan structure in a sample from an individual or patient can be compared with a reference level / amount, and the comparison can be performed automatically by a computer program that executes a comparison algorithm. The computer program that performs the evaluation will provide the desired assessment in an appropriate output format. For computer-assisted comparisons, the value of the measured amount can be compared with a value corresponding to an appropriate reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired assessment in an appropriate output format. For computer-assisted comparison, the value of the determined quantity can be compared with the value corresponding to an appropriate reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e. automatically provide the required assessment in an appropriate output format.

[0266] The terms "glycan structure" or "glycan" are used interchangeably. In the present disclosure, the glycans studied are N-glycans, so the terms glycan and N-glycan are used interchangeably herein. Glycans or glycan structures are composed of various types of carbohydrates. The glycan structure can, for example, be bound to an amino acid, such as to the amino acid asparagine. In this example, a glycan bound to asparagine at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1) was analyzed. The glycan structures or glycans disclosed herein serve as biomarkers or markers for HCC (e.g., early HCC).

[0267] The term "glycopeptide" is used to refer to a peptide or a peptide fragment of a larger polypeptide comprising an amino acid covalently attached to a glycan. In the present disclosure, the glycopeptide preferably analyzed is a peptide sequence derived from the β-chain of haptoglobin, which comprises the amino acid asparagine (N) at position 207 of the β-chain of haptoglobin (SEQ ID NO: 1). Preferred embodiments of the peptide portion of the glycopeptide preferably detected or quantified are disclosed elsewhere herein.

[0268] Polysaccharides are generally referred to herein by the general formula. In the text, we refer to the polysaccharide composition as follows: HexNAc(x)Hex(x)Fuc(x)NeuAc(x). The numbers in brackets represent the number of monomers in the polysaccharide. This nomenclature is well known to those skilled in the art of polysaccharide biology. HexNAc can be GlcNAc or GalNAc (GlcNAc in the embodiment) and Hex can be Glu or Gal (Gal in the embodiment). HexNAc means N-acetylglucosamine, 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).

[0269] Preferred glycans are represented by specific formulas or using schematic representations 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 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, for example, Figure 4 The glycan structure at N207 of haptoglobin is depicted in FIG.

[0270] In an embodiment of the present invention, the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) may have Formula 2:

[0271]

[0272] In a preferred embodiment of the present invention, the glycan HexNAc(4)Hex(5)NeuAc(2) may have Formula 3:

[0273]

[0274] In a preferred embodiment of the present invention, the glycan HexNAc(5)Hex(5)NeuAc(1) may have Formula 4:

[0275]

[0276] In a preferred embodiment of the present invention, the glycan HexNAc(6)Hex(7)Fuc(1)NeuAc(4) may have formula 2, the glycan HexNAc(4)Hex(5)NeuAc(2) may have formula 3 and the glycan HexNAc(5)Hex(5)NeuAc(1) may have formula 4.

[0277] As used in any one of Formulas 2 to 4 and any other formula herein, GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, and NeuAc means N-acetyl-neuraminic acid (sialic acid). Lines represent covalent bonds between monosaccharides. Dashed lines indicate if the glycan is part of a glycopeptide or glycoprotein, through which monosaccharide the glycan is attached to the peptide or protein, respectively (e.g., N207 of SEQ ID NO: 1). Dashed lines used in the context of NeuAc (or Neu5Ac used as a synonym) indicate that NeuAc can be attached to either sugar.

[0278] As used herein, the term "reference amount" (or "reference level") of an analyte (e.g., a glycan structure or glycopeptide) refers to an independently determined predetermined amount of the analyte. As will be appreciated by those skilled in the art, a reference amount is predetermined and is set to meet conventional requirements in terms of specificity and / or sensitivity, for example, for detecting HCC (e.g., early HCC). Therefore, a reference amount may be selected so that it indicates HCC (e.g., early HCC). The requirements for detecting HCC may vary, for example, from regulatory agencies to regulatory agencies. For example, it may be necessary to set the assay sensitivity or specificity to certain limits, for example, to 80%, 90%, 95%, or 98%, respectively. These requirements may also be defined according to positive or negative predictive values. For any selected requirement, for example, respectively with respect to sensitivity or specificity levels, a reference range (if the assessment value and the reduction value indicate an abnormal state) or a reference level or a cutoff level (if the assessment value or the reduction value indicate an abnormal state) may be determined by those skilled in the art. Similarly and according to the same principle, a reference ratio may be determined.

[0279] As used herein, the term "reference value" (e.g., in the context of a reference value for a score) relates to an independently determined predetermined value for each parameter (e.g., score). As will be appreciated by those skilled in the art, the reference value is predetermined and is set to meet conventional requirements in terms of specificity and / or sensitivity, for example, for detecting HCC (e.g., early HCC). Thus, the reference value may be selected so that it indicates HCC (e.g., early HCC). The above content regarding reference amounts also applies with appropriate modifications.

[0280] The reference amount or reference value for the score is usually determined in one reference sample or a reasonable number of reference samples. Reference samples are also referred to as control samples. In general, reference samples are obtained from individuals or groups of individuals known to suffer from a given disease or known to be at risk of a given disease; or from individuals or groups of individuals known not to suffer from a given disease, i.e., "normal" or "healthy" individuals. The marker concentration of the sample is usually directly or indirectly related to the diagnosis, and the marker level is used, for example, to determine whether the individual is at risk of HCC. According to the intended diagnostic use, an appropriate reference sample is selected and a control or reference value of the marker is established therein. The technician will understand that, in one embodiment, such reference samples are obtained from a reference population that is age-matched and free of confounding diseases. It is also clear to the technician that the absolute marker value established in a reference sample or reference sample set (e.g., forming a reference population) will depend on the assay used. Preferably, samples of 100 well-characterized individuals from an appropriate reference population are used to establish a reference amount or reference value. Also preferably, the reference population can be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a reference population that is often 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.

[0281] For the detection of HCC in clinical routine, it is of utmost importance to obtain an indication of the possible development or presence of HCC in those patients with cirrhosis or other HCC risk factors. Thus, in an embodiment, the reference level is determined in a reference sample from a patient with cirrhosis.

[0282] In embodiments, reference populations from which control or reference samples can be obtained include samples obtained from control subjects (eg, healthy individuals or subjects with liver disease and increased risk of developing HCC) and subjects suffering from HCC (eg, early-stage HCC).

[0283] As is known to those skilled in the art, any method or use for detecting HCC or assisting in detecting HCC may include the step of detecting HCC or assisting in detecting HCC. Such assisting detection or detection is usually achieved based on the comparison step of such method.

[0284] Alpha-fetoprotein is a glycoprotein, and various glycosylation forms of AFP have been described. Lectins can be used for the analysis of glycoproteins. By using the selective binding ability of lectins to the sugar chain structure of glycoproteins, it is possible to separate and concentrate marker glycoprotein parts with specific sugar chain structures. In the case of AFP, lectins derived from lentil lectin-A (LCA) have been widely used. The lentil lectin (LCA) reactive alpha-fetoprotein part (AFP-L3) is particularly increased in patients with HCC. Many attempts have been made to specifically measure AFP-L3, such as by affinity electrophoresis using LCA, lectin-based ELISA, or by antibodies that specifically bind to the L3 form of AFP.

[0285] Vitamin K deficiency / antagonist-II induced protein (PIVKA-II), also known as des-γ-carboxyprothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and can be used alone or in combination with AFP as an alternative HCC biomarker. Prothrombin has 10 potential γ-carboxylation sites, and various forms of PIVKA-II with different levels of insufficient carboxylation exist in the circulation. Different assays for PIVKA-II can detect different groups of PIVKA-II forms, and the specificity / sensitivity of PIVKA-II may vary depending on the assay used, and has limited utility in detecting early HCC. As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding properties.

[0286] As used herein, the term "specific binding" or "specifically binds" refers to a binding reaction in which the binding pair molecules appear to bind to each other under conditions in which they do not significantly bind to other molecules.

[0287] The term "specific binding" or "specifically binds" when referring to a protein or peptide as an antibody or binding agent means that the binding agent binds to the protein or peptide with a specific binding activity of 10 -7 M or lower K D The term "specific binding" or "specifically binds" preferably refers to the binding reaction of a target molecule to a target molecule. D For 10 -8 M or less, or even more preferably 10 -9 The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the binding agent that is specific for the target molecule. Preferably, the level of binding to molecules other than the target molecule results in a binding affinity that is only 10% or less of the affinity for the target molecule, more preferably only 5% or less. In other words, the K for binding to the target molecule is D Preferably, the K of binding to non-target proteins is higher than that of binding to non-target proteins.D At least 10 times lower, more preferably at least 20 times lower.

[0288] The methods and uses of the present disclosure provide valuable information regarding the presence of HCC in a subject. In certain embodiments, the detection performed by the methods and uses of the present invention is used to provide a treatment recommendation for a subject.

[0289] It should be understood that the methods and uses of the present disclosure can be performed remotely from the subject or their attending physician, and in fact, these methods and uses can be performed offshore and the results transmitted back. In specific embodiments, the results of the methods of the present invention (e.g., determining whether the subject has HCC) are provided to a third party, such as the subject or their attending physician, a laboratory or a health center.

[0290] Here, elements of the present invention are described. These elements are listed as aspects with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to create other embodiments. Specifically, the embodiments disclosed in the context of one aspect are equally applicable to other aspects after appropriate modifications. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of the elements described in this application should be considered to be disclosed by the specification of the application.

[0291] The entire disclosures of all references cited in this specification and the disclosures specifically mentioned in this specification are incorporated herein by reference.

[0292] The following examples and figures are provided to assist in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention. A description of the amino acid sequences disclosed herein is also provided.

[0293] Sequence Description

[0294] SEQ ID NO: 1 shows the amino acid sequence of human haptoglobin; N207 is printed in bold and underlined:

[0295]

[0296] (Sequence Listing; Uniprot P00738, Version 221).

[0297] SEQ ID NO: 2 shows the amino acid sequence of the peptide portion included 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 and the Asn residue corresponding to N207 of SEQ ID NO: 1 is printed in bold and underlined:

[0298]

[0299] SEQ ID NO:3 shows the amino acid sequence of a peptide included in an exemplary glycopeptide for detecting a glycan structure at position N184; the Asn residue corresponding to N184 of SEQ ID NO:1 is printed in bold and underlined:

[0300]

[0301] SEQ ID NO:4 shows the amino acid sequence of a peptide included in an exemplary glycopeptide for detecting a glycan structure at position N211; the Asn residue corresponding to N211 of SEQ ID NO:1 is printed in bold and underlined:

[0302]

[0303] SEQ ID NO:5 shows the amino acid sequence of a peptide included in an exemplary glycopeptide for detecting a glycan structure at position N241; the Asn residue corresponding to N241 of SEQ ID NO:1 is printed in bold and underlined:

[0304] BRIEF DESCRIPTION OF THE DRAWINGS

[0305] Figure 1 : Schematic diagram representing the various forms of haptoglobin: The three types of haptoglobin contain (always together with the β-chain) two α1-chains (1-1); the haptoglobin α1-chain and one α2-chain (2-1) or two α2-chains (2-2).

[0306] Figure 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 following the SNFG (Glycan Notation Nomenclature) system (PMID 26543186, Glycobiology 25: 1323–1324, 2015). Connections are indicated by black lines. Right: Symbolic depiction of glycan structure: Monosaccharide notations follow SNFG.

[0307] Figure 3 : Box plot of various glycopeptides at position N207 of the β-chain of haptoglobin (SEQ ID NO: 1) indicating HCC: Site-specific glycan analysis revealed that a considerable number of glycopeptides were elevated or decreased in HCC. The amounts of Hp glycopeptides were compared between the control, early, and late cohorts. Compared with the control, some glycopeptides were specifically upregulated in early HCC (compound 126), some were upregulated in both early and late HCC (compound 140 and compound 131), and some of them were downregulated in early and late HCC (compound 172). The statistically significant difference between the control (C) early HCC (ESH) and late HCC (LSH) was determined by the Wilcoxen (Mann-Whitney U) test. See the glycopeptide structure for Figure 4 .

[0308] Figure 4 : Overview of up-regulated / down-regulated glycopeptides on glycosylation site N-207. Monosaccharide symbols follow the SNFG (Glycan Symbol Nomenclature) system (PMID 26543186, Glycobiology 25:1323–1324, 2015). Hp glycopeptides are listed on the left for distinguishing early HCC cases from controls. In the right column, these glycopeptides showed differences between early and late HCC and controls, while no glycans were found on glycosylation site N-207 that differed only between late HCC and controls. Only glycopeptides with AUC>70% are shown. The direction of expression change (decrease or increase) is indicated by up (regulation) or down (regulation).

[0309] Figure 5 Receiver operating curve (ROC): Several Hp glycopeptides at site N-207 have high potential for clinical application in detecting early HCC. This is evident from the displayed ROC and given area under the curve (AUC) values. The specificity and sensitivity values ​​are sensitivity and specificity at a cutoff value of 0.9, respectively.

[0310] Figure 6 : Overview of up-regulated / down-regulated glycopeptides at all haptoglobin glycosylation sites (N-184, N-207, N-211, and N-241). Monosaccharide symbols follow the SNFG (Glycan Symbol Nomenclature) system (PMID 26543186, Glycobiology 25:1323–1324, 2015). Hp glycopeptides are listed on the left to distinguish early HCC cases from controls. The glycopeptides shown in the middle column are present in early and late HCC, and the glycopeptides shown in the right column differ in late HCC from controls. Only glycopeptides with AUC>70% are shown. The direction of expression change (decrease or increase relative to control) is indicated by up (regulation) or down (regulation).

[0311] Examples

[0312] Example 1

[0313] 1.1 Research composition

[0314] EDTA plasma samples were obtained from 57 controls representing chronic liver diseases including HBV, HCV, and cirrhosis, as well as HCC patients including 33 early-stage HCC subjects and 32 advanced-stage HCC subjects (see Table 1 for demographic information).

[0315] The staging classification of the samples was based on the Barcelona Liver Cancer (BCLC, Llovet JM, Brú C, Bruix J, Semin Liver Dis. 1999; 19(3): 329-38) method, in which BCLC stages 0 and A were classified as early HCC and BD stages were classified as advanced HCC. After plasma preparation, the samples used in this analysis were stored at -80°C until analysis as described below. Repeated freezing and thawing of the samples was avoided.

[0316]

[0317] Table 1: Summary of demographic variables grouped according to clinical status.

[0318] 1.2 Hp immunoprecipitation from plasma

[0319] In the first step, immune capture beads were prepared. 10 mg of streptavidin (SA) coated latex beads were co-incubated with 150 μg of biotin (Bi) labeled mouse monoclonal antibody against Hpβ-chain MAK<haptoglobin>M-1.1.13-F(ab′)2-Bi (Roche DiagnosticsGmbH, Mannheim, Germany) at RT on a rotator for 1 h. 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 the F(ab′)2 fragment was applied to the beads, the resulting coated beads were washed 3 times with PBS. Fifty microliters of each plasma sample were incubated with 5 μl 20 mM DTT (dithiothreitol) at 37°C for 1 h. The samples were then treated with 10 μl 50 mM IAM (iodoacetamide) for 30 min at RT. Thereafter, the samples were diluted with 2 ml PBS and incubated with MAK<haptoglobin>M-1.1.13-F(ab′)2-Bi-SA-beads (antibody-coupled beads from the previous step). After incubation on a rotator for 2 h at RT, the beads were incubated using a two-step PBST (PBS buffer + 0.1% Tween ) wash and two PBS buffer washes to wash away unbound proteins. Hp bound to the antibody-coated beads was eluted from the beads in two steps by incubating the beads in 500 μl glycine buffer (0.2 M glycine; pH: 2.6). Two-step elution facilitates recovery. To neutralize the pH, 200 μl NaOH (1N) was added to the eluate after each elution step.

[0320] 1.3 FASP digestion

[0321] The two eluted HP fractions from the previous step were combined and loaded onto a TM Membrane-10K (PALL, US) filter The samples were centrifuged at 10.000 g for 20 min on a centrifugal device (cutoff: 10 kDa). In this step, molecules, such as proteins with a molecular weight of 10.000 or more, are retained on the filter, while small molecules, such as salts, pass through the filter and are removed. Then, 75 ng of heavy haptoglobin (recombinant Hp with isotope-labeled heavy Lys and Arg) protein was added to each sample / filter as an internal standard. 50 μl of denaturing buffer (1 mg / ml PPS (3-[3-(1,1-diheptyloxyethyl) pyridin-1-yl] propane-1-sulfonate) in 50 mM ammonium bicarbonate) and 5 μl of DTT (10 mM) were then added to the samples, and the samples were incubated at 50°C for 30 min. In the next step, 5 μl of IAM (55 mM) was added to the samples, and they were incubated at 37°C in the dark for 30 min. The sample was centrifuged at 10.000 g for 20 min to wash away the buffer and washed once with 100 μl ABC (ammonium bicarbonate, 50 mM) buffer. Then, 6 μg of trypsin was first added to 50 μl ABC buffer to digest the protein retained on the filter for 3 h at 37 ° C. The filter was incubated at 95 ° C for 10 min to stop the trypsin digestion. 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.

[0322] 1.4 LC-MS / MS analysis

[0323] Twenty microliters of peptides obtained by enzymatic digestion as described in 1.3 were injected into a LC-MS / MS system (HF-X mass spectrometer (Thermo Fisher Scientific, Germany) coupled to a Vanquish (Thermo Fisher Scientific, Germany) UHPLC system). The peptides were purified by reverse phase chromatography on a C18 column (Waters, XSelect CSH C18 column, 3.5μm, 2.1mm X 150mm) to separate peptides. The flow rate of LC was 320μl / min and the gradient was set as follows: 0%-30%B (0-30min), 30%-80%B (30min-31min), 80%B (31min-36min), 80%-0%B (36min-37min) and 0%B (37min-42min), where eluent A and eluent B were H2O with 0.1% formic acid and acetonitrile with 0.1% formic acid, respectively. The separated peptides were ionized by an electrospray ionization (ESI) source and analyzed in positive ion mode and data-dependent acquisition method. Full scan MS spectra were collected in the range of 300m / z–2000m / z at 60000 resolution, 10e6 automatic gain control (AGC), and 50ms injection time. The first 5 most intense peaks in this scan (ie, the survey scan) were selected for fragmentation using high energy collision dissociation (HCD) at a normalized collision energy of 28%, a resolution of 15000, 1e5 AGC, and an injection time of 150 ms.

[0324] 1.5AFP and PIVKA-II detection

[0325] AFP and PIVKA-II were measured using microchip capillary electrophoresis and liquid crystal binding assays performed on an uTASWako i30 autoanalyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan).

[0326] 1.6 Data Analysis

[0327] The raw files acquired from the mass spectrometer were processed by the Byonic (Protein Metrics, CA, US) search engine embedded in Proteome Discoverer 2.2 (Thermo Fisher Scientific). The data set was searched against the Uniprot haptoglobin protein sequence (P00738). To identify glycopeptides, the Byonic curated database was used. The Byonic / Proteome Discoverer configuration was as follows: the mass tolerance for MS1 was set to 10 ppm, and the mass tolerance for MS2 was set to 20 ppm. GluC and trypsin were set as proteases, allowing two missed cleavages. Carbamidomethylation of cysteine ​​was set as a fixed modification, while methionine oxidation and glycosylation on asparagine were specified as variable modifications. The results were filtered with a 1% false discovery rate (FDR) and a confidence threshold of Byonic score >100. The glycopeptide composition with significant differences between the cohort groups and an AUC greater than 0.7 was manually checked. In these cases, we examined retention time, charge state, glycan oxonium ions, and isotope patterns and compared them with the predicted isotope patterns of the proposed glycopeptides. In the 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 the corresponding monomers 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).

[0328] The schematics shown herein are drawn according to the monosaccharide symbols of the SNFG (Syntax Nomenclature for Glycans) system (PMID 26543186, Glycobiology 25:1323–1324, 2015), as detailed in NCBI (Ajit Varki et al., Symbol Nomenclature for Graphical Representations of Glycans, Glycobiology, Vol. 25, No. 12, December 2015, pp. 1323-1324, https: / / doi.org / 10.1093 / glycob / cwv091 and Sriram Neelamegham et al., The SNFG Discussion Group, Updates to the Symbol Nomenclature for Glycans guidelines, Glycobiology, Volume 29, Issue 9, September 2019, Pages 620–624, https: / / doi.org / 10.1093 / glycob / cwz045). The peak areas of glycopeptide XICs were automatically integrated by Proteome Discoverer and used as relative quantitative values.

[0329] 1.7 Statistical analysis

[0330] The abundance of glycopeptides in the samples was normalized to the abundance of the top three peptides spiked with heavy Hp to correct for possible changes in processing, digestion, or MS measurements. Missing values ​​for a certain glycopeptide (i.e., glycopeptides below the detection limit) were replaced by the minimum amount of the glycopeptide in the data set. The significance of glycopeptide differences 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, and the FDR control was 20%. To evaluate the diagnostic value of glycopeptides, receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) values ​​were calculated by the DeLong method using R software (version 3.5.2) obtained from https: / / www.R-project.org (Elisabeth R. DeLong, David M. DeLong, and Daniel L. Clarke-Pearson (1988), Biometrics 44, 837–845)

[0331] 2. Results

[0332] 2.1 Glycan properties for HCC diagnosis

[0333] Global analysis of glycan features, such as fucosylation, provides insights into major changes in Hp glycosylation patterns after HCC progression. However, different Hp sugar sites vary in the types and abundance of glycoforms. Therefore, we investigated whether Hp glycosylation site- and glycoform-specific analysis could indicate HCC. We compared site-specific glycopeptide levels between controls, early, and late HCC. Our observations revealed that certain glycopeptides were significantly upregulated in early HCC compared with controls with an AUC of 0.70 or higher and could be clearly assigned to specific glycan structures ( Figure 6 This group of glycopeptides includes highly branched glycopeptides (see Figure 4 , Compound 126 or 140), fucosylated glycopeptides (see Figure 4 , compounds 126, 131 or 140) and highly mannosylated glycopeptides (see Figure 6 , compounds 58, 60, 24 and 27), and sialylated glycopeptides (see Figure 4 and 6 , compound 126). In addition to the early HCC-specific biomarkers, we also found fourteen other glycopeptides that were significantly more highly expressed in early and late HCC and provided an AUC of 0.70 or higher in distinguishing early HCC patients from CLD controls ( Figure 6 ).

[0334] In addition to the upregulated glycopeptides in HCC, we also observed that two glycopeptides were significantly downregulated in early and late HCC compared with CLD ( Figure 4 and 6 ), which are located at the N207 glycosylation site. One of the glycoforms is a biantennary glycan with two sialic acids (HexNAc(4)Hex(5)NeuAc(2)), which is one of the most abundant glycans. Another glycoform is a bisected biantennary glycan with one sialic acid (HexNAc(5)Hex(5)NeuAc(1)). These downregulated glycopeptides can be used to establish ratios with upregulated glycopeptides (e.g., compound 126) to further improve robustness by providing an internal control. In total, we identified 29 glycan structures that distinguished controls from early and late HCC with AUCs above 70% ( Figure 6 ).

[0335] To date, the best glycopeptide for detecting early HCC is compound 126 on N207 (see Figure 6 ). Therefore, among all the glycosylation sites analyzed in this paper, haptoglobin glycosylation at position N207 seems to be the most suitable for detecting early HCC. Figure 6 Data are shown for all altered glycopeptides, using an AUC of 0.70 or greater as a cutoff.

[0336] The results, glycan structures, peptide sequences and m / z values ​​of the detected glycopeptides are summarized in Tables 2 and 3 below.

[0337] These results consistently revealed that glycopeptide analysis of Hp, especially at position N207 (but also at positions N184, N211, and N241), could provide sugar biomarkers with better clinical value than established biomarkers for early HCC diagnosis.

[0338]

[0339] Table 2: Glycan structures, peptide sequences and m / z values ​​of detected glycopeptides significantly different between early and late HCC and controls. AUC [%] and the direction of expression changes are indicated for each examined group. *Methionine residues may be oxidized.

[0340]

[0341]

[0342] Table 3: Glycan structures, peptide sequences and m / z values ​​of detected glycopeptides significantly different between early and late HCC and controls. AUC [%] and the direction of expression changes are indicated for each examined group. *Methionine residues may be oxidized.

[0343] 2.2 Top glycans for detecting (early) HCC

[0344] The results showed that the Hp glycopeptide compound 126 (i.e., HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207) in particular has a high potential for clinical application in HCC detection, especially in early detection of HCC. Figure 5 This is evident from the receiver operating curves (ROC) shown in and the area under the curve (AUC) values ​​given for each of these glycopeptides. Surprisingly, for this glycopeptide the calculated AUC already exceeds the AUC values ​​for AFP and PIVKA-II, respectively, and their combination.

[0345] 2.3 Marker combination

[0346] In order to analyze the added value of compound 126 to PIVKA-II and / or AFP, a logistic regression model consisting of compound 126, PIVKA-II and / or AFP was established. In these models, logarithmic transformation was applied to the markers to reduce the skewness of the marker distribution. For all compounds, these multivariate models were constructed and the performance of the combined AUC form was compared. The results of compound 126 are summarized in Table 4 below. Models other than logistic regression (or logistic regression models with interaction terms between variables) were unable to significantly improve the performance of the aforementioned logistic regression models.

[0347]

[0348] Table 4: The combination of glycopeptide compound 126 improves the clinical value of AFP and PIVKA-II biomarkers. In our cohort, the AUC values ​​of AFP and PIVKA-II for early diagnosis of HCC were 83% and 88%, respectively. Combining these two biomarkers can increase the AUC to 94%. The most important regulated glycopeptide in our study (compound 126) can increase the AUC of the combination of AFP and PIVKA-II to 97%.

Claims

1. An in vitro method for assisting in detecting hepatocellular carcinoma (HCC) in a subject, the method comprising the following steps: a) determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from said subject; and b) (i) comparing the amount of the N-glycan structure determined in a) with a reference amount of the N-glycan structure, wherein an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position 207 of haptoglobin in the sample of the subject relative to the reference amount of such N-glycan structure indicates HCC; or (ii) 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 a), and comparing the determined score for detecting HCC with a reference value for the score indicating HCC.

2. An in vitro method for assisting in detecting hepatocellular carcinoma (HCC) in a subject, the method comprising the following steps: a) determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. of the β-chain of haptoglobin having the sequence given 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) or HexNAc(5)Hex(5)NeuAc(1) at position N207 of the haptoglobin (i.e. of the β-chain of the haptoglobin having the sequence given in SEQ ID NO: 1) in the sample obtained from the subject, c) determining a score for detecting HCC by dividing a) by b) or vice versa, said score taking into account or consisting of the ratio of the amounts of the two glycan structures determined in a) and b), and d) comparing the score determined in c) with a reference score value indicative of HCC (eg, early stage HCC).

3. 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 wherein the N-glycan portion is HexNAc(6)Hex(7)Fuc(1)NeuAc(4), and wherein the N-glycan portion is attached to the N in position 5 of SEQ ID NO:

2.

4. The glycopeptide according to claim 3, wherein the glycopeptide has a structure as shown in Formula 1 5. Use of a glycopeptide as defined in claim 3 or 4 for aiding detection of HCC, in an embodiment early stage HCC.

6. The method of 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 comprising N207 of haptoglobin and the glycan structure attached thereto, wherein in an embodiment, the peptide portion of the glycopeptide has an amino acid sequence of SEQ ID NO:

2.

7. The method according to any one of claims 1, 2 or 6, wherein 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 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, 6 and 7, 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 according to claim 3 or 4 or corresponds to the amount of the glycopeptide according to claim 3 or 4.

9. A method for detecting and / or quantifying the glycopeptide according to claim 3 or 4, the method comprising the following steps: a) purifying haptoglobin from a sample to be analyzed; b) digesting the haptoglobin obtained in step a) by GluC and trypsin, and c) detecting the glycopeptide obtained in b), thereby detecting the glycopeptide according to claim 3 or 4.

10. A clinical workflow for screening a subject for hepatocellular carcinoma (HCC), wherein the clinical workflow comprises the following steps: a) determining the amount of the glycan structure HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin (i.e. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from said subject; and b) determining the amount of AFP and / or determining the amount of PIVKA-II in a sample obtained from said subject, c) combining the quantities determined in a) and b) into a combined value and comparing the combined value with a reference value for the combined value; d) Additionally using the results of the ultrasound examination, wherein a positive ultrasound result for HCC and / or a combined value of a change relative to a reference value is indicative of HCC.

11. A computer-implemented method for aiding in detecting 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., of the β-chain of haptoglobin having the sequence given 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 the glycan structure, wherein an increased amount of HexNAc(6)Hex(7)Fuc(1)NeuAc(4) at position N207 of haptoglobin in the sample of the subject relative to the reference amount of such glycan structure is indicative of HCC, or (ii) calculating a score for detecting 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 with a reference value for the score indicating HCC; and c) assisting in detecting whether the subject has HCC based on the comparison in b).

12. A computer-implemented method for assisting in detecting HCC in a subject, the method comprising the steps of: 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. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from said 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 the ratio of the amounts of the two glycan structures comprising a) and b) (or vice versa) or the fraction consisting thereof, and d) comparing the score calculated in c) with a reference value for the score indicative of HCC.

13. The computer-implemented method of claim 11 or 12, wherein the method further comprises receiving data comprising the amount of PIVKA-II and / or the amount of AFP in the sample or another sample from the same subject, and 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 comprising instructions which, when said program is executed by a computer, cause said computer to perform the computer-implemented method according to any one of claims 11 to 13.

15. The method according to any one of claims 1, 2, 6, 7, 8 and 10 to 13, the use according to claim 5 or the clinical workflow according to claim 10, wherein the HCC is early stage HCC.

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