Glycan structures of haptoglobin as hepatocellular carcinoma biomarkers

Abstract

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

Description

Technical Field

[0001] The present invention relates to an in vitro method for assisting in the detection of hepatocellular carcinoma (HCC) in a subject. The method may include determining the amount of one or more N-glycan structures attached to 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 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's 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 attached to haptoglobin or a glycopeptide derived from haptoglobin with AFP and / or PIVKA-II in detecting 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 leading histological type of primary liver cancer worldwide, accounting for 70% to 85% of the total burden. Underlying liver diseases such as liver fibrosis and cirrhosis are known to be 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.

[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 HCC patients will drop 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: official journal of the American Society of Clinical Oncology. 2009; 27(9): 1485-91). Therefore, the development of a non-invasive HCC test for early diagnosis is crucial to reducing mortality and increasing 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 based on each lesion and are costly. On the other hand, in recent years, efforts have been made to discover new blood biomarkers that can be used to monitor projects for early detection of HCC in high-risk patients (Yang JD. Detect or not to detect very early stage hepatocellular carcinoma? The western perspective. Clin Mol Hepatol. 2019; 25 (4): 335-43).

[0006] Some current medical guidelines recommend surveillance of 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 in 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 many other tumor-related biomarkers, Hp has occasionally been mentioned as a possible candidate biomarker in the field of HCC (Tai CS, Lin YR, Teng TH, Lin PY, Tu SJ, Chou CH, Huang YR, Huang WC, Weng SL, Huang HD, Chen YL, Chen WL. Haptoglobin expression correlates with tumor differentiation and five-year overall survival rate in hepatocellular carcinoma. PLoS ONE 2017; 12(2): e0171269). Aberrant glycosylation of serum haptoglobin, especially at glycosylation 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 in nonalcoholic steatohepatitis and hepatocellular carcinoma. The study also included mass spectrometry-based glycan assessment at amino acid residue positions 184, 207, 211, and 241 of haptoglobin (Ramachandran, Gege Xu, Hector H. Huang, Rachel Rice, Bo Zhou, Klaus Lindpaintner, and Daniel Serie Journal of Proteome Research 2022 21(4), 1083-1094. DOI: 10.1021 / acs.jproteome.1c00965).

[0009] Alpha-fetoprotein (AFP) is the most established blood biomarker for HCC. However, even AFP exhibits suboptimal sensitivity and specificity for early detection of HCC. In addition, it has been reported 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 utilizing the selective binding ability of lectins to the sugar chain structure of glycoproteins, marker glycoprotein fractions with specific sugar chain structures can be separated and concentrated. With regard to AFP, lectins derived from lentil lectin-A (LCA) have been widely used. The lentil lectin (LCA) reactive fraction of α-fetoprotein (AFP-L3) is specifically increased in patients with HCC. Many attempts have been made to specifically measure AFP-L3, such as by affinity electrophoresis using LCA, ELISA based on lectins, or by antibodies specifically binding 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 proteolysis and is very reliable. Parallel reaction monitoring (PRM) technology uses a mass spectrometer equipped with a trap and a time-of-flight mass analyzer to obtain product ion spectra of peptides, thereby allowing quantitative and qualitative analysis of peptides 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 one and the same amino acid position of a protein can have various structures and show heterogeneity. It is also known that sugar structures can vary 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 were able to better distinguish the cancer status of normal subjects from hepatocellular carcinoma patients than non-glycopeptides (Kim H, Kim K, Jin J, Park J, Yu SJ, Yoon JH, Kim Y. Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Formin Hepatocellular Carcinoma. PLOS One, 2014, 9: e110366). In EP3 415 918, various glycopeptides of AFP-L3 have been analyzed by mass spectrometry and used 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-gamma carboxyprothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and is used alone or in combination with AFP as an alternative HCC biomarker. There are 10 potential gamma-carboxylation sites for prothrombin, and various forms of PIVKA-II with varying levels of incomplete carboxylation exist in the circulation. Different assays for PIVKA-II can detect a collection of different 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 diagnosis of patients at risk. However, many recent publications clearly indicate that the inclusion of input variables, namely sex, age, AFP and decarboxy prothrombin (DCP = PIVKA-II) (= GAAD-score) or the score of 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 to further improve in vitro methods to assist in the detection of HCC.

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

[0019] The present inventors have discovered that analysis of the glycan structure attached to the β-chain of haptoglobin can overcome some of the problems of current in vitro diagnostic methods to aid in the detection of HCC.

[0020] In particular, the present inventors have identified a glycan structure at position N184 of the β-chain of haptoglobin (ie at the amino acid asparagine in position 184 of SEQ ID NO: 1) as a good biomarker for detecting HCC, also in particular early stage HCC.

[0021] 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 N184 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.

[0022] In a specific embodiment, the one or more glycan structures attached to N184 are the glycan structures HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0023] 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: The invention relates to a method for detecting HCC (e.g., early HCC) in a subject comprising the steps of: (i) determining the amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of the β-chain of the haptoglobin having the sequence given in IDNO:1, and (ii) (ii) determining a score for detecting HCC (e.g., early HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of the haptoglobin having the sequence given in IDNO:1, and (iii) comparing the determined score for detecting HCC (e.g., early HCC) with a reference value for the score indicating HCC (e.g., early HCC).

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

[0025] In a specific embodiment of the third aspect of the present disclosure, provided herein is a glycopeptide of Formula 1:

[0026]

[0027] Wherein GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetylneuraminic acid (sialic acid). Lines represent covalent bonds. The glycans in the glycopeptide are covalently attached as N-glycans to the asparagine side chain corresponding to N184 of haptoglobin via the GlcNac of the indicated glycans. (i.e., N184 of the β-chain of haptoglobin of SEQ ID NO: 1). In an embodiment, the methionine in position 1 of SEQ ID NO: 2 can be oxidized.

[0028] The glycopeptide comprises a peptide having an amino acid sequence as described in SEQ ID NO: 2. An alternative graphical representation of a glycopeptide having the same structure as that shown in Formula 1 above is as follows: Figure 2 As shown in the picture on the right.

[0029] In a fourth aspect, the invention relates to the use of the glycopeptide of the third aspect (eg, the glycopeptide of Formula 1, see above) in detecting HCC.

[0030] According to the fifth aspect of the present disclosure, a method for detecting the glycan structure at position N184 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 generated in b) at position N184 of haptoglobin having a glycan structure attached thereto, thereby detecting the glycan structure at position N184.

[0031] In a sixth aspect, the present disclosure 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 amount of the N-glycan structure at position N184 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 HCC biomarkers (e.g., the amount of AFP and / or the amount of PIVKA) in the sample obtained from the subject other than the N-glycan structure at position N184 in a), c) combining the amounts determined in a) and b) into a combined value (e.g., a score for detecting HCC such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0032] In a seventh aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0033] a) receiving data comprising the amount of a glycan structure at position N184 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

[0034] b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at position N184 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 N184 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).

[0035] In an eighth aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0036] a) receiving data comprising the amount of one or more glycan structures (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2)) at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from the subject

[0037] b) receiving data comprising the amount of PIVKA-II and / or AFP in a sample obtained from said subject,

[0038] c) calculate the fraction taking into account the quantities determined in a) and b), and

[0039] d) comparing the score calculated in c) with a reference value of said score indicative of HCC (eg, early stage HCC).

[0040] 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 the seventh or eighth aspect.

[0041] Furthermore, there is provided a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to the seventh or eighth aspect.

[0042] A data processing system is also provided, comprising: a receiving unit configured to receive the data defined in the seventh or eighth aspect; and a processing unit configured to perform the calculation and / or comparison steps and / or any determination / assessment steps in the seventh or eighth aspect; and optionally, an output unit configured to output an assessment result. DETAILED DESCRIPTION

[0043] 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 create additional aspects and embodiments.

[0044] 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 N184 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.

[0045] 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 N184 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.

[0046] The method of the first aspect of the invention is based on the discovery that the glycosylation pattern at position 184 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.

[0047] In an embodiment of the first aspect of the present invention, the determined amount of one or more glycan structures at position N184 of haptoglobin may not be individually compared with the reference amount of the corresponding glycan structure, but the determined amount of one or more glycan structures at position N184 of haptoglobin may be considered to calculate a score for determining HCC (e.g., early HCC). The score may be compared with a corresponding reference value for the score that determines the presence of HCC (e.g., early HCC).

[0048] Therefore, the present invention provides 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 a glycan structure at position N184 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.

[0049] In an embodiment, the glycan structure at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) determined according to the method of the present disclosure is selected from the group consisting of: HexNAc(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), HexNAc (5) Hex (6) Fuc (1) NeuAc (1), HexNAc (5) Hex (6) Fuc (3), HexNAc (4) Hex (5) Fuc (3) NeuAc (2), HexNAc (4) Hex (5) Fuc (1) NeuAc (1), HexNAc (5) Hex (6) Fuc (1) NeuAc (3) and HexNAc (6) Hex (7) NeuAc (1). In an embodiment, the haptoglobin protein (i.e., the protein having SEQ ID NO: 1) is determined according to the method of the present disclosure. The glycan structure at position N184 of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) is selected from the group consisting of: HexNAc(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In an embodiment, the glycan structure at position N184 of the β-chain of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) determined according to the method of the present disclosure is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0050] In embodiments, an increased amount of a determined glycan structure at position N184 of haptoglobin (ie, of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) relative to a reference amount may indicate HCC (eg, early-stage HCC).

[0051] In embodiments in which the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin is determined, an increase in the amount of this glycan structure at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) relative to a reference amount can indicate HCC (e.g., early HCC).

[0052] In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin (ie of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) is determined to be HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0053] 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 N184 of haptoglobin with AFP and / or PIVKA-II increases the diagnostic utility for HCC (e.g., early HCC).

[0054] In a second aspect, the present disclosure 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(5)Hex(6)Fuc(1)NeuAc(2) at position 184 of the 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of the 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.

[0055] The method of the second aspect is based on the following discovery: the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position 184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) has been found to be increased in samples obtained from subjects with HCC (particularly early HCC) relative to control samples representing chronic liver diseases (including HBV, HCV and cirrhosis). As shown in the attached examples, the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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), which shows the highest AUC in the detection of HCC (combined early and late HCC) of all identified N-glycan structures (including all glycan structures at position N184 and other glycosylation sites). The structure of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) can be expressed as Figure 2 Thus, in an embodiment, the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may have the following structure: Figure 2The glycan structure depicted in the glycan portion of the glycopeptide in or the glycan structure of Formula 2 as referred to elsewhere herein.

[0056] What was said herein with respect to the method according to the first aspect of the invention applies mutatis mutandis.

[0057] 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin in the sample of the subject relative to the reference amount of such N-glycan structure indicates HCC (e.g., early HCC).

[0058] If a reference amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) is selected so that it represents the amount of said N-glycan structure in healthy subjects and / or subjects with non-cancerous chronic liver diseases (e.g. selected from the group consisting of HBV, HCV and cirrhosis), then an increased amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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).

[0059] Thus, in embodiments, an increase in the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) is indicative of HCC (eg, early-stage HCC).

[0060] In an embodiment, the second aspect of 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 amount of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin determined in a); and c) comparing the determined score for detecting HCC (e.g., early 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).

[0061] In embodiments, the score may be configured such that higher values ​​of the score indicate an increased risk for HCC. In these embodiments, the score value will increase with increasing levels of the N-glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin.

[0062] Determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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 reflecting the absolute or relative amount of haptoglobin in the sample that is glycosylated by the corresponding glycan structure at position N184 of haptoglobin.

[0063] Examples for determining the amount of a glycan structure such as the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) are disclosed below.

[0064] In an embodiment, the level of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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 N184 and the glycan structure, for example via mass spectrometry.

[0065] In an embodiment, the glycopeptide having the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) used for determining the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) comprises the peptide sequence of MVSHHNLTTGATLINE (SEQ ID NO: 2), wherein the N in position 6 of SEQ ID NO: 2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1). In a specific embodiment, the peptide portion of the glycopeptide may consist of MVSHHNLTTGATLINE (SEQ ID NO: 2). Thus, the amount of the glycopeptide of Formula 1 (see elsewhere herein) can be determined to determine the amount of the glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin. Depending on the sample preparation and method used for determination, the methionine in position 1 of SEQ ID NO:2 may be oxidized when measuring the level of the glycopeptide.

[0066] In the embodiments, the glycopeptide having the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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 the N-glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin has an m / z of 1118,2098, a charge of 4, and a glycopeptide mass of 4469,8163 Da.

[0067] In other words, for determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO:1), a glycopeptide can be analyzed, wherein the glycopeptide can comprise the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO:2), wherein the N in position 6 of SEQ ID NO:2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO:1), and wherein the glycan structure is HexNAc(5)Hex(6)Fuc(1)NeuAc(2). In a specific embodiment, the peptide portion of the glycopeptide can consist of MVSHHNLTTGATLINE (SEQ ID NO:2). Therefore, for determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin, a glycopeptide of formula 1 (see elsewhere herein) can be analyzed.

[0068] In an embodiment, for determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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 MVSHHNLTTGATLINE (SEQ ID NO: 2) can be analyzed, wherein the N in position 6 of SEQ ID NO: 2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1), and wherein the glycan is HexNAc(5)Hex(6)Fuc(1)NeuAc(2). Such a glycopeptide can be a glycopeptide of formula 1 (see elsewhere herein) or Figure 2The glycopeptide shown. 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin may have an m / z of 1118,2098, a charge of 4 and a glycopeptide mass of 4469,8163 Da. In an embodiment, the glycopeptide may comprise the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO: 2) as a peptide portion and HexNAc(5)Hex(6)Fuc(1)NeuAc(2) as a glycan structure attached to N in position 6 of SEQ ID NO: 2, wherein the N in position 6 of SEQ ID NO: 2 corresponds to the N at position 184 of the β-chain of the haptoglobin (SEQ ID NO: 1) sequence, and the m / z may be 1118,2098927.9544, the charge is 5 4, and the glycopeptide mass is 4469,81634636.8124 Da.

[0069] In order to reduce the impact of sample processing and the difference in the absolute amount of haptoglobin between individuals, determining the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e., the β-chain of haptoglobin having the sequence given in SEQ ID NO:1) can mean determining the relative amount relative to the amount of a second analyte or group of analytes. Such a second analyte can be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide or glycopeptide can be externally incorporated into the sample prior to the determination step, or can be a peptide or glycopeptide of haptoglobin contained in the sample. The incorporated peptide can be labeled with a heavy isotope.

[0070] 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 N184 of haptoglobin with at least AFP increases the diagnostic utility for HCC (e.g., early HCC).

[0071] In a third aspect, the invention relates to an isolated glycopeptide having a peptide portion and an N-glycan portion, wherein the peptide portion comprises or consists of the amino acid sequence MVSHHNLTTGATLINE (SEQ ID NO: 2), and wherein the N-glycan portion is HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and wherein the N-glycan portion is attached to the N in position 6 of SEQ ID NO: 2. In embodiments, the methionine in position 1 of SEQ ID NO: 2 can be oxidized.

[0072] In a preferred embodiment, the glycopeptide is a glycopeptide of Formula 1:

[0073]

[0074] Wherein GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetylneuraminic acid (sialic acid). Lines represent covalent bonds. The glycans in the glycopeptide are covalently attached as N-glycans to the asparagine side chain corresponding to N184 of haptoglobin via the GlcNac of the indicated glycans. (i.e., N184 of the β-chain of haptoglobin of SEQ ID NO: 1).

[0075] The isolated glycopeptide of Formula 1 is shown only as a graphical replacement representation in Figure 2 (Right picture). Figure 2 The figure on the right is a representation according to the SNFG (Symbol Nomenclature for Glycans) system (Ajit Varki et al., Symbol Nomenclature for Graphical Representations of Glycans, Glycobiology, Volume 25, Issue 12, December 2015, Pages 1323-1324, https: / / doi.org / 10.1093 / glycob / cwv091 and Sriram Neelamegham et 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)

[0076] As shown throughout this disclosure, the glycopeptide of Formula 1 holds great promise for the detection of HCC (eg, early-stage HCC). It is found herein to be a reliable biomarker for the detection of both early-stage and late-stage HCC.

[0077] Therefore, in a fourth 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).

[0078] The embodiments of the method according to the first and second aspects are applicable mutatis mutandis for this purpose.

[0079] According to the fifth aspect of the present disclosure, a method for detecting the glycan structure at position N184 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 generated in b) at position N184 of haptoglobin having a glycan structure attached thereto, thereby detecting the glycan structure at position N184.

[0080] In an embodiment, the glycan structure at position N184 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(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), (HexNAc(3)Hex(4)NeuAc(1), H exNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc(2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In the embodiments, the haptoglobin (i.e., having SEQ ID The glycan structure at position N184 of the β-chain of haptoglobin of the sequence given in NO:1 is selected from the group consisting of: HexNAc(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0081] 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 N184 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 such methods.

[0082] The attached examples also specifically show that after purification and digestion and an appropriate detection method (preferably LC-MS), the glycopeptide according to the fourth aspect of the present disclosure can be reliably determined with good sensitivity. Therefore, in an embodiment, a method for detecting the glycan structure of a glycopeptide according to the fourth 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 the glycopeptide according to the fourth aspect (e.g., a glycopeptide of Formula 1).

[0083] In clinical routine, the diagnosis of HCC, especially early HCC, is very challenging, and many HCC cases are diagnosed too late and can no longer 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 / fat. Nevertheless, advanced HCC is usually first discovered via ultrasound.

[0084] It is expected that the glycan structures disclosed in the present invention will significantly improve the detection of HCC, especially the detection of early HCC.In one embodiment, the method for detecting HCC disclosed in the present invention is a method for detecting early HCC (ie, early HCC).

[0085] The glycan structures of the present invention can also be used in combination with other biomarkers for detecting HCC (eg, AFP).

[0086] In a sixth aspect, the present disclosure 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 amount of the N-glycan structure at position N184 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 HCC biomarkers other than the N-glycan structure at position N184 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 such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0087] The embodiments of the aforementioned aspects apply mutatis mutandis.

[0088] HCC biomarkers are biomarkers indicative of HCC (eg, early stage HCC). In a specific example, the HCC biomarker may be AFP or PIVKA-II.

[0089] In an embodiment of the sixth aspect of the present invention, there is provided 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 amount of the N-glycan structure at position N184 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 such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0090] In an embodiment of the method of the sixth 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 N184 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 such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0091] In an embodiment of the method of the sixth 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 N184 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 such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0092] In an embodiment of the method of the sixth 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 N184 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 such as early HCC), and comparing the combined value with a reference value for the combined value, wherein a changed combined value indicates HCC (e.g., early HCC).

[0093] In an embodiment, the glycan structure at position N184 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(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), HexNAc(3)Hex(4)NeuAc(1). euAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc( 2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In an embodiment, the glycan structure at position N184 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(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0094] What has been said elsewhere herein (eg, in other aspects and in the context below) regarding determining the amount of individual glycan structures applies mutatis mutandis. Thus, the amount of a glycan structure may be determined by determining the amount of a glycopeptide (eg, via mass spectrometry).

[0095] The samples used for determination in a) and b) can be the same or can be different samples obtained from a subject. Preferably, the samples are obtained from a subject simultaneously. In a specific embodiment, the sample used for determination in a) is the same as the sample used for determination in b).

[0096] In an embodiment, a "combined value" may mean determining a score indicating HCC (e.g., early HCC) that takes into account the amounts determined in a) and b). The score may further take into account more biomarkers. The score may alternatively and additionally further take into account the subject's clinical data (e.g., age, sex, smoking status, cancer history, family cancer history, and the presence of pre-existing liver disease).

[0097] An exemplary but non-limiting manner for calculating a combined value or score is disclosed below.

[0098] 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 on an uTASWako i30 automated analyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) are performed according to the manufacturer's instructions. For example, AFP can also be measured using AFP (Material No.: 044817981190) can be used for detection. PIVKA-II (Material No.: 08333602190) was used for detection.

[0099] In the method according to the sixth aspect, the N-glycan structure is preferably HexNAc(5)Hex(6)Fuc(1)NeuAc(2), and even more preferably 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) at position N184 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 these amounts can be calculated and such ratio can be used to calculate the combined value.

[0100] In a seventh aspect, provided herein is a computer-implemented method for aiding in detecting HCC (e.g., early-stage HCC) in a subject, the method comprising:

[0101] a) receiving data comprising the amount of a glycan structure at position N184 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

[0102] b) i) comparing the amount of the glycan structure received in a) with a reference amount of the N-glycan structure at position N184 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 N184 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).

[0103] The embodiments of the aforementioned aspects apply mutatis mutandis.

[0104] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0105] In embodiments, the method may include outputting (eg, via a display) whether the subject has HCC (eg, early-stage HCC).

[0106] In an embodiment, the glycan structure at position N184 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(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3), HexNAc(3)Hex(4)NeuAc(1). euAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(3), HexNAc(4)Hex(5)Fuc(3)NeuAc( 2), HexNAc(4)Hex(5)Fuc(1)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(3) and HexNAc(6)Hex(7)NeuAc(1). In an embodiment, the glycan structure at position N184 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(5)Hex(6)Fuc(1)NeuAc(2); HexNAc(2)Hex(8), HexNAc(2)Hex(9), HexNAc(5)Hex(6)Fuc(3)NeuAc(1), HexNAc(4)Hex(5)Fuc(3). In a particularly preferred embodiment, the glycan structure at position N184 of haptoglobin is HexNAc(5)Hex(6)Fuc(1)NeuAc(2).

[0107] In an eighth aspect, a computer-implemented method for aiding in detecting hepatocellular carcinoma (HCC) in a subject is provided, the method comprising the steps of:

[0108] a) receiving data comprising the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin (i.e. of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in a sample obtained from said subject and

[0109] b) i) comparing the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) received in a) with a reference amount of the glycan structure, wherein an increase in the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin in the sample of the subject relative to the reference amount of such glycan structure indicates HCC, or (ii) calculating a score for detecting HCC (e.g., early HCC) taking into account the amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin received in a), and comparing the calculated score for detecting HCC (e.g., early HCC) with a reference value for the score indicating HCC (e.g., early HCC).

[0110] The embodiments of the aforementioned aspects apply mutatis mutandis.

[0111] In an embodiment, the method may include assisting in detecting HCC (eg, early stage HCC) based on i) or ii) in b).

[0112] In embodiments, the method may include outputting (eg, via a display) whether the subject has HCC (eg, early-stage HCC).

[0113] If there are two or more steps of receiving data in the computer-implemented method disclosed herein, the skilled person will readily understand that this includes both: (i) embodiments in which a single data set includes all of the received data in two or more steps and (ii) embodiments in which two or more separate data sets are received.

[0114] Any of the above computer-implemented methods may optionally include the following steps: outputting whether the subject has or is suspected of having HCC (e.g., early HCC) and / or whether further clinical examination of HCC (e.g., early HCC) is required based on the comparing step. The output may be via a display.

[0115] Also disclosed herein is a computer program product comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to the seventh or eighth aspect.

[0116] Furthermore, there is provided a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to perform the computer-implemented method according to the seventh or eighth aspect.

[0117] A data processing system is also provided, comprising: a receiving unit configured to receive the data defined in the seventh or eighth aspect; and a processing unit configured to perform the calculation and / or comparison steps and / or any determination / assessment steps in the seventh or eighth aspect; and optionally, an output unit configured to output an assessment result.

[0118] The above aspects relate to the glycan structure at position N184 of the β-chain of haptoglobin.As is apparent from the appended examples, many other glycan structures at positions N207, N211 or N241 that can be used to detect HCC are also identified herein.

[0119] The corresponding glycan structures at position N207 of haptoglobin are: HexNAc(6)Hex(7)Fuc(1)NeuAc(4); HexNAc(4)Hex(5)NeuAc(2); HexNAc(5)Hex(5)NeuAc(1); HexNAc(5)Hex(6)Fuc(1)NeuAc(1); HexNAc(5)Hex(6)Fuc(1)NeuAc(2); and HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0120] In addition, the corresponding glycan structures at position N207 are provided in Tables 2 and 3.

[0121] The corresponding glycan structures at position N211 of haptoglobin are: HexNAc(6)Hex(7)NeuAc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2), HexNAc(6)Hex(7)Fuc(1)NeuAc(1).

[0122] In addition, the corresponding glycan structures at position N211 are provided in Tables 2 and 3.

[0123] 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), HexNAc(4)Hex(5)Fuc(1)NeuAc(1).

[0124] In addition, the corresponding glycan structures at position N241 are provided in Tables 2 and 3.

[0125] Therefore, all aspects and embodiments as described herein for N184 of haptoglobin disclose mutatis mutandis for N207, N211 and N241 of haptoglobin. In addition, all aspects and embodiments disclosed herein for a specific glycan structure at position N184 also disclose mutatis mutandis for any of the above glycan structures at positions N207, N211 and N241.

[0126] For detecting the amount of glycan structure at position N207, a PCR reaction with a NLFL N A glycopeptide of a peptide portion of HSE (SEQ ID NO: 3) or consisting thereof, wherein the N in position 5 corresponds to N207.

[0127] For detecting 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.

[0128] For detecting 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.

[0129] 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 invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of ambiguity, this patent specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0130] The following definitions and examples apply to this disclosure as a whole and in particular to all aspects and embodiments of the invention.

[0131] 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.

[0132] The word "comprise" and variations such as "comprising" and "including" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0133] The use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

[0134] The term "and / or" should be understood to mean either or both of the alternatives.

[0135] 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 use of the term "about" when used in conjunction with a stated value or range means slightly greater than 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. Such values ​​are therefore encompassed by the scope of claims reciting the term "about" or "approximately".

[0136] 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 individual health or disease. Therefore, sometimes the more general term "marker" is also used when discussing more general terms and definitions below. The term marker also includes a polysaccharide structure or polysaccharide or glycopeptide as analyzed in the present disclosure.

[0137] The term "in vitro method" is used to indicate that the method is performed outside a living organism, preferably in a body fluid, isolated tissue, organ or cell. An in vitro method may also be referred to as an ex vivo method.

[0138] Hepatocellular carcinoma (HCC) is the leading histological type of primary liver cancer worldwide, accounting for 70% to 85% of the total burden. Underlying liver diseases such as liver fibrosis and cirrhosis are known to be 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 5-year survival rate of HCC patients will drop 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).

[0139] Haptoglobin is an acute phase protein. It is synthesized in the liver and secreted into the plasma and has a rather complex biochemistry and diverse biological functions. Figure 1 As shown, haptoglobin can form dimers (or even polymers) via 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 0.3 mg / ml to 3 mg / ml. Haptoglobin binds to free hemoglobin and thereby protects against oxidative stress. It also plays a role in the regulation of immune responses by binding to resting and activated CD4+ and CD8+ T cells.

[0140] The β-chain of haptoglobin does not show genetic polymorphism. As used herein, the β-chain of haptoglobin preferably has the sequence given in SEQ ID NO:1. Haptoglobin can undergo secondary modification, for example in the form of glycosylation. The β-chain of haptoglobin has four N-glycosylation sites (asparagine (N)), respectively at amino acid positions 183, 207, 211 and 241.

[0141] As used herein and if not mentioned otherwise, the expression "at position N184 of haptoglobin" refers to position N184 of the β-chain of haptoglobin (eg having the sequence given in SEQ ID NO: 1).

[0142] 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 HCC assessment performed according to the present invention is preferred, but may not be correct for 100% of the investigated subjects (for substantially every diagnostic method). The term generally requires that a statistically significant portion of the 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.

[0143] 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 HCC have an amount of biomarkers above the reference level, and not all healthy individuals have a level of biomarkers 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 a marker combination comprising the marker gives a certain probability that the individual whose sample has been analyzed has a certain clinical state (e.g., suffering from HCC), for example, at a given level of specificity or a given level of sensitivity. 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.

[0144] 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 (such as glycan structure data) to detect or exclude the presence of HCC. The final diagnosis of HCC is usually performed after surgery by tissue biopsy or by 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.

[0145] In an embodiment of the present invention, “assisting the detection of hepatocellular carcinoma HCC” may be “assisting the detection of early HCC.” As shown in the attached examples, certain glycans at position 184 of the β-chain of haptoglobin exhibit particularly good performance in detecting early HCC.

[0146] 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.

[0147] 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.

[0148] In the aspects and embodiments set forth above, certain marker values ​​(e.g., the amount of glycan structures or the amount of glycopeptides) can be combined with amounts (e.g., scores) determined for one or more other biomarkers. This combination is performed using standard mathematical / statistical methods.

[0149] 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 to be correctly identified (or distinguishes one condition from another). The value is usually 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 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 whether the patient has HCC or does not have HCC.

[0150] The present invention uses the terms "HCC", "early HCC" and "advanced HCC".

[0151] As used herein, "early HCC", "early stage HCC" or "early HCC" 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).

[0152] As used herein, "late HCC", "late stage HCC" or "advanced HCC" 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).

[0153] "HCC" refers to any form of HCC, including early-stage and advanced-stage HCC.

[0154] The BCLC classification is recognized by the American Association for the Study of Liver Diseases, the American Gastroenterological Association, the European Association for the Study of the Liver, and the European Organization for Research and Treatment of Cancer as the standard system for the management of HCC. According to the BCLC staging, 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.), who are diagnosed with one asymptomatic nodule measuring less than 2 cm without vascular invasion or satellites. BCLC A stage (defined as early stage disease) includes patients with Child-Pugh A or B status, which are diagnosed as having a nodule of any size or up to three nodules measuring <3cm. In the context of the present disclosure, BCLC 0 stage and A stage are defined as early stage HCC group. BCLC B stage (defined as mid-term disease) corresponds to patients with Child-Pugh A or B status, which are diagnosed as multiple nodules, without vascular invasion or extrahepatic metastasis. Patients with Child-Pugh A or B, vascular invasion or extrahepatic metastasis and cancer-related symptoms (PS1-2) are classified as suffering from BCLC C disease (defined as advanced stage disease). Finally, patients with Child-Pugh C level, in any tumor stage and with cancer-related symptoms (PS>2) are classified as belonging to BCLC D disease (defined as terminal stage disease). In the context of the present disclosure, patients with BCLC B stage, C stage and D stage are defined as advanced stage HCC group.

[0155] 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 HCC signs, symptoms or other indicators. 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 may serve as a control. In an embodiment, it may be known that the subject is at risk of developing HCC, for example, by long-term drinking, hepatitis B and / or hepatitis C infection, nonalcoholic fatty liver disease, Wilson's disease, hereditary hemochromatosis, α1-antitrypsin deficiency, primary biliary cirrhosis, autoimmune hepatitis and other risk factors.

[0156] 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 is screened for (the presence of) HCC as part of routine oncology monitoring.

[0157] 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.

[0158] If the subject is known to have chronic liver disease, viral or non-viral hepatitis, and / or cirrhosis, the subject may be at risk for developing HCC.

[0159] 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 for the presence or absence of HCC.

[0160] As used herein, the terms "sample", "subject's sample", "patient sample" or "individual 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 can preferably 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 target markers. The patient sample is discarded after analysis. The patient sample is used only for the in vitro method of the present invention, and no material from the patient sample is transferred back into the patient's body.

[0161] As used herein, the term "determining" the amount of N-glycan structures or the amount of glycopeptides refers to measuring the amount or level of said N-glycan structures or said glycopeptides. The level or amount of N-glycan structures or glycopeptides in a sample is determined by any suitable method, for example, as known in the art or by using the methods described herein.

[0162] In the context of various aspects of the present invention, determining the amount of a glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) or any of the other glycan structures mentioned herein) at position N184 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 reflecting the absolute or relative amount of haptoglobin in the sample that is glycosylated by the corresponding glycan structure at position N184 of haptoglobin. 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 N184 corresponds to the amount of a glycopeptide comprising the glycan structure at N184. Such glycopeptides may be generated by hydrolyzing the haptoglobin in a sample (e.g., using a protease, such as a protease described below).

[0163] In an embodiment of the present disclosure, determining the amount of a glycan structure (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2)) at position N184 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 allows reducing the effects of sample processing and differences in the absolute amount of haptoglobin between individuals. The second analyte can be a haptoglobin peptide or a haptoglobin glycopeptide. The peptide can be incorporated into the sample prior to the determination step or can be a peptide or glycopeptide of haptoglobin contained in the sample. The incorporated peptide can be labeled with a heavy isotope.

[0164] In the present disclosure, determining the amount of the glycan structure at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) in the sample obtained from the subject may include generating a glycopeptide from the haptoglobin contained in the sample, the glycopeptide comprising position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) and the glycan structure attached thereto (if present), and determining the amount of the glycopeptide comprising the target glycan, for example, via mass spectrometry. The amount of the corresponding glycan structure at position N184 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 N184.

[0165] In an exemplary embodiment, determining the amount of the glycan structure at position N184 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 so as to generate a glycopeptide comprising position N184 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 N184 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 N184 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 N184.

[0166] In the present disclosure, determining the amount of the glycan structure at position N184 of haptoglobin (i.e., of the β-chain of haptoglobin having the sequence given in SEQ ID NO: 1) may, for example, include 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 N184 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 N184 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 N184.

[0167] As the skilled person will appreciate, other methods can be used to determine the glycan structure at N184 of haptoglobin. In such analysis, it is only necessary to ensure that this glycan structure is attached or has been attached to N184 of the β-chain of haptoglobin. Non-limiting examples of such methods are disclosed in Oh MJ, Lee SH, Kim U, An HJ. Mass Spectrom Rev. 2021. Similar methods can be used to detect glycan structures at positions N 207, N211 and / or N241 of the β-chain of haptoglobin.

[0168] In an embodiment, a glycopeptide comprising position N184 of haptoglobin used to determine the amount of a glycan structure may comprise the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO: 2), wherein the N in position 6 of SEQ ID NO: 2 corresponds to the N at position 184 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 may consist of MVSHHNLTTGATLINE (SEQ ID NO: 2). In an embodiment, the glycopeptide may be oxidized, particularly at methionine. The skilled person will appreciate that the occurrence of such oxidation may depend on sample processing and the method used to detect such glycopeptides.

[0169] In an embodiment, the glycan structure whose amount is determined is part of a glycopeptide, wherein the glycopeptide comprises the peptide sequence MVSHHNLTTGATLINE (SEQ ID NO:2), and wherein the N in position 6 of SEQ ID NO:2 corresponds to the N at position 184 of the β-chain of haptoglobin (SEQ ID NO:1).

[0170] In the context of the present invention, the level or amount of an N-glycan structure at position N184 of the β-chain of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) or a glycopeptide (e.g., a glycopeptide of Formula 1) 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 at position N184 of the β-chain of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) or a corresponding glycopeptide (e.g., a glycopeptide of Formula 1) includes purifying haptoglobin (at least comprising the β-chain) prior to making such determination. Preferably, purification of haptoglobin is achieved via 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 various publications in different immunological methods. Non-limiting examples are: Abcam, #AB13429, clone HG-36; Abnova, #MAB12976, clone 2F4; Acris Antibodies, #UM500010, clone UMAB10; Novus Biologicals, #NBP2-03008, clone OTI4H5; OriGene, #TA00399, clone OTI2B8; Thermo Fisher Scientific, #HYB 170-06-02, clone 9G10. In a specific embodiment of the method according to the present disclosure, the step of determining one or more N-glycan structures at position N184 of the β-chain of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) or the corresponding glycopeptide (e.g., the glycopeptide of Formula 1) comprises purifying haptoglobin (at least comprising the β-chain) via a combination of a biotinylated anti-haptoglobin monoclonal antibody and streptavidin-coated beads (SA-beads). In such a purification step, the sample can be incubated with a 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).

[0171] In an embodiment of the present disclosure, the step of determining the amount of (or one or more) N-glycan structures at position N184 of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) comprises chemically hydrolyzing or biochemically / enzymatically cleaving / hydrolyzing the haptoglobin into peptides / glycopeptides. Various methods for hydrolyzing polypeptides by chemicals are known and can be used. In an embodiment, the haptoglobin is enzymatically cleaved into peptides / glycopeptides.

[0172] In an embodiment of the present disclosure, the step of determining the amount of (or one or more) N-glycan structures at position N184 of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) comprises enzymatically cleaving 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.

[0173] As described above, (or one or more) N-glycans at position 184 of haptoglobin (e.g., HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) or a glycopeptide comprising N184 (e.g., a glycopeptide of Formula 1) can be measured by mass spectrometry (MS). Various MS methods are known. The discovery and quantification of glycopeptides can be accomplished by high-resolution data-dependent mass spectrometry (Mol Cell Proteomics. 2014 Jan;13(1):329–338). The mass spectrometer selects the peptide with the highest intensity that enters the mass spectrometer at a given time, and then sequentially selects the species with the highest intensity for gas phase pyrolysis, and the obtained fragments are subsequently analyzed. The fragments carry sequence information of the peptide and glycan structure, and computer-assisted analysis is performed by comparing the obtained fragment spectrum with the predicted spectrum (derived from a database). In rare cases, manual inspection of isotope patterns is required to distinguish similar structures.

[0174] Highly sensitive identification of glycopeptides can also be accomplished by triple quadrupole mass spectrometry in single reaction monitoring mode (SRM) (Mol Cell Proteomics. 2013 Apr;12(4):1005–1016.). In this technique, the mass spectrometer selects only the target peptide. This peptide is fragmented in the gas phase, and unique and representative fragments are identified and quantified.

[0175] For the MS analysis of glycan structures or glycopeptides, quite a few processing steps are required. These processing steps may cause considerable changes, particularly in the measurement of (absolute) levels or quantities. In order to compensate for the differences in sample processing, an internal standard (usually the target analyte, but in the form of isotope labeling) is usually incorporated into the sample. Sample processing 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 processing. Therefore, it may be advantageous to determine the amount of a glycan structure or glycopeptide relative to another glycan structure or glycopeptide, respectively. In an embodiment, the ratio can be the ratio between the glycan structure increased at the same position of a polypeptide and the glycan structure reduced. Specific but non-limiting examples of such ratios are described elsewhere herein.

[0176] 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 corresponding to or proportional to the amount or concentration of the analyte in the sample; or any relative measure, i.e., a measure representing the amount or concentration of the analyte relative to a reference amount or concentration, respectively. A reference amount or concentration can be the amount of an internal control (e.g., a standard glycopeptide, another glycopeptide or peptide detected in a sample) measured simultaneously with the target analyte using the same method.

[0177] In embodiments, the amount of the glycan structure at position N184 of haptoglobin in the sample can be the amount of a glycopeptide comprising the glycan structure at N184 determined from the sample. In these embodiments, the method can include generating a glycopeptide from haptoglobin (e.g., via protease digestion as described elsewhere). In embodiments, the peptide portion of the glycopeptide can be as described above.

[0178] In an embodiment, the amount of a glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal of a non-glycosylated peptide (e.g., from haptoglobin), respectively. In an embodiment, the amount of a glycopeptide can be the amount or raw signal of the glycopeptide divided by the amount or raw signal of all detected glycopeptides (e.g., of haptoglobin), respectively.

[0179] The term "amount of change" refers to the fact that an amount is outside a reference range, that is, is higher than a certain reference amount or is lower than a certain reference amount.

[0180] The term "elevated" or "increased" level or amount of a marker refers to an amount or level of such marker in the study sample that is higher than the amount or level of such marker in a reference or control sample (i.e., does not include the amount or level of such marker in the study sample).

[0181] The term "reduced" level or amount of a marker refers to an amount or level of such marker in the study sample that is lower than the amount or level of such marker in a reference or control sample (ie, does not include the amount or level of such marker in the study sample).

[0182] 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 further parameter (such as, for example, the amount of a second glycan structure, the amount of one or more other biomarkers, or a clinical parameter). 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 should be configured so that HCC (e.g., early HCC) can be indicated.

[0183] "Take into account" as used in the context of a fraction includes embodiments where only the specifically listed quantities and parameters are taken into account as well as embodiments where other, non-listed parameters are taken into account.

[0184] 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.

[0185] Determining or calculating the score includes any mathematical combination of the amount of glycan structures or glycopeptides as mentioned herein with further parameters (see above). Thus, the score can be calculated by mathematically combining the various parameters (including or consisting of the determined or received amount of the glycan or corresponding glycopeptide attached to N184 of haptoglobin). These levels can be used as is or can be mathematically transformed (e.g., by a logarithmic transformation, such as a log2 or log 10 transformation) for determining the score. The score can take into account one or more other factors in addition to the level of PSA-glycoform species, including but not limited to the presence or level of one or more other biomarkers in the sample and one or more clinical parameters of the subject (e.g., tumor histology, smoking status, disease stage and / or age).

[0186] In certain embodiments of the present invention, the score for detecting HCC can be obtained by weighted calculation or can include weighted calculation. This means that biomarkers (e.g., the amount of the glycan structure at position N184 of haptoglobin) can be given different weights. For example, when the score considers the amount of the first glycan structure at position N184 of haptoglobin in the sample and the amount of another glycan structure at position N184 of haptoglobin, the score can be calculated by the following equation:

[0187] Score = a*[amount of the first glycan structure] + b*[amount of the second glycan structure], where a and b represent weighting factors. Preferably, the weighting 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 weighting factors or coefficients can be obtained by a machine learning method applied to a training data set of samples obtained from a reference population as defined herein.

[0188] A person skilled in the art knows that the score and the corresponding reference value of the score can be optimized based on a reference population (eg, any reference population as defined herein or as disclosed in the accompanying Examples).

[0189] 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 a 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.

[0190] Comparing the binary score to a reference value of the cutoff binary score means comparing the first value of the determined binary score to the first value of the reference value of the binary score and the second value of the determined binary score to the second value of the reference value of the binary score. If a binary score is established based on the amount of the first glycan structure at position N184 of haptoglobin and the amount of the glycan structure at position N184 of haptoglobin, HCC can be detected only when both amounts are indicative of HCC relative to the reference value of which they form part of the binary score.

[0191] In aspects and embodiments using two or more markers (or even more general input parameters, which may also include clinical parameters such as age or gender), they may be combined by an appropriate algorithm derived from multivariate analysis of the available data (e.g., logistic regression), preferably in an exhaustive search for feature selection. In such a combination / calculation, a multivariate score is calculated. Other methods may also be used to combine biomarkers / input values ​​into fractions, such as selected from DA (i.e., linear, quadratic, regularized discriminant analysis), kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classification), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forests, Boosting).

[0192] 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 indicative of the performance of the test. For example, if the sensitivity value of a biomarker combination is 80%, 80 out of 100 patients with the disease can be correctly identified; if its specificity value is 80%, 80 out of 100 patients without the disease can be accurately tested negative for the disease.

[0193] In the same manner as previously described, ROC curves can be used to assess the performance of discriminating between patients and controls using the aforementioned logistic regression model.

[0194] The present disclosure, particularly in the context of the methods disclosed herein, refers to comparing the amount or level of a glycan structure, glycopeptide or score with a reference amount / level or score for the glycan structure, glycopeptide or score. It should be understood that such comparisons as used herein generally refer to the comparison of corresponding parameters, amounts, values ​​or scores, 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 such a 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 automatically performed by a computer program that performs a comparison algorithm. The computer program that performs the evaluation will provide the required assessment in an appropriate output format. For computer-assisted comparisons, the value of the determined 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 a value corresponding to an appropriate reference stored in a database by the 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.

[0195] 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. Glycan structures can, for example, be bound to amino acids, for example, amino acid asparagine. In the case of the present invention, glycans bound to asparagine at position 184 of the β-chain of haptoglobin (SEQ ID NO: 1) were analyzed. The glycan structures or glycans disclosed herein serve as biomarkers or markers for HCC (e.g., early HCC).

[0196] 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 184 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.

[0197] Polysaccharides are generally referred to herein by the general formula. In this article, we refer to polysaccharide compositions 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-acetylhexosamine, GlcNAc means N-acetylglucosamine, GalNAc means N-acetylgalactosamine, Hex means hexose, Man means mannose, Glu means glucose, Gal means galactose, Fuc means fucose, and NeuAc or Neu5Ac means N-acetylneuraminic acid (sialic acid).

[0198] Preferred glycans are indicated by specific formulas or using schematic diagrams according to the SNFG (Glycan Symbol Nomenclature) system (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, Vol. 29, No. 9, September 2019, pp. 620–624). Exemplary glycans are those of Formula 2.

[0199] In an embodiment of the present invention, the glycan HexNAc(5)Hex(6)Fuc(1)NeuAc(2) may have Formula 2:

[0200]

[0201] As used in the formulas herein (e.g., Formulas 1 and 2), GlcNAc means N-acetylglucosamine, Man means mannose, Gal means galactose, Fuc means fucose, and NeuAc means N-acetylneuraminic acid (or sialic acid). Lines represent covalent bonds between monosaccharides. Low-level GlcNac is a monosaccharide, and if the polysaccharide is part of a glycopeptide or glycoprotein, the polysaccharide is attached to a peptide or protein (e.g., N184 of SEQ ID NO: 1) via the low-level GlcNac. The brackets used in the context of NeuAc (or Neu5Ac used as a synonym) indicate that NeuAc can be attached to any of the three galactose residues.

[0202] As used herein, the term "reference amount" (or "reference level") of any analyte (e.g., a glycan structure or glycopeptide) refers to an independently determined predetermined amount of the analyte. As will be appreciated by the technician, the reference amount is predetermined and is set to meet the conventional requirements in terms of specificity and / or sensitivity, for example, for detecting HCC (e.g., early HCC). Therefore, the reference amount can be selected so that it indicates HCC (e.g., early HCC). The requirements for detecting HCC may vary, for example, from regulatory agency to regulatory agency. 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 can 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 evaluation value and the reduction value indicate an abnormal state) or a reference level or a cutoff level (if the evaluation value or the reduction value indicate an abnormal state) can be determined by a technician. Similarly and according to the same principle, a reference ratio can be determined.

[0203] As used herein, the term "reference value", for example in the context of a reference value for a score, refers to an independently established predetermined value of each parameter (e.g., score). As will be appreciated by the skilled person, 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 applies mutatis mutandis.

[0204] The reference amount or reference value for the score is usually determined in a 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 condition or known to be at risk of a given condition; or obtained from individuals or groups of individuals known not to suffer from a given condition, 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., from a reference population) will depend on the assay used. Preferably, a reference amount or reference value is established using samples of 100 fully characterized individuals from an appropriate reference population. Also preferably, the reference population can be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a frequently used reference population for establishing control or reference amounts or values. In one embodiment, the reference level or value is determined in a reference sample from a healthy individual.

[0205] For detecting HCC in clinical routine, it is of utmost importance to obtain signs of the possible development or presence of HCC in those patients with cirrhosis or other HCC risk factors.Therefore, in an embodiment, a reference level is determined in a reference sample from a patient with cirrhosis.

[0206] In embodiments, a reference population from which control or reference samples can be obtained includes samples obtained from control subjects (e.g., healthy individuals or subjects with liver disease that have an increased risk of developing HCC) and subjects with HCC (e.g., early-stage HCC).

[0207] As will be understood by the skilled person, any method or use for detecting HCC or assisting in detecting HCC may include a step of detecting HCC or assisting in detecting HCC. Such assisting detection or detection is usually achieved based on a comparison step of such a method.

[0208] Alpha-fetoprotein is a glycoprotein, and various glycosylation forms of AFP have been described. Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the sugar chain structure of glycoproteins, marker glycoprotein fractions with specific sugar chain structures can be separated and concentrated. With respect to AFP, lectins derived from lentil agglutinin-A (LCA) have been widely used. The lentil agglutinin (LCA) reactive fraction of α-fetoprotein (AFP-L3) is specifically increased in patients with HCC. Many attempts have been made to specifically measure AFP-L3, such as by affinity electrophoresis using LCA, ELISA based on lectins, or by antibodies that specifically bind to the L3 form of AFP.

[0209] Vitamin K deficiency / antagonist-II induced protein (PIVKA-II), also known as des-gamma carboxy prothrombin (DCP), is an abnormal form of prothrombin protein that is elevated in HCC patients and is used alone or in combination with AFP as an alternative HCC biomarker. Prothrombin has 10 potential γ-carboxylation sites, and various forms of PIVKA-II with different levels of incomplete carboxylation exist in the circulation. Different assays for PIVKA-II can detect a collection of different 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.

[0210] 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 where they do not significantly bind to other molecules.

[0211] The term "specific binding" or "specifically binds" when referring to a protein or peptide that is an antibody or binding agent refers to a binding agent that binds to a protein or peptide at a rate of 10 -7 M or smaller K D The term "specific binding" or "specifically binds" preferably refers to the binding reaction of the target molecule to the corresponding 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, preferably, the K of binding to the target molecule isD The binding to non-target proteins is at least 1 / 10, more preferably at least 1 / 20.

[0212] 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 treatment recommendations for a subject.

[0213] It should be understood that the methods and uses of the present disclosure can be performed away from the subject or their attending physician, in fact, they can be performed offshore and the results can be transmitted back. In a specific embodiment, the results of the methods of the present invention (e.g., determining whether the subject suffers from HCC) are provided to a third party, such as the subject or his attending physician, laboratory or health center.

[0214] In this article, elements of the present invention are described. These elements are listed as aspects together with specific embodiments, however, it should be understood that they can be combined in any way and in any number to create other aspects and embodiments. In particular, the embodiments disclosed in the context of one aspect are applicable to other aspects after necessary modifications. The examples and preferred embodiments of the various descriptions 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.

[0215] The entire disclosures of all references cited in this specification and the disclosures specifically mentioned in this specification are incorporated herein by reference.

[0216] The following examples and figures are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that the procedures set forth may be modified without departing from the spirit of the present invention. A description of the amino acid sequences disclosed herein is also provided.

[0217] Description of the sequence

[0218] SEQ ID NO: 1 shows the amino acid sequence of human haptoglobin; N184 is printed in bold and underlined:

[0219]

[0220]

[0221] (Sequence Listing; Uniprot P00738, Version 221).

[0222] SEQ ID NO:2 shows the amino acid sequence of the peptide portion contained in an exemplary glycopeptide (e.g., a glycopeptide of Formula 1) for detecting a glycan structure at position N184; the sequence corresponds to amino acid positions 179 to 194 of SEQ ID NO:1 and the Asn residue corresponding to N184 of SEQ ID NO:1 is printed in bold and underlined:

[0223]

[0224] SEQ ID NO:3 shows the amino acid sequence of a peptide included in an exemplary glycopeptide for detecting a glycan structure at position N207; the Asn residue corresponding to N207 of SEQ ID NO:3 is printed in bold and underlined:

[0225]

[0226] 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:4 is printed in bold and underlined:

[0227]

[0228] 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:5 is printed in bold and underlined:

[0229] BRIEF DESCRIPTION OF THE DRAWINGS

[0230] Figure 1 : Schematic representation of the various forms of haptoglobin: The three types of haptoglobin contain two α1-chains of haptoglobin (1-1); an α1-chain and one α2-chain (2-1) or two α2-chains (2-2) (always together with a β-chain).

[0231] Figure 2 : Structure of Formula 1 (Compound 41): HexNAc (5) Hex (6) Fuc (1) NeuAc (2) at position N-184. Left: Glycan structure drawn using monosaccharide abbreviations following the SNFG (Glycan Symbol Nomenclature) system (PMID 26543186, Glycobiology 25: 1323–1324, 2015). Connections are depicted by black lines. Right: Symbolic depiction of glycan structure: Monosaccharide symbols follow SNFG.

[0232] Figure 3: Box-Blot of various glycopeptides at position N184 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 amount of Hp glycopeptides was compared between the control, early and late HCC cohorts. Statistically significant differences between the control (C), early HCC (ESH) and late HCC (LSH) were determined by the Wilcoxen (Mann-Whitney U) test.

[0233] Figure 4 : Summary of upregulated glycopeptides at glycosylation site N-184. Monosaccharide symbols follow the SNFG (Synsaccharide Symbol Nomenclature) system (PMID 26543186, Glycobiology 25: 1323–1324, 2015). Only the five glycopeptides with the greatest difference between early and late HCC and controls are shown.

[0234] Figure 5 : Receiver operating curve (ROC): Diagnostic performance of the compound HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N-184 for detecting early and late stage HCC is shown. Specificity and sensitivity values ​​are for sensitivity and specificity with a cut-off value of 0.9, respectively.

[0235] 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 that distinguish early HCC cases from controls are listed on the left. The glycopeptides shown in the middle column are present in both early and late HCC, and those shown in the right column distinguish late HCC from controls. Only glycopeptides with AUC>70% are shown. The direction of expression change (reduced or increased relative to the control) is indicated by up (regulated) or down (regulated).

[0236] Examples

[0237] Example 1

[0238] 1.1 Study composition

[0239] EDTA-plasma samples were obtained from 57 controls representing chronic liver diseases including HBV, HCV, and cirrhosis, and HCC patients including 33 individuals with early-stage HCC and 32 individuals with advanced-stage HCC (see Table 1 for demographic information).

[0240] 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, where BCLC stages 0 and A are classified as early HCC and stages B to D are classified as advanced HCC. After plasma preparation, the samples used in this analysis have been stored at -80°C until analysis as described below. Repeated freezing and thawing of the samples has been avoided.

[0241]

[0242] Table 1: Summary of demographic variables grouped according to clinical status.

[0243] 1.2 H. pylori immunoprecipitation from plasma

[0244] The first step is to prepare immune capture beads. 10 mg of streptavidin (SA) coated latex beads were incubated together with 150 μg of biotin (Bi) labeled F(ab')2 fragment MAK<haptoglobin>M-1.1.13-F(ab')2-Bi (Roche Diagnostics GmbH, Mannheim, Germany) of the mouse monoclonal antibody against Hpβ-chain on a rotator at room temperature (RT) for 1 hour. 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 coated on 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 hour. The samples were then treated with 10 μl 50 mM IAM (iodoacetamide) at room temperature for 30 minutes. Afterwards, the sample was 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 for 2 h at room temperature on a rotator, two steps of PBST (PBS buffer + 0.1% Tween ) and two steps of PBS buffer to wash away unbound proteins. Hp bound to the antibody-coated beads was eluted from the beads by incubating the beads in 500 μl of glycine buffer (0.2 M glycine; pH: 2.6) in two steps. Two-step elution facilitates recovery. To naturalize the pH, 200 μl of NaOH (1N) was added to the eluate after each elution step.

[0245] 1.3 FASP digestion

[0246] The two HP fractions eluted in the previous step were combined and loaded onto an OmegaTM Membrane-10K (PALL, US) filter (cut-off value: 10 kDa) The samples were centrifuged at 10.000 g for 20 min on a centrifuge device. In this step, molecules (e.g., proteins with a molecular weight of 10.000 or more) are retained on the filter, while small molecules (e.g., salts) pass through the filter and are removed. Then, 75 ng of heavy haptoglobin (recombinant Hp with isotope-labeled heavy 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 off the buffer and washed once with 100 μl ABC (ammonium bicarbonate, 50 mM) buffer. Then, the protein retained on the filter was first digested by 6 μg trypsin added in 50 μl ABC buffer at 37 ° C for 3 h. The trypsin digestion was stopped by incubating the filter at 95 ° C for 10 min. For the second digestion, 10 μg 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.

[0247] 1.4 LC-MS / MS analysis

[0248] Twenty microliters of peptides obtained by enzymatic digestion as described in 1.3 were injected into the 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 a C18 column (Waters, XSelect CSH C18 column, Peptides were separated by reverse phase chromatography on a 3.5 μm, 2.1 mm × 150 mm (100 μl / min). The flow rate of the LC was 320 μl / min, and the gradient was set as follows: 0% to 30% B (0 min to 30 min), 30% to 80% B (30 min to 31 min), 80% B (31 min to 36 min), 80% to 0% B (36 min to 37 min), and 0% B (37 min to 42 min), where eluents A and B were 0.1% formic acid in H2O and 0.1% formic acid in acetonitrile, respectively. The separated peptides were ionized by an electrospray ionization (ESI) source and analyzed in positive ion mode and data-dependent acquisition methods. Full scan MS spectra were collected in the range of 300 m / z to 2000 m / z at a resolution of 60000, an automatic gain control (AGC) of 10e6, and an injection time of 50 ms. The five most intense peaks from 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 15,000, 1e5 AGC, and an injection time of 150 ms.

[0249] 1.5AFP and PIVKA-II determination

[0250] AFP and PIVKA-II were measured using microchip capillary electrophoresis and liquid phase binding assays on an uTASWako i30 autoanalyzer (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions.

[0251] 1.6 Data Analysis

[0252] 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). For the identification of 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, and two false cleavages were allowed. Carbamidomethylation of cysteine ​​was set as a fixed modification, and 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 that was significantly different in the cohort groups and had an AUC greater than 0.7 was manually checked. In these cases, we examined retention time, charge state, glycan oxonium ions, and isotope patterns compared to the predicted isotope patterns of the proposed glycopeptides. In this article, we refer to the glycan compositions as follows: HexNAc(x)Hex(x)Fuc(x)NeuAc(x), where the numbers in parentheses represent the number of individual monomers present in the glycan structure. HexNAc means N-acetylhexosamine, Hex means hexose, Fuc means fucose, and NeuAc or Neu5Ac means N-acetylneuraminic acid (sialic acid). The schematic diagram is based on monosaccharide symbols following the SNFG (Syntax Nomenclature) system (PMID 26543186, Glycobiology 25: 1323–1324, 2015), and for details, see NCBI (Ajit Varki et al., Symbol Nomenclature for Graphical Representations of Glycans, Glycobiology, Volume 25, Issue 12, December 2015, Pages 1323–1324, https: / / doi.org / 10.1093 / glycob / cwv091 and Sriram Neelamegham et 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 quantification values.

[0253] 1.7 Statistical analysis

[0254] The abundance of glycopeptides in the sample was normalized to the abundance of the first three peptides spiked with heavy Hp to correct for possible changes in processing, digestion or MS measurement. The missing value of a certain glycopeptide (i.e., a glycopeptide below the detection limit) was replaced by the minimum value of the glycopeptide in the data set. The Wilcoxen (Mann-Whitney U) test was used to test the significance of glycopeptide differences between clinical groups by calculating the p value. To correct for multiple testing, the Benjamini-Hochberg correction was used, and the FDR control was 20%. In order to evaluate the diagnostic value of glycopeptides, receiver operating characteristic (ROC) curves were prepared, and the area under the curve (AUC) values ​​were calculated by the DeLong method using R software (version 3.5.2, available at https: / / www.R-project.org) (Elisabeth R. DeLong, David M. DeLong and Daniel L. Clarke-Pearson (1988), Biometrics 44, 837–845).

[0255] 2. Results

[0256] 2.1 Glycan properties for HCC diagnosis

[0257] Global analysis of glycan features (e.g., fucosylation) provides insight into the major changes in Hp glycosylation patterns as HCC progresses. However, the types and abundance of glycoforms at different Hp glycosylation sites vary. Therefore, we investigated whether Hp glycosylation site- and glycoform-specific analysis could be indicative of 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 set of glycopeptides includes highly branched, fucosylated (compounds 126, 131 or 140) and highly mannosylated (compounds 58, 60, 24 and 27) and sialylated (compound 126) glycopeptides.

[0258] In addition, for early HCC-specific biomarkers, we have discovered fourteen other glycopeptides that were significantly more highly expressed in both early and late HCC and provided an AUC of 0.70 or higher in distinguishing early HCC patients from cirrhotic controls ( Figure 6Among them, compound 41 showed the best AUC. As HCC progressed from early stage to late stage, these glycopeptides were expressed more. Figure 4 The top five Hp glycopeptides that were significantly more highly expressed in both early and advanced HCC are listed.

[0259] In addition to the glycopeptides upregulated in HCC, we also observed two glycopeptides ( Figure 6 ), which are located at the N207 glycosylation site. One of these 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 bisecting 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 or compound 41) to further improve robustness by providing an internal control. In total, we identified 29 glycan structures that distinguished controls from early and advanced HCC with an AUC higher than 70% ( Figure 6 ).

[0260] To date, the best glycopeptide for detecting early and late HCC is compound 41 on N184 (see Figure 4 and 5 ). Therefore, among all the glycosylation sites analyzed in this paper, haptoglobin glycosylation at position N184 seems to be the most suitable for detecting all stages of HCC. Figure 4 and 6 Data for all altered glycopeptides are presented, using an AUC of 0.70 or greater as a cutoff.

[0261] The results, glycan structures, peptide sequences and m / z values ​​of the detected glycopeptides are summarized in Tables 2 and 3 below.

[0262] These results consistently revealed that glycopeptide analysis of Hp, especially at position N184 (but also at positions N207, N211, and N241) can provide sugar biomarkers with better clinical value than established biomarkers for early HCC diagnosis.

[0263]

[0264] Table 2: Glycan structures, peptide sequences and m / z values ​​of the tested glycopeptides that significantly distinguished early and late HCC from controls. AUC [%] and the direction of expression change are indicated for each examined group. * Potential oxidation of methionine residues

[0265]

[0266] Table 3: Glycan structures, peptide sequences and m / z values ​​of the detected glycopeptides that significantly distinguished early and advanced HCC from controls. AUC [%] and direction of expression change are indicated for each examined group. * Potential oxidation of methionine residues.

[0267] 2.2 Optimal glycans for detecting (early or early and late) HCC

[0268] The results indicate that in particular the Hp glycopeptide compound 41 (i.e. HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184) has a high potential for clinical application in HCC detection, especially for early-stage HCC. This is evident from the area under the curve (AUC) values ​​given for each of these glycopeptides. Surprisingly, for this glycopeptide, the calculated AUC already exceeds the AUC value for AFP, and the glycopeptide improves the performance of each individual protein biomarker and their combination. Table 2 and Figure 6 Other Hp glycopeptide compounds with good diagnostic potential for early HCC were shown in .

[0269] In addition, many glycans with good performance in identifying early HCC and late HCC cases were identified. In particular, compound 41 has high clinical application potential. This Hp glycopeptide is a glycopeptide with the best AUC for early HCC, and it also shows a significant AUC for late HCC. Table 2 and Figure 4 and 6 Other Hp glycopeptide compounds with good diagnostic potential for early and advanced HCC were shown in .

[0270] 2.3 Marker combination

[0271] In order to analyze the value of adding compound 41 to PIVKA-II and / or AFP, a logistic regression model consisting of compound 41, 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 marker combinations are summarized in Table 4. Models other than logistic regression (or logistic regression models with interaction terms between variables) were unable to significantly improve the performance of the above logistic regression models.

[0272]

[0273] Table 4: The combination of glycopeptide compound 41 improves the clinical value of AFP. In our cohort, the AUC value of AFP for early diagnosis of HCC was 83%. The combination of these two biomarkers improved the AUC to 87%, and the combination of glycopeptide with AFP and PIVKA-II improved the AUC to 93%.

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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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(5)Hex(6)Fuc(1)NeuAc(2) at position 184 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin determined in a), and comparing the determined score for detecting HCC with a reference value for the score indicating HCC.

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

2.

3. The glycopeptide according to claim 2, wherein the glycopeptide has a structure as shown in Formula 1:

4. Use of a glycopeptide as defined in claim 2 or 3 for aiding detection of HCC, in an embodiment early stage HCC. 5 . The method of claim 1 , wherein the method further comprises determining 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 AFP.

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

7. The method of claim 6, wherein the method comprises purifying haptoglobin from the sample prior to hydrolyzing the haptoglobin.

8. The method according to any one of claims 1 and 5 to 7, wherein mass spectrometry is used to determine the amount of the glycan structure HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin.

9. A method for detecting and / or quantifying the glycopeptide according to claim 2 or 3, 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 2 or 3.

10. 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 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 HexNAc(5)Hex(6)Fuc(1)NeuAc(2) received in a) with a reference amount of said glycan structure, wherein an increased amount of HexNAc(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin in the sample of said subject relative to said 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(5)Hex(6)Fuc(1)NeuAc(2) at position N184 of haptoglobin received in a), and comparing the calculated score for detecting HCC with a reference value for said score indicating HCC; and c) assisting in determining whether the subject has HCC based on the comparison in b).

11. The computer-implemented method of claim 10, wherein the method further comprises receiving data comprising 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 AFP.

12. A computer program product comprising instructions which, when said program is executed by a computer, cause said computer to perform a computer-implemented method according to claim 10 or 11.

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

14. The method according to any one of claims 1, 5 to 8, 10, 11 and 13, wherein the sample is a blood sample, in particular serum or plasma.

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