CN115078719A

CN115078719A - IgG sugar chain marker for colon cancer screening and diagnosis and application thereof

Info

Publication number: CN115078719A
Application number: CN202110268879.8A
Authority: CN
Inventors: 顾建新; 任士芳; 顾勇
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2022-09-20

Abstract

Disclosed herein are an IgG sugar chain marker for colon cancer screening and diagnosis and use thereof. In particular, the present disclosure relates to a product (e.g., kit, device, operable system and/or combination thereof) for colon cancer screening, colon cancer diagnosis and/or colon cancer disease progression judgment in a subject, the product comprising reagents, instruments and/or systems for determining abundance of alpha-1, 3 galactosylated fucose-type N-sugar chains (F (6) a2[3] G1) and/or derivatives thereof (e.g., F (6) a2[3] G1S1) on blood IgG surface. The present invention also relates to the use of a substance for detecting the abundance of F (6) A2[3] G1 and/or its derivatives on the surface of blood IgG in the manufacture of a product for use in the early diagnosis, risk assessment, disease monitoring and/or efficacy assessment of colon cancer in a subject.

Description

IgG sugar chain marker for colon cancer screening and diagnosis and application thereof

Technical Field

The invention belongs to the field of biotechnology and medicine. In particular to an N-sugar chain tumor marker, namely alpha-1, 3 galactosyl fucose type N-sugar chain and a derivative structure sugar chain thereof, which can be used for clinical screening and diagnosis of colon cancer, and the application of the change thereof in screening, diagnosis and prognosis evaluation of colon cancer.

Background

Colon cancer is one of the three most common cancers of worldwide morbidity and mortality, and is also the second most common cancer in men and the third most common cancer in women. The incidence of the malignant disease is higher in developed countries than in developing countries.

Although the long-term survival rate of patients is improved due to the diversified development of colon cancer treatment measures in the last 30 years, at least 50% of cancer patients have poor prognosis such as metastasis after diagnosis, and the total survival rate in 5 years is only 12.5%. At present, the pathogenesis of colon cancer is still lack of clear description, so that an effective treatment means is lacked.

The effective mode of cancer control is "early three", namely: early detection, early diagnosis and early treatment. The World Health Organization (WHO) specifies: early detection is key to improving the rate of cancer treatment. As long as it is found early, 90% of cancers are completely cured. If the early detection, early diagnosis and early treatment of cancer are carefully done, the cancer death rate can be reduced by about one third. However, to achieve the prevention and treatment of cancer at the third morning, a screening method which is high in sensitivity, simple and convenient to use, painless, low in price and easy to accept must be adopted, so that the screening method can be popularized in a large area of people, and more early-stage cancer patients and cancer high-risk people can be found in a plurality of asymptomatic people.

Accordingly, early diagnosis and screening of colon cancer have a very important clinical role, and for example, early screening and early intervention are among the most important factors affecting survival of colon cancer patients. The 5-year survival rate of the early-diagnosed patient can reach more than 95 percent. Unfortunately, the current techniques of early diagnosis or screening often suffer from invasiveness and complexity or lack of accuracy, which can delay the optimal period of early screening for patients.

Most colon cancers are derived from Advanced Adenomas (AA), and early detection and resection of these adenomas can reduce the incidence and mortality of colon cancer. Colonoscopy is the gold standard for colon cancer screening due to its high sensitivity and specificity, but also suffers from drawbacks such as complications, high cost and invasiveness, low patient compliance, etc. Therefore, non-invasive screening is of paramount importance.

In recent years, the main non-invasive methods for colon cancer diagnosis are the guaiac stool occult blood test (gfobet) and the immunochemical stool occult blood test (FIT). The guaiac stool occult blood test (gfobet) detects peroxidase activity in the hemoglobin heme subunit and is therefore not specific for human hemoglobin, theoretically requiring dietary restriction several days prior to performing the test to eliminate the effects of diet. Immunochemical stool occult blood test (FIT) is a non-invasive test based on specific monoclonal or polyclonal antibodies to human hemoglobin and therefore does not require dietary changes. FIT screens patients with a high average risk of developing colon cancer, but has limited sensitivity to detect advanced colon adenomas or stage I colon cancer. On the other hand, FIT varies widely in sensitivity from 5.4% to nearly 98% with specificity ranging from 77% to 99%, which results in a large number of false negative test results with missed diagnosis in some cases.

In recent research, serum and stool are mainly used as research objects, and markers which are high in specificity and sensitivity and can distinguish different stages of colon cancer are searched at the genetic material level and the protein level respectively. Serum is the most suitable object for developing noninvasive tumor markers at present stage as a human body sample which is easy to obtain. At present, the existing tumor serological markers comprise carcinoembryonic antigen (CEA), cancer antigen 19-9(CA19-9), CA125, Alpha Fetoprotein (AFP) and the like, but the clinical diagnosis effect is poor due to the insufficient sensitivity and specificity.

In conclusion, it is important to develop efficient specific biomarkers that can be used for early detection and risk assessment of colon cancer.

Disclosure of Invention

It is the present application that provides a highly specific biomarker that can be used for colon cancer screening, risk assessment, and diagnostic and progress assessment.

In a first aspect of the present application, there is provided a product (e.g., a kit, a device, an operable system, and/or a combination thereof) for use in screening, diagnosing, and/or determining the progression of colon cancer in a subject, the product comprising:

(A) a reagent, an apparatus and/or a system for measuring the abundance of an alpha-1, 3 galactosylated fucose-type N-sugar chain F (6) A2[3] G1 and/or a derivative thereof on the IgG surface of blood,

the structure of the F (6) A2[3] G1 is shown as follows:

the derivatives are natural derivatives having an additional sialyl sugar group attached to the terminal Gal of F (6) A2[3] G1, for example F (6) A2[3] G1S1, the structure of which is shown below:

for example, the reagent, apparatus and/or system is a reagent, apparatus and/or system for one or more methods selected from the group consisting of: ultra Performance Liquid Chromatography (UPLC); matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-MS, fast atom bombardment mass spectrometry FAB-MS and electrospray mass spectrometry ES-MS; liquid chromatography; capillary electrophoresis chromatography and methods based thereon; liquid chromatography-mass spectrometry; sugar chip technology; nuclear magnetic resonance NMR, preferably Ultra Performance Liquid Chromatography (UPLC);

(B) optionally, a module and/or processor for calculating the abundance of F (6) A2[3] G1 and/or derivatives thereof;

(C) optionally, the system further comprises means and/or a processor for determining whether the subject suffers from colon cancer based on the abundance of F (6) A2[3] G1 and/or derivatives thereof.

In some embodiments, the blood is selected from: serum, plasma and whole blood, more preferably serum.

In some embodiments, the product further comprises one or more selected from the group consisting of:

a) reagents and/or instruments for collecting and/or processing a subject blood sample;

b) reagents and/or instruments for separating and/or purifying serum and/or plasma;

c) reagents and/or apparatus for the separation and/or purification of serum/plasma IgG;

d) a reagent and/or an apparatus for separating, purifying and/or enriching N-sugar chains on the surface of serum IgG,

for example, a reagent and/or an apparatus for a method for separating and purifying an N sugar chain selected from the group consisting of: solid phase extraction of porous graphitized carbon PGC (preferably, PGC is activated with acetonitrile/water trifluoroacetic acid (80: 19.9: 0.1), PGC is equilibrated with water/trifluoroacetic acid (99.9: 0.1), and sugar chain is eluted with a mixed solvent of acetonitrile/water trifluoroacetic acid (25: 74.95: 0.05), polysaccharide purification column, lectin affinity method (e.g., SLAC), capillary electrophoresis, and high performance liquid chromatography;

e) a database, module and/or processor for storing and/or processing the subject's serum F (6) A2[3] G1 and/or derivatives abundance values thereof;

f) means and/or a processor for providing a decision threshold;

g) means and/or a processor for comparing the abundance of F (6) a2[3] G1 and/or derivatives thereof in a subject to a control to determine whether the subject has a precancerous lesion of colon cancer or suffers from colon cancer or its progression;

h) a module and/or processor for providing a diagnosis and/or test result and/or report;

j) the instruction or the instruction manual records one or more of the following applications and judgment modes:

when the abundance of F (6) A2[3] G1 and/or derivatives thereof reaches or exceeds a predetermined threshold, determining that the subject and/or subject has or is at risk of having colon cancer (e.g., has a precancerous lesion of colon cancer);

(ii) for the benefit/disadvantage determination: when the abundance of F (6) A2[3] G1 and/or the derivative thereof reaches or is higher than a preset threshold value, judging that the subject has colon cancer or is at risk of having colon cancer;

(iii) for colon carcinogenesis anticipation in a subject population (e.g., high risk population): when the abundance of F (6) A2[3] G1 and/or derivatives thereof reaches or exceeds a predetermined threshold, indicating that the subject is more likely to develop colon cancer than other subjects;

(vi) for cancer patient condition monitoring: when the abundance of F (6) A2[3] G1 and/or derivatives thereof is higher than that of F (6) A2[3] G1 and/or derivatives thereof obtained in the previous test, reaches or is higher than a preset range (for example, 5-50% or any point or sub-range in the range), the cancer patient is indicated to have developed or further worsened;

(v) for efficacy assessment: the abundance of F (6) A2[3] G1 and/or derivatives thereof is measured and calculated at various time points before and after or during treatment, and poor therapeutic efficacy of the therapy and/or drug is indicated when the abundance of F (6) A2[3] G1 and/or derivatives thereof is at or above a predetermined range (e.g., 5% to 50% or any point or subrange within this range) above the corresponding abundance obtained from the previous test.

In some embodiments, the threshold value in (i) - (v) is 0.4-0.8, more preferably 0.5-0.7;

in some embodiments, the optimal threshold value may be determined based on the point of maximum slope in the ROC curve or the value that maximizes sensitivity and specificity, and a preferred threshold range may be a range of ± 1-15% (including any numerical point or subrange within that range) of the optimal threshold value.

In some embodiments, the abundance of F (6) A2[3] G1 and/or derivatives thereof is its relative abundance to a reference, e.g., F (6) A2[3] G1 and/or derivatives thereof relative to all sugar chains on a blood IgG surface or relative abundance to an added internal standard (e.g., a sugar standard);

for example, the relative abundance of F (6) A2[3] G1 and/or derivatives thereof may be calculated using a formula selected from the group consisting of:

X＝B/(B+C ₁ +……+C _n )＝B/A

in the formula:

x: the relative abundance of the alpha-1, 3-galactosylated fucose-type N-sugar chain F (6) A2[3] G1 and/or derivatives thereof;

a: total abundance of N sugar chains on all serum IgG surfaces;

b: abundance of alpha-1, 3-galactosylated fucose-type N-sugar chain F (6) A2[3] G1 and/or derivatives thereof;

C ₁ ～C _n : except for F (6) A2[3]]The abundance of each of the remaining sugar chains other than G1 and/or its derivative; or

And other transformation forms such as percentage, reciprocal and logarithm.

In some embodiments, the early screening is selected from: screening precancerous lesion and diagnosing early cancer.

In some embodiments, the product is used in a detection method comprising the steps of:

(A') measuring the abundance of IgG surface sugar chains F (6) A2[3] G1 and/or a derivative thereof in a subject blood sample;

(B') calculating the relative abundance (e.g., the ratio of abundance of all sugar chains, or the abundance ratio with respect to an internal standard) of F (6) A2[3] G1 and/or its derivatives;

(C ') determining whether the subject has colon cancer based on the relative abundance of F (6) A2[3] G1 and/or derivatives thereof, assessing the subject's risk of having colon cancer, monitoring the condition of the disease, and/or assessing the efficacy of the therapy.

In some embodiments, the method further comprises one or more steps selected from the group consisting of:

(a') collecting and/or processing a subject blood sample;

(b') isolating and/or purifying serum and/or plasma;

(c') isolating and/or purifying serum/plasma IgG;

(d') isolating, purifying and/or enriching serum IgG surface N-sugar chains;

(e') the abundance of N-sugar chains F (6) A2[3] G1 and/or derivatives thereof on the IgG surface of the serum of the subject of storage and/or treatment;

(f') providing a decision threshold;

(G ') comparing the abundance or relative abundance of F (6) A2[3] G1 and/or derivatives thereof in a subject to a control to determine whether the subject has colon cancer, to assess the subject's risk for developing colon cancer, to monitor disease and/or to assess efficacy;

(h') providing a diagnosis and/or test result and/or report;

(j') detecting other colon cancer markers and combining the detection of said other colon cancer markers with the detection of abundance of F (6) A2[3] G1 and/or derivatives thereof.

In some embodiments, the product further comprises a kit, device, system and/or combination thereof for detecting other colon cancer markers,

for example, the indicator is selected from one or more of the following:

(i) carcinoembryonic antigen (CEA);

(ii)A2G2：

(iii)A2G2S1：

for another example, the indicator is selected from: basic condition of the patient, such as age, sex, family history; the clinical manifestations of colon cancer, including local manifestations such as lumps and secondary symptoms, occult blood in stool, pathological secretion, and systemic manifestations such as fever, infection, anemia, emaciation, hypodynamia, dyscrasia; specific physicochemical examination indexes including specific image examination such as nuclear magnetism, CT, B-ultrasound, and endoscope; tumor biochemical indicators, such as AFP, CEA, PSA, SF, TSGF, POA, PROGRP, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, NSE, SCC, AFU, EBV-VCA, etc.

In another aspect of the present application, there is provided a use of a substance (e.g., a reagent, an apparatus, a module and/or a processor) for detecting abundance of α -1,3 galactosylated fucose-type N-sugar chains (F (6) a2[3] G1) and/or derivatives thereof (e.g., F (6) a2[3] G1S1) on IgG surface of blood in the manufacture of a product (e.g., a kit, a device, a system and/or a combination thereof) for early diagnosis, risk assessment, disease monitoring and/or efficacy assessment of colon cancer in a subject, the substance comprising one or more selected from the group consisting of:

(A) a substance (e.g., a reagent, an instrument, a module, and/or a processor) for determining the abundance of F (6) A2[3] G1 and/or a derivative thereof on a blood IgG surface;

for example, reagents, instruments, modules and/or processors for one or more methods selected from the group consisting of: ultra Performance Liquid Chromatography (UPLC); matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI), fast atom bombardment mass spectrometry (FAB-MS) and electrospray mass spectrometry (ES-MS); liquid chromatography; liquid chromatography-mass spectrometry; sugar chip technology; nuclear magnetic resonance NMR, preferably Ultra Performance Liquid Chromatography (UPLC);

(B) optionally, a module or processor for calculating the relative abundance of F (6) A2[3] G1 and/or derivatives thereof (e.g., ratio of all sugar chains on blood IgG surface or ratio to internal standard);

(C) optionally, a means (e.g., a module or a processor) for determining whether the subject has colon cancer based on the abundance or relative abundance of F (6) A2[3] G1 and/or a derivative thereof, and performing a risk assessment, disease monitoring and/or efficacy assessment of the subject's colon cancer.

In some embodiments, the product is as described herein.

In another aspect of the present application, there is provided a method for drug and/or therapy screening, the method comprising:

(A') determining the abundance of IgG surface alpha-1, 3 galactosylated fucose-type N-sugar chains F (6) A2[3] G1 and/or derivatives thereof in the blood sample at different time points before, after and/or during administration and/or treatment of the subject;

(B') calculating the relative abundance of F (6) A2[3] G1 and/or derivatives thereof, e.g., F (6) A2[3] G1 and/or derivatives thereof, relative to all sugar chains on the surface of the blood IgG, relative to an added internal standard (e.g., a standard for sugars);

(C ") assessing the relative abundance of F (6) a2[3] G1 and/or derivatives thereof to determine the effectiveness of the drug and/or therapeutic method, wherein when the relative abundance of F (6) a2[3] G1 and/or derivatives thereof is at or above a predetermined range (e.g., 5% to 50% or any point or subrange within the range) above the corresponding relative abundance of the previous test, the drug and/or therapeutic method is indicated to be not therapeutically effective; otherwise, the medicine and/or the treatment method are indicated to have good curative effect.

In other aspects of the present application, methods are provided for early screening for colon cancer, diagnosis of colon cancer, and/or diagnosis of the progression of a colon cancer condition in a subject, the methods comprising determining the abundance of alpha-1, 3 galactosylated fucose-type N-sugar chains (F (6) A2[3] G1) and/or derivatives thereof on the IgG surface of the subject's blood.

In other aspects of the present application, methods for early screening for colon cancer, diagnosis of colon cancer, and/or diagnosis of the progression of a colon cancer condition in a subject are provided, the methods comprising detecting a blood sample from the subject using the products (e.g., kits, devices, operable systems, and/or combinations thereof) of the present application.

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.

FIG. 1: the relative expression levels of alpha-1, 3 galactosylated fucose-type N-sugar chains F (6) A2[3] G1 on 73 healthy human serum IgG were compared with those of 47 pre-colon cancer patients and 95 colon cancer patients.

Represents P < 0.0001.

FIG. 2: ROC analysis curve was made using the relative expression level of alpha-1, 3 galactosylated fucose type N-sugar chain as a diagnostic index. The AUC of the control group and the precancerous group is 0.8359, and the 95% confidence interval is 0.8359-0.9218; AUC of the control group and the tumor group is 0.9135, and the 95% confidence interval is 0.8643-0.9626.

FIG. 3: the classical index CEA alone and in combination with the indices of the present application were compared to ROC curves for pre-cancerous samples:

a: ROC curve (AUC 0.467) of classic index CEA diagnosis precancerous sample;

b: the classical index CEA and the index of the application jointly diagnose the ROC curve of the precancerous sample (AUC is 0.938, and the 95% confidence interval is 0.777-0.938).

FIG. 4 is a schematic view of: the alpha-1, 3-galactosylated fucose type N-sugar chain F (6) A2[3] of the present application]The structure and detection effect of G1 on precancerous lesion of colon cancer are similar to those of Gal in CN 105277718A ("150046") in the prior art ₁ And (3) comparing the detection effects of the structure and detection indexes Gal ratio:

a: 150046 mono-galactosylated fucose type N-sugar chain Gal ₁ Structure and alpha-1, 3 galactan of the present applicationGlycosylated fucose type N-sugar chain F (6) A2[3]]Structural comparison of G1. The former is a mixture of alpha-1, 3 and alpha-1, 6 isomers, and the latter is an alpha-1, 3 isomer thereof;

b: 150046 comparison of Gal ratio index (left) with the present application F (6) A2[3] G1 index (right) in the detection of precancerous lesions in colon cancer, wherein the Gal ratio index in 150046 is calculated as follows:

gal ratio is Gal ₀ /(Gal ₁ +Gal ₂ ) Represents the terminal galactosylation level of the IgG surface dual-antenna complex N sugar chain in the blood sample of the subject, and the terminal galactosylation level comprises: gal ₀ IgG surface terminal no galactose linkage dual antenna complex N-sugar chain level; gal ₁ The double-antenna complex N-carbohydrate chain with 1 galactose connected to the tail end is horizontal; gal ₂ The double-antenna complex N-carbohydrate chain with 2 galactose connected at the tail end is horizontal.

FIG. 5 is a schematic view of: the derivatized structures of alpha-1, 3 galactosylated fucose-type N-sugar chains are designated F (6) A2[3] G1S1 and the structures of two other nonfucosylated sugar chains on IgG are designated A2G2 and A2G2S1, respectively.

FIG. 6: ROC plot of 9 sugar chains on Ig as an early screening indicator for colon cancer, wherein F (6) A2[3] G1S1 showed excellent diagnostic effect (upper left panel of FIG. 6).

FIG. 7: ROC graph of optimal early screening effect (control group and pre-cancer sample group) by combination of several sugar chain joint indicators:

a: ROC curve diagram of F (6) A2[3] G1+ A2G2+ F (6) A2[3] G1S1+ A2G2S1 combined index;

b: an ROC curve graph of the combined index of F (6) A2[3] G1+ A2G2S 1;

c: an ROC curve chart of the combined index of F (6) A2[3] G1+ A2G2+ A2G2S 1;

the AUC of the combined index is more than 0.9, which is obviously superior to that of the single index.

FIG. 8: a spectrum of sugar chains on IgG, in which characteristic peaks of alpha-1, 3 galactosylated fucose-type N-sugar chain peaks are labeled.

FIG. 9: ROC profile of serum alpha-1, 3-galactosylated fucose-type N-sugar chains F (6) A2[3] G1 of healthy humans and patients with ovarian precancerous lesions.

In the above figures:

■ ═ acetylglucosamine; sialic acid; ● ═ galactose; ● high mannose;

fucose.

Detailed Description

The aim of the method is to evaluate and predict a tested object by detecting the change of expression quantity of combined application of alpha-1, 3 galactosylated fucose type N-sugar chain and/or derivative structure sugar chain on IgG in a human blood sample (such as serum), judge whether the tested object is in a colon cancer precancerous lesion period and serve as a tumor marker for clinical early screening of colon cancer. By adopting the single sugar chain F (6) A2[3] G1 and/or the derivative thereof (F (6) A2[3] G1S1) as a detection index or combining the detection index with other available indexes (such as classical indexes CEA, A2G2, A2G2S1 and the like), the colon cancer early-stage screening and diagnosis can be effectively carried out in a targeted manner, and the application prospect is wide.

Definition of related terms

All numerical ranges provided herein are intended to expressly include all numbers falling between the endpoints of the ranges and numerical ranges therebetween. The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

As used herein, the term "α -1, 3-galactosylated fucose-type N-sugar chain" means "F (6) A2[3] G1" having the structure shown below:

as can be seen from the above formula, the galactosyl terminal modification in the sugar chain is located on the 1, 3-arm side of the sugar chain, which is distinguishable from isomers containing the galactosyl terminal modification on the 1, 6-arm side and mixtures of the two. No study prior to the present application was made on differences in the properties of the 1,3 and 1,6 isomers or combinations thereof.

As used herein, the term "F (6) A2[3] G1 derivative" or "derivative sugar chain" refers to a natural derivative having an additional sialyl sugar group attached to the terminal Gal of F (6) A2[3] G1. For example, the derivative is F (6) A2[3] G1S1 with terminal sialylation, the structure of which is shown below:

as used herein, the term "abundance" refers to the amount of N sugar chains on the IgG surface of the subject's blood. For example, the abundance of F (6) A2[3] G1 refers to the level of F (6) A2[3] G1 which is an α -1, 3-galactosylated fucose-type N-sugar chain on the IgG surface of the subject's blood. F (6) A2[3] G1S1 abundance means the level of F (6) A2[3] G1S1 which is an α -1,3 galactosylated fucose-type N-sugar chain on the IgG surface of the subject' S blood. These levels can be detected by various means known in the art.

The term "relative abundance" as used herein refers to the ratio of the abundance of F (6) A2[3] G1 or a derivative thereof to a reference, for example: the relative abundance of F (6) A2[3] G1 with respect to all sugar chains on the blood IgG surface (i.e., the ratio of the abundance of F (6) A2[3] G1 to the total sugar chain abundance), with respect to an added internal standard (e.g., a standard for sugars); the relative abundance of F (6) A2[3] G1S1 with respect to all sugar chains on the blood IgG surface (i.e., the ratio of the abundance of F (6) A2[3] G1S1 to the abundance of total sugar chains), with respect to an added internal standard (e.g., a standard for sugars); alternatively, the sum of the abundances of F (6) A2[3] G1 and F (6) A2[3] G1S1 is relative to the relative abundance of all sugar chains on the blood IgG surface (i.e., [ F (6) A2[3] G1 abundance + F (6) A2[3] G1S1 abundance ]/total sugar chain abundance), relative abundance to an added internal standard (e.g., a standard of sugars). The relative abundance subject and calculation may be adjusted as desired.

As used herein, the terms "specificity", "sensitivity", "rate of compliance" are all synonymous with the corresponding medical statistical terms. "specificity" (specificity) refers to the proportion of cases diagnosed as "non-cancer" that are negative by the kit. "sensitivity" (sensitivity) refers to the proportion of cases with pathological diagnosis "cancer" that are positive by the kit. The "coincidence rate" (acuracy) refers to the ratio of the sum of the true positive sample amount and the true negative sample amount to the total sample amount, and reflects the degree of coincidence of the detection result of the kit with the true condition of whether the subject suffers from cancer.

As used herein, the terms "threshold", "cut-off value", "cutoff value", "cut-off value" and "reference value", which are used interchangeably, refer to a criterion for determining a detection result, i.e., a cut-off value, above which a detection result is deemed positive and below which a detection result is deemed negative.

As used herein, the term "healthy person/subject" refers to a person/subject that is free of any known benign and malignant disease, normal in appearance, and free of any visible symptoms of disease.

As used herein, the term "pre-cancerous lesion" refers to a patient diagnosed as not being cancerous but suffering from a benign associated disease by pathological diagnosis (e.g., clinical biochemistry, imaging, or histopathological reporting), such as a patient diagnosed as not being colon cancer but suffering from a benign associated disease of the colon, such as inflammation, colon polyps, etc., in a colon cancer test, which is at high risk of developing cancer.

The term "colon cancer patient" as used herein refers to a patient diagnosed with malignant colon cancer as reported by histopathology. Preferably, colon cancer or a risk thereof is detected at a precancerous lesion stage, an early stage.

As used herein, the term "high risk group" or "high risk group" refers to a group with a high risk of developing a tumor, especially a group that is screened according to the conditions of age and living habits without developing a tumor, and does not use clinical criteria as a basis for classification. The probability of developing tumors in the high risk population of tumors is much higher than in the general population.

As used herein, "high risk group of colon cancer" refers to those above the age of forty who exhibit any one of the following:

1. the first-degree relatives have a history of colon cancer;

2. a history of cancer or intestinal adenoma and polyps;

3. those positive in the large intestine occult blood test;

4. two or more of the following manifestations: mucous bloody stool, chronic diarrhea, chronic constipation, and chronic appendicitis.

For the group of high risk people or the suspected colon cancer patients, further auxiliary examination should be carried out to achieve the purpose of early screening or early diagnosis. For example, a high risk population may include individuals with pre-cancerous lesions of colon cancer.

As used herein, the term "malignancy screening" refers to: the healthy population/population is periodically examined by specific detection methods to identify those with a healthy appearance and those with subclinical symptoms, and it is expected that precancerous lesions or early stage patients can be detected by further diagnostic procedures, and early intervention and/or treatment can be performed to prevent the occurrence of diseases or alleviate disability and death caused by diseases, maintain a good health level of patients, or improve prognosis and quality of life.

As used herein, the term "early diagnosis of malignancy" refers to: the diagnosis and treatment method specially for high risk tumor or early stage tumor patient aims at early detection and early intervention and early treatment to reduce pain, spirit and economic burden of the patient and to make the tumor patient recover early through early diagnosis and treatment of tumor.

Blood (including serum, plasma and whole blood) IgG and its separation and purification

In embodiments of the invention, a subject's blood sample may be collected and whole blood, serum and/or plasma may be isolated and stored using conventional methods known in the art. Tests of the invention can be performed using fresh or cryopreserved blood, serum or plasma. The blood sample may be obtained from various mammalian subjects, preferably human, monkey, dog, horse, cow, sheep, pig, mouse, rabbit, and the like. It is understood by those skilled in the art that due to the difference in composition, the abundance of sugar chains may be slightly different when the detection is performed using a plasma sample and a serum sample of a subject, respectively. Serum samples are preferred in the present invention for the purpose of facilitating quantification and comparison.

Methods for isolating IgG are known to those of ordinary skill in the art and include, but are not limited to: IgG purification column, salting out method, organic solvent precipitation method, polyethylene glycol substitution method, liquid chromatography, affinity chromatography (such as protein A affinity method, polyamide composite membrane affinity method), as long as the method does not damage IgG connected to the N-sugar chain. IgG isolation kits are commercially available, for example from Thermo Fisher Scientific.

In one embodiment of the present invention, an IgG purification column is used to separate IgG from a sample, preferably a protein a purification column, and weakly basic binding buffer and weakly acidic elution buffer are used in conjunction with the purification column, more preferably a high throughput purification column, such as a purification column that can process 96 samples simultaneously.

IgG surface N-sugar chain and its separation, purification and analysis

The N sugar chains can be isolated from IgG using methods known in the art, including but not limited to: enzymatic methods, e.g. using glycosidases, preferably the glycosidase PNGase F; chemical methods, for example, use glycoprotein hydrazinolysis reagents, such as ADM0155A hydrazinolysis kit.

After separation of the N sugar chains from the IgG, the N sugar chains can be separated and/or purified using methods known in the art, including but not limited to: porous graphitized carbon PGC solid phase extraction, polysaccharide purification column, lectin affinity method (such as continuous lectin affinity chromatography SLAC), capillary electrophoresis, high performance liquid chromatography, etc.

In one embodiment of the present invention, the N sugar chains are separated by a porous graphitized carbon PGC solid phase extraction method in which PGC is activated with acetonitrile/water/trifluoroacetic acid of 80:19.9:0.1, PGC is equilibrated with water/trifluoroacetic acid of 99.9:0.1, and sugar chains are eluted with a mixed solvent of acetonitrile/water/trifluoroacetic acid of 25:74.95:0.05 (the ratio is a volume ratio).

The abundance of F (6) A2[3] G1 and/or derivatives thereof (e.g., F (6) A2[3] G1S1) on IgG surfaces can be determined using methods known in the art, so long as the method quantifies the level. The methods include, but are not limited to: ultra high performance liquid chromatography (UPLC); liquid chromatography; liquid chromatography-mass spectrometry; nuclear magnetic resonance NMR or any combination of the above.

In the present invention, ultra-high performance liquid chromatography (UPLC) is preferably used to rapidly detect the abundance of F (6) A2[3] G1 and/or its derivatives (e.g., F (6) A2[3] G1S1) on IgG surface at high throughput. The technology has the following advantages:

1) the method realizes accurate and comprehensive detection of the dissociated N-sugar chains, and compared with Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry, the method can accurately distinguish isomers and comprehensively detect the sugar chains;

2) the method for detecting the sugar chain on the IgG has the characteristics of stability and high efficiency, the detection characteristic of high sensitivity can be achieved by a small amount of samples, the calculation and analysis are easy, the operation is simple, and the data is visual;

3) the method avoids deviation generated in the operation processes of parallel sample pretreatment and the like, reduces the error of the parallel sample entering mass spectrum detection, and ensures high repeatability and accuracy of analysis. In conclusion, the characteristics enable the index to have the characteristics of rapidness, flux and accuracy, and provide conditions for the index to be used as a potential clinical diagnosis index.

Data analysis method

In the present invention, the abundance/relative abundance of the α -1,3 galactosylated fucose-type N-sugar chain F (6) A2[3] G1 and/or its derivatives (e.g., F (6) A2[3] G1S1) is used as an index for judgment of precancerous lesion of colon cancer, early diagnosis, risk assessment, disease monitoring and/or efficacy assessment. The relative abundance can be calculated by using different calculation formulas as required, for example, the following formulas or other transformation forms such as percentage, reciprocal, logarithm, etc:

X＝B/(B+C ₁ +……+C _n )＝B/A

in the formula:

x: the relative abundance of alpha-1, 3-galactosylated fucose-type N-sugar chains F (6) A2[3] G1 and/or derivatives thereof (e.g., F (6) A2[3] G1S 1);

a: the total abundance of N sugar chains on all serum IgG surfaces;

b: abundance of alpha-1, 3-galactosylated fucose-type N-sugar chains F (6) A2[3] G1 and/or derivatives thereof (e.g., F (6) A2[3] G1S 1);

C ₁ ～C _n : except for F (6) A2[3]]G1 and/or derivatives thereof (e.g. F (6) A2[3]]G1S1) and the abundance of each of the remaining sugar chains; or

And other transformation forms such as percentage, reciprocal, logarithm and the like.

In addition to the relative abundance with respect to the total abundance of N sugar chains, other relative abundances, such as relative abundance with respect to an added internal standard (e.g., a standard of sugars), may be employed as desired and under conditions.

The abundances of sugar chains used to generate the relative abundances are preferably all derived from the same sample, the same assay, and/or the same profile. Therefore, the index can avoid deviation generated in the operation processes of pretreatment and the like of the parallel samples, thereby reducing the error of the parallel samples in mass spectrum detection, ensuring high reproducibility and accuracy of analysis, and ensuring that the method can be applied to clinical tumor screening, risk assessment, disease monitoring and/or curative effect assessment.

Based on the common knowledge in the art, a person skilled in the art can plot corresponding ROC curves according to the detection data of different object groups, for example, plot corresponding ROC curves according to the data of different groups in the embodiment of the present invention. Each ROC curve provides a series of thresholds and corresponding sensitivities and specificities, according to techniques well known in the art. Thus, using these ROC curves, one skilled in the art can easily ascertain the ability of the test to identify disease, i.e., the sensitivity, specificity and concordance rate for diagnosing the corresponding cancer species, when an arbitrary threshold (i.e., cut-off value) is chosen.

The calculation of the screening result (calculation of the threshold) depends on the specificity and sensitivity. The calculation method of the threshold includes but is not limited to:

the method comprises the following steps: and selecting the point with the maximum slope in the ROC curve, and taking the corresponding relative abundance as a threshold value.

The method 2 comprises the following steps: maximizing the value of sensitivity and specificity, [ sensitivity% - (1-specificity%)] _max The corresponding abundance is the optimal threshold (maximum joden index).

With respect to the threshold value as a boundary, a positive result is judged above the value and a negative result is judged below the value.

In the present invention, the preferable threshold value range is 0.4 to 0.8, and preferably 0.7. As will be understood by those skilled in the art, the selection of the threshold and the corresponding parameters such as sensitivity, specificity and coincidence rate will vary depending on the population to be tested, but are all included in the threshold range defined in the present invention. Those skilled in the art can select an appropriate preferred threshold from the ROC curve or threshold range disclosed in the present invention according to the actual application requirements, such as the requirements for specific tumor species, sensitivity, specificity, etc. The optimal threshold value may be determined followed by a range of + -1-15% (including any number points or subranges within the range).

F(6)A2[3]G1 and/or derivatives thereof (e.g. F (6) A2[3]]G1S1) index

The F (6) A2[3] G1 and/or derivatives thereof (such as F (6) A2[3] G1S1) index can be applied to colon cancer screening, early diagnosis, prognosis evaluation, risk evaluation, disease monitoring and/or curative effect evaluation and the like.

These applications include, but are not limited to:

1) applying F (6) A2[3] G1 and/or its derivative indicators to tumor screening test: when the abundance/relative abundance of F (6) A2[3] G1 and/or its derivatives reaches or exceeds a predetermined threshold, the subject is determined to have or be at risk of having colon cancer (e.g., having a precancerous lesion of colon cancer).

2) F (6) A2[3] G1 and/or its derivative index is applied to the judgment of health and malignancy: when the abundance/relative abundance of F (6) A2[3] G1 and/or its derivatives reaches or exceeds a predetermined threshold, the subject is determined to have or be at risk of having colon cancer.

3) Use of F (6) A2[3] G1 and/or its derivative indicators for prognostic monitoring: (ii) when the abundance/relative abundance of F (6) a2[3] G1 and/or a derivative thereof reaches or is above a predetermined threshold, then the subject is indicated that the colon cancer is or is at high risk of recurrence;

4) f (6) A2[3] G1 and/or derivatives thereof are used for high-risk group colon cancer occurrence prediction: when the abundance/relative abundance of F (6) A2[3] G1 and/or its derivatives reaches or exceeds a preset threshold, indicating that the possibility of colon cancer in the subject is higher than that in other subjects;

5) use of F (6) A2[3] G1 and/or derivatives thereof as indicators for monitoring disease in a patient with colon cancer: when the abundance/relative abundance of F (6) A2[3] G1 is higher than that of F (6) A2[3] G1 and/or the derivative thereof obtained in the previous test, reaches or is higher than a preset range (for example, 5-50 percent or any point or sub-range in the range), the condition of the colon cancer patient is developed or further worsened;

6) use of F (6) A2[3] G1 and/or its derivative markers for efficacy assessment: determining the abundance/relative abundance of F (6) A2[3] G1 and/or derivatives thereof before and after treatment or at various time points during treatment, wherein when the abundance/relative abundance of F (6) A2[3] G1 and/or derivatives thereof is higher than the abundance/relative abundance of F (6) A2[3] G1 and/or derivatives thereof obtained in the previous test, the abundance/relative abundance is within or above a predetermined range (e.g., 5% to 50% or any point or subrange within the range), the therapy and/or drug is indicated to have poor therapeutic effect.

Method and product

Based on the techniques described above, methods and products (e.g., kits, devices, and/or systems) for screening, early diagnosis, prognosis evaluation, risk evaluation, disease monitoring, and/or efficacy evaluation of colon cancer are provided.

The method of the invention comprises the following steps:

(A') determining the abundance of IgG surface F (6) A2[3] G1 and/or derivatives thereof in a subject blood sample;

(B') optionally, calculating the relative abundance of F (6) A2[3] G1 and/or its derivatives in total N sugar chains on the IgG surface in the blood sample;

(C') determining whether the subject suffers from colon cancer, performing a prognostic assessment and/or risk assessment of suffering from colon cancer, monitoring the disease state and/or assessing the efficacy of a treatment based on the abundance/relative abundance ratio of F (6) A2[3] G1 and/or derivatives thereof.

The threshold value in the method of the invention can be in the range of 0.4-0.8, preferably 0.7. The threshold ranges may be adjusted from population to population, for example, based on the point of maximum slope in the ROC curve or the value that maximizes sensitivity and specificity.

Furthermore, the method of the present invention may optionally comprise one or more steps selected from the group consisting of:

(a') collecting and/or processing a subject blood sample;

(b') isolating and/or purifying serum and/or plasma;

(c') isolating and/or purifying serum/plasma IgG;

(d') isolating, purifying and/or enriching serum IgG surface N-sugar chains;

(e') storing and/or processing the abundance of F (6) A2[3] G1 and/or its derivatives and/or the relative abundance of the abundance to the total N sugar chain abundance on the IgG surface of the subject serum;

(f') providing a decision threshold;

(G') comparing the abundance or relative abundance of F (6) A2[3] G1 and/or derivatives thereof in the subject to a control to determine whether the subject is suffering from colon cancer, to monitor the subject for disease, and/or to assess the efficacy of the treatment;

(h') providing a diagnosis and/or test result and/or report.

The method of the invention is preferably a high throughput detection method, for example 96, 192, 288, 384, 480, 576 samples can be processed simultaneously.

Accordingly, the invention also provides products for colon cancer screening, early diagnosis, prognosis evaluation, risk evaluation, disease monitoring and/or efficacy evaluation. The product may be, for example, a kit, device, system, and/or combination thereof. Applications of the products and methods of the present application include diagnostic or prescreening as well as aids in existing diagnostic or prescreening products and methods. The products and methods of the present application can also be used to differentiate between precancerous variant types, such as distinguishing between a colon cancer precancerous lesion and an ovarian cancer precancerous lesion.

Joint detection

Also provided herein are combinations of indices of IgG surface F (6) A2[3] G1 and/or derivatives thereof in a subject blood sample with other indices (e.g., sugar chain indices, classical indices) and uses thereof. For example, kits, devices, systems and/or combinations thereof for detecting other colon cancer markers are also included in the products of the present application. For example, the index is selected from one or more of the following sugar chain markers:

carcinoembryonic antigen (CEA),

The combined detection of the indicators may have the effect of improving the sensitivity and/or specificity and/or compliance rate etc. of the product/method. Preferably the effect is a synergistic effect.

Exemplary embodiments

In some embodiments of the present application, the sugar chain index may be determined or used using a method including one or more steps such as:

(A) human serum IgG enrichment and purification

The method may be carried out, for example, by:

1)70 μ L of serum was mixed with 100 μ L of 1 XPBS and incubated with protein G column for 45 min;

2) adding 400 μ L of 1 × PBS, centrifuging for 2 min on a 500-rotation centrifuge for washing off impurities, and repeating the operation for three times;

3) mu.L of Tris-HCl (7.5. mu.L) was added to the new collection plate to equilibrate the pH, and 200. mu.L of glycine was used to elute IgG adsorbed on the column to the plate, resulting in total IgG in the serum.

(B) Dissociation, derivatization and enrichment of N-sugar chains on IgG

This can be carried out, for example, by a method comprising the following steps:

1) to the obtained IgG was added 6. mu.L of PNGase F, and incubated overnight at 37 ℃;

2) loading a proper amount of porous graphitized carbon into a 96-well plate containing a PVDF membrane, and simultaneously preparing an aqueous solution A of 80% acetonitrile containing 0.1% TFA, an aqueous solution B of 0.1% TFA and an aqueous solution C of 25% acetonitrile containing 0.05% TFA;

3) sequentially rinsing the 96-well plate by using the solution A and the solution B, and repeatedly loading an overnight IgG sample for three times and centrifuging;

4) washing with 100 μ L water twice, and eluting sugar chain sample with 50 μ L C solution in clean 96-well plate to obtain high purity IgG N-sugar chain;

5) spin-drying the sugar chain sample, adding 3 μ L2-AB derivatization reagent, and reacting at 60 deg.C in dark for 2 hours;

6) the reaction was terminated by adding 50. mu.L of water, and a sugar chain liquid sample was collected through a PVDF membrane.

(C) The sugar chain sample is analyzed by an analyzing instrument or system,

this can be done using a method comprising, for example, the following instruments and/or steps:

ultra Performance Liquid Chromatography (UPLC) is used; or matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS); or electrospray mass spectrometry ESI-MS; or a sugar chip technology detection method; wherein, the method of ultra-high performance liquid chromatography is preferably used for detecting glycosylation modification;

(D) the relative expression level of the alpha-1, 3 galactosylated fucose-type N-sugar chain was calculated and analyzed.

1) the relative expression amount of the α -1,3 galactosylated fucose-type N-sugar chain is X: summing the peak areas of all sugar chain peak signals detected in the liquid chromatogram on the corresponding software to obtain A, wherein the peak area of the alpha-1, 3 galactosylated fucose type N-sugar chainB, the remaining peak signal areas of the sugar chains detected are C in order ₁ ～C _n

X＝B/(B+C ₁ +.......C _n )＝B/A；

2) Carrying out ROC analysis on the relative expression quantity of the alpha-1, 3 galactosylated fucose type N-sugar chain between a healthy group and a precancerous lesion group by using MedCalc software to obtain the prediction probability of the index;

3) AUC values in 2) were obtained.

4) And obtaining other structural sugar chain index AUC values in the same calculation mode, and analyzing the diagnosis model of the combined index by using a binary logistic regression model.

(E) Combining alpha-1, 3 galactosylated fucose-type N-sugar chain with other sugar chain index or classical index (such as CEA)

The diagnosis model of the combined index can be analyzed by adopting an ROC curve and combining a binary logistic regression model to predict the precancerous lesion.

The technical scheme of the application has the advantages

Advantages of the present application include, but are not limited to:

1. compared with the classic CEA index, the CEA has a larger excellent effect. The noninvasive marker can be used for efficiently, sensitively and specifically detecting the patient with the precancerous lesion of the colon cancer or the early canceration, is favorable for clinical early intervention, and greatly improves the survival rate of the patient.

2. The indicators of the present application can be directed to normal and pre-cancerous (as distinguished from normal and neoplastic) groups, which are in a pre-stage relative to the neoplastic group and do not develop into mature tumors (e.g., stage I tumors).

3. Data in the application show that the new index has more remarkable discrimination compared with the known index, and has certain advantages. The combination of the new index and several other sugar chain structures or the combination of the existing mature marker has good effect in early prediction of colon cancer, and greatly improves the early screening capability of clinical colon cancer.

4. In addition to early screening capabilities, the indicators or combination of indicators of the present application can be effectively used in the diagnosis of colon cancer, as well as in the judgment of colon cancer development.

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications, alterations and adaptations to the present invention, and such modifications and alterations are intended to be within the scope of the present invention.

The experimental methods in the following examples, in which specific conditions are not specified, may be performed by a conventional method in the art or according to the conditions suggested by the supplier. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 serum IgG enrichment purification

A. Sample source

Blood samples from healthy persons (without any known benign or malignant disease, normal appearance, no visible disease symptoms) and from patients with pre-colon cancer (specifically pre-cancerous), and from patients with colon cancer (specifically pathologically diagnosed with colon cancer) were obtained from hospitals such as third-class A and outpatients (east Hospital, Shanghai). Wherein, the patients with pre-colon cancer lesions meet the following characteristics: tumor diameter >1cm, with severe high grade neoplasia, tubular villous, sessile serrated or traditional serrated tissue or adenomas of any size over 3.

B. Sample preservation

Collecting serum sample of patient to be detected, storing at-80 deg.C for use if necessary, and repeatedly freezing and thawing for no more than three times.

C. Detection operation

Both serum detection and data processing were performed under double-blind conditions.

D. Reagent and method for enriching and purifying human serum IgG

1. mu.L of serum and 100. mu.L of 1 XPBS were mixed and incubated for 45 minutes at room temperature with a protein G column (from Bestchrom, AA104307, diluted 1: 1 with 20% ethanol);

2. adding 400 μ L of 1 × PBS, centrifuging for 2 min on 500-rotation centrifuge, removing impurities, and repeating for three times;

3. to the new collection plate, 7.5. mu.L of Tris-HCl was added to balance the pH (pH 9.0), and IgG adsorbed on the column was eluted to the plate with 200. mu.L of 0.1M glycine aqueous solution to obtain total IgG in serum.

Example 2 dissociation, derivatization and enrichment of N-sugar chains on IgG

Sample origin, classification and preservation are as described in example 1. Both serum detection and data processing were performed under double-blind conditions.

Reagents and methods related to dissociation, derivatization, and enrichment of N-sugar chains on IgG are as follows:

1. to the IgG obtained in example 1, 6. mu.L of PNGase F (500U/mL) enzyme solution (purchased from New England Biolabs, P0709S) was added and incubated overnight at 37 ℃;

2. loading a proper amount of porous graphitized carbon into a 96-well plate containing a PVDF membrane, and preparing a solution A (80% acetonitrile aqueous solution containing 0.1% TFA), a solution B (0.1% TFA aqueous solution) and a solution C (25% acetonitrile aqueous solution containing 0.05% TFA);

3. sequentially rinsing the 96-well plate with A, B solution, repeatedly loading the overnight IgG sample in the step 1 for three times, and centrifuging;

4. rinsing the centrifugal plate twice with 100. mu.L of deionized water, and then eluting the sugar chain sample with 50. mu. L C solution in a clean 96-well plate to obtain high-purity N-sugar chains on IgG;

5. spin-drying all sugar chain samples, adding 3 μ L2-aminobenzamide (2-AB) solution derivatization reagent (50mg 2-AB +60mg NaBNCH) ₃ Dissolving in 1mL DMSO and AcOH), and reacting at 60 ℃ in a dark place for 2 hours;

6. the reaction was terminated by adding 50. mu.L of water, and a sugar chain liquid sample was collected through a PVDF membrane.

Example 3 analysis of IgG surface sugar chains and calculation of relative expression amount

Analysis of sugar chains on IgG surface

The analysis of the sugar chains on the surface of IgG can be performed by ultra high performance liquid chromatography (UPLC); or matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-MS; or electrospray mass spectrometry ESI-MS; or a sugar chip technology detection method; among them, the ultra high performance liquid chromatography is preferable for detecting the glycosylation modification.

In the embodiment, an Shimadzu ultra-high performance liquid chromatography (UPLC) analysis is adopted, and the specific operation steps are as follows:

the sugar chain sample purified as described in example 2 was transferred to a liquid phase injection vial and analyzed using a Nexera UPLC LC-30A high performance liquid chromatograph (Shimadzu), which was controlled by Labsolution software.

1) Flow phase preparation conditions:

mobile phase a ═ 100mM ammonium formate: mixing 3.75mL formic acid solution with 800mL water, adding ammonia water to adjust pH to 4.5 with error of 0.05 under the action of magnetic stirrer, and adding H ₂ O is added to the volume of 1L, mixed evenly and filtered by a filter membrane of 0.22 mu m and transferred into a mobile phase bottle;

mobile phase B100% CAN (in another mobile phase bottle).

2) Setting the liquid chromatographic analysis conditions: the fluorescence detector excitation wavelength was 330nm, the emission wavelength was 420 nm; amino chromatographic column (BEH Amide columns 2.1X 150mm,1.7 μm), column temperature set at 70 deg.C, sample injection volume of 2 μ L, flow rate of 0.4mL min ^-1 (ii) a Continuous sample injection was automated and took 46min per sample.

FIG. 8 shows a spectrum of sugar chains on IgG, in which characteristic peaks of alpha-1, 3-galactosylated fucose-type N-sugar chain peaks are labeled.

3) Spectral peak assignment and data processing

And (3) carrying out chromatogram preprocessing and signal extraction by using Labsolution software according to the obtained liquid chromatogram data, and deriving the area value of the peak signal to be processed into Excel. And exporting the peak areas into an Excel document, and calculating the relative abundance of the single peak by adopting a normalization method, wherein the relative abundance algorithm of the single peak is each peak area/the total area of all peaks.

B. Calculation of relative expression amount of alpha-1, 3-galactosylated fucose-type N-sugar chain

The relative expression amount of the α -1,3 galactosylated fucose-type N-sugar chain was calculated by the following formula:

X＝B/(B+C ₁ +……+C _n )＝B/A

in the formula:

x: relative expression amount of alpha-1, 3 galactosylated fucose-type N-sugar chain;

a: the total peak area of all sugar chain peak signals detected in the liquid chromatogram on the corresponding software;

b: peak area of alpha-1, 3 galactosylated fucose type N-sugar chain;

C ₁ ～C _n : the peak signal area of each sugar chain was detected for the other non- α -1,3 galactosylated fucose type N-sugar chains.

The relative expression of the derivative F (6) A2[3] G1S1 was calculated using a similar formula.

Example 4 comparison of the abundance of alpha-1, 3 galactosylated fucose-type N-sugar chains in serum

The sugar chain abundance analysis and comparison of alpha-1, 3 galactosylated fucose type N-sugar chains on serum IgG of healthy people, patients with pre-colon cancer pathological changes and patients with colon cancer malignant tumors show that the index has excellent resolving power on samples and early screening capability.

The specific research method is as follows: the data of three groups of samples were obtained by the data processing and analysis method described in example 3, and statistical analysis and mapping were performed by GraphPad prism 6 software, N-sugar chain difference comparison was performed by Student's t test, P <0.05 was a criterion for judging statistical significance, and the results shown in fig. 1 were obtained.

The results show that: it was found that the abundance of α -1,3 galactosylated fucose-type N-sugar chains was significantly increased in the precancerous lesion group and the colon cancer group (P <0.0001) compared to the healthy group, and that a significant increase was already exhibited in the precancerous lesion period (P <0.0001), and therefore, this index could be used as an index for early screening of colon cancer.

After the sugar chain abundance in the tumor such as ovarian cancer and the canceration lesion sample thereof is tested, the abundance of the alpha-1, 3 galactosyl fucose type N-sugar chain is specifically improved in the colon cancer and the precancerosis thereof. The results suggest that the index has certain specificity for judging colon cancer and precancerous lesions thereof.

Example 5 Effect of alpha-1, 3-galactosylated fucose type N-sugar chain F (6) A2[3] G1 on early screening and diagnosis of colon cancer

1. Serum samples from the subjects to be tested were collected, stored at-80 ℃ if necessary, and freeze-thawed repeatedly no more than three times as described in example 1.

2. The serum glycosylation modification was examined according to the examination procedures described in the examples, and the relative expression level of the alpha-1, 3 galactosylated fucose type N-sugar chain was calculated, and the prediction probability was obtained by using MedCalc software.

3. Analysis gave a jotan index of 0.7.

4. When the john index is 0.7, performing ROC analysis according to the object condition; the specificity of alpha-1, 3 galactosylated fucose N-sugar chain F (6) A2[3] G1 in early diagnosis of colon cancer is 100%, the sensitivity is 70.2%, and the AUC is 0.8359 (as shown in FIG. 2), which can effectively distinguish precancerous lesion groups.

5. The data show that the AUC of healthy and tumor patients is 0.9135, which has excellent differentiation effect, suggesting that α -1,3 galactosylated fucose-type N-sugar chain F (6) a2[3] G1 also has excellent potential in tumor diagnosis.

6. The samples were tested using the methods and criteria of CN 105277718A ("150046") and ROC curves were plotted for comparison with the criteria of the present application (results are shown in fig. 4).

The results showed the Gal ratio index in 150046 (Gal ratio ═ Gal) ₀ /(Gal ₁ +Gal ₂ )，Gal ₀ 、Gal ₁ And Gal ₂ Gal-containing levels of dual-antenna complex N-sugar chains without galactose linkage, with mono-galactose linkage and with digalactose linkage, respectively, at the IgG surface terminals) ₁ However Gal ₁ Is a mixture of isomers of alpha-1, 3 and alpha-1, 6 galactosylated fucose-type N-sugar chains and appears as a constituent in the Gal index, i.e., not as a separate index; moreover, the effect of the Gal ratio index on judging the precancerous lesion of the colon cancer is obviously inferior to that of F (6) A2[3] of the application]G1 (only α -1,3 isomer).

The results indicate that the index of the present application is significantly different from the index of 150046, and that a significantly excellent effect is produced in the judgment of precancerous lesions of colon cancer.

Example 6 Effect of analogs of alpha-1, 3-galactosylated fucose type N-sugar chain F (6) A2[3] G1 on early screening of colon cancer

In order to develop more markers useful for early screening of colon cancer, the role of analogs of alpha-1, 3 galactosylated fucose-type N-sugar chain F (6) A2[3] G1 in early screening of colon cancer was studied. The analogues studied included: f (6) A2[3] G1S1, A2G2 and A2G2S1 shown in FIG. 5 and GP1, GP6, GP8, GP13, GP15 and GP22 shown in FIG. 6.

The method described in example 5 was used to test the effect of each of the analogue indices in colon cancer early screening to obtain an ROC curve. The results are shown in FIG. 6.

The results show that: sialylated sugar chains F (6) a2[3] G1S1 of F (6) a2[3] G1, which are naturally downstream-derived on the basis of the index α -1, 3-galactosylated fucose-type N-sugar chains F (6) a2[3] G1, have significant diagnostic efficacy, and can effectively distinguish between the healthy group and the precancerous group (AUC 0.834 in ROC curve); the other two nonfucosylated sugar chains A2G2 and A2G2S1 also have good screening effects, and the AUC values are both more than 0.800. However, not all analogs have the same screening effect, GP1, GP6, GP8, GP13, GP15 and GP22 do not show excellent effects similar to F (6) A2[3] G1, F (6) A2[3] G1S1, A2G2 and A2G2S 1.

Example 7 Effect of a combination of alpha-1, 3-galactosylated fucose-type N-sugar chains F (6) A2[3] G1 and the like on early screening of colon cancer

The F (6) A2[3] G1, H5N4F0, F (6) A2[3] G1S1 and H5N4F0S1 sugar chains, which were demonstrated to have excellent screening effects in examples 5 and 6, were randomly combined, each of which included the F (6) A2[3] G1 sugar chain, to give different combinations of joint index diagnoses.

The AUC value of each sugar chain index was obtained by the calculation method described in the previous example, and the diagnostic model of the combined index was analyzed using a binary logistic regression model. The results are shown in FIG. 7.

The results show that: AUC of each combined index exceeds 0.9, so that the excellent early screening purpose is achieved, and a healthy group and a precancerous lesion group can be obviously distinguished.

Example 8 Effect of a combination of alpha-1, 3-galactosylated fucose type N-sugar chains F (6) A2[3] G1 and the classical index CEA in early screening of colon cancer

After blood samples of 51 healthy groups and 37 colon cancer precancerous lesion groups were obtained, clinical case-related data were reviewed to obtain the value of CEA in each sample, and ROC graphs were prepared using GraphPad prism 6 software (fig. 3A). The results show that: AUC 0.467. It can be seen that the classical index CEA has poor ability of distinguishing early colon cancer lesions.

The combination of F (6) A2[3] G1, which has been proved to have excellent screening effect, and the classical index CEA is used for early screening diagnosis of colon cancer. The AUC value of each sugar chain index was obtained by the calculation method described in the previous example, and the diagnostic model of the combined index was analyzed using a binary logistic regression model. The results are shown in FIG. 3B.

The results show that: the AUC of the combined index is 0.938, has good distinguishing effect, is far better than the diagnosis efficiency of single CEA, and can be effectively used for early screening of colon cancer.

Example 9 detection of serum alpha-1, 3-galactosylated fucose type N-sugar chains F (6) A2[3] G1 in healthy humans and patients with ovarian precancerous lesions

The method in the foregoing examples was used to perform sugar chain detection on blood samples of healthy persons (subjects without any known benign and malignant diseases, normal appearance, and any visible disease symptoms), and blood samples of patients with ovarian precancerous lesions (patients with pre-malignant lesions) both from hospitals such as Tertiary A and outpatients, Shanghai Oriental Hospital), to obtain the relevant ROC curves.

FIG. 9 is a ROC graph showing the results of the serum α -1, 3-galactosylated fucose-type N-sugar chains F (6) A2[3] G1 of healthy persons and patients with ovarian precancerous lesions. The results show that this index does not show a differentiating effect in patients with ovarian precancerous lesions.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A product (e.g., kit, device, operable system, and/or combination thereof) for colon cancer screening, colon cancer diagnosis, and/or colon cancer condition progression judgment in a subject, the product comprising:

the structure of F (6) A2[3] G1 is shown as follows:

(C) optionally, means and/or a processor for determining whether the subject suffers from colon cancer based on the abundance of F (6) A2[3] G1 and/or derivatives thereof;

preferably, the blood is selected from: serum, plasma and whole blood, more preferably serum.

2. The product of claim 1, further comprising one or more selected from the group consisting of:

for example, a reagent and/or an apparatus for a method of separating and purifying an N sugar chain selected from the group consisting of: solid phase extraction of porous graphitized carbon PGC (preferably, PGC is activated with acetonitrile/water trifluoroacetic acid (80: 19.9: 0.1), PGC is equilibrated with water/trifluoroacetic acid (99.9: 0.1), and sugar chain is eluted with a mixed solvent of acetonitrile/water trifluoroacetic acid (25: 74.95: 0.05), polysaccharide purification column, lectin affinity method (e.g., SLAC), capillary electrophoresis, and high performance liquid chromatography;

f) means and/or a processor for providing a decision threshold;

j) instructions or instructions for use in which one or more of the following applications and decisions are described:

(iii) colon carcinogenesis anticipation for a subject population (e.g., a high risk population): when the abundance of F (6) A2[3] G1 and/or derivatives thereof reaches or exceeds a predetermined threshold, indicating that the subject is more likely to develop colon cancer than the other subjects;

(v) for efficacy assessment: determining and calculating the abundance of F (6) A2[3] G1 and/or derivatives thereof at various time points before and after or during treatment, wherein a value of F (6) A2[3] G1 and/or derivatives thereof that is higher than the corresponding abundance value from the previous test by or above a predetermined range (e.g., 5% to 50% or any point or subrange within the range) indicates poor therapeutic efficacy of the therapy and/or drug;

preferably, the threshold value in (i) to (v) is 0.4 to 0.8, more preferably 0.5 to 0.7;

preferably, the optimal threshold value may be determined based on the point of maximum slope or the value that maximizes sensitivity and specificity in the ROC curve, and the preferred threshold value range may be the range of + -1-15% (including any point or subrange of values within this range) of the optimal threshold value.

3. A product according to claim 1 or claim 2 wherein the abundance of F (6) A2[3] G1 and/or derivatives thereof is relative to a reference, for example F (6) A2[3] G1 and/or derivatives thereof, relative to all sugar chains on a blood IgG surface or relative to an added internal standard (e.g. a sugar standard);

for example, the relative abundance of F (6) A2[3] G1 and/or derivatives thereof is calculated using a formula selected from the group consisting of:

X＝B/(B+C ₁ +……+C _n )＝B/A

in the formula:

x: the relative abundance of the alpha-1, 3-galactosylated fucose-type N-sugar chain F (6) A2[3] G1 or derivatives thereof;

a: total abundance of N sugar chains on all serum IgG surfaces;

And other transformation forms such as percentage, reciprocal and logarithm.

4. The product of claim 1, wherein the early screening is selected from the group consisting of: screening precancerous lesion and diagnosing early cancer.

5. The product of claim 1, wherein the product is used in a test method comprising the steps of:

(B') calculating the relative abundance (e.g., the abundance ratio of all sugar chains, or the abundance ratio with respect to an internal standard) of F (6) A2[3] G1 and/or a derivative thereof;

6. The product of claim 5, wherein the method further comprises one or more steps selected from the group consisting of:

(a') collecting and/or processing a subject blood sample;

(b') isolating and/or purifying serum and/or plasma;

(c') isolating and/or purifying serum/plasma IgG;

(d') isolating, purifying and/or enriching serum IgG surface N-sugar chains;

(f') providing a decision threshold;

(G ') comparing the abundance or relative abundance of F (6) A2[3] G1 and/or derivatives thereof in the subject to a control to determine whether the subject has colon cancer, to assess the subject's risk of having colon cancer, to monitor the condition of the subject, and/or to assess the efficacy of the subject;

(h') providing a diagnosis and/or test result and/or report;

(j') detecting other colon cancer markers and combining the detection of said other colon cancer markers with the abundance detection of F (6) A2[3] G1.

7. The product of claim 1, further comprising a kit, device, system and/or combination thereof for detecting other colon cancer markers,

for example, the indicator is selected from one or more of the following:

(i) carcinoembryonic antigen (CEA);

(ii) the IgG surface dual-antenna galactosylated nonfucose type N-sugar chain A2G2 has the structure shown below:

(iii) an IgG surface dual-antenna galactosylated nonfucose type N-sugar chain derivative A2G2S1 having the structure shown below, wherein sialyl group is linked to any of the galactose groups through α -2,6 bond:

for another example, the indicator is selected from: basic condition of the patient, such as age, sex, family history; the clinical manifestations of colon cancer, including local manifestations such as lumps and secondary symptoms, occult blood in stool, pathological secretion, and systemic manifestations such as fever, infection, anemia, emaciation, hypodynamia, dyscrasia; specific physicochemical examination indexes including specific image examination such as nuclear magnetism, CT, B-ultrasound, and endoscope; biochemical markers of tumor such as AFP, PSA, SF, TSGF, POA, PROGRP, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, NSE, SCC, AFU, EBV-VCA, etc.

8. Use of a substance (e.g. reagent, instrument, module and/or processor) for detecting abundance of a-1, 3 galactosylated fucose-type N-sugar chain F (6) a2[3] G1 and/or derivatives thereof on IgG surface of blood in the manufacture of a product (e.g. kit, device, system and/or combination thereof) for early diagnosis, risk assessment, disease monitoring and/or efficacy assessment of colon cancer in a subject, the substance comprising one or more selected from the group consisting of:

(A) a substance (e.g., a reagent, an instrument, a module, and/or a processor) for determining the abundance of F (6) A2[3] G1 and/or its derivatives on an IgG surface of blood;

for example, a reagent, an instrument, a module and/or a processor for use in one or more methods selected from the group consisting of: ultra Performance Liquid Chromatography (UPLC); matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI), fast atom bombardment mass spectrometry (FAB-MS) and electrospray mass spectrometry (ES-MS); liquid chromatography; liquid chromatography-mass spectrometry; sugar chip technology; nuclear magnetic resonance NMR, preferably Ultra Performance Liquid Chromatography (UPLC);

9. Use according to claim 8, wherein the product is as defined in any one of claims 1 to 7.

10. A method for drug and/or therapy screening, the method comprising:

(A') determining the abundance of IgG surface alpha-1, 3 galactosylated fucose-type N-sugar chains F (6) A2[3] G1 and/or derivatives thereof (e.g., F (6) A2[3] G1S1) in the blood sample at different time points before, after and/or during administration and/or treatment of the subject;

(C ") assessing the effectiveness of the drug and/or therapeutic method based on the relative abundance of F (6) a2[3] G1 and/or derivatives thereof, wherein poor treatment of the drug and/or therapeutic method is indicated when the relative abundance of F (6) a2[3] G1 and/or derivatives thereof is at or above a predetermined range (e.g., 5% to 50% or any point or subrange within the range) above the corresponding relative abundance of the previous test; otherwise, the medicine and/or the treatment method are indicated to have good curative effect.