CN115184611A

CN115184611A - Endometrial cancer stratification related marker and application thereof

Info

Publication number: CN115184611A
Application number: CN202210917174.9A
Authority: CN
Inventors: 张泽建; 王瑾晖; 曹桢
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2022-08-01
Filing date: 2022-08-01
Publication date: 2022-10-14
Anticipated expiration: 2042-08-01
Also published as: CN115184611B

Abstract

The invention belongs to the fields of biomedicine and medical detection, and particularly relates to a marker related to endometrial cancer patient stratification and application thereof. In particular, the use of THy and/or TC to differentiate patients with highly and poorly differentiated endometrial cancer is provided, together with methods and related products for differentiating patients with highly or poorly differentiated endometrial cancer using THy and/or TC.

Description

Endometrial cancer stratification related marker and application thereof

Technical Field

The invention belongs to the fields of biomedicine and medical detection, and particularly relates to a endometrial cancer layering related marker and application thereof.

Background

In recent years, along with the great change of living habits and dietary structures of people, the endometrial cancer also shows the trend of increasing morbidity and gradually younger sick people, is one of three malignant tumors of a female reproductive system, and seriously threatens the physical and mental health of women. The traditional view points that the prognosis of endometrial cancer is good and the cure rate is high. However, statistics over the past 20 years show that 13-17% of patients with endometrial cancer still have recurrence after surgery and the window phase is usually within three years of surgery, with nearly half of patients with recurrence showing distant metastasis, with a 2-fold increase in mortality. In summary, endometrial cancer usually presents symptoms early, progresses slowly, and has a better prognosis; but once the disease recurs, the treatment is difficult and the death rate is high; for patients with endometrial cancer, the pathological stratification should be fully emphasized, and a treatment scheme is comprehensively specified by combining the age, fertility requirements, physical conditions and other factors of the patients so as to improve the survival rate of the patients.

There are no conventional biomarkers of endometrial cancer with the aim of stratification in the clinic, and the biomarkers for screening diagnosis of endometrial cancer that have been evaluated include: tumor markers such as CA125 and HE4, (2) individual proteins and proteomes, (3) enzymes, (4) metabolites, and (5) miRNAs. Most of these candidate biomarkers, which have been identified as single molecules or groups of molecules, are currently in the first stage of biomarker development.

Glycosylation is one of the most common and important modifications of proteins post-translationally. The sugar chain of glycoprotein is involved in many key physiological and pathological processes such as canceration process, cancer progression and cancer metastasis, abnormal glycosylation is considered as one of cancer markers, and the glycoprotein secreted from cancer cells can reflect the mechanism of glycosylation change of cancer cells. In addition, systemic changes in cancer also cause changes in the glycosylation of immunoglobulins secreted by plasma cells and acute phase proteins synthesized by the liver and glycoprotein function. Sugar chains are potential biomarkers in the blood circulation of cancer patients associated with systemic disorders. In view of this, the analysis of full serum glycoprotein glycosylation of endometrial cancer patients with high and low differentiation provides an important way for finding a layered marker with high accuracy and good specificity.

Disclosure of Invention

The total serum N-sugar spectrum analysis is carried out on endometrial cancer patients with high differentiation and low differentiation to obtain derivative sugar chain characteristics THy and TC which have remarkable difference among the endometrial cancer patients with high differentiation and low differentiation, the AUC values of the patients with high differentiation and low differentiation are respectively greater than 0.8, the THy and/or TC can show better layering effect, and the derivative sugar chain characteristics THy and TC can be used as a new serum biomarker for distinguishing the endometrial cancer patients with high differentiation and low differentiation.

Also provided are methods and related products for differentiating patients with highly or poorly differentiated endometrial cancer using THy and/or TC.

The specific technical scheme is as follows:

layered applications

In one aspect, the invention provides the use of a reagent and/or apparatus for the detection of THy and/or TC in the manufacture of a product for the treatment of patients with high and low differentiation of endometrial cancer.

The measurement/calculation methods of THy, TC as used in the present invention are well known in the art and may be measured/calculated by any measurement method commonly used in the art without limitation. The THy refers to the total heterozygote sugar chain, and the numerical value of the THy is obtained by calculating the ratio of the relative expression quantity of all heterozygote sugar chains in the relative expression quantity of all heterozygote sugar chains after measuring the relative expression quantity of all heterozygote sugar chains; the TC is a value obtained by measuring the relative expression level of all the glycoforms and calculating the ratio of the relative expression level of all the complex carbohydrate chains to the relative expression level of all the glycoforms. More specifically, the ways of measuring THy and TC are as described in the specific embodiments of the present invention.

As used in the present invention, the calculation formula for THy is:

As used in the present invention, the calculation formula for TC is:

In the above formula, hy = heterozygote; t = in all glycoforms; c = complex glycoform; f = deoxyribose (fucose); e = α 2,6-linked sialic acid; l = α 2,3-linked sialic acid; h = hexose (mannose or galactose); n = N-acetylglucosamine (N-GlcNAc).

The above calculation may be calculated by software such as Rstudio, flexAnalysis, massytols, glycoWorkbench, etc., or manually.

The "sugar type (e.g., the above H5N3, H5N3F1, H6N3, H5N3L1, etc.)" referred to herein means the content percentage thereof, which is calculated by the ratio of the peak area of the single sugar chain to the peak areas of all the sugar chains detected, also referred to as a relative expression amount.

As used herein, the differentiation of high and low differentiation of endometrial cancer is determined by post-operative pathology.

Preferably, the reagent for detecting THy and/or TC includes, but is not limited to, any one or more of the following: sugar chain releasing reagent, N-sugar chain derivatization reagent, enrichment and purification reagent, and reagent used in mass spectrometry.

Preferably, the sugar chain-releasing agent includes an agent used in the following method: enzymatic methods, for example with endoglycosidases; chemical methods, e.g., using beta elimination reactions, glycoprotein hydrazinolysis reagents; a combination of enzymatic and chemical methods may also be used to release the sugar chains.

Preferably, the endoglycosidase comprises EndoS2, PNGase F (N-carbohydrase F or peptide N-glycosidase F).

Preferably, the endoglycosidase is PNGase F.

Preferably, the mass spectrometry method comprises: matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), matrix-assisted laser desorption ionization-quaternary ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS)), fast atom bombardment mass spectrometry (FAB-MS), electrospray mass spectrometry (ESI-MS), multi-stage mass spectrometry, high Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry combined method (LC-MS).

Preferably, the apparatus for detecting THy and/or TC includes, but is not limited to, a mass spectrometer.

Preferably, the mass spectrometer generally comprises an ion source, an analyzer and a collector; in particular, the mass spectrometer typically includes a sample introduction system, an ion source, a mass analyzer, an ion detector, and a data acquisition and control system, among others.

Preferably, the THy and/or TC are detected on a sample from a subject, including, but not limited to, blood (whole blood, whole serum or plasma), interstitial fluid, cells, tissue, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

Preferably, the sample is whole serum.

In one embodiment of the invention, a Cotton HILIC SPE separation column is used for enriching and purifying the N-sugar chains, wherein water is used for activating the separation column, and the following steps are adopted: acetonitrile =15:85 The separation column was equilibrated with the solution (volume ratio), and the sugar chains were eluted with pure water.

Method

In another aspect, the invention provides a method of differentiating patients with high and low endometrial cancer, said method comprising the step of determining endometrial cancer stratification in a subject based on the value of THy and/or TC in said subject.

Preferably, the steps include:

1) A sample of the subject is collected and,

2) The value of THy and/or TC is measured,

3) And judging endometrial cancer stratification.

Preferably, the sample includes, but is not limited to, blood (whole blood, whole serum or plasma), interstitial fluid, cells, tissue, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

Preferably, the sample is whole serum.

Product(s)

In another aspect, the present invention provides a machine-readable storage medium for differentiating patients with endometrial cancer high differentiation and low differentiation, the machine-readable storage medium having stored thereon computer instructions, which when executed, perform the following:

and (d) determining endometrial cancer stratification in the subject based on the value of THy and/or TC in the subject.

Preferably, the computer instructions when executed perform the following:

1) A sample of the subject is collected and,

2) A value for THy and/or TC is obtained,

3) And judging endometrial cancer stratification.

Preferably, the method used to measure the value of THy and/or TC comprises: matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), matrix-assisted laser desorption ionization-quaternary ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS)), fast atom bombardment mass spectrometry (FAB-MS), electrospray mass spectrometry (ESI-MS), multi-stage mass spectrometry, high Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry combined method (LC-MS).

In another aspect, the invention provides a system for differentiating between highly and poorly differentiated patients with endometrial cancer, the system comprising a computing device for determining the stratification of endometrial cancer in a subject based on the value of THy and/or TC in the subject.

Preferably, any one or more of the following devices can also be arranged in the system:

(1) A collection device for collecting and processing a patient sample;

(2) A detection device for mass spectrometry detection;

(3) A data processing device for calculating THy and/or TC value according to the detection result;

(4) An output device for outputting the endometrial cancer stratification of the subject.

Preferably, the calculation may be performed by any one or more of Rstudio, flexAnalysis, massyTools, glycoWorkbech software.

In another aspect, the invention provides a device for differentiating patients with endometrial cancer high and low differentiation, comprising:

at least one processor;

at least one memory for storing at least one program;

when the at least one program is executed by the at least one processor, the at least one processor may be configured to perform the methods of the present invention for patients with high and low differentiation of endometrial cancer.

Implementation of the "methods, systems, apparatus" described herein may include performing or completing selected tasks manually, automatically, or a combination thereof.

The layered marker (the hierarchical marker) for judging the endometrial cancer layering of the subject provides a new way for detection or risk assessment of endometrial cancer layering, can be used for confirmation of endometrial cancer layering, and avoids delay of confirmation or treatment of high-differentiation endometrial cancer depending on conventional detection of symptoms, imaging and the like. Other major advantages of the present application include:

(a) The biomarker is used for judging endometrial cancer stratification of a subject, has the advantages of high sensitivity and high specificity, and has important application value.

(b) The marker is used for judging the endometrial cancer stratification of a subject and has the characteristic of good repeatability.

Drawings

Figure 1 is a graphical representation of glycan naming and classification. Wherein, F = deoxyribose (fucose); e = α 2,6-linked sialic acid; l = α 2,3-linked sialic acid; h = hexose (mannose or galactose); n = N-acetylglucosamine (N-GlcNAc).

Figure 2 is the potency of THy or TC in patients with high and low differentiation of endometrial cancer, a: TC, B: THy.

Figure 3 is the efficacy of THy and TC in combination in patients with high and low differentiation of endometrial cancer.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 high differentiation and Low differentiation of endometrial cancer

Full serum samples of high and low differentiation of Endometrial Cancer (EC) were collected and the participant information is tabulated in table 1 below.

The gold standard for judging high differentiation and low differentiation is tissue pathology after operation, and high differentiation and low differentiation are determined according to the pathology after operation. High differentiation is usually associated with a good prognosis, low differentiation is usually associated with a poor prognosis, and the risk of metastasis and relapse is high.

TABLE 1 participant information

	Highly differentiated endometrial cancer	Hypodifferentiation of endometrial cancer
				n＝23	n＝11
Age, mean (standard deviation)	56(9)	60(6)
			CA125, U/mL, median (interquartile range)	17.7(12.3-30.2)	13.1(8.5-44.5)
CA199, U/mL, median (interquartile range)	17.6(12.6-33.7)	6.3(4.6-43.8)
			CEA, U/mL, median (interquartile range)	1.5(1.1-2.6)	1.9(1.2-3.6)

Experimental method

1. N-sugar chain released by glycosidase

The N-sugar chains were released from the whole serum/plasma glycoprotein upstream using the glycosidase PNGase F. The method comprises the following specific steps: taking 5. Mu.L of serum from each sample, adding 10. Mu.L of 2% SDS, incubating at 60 ℃ for 10 minutes; then, 10. Mu.L of the enzymatic hydrolysate (containing 2% NP-40,2.5 XPBS and 1U of PNGase F) was added thereto, and incubated at 37 ℃ for 12 to 16 hours.

2. Derivatization of N-sugar chains

The N-sugar chain obtained by the above-mentioned dissociation is derivatized by a known derivatization technique, and sialic acids in α 2,3 and α 2,6 linkages can be distinguished by the derivatization. The method comprises the following specific steps: mu.L of the above-mentioned serum after the enzymatic digestion was added with 20. Mu.L of a derivatization reagent (250 mM EDC and 250mM HOBt, in absolute ethanol) and incubated at 37 ℃ for 60 minutes.

3. Enrichment and purification of N-sugar chain HILIC-SPE

The derivatized sugar chain obtained above was enriched and purified by HILIC-SPE. HILIC uses cotton thread as stationary phase, the cotton thread is filled in 20 μ L of gun head to make purification cartridge, firstly, the cartridge is activated 3 times with 15 μ L of ultrapure water (MQ); then, the column was equilibrated 3 times with 15 μ L of 85% Acetonitrile (ACN); adding the derivatized sugar chain mixed solution into a column, and loading for 30 times to ensure that the derivatized N-sugar chain is adsorbed on the column as completely as possible; the column was then rinsed 3 times with 15 μ L of 85% acetonitrile +1% trifluoroacetic acid (TFA) and then with 15 μ L of 85% acetonitrile for 3 times; finally, the sugar chains were eluted in 10 μ L MQ.

4. Mass spectrometry of N-sugar chains

Prior to detection, the mass spectrometer was calibrated with a Peptide fragment mixture Standard (Bruker Peptide Calibration Standard II) of known molecular mass. The substrate super-DHB was dissolved in a solution of 1mM NaOH in 50% acetonitrile (water) at a concentration of 5mg/mL. mu.L of the purified N-sugar chains was spotted on a mass spectrometry target plate, and then 1. Mu.L of the matrix solution was dropped on the sample and air-dried at room temperature. MALDI-TOF MS is used for analysis, a Smartbeam 3D laser source is arranged in mass spectrum, signal ions are collected in a positive ion Reflection (RP) mode, flexControl software is used for control, and the m/z range is set as follows when a sample is detected: 1000 to 5000. The spectrogram acquisition is set as follows: for each sample point on the mass spectrum target plate, the laser completely randomly acquires signals within the range of the sample point, 10K laser shots are accumulated, and a mass spectrum is acquired, wherein the laser frequency is 5000Hz.

5. Data preprocessing and statistical analysis

The collected mass spectra were preprocessed using FlexAnalysis and MassyTools software and exported to Microsoft Excel for further analysis. Mass spectrum data are analyzed by sugar chain analysis function auxiliary artificial analysis of GlycoWorkBench, and sugar chain structure identification is mainly based on mass-to-charge ratio, secondary mass spectrum fragment attribution and published documents. The single directly detected sugar chain quantification was obtained from the peak area of the single sugar chain/peak area of all sugar chains detected and the directly detected sugar chains are shown in the table below.

TABLE 2 sugar chains directly detected

All ionic forms being gana, i.e. [ M ] ⁺ Na] ⁺ . H = hexose (mannose or galactose); n = N-acetylglucosamine (N-GlcNAc); f = deoxyribose (fucose); l = α 2,3-linked sialic acid; e = α 2,6-linked sialic acid.

In addition to the directly detected sugar chain structure, derived sugar chain characteristics (derived sugar chains) were calculated from the directly detected N-sugar chains by Rstudio in terms of their structural characteristics and biological relevance. The derived sugar chain characteristics include: the number of antennas of complex N-sugar chains (a), the level of fucosylation (F), the level of bisected sugar chains (B), the level of terminal galactosylation (G), the level of sialylation (S), and the like.

The relationship between high-low differential EC patients is evaluated by statistical test, regression analysis and the working characteristic curve of the testee. The mass spectrometric data quality of the study cohort was evaluated by the standards randomly distributed on the target plate during the sample detection and calculating the mean, coefficient of variation and standard deviation of each sugar chain of the resulting plurality of standards.

6. Quality control

The Top30 obtained for the quality control sample had an average CV value of 4.49% for the sugar chains, indicating that the data obtained in the present invention are reliable.

Results of the experiment

We found by logistic regression (age-corrected) that sugar chains have significant correlation with EC differentiation type (high differentiation or low differentiation), while only THy, TC among derived sugar chain characteristics showed differences in EC with high and low differentiation. In EC patients, poorly differentiated was negatively correlated with THy (total heterozygote sugar chains) (p =0.018 or = 0.11), while poorly differentiated was positively correlated with TC (total complex sugar chains) (p =0.035 or = 4.86) (see table 3.

TABLE 4 derived sugar chain characteristics relating to EC differentiation types

Furthermore, we found that the AUC value for THy distinguishing poorly differentiated ECs from highly differentiated ECs was 0.846 (95% ci 0.710-0.982) as analyzed by the ROC curve (fig. 2). The AUC value for TC distinguishing poorly differentiated ECs from highly differentiated ECs was 0.814 (95% CI 0.666-0.963). As shown in FIG. 3, the AUC value for THy in combination with TC distinguishing poorly differentiated ECs from highly differentiated ECs was 0.838 (95% CI 0.697-0.979).

Claims

1. Application of reagent and apparatus for detecting THy and/or TC in preparation of products for differentiating high-differentiation and low-differentiation endometrial cancer patients.

2. Use as claimed in claim 1, wherein the reagent for detecting THy and/or TC comprises any one or more of: sugar chain releasing reagent, N-sugar chain derivatization reagent, enrichment and purification reagent, and reagent used in mass spectrometry.

3. The use according to claim 2, wherein the sugar chain-releasing agent comprises an agent used in an enzymatic method and/or a chemical method.

4. The use according to claim 3, wherein the enzyme in the enzymatic method is an endoglycosidase comprising Endos2, PNGase F.

5. The use according to claim 4, wherein the endoglycosidase is PNGase F.

6. The use of claim 2, wherein the mass spectrometry method comprises: matrix-assisted laser desorption ionization mass spectrometry, matrix-assisted laser desorption ionization-flight time mass spectrometry, matrix-assisted laser desorption ionization-quaternary ion trap-flight time mass spectrometry, fast atom bombardment mass spectrometry, electrospray mass spectrometry, multistage mass spectrometry, high performance liquid chromatography, and liquid chromatography-mass spectrometry.

7. A method of identifying a sample as belonging to a highly or poorly differentiated endometrial cancer patient, the method comprising the step of determining that the sample belongs to a highly or poorly differentiated endometrial cancer patient based on the value of the subject's THy and/or TC;

preferably, the steps include:

1) A sample of the subject is collected and,

2) A value for THy and/or TC is obtained,

3) And judging endometrial cancer stratification.

8. A machine-readable storage medium of a patient with high and low differentiation of endometrial cancer, said machine-readable storage medium having stored thereon a plurality of computer instructions that, when executed, perform the process of:

judging the endometrium of the subject according to the THy and/or TC value of the subject;

preferably, the computer instructions when executed perform the following:

1) A sample of the subject is collected and,

2) A value for THy and/or TC is obtained,

3) Judging endometrial cancer stratification;

preferably, the method used to obtain the value of THy and/or TC comprises detection using any one of the following methods: matrix-assisted laser desorption ionization mass spectrometry, matrix-assisted laser desorption ionization-flight time mass spectrometry, matrix-assisted laser desorption ionization-quaternary ion trap-flight time mass spectrometry, fast atom bombardment mass spectrometry, electrospray mass spectrometry, multistage mass spectrometry, high performance liquid chromatography, and liquid chromatography-mass spectrometry.

9. A system for differentiating patients with high and low endometrial cancer, the system comprising a computing device for determining endometrial cancer stratification in a subject based on the value of THy and/or TC in the subject;

(1) A collection device for collecting and processing a patient sample;

(2) A detection device for mass spectrometry detection;

(3) A data processing device for calculating the value of THy and/or TC according to the detection result;

(4) An output device for outputting the endometrial cancer stratification of the subject;

preferably, the calculation may be performed by any one or more of the following Rstudio, flexAnalysis, massyTools, glycoWorkBench software.

10. A device for differentiating patients with endometrial cancer high and low differentiation, comprising:

at least one processor;

at least one memory for storing at least one program;

when executed by the at least one processor, cause the at least one processor to implement a method of determining endometrial cancer stratification in a subject based on a value of THy and/or TC in the subject.