CN110927263A - Method for predicting platinum chemotherapy sensitivity of ovarian cancer patient by combining serum glycosylation modification with CA125 - Google Patents

Abstract

The invention carries out prognosis evaluation on the sensitivity of the ovarian cancer patient to platinum chemotherapeutic drugs by detecting glycosylation modification in serum before the ovarian cancer patient is treated and utilizing an a 2, 3-sialic acid N-sugar chain combined with CA 125. The detection object of the invention is serum of a patient with ovarian cancer before treatment; detecting the relative expression quantity of the a 2, 3-sialyl N-sugar chain after the glycosylation modification of the serum by using an analytical instrument, combining the content of CA125 in the serum of a patient, and performing binary logistic regression analysis by using SPSS software to obtain a prediction probability; taking 0.24 as a critical value, if the prediction probability is more than 0.24, the patient is a drug-resistant patient, and the patient is less than 0.24, and the patient is a sensitive patient. The method has the advantages that the appropriate marker is used for predicting the chemotherapy effect of the drug on the patient, so that the method is beneficial to selecting a more appropriate treatment scheme for the patient, and further the survival rate of the ovarian cancer patient is improved.

Description

Method for predicting platinum chemotherapy sensitivity of ovarian cancer patient by combining serum glycosylation modification with CA125

Technical Field

The invention relates to the field of biotechnology.

Background

Ovarian cancer is the most lethal gynecological tumor. At this stage, serum CA125 and ultrasound are the most common detection methods; the treatment scheme adopted by most patients is combined chemotherapy of tumor body extinction. At present, the first-line chemotherapy drug for ovarian cancer is platinum combined with paclitaxel. Although most patients are sensitive to platinum-based drugs during primary chemotherapy, most patients relapse with concomitant drug resistance, and ovarian cancer patients have poor five-year survival and poor prognosis. Clinically, the sensitivity of patients to drugs is usually differentiated by taking 6 months as a time node. The time from the end of the platinum chemotherapy to the relapse is defined as a drug-resistant patient if the time is less than 6 months; if it is more than 6 months, it is defined as sensitive patient. If the chemotherapy effect of the platinum drugs on the patients is predicted by using the appropriate markers before chemotherapy, the selection of a more appropriate treatment scheme for the patients is facilitated, and the survival rate is further improved.

Glycosylation modification is the most common post-transcriptional translation modification, almost participates in all physiological and pathological processes, a large number of reports prove that the glycosylation modification plays an important role in tumorigenesis and development, and the glycosylation modification is reported to participate in a drug resistance mechanism of ovarian cancer. The glycosylation modification of the serum protein of the ovarian cancer patients is changed before and after chemotherapy. The glycosylation modification has strong advantages as a marker, can make up the defects of protein markers, such as a tumor marker CA125 used at the present stage of ovarian cancer is also highly expressed in the serum of a plurality of gynecological benign disease patients, and the combination of CA125 and Tn sugar chains on the CA125 can effectively distinguish benign ovarian diseases from malignant tumors, and for example, fucosylated alpha-fetoprotein is a more effective liver cancer early diagnosis marker of international certification.

Some researchers have attempted to find markers that differentiate susceptibility of ovarian cancer patients to chemotherapy to assess the treatment regimen of the patients prior to treatment, but to date, no suitable markers have been found and no studies have been reported that utilize glycosylation modifications to differentiate susceptibility of ovarian cancer patients prior to treatment.

Disclosure of Invention

The invention aims to carry out prognostic evaluation on the sensitivity of ovarian cancer patients to platinum chemotherapeutic drugs by detecting glycosylation modification in serum before ovarian cancer patients are treated and utilizing an a 2, 3 sialic acid N-sugar chain combined with CA 125. The detection object of the invention is serum before treatment of ovarian cancer patients; the detection comprises the following steps:

(A) reagents and methods related to sugar chain dissociation, derivatization, and enrichment:

1) adding 5 muL serum into 10 mul 2% SDS, and denaturing at 60 ℃ for 10 minutes;

2) add 10 μ L of 2% NP-40 containing 0.5 mU PNGase F for overnight incubation at 37 ℃;

3) adding 20 muL of derivatization mixture including 250mM EDC and 250mM HOBt into 2 muL of the solution, and reacting for 1 hour at 37 ℃;

4) taking out the derivatization sample, adding 22 muL acetonitrile, and placing at-20 ℃ for precipitating protein for 15 minutes;

5) filling HILIC SPE absorbent cotton in a 20 mu L gun head to prepare a HILIC SPE purification small column, and activating the purification small column with 10 mu L ultrapure water for three times; then, the purifying column was equilibrated three times with 10 μ L of an aqueous solution containing 85% acetonitrile; the sample is pumped back and forth for 40 times to ensure that the sugar chains are completely adsorbed on the purification small column; finally eluting with 10 muL of ultrapure water to obtain a sample;

(B) analyzing the sample with an analytical instrument or system: adopting matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-MS; or fast atom bombardment mass spectrometry FAB-MS; or electrospray mass spectrometry ESI-MS; liquid chromatography; or a combination of liquid chromatography-mass spectrometry; or a sugar chip technology detection method; or nuclear magnetic resonance detection (NMR); wherein, the matrix-assisted laser desorption-time-of-flight mass spectrometry MALDI method is preferably used for detecting glycosylation modification;

(C) analyzing the expression of a 2, 3 sialic acid, comprising the steps of:

1) the relative amount of a 2, 3 sialic acid is defined as S: summing the peak heights of all sugar chains detected in the mass spectrum to A, and each peak height of N-sugar chains containing an a 2, 3-sialyl type is B_nWherein the number of galactose atoms contained in each of the N-sugar chains containing an a 2, 3-sialyl group is C_nAnd D represents a number of a 2, 3 sialic acids_n；

S=（D₁/C₁）*B₁+ （D₂/C₂）*B₂+ （D₃/C₃）*B₃+...+ （D_n/C_n）*B_n；

2) Combining the relative expression quantity of the a 2, 3-sialyl N-sugar chain with the content of CA125, and performing binary logistic regression analysis by using SPSS software to obtain the combined prediction probability of the two indexes;

3) after the prediction probability of 2) is obtained, taking 0.24 as a critical value, the patient with the prediction probability larger than the critical value is a drug-resistant patient, and the patient with the prediction probability smaller than the critical value is a sensitive patient.

The invention has the advantages that the appropriate marker is used for predicting the chemotherapy effect of the platinum drug on the patient, so that the treatment scheme more suitable for the patient can be selected, and the survival rate of the ovarian cancer patient can be further improved.

Drawings

FIG. 1 is a ROC analysis curve for distinguishing drug-resistant and sensitive patients using an a 2, 3-sialyl N-sugar chain in combination with CA125

FIG. 2 shows an a 2, 3-sialyl N-oligosaccharide structure

The designation of the numbers in the figures is: the rectangular frame is marked with an a 2, 3 sialic acid moiety

。

Detailed Description

1) Serum samples were collected from patients with ovarian cancer prior to treatment. 1 year later, follow-up was performed on a platinum-based chemotherapy regimen, wherein 16 patients relapsed within half a year after platinum chemotherapy was completed and were chemotherapy-resistant patients; the 48 patients relapsed after half a year, and were chemotherapy-sensitive patients.

2) According to the test steps of the invention, the glycosylation modification of the serum of the patient is detected, the relative expression amount of the N-sugar chain of the 2, 3-sialyl is calculated, and the prediction probability is obtained by combining the content of CA125 in the serum of the patient and performing binary logistic regression analysis by using SPSS software.

3) Taking 0.24 as a critical value, if the prediction probability is more than 0.24, the medicine is classified as a chemotherapy resistant medicine group, and the medicine is classified as a chemotherapy sensitive group, wherein the prediction probability is less than 0.24.

4) When the critical value is 0.24, carrying out ROC analysis according to the actual drug resistance condition of the patient; the specificity of the a 2, 3-sialyl N-oligosaccharide in combination with CA125 for distinguishing chemotherapy-resistant and sensitive patients was 87.5%, the sensitivity was 79.2%, and AUC was 0.85 (as shown in FIG. 1); can effectively distinguish the chemotherapy sensitivity of the patient to the platinum drugs. For patients with platinum chemotherapy resistance, the chemotherapy regimen can be adjusted to prepare for subsequent treatment.

FIG. 1 is a graph showing ROC analysis using an a 2, 3-sialyl N-sugar chain in combination with CA125 to distinguish between patients resistant and sensitive to platinum chemotherapy. When the cut-off value was 0.24, the AUC was 0.85, the specificity was 87.5%, and the sensitivity was 79.2%.

An a 2, 3-sialyl N-sugar chain structure:

neu5Ac a 2-3 is an a 2, 3 is a sialic acid moiety;

。

Claims (1)

1. a method for predicting platinum chemotherapy sensitivity of ovarian cancer patients by using serum glycosylation modification and CA125, comprising the following steps:

(A) reagents and methods related to sugar chain dissociation, derivatization, and enrichment:

1) adding 10ul of 2% SDS into 5 muL of serum, and denaturing at 60 ℃ for 10 minutes;

2) add 10 μ L of 2% NP-40 containing 0.5 mU PNGase F for overnight incubation at 37 ℃;

3) adding 20 muL of derivatization mixture including 250mM EDC and 250mM HOBt into 2 muL of the solution, and reacting for 1 hour at 37 ℃;

4) taking out the derivatization sample, adding 22 muL acetonitrile, and placing at-20 ℃ for precipitating protein for 15 minutes;

5) filling HILIC SPE absorbent cotton in a 20 mu L gun head to prepare a HILIC SPE purification small column, and activating the purification small column with 10 mu L ultrapure water for three times; then, the purifying column was equilibrated three times with 10 μ L of an aqueous solution containing 85% acetonitrile; the sample is pumped back and forth for 40 times to ensure that the sugar chains are completely adsorbed on the purification small column; finally eluting with 10 muL of ultrapure water to obtain a sample;

(B) analyzing the sample with an analytical instrument or system: adopting matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-MS; or fast atom bombardment mass spectrometry FAB-MS; or electrospray mass spectrometry ESI-MS; liquid chromatography; or a combination of liquid chromatography-mass spectrometry; or a sugar chip technology detection method; or nuclear magnetic resonance detection (NMR); wherein, the matrix-assisted laser desorption-time-of-flight mass spectrometry MALDI method is preferably used for detecting glycosylation modification;

(C) analyzing the expression of a 2, 3 sialic acid, comprising the steps of:

1) the relative amount of a 2, 3 sialic acid is defined as S: summing up all the sugar chain peaks detected in the mass spectrum to A, each N-sugar chain containing an a 2, 3-sialic acid type has a peak height of B_nWherein the number of galactose atoms contained in each of the N-sugar chains containing an a 2, 3-sialyl group is C_nAnd D represents a number of a 2, 3 sialic acids_n；

S=（D₁/C₁）*B₁+ （D₂/C₂）*B₂+ （D₃/C₃）*B₃+...+ （D_n/C_n）*B_n；

2) Combining the relative expression quantity of the a 2, 3-sialyl N-sugar chain with the content of CA125, and performing binary logistic regression analysis by using SPSS software to obtain the combined prediction probability of the two indexes;

3) after the prediction probability of 2) is obtained, taking 0.24 as a critical value, the patient with the prediction probability larger than the critical value is a drug-resistant patient, and the patient with the prediction probability smaller than the critical value is a sensitive patient.

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