RU2560703C1 - Method for detecting epithelial dysplasia of gastric mucosa in patients suffering atrophic gastritis or gastric adenoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to gastroenterology, and can be used for the detection of epithelial dysplasia of the gastric mucosa in the patients suffering atrophic gastritis or gastric adenoma. That is ensured by measuring the interferon-γ concentration in patient's blood cell supernatant after incubation with or without carcino-embryonic antigen (CEA). An index of CEA effect (CEA IE) on IFNγ production by blood cells is calculated by formula: CEA IE IFNγ=A/B, wherein A is the IFNγ concentration in the cell supernatant after the CEA incubation, B is the the IFNγ concentration in the cell supernatant without the CEA incubation. If the CEA IE IFNγ is less than 2.40 standard units, the presence of epithelial dysplasia of the gastric mucosa is stated in the patient, whereas the CEA IE IFNγ being 2.40 standard units or more, the absence of epithelial dysplasia of the gastric mucosa is stated in the patient.

EFFECT: using the given method enables the timely detection of epithelial dysplasia of the gastric mucosa (cellular atypism) and forming a risk group of developing gastric cancer.

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Description

The invention relates to medicine, namely to the diagnosis of dysregenerative processes in the epithelium of the gastric mucosa.

The following methods are known for detecting dysplasia (atypical cells) of the epithelium of the gastric mucosa in atrophic gastritis and gastric adenomas, which are referred to as precancerous conditions:

1. Endoscopic biopsy, in which the patient takes the tissue of the gastric mucosa - a biopsy specimen that is stained and under the microscope determines the presence or absence of atypical cells. The method is quite invasive. In addition, the probability of obtaining material from areas of the stomach that do not have atypical cells is increased, which can lead to false negative results [Lee SB, Kang HY, Kim Y, Ahn DH. The diagnostic accuracy of endoscopic biopsy for gastric dysplasia // J Gastric Cancer. 2010. Vol. 10, No. 4. - P. 175-181].

2) As in the previous method, take a biopsy sample of the gastric mucosa, but determine the expression of Toll-like receptors, which are markers of dysplasia in biopsy samples of the gastric mucosa. The disadvantage of this method is the increased invasiveness and the likelihood of obtaining a biopsy from areas of the stomach that do not have dysplasia, which can lead to false negative results [Pimentel-Nunes PI, Afonso L, Lopes Ρ, Roncon-Albuquerque R Jr, Gonçalves Ν, Henrique R, Moreira- Dias L, Leite-Moreira AF, Dinis-Ribeiro M. Increased expression of toll-like receptors (TLR) 2, 4 and 5 in gastric dysplasia // Pathol Oncol Res. 2011. - Vol. 17, No. 3. - P. 677-683].

3) In this paper [Jang JS, Choi SR, Han SY, Roh MH, Lee JH, Lee SW, Jeung JS, Kim MC, Son YK, Baek YH. Predictive significance of serum IL-6, VEGF, and CRP in gastric adenoma and mucosal carcinoma before endoscopic submucosal dissection // Korean J Gastroenterol. 2009. - Vol. 54, No. 2. - P. 99-107] it was shown that in patients with gastric dysplasia in blood serum levels of IL-6, VEGF and C-reactive protein are increased (prototype).

The method is characterized by low specificity, since so many pathological processes, in particular inflammation of various localization, can lead to an increase in the levels of these factors in serum, and also many body cells can be their sources: monocytes-macrophages, lymphocytes, granulocytes, hepatocytes, keratinocytes, fat cells and the like

Disclosure of invention

The proposed method for detecting gastric mucosa epithelial dysplasia in a patient with atrophic gastritis or with gastric adenoma is characterized by determining the concentration of interferon-γ in the patient’s blood cell supernatant after their incubation with and without cancer-embryonic antigen (CEA), the effect index of CEA is calculated (IV CEA) for the production of IFNγ by blood cells according to the formula: IV CEA IFNγ = A / B, where A is the concentration of IFNγ in the cell supernatant after incubation with CEA, B is the concentration of IFNγ in the cell supernatant without incubation with CEA and when a value of IV CEA IFNγ of less than 2.40 conventional units concludes that there is dysplasia of the epithelium of the gastric mucosa in the patient, and with a value of IV CEA IFNγ equal to or greater than 2.40 conventional units, they conclude that there is no dysplasia of the epithelium of the gastric mucosa in the patient.

The technical result of the proposed method:

1) high specificity, due to the determination of the interferon-γ-producing function of blood ICC in response to incubation with CEA, thereby eliminating the non-specific element inherent in the prototype;

2) low invasiveness due to taking blood from a patient’s finger or vein (and not a biopsy from the stomach);

3) the high efficiency of the proposed method associated with high sensitivity (87.5%);

4) a very good quality model (diagnostic method), determined using ROC analysis with the calculation of AUC (Area Under Curve) using the trapezoidal method. The AUC of the proposed method is equal to 0.854, which, as mentioned above, means "very good quality" diagnostic method;

5) the proposed method allows timely detection of dysplasia of the epithelium of the gastric mucosa (cellular atypism) and form a risk group for the development of gastric cancer.

The implementation of the invention

A patient with atrophic gastritis or with adenoma of the stomach takes blood from a vein or finger in an amount of 1 ml. Blood is placed in a sterile vial with 4 ml of culture medium (sterile culture medium DMEM). Gently mix the contents of the vial, then under sterile conditions take two samples from it. One 0.980 ml sample is placed in a sterile vial containing 0.02 ml CEA (Canceroembryonic Antigen Human Colon Adenocarcinoma Cell Line) manufactured by Calbiochem, Germany, with a concentration of CEA of 2 μg / ml. Another 0.980 ml sample is also placed in a sterile vial containing 0.02 ml of DMEM growth medium (internal control). The concentration of cells in the samples and the volumes of the samples in both sterile vials are the same.

The contents of the vials are gently mixed and placed in a thermostat at 37 ° C for 24 hours. During the first hour of incubation, the contents of the vials are gently mixed once. After incubation by centrifugation at 3000 rpm for 10 minutes, blood cells are precipitated and the supernatant is taken from a sample of cells incubated with CEA (to determine IFNγ production under the influence of CEA) and from a cell sample without CEA (to determine spontaneous IFNγ production). Using a standard test system for determining IFNγ (manufactured by Vector-Best CJSC), the concentration of IFNγ in both samples is determined. Calculate the effect index of CEA (IV CEA) on the production of IFNγ by blood cells according to the formula: IV CEA IFNγ = A / B, where A is the concentration of IFNγ in the cell supernatant after incubation with CEA, B is the concentration of IFNγ in the cell supernatant without incubation with CEA . IV CEA IFNγ expressed in arbitrary units. With a value of IV REA IFNγ of less than 2.40 conventional units, a conclusion is made about the presence of dysplasia of the gastric mucosa epithelium in a patient, and with a value of IV CEA IFNγ equal to or greater than 2.40 conventional units, a conclusion is drawn that there is no dysplasia of epithelium of the gastric mucosa in a patient.

The rationale for the method

We examined 34 people with a diagnosis of atrophic gastritis and with a diagnosis of adenoma. Dysplasia was found in 16 people, lack of dysplasia - in 18 people. The diagnosis in these patients was made on the basis of generally accepted clinical, laboratory and instrumental studies. The presence of gastric mucosa epithelial dysplasia was determined by histopathological examination of biopsy specimens.

Blood was taken from patients and in the above-described way using standard test systems manufactured by Vector-Best CJSC to determine cytokines, IEV CE was determined for the production of tumor necrosis factor, interleukin 1-β, receptor antagonist interleukin 1-β-interleukin 1-RA, interleukin 6, interleukin 8 and IFNγ.

It turned out that only IV CEA for the production of IFNγ by blood cells allows us to detect dysplasia of the epithelium of the gastric mucosa with atrophic gastritis and gastric adenoma.

The study showed that in 87.5% of patients with dysplasia of IV REA, IFNγ was less than 2.40 conventional units, and in 100% of the patients in the surveyed group without dysplasia of IV REA, IFNγ was equal to or higher than 2.40 conventional units.

An IEA CEA IFNγ of 2.4 was obtained from a study of a sample of 16 patients with dysplasia and 18 patients with no dysplasia. In 14 out of 16 patients with dysplasia (87.5%), the value of IV CEA IFNγ was lower than 2.4, and in the other two it was higher. In all 18 examined patients without dysplasia, RE CE IFNγ was equal to or exceeded 2.4 (100%).

The statistical significance of the differences was estimated using the exact Fisher method, p = 0.0004.

To obtain a numerical value of the clinical significance of the proposed method for detecting gastric mucosa epithelial dysplasia with atrophic gastritis and gastric adenoma, we used a ROC analysis with the determination of AUC (Area Under Curve) indicator (https://ru.wikipedia.org/wiki/ROC-%D0 % BA% D1% 80% D0% B8% D0% B2% D0% B0% D1% 8F), which was calculated using the trapezoid method. The quality of the test was judged by an expert scale for AUC values: 0.9-1.0 - excellent model quality, 0.8-0.9 - very good model quality, 0.7-0.8 - good model quality, 0, 6-0.7 - average model quality, 0.5-0.6 - unsatisfactory model quality (http://www.basegroup.ru/library/analysis/regression/logistic/).

The proposed method for detecting dysplasia of the epithelium of the gastric mucosa with atrophic gastritis and gastric adenoma showed very good quality, because AUC was 0.854.

Clinical examples

1. Patient E.V., 49 years old, diagnosed with chronic atrophic gastritis, IV CEA IFNγ in a patient was 2.2 conventional units. Conclusion of the pathomorphologist: dysplasia, which corresponds to the current WHO classification - high grade intraepithelial neoplasia / dysplasia.

2. Patient D, about 72 years old, the diagnosis of chronic atrophic gastritis. IV REA IFNγ in the patient was 13.4 conventional units. The conclusion of the pathomorphologist: the absence of dysplasia, which corresponds to the current WHO classification - the absence of intraepithelial neoplasia / dysplasia.

3. Patient M-o, 61 years old, diagnosed with gastric adenoma. IV CEA IFNγ in the patient was equal to 1.0 conventional unit. Conclusion of the pathomorphologist: dysplasia, which corresponds to the current WHO classification - high grade intraepithelial neoplasia / dysplasia.

Claims (1)

A method for detecting gastric mucosa epithelial dysplasia in patients with atrophic gastritis or with gastric adenoma, characterized in that the concentration of interferon-γ in the patient’s blood cell supernatant after incubation with and without cancer-embryonic antigen (CEA) is determined, the effect index of CEA is determined (IV CEA) for the production of IFNγ by blood cells according to the formula: IV CEA IFNγ = A / B, where A is the concentration of IFNγ in the cell supernatant after incubation with CEA, B is the concentration of IFNγ in the cell supernatant without incubation with CEA and at AND CEA IFNγ least 2.40 cu conclude dysplasia of the epithelium of the gastric mucosa in a subject, and when the magnitude of IW CEA IFNγ, equal to or greater than 2.40 arbitrary units concluded the absence of dysplasia of the epithelium of the gastric mucosa of the patient.