CN111936858B - Gastric cancer very early cell marker and gastric precancerous lesion early cell marker and application thereof in diagnostic kit - Google Patents
Gastric cancer very early cell marker and gastric precancerous lesion early cell marker and application thereof in diagnostic kit Download PDFInfo
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Abstract
The present inventors have found that a cell population which begins to appear at the stage of low-grade dysplasia in the stomach, has specific molecular characteristics, and is highly at risk of canceration (designated as "stomach cancer very early cells") can be used as a marker for very early diagnosis of stomach cancer. The molecular marker of the stomach cancer very early cell is applied to the preparation of a kit for realizing the early diagnosis of the stomach cancer or other digestive system tumors based on stomach tissues or blood samples. The stomach cancer very early cell marker can also distinguish the postoperative recurrence risk of the stomach cancer and the risk of the tumor occurrence of digestive system organs such as intestines, pancreas, esophagus and the like. In addition, the present inventors have found that a cell population having a specific molecular characteristic, which appears in the early stage of gastric precancerous lesions, can be used as a marker for early diagnosis of gastric precancerous lesions, and is also integrated into a kit. The invention can be used for clinically diagnosing the occurrence of gastric cancer and other digestive tract tumors in early stage, can also be used as an intervention target for preventing and treating the digestive tract tumors, and has good application prospect.
Description
Technical Field
The invention relates to a cell marker for assisting in the very early diagnosis of gastric cancer and other digestive system tumors, a determination system and a determination method thereof, and also relates to a cell marker for the early diagnosis of gastric precancerous lesions, a determination system and a determination method thereof.
Background
Gastric cancer is a common malignant tumor of the digestive system and has high mortality rate. Most gastric cancers occur through a series of precancerous lesions, including intestinal metaplasia and dysplasia. Patients with precancerous lesions are at approximately 3 times the risk of developing gastric cancer as compared to patients with non-precancerous lesions [1 ]. Early diagnosis of gastric precancerous lesions is of great significance for preventing gastric cancer.
Besides precancerous lesions of gastric cancer, the very early diagnosis of gastric cancer is also of great significance for the prevention and treatment of gastric cancer. Studies have shown that the 5-year postoperative survival rate of patients with gastric precancer can reach 96% [2 ]. However, the diagnosis of stomach precancer still mainly includes imaging, endoscopy and histopathology, and has certain limitations. The change of cell levels cannot be accurately observed in the diagnosis based on the imaging, and key biological characteristics are easy to omit; while diagnostic modalities based on histopathological observations and conventional tumor marker molecule expression may be limited in sensitivity and specificity. For example, CEACAM5, a classical gastric cancer marker, shows high expression in gastric cancer cells and other types of cells beside the gastric cancer (such as intestinal cells and normal gastric epithelial cells), and thus is difficult to be a specific marker for early screening of gastric cancer. More importantly, the key transformation point of the gastritis cancer transformation, namely the 'canceration starting point', is difficult to define due to the long transformation time of the gastritis cancer, which is a key difficulty for the early diagnosis of the gastric cancer and also a key bottleneck for limiting the early diagnosis of the clinical gastric cancer. Therefore, the identification of the high-precision gastric cancer very early cell marker is of great significance for defining the 'canceration starting point' of the transformation of the gastritis cancer and realizing the clinical very early diagnosis of the gastric cancer.
Similar to the development of gastric cancer, the development of tumors in other organs of the digestive system, such as the intestine, esophagus and pancreas, is closely associated with malignant transformation of inflammation and also undergoes precancerous pathological processes including dysplasia. Therefore, finding out the early cancer marker common to the digestive system tumors by using the identification of the stomach cancer very early cell marker as an entry point is also very important for improving the early diagnosis rate of the digestive system tumors.
Disclosure of Invention
In the present invention, the present inventors found that a group of cells (which the inventors named "stomach cancer very early cells") that begin to appear at the stage of low-grade dysplasia in the stomach, have specific molecular characteristics (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B), and have a high risk of carcinogenesis can be used as a marker for very early diagnosis of stomach cancer. The stage at which the cell population appears is defined by the inventors as the key point for the transformation of precancerous gastric disease into gastric cancer, i.e. "the onset of cancer". In addition, the inventor also finds that the gastric cancer very early cell marker can distinguish postoperative recurrence risks of gastric cancer and evaluate the risk of transforming the precancerous lesions of digestive system organs such as intestines, pancreas and esophagus into cancer. In the invention, the molecular characteristics of the gastric cancer very early cell marker are applied to the preparation of a kit for realizing early diagnosis of gastric cancer or other digestive system tumors based on gastric tissues or blood samples. In addition, the present inventors have found that a cell population having a specific molecular characteristic (HES6) which begins to appear at an early stage of gastric precancerous lesion can be used as a marker for early diagnosis of gastric precancerous lesion and also be integrated into a kit. The kit is expected to provide effective basis for digestive system tumor patients to take relevant treatment measures or decisions, and has good clinical application prospect.
According to an aspect of the present invention, there is provided use of an agent for detecting an expression level of a marker in the preparation of a product for determining very early gastric cancer, wherein the marker comprises at least one selected from the group consisting of KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT 6B.
According to another aspect of the present invention, there is provided a product for determining the cell content of very early gastric cancer, thereby diagnosing the very early gastric cancer. Wherein the product comprises a reagent for detecting the expression level of a marker in a sample, wherein the marker comprises at least one selected from the group consisting of KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT 6B.
According to another aspect of the invention, the application of the reagent for detecting the expression level of the HES6 in the preparation of products for determining the level of gastric precancerous lesions is provided.
According to another aspect of the present invention, there is provided a product for determining the level of gastric precancerous lesions, which comprises an agent for detecting the expression level of HES6 in a sample.
The invention mainly comprises the following aspects:
1) the invention provides a new application of 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in the early diagnosis of gastric cancer.
2) The invention also provides a new application of the molecule (HES6) serving as a characteristic of a cell population in an early stage of gastric precancerous lesion in early diagnosis of gastric precancerous lesion.
The invention provides a change characteristic of 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) from precancerous change to early cancer in tissues such as pancreas, intestine and esophagus, and a new application of the molecule in early diagnosis of pancreatic cancer, intestinal cancer and esophageal cancer.
The invention provides a new application of the correlation between the expression levels of 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) and the survival risk of postoperative recurrence of gastric cancer, and the application of the correlation to judgment of the recurrence risk of postoperative gastric cancer.
3) The invention provides a detection kit, which can be used for Immunohistochemistry (IHC) staining the protein molecules to obtain the expression condition of the protein molecules in a stomach tissue to be detected, and further evaluating the content of gastric precancerous lesion early-stage cells and early cancer cells in the tissue to be detected; can also be used for detecting the expression of the protein molecules in blood by enzyme-linked immunosorbent assay (ELISA).
4) The invention provides a system and a method for early diagnosis of precancerous lesions of gastric cancer, pancreatic cancer and intestinal cancer and esophageal cancer and evaluation of postoperative recurrence risk of gastric cancer. According to the expression condition of the cell marker protein in the tissue or blood, the system evaluates the number of gastric precancerous lesion cells and early cancer cells in the gastric tissue, and further provides the risk of the patient to be detected suffering from gastric precancerous lesion and gastric cancer and the risk of postoperative recurrence of gastric cancer. The system can also give the risk of the patient to suffer from other digestive system tumors such as intestinal cancer, esophageal cancer, pancreatic cancer and the like according to the expression condition of the cell marker protein in the tissues or blood of the patient. The system comprises the following two parts:
a detection kit (20) for respectively immunohistochemically staining the expression levels (11) of the cell markers HES6(10) at the early stage of gastric precancerous lesions and 13 gastric cancer very early stage cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) so as to obtain the expression condition of the cells in tissues to be detected; and the method can also be used for obtaining the content of the gastric cancer very early cell marker in a blood sample of a patient to be detected through enzyme-linked immunosorbent assay detection.
And if the tissue sample is the tissue sample to be detected, a gastric precancerous lesion and gastric cancer very early cell counting device (30) is used for determining the proportion fraction (31) of the positive cells of a gastric precancerous lesion early cell marker HES6 and the proportion fraction (32) of the positive cells of a gastric cancer very early cell marker KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT 6B. If the sample is a blood sample to be detected, the counting device calculates an average value to further determine the total expression level (33) of KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B in serum.
The system gives the risk index of the patient suffering from the gastric precancerous lesion and the risk index of the patient suffering from the gastric precancerous lesion according to the proportion of cells in the early stage of the gastric precancerous lesion to the total number of cells in the whole tissue to be detected; the risk index of the patient suffering from the gastric cancer and the postoperative recurrence risk index of the gastric cancer can be given according to the proportion of the extremely early gastric cancer cells to the total number of the cells in the whole tissue to be detected; the risk indexes of esophageal cancer, pancreatic cancer and intestinal cancer can be respectively evaluated according to the expression level of the stomach cancer very early cell marker molecules in the tissues of esophagus, pancreas and intestinal tract. Meanwhile, the total expression level of the marker molecules of the stomach cancer very early cells in serum can also be used for evaluating the risk index of the patient to be tested for suffering from digestive system tumors
5) The invention provides application of a reagent for respectively detecting expression levels of the cell marker HES6(10) at the early stage of gastric precancerous lesion and 13 gastric cancer very early stage cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) so as to obtain the expression condition of the cell marker in tissues/blood to be detected in preparation of a composition for early diagnosis of gastric precancerous lesion and/or gastric cancer and other digestive system tumors.
Drawings
FIG. 1 is a schematic diagram of a system for assisted early diagnosis of gastric precancerous lesions and gastric cancer and other digestive system tumors based on cell markers, according to an embodiment of the present invention.
FIG. 2A is a schematic diagram illustrating the demonstration of the specific expression of the cell marker HES6 in a specific cell type of pre-gastric lesion, according to an exemplary embodiment of the present invention.
Fig. 2B is a proportion distribution of positive cells labeled by the pre-gastric lesion early stage cell marker (HES6) and positive cells labeled by the pre-gastric lesion late stage marker MUC2 in pre-gastric lesion tissues in an example according to the present invention.
FIG. 2C is a graph of the proportion of positive cells labeled by the gastric precancerous lesion early stage cell marker (HES6) in stomach tissue at four stages, a non-precancerous lesion stage, a mild precancerous lesion stage, a severe precancerous lesion stage, and a gastric cancer early stage, in an example according to the present invention.
FIG. 2D is the proportion distribution of positive cells marked by the pre-gastric cancer lesion cell marker (HES6) in normal stomach tissue as well as pre-gastric cancer lesion tissue in an example according to the present invention.
Figure 2E is a schematic diagram illustrating partial co-expression in tissue of the cellular marker HES6 with the classic pre-gastric lesion marker MUC2, according to an exemplary embodiment of the present invention.
FIG. 2F is a schematic diagram illustrating the appearance of the cellular marker HES6 in tissue with the proliferation of the cellular marker KI67, but without co-expression, according to an exemplary embodiment of the present invention.
FIG. 3A is the expression pattern of 13 early carcinoma cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in the stomach early carcinoma tissue in example according to the present invention.
FIG. 3B is the expression pattern of 13 early cancer cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent gastritis (19 cases), pre-gastric lesions (39 cases), and early gastric cancer (19 cases) samples according to example of the present invention.
FIG. 3C is the expression pattern of 13 early cancer cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in blood samples from independent gastritis, gastric cancer patients according to example of the present invention.
Fig. 3D is an expression pattern of five representative marker molecules (KLK10, KRT17, TIMP1, MAP17, and KLK6) of stomach cancer very early cells in normal stomach tissue, dysplasia (precancerous lesion), and early stage stomach cancer in the example according to the present invention.
Fig. 3E is the proportion distribution of positive cells labeled by the gastric cancer very early cell representative marker (KLK10) in pre-gastric lesion, early gastric cancer, and advanced gastric cancer in the examples according to the present invention.
FIG. 4 is a graph illustrating the association of representative components of gastric cancer very early cell markers (KLK10, SLC11A2, KLK7 and TIMP1) with the risk of postoperative recurrence of gastric cancer, according to an exemplary embodiment of the present invention.
FIG. 5A is the expression pattern of 13 gastric cancer very early cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent normal intestinal tissue (38 cases), precancerous lesions (29 cases), and intestinal cancer (27 cases) samples according to example of the present invention.
FIG. 5B is the expression pattern of 13 gastric cancer very early cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent normal esophageal (5 cases), precancerous (4 cases), and esophageal cancer (3 cases) samples according to example of the present invention.
Fig. 5C is an expression pattern of representative molecules (ECM1, KRT17, TIMP1, and MMP7) in the stomach cancer very early cell marker in independent normal pancreas (8 cases), intraepithelial neoplasia (6 cases), and pancreatic cancer (12 cases) samples according to the example of the present invention.
Detailed Description
Fig. 1 is a schematic diagram of a gastric precancerous lesion and gastric cancer-assisted early diagnosis system based on cell markers according to an embodiment of the present invention.
Specifically, the invention firstly provides a detection kit based on 14 molecules. The kit comprises reagents for detecting molecular expression levels of KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, KRT6B and the like, and also comprises blank liquid, diluent liquid, antigen repairing liquid and the like. In a preferred embodiment, the reagent is an antibody for an immunohistochemical detection method, specifically as shown in table 1. The reagent can also be an antibody for Western Blot or ELISA detection method.
TABLE 1 immunohistochemical detection antibody List and formulated concentration ratio for molecules to be tested
The invention also provides an immunohistochemical detection method for the expression level of the 13 molecules in the stomach tissue. In one embodiment of the detection method, in addition to the above-described reagents contained in the kit, the user may self-formulate or purchase the following reagents:
(1) distilled or deionized water;
(2)3％H2O2；
(3) xylene;
(4) 75%, 85%, 95% alcohol and absolute ethyl alcohol;
(5)10mM TBS solution (pH 7.2-7.4): 1.21g of trihydroxyaminomethane, 7.6g of sodium chloride, 800mL of distilled water, adjusting the pH value to 7.2-7.4 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(6)10mM pH6.0 citrate buffer: 0.38g of citric acid, 2.45g of trisodium citrate, 900mL of distilled water, adjusting the pH value to 6.0 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(7) a hematoxylin solution;
(8) a neutral resin.
Example 1
In order to verify the effects of the invention in assisting early diagnosis of gastric precancerous lesions and digestive system tumors and predicting recurrence risk of gastric cancer, the inventor independently analyzes gastric precancerous lesions, gastric precancerous cancer, gastric cancer recurrence, other digestive system tumors (intestinal cancer, esophageal cancer and pancreatic cancer) and typical cases of precancerous lesions.
Diagnosis of cells in very early stage of gastric precancerous lesions
First, the inventors evaluated the expression of the early-stage gastric precancerous lesion cell marker HES6 in different types of cells of independent pre-gastric lesion samples. As shown in FIG. 2A, HES6 was specifically expressed in the precancerous lesion cells of gastric cancer at a high level.
Second, the inventors evaluated the co-expression of the very early gastric precancerous lesion cell marker HES6 with the late marker MUC2 in separate pre-gastric lesion samples. The results are shown in fig. 2B, where HES6 and MUC2 are in mutually exclusive expression patterns, suggesting that HES 6-labeled cells do not appear late in precancerous lesions.
Thirdly, the inventor evaluates the proportion distribution of the cells in the extreme early stage of the stomach precancerous lesion in the stomach tissue samples in four stages, namely a non-precancerous lesion stage, a mild precancerous lesion stage, a severe precancerous lesion stage, early cancerization and the like. As a result, as shown in FIG. 2C, cells in the very early stage of gastric precancerous lesion begin to appear in the stage of mild precancerous lesion, and the proportion thereof gradually increases as the degree of precancerous lesion increases.
Again, expression of HES6 in pre-gastric lesion samples was obtained according to the pre-gastric lesion cell markers (10) in the immunohistochemical test kit (20) of the present invention. The kit uses immunohistochemical staining (IHC) to measure the expression level of the marker. As a complementary analysis, the inventors co-stained the classic marker MUC2 of gastric precancerous lesions and the cell proliferation marker KI67 in the sample with fluorescence. Paraffin-embedded surgical specimens were fixed with 10% formalin buffer and the tissue sections were 4 μm/piece.
The kit in this example comprises the following components:
(1) reagent A: sealing liquid, 10% goat serum;
(2) and (3) reagent B: diluted ready-to-use anti-HES 6 primary antibody;
(4) and (3) reagent C: anti-goat biotinylated secondary antibody;
(5) and (3) reagent D: streptavidin-labeled HRP;
(6) and (3) reagent E: concentrating DAB substrate solution by 20 times;
(7) and (3) reagent F: concentrating DAB substrate buffer solution by 20 times;
(8) reagent G: the DAB chromogenic solution was concentrated 20-fold.
According to an embodiment of the present invention, the reagent B is originally packaged in the container and is dispensed with the ready-to-use antibody, and the dilution ratio is 1: 200 of a carrier; reagent G is originally packaged and is imported to split the ready-to-use antibody, and the dilution multiple is 1: 400, respectively; the reagent A, C, D, E, F, G is packaged by original package.
According to one embodiment of the present invention, in addition to the above-mentioned reagents contained in the kit, the user may self-prepare or purchase the following reagents:
(1) distilled or deionized water;
(2)3％H2O2；
(3) xylene;
(4) 75%, 85%, 95% alcohol and absolute ethyl alcohol;
(5)10mM TBS solution (pH 7.2-7.4): 1.21g of trihydroxyaminomethane, 7.6g of sodium chloride, 800mL of distilled water, adjusting the pH value to 7.2-7.4 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(6)10mM pH6.0 citrate buffer: 0.38g of citric acid, 2.45g of trisodium citrate, 900mL of distilled water, adjusting the pH value to 6.0 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(7) a hematoxylin solution;
(8) a neutral resin.
The expression of the markers in the tissues is detected by using the kit:
(1) tissue embedding: fixing a tissue specimen to be detected with 10% neutral formalin for 2h, repeatedly washing with running water to remove a fixing solution, putting the specimen into 75% alcohol overnight, then performing gradient dehydration with alcohol, wherein the alcohol content is 75% for 1h, the alcohol content is 85% for 1h, the alcohol content is 95% for 1h, and the absolute ethyl alcohol is used for 2 times, each time is 1.5h, then putting the specimen into xylene to be soaked for 1.5h, soaking the specimen in wax in a 60 ℃ oven for 1h for embedding, and preserving the specimen at 4 ℃ for later use after cooling;
(2) paraffin section: trimming a wax block, adjusting a slicer (SLEE paraffin slicer CUT5062), setting the slice thickness to be 3-4 mu m, continuously slicing, floating and flattening in warm water at 60 ℃, and flatly paving on a glass slide coated with cationic resin;
(3) baking slices: placing the slices to be sliced on a slicing frame, and baking for at least 1h in a constant-temperature oven at 60 ℃;
(4) dewaxing: dewaxing the slices in a container containing xylene for 3 times (i.e. xylene I, xylene II and xylene III) each for 10 min;
(5) hydration: hydrating the slices with descending ethanol, wherein the ethanol content is 5min, 95% ethanol 2 times (2 min each time), and 85% ethanol 2 min; 75% ethanol for 2min, and distilled water for 1 min;
(6) antigen retrieval: adding 1000ml of citric acid buffer solution into a pressure cooker, immersing the slicing frame with slices into the buffer solution, restoring at high temperature and high pressure for 2min and 45 sec, and washing with TBS for 3 times, each time for 2 min;
(7) dripping 3% H2O2 on the slices, standing at room temperature for 15min, washing with TBS for 3 times, each time for 2 min;
(8) and (3) sealing: dripping the reagent A on the section, completely covering the tissue section, incubating at room temperature for 10min, and sucking the liquid without washing;
(9) adding a primary antibody: dripping reagent B (anti-Hes 6 primary antibody) into different slices respectively, covering the tissue slices completely, and incubating at 37 deg.C for 2hr or 4 deg.C overnight;
(10) washing: TBS-T wash (3X 5 min);
(11) adding a secondary antibody: reagent C (biotinylated secondary antibody is dripped) and is required to completely cover the tissue slices, and the tissue slices are incubated in a 37 ℃ wet box for 30 min;
(12) washing: TBS washing for 5min 3 times;
(13) adding HRP-SA: dropwise adding a reagent D (streptavidin-labeled HRP) to completely cover the tissue slices, and incubating for 30min in a 37 ℃ wet box;
(14) washing: TBS washing for 5min 3 times;
(15) preparing a DAB color developing solution: taking a dyed slice as an example, taking 2.5ul of reagent E into 50ul of distilled water to be mixed uniformly, then respectively adding 2.5ul of reagent F and 2.5ul of reagent G into the liquid, and mixing uniformly;
(16) color development: dripping the DAB color developing solution on the section, wherein the tissue section needs to be completely covered, observing and developing under a microscope, and washing with distilled water to stop developing;
(17) counterdyeing: counterstaining with hematoxylin for 3min, and differentiating with hydrochloric acid and ethanol;
(18) sealing: soaking in 75% ethanol for 2min, 85% ethanol for 2min, 95% ethanol for 2min, soaking in anhydrous ethanol for 2min, soaking in xylene for 15min, replacing xylene, soaking for 15min, and sealing with neutral resin;
(19) and (4) interpretation of results: the stained tissue sections to be tested were observed under a microscope, the positive results were stained in the form of brown yellow particles, and 5 high-power fields (10 × 40) were randomly selected to estimate the proportion of positive cells.
FIG. 2D shows the expression of the cellular marker HES6 in normal stomach tissue, pre-cancerous stomach tissue. It can be seen that HES6 positive cells were 0 in normal stomach tissue, while their proportion increased to 10% in pre-gastric lesions.
Finally, as a further example material, fig. 2E and 2F show the co-expression pattern between the cellular marker HES6 in independent precancerous lesion samples and the classical precancerous lesion marker MUC2 and the proliferation cell marker KI67, respectively. . As can be seen in fig. 2E, HES6 was partially co-expressed with the classic pre-gastric lesion marker MUC 2; as can be seen in fig. 2F, HES6 encircled the proliferating cell marker KI67 in tissue. Therefore, the HES6 positive cells were considered to be cells in the early stage of precancerous lesions of gastric cancer.
Diagnosis of stomach cancer in stomach tissue and blood sample
First, the inventors evaluated the expression of 13 gastric cancer very early cell marker molecules (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in different cell types in individual early gastric cancer samples. As a result, as shown in FIG. 3A, these cell markers were specifically expressed in cancer cells at high levels.
Next, the inventors evaluated the expression of 13 gastric cancer very early cell markers (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent gastritis (19 cases), pre-gastric lesions (39 cases), and early gastric cancer (19 cases) samples. The results are shown in fig. 3B, where these cell markers are expressed in significantly lower amounts in gastritis than in the dysplastic stage (precancerous lesions) and in the gastric precancerous stage. The mean expression of the molecules is used as a molecular marker (signature), and the molecular marker shows a gradually increasing change trend in the transformation of gastritis-low-grade dysplasia-high-grade dysplasia-early gastric cancer, so that the fact that the high expression of the molecules in gastric tissues is closely related to the generation of gastric cancer is suggested, and the molecular marker can be used as an index for evaluating the generation risk of gastric cancer. The sample data involved in this validation is from public data (GSE 55696).
Again, the inventors evaluated the expression of 13 gastric cancer very early cell markers (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in blood samples of independent gastritis, gastric cancer patients. The results are shown in fig. 3C, where the expression levels of these cell markers in the serum of gastric cancer patients were significantly increased compared to the normal control samples. The mean expression level of the above molecules was used as a molecular marker (signature), and it was found that the value was significantly increased in cancer samples as compared with normal samples. It is suggested that the high expression of the above molecules in serum is also closely related to the occurrence of gastric cancer, and can be used as an index for evaluating the risk of gastric cancer. The sample data involved in this validation is from public data (GSE 64916).
Example 2
The present inventors obtained the expression of five representative components (KLK10, KRT17, TIMP1, MAP17 and KLK6) in early cancer cell markers in normal stomach tissue, precancerous lesions of stomach, early cancerous lesions and progressive cancerous lesions, based on the precancerous lesion cell markers (11) in the immunohistochemical test kit (20) of the present invention. The kit uses immunohistochemical staining (IHC) to measure the expression level of the marker. Paraffin-embedded surgical specimens were fixed with 10% formalin buffer and the tissue sections were 4 μm/piece.
The kit in this example comprises the following components:
(1) reagent A: sealing liquid, 10% goat serum;
(2) and (3) reagent B: diluted ready-to-use anti-Klk 10 primary antibody (any one of KRT17, TIMP1, MAP17 and KLK6 antibody in Table 1 may be used);
(4) and (3) reagent C: anti-goat biotinylated secondary antibody;
(5) and (3) reagent D: streptavidin-labeled HRP;
(6) and (3) reagent E: concentrating DAB substrate solution by 20 times;
(7) and (3) reagent F: concentrating DAB substrate buffer solution by 20 times;
(8) reagent G: the DAB chromogenic solution was concentrated 20-fold.
According to an embodiment of the present invention, the reagent B is originally packaged in the container and is dispensed with the ready-to-use antibody, and the dilution ratio is 1: 200 of a carrier; reagent G is originally packaged and is imported to split the ready-to-use antibody, and the dilution multiple is 1: 400, respectively; the reagent A, C, D, E, F, G is packaged by original package.
In addition to the above reagents contained in the kit, the inventors also self-formulated the following reagents (these reagents are also commercially available):
(1) distilled or deionized water;
(2)3％H2O2；
(3) xylene;
(4) 75%, 85%, 95% alcohol and absolute ethyl alcohol;
(5)10mM TBS solution (pH 7.2-7.4): 1.21g of trihydroxyaminomethane, 7.6g of sodium chloride, 800mL of distilled water, adjusting the pH value to 7.2-7.4 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(6)10mM pH6.0 citrate buffer: 0.38g of citric acid, 2.45g of trisodium citrate, 900mL of distilled water, adjusting the pH value to 6.0 by concentrated hydrochloric acid, and finally fixing the volume to 1000 mL;
(7) a hematoxylin solution;
(8) a neutral resin.
The expression of the markers in the tissues is detected by using the kit:
(1) tissue embedding: fixing a tissue specimen to be detected with 10% neutral formalin for 2h, repeatedly washing with running water to remove a fixing solution, putting the specimen into 75% alcohol overnight, then performing gradient dehydration with alcohol, wherein the alcohol content is 75% for 1h, the alcohol content is 85% for 1h, the alcohol content is 95% for 1h, and the absolute ethyl alcohol is used for 2 times, each time is 1.5h, then putting the specimen into xylene to be soaked for 1.5h, soaking the specimen in wax in a 60 ℃ oven for 1h for embedding, and preserving the specimen at 4 ℃ for later use after cooling;
(2) paraffin section: trimming a wax block, adjusting a slicer (SLEE paraffin slicer CUT5062), setting the slice thickness to be 3-4 mu m, continuously slicing, floating and flattening in warm water at 60 ℃, and flatly paving on a glass slide coated with cationic resin;
(3) baking slices: placing the slices to be sliced on a slicing frame, and baking for at least 1h in a constant-temperature oven at 60 ℃;
(4) dewaxing: dewaxing the slices in a container containing xylene for 3 times (i.e. xylene I, xylene II and xylene III) each for 10 min;
(5) hydration: hydrating the slices with descending ethanol, wherein the ethanol content is 5min, 95% ethanol 2 times (2 min each time), and 85% ethanol 2 min; 75% ethanol for 2min, and distilled water for 1 min;
(6) antigen retrieval: adding 1000ml of citric acid buffer solution into a pressure cooker, immersing the slicing frame with slices into the buffer solution, restoring at high temperature and high pressure for 2min and 45 sec, and washing with TBS for 3 times, each time for 2 min;
(7) dripping 3% H2O2 on the slices, standing at room temperature for 15min, washing with TBS for 3 times, each time for 2 min;
(8) and (3) sealing: dripping the reagent A on the section, completely covering the tissue section, incubating at room temperature for 10min, and sucking the liquid without washing;
(9) adding a primary antibody: dripping reagent B into different slices respectively, covering tissue slices completely, and incubating at 37 deg.C for 2hr or 4 deg.C overnight;
(10) washing: TBS-T wash (3X 5 min);
(11) adding a secondary antibody: reagent C (biotinylated secondary antibody is dripped) and is required to completely cover the tissue slices, and the tissue slices are incubated in a 37 ℃ wet box for 30 min;
(12) washing: TBS washing for 5min 3 times;
(13) adding HRP-SA: dropwise adding a reagent D (streptavidin-labeled HRP) to completely cover the tissue slices, and incubating for 30min in a 37 ℃ wet box;
(14) washing: TBS washing for 5min 3 times;
(15) preparing a DAB color developing solution: taking a dyed slice as an example, taking 2.5ul of reagent E into 50ul of distilled water to be mixed uniformly, then respectively adding 2.5ul of reagent F and 2.5ul of reagent G into the liquid, and mixing uniformly;
(16) color development: dripping the DAB color developing solution on the section, wherein the tissue section needs to be completely covered, observing and developing under a microscope, and washing with distilled water to stop developing;
(17) counterdyeing: counterstaining with hematoxylin for 3min, and differentiating with hydrochloric acid and ethanol;
(18) sealing: soaking in 75% ethanol for 2min, 85% ethanol for 2min, 95% ethanol for 2min, soaking in anhydrous ethanol for 2min, soaking in xylene for 15min, replacing xylene, soaking for 15min, and sealing with neutral resin;
(19) and (4) interpretation of results: the stained tissue sections to be tested were observed under a microscope, the positive results were stained in the form of brown yellow particles, and 5 high-power fields (10 × 40) were randomly selected to estimate the proportion of positive cells.
The expression of five representative marker molecules (KLK10, KRT17, TIMP1, MAP17, and KLK6) in stomach cancer very early cells in normal stomach tissue, dysplasia (precancerous lesions), and early stage stomach cancer is shown in fig. 3D. Among them, the cells of the stomach cancer in the very early stage identified by the representative marker molecules in the precancerous lesion of the stomach cancer are circled. It can be seen that the very early gastric cancer cells begin to appear in the dysplastic stage and persist in the early cancer.
Furthermore, the content of cells in the very early stage of gastric cancer in each stage is calculated by the precancerous lesion and early cancer cell counting device (32) in the early diagnosis system of the present invention. The proportion distribution of positive cells marked by the gastric cancer very early cell representative marker (KLK10) in pre-gastric lesion, early gastric cancer, and progressive gastric cancer is shown in fig. 3E. It can be seen that KLK10 positive cells were not present in the pre-gastric lesion stage samples, whereas the proportion was 5% in the early gastric cancer samples and increased to 35% in the advanced samples.
Application of gastric cancer very early cell marker molecule in evaluation of postoperative recurrence risk of gastric cancer
The inventors evaluated the association of representative components in the gastric cancer very early cell markers (KLK10, SLC11a2, KLK7 and TIMP1) with the risk of postoperative recurrence of gastric cancer. These data were derived from 431 gastric cancer patients who recorded time to relapse after surgery. As shown in FIG. 4, the above-mentioned cell markers were significantly correlated with the risk of recurrence and survival after gastric cancer (p < 0.05). The mean expression quantity of the stomach cancer very early cell marker molecules in a sample is used as a molecular marker (signature), and the signature is found to be remarkably related to postoperative recurrence of the stomach cancer (p <0.01), so that the stomach cancer very early cell marker can be used as a marker for evaluating the recurrence risk of the stomach cancer. The sample data involved in this validation is from public data (TCGA).
Application of stomach cancer very early cell marker molecule in assessing pancreatic cancer, intestinal cancer and esophageal cancer risks
The present inventors have also validated the use of gastric cancer very early cell marker molecules (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) to assess the risk of transformation of other organs of the digestive system (pancreas, esophagus and intestine) from precancerous lesions into cancer.
1. Application of a gastric cancer very early cell marker molecule in assessing intestinal cancer risk. The present inventors evaluated the expression of 13 gastric cancer very early cell markers (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent normal intestinal tissue (38 cases), precancerous lesions (29 cases), and intestinal cancer (27 cases) samples. As a result, as shown in fig. 5A, the expression level of these gastric cancer very early cell markers in normal intestinal tissues was significantly lower than that in the dysplasia stage (pre-intestinal cancer lesion) and intestinal cancer stage (p < 0.05). The mean value of the expression amount of the molecules in a sample is used as a molecular marker (signature), and the molecular marker shows a gradually increasing change trend in the transformation process of normal intestine, low-grade dysplasia, high-grade dysplasia and intestinal cancer, and indicates that the high expression of the molecules in intestinal tissues is closely related to the generation of the intestinal cancer and can be used as an index for evaluating the risk of the intestinal cancer. The sample data involved in this validation comes from public data (GSE 37364).
2. Application of gastric cancer very early cell marker molecules in evaluating esophageal cancer risks.
Next, the inventors evaluated the expression of 13 gastric cancer very early cell markers (KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent normal esophageal (5 cases), precancerous (4 cases), and esophageal cancer (3 cases) samples. As a result, as shown in FIG. 5B, the expression level of these gastric cancer very early cell markers in normal esophagus was lower than that in the dysplastic stage (precancerous lesion) and in the esophageal cancer stage. The high expression of the molecule in the esophagus is suggested to have close correlation with the generation of esophageal cancer, and the molecule can be used as an index for evaluating the risk of esophageal cancer generation. The sample data involved in this validation is from public data (GSE 19529).
3. Application of representative molecules in stomach cancer very early cell markers in assessing pancreatic cancer risks.
The present inventors evaluated the expression of representative molecules (ECM1, KRT17, TIMP1, and MMP7) in the gastric cancer very early cell markers in independent normal pancreatic (8 cases), intraepithelial neoplasia (6 cases), and pancreatic cancer (12 cases) samples. As a result, as shown in fig. 5C, the expression level of these representative marker molecules for the very early gastric cancer cells was significantly lower in normal pancreatic tissues than in the intraepithelial neoplastic stage (precancerous lesion) and pancreatic cancer. The mean expression of the molecules is used as a molecular marker (signature), and the molecular marker is found to show a gradually increasing change trend in the transformation process of normal pancreas-pancreatic intraepithelial neoplasia-pancreatic cancer, which indicates that the high expression of the molecules in pancreatic tissues has close correlation with the occurrence of pancreatic cancer and can be used as an index for evaluating the occurrence risk of pancreatic cancer. The sample data involved in this validation is from public data (GSE 43288).
Reference documents:
1.de Vries AC,van Grieken NCT,Looman CWN,Casparie MK,de Vries E,Meijer GA,Kuipers EJ.(2008)Gastric cancer risk in patients with premalignant gastric lesions:A nationwide cohort study in the Netherlands.GASTROENTEROLOGY,134,945-952.
2.ITOH H,OOHATA Y,NAKAMURA K,NAGATA T,MIBU R,NAKAYAMA F.(1989)COMPLETE 10-YEAR POSTGASTRECTOMY FOLLOW-UP OF EARLY GASTRIC-CANCER.AMERICAN JOURNAL OF SURGERY,158,14-16.
Claims (5)
1. use of an agent for detecting the expression level of a marker in the manufacture of a product for determining dysplasia, thereby determining the risk of gastritis-gastric precancerous lesion transition, wherein the marker comprises KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B, wherein the dysplasia comprises low-grade and/or high-grade dysplasia.
2. Use according to claim 1, characterized in that:
the reagent utilizes immunohistochemical staining or enzyme-linked immunosorbent to measure the expression level of the marker.
3. Use according to claim 1 or 2, characterized in that:
the product is one selected from a chip, a preparation and a kit.
4. A product for determining dysplasia, which comprises a reagent that detects the expression level of a marker in a sample, wherein the marker comprises KLK10, SLC11a2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B, wherein the dysplasia comprises low-grade dysplasia and/or high-grade dysplasia.
5. The product of claim 4, wherein the product is one selected from a chip, a formulation, and a kit.
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Application publication date: 20201113 Assignee: Beijing Timely Biotechnology Co.,Ltd. Assignor: TSINGHUA University Contract record no.: X2022110000081 Denomination of invention: A very early cell marker for gastric cancer and early cell marker for precancerous lesions of gastric cancer and its application in diagnostic kits Granted publication date: 20210803 License type: Common License Record date: 20221118 |