WO2013046760A1 - Stomach cancer inspection method and inspection kit - Google Patents

Abstract

The problem addressed by the present invention is to provide a stomach cancer inspection method that is more convenient and has high sensitivity and specificity. The present invention provides a stomach cancer or Helicobacter pylori bacterial infection inspection method including a step for measuring trefoil factor family (TFF) levels in the blood serum of a subject.

Description

Test method and test kit for gastric cancer

The present invention relates to a method for examining gastric cancer including a step of measuring the amount of trefoil factor family (TFF) protein in the serum of a subject.

Conventionally, gastric cancer screening is performed by gastric fluoroscopy or gastroscopy with barium. In Japan, screening by barium or gastroscopy is obligatory for those over 40 years old, but the consultation rate is still low. One cause of this is the complexity and pain of inspection techniques and physical burden.
Moreover, there is a pepsinogen method as a gastric cancer screening method using blood (Non-Patent Document 1), but the sensitivity and specificity are not sufficient. Further, the pepsinogen method measures the ratio of pepsinogen I and pepsinogen II in serum, and the cost increases because two factors must be measured.

Gastric cancer is still the fourth leading cause of cancer death worldwide. The possibility of cure depends on the stage of gastric cancer, and early detection is the most effective means of reducing mortality from gastric cancer. Therefore, there is a need for a method for examining gastric cancer that is simpler and more sensitive and specific.

For example, it is useful to have a marker that can be detected in a blood sample. There are reports on proteins whose expression increases in gastric cancer tissues, but in order to be detected in blood samples, they must enter the blood circulation from gastric cancer tissues and not be metabolized in the liver, which is useful so far No marker has been found.

Miki K. et al. Adv Exp Med Biol 1995; 362: 139-143. Plaut AG. N Engl J Med 1997; 336: 506-507 Ribieras S. et al. Biochim Biophys Acta 1998; 1378: F61-F77 Kjellev S. Cell Mol Life Sci 2009; 66: 1350-1369 Wong WM. Et al. Gut 1999; 44: 890-895

It is an object of the present invention to provide a method for examining gastric cancer that is simpler and has higher sensitivity and specificity.

As a result of repeated researches to solve the above problems, the present inventors have found that the concentration of trefoil factor family (TFF) in serum is significantly different between gastric cancer patients and non-gastric cancer patients. And it discovered that a test | inspection with high specificity could be performed, and came to complete this invention.
That is, the present invention
[1] A method for examining gastric cancer or Helicobacter pylori infection, which comprises a step of measuring a trefoil factor family (TFF) level in the serum of a subject;
[2] A method for examining gastric cancer, wherein the TFF is TFF1 or TFF3;
[3] A method for testing Helicobacter pylori infection, wherein the TFF is TFF1;
[4] The method according to any one of [1] to [3] above, wherein the step of measuring the serum TFF level is performed by immunoassay using an anti-TFF protein antibody;
[5] The method according to any one of [1] to [4] above, wherein the method for examining gastric cancer has a cutoff value of serum TFF1 level of 0.1 ng / mL to 10 ng / mL;
[6] A method for examining gastric cancer according to any one of [1] to [5] above, wherein the serum TFF3 level has a cutoff value of 1 ng / mL to 15 nm / mL;
[7] A kit for testing gastric cancer or Helicobacter pylori infection, comprising an anti-TFF antibody;
[8] The kit according to [7], wherein the anti-TFF antibody is an anti-TFF1 antibody or an anti-TFF3 antibody.

According to the test method of the present invention, gastric cancer with high sensitivity and specificity can be tested by a simple and painless method of measuring the TFF concentration in serum prepared from the blood of a subject. In particular, if TFF1 and TFF3 are used, the sensitivity and specificity are significantly higher than those of the conventional pepsinogen method, and the cost can be reduced as compared with the pepsinogen method that measures the amount of two types of markers. Such a test is considered to increase the consultation rate, leading to early detection of gastric cancer, and possibly reducing the mortality from gastric cancer.

FIG. 1 shows the results of measuring the serum TFF1, TFF2, and TFF3 levels of gastric cancer patient group and control group (positive or negative for H pylori infection) by ELISA. FIG. 2 shows the results of ROC analysis based on the measurement results of FIG. FIG. 2A is an ROC curve for serum TFF levels for determining the presence or absence of H pylori infection. FIG. 2B is an ROC curve for serum TFF levels to distinguish gastric cancer patients from non-gastric cancer patients. FIG. 2C is an ROC curve for serum TFF levels for discriminating H pylori infection negative gastric cancer patients from H pylori infection negative non-gastric cancer patients. FIG. 2D is an ROC curve for serum TFF levels for distinguishing H pylori positive gastric cancer patients from H pylori positive non-gastric cancer patients. FIG. 3 shows the results of determining the sensitivity, specificity, and odds ratio of the gastric cancer test method using serum TFF1-3 levels, pepsinogen I / II, and anti-H pylori IgG as indices. FIG. 4 shows the results of comparing the serum TFF3 level with the age-matched control group for the second cohort (30s-60s) of gastric cancer patients. FIG. 5 is an ROC curve for each of the pepsinogen method, serum TFF3 level, and a combination of these methods for discriminating gastric cancer patients. FIG. 6A shows the results of measuring serum TFF1 levels in differentiated gastric cancer patients and undifferentiated gastric cancer patients. FIG. 6B shows the results of measuring serum TFF2 levels in differentiated gastric cancer patients and undifferentiated gastric cancer patients. FIG. 6C shows the results of measuring serum TFF3 levels in differentiated gastric cancer patients and undifferentiated gastric cancer patients. FIG. 7 shows the results of measurement of serum TFF levels before and after one week from gastrectomy in gastric cancer patients. FIG. 8 shows the results of measuring the serum TFF level after 3 months or more have passed since gastrectomy in a stomach cancer patient.

(Test method for gastric cancer and Helicobacter pylori infection)
One aspect of the method for examining gastric cancer according to the present invention includes a step of measuring the amount of TFF protein in the serum of a subject.
TFF is a family consisting of three stable proteins, TFF1, TFF2, and TFF3, each of 12 kDa to 22 kDa, and is known to be secreted from the digestive tract of mammals (Non-Patent Documents 2 to 5). .
TFF was named after the three loop structures that TFF1-3 have in common. This loop structure makes TFF extremely stable against proteolysis and has excellent acid resistance and heat resistance. TFF1-3 are widely expressed in a tissue-specific manner in the gastrointestinal tract. TFF1 is expressed in the surface mucous cells of the gastric mucosa, TFF2 is expressed in mucus neck cells, deep antral gland cells, and Brunner's gland in the duodenum, and TFF3 is expressed in goblet cells of the small and large intestines .

Gastric cancer follows chronic atrophic gastritis caused by chronic Helicobacter pylori infection. Mucosal histological changes in chronic atrophic gastritis include foveolar hyperplasia, Spasmolytic polypeptide (TFF2) -expressing metaplasia (SPEM), and secreted atrophy with intestinal metaplasia.
Foveolar hyperplasia is an extension of a gastric pit consisting of pit surface mucus cells that originally express TFF1. SPEM is an antral phenotype lineage characterized by TFF2-positive cells in the stomach fundus. SPEM is common in the gastric mucosa surrounding gastric cancer, and TFF2 is positive in 58% of early gastric cancer. Intestinal metaplasia is characterized by intestinal phenotype cells that occur in the gastric mucosa and is considered to be a precancerous lesion of intestinal gastric cancer. TFF3 is also expressed in the intestinal metaplasia of the stomach in addition to the aforementioned goblet cells of the small and large intestines.

As shown in the examples described later, serum TFF1 levels tend to be significantly higher in the positive group than in the Helicobacter pylori infection negative group, and higher in the gastric cancer patient group than in the non-gastric cancer patient group. It was seen. Thus, serum TFF1 levels are thought to be particularly useful for testing for Helicobacter pylori infection. The test for Helicobacter pylori is generally carried out by a method using an anti-Helicobacter pylori antibody. However, Helicobacter pylori dies when atrophic gastritis worsens and cannot be detected by this method. If detected by the serum TFF1 level, it is possible to detect the presence or absence of infection even after such Helicobacter pylori dies.
TFF1 exhibits high sensitivity, specificity, and odds ratio in discrimination between Helicobacter pylori infection negative groups and gastric cancer patients, and is also useful for gastric cancer testing.

Serum TFF2 and serum TFF3 were significantly higher in the positive group than in the Helicobacter pylori infection negative group, and significantly higher in the gastric cancer patient group than in the non-gastric cancer patient group. In particular, TFF3 shows high sensitivity, specificity, and odds ratio in discrimination between non-gastric cancer patients and gastric cancer patients regardless of the presence or absence of Helicobacter pylori infection, and is useful for gastric cancer testing.

As the serum used in the test according to the present invention, one prepared from blood collected from a subject according to a conventional method can be used.

The step of measuring TFF in serum can be performed using any method for detecting and measuring a specific protein in a liquid. For example, immunoassay (including agglutination and turbidimetry), Western blotting , Surface plasmon resonance (SPR) method and the like, but are not limited thereto. An immunoassay that measures the amount of TFF using an antigen-antibody reaction between an anti-TFF antibody and TFF is particularly simple and preferred.

The immunoassay uses a detectably labeled anti-TFF antibody and / or an antibody (secondary antibody) against the detectably labeled anti-TFF antibody. Depending on the antibody labeling method, enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay (CLIA), fluorescent enzyme immunoassay (FLEIA), chemiluminescent enzyme They are classified into immunoassay (CLEIA), electrochemiluminescence immunoassay (ECLIA) and the like, and any of these can be used in the method of the present invention.
In the ELISA method, enzymes such as peroxidase and alkaline phosphatase, in the RIA method, radioactive substances such as ¹²⁵ I, ¹³¹ I, ³⁵ S and ³ H, in the FPIA method, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine Fluorescent substances such as isothiocyanate and near-infrared fluorescent materials, and CLIA methods use antibodies labeled with luminescent substances such as luciferase, luciferin, and aequorin. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can also be detected.
In an immunoassay, anti-TFF antibody can be detected by labeling with biotin and binding avidin or streptavidin labeled with an enzyme or the like.

Among immunoassays, the ELISA method using an enzyme label is preferable because it can easily and rapidly measure an antigen.
The ELISA method includes a competitive method and a sandwich method. In the competitive method, an anti-TFF antibody is immobilized on a solid phase carrier such as a microplate, and a serum sample and enzyme-labeled TFF are added to cause an antigen-antibody reaction. Once washed, it reacts with the enzyme substrate, develops color, and the absorbance is measured. If the serum sample has a large amount of TFF, the color development becomes weak. If the serum sample has a small amount of TFF, the color development becomes strong. Therefore, the TFF level can be obtained using a calibration curve.
In the sandwich method, an anti-TFF antibody is immobilized on a solid phase carrier, a serum sample is added and reacted, and then an anti-TFF antibody that recognizes another epitope labeled with an enzyme is further added and reacted. After washing, the amount of TFF can be determined by reacting with the enzyme substrate, causing color development, and measuring the absorbance. In the sandwich method, an antibody immobilized on a solid phase carrier is reacted with TFF in a serum sample, an unlabeled antibody (primary antibody) is added, and an antibody against the unlabeled antibody (secondary antibody) is enzyme-labeled. Further, it may be added.
When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used as the enzyme substrate. In this case, p-nitropheny phosphate (NPP) or the like can be used.

In the present specification, the “solid phase carrier” is not particularly limited as long as it can immobilize the antibody, and is made of a microtiter plate made of glass, metal, resin, etc., substrate, beads, nitrocellulose membrane, nylon membrane, PVDF Examples include a membrane, and the target substance can be immobilized on these solid phase carriers according to a known method.

In the immunoassay, an agglutination method is also preferable as a method for easily detecting a trace amount of protein. Examples of the aggregation method include a latex aggregation method in which latex particles are bound to an antibody.
When anti-TFF antibody is bound to latex particles and mixed with a serum sample that has been appropriately treated, if TFF is present, antibody-bound latex particles aggregate. Therefore, the concentration of the antigen can be determined by irradiating the sample with near-infrared light and quantifying the aggregate by measuring the absorbance (turbidimetric method) or the scattered light (Hipple method).

Anti-TFF antibodies can be prepared according to known methods for both monoclonal antibodies and polyclonal antibodies. The monoclonal antibody is, for example, an antibody-producing cell isolated from a non-human mammal immunized with each of TFF1 to 3 or a fragment thereof, and this is fused with a myeloma cell to produce a hybridoma, and the antibody produced by this hybridoma Can be obtained by purification. Polyclonal antibodies can also be obtained from the sera of animals immunized with each of TFF1-3 or fragments thereof. An existing antibody may be used as the anti-TFF antibody.

In this specification, “gastric cancer” is used in the usual sense, and includes gastric cancer in any state regardless of pathological classification, morphology, depth of advance, stage indicated by progression, and the like.

In the present specification, “inspection” means to examine a sample collected from a subject in order to obtain information necessary for diagnosis, and the inspection method of the present invention can be performed by, for example, an inspection company.

One aspect of the test method of the present invention is a gastric cancer test method comprising a step of measuring serum TFF1 level, wherein the cutoff value of serum TFF1 level is 0.1 ng / mL to 10 ng / mL, 0.3 ng / mL to 8 ng / mL, 0.5 ng / mL to 5 ng / mL, 0.8 ng / mL to 1.2 ng / mL, and the like. By setting the cut-off value within this range, it is possible to make a test with sufficiently high sensitivity, specificity, and odds ratio.

One aspect of the test method of the present invention is a gastric cancer test method comprising a step of measuring serum TFF3 level, wherein the cutoff value of serum TFF3 level is 1 ng / mL to 15 ng / mL, 2 ng / mL to 10 ng / mL 3 ng / mL to 5 ng / mL, 3.4 ng / mL to 3.8 ng / mL, and the like. By setting the cut-off value within this range, it is possible to make a test with sufficiently high sensitivity, specificity, and odds ratio.

The inspection method according to the present invention may be combined with a conventionally used pepsinogen method. Here, the pepsinogen method is a method for measuring pepsinogen I and II in serum using an anti-pepsinogen antibody, and is positive when the serum pepsinogen I level is less than 70 ng / mL and the serum pepsinogen I / II ratio is less than 3. Determined.
As shown in the Examples described later, sensitivity and specificity can be further increased by combining the method for measuring serum TFF3 level and the pepsinogen method.

(Diagnosis method for gastric cancer and Helicobacter pylori infection)
In addition, this invention also includes the diagnostic method of gastric cancer or Helicobacter pylori infection including the process of measuring the trefoil factor family (TFF) level in the serum of a subject.
Here, “diagnosis” means that the medical practitioner determines whether the subject suffers from a specific disease based on the test result or the like.
Of the terms used for the method for diagnosing gastric cancer and Helicobacter pylori infection according to the present invention, the terms used in the aforementioned test method according to the present invention are synonymous with them, and the description thereof is omitted here.

(Gastric cancer test kit)
The test kit for gastric cancer according to the present invention is a kit for performing a test for gastric cancer using the test method described above, and includes at least one of an anti-TFF1 antibody, an anti-TFF2 antibody, and an anti-TFF3 antibody.
The test kit of the present invention includes reagents and devices necessary for measuring serum TFF levels by immunoassay using an antigen-antibody reaction between an anti-TFF antibody and TFF.

One embodiment of the test kit is for measuring TFF by the sandwich method; a microtiter plate; an anti-TFF antibody for capture; an anti-TFF antibody labeled with alkaline phosphatase or peroxidase; and an alkaline phosphatase substrate (NPP Or a substrate of peroxidase (DAB, TMB, OPD, etc.).
The capture antibody and the labeled antibody recognize different epitopes.
In such a kit, first, a capture antibody is immobilized on a microtiter plate, a serum sample is appropriately diluted and added thereto, and then incubated, and the sample is removed and washed. Next, the labeled antibody is added and then incubated, and the substrate is added to cause color development. The TFF level can be determined by measuring the color development using a microtiter plate reader or the like.

Another embodiment of the test kit is for measuring TFF by a sandwich method using a secondary antibody; a microtiter plate; an anti-TFF antibody for capture; an anti-TFF antibody as a primary antibody; a secondary The antibody includes an anti-TFF antibody labeled with alkaline phosphatase or peroxidase; and an alkaline phosphatase (NPP or the like) or a substrate of peroxidase (DAB, TMB, OPD or the like).
The capture antibody and the primary antibody recognize different epitopes.
In such a kit, first, a capture antibody is immobilized on a microtiter plate, a serum sample is appropriately diluted and added thereto, and then incubated, and the sample is removed and washed. Subsequently, the primary antibody is added to incubate and wash, and the enzyme-labeled secondary antibody is further added and incubated, and then the substrate is added to cause color development. The TFF level can be determined by measuring the color development using a microtiter plate reader or the like. By using a secondary antibody, the reaction is amplified and the detection sensitivity can be increased.

Another embodiment of the test kit includes a microtiter plate; an anti-TFF antibody as a primary antibody; an anti-TFF antibody labeled with alkaline phosphatase or peroxidase; and an alkaline phosphatase or peroxidase substrate.
According to such a kit, first, a microtiter plate is coated with a sample diluted to an appropriate concentration, and a primary antibody is added. After incubation and washing, an enzyme-labeled secondary antibody is added, incubation and washing are performed, and a substrate is added to cause color development. The TFF level can be determined by measuring the color development using a microtiter plate reader or the like.

It is preferable that each test kit further contains necessary buffer solution, enzyme reaction stop solution, microplate reader and the like.

Labeled antibodies are not limited to enzyme-labeled antibodies, but include radioactive substances ( ²⁵ I, ¹³¹ I, ³⁵ S, ³ H, etc.), fluorescent substances (fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, Near-infrared fluorescent materials, etc.), luminescent substances (luciferase, luciferin, aequorin, etc.), nanoparticles (gold colloid, quantum dots), etc. Alternatively, a biotinylated antibody can be used as a labeled antibody, and labeled avidin or streptavidin can be added to the kit.

Still another embodiment of the test kit of the present invention includes one for measuring the TFF level by the latex agglutination method. This kit contains anti-TFF antibody-sensitized latex, a serum sample and anti-TFF antibody are mixed, and the clump is quantified by an optical method. It is also preferable that an agglutination reaction plate for visualizing the agglutination reaction is included in the kit.
The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited thereto. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

1. Example 1
<Subject>
The subjects were 183 patients with gastric cancer who were treated from February 2006 to September 2008 in gastroesophageal surgery at the University of Tokyo Hospital. Blood samples were taken before treatment. Patients were categorized by early or advanced cancer, histology (differentiated, undifferentiated), number, depth, tumor size, lymph node metastasis, and clinical stage. The control group consisted of 280 healthy male and female donors who had undergone a medical examination at NTT Kanto Central Hospital from September to November 2006. The characteristics of patients and donors are shown in the table below.

As a second cohort to validate results, serum levels of TFF3 and pepsinogen were measured in 59 gastric cancer patients treated at Gastroesophageal Surgery at the University of Tokyo Hospital from August 2009 to March 2010 did. The age-matched control group consisted of 45 healthy male and female donors who had undergone a medical examination at NTT Kanto Central Hospital from January to April 2011. Patients in their 30s to 60s in the second cohort Compared with 15 people.
The group of patients before and after bariatric surgery was 20 patients who underwent bypass surgery by Roux-en-Y method at Vanderbilt University Medical Center.

<Measurement of serum TFF level>
Human TFF1, TFF2, and TFF3 expression plasmids were constructed and expressed according to conventional methods, and recombinant human TFF1, TFF2, and TFF3 were purified. This was used to immunize rabbits to obtain antisera against human TFF1, TFF2, and TFF3, respectively.
Serum TFF1, TFF2, and TFF3 levels were measured by ELISA using this antiserum. The sensitivity was 7 pg / mL for TFF1, 30 pg / mL for TFF2, and 30 pg / mL for TFF3. Each anti-TFF antibody reacted specifically and showed no cross-reactivity to other TFFs.

<Measurement of Serum Helicobacter Pylori IgG Level and Serum Pepsinogen Level>
The Helicobacter Pylori IgG level in serum was measured with Helicobacter Pylori IgG ELISA Kit (manufactured by Biohit Plc), and the infection state to H pylori was examined. Pepsinogen I level was measured with Pepsinogen I ELISA Kit (Biohit Plc.), And pepsinogen II level was measured with Pepsinogen II ELISA Kit (Biohit Plc.). All samples were analyzed in duplicate. When the IgG level exceeded 9.9 U / mL, it was diagnosed as being infected with H pylori.

<Statistical analysis>
All statistical analyzes were performed with JMP7 software (SAS Institute, Inc.). Mean t-test was used for comparison between the two groups. In each evaluation, an ROC curve (receiver operating characteristic curve) was used in order to obtain a cut-off value that can discriminate the state of the stomach. The discriminability was evaluated by the area under the ROC curve (area under the ROC curve). Sensitivity, specificity, and odds ratio were determined using the cut-off values thus determined. Subsequently, the median 95% confidence interval was determined. A two-sided p value of less than 0.05 was considered statistically significant.

<Result>
1. Serum TFF level FIG. 1 shows the serum TFF1, TFF2, and TFF3 levels of the stomach cancer patient group and the control group (H pylori infection positive group and negative group). H pylori infection was determined by serum anti-H pylori IgG levels.

1-1. TFF1 (Figure 1 left)
In the control group negative for H pylori infection, the serum TFF1 level was 0.57 ± 0.29 (median 0.51, range 0.18-3.10) ng / mL. In the control group positive for H pylori infection, the serum TFF1 level was 2.51 ± 1.52 (median 2.43, range 0.36-6.94) ng / mL, which was significantly higher than the H pylori negative control group. The serum TFF1 level in the gastric cancer patient group was 3.35 ± 3.06 (median 2.37, range 0.31-19.1) ng / mL, which was significantly higher than the H pylori negative control group. Compared with the H pylori positive control group, the serum TFF1 level of the gastric cancer patient group tended to be higher, but no significant difference was obtained.

1-2. TFF2 (in Fig. 1)
In the control group negative for H pylori infection, the serum TFF2 level was 2.88 ± 1.04 (median 2.7, range 0.53-7.2) ng / mL. In the control group positive for H pylori infection, the serum TFF2 level was 5.15 ± 2.41 (median 4.83, range 1.51-14.5) ng / mL, which was significantly higher than the H pylori negative control group. The serum TFF2 level in the gastric cancer patient group was 8.79 ± 16.2 (median 6.36, range 0.78-210) ng / mL, which was significantly higher than any of the control groups.

1-3. TFF3 (right in Fig. 1)
In the control group negative for H pylori infection, the serum TFF3 level was 2.72 ± 0.80 (median 2.56, range 1.22-5.30) ng / mL. In the control group positive for H pylori infection, the serum TFF3 level was 3.05 ± 1.10 (median 2.79, range 1.40-6.25) ng / mL, which was significantly higher than the H pylori negative control group. The serum TFF1 level in the gastric cancer patient group was 6.44 ± 6.19 (median 5.02, range 1.85-74.4) ng / mL, which was significantly higher than any of the control groups.
There were no significant differences in serum TFF3 levels between the 7 patients who developed the second cancer during the follow-up period and other patients.

2. Serum TFF level as marker for H pylori infection To examine the accuracy of diagnosis of H pylori infection using serum TFF level, ROC analysis was performed. The result is shown in FIG. 2A. FIG. 2A shows the ROC curve of the serum TFF level when comparing the H pylori infection positive group and the H pylori infection negative group determined by the serum anti-H pylori IgG level. The areas under the curves for TFF1, TFF2, and TFF3 were 0.952, 0.811, and 0.587, respectively.
The area under the ROC curve for TFF1 was 0.95. When the cutoff value was 1.0 ng / mL, the odds ratio of serum TFF1 level was 141.3, sensitivity was 87.8%, specificity was 95.6%, and serum TFF1 level correlated well with H pylori infection. The ROC curves for TFF2 and TFF3 did not show high sensitivity and specificity for H pylori infection.

3. Serum TFF levels as a predictor of gastric cancer ROC analysis was performed to examine the accuracy of gastric cancer diagnosis using serum TFF levels and pepsinogen I / II ratio. In the pepsinogen method, positive is determined when the serum pepsinogen I level is less than 70 ng / mL and the serum pepsinogen I / II ratio is less than 3.
FIG. 2B is an ROC curve showing the discrimination ability of gastric cancer patients by serum TFF level and pepsinogen I / II ratio. The areas under the ROC curves for TFF3, TFF1, pepsinogen I / II ratio, and TFF2 were 0.89, 0.84, 0.76, and 0.74, respectively. The positive predictive values of TFF1, TFF2, TFF3, and pepsinogen I / II ratio were 0.65, 0.67, 0.71, and 0.61, respectively, and the negative predictive values were 0.92, 0.81, 0.90, and 0.87, respectively.
The results of the ROC analysis show that TFF3 and TFF1 are more accurate indicators than the pepsinogen I / II ratio.

Next, ROC analysis was performed by dividing into a H pylori infection negative group and a positive group.
FIG. 2C shows the results of ROC analysis when a group of H pylori-negative gastric cancer patients is compared with a non-gastric cancer patient who is H pylori-negative. Compared to FIG. 2B, the area under the curve increased in all curves, but in particular, TFF3 and TFF1 were recognized as better markers than TFF2 and pepsinogen I / II. The positive predictive values for TFF1, TFF2, TFF3, and pepsinogen I / II ratio were 0.92, 0.84, 0.78, and 0.81, and the negative predictive values were 0.93, 0.87, 0.94, and 0.89, respectively.
FIG. 2D shows the results of ROC analysis when comparing H pylori positive gastric cancer patient groups with H pylori positive non-gastric cancer patients. The area under the curve of TFF1 and pepsinogen I / II was small, suggesting that it is not a powerful gastric cancer marker when H pylori infection is positive. The area under the curve for TFF2 was also small. On the other hand, the area under the curve of TFF3 was large, indicating that TFF3 can be a useful marker for detecting gastric cancer regardless of the presence or absence of H pylori infection. The positive predictive values for TFF1, TFF2, TFF3, and pepsinogen I / II ratio were 0.62, 0.81, 0.78, and 0.61, respectively, and the negative predictive values were 0.68, 0.59, 0.81, and 0.51.

FIG. 3 shows sensitivity, specificity, and odds ratio for TFF, pepsinogen method, and anti-H pylori IgG. Cut-off values were 1.0 ng / mL for serum TFF1, 4.0 ng / mL for serum TFF2, and 3.6 ng / mL for serum TFF3.
The odds ratio of serum TFF1 level was 18.1 (10.5-31.0), sensitivity was 89.6%, and specificity was 67.7%. The odds ratio of serum TFF3 level was 18.1 (11.2-29.2), sensitivity was 80.9%, and specificity was 81.0%. TFF1 and TFF3 showed significantly higher odds ratios than the pepsinogen method.

Since the average age of the gastric cancer patient group was higher than that of the control group, the serum TFF3 level in each age group was compared. Serum RFF3 levels showed high odds ratios even when the patient and control groups were categorized by age.
To verify that serum TFF3 levels are high in gastric cancer patients, as a second cohort, 15 patients in their 30s and 60s were compared and analyzed with 45 age-matched control groups. The results are shown in FIG. Patients' serum TFF3 levels were also significantly higher compared to the age-matched control group (P <0.001).

4). Combinations of serum TFF3 level and pepsinogen method Among the 182 patients with gastric cancer, the number of patients diagnosed as positive or negative with serum TFF3 level and pepsinogen method is shown in the table below.

Of the 182 patients, 85 were not diagnosed with gastric cancer by the pepsinogen method. However, when combined with serum TFF3 levels, 69 of the 85 are diagnosed with possible gastric cancer. On the other hand, 8 out of 182 were negative at TFF3 level, but were diagnosed as positive by the pepsinogen method.
To verify this result, 59 gastric cancer patients were analyzed in the second cohort by serum TFF3 levels and the pepsinogen method. With the pepsinogen method, 27 of 59 patients were negative, but when combined with the TFF3 method, 26 of the 27 patients were positive. FIG. 5 shows ROC curves for the pepsinogen method only, the TFF3 method only, and combinations thereof. The positive and negative predictive values were 0.608 and 0.874 for the pepsinogen method, 0.709 and 0.895 for the TFF3 method, and 0.828 and 0.878, respectively, when both were combined.

5. Serum TFF levels and gastric cancer histology To examine the effects of gastric cancer on TFF serum levels, the difference in serum TFF levels by gastric cancer histology was measured. The results are shown in FIG. TFF1 and TFF2 levels were significantly lower in differentiated gastric cancer patients compared to undifferentiated gastric cancer patients (TFF1: P = 0.018, TFF2: P = 0.016). On the other hand, there was no significant difference in serum TFF3 levels between differentiated and undifferentiated patients (P = 0.312). There were no differences in serum TFF levels due to other pathological classifications of gastric cancer (number, depth, size, lymph node metastasis, clinical stage) (data not shown).

6). Serum TFF levels before and after gastrectomy To investigate the cause of elevated serum TFF levels, serum TFF levels before and after gastric cancer resection were compared. Forty-six patients were measured for serum TFF1, TFF2, and TFF3 levels before and one week after surgery. Nine patients underwent total gastrectomy, 32 underwent pyloric gastrectomy, and 5 underwent cardiotomy.
FIG. 7 shows the distribution of serum TFF levels before and after gastrectomy. Serum TFF1 and serum TFF2 levels were less than half a week after surgery. On the other hand, there was no significant change in serum TFF3 level. One week after surgery, the patient is still in an acute inflammatory condition, and serum TFF levels were measured for more than 3 months thereafter, confirming that TFF1 and TFF2 levels remained low and TFF3 levels remained high (FIG. 8).
To verify that postoperative TFF3 levels may be due to changes in physiological food flow, 20 patients who underwent Roux-en-Y bypass surgery were treated with serum before and after surgery. TFF3 level was measured. Serum TFF3 levels were 7.13 ± 8.18 ng / mL before surgery and 5.32 ± 2.04 ng / mL after surgery. The patient showed higher TFF3 levels compared to the healthy control group, but no post-upregulation was seen.

2. Example 2
From October 2007 to December 2009, patients with gastric cancer who underwent endoscopy before surgery at Toshiba Hospital were included. Those with other cancers or serious illnesses were excluded from the subjects. The control group consisted of healthy men and women who underwent a medical examination at the Health Medical Center of Nomura Hospital from December 2008 to December 2009.
In the age / sex match group, the ratio of the patient group and the control group was 1: 3, the age was matched within 3 years in the patient group and the control group, and the sex was completely matched.
The serum TFF1 to 3 levels of the patient group and the control group measured by the same method as in Example 1 are as shown in the table below. The P values are both less than 0.0001, and there is a significant difference between the patient group and the control group. It was.

When the ROC analysis was performed on the result of TFF3 in the same manner as in Example 1, the area under the curve was 0.812.
The sensitivity and specificity were determined for TFF3 with a cutoff value of 7 ng / mL, which was 66.1% (63.6-68.6%) and 91.7% (90.1% -93.1%), respectively.
On the other hand, the sensitivity and specificity of the pepsinogen I / II ratio for this patient group and the control group were 67.2% (64.7-69.6%) and 81.7% (79.6-83.6%).
The TFF3 method was about 10% more specific and about 25% more positive than the pepsinogen method.

In addition, the sensitivity of the gastric cancer patient group was determined by dividing the cancer progression into the cancer progression and tissue type. The results are shown in the table below.

Claims (8)

A test method for gastric cancer or Helicobacter pylori infection, comprising a step of measuring a trefoil factor family (TFF) level in the serum of a subject. The method according to claim 1, wherein the TFF is TFF1 or TFF3. The method according to claim 1, wherein the TFF is TFF1. The method according to any one of claims 1 to 3, wherein the step of measuring the serum TFF level is performed by an immunoassay using an anti-TFF protein antibody. The method according to any one of claims 1 to 4, wherein the method is a method for examining gastric cancer, wherein the cutoff value of the serum TFF1 level is 0.1 ng / mL to 10 ng / mL. 6. The method according to any one of claims 1 to 5, which is a method for examining gastric cancer, wherein the cutoff value of serum TFF3 level is 1 ng / mL to 15 nm / mL. A test kit for gastric cancer or Helicobacter pylori infection containing an anti-TFF antibody. The kit according to claim 7, wherein the anti-TFF antibody is an anti-TFF1 antibody or an anti-TFF3 antibody.

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