WO2019069980A1 - Colorectal cancer detection method - Google Patents

Abstract

The present application provides a method for detecting colorectal cancer in a subject, said method including measuring the amount of immunoglobulin G (IgG) that is bound to red blood cells in a sample derived from the subject.

Description

Method of detecting colon cancer

The present invention relates to a method for detecting colon cancer.

Colorectal cancer is a generic term for carcinomas that occur in the large intestine (colon, rectum, anus). Methods of detection of colorectal cancer include fecal occult blood test and endoscopy, which have been shown to be useful in randomized controlled trials. However, since the fecal occult blood test detects cancer based on hemorrhage, it may miss colon cancer that does not accompany hemorrhage, and for the subject, it is complicated to collect stool prior to the test. Endoscopic examination is generally performed as a detailed examination of colon cancer, but it requires food restriction and pretreatment with laxative before the examination, and subjects such as inserting an endoscope into the intestine and biopsy accompanied by invasion, etc. The burden on the
Although a method for detecting DNA from blood and stools (Non-patent Document 1) has been developed as a method for detecting colon cancer, this method is economically costly, and its usefulness has not been confirmed. . In addition, a method of measuring the amount of autoantibodies by enzyme-linked immunosorbent assay (ELISA) method using the fact that autoantibodies against abnormal proteins expressed by colon cancer cells are produced (Non-patent Document 2) However, it is necessary to identify which abnormal protein is detected at the same time as the measurement of the antibody against a single abnormal protein is insufficient, and the operation is very complicated.

There is a need for a simple, low-cost and feasible method for detecting colorectal cancer which substitutes or complements the above method. In particular, since a burden on a subject is large in endoscopy, a simple method for detecting colorectal cancer that can be used to appropriately screen a subject who should undergo endoscopy is desired.

The disclosures of the prior art documents cited herein are all incorporated herein by reference.

An object of the present invention is to provide a simple method for detecting colorectal cancer that can replace or complement conventional detection methods.

The inventors of the present invention conducted intensive studies to solve the above problems and found that in colon cancer patients, the amount of immunoglobulin IgG bound on the erythrocyte membrane is significantly higher than in other cancer patients and healthy people. The present invention has been achieved.

In one embodiment, the present application provides a method of detecting colon cancer in a subject, comprising measuring the amount of immunoglobulin G (IgG) bound to red blood cells in a sample from the subject.

In yet another embodiment, the present application provides a detection kit for colon cancer, which comprises a labeled anti-IgG antibody for measuring the amount of IgG bound to erythrocytes.

The result of having measured the quantity of IgG couple | bonded with the erythrocyte in the colon cancer patient group, the stomach cancer patient group, and the healthy subject group by the flow cytometry method is shown. It is the ROC curve obtained by the logistic regression etc. which used the result of having measured the quantity of IgG couple | bonded with the erythrocyte in the colon cancer patient group and the healthy subject group by the flow cytometry method. The results of the Coombs test for a group of healthy people are shown. The results of the Coombs test on gastric cancer patient groups are shown. The results of the Coombs test for a group of colon cancer patients are shown.

As described in the following examples, the inventor has clarified the following matters as a result of measuring the amount of IgG bound to erythrocytes in blood samples of patients with colon cancer, stomach cancer and healthy subjects using flow cytometry. I made it.
(1) The amount of IgG bound to erythrocytes of colorectal cancer patients is significantly higher than the amount of IgG in healthy subjects.
(2) The amount of IgG bound to erythrocytes of colorectal cancer patients is significantly higher than that of gastric cancer patients.
(3) There is no significant difference between the amount of IgG in gastric cancer patients and the amount of IgG in healthy subjects.

In addition, as described in the examples below, also in the Coombs test, which is a test for detection of IgG bound to erythrocytes, in colon cancer patients, IgG bound to erythrocytes in comparison with healthy subjects and gastric cancer patients The trend of large amount was supported.
From these findings, it became clear that IgG bound to erythrocytes is useful for detection of colon cancer.

Anion exchanger 1 (AE1, also called band 3) is a membrane transport protein abundant in erythrocyte membranes. Usually, AE1 detects the carbon dioxide dissolved in blood on the erythrocyte membrane and functions to accelerate the release of oxygen from the hemoglobin in the erythrocyte. It is known that this AE1 is ectopically expressed in colon cancer cells (Non-patent Document 3).

Autoimmune hemolytic anemia (AIHA) is a pathological condition in which autoantibodies that react with antigens on erythrocyte membranes are produced, erythrocytes are damaged as a result of antigen-antibody reaction, and erythrocyte life is shortened significantly (hemolysis), resulting in anemia. .

As a result of studies in colorectal cancer patients having AIHA, the present inventors elucidated that AIHA in such colorectal cancer patients is caused by the production of autoantibodies against AE1. That is, it was revealed for the first time that autoantibodies (IgG) against AE1 are being produced in colon cancer patients. This research result is unpublished at the time of filing this application.

Normally, red blood cells circulate in the body while being exposed to oxidative stress, are aged, and are finally captured by macrophages. One of the mechanisms defining the life span of this red blood cell is opsonization by the reaction of AE1 with a natural antibody in the serum that recognizes it. A natural antibody (anti-AE1 IgG) in serum binds to AE1 on the cell membrane of senescent red blood cells, and senescent red blood cells are fed by macrophages having a receptor for the antibody (non-patent documents 4 and 5).

In addition to natural antibodies, autoantibodies bind to AE1 present in the erythrocyte membrane of colon cancer patients based on the finding that autoantibodies (IgG) against AE1 are produced in colon cancer patients, and as a result colon cancer patients It is inferred that IgG bound to the erythrocyte membrane is increasing. The estimation of the mechanism does not limit the present invention.

In one embodiment, the invention provides a method of detecting colon cancer in a subject comprising measuring the amount of IgG bound to red blood cells in a sample from the subject.

The subject in the method of the present invention is not particularly limited, and is preferably a mammal, more preferably a human. Such subjects may include, for example, those who have not been diagnosed with cancer (eg, those who have no disease being treated, or patients with diseases other than cancer) and those who have cancer other than colon cancer.

In the method of the present invention "detection" means to relate the amount of IgG bound to erythrocytes to the possibility of colon cancer in a subject. For example, based on the amount of IgG bound to erythrocytes, it is determined that the subject has or is likely to have colon cancer, or that cancer other than colon cancer has metastasized to colon or is likely to have it. Means

That is, the method of the present invention can be used to examine the possibility of having colon cancer, for example, at a health checkup, in persons who have not been diagnosed with cancer. In those who have cancer other than colon cancer, the method of the present invention can be used to investigate the possibility that the cancer has metastasized to the large intestine.

The sample derived from the subject used in the method of the present invention is not particularly limited as long as it is collected from the subject and contains red blood cells. Preferred examples include blood and lymph, and more preferably blood.

In the method of the present invention, the state is not particularly limited as long as “red blood cells” have a binding site of IgG, and examples thereof include red blood cells in which red blood cells are hemolyzed in addition to red blood cells. It may be a ghost. Red blood cells or erythrocyte ghosts may be disrupted to obtain the amount of IgG in the state of erythrocyte membranes obtained.

In the method of the present invention, the method for measuring the amount of IgG bound to erythrocytes is not particularly limited, and can be carried out by methods known to those skilled in the art. As long as the purpose of the present method for detecting colon cancer can be achieved, the method of measuring the amount of IgG bound to the red blood cells may be a quantitative method or a qualitative method. It is desirable to have. Examples of the method of measuring the amount of the IgG include immunological methods, such as radioimmunoassay (RIA), immunofluorescence assay (FIA), immunoluminescence assay, enzyme immunoassay (eg, Enzyme immunoassay) Immunoassay (EIA), Enzyme-linked Immunosorbent assay (ELISA), immunochromatography, latex particle agglutination), flow cytometry (with immunological techniques), Western blotting, and the like. Examples of preferred methods include ELISA and flow cytometry. Specific operating conditions of each measurement method can be appropriately set based on the knowledge of those skilled in the art.

Radioactive substances that can be used for labeling in radioimmunoassay (RIA) include, for example, ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, ³⁵ S, and ³² P.
Examples of fluorescent substances that can be used for labeling in immunofluorescence assay (FIA) include Eu (Europium), FITC, TMRITC, Cy3, PE, Texas-Red, and the like.
Examples of luminescent materials that can be used for labeling in immunoluminescence measurement include luminol, luminol derivatives, luciferin and the like.
Examples of enzymes that can be used for labeling by enzyme immunoassay or western blotting include horseradish peroxidase (HRP), alkaline phosphatase (ALP), glucose oxidase (GO) and the like.
As a fluorescent dye or fluorescent substance that can be used for labeling by flow cytometry, FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allophycocyanin), PerCP-Cy5.5, PE-Cy7, APC-H7 and the like can be mentioned.
Furthermore, a biotin-avidin system can also be used to bind an antibody or an antigen to these labeled substances.

In one of the method embodiments of the invention, anti-IgG antibodies may be used when immunological methods are used. Anti-IgG antibodies can be prepared by methods known to those skilled in the art, and may be commercially available. An anti-IgG antibody labeled with the above-mentioned labeling substance can be used, and such an anti-IgG antibody can be prepared by methods well known to those skilled in the art, and may be commercially available.
The anti-IgG antibody may be a polyclonal antibody or a monoclonal antibody, but in view of high sensitivity, the polyclonal antibody is desirable. Examples of preferred anti-IgG antibodies include polyclonal anti-human IgG-FITC (manufactured by DAKO, code-Nr. F0202).

(Flow cytometry method)
In one embodiment of the method of the present invention, when the sample derived from the subject is blood, and flow cytometry is employed, for example, blood to plasma component layer, prior to flow cytometry. The white blood cell layer and the platelet layer are removed, and phosphate buffered saline (PBS) is added to the red blood cell layer to obtain a red blood cell suspension, which can be treated with, for example, an anti-IgG antibody labeled with FITC. The method for treating red blood cells with anti-IgG antibody is not particularly limited, and various treatment conditions such as the amount of red blood cells, treatment time of anti-IgG antibody, treatment temperature and the like may be appropriately determined according to flow cytometry conditions etc. it can. The fluorescent dye and the fluorescent substance can be appropriately selected, for example, from the above examples, depending on the number of red blood cells to be detected, the setting of conditions of flow cytometry, and the like. By these operations, a suspension of red blood cells treated with anti-IgG antibody is obtained.

Next, flow cytometry is performed using a suspension of red blood cells treated with anti-IgG antibody as a sample. The operating method, operating conditions, and analysis conditions of the flow cytometer can be appropriately adjusted according to the flow cytometer and sample to be used.

For example,
(1) Develop with the forward scattered light pulse area (FSC-A) and the side scattered light pulse area (SSC-A) and gate to the red blood cell region.
(2) The cells in the gated area can be developed with a histogram, and measurement can be performed by measuring the mean fluorescence intensity (MFI) of the fluorescent dye added to the antibody.
MFI is also measured for a suspension of erythrocytes similarly treated with an isotype control antibody (which may be appropriately selected according to measurement conditions etc.), (MFI with anti-IgG antibody)-(MFI with isotype control antibody) = measurement It may be a value (MFI).

Note that “gating” refers to selecting only a cell population of interest in a sample and creating a histogram of only the cells (also referred to as “setting a gate”).

In one of the method embodiments of the present invention, the method may further comprise the step of correlating the amount of IgG bound to red blood cells with the likelihood of colon cancer in the subject.

When the amount of IgG bound to erythrocytes is quantitatively measured, the determination of the colon cancer potential of the subject may be performed by, for example, comparing the measured value of the IgG with a reference value or a preset cutoff value. It can be done by

In the method of the present invention, the reference value is a measurement value of IgG bound to red blood cells in a sample (normal sample) derived from a non-colorectal cancer person to be compared. Examples of non-colorectal cancer persons include healthy persons (persons with no disease being treated), persons with diseases other than cancer, and persons with cancer other than colon cancer and have not been pointed out for anemia Is preferred. It is preferable to use an average value of samples of a plurality of persons (more is preferable) for calculation of the reference value, for example, 2 or more persons, preferably 5 or more persons. When the measured value of IgG bound to erythrocytes of the subject is higher than the reference value, for example, 102% or more, 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 140% or more, 150% or more of the reference value If it is 160% or more, or 200% or more, it can be determined that the subject has, for example, colon cancer or that cancer has metastasized to the colon. Alternatively, the subject has, for example, a colon cancer when the measured value of IgG bound to erythrocytes of the subject is within a range consisting of a combination of appropriately selecting the upper limit value and the lower limit value below with respect to the reference value. Or cancer has metastasized to the large intestine.
Upper limit: 102%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% ;
Lower limit values: 101%, 102%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 200%, 300%, 400%, 500%, 600%.

The cutoff value can be determined, for example, as follows. First, the amount of IgG bound to red blood cells in a sample from a colorectal cancer patient is measured. At this time, it is preferable that the number of colorectal cancer patients to be targeted is large, for example, 2 or more, 5 or more, 10 or more, 20 or more, or 100 or more. It is also preferable to measure the amount of IgG bound to red blood cells in two or more non-colorectal cancer-derived samples (normal samples), and the number of non-colorectal cancer subjects targeted is, for example, two or more, There are 5 or more, 10 or more, 20 or more, or 100 or more. Then, the cutoff value of the amount of IgG bound to red blood cells is determined by statistical processing from the whole including both the sample group derived from colon cancer patients and the normal sample group. Examples of statistical processing include Youden Index and the like.
When the measured value of IgG bound to erythrocytes of the subject is higher than the cut-off value, the subject may be judged to have, for example, colon cancer or that cancer has metastasized to the colon.

In one embodiment of the method of the invention, if the amount of IgG bound to red blood cells in the blood sample is determined by flow cytometry, the amount of IgG can be calculated as the mean fluorescence intensity and has colon cancer The cutoff value that distinguishes (or is likely to have) a person who does not have (or is likely to have) colon cancer is, for example, an average fluorescence intensity of 20 or more, 25 or more, 30 or more, 35 It may be 40 or more, 45 or more, 50 or more, 60 or more, or 65 or more. Alternatively, the cutoff value may be included within the following range: mean fluorescence intensity 20-65, 25-60, or 30-55, 32-40. In addition, the cutoff value may be included in a range consisting of a combination of appropriately selecting the following upper limit value and lower limit value.
Upper limit: average fluorescence intensity 65, 60, 55, 45, 40, 35, 32.3, 32;
Lower limit: average fluorescence intensity 40, 32.3, 32, 30, 25, 20.
An example of a preferred cutoff value is mean fluorescence intensity 32.3.

In one embodiment of the method of the present invention, the step of correlating the amount of IgG bound to erythrocytes with the likelihood of colon cancer in the subject comprises measuring the amount of IgG bound to erythrocytes from the same subject measured over time. You may do by comparing. For example, relative to the amount of said IgG measured in advance, compared with the amount of said IgG measured at intervals (eg after 1, 2, 3, 6, 12, 18 months, 2 years, 3 years) If there is a significant increase, the subject can be determined to have, for example, colon cancer or that cancer has metastasized to the colon.

In one embodiment, the present application provides a detection kit for colon cancer comprising a labeled anti-IgG antibody for measuring the amount of IgG bound to erythrocytes. The labeled anti-IgG antibody contained in the present detection kit is, for example, labeled with the labeling substance exemplified above according to the measuring method.

The kit of the present invention may contain, in addition to the labeled anti-IgG antibody, a container for a reagent or a test sample, and a label for which the kit has been shown to be used for detection of colon cancer. In addition, it may further include instructions for use in the method of detecting colon cancer using the labeled anti-IgG antibody.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

<Example 1> Measurement of the Amount of IgG Bound to Erythrocytes by Flow Cytometry There are no colorectal cancer patients (23), gastric cancer patients (11), and healthy people (20 years of age or older) Blood was collected from (5 persons) who did not indicate anemia, and the amount of IgG bound to erythrocytes was measured by the following method.

(Test method)
1. Preparation of erythrocyte suspension (1) The collected blood (2 mL) was placed in an EDTA blood collection tube and allowed to stand to remove plasma, white blood cells and platelets.
(2) The obtained red blood cells were transferred to a 15 mL Falcon tube.
(3) Phosphate buffered saline was added to a total volume of 14 mL, and the mixture was properly mixed by inversion and stirred so that pellets could not be formed.
(4) Centrifugation at 3000 rpm for 5 minutes at 4 ° C., and the supernatant was removed with an aspirator.
(5) The above (3) and (4) were repeated twice more to wash red blood cells.
(6) 100 μL of the washed erythrocyte pellet was added to a 15 mL Falcon tube containing 10 mL of phosphate buffered saline, and mixed gently by pipetting to make a 1% erythrocyte suspension.

2. Incubation with antibody (1) 100 μL of the 1% erythrocyte suspension obtained above was dispensed into two FACS tubes.
(2) 10 μL each of an anti-human IgG polyclonal antibody of DAKO (DAKO polyclonal anti-human IgG-FITC code-Nr. F0202) was added to one side, and an isotype control antibody (abcam Rabbit IgG (FITC) ab37406) to the other side.
(3) After vortexing the resulting mixture gently, each tube was shielded from light and incubated at room temperature for 30 minutes.
(4) Then, 3 to 4 mL of phosphate buffered saline warmed to 37 ° C. was added to each tube for washing, and the supernatant was removed by centrifugation at 1500 rpm for 5 minutes. The resulting pellet was loosened and washing and centrifugation repeated one more time.
(5) The obtained pellet was loosened and phosphate buffered saline was added to make the total amount to about 500 μL.
(6) This was subjected to flow cytometry analysis.

3. Using flow cytometry (1) manufactured by Becton Dickinson FACS CANT ^(TM) II, and developed with the forward scattered light pulse area (FSC-A) and side scattered light pulse area (SSC-A), the erythrocyte Gated.
(2) The cells in the gated area were developed with a histogram, and the mean fluorescence intensity (MFI) of the fluorescent dye added to the antibody was measured.
(3) (MFI with anti-human IgG polyclonal antibody)-(MFI with isotype control antibody) was calculated and used as the measurement value (MFI).

(Test method)
The measurement results are shown in FIG.
The ROC curve which drew logistic regression as a platform is shown in FIG. 2 using the measurement result of a colon cancer patient group and a healthy subject group. "Sensitivity-(1- specificity)" was calculated, and the value (cutoff value) at which this value became the largest was 32.3.

<Example 2> Coombs test Colorectal cancer patients (23), gastric cancer patients (11), and healthy persons (persons 20 years of age or older who have no disease currently being treated and have not been pointed out for anemia) (5 Blood was collected from the n.) And IgG bound to erythrocytes was detected by agglutination reaction.

Classification of the agglutination reaction is as shown in Table 1.
The results are shown in FIG.

(Test method)
1. Test Tube Method (1) Put one drop of subject's red blood cells in 10 mL of phosphate buffered saline.
(2) After mixing, centrifuge at 3,000 rpm for 2 to 5 minutes.
(3) Remove the supernatant and resuspend the pellet in 10 mL of phosphate buffered saline.
(4) After mixing, centrifuge at 3,000 rpm for 2 to 5 minutes.
(5) Repeat (3) and (4) three times.
(6) After removing the supernatant as much as possible, make 3-5% erythrocyte suspension with phosphate buffered saline.
(7) Prepare one test tube for anti-IgG antibody, one for anti-C3d antibody, and one for negative control.
(8) The above 3 to 5% erythrocyte suspension is dropped one by one into each test tube and washed once with an automatic blood cell washing apparatus (manufactured by HITACHI himac MC450).
Add 2 drops of gamma-clone anti-IgG (manufactured by IMMUCOR), ortho-bioclone anti-C3d (manufactured by Ortho Clinical Diagnostics, Inc.), or phosphate buffered saline (negative control) to each test tube. .
(9) After mixing, centrifuge at 3,000 to 3,500 rpm for 15 to 30 seconds.
(10) While shaking the test tube gently, determine the presence or absence of aggregation.
(11) In the case of anti-IgG negative, 1 drop of Checcell ^{(registered trademark)} (Weak) (manufactured by IMMUCOR) is added and centrifuged for determination.

2. Column Method (1) Prepare two test tubes for adjusting blood cells of the subject, and dispense 500 μL each of phosphate buffered saline.
(2) Add 20 μL of the subject's red blood cells washed 4 times by the same method as the test tube method on one side and 20 μL of the non-washed subject's red blood cells on the other side to prepare a washed / unwashed 3-5% red blood cell suspension .
(3) Ortho Bioview Anti-IgG cassette (Ortho Clinical Diagnostics, Inc.) is prepared, and the cassette is filled with washed and unwashed.
(4) Peel off the foil on the top of the cassette tube to be used.
(5) Add 10 μL of washed and unwashed 3 to 5% blood cell suspension to the reaction vessel of each cassette tube.
(6) Centrifuge the cassette with a dedicated centrifuge (after 2 minutes at 795 rpm, 3 minutes at 1,510 rpm).
(7) The columns of each cassette tube are observed from the front and the rear to observe and determine the presence or absence of aggregation or hemolysis.

Claims (10)

1. A method of detecting colon cancer in a subject, comprising measuring the amount of immunoglobulin G (IgG) bound to red blood cells in a sample derived from said subject.
2. The method of claim 1, further comprising correlating the amount of IgG bound to red blood cells with the likelihood of colon cancer in the subject.
3. The method according to claim 1 or 2, wherein the amount of immunoglobulin G (IgG) bound to the red blood cells is measured by an immunological method.
4. The amount of immunoglobulin G (IgG) bound to the red blood cells is determined by radioimmunoassay (RIA), immunofluorescence assay (FIA), immunoluminescence assay, enzyme immunoassay, flow cytometry, and western blotting The method according to any one of claims 1 to 3, which is measured by a method selected from
5. The method according to any one of claims 1 to 4, comprising using a labeled anti-IgG antibody.
6. The method according to any one of claims 1 to 5, wherein the amount of immunoglobulin G (IgG) bound to the red blood cells is measured by flow cytometry.
7. The amount of IgG bound to red blood cells is obtained as the mean fluorescence intensity, and if the obtained mean fluorescence intensity is higher than the cutoff value, it is likely to have colon cancer, or the cancer has metastasized to the colon The method according to claim 6, which indicates that there is a high possibility of
8. The method according to claim 7, wherein the cutoff value is an average fluorescence intensity of 20 to 65.
9. An agent comprising a labeled anti-IgG antibody for use in the method according to any one of claims 1-8.
10. A kit for detecting colon cancer comprising a labeled anti-IgG antibody for measuring the amount of IgG bound to erythrocytes.

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