JP2023083882A - Method for predicting cancerization of oral leukoplakia and method for determining presence or absence of invasion of oral squamous cell carcinoma - Google Patents

Abstract

To provide a method for predicting cancerization of oral leukoplakia and a method for determining the presence or absence of invasion of oral squamous cell carcinoma.SOLUTION: Provided is a method for predicting the cancerous transformation of oral leukoplakia, including predicting that the oral leukoplakia will become cancerous if the level of CHI3L1 localized in the nucleus is higher than a reference value, but will not become cancerous if it is low compared to the reference value, in cells of diseased tissue obtained from a subject having the oral leukoplakia. Also, provided is a method for determining the presence or absence of invasive oral squamous cell carcinoma, including determining that the oral squamous cell carcinoma has not invaded if the level of CHI3L1 localized in the nucleus is higher than the reference value, but the oral squamous cell carcinoma has invaded if it is low compared to the reference value, in cells of diseased tissue obtained from a subject having the oral squamous cell carcinoma.SELECTED DRAWING: Figure 6

Description

The present disclosure relates to a method for predicting canceration of oral leukoplakia and a method for determining the presence or absence of invasion of oral squamous cell carcinoma.

Oral leukoplakia is a keratinized white lesion in the oral mucosa, gingiva, and tongue, and is a typical precancerous lesion of oral cancer. Its cancer rate is estimated to be 2-18%, while about 18% of tongue cancers are preceded by leukoplakia. Oral leukoplakia is characterized clinically by vitiligo that cannot be removed by abrasion and is not another diagnosable disease, and histopathologically varies from squamous hyperplasia to dysplasia. Epithelial dysplasia in oral leukoplakia is said to be frequently malignant.

Histopathologic examination of oral leukoplakia usually uses hematoxylin-eosin staining to determine epithelial histology. Anti-keratin antibodies such as ck13 and ck17 are also used to differentiate intraepithelial neoplasia and epithelial dysplasia of squamous epithelium. However, it is difficult to completely distinguish between normal epithelium and dysplasia with these staining methods, and there is an urgent need to establish an examination method that can detect signs of cancer from dysplasia at an early stage.

Chitinase 3-like protein 1 (CHI3L1) is a mammalian chitinase (chitin hydrolase) without enzymatic activity and is produced by various cells such as epithelial cells, chondrocytes, macrophages and neutrophils. It has been clarified that CHI3L1 induces chronic inflammation, epithelial dysplasia, and canceration, and there are also reports of its expression in colon cancer and breast cancer.

One of the purposes of the present disclosure is to provide a method for predicting canceration of oral leukoplakia. One object of the present disclosure is to provide a method for determining the presence or absence of invasion of oral squamous cell carcinoma.

The present inventors have found that in cases of oral leukoplakia with histopathological dysplasia, expression of CHI3L1 in the nucleus is observed, and in cases without dysplasia, expression in the nucleus is not observed. , and in oral squamous cell carcinoma confined to the epithelium without invasion, CHI3L1 is specifically expressed in the nucleus. It was found that no expression was observed in

Therefore, in one aspect, predicting that oral leukoplakia will become cancerous when the level of CHI3L1 localized in the nucleus is higher than the reference value in cells of diseased tissue obtained from a subject with oral leukoplakia, Methods are provided for predicting canceration of oral leukoplakia, including predicting that oral leukoplakia will not become cancerous when the level of CHI3L1 localized in the nucleus is low compared to a reference value.

In one embodiment, if the level of CHI3L1 localized in the nucleus is higher than a reference value in cells of diseased tissue obtained from a subject with oral squamous cell carcinoma, it is determined that oral squamous cell carcinoma has not invaded. and determining the presence or absence of infiltration of oral squamous cell carcinoma, comprising determining that oral squamous cell carcinoma has infiltrated when the level of CHI3L1 localized in the nucleus is lower than the reference value. provided.

According to the disclosure of the present application, canceration of oral leukoplakia can be predicted, or the presence or absence of invasion of oral squamous cell carcinoma can be determined.

The evaluation criteria for CHI3L1 expression in immunohistochemically stained images are shown. FIG. 2A shows CHI3L1-stained images of early oral squamous cell carcinoma tissue. FIG. 2B shows CHI3L1-stained images of advanced oral squamous cell carcinoma tissue. The ratio of the cytoplasmic and nuclear localization scores of CHI3L1 in oral squamous cell carcinoma tissues is shown. FIG. 4A shows a CHI3L1-stained image of an oral leukoplakia tissue showing epithelial dysplasia. FIG. 4B shows a CHI3L1-stained image of an oral leukoplakia tissue without epithelial dysplasia. FIG. 3 shows the ratio of CHI3L1 cytoplasmic and nuclear localization scores in oral leukoplakia tissues. Fluorescence intensity of CHI3L1 in the cytoplasm and nucleus of oral squamous cell carcinoma tissue and oral leukoplakia tissue is shown. The nuclear/cytoplasmic ratio of CHI3L1 fluorescence intensity in oral squamous cell carcinoma tissue and oral leukoplakia tissue is shown.

Unless otherwise specified, the terms used herein have meanings as commonly understood by those of ordinary skill in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. Listed below are definitions for some terms used herein, which definitions supersede general understanding herein.

I. Method for Predicting Cancerization of Oral Leukoplakia In this method, cells of diseased tissue obtained from a subject with oral leukoplakia are used. Oral leukoplakia is a white patchy lesion caused by hyperkeratosis of the oral mucosa and is defined as "prominent white lesions of the oral mucosa not characterized by any other disease". Histopathologically, it includes epithelial hyperkeratosis (hyperkeratosis, acanthocytosis, or hyperkeratosis) and epithelial dysplasia. Cells may be collected from diseased tissue of a subject, or cells may be collected from diseased tissue collected from a subject. Diseased tissue may be collected from a subject and the cells contained therein may be observed. Although a plurality of cells are usually used, a single cell may be used.

Chitinase 3-like protein 1 (CHI3L1), also called YKL-40, is a secreted glycoprotein of approximately 40 kDa. An example of the amino acid sequence of human CHI3L1 is disclosed in Protein Accession:P36222. Chitinase is a hydrolase that degrades the glycosidic bond of chitin, but CHI3L1 has no enzymatic activity. CHI3L1 protects cells from apoptosis and plays important roles in cell proliferation and differentiation, angiogenesis, inflammation, extracellular matrix remodeling and innate immune responses.

Regarding the present disclosure, CHI3L1 may contain a sequence in which one or several amino acids are deleted, substituted or added in the original amino acid sequence as long as its function is maintained. By "several" is meant preferably 2 to 7, more preferably 2 to 5 and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues.

In addition, as long as the function is maintained, CHI3L1 has an amino acid sequence of at least about 60% when calculated using BLAST or the like (for example, when BLAST default parameters, ie, initial parameters) are used. % or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98% or about 99% or more It may contain amino acid sequences with identity.

The level of CHI3L1 localized in the nucleus may be the amount of CHI3L1 localized in the nucleus, and is the relative value of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm. good too. For example, the level of CHI3L1 localized in the nucleus can be the ratio of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm. The amount of CHI3L1 can be indicated, for example, by the signal intensity of the label described below.

The level of CHI3L1 may be obtained by scoring the amount of CHI3L1 localized in the nucleus. For example, the signal intensity of a label indicating the amount of CHI3L1 localized in the nucleus can be scored on the basis of 0: negative, 1: weakly positive, 2: positive, 3: strongly positive. Alternatively, for example, the maximum amount of CHI3L1 localized in the nucleus is 100%, 0% or more and less than 25% score 1, 25% or more and less than 50% score 2, 50% or more and less than 75% can be scored on the basis of a score of 3 and 75% or more and 100% or less as a score of 4. The amount of CHI3L1 localized to the cytoplasm may optionally be used for scoring. For example, the ratio of the score for CHI3L1 localized in the nucleus to the score for CHI3L1 localized in the cytoplasm can be used.

For example, the amount of CHI3L1 can be measured by contacting the sample with a reagent that specifically binds to CHI3L1 and measuring the amount of reagent bound to CHI3L1 in the nucleus and optionally in the cytoplasm. The sample is cells of diseased tissue obtained from a subject. For example, the cells may be observed by cutting the diseased tissue into sections, or the cells of the diseased tissue may be fixed on a substrate such as a slide glass and observed. Reagents can be, for example, antibodies, RNA, DNA, polypeptides or aptamers. These reagents may be fragments, derivatives or analogues as long as they can specifically bind to CHI3L1. General techniques for in vitro detection of substances in samples, such as sample fixation, permeabilization, etc., are well known in the art.

A reagent can be labeled with a detectable substance. Examples of detectable substances include radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzymatic labels, biotin, electron-dense substances easily visualized by electron microscopy, such as ferritin or colloidal gold. . A substance that specifically binds to the reagent and carries a label, such as a secondary antibody, may be used.

The label is visualized under a microscope such as an upright microscope, fluorescence microscope, etc., photographed with a CCD camera or the like, and the signal intensity of the label within the nucleus and optionally within the cytoplasm is measured. Image processing software such as ImageJ software (NIH, Bethesda, MD, USA) can be used to measure signal intensity.

Usually, the level of CHI3L1 localized in the nucleus is measured in multiple cells and statistically processed. For example, the level of CHI3L1 localized in the nucleus is measured in all cells or CHI3L1-expressing cells observed in a fixed field of view of a microscope, and the average value is calculated. There may be one field of view or multiple fields of view. Measured levels of CHI3L1 may be corrected by endogenous controls such as housekeeping gene measurements, nuclear staining measurements, and the like. For statistical analysis, for example, statistical analysis software such as Prism can be used. Alternatively, CHI3L1 levels may be assessed visually.

Automated imaging and image analysis techniques, such as high content analysis (HCA) techniques, may be used to measure levels of CHI3L1. HCA techniques are known in the art and involve automated imaging of large numbers of cells followed by quantitative image analysis. Synonyms of HCA include high content imaging and high content screening. Robotic or automated equipment or microfluidic devices may be used to speed up the measurements.

In the examples described later, it was found that the level of CHI3L1 localized in the nucleus is high in cases of oral leukoplakia with histopathological dysplasia and low in cases without dysplasia. rice field. Oral leukoplakia with dysplasia is known to have a high frequency of canceration. Therefore, in this method, oral leukoplakia is predicted to become cancerous when the level of CHI3L1 localized in the nucleus is higher than the reference value, and the level of CHI3L1 localized in the nucleus is compared with the reference value. It is predicted that oral leukoplakia will not become cancerous if the level is low.

When the level of CHI3L1 localized in the nucleus is equivalent to the reference value, whether oral leukoplakia is predicted to become cancerous or not can be arbitrarily set according to the purpose of prediction. Therefore, in one embodiment, oral leukoplakia is predicted to become cancerous when the level of CHI3L1 localized in the nucleus is equal to or higher than the reference value, and when the level of CHI3L1 localized in the nucleus is less than the reference value It is predicted that oral leukoplakia will not become cancerous. In another embodiment, if the level of CHI3L1 localized in the nucleus is higher than the reference value, oral leukoplakia is predicted to become cancerous, and if the level of CHI3L1 localized in the nucleus is lower than the reference value, the oral cavity I predict that leukoplakia will not become cancerous.

In some embodiments, the reference value is a cutoff value for the level of CHI3L1. The cut-off value is a value that allows a group of patients with oral leukoplakia to be divided into a group of patients with dysplasia and a group of patients without dysplasia with a statistically significant difference. A cutoff value can be set by a known method using various statistical analysis techniques. For example, the level of CHI3L1 localized in the nucleus in samples obtained from a group of patients with oral leukoplakia with dysplasia, and A cut-off value can be set by statistically processing the level of CHI3L1 localized to . Statistical significance can be analyzed by known test methods such as Chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, Cox proportional hazards. Statistical analysis software such as Prism can be used to set the cutoff value.

Cutoff values may be set based on sensitivity and/or specificity. Preferably, the cutoff value exhibits both high sensitivity and high specificity. Here, sensitivity means the true positive rate. Moreover, specificity means a true negative rate. For example, the level of CHI3L1, which shows a high positive rate in a group of patients with oral leukoplakia with dysplasia and a high negative rate in a group of patients with oral leukoplakia without dysplasia, is set as a cutoff value. can.

For example, the cutoff value can be set by ROC analysis (receiver operating characteristic analysis), which is commonly used as a technique for examining the usefulness of diagnostic tests. In the ROC analysis, an ROC curve is created by plotting the sensitivity at each cutoff value on the vertical axis and the false positive rate (1-specificity) on the horizontal axis. The ROC curve is a diagonal straight line in a test with no diagnostic ability, but the more the diagnostic ability is improved, the more the ROC curve draws an arc in the upper left. It can be said that the cutoff value that gives the point on the ROC curve with the smallest distance from the upper left corner has excellent sensitivity and specificity. A cutoff value can also be set based on the Youden index. Specifically, sensitivity and specificity were determined from the levels of CHI3L1 in a group of patients with oral leukoplakia with dysplasia and a group of patients with oral leukoplakia without dysplasia. Create a ROC curve using software. Then, the value at which the sensitivity and specificity are as close to 100% as possible is obtained, and that value can be used as the cutoff value.

In addition, for example, the diagnostic efficiency (i.e., cases in which patients with oral leukoplakia with dysplasia were correctly diagnosed as “with dysplasia” and patients with oral leukoplakia in which dysplasia was not recognized were diagnosed with “no dysplasia”) The ratio of the total number of correctly diagnosed cases to the total number of cases) is obtained, and the level of CHI3L1 at which the highest diagnostic efficiency is calculated can be used as the cutoff value.

Cut-off values can also be set for patient groups subgrouped according to characteristics. For example, cutoff values may be set according to gender, age group, or race.

When scoring the amount of CHI3L1 localized in the nucleus, a specific score may be used as a reference value. For example, if the score of CHI3L1 localized in the nucleus is 1 (weakly positive) or more, it is predicted that oral leukoplakia will become cancerous, and if it is less than 1, it is predicted that oral leukoplakia will not become cancerous. .

In one embodiment, the predicted result of the method can be provided as information for diagnosis.
In one embodiment, a method for predicting cancerous transformation of oral leukoplakia, comprising:
(1) obtaining cells of diseased tissue from a subject with oral leukoplakia;
(2) measuring the level of CHI3L1 localized in the nucleus in cells of the diseased tissue; and
(3) When the level of CHI3L1 localized in the nucleus is higher than the reference value, oral leukoplakia is predicted to become cancerous, and the level of CHI3L1 localized in the nucleus is lower than the reference value to predict that oral leukoplakia will not become cancerous in
A method is provided comprising:

In one embodiment, oral leukoplakia is treated and/or cancerous prevention is performed in a subject whose oral leukoplakia is predicted to become cancerous by this method. For example, surgical resection of diseased tissue is performed.

At least part of the methods described above may be executed by a computer. For example, a test operator such as a medical professional appropriately processes cells of lesion tissue obtained from a subject with oral leukoplakia, and sets the cells in an image analysis device. The computer causes the image analyzer to measure the level of CHI3L1 localized in the nucleus and acquires the measurement results. The computer predicts whether or not the subject's oral leukoplakia will become cancerous based on the obtained levels of CHI3L1 localized in the nucleus. The computer outputs the prediction results so obtained, allowing the tester to obtain information about the subject. Thus, for example, a program is provided that causes a computer to perform a method that utilizes the obtained levels of CHI3L1 localized in the nucleus. The program may also cause the computer to perform a method of measuring levels of CHI3L1 localized in the nucleus.

Thus, for example
(1) obtaining image data showing the localization of CHI3L1 in cells of diseased tissue obtained from a subject with oral leukoplakia;
(2) measuring the level of CHI3L1 localized in the nucleus;
(3) When the level of CHI3L1 localized in the nucleus is higher than the reference value, oral leukoplakia is predicted to become cancerous, and the level of CHI3L1 localized in the nucleus is lower than the reference value predicting that oral leukoplakia will not become cancerous, and
(4) A program is provided for causing a computer to execute the step of outputting the obtained prediction result.

A computer-readable recording medium recording the program is also provided. A recording medium is not particularly limited, and may be a magnetic disk, an optical disk, a flash memory device, or the like.

In another aspect, a kit is provided for predicting canceration of oral leukoplakia, comprising a reagent that specifically binds to CHI3L1. Reagents may be provided dissolved in water or a suitable buffer, eg, phosphate-buffered saline (PBS), or lyophilized, contained in suitable containers. Suitable containers include bottles, vials, syringes, test tubes, plates, membranes and the like. The container may be made from a variety of materials such as glass, plastic, and the like. The kit may further contain other components and reagents necessary for detection of CHI3L1. For example, the kit can further include labeled secondary antibodies, chromogenic substrates, blocking solutions, wash buffers, and the like. The kit may further include other materials desirable from a commercial and user standpoint, such as a package insert containing instructions for use.

In one aspect, a reagent that specifically binds to CHI3L1 for predicting canceration of oral leukoplakia is provided.
In one aspect, use of a reagent that specifically binds to CHI3L1 for manufacturing a kit for predicting canceration of oral leukoplakia is provided.

II. Method for Determining the Presence or Absence of Invasion of Oral Squamous Cell Carcinoma In this method, cells of diseased tissue obtained from a subject with oral squamous cell carcinoma are used. Oral squamous cell cancer includes, for example, tongue cancer, floor of the mouth cancer, gingival cancer, buccal mucosa cancer, and hard palate cancer. Cells may be collected from diseased tissue of a subject, or cells may be collected from diseased tissue collected from a subject. Diseased tissue may be collected from a subject and the cells contained therein may be observed. Although a plurality of cells are usually used, a single cell may be used.
The level of CHI3L1 localized in the nucleus is the same as in I above.

In the examples described later, it was found that the level of CHI3L1 localized in the nucleus is high in oral squamous cell carcinoma cases where the cancer is localized in the epithelium and low in cases where the cancer invades below the basal layer. served. Therefore, in this method, when the level of CHI3L1 localized in the nucleus is higher than the reference value, it is determined that the oral squamous cell carcinoma has not invaded, and the level of CHI3L1 localized in the nucleus is the reference value. It is determined that oral squamous cell carcinoma is infiltrating when it is low compared to .

If the level of CHI3L1 localized in the nucleus is equivalent to the reference value, whether it is determined that the oral squamous cell carcinoma is not invading or that it is infiltrating is determined arbitrarily depending on the purpose of determination. Can be set. Therefore, in one embodiment, it is determined that oral squamous cell carcinoma has not invaded when the level of CHI3L1 localized in the nucleus is equal to or higher than the reference value, and the level of CHI3L1 localized in the nucleus is less than the reference value It is determined that oral squamous cell carcinoma infiltrates when . In another embodiment, it is determined that oral squamous cell carcinoma has not invaded when the level of CHI3L1 localized in the nucleus is higher than the reference value, and the level of CHI3L1 localized in the nucleus is below the reference value. In some cases, it is determined that oral squamous cell carcinoma is infiltrating.

In some embodiments, the reference value is a cutoff value for the level of CHI3L1. The cut-off value is a value that allows the oral squamous cell carcinoma patient group to be divided into a non-infiltrating patient group and a cancer-infiltrating patient group with a statistically significant difference. The cut-off value can be set by a known method using various statistical analysis methods, as in I above. For example, levels of CHI3L1 localized in the nucleus in samples obtained from a group of patients with non-invasive oral squamous cell carcinoma and A cut-off value can be set by statistically processing the level of localized CHI3L1.

When setting the cut-off value based on sensitivity and/or specificity, for example, a group of patients with non-invasive oral squamous cell carcinoma showing a high positive rate and a group of patients with invasive oral squamous cell carcinoma The level of CHI3L1 that shows a high negative rate at 2000 can be set as a cut-off value.

When setting the cutoff value by ROC analysis, for example, sensitivity and specificity are determined from the levels of CHI3L1 in a group of patients with non-infiltrating oral squamous cell carcinoma and a group of patients with infiltrating oral squamous cell carcinoma, and these Based on the values, a ROC curve is created using commercially available analysis software, the value when the sensitivity and specificity are as close to 100% as possible is obtained, and this value can be used as the cutoff value.

When setting the cut-off value according to the diagnostic efficiency, for example, patients with oral squamous cell carcinoma without The ratio of the total number of cases correctly judged to be "presence" to the total number of cases is obtained, and the level of CHI3L1 at which the highest diagnostic efficiency is calculated can be used as the cutoff value.

Cut-off values can also be set for patient groups subgrouped according to characteristics. For example, cutoff values may be set according to gender, age group, or race.

When scoring the amount of CHI3L1 localized in the nucleus, a specific score may be used as a reference value. For example, if the score of CHI3L1 localized in the nucleus is 1 (weakly positive) or more, it is determined that oral squamous cell carcinoma has not invaded, and if it is less than 1, oral squamous cell carcinoma has invaded. determined to be In another example, if the score of CHI3L1 localized in the nucleus is 2 (positive) or more, it is determined that oral squamous cell carcinoma has not invaded, and if it is less than 2, oral squamous cell carcinoma is Determined to be infiltrated.

In one embodiment, the results of the determination by this method can be provided as information for diagnosis.
In one embodiment, a method for determining the presence or absence of invasion of oral squamous cell carcinoma,
(1) obtaining cells of diseased tissue from a subject with oral squamous cell carcinoma;
(2) measuring the level of CHI3L1 localized in the nucleus in cells of the diseased tissue; and
(3) When the level of CHI3L1 localized in the nucleus is higher than the reference value, it is determined that the oral squamous cell carcinoma has not invaded, and the level of CHI3L1 localized in the nucleus is compared with the reference value. Determining that oral squamous cell carcinoma is infiltrating if it is low,
A method is provided comprising:

In one embodiment, oral squamous cell carcinoma is treated according to the presence or absence of invasion in a subject for whom the presence or absence of oral squamous cell carcinoma invasion has been determined by the method. Usually, the diseased tissue and its surroundings are resected regardless of the presence or absence of invasion, but if it is determined that there is infiltration, the area around the lesion is widely resected, and if it is determined that there is no invasion, the resection area around the lesion can be reduced.

At least part of the methods described above may be executed by a computer. For example, a test operator such as a medical professional appropriately processes cells of diseased tissue obtained from a subject with oral squamous cell carcinoma as necessary and sets them in an image analysis device. The computer causes the image analyzer to measure the level of CHI3L1 localized in the nucleus and acquires the measurement results. The computer determines whether or not the oral squamous cell carcinoma of the subject is infiltrating based on the obtained level of CHI3L1 localized in the nucleus. The computer outputs the results of the determination thus obtained, enabling the tester to obtain information about the subject. Thus, for example, a program is provided that causes a computer to perform a method that utilizes the obtained levels of CHI3L1 localized in the nucleus. The program may also cause the computer to perform a method of measuring levels of CHI3L1 localized in the nucleus.

Thus, for example
(1) obtaining image data showing the localization of CHI3L1 in cells of diseased tissue obtained from a subject with oral squamous cell carcinoma;
(2) measuring the level of CHI3L1 localized in the nucleus;
(3) When the level of CHI3L1 localized in the nucleus is higher than the reference value, it is determined that the oral squamous cell carcinoma has not invaded, and the level of CHI3L1 localized in the nucleus is compared with the reference value. determining that the oral squamous cell carcinoma is infiltrating if the
(4) A program is provided for causing a computer to execute the step of outputting the obtained prediction result.

A computer-readable recording medium recording the program is also provided. The details of the recording medium are as described in I above.

In another aspect, a kit is provided for determining the presence or absence of invasion of oral squamous cell carcinoma, comprising a reagent that specifically binds to CHI3L1. The details of the kit are as described in I above.

In one aspect, a reagent that specifically binds to CHI3L1 for determining the presence or absence of invasion of oral squamous cell carcinoma is provided.
In one aspect, use of a reagent that specifically binds to CHI3L1 for manufacturing a kit for determining the presence or absence of invasion of oral squamous cell carcinoma is provided.

For example, the following embodiments are provided.
[1] In cells of diseased tissue obtained from a subject with oral leukoplakia, if the level of CHI3L1 localized in the nucleus is higher than the reference value, oral leukoplakia is predicted to become cancerous, A method of predicting canceration of oral leukoplakia, comprising predicting that oral leukoplakia will not become cancerous when the level of localized CHI3L1 is low compared to a reference value.
[2] The method according to item 1, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus.
[3] The method according to item 1, wherein the level of CHI3L1 localized in the nucleus is the ratio of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm.
[4] The method according to any one of items 1 to 3, comprising the step of measuring the amount of CHI3L1 localized in the nucleus.
[5] The method according to any one of items 1 to 3, comprising the step of measuring the amount of CHI3L1 localized in the cytoplasm.
[6] The method according to any one of items 1 to 5, wherein the level of CHI3L1 is measured by immunostaining.
[7] The method according to any one of items 1 to 6, wherein the reference value is a CHI3L1 level cutoff value.

[8] When the level of CHI3L1 localized in the nucleus is higher than the reference value in the cells of the diseased tissue obtained from the oral squamous cell carcinoma subject, it is determined that the oral squamous cell carcinoma has not invaded. A method for determining the presence or absence of invasion of oral squamous cell carcinoma, comprising determining that oral squamous cell carcinoma has infiltrated when the level of CHI3L1 localized in the nucleus is lower than a reference value.
[9] The method according to item 8, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus.
[10] The method according to item 8, wherein the level of CHI3L1 localized in the nucleus is the ratio of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm.
[11] The method according to any one of items 8 to 10, comprising the step of measuring the amount of CHI3L1 localized in the nucleus.
[12] The method according to any one of items 8 to 11, comprising the step of measuring the amount of CHI3L1 localized in the cytoplasm.
[13] The method according to any one of items 8 to 12, wherein the level of CHI3L1 is measured by immunostaining.
[14] The method according to any one of items 8 to 13, wherein the reference value is a CHI3L1 level cutoff value.

All documents cited herein are hereby incorporated by reference.
The above description is all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical spirit thereof. EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

Test 1: immunohistochemical staining
Experimental method 1. Preparation of Specimen Slides Formalin-fixed paraffin-embedded sections of oral squamous cell carcinoma tissue and oral leukoplakia tissue possessed by the Dental Center of Kurume University Hospital were used. Tissue sections were cut at 4 μm and attached to glass slides.

2. Deparaffinization (1) Heat Treatment Slides were heated in an incubator at 60° C. for 30 minutes.
(2) Xylene treatment Slides were immersed in xylene for 5 minutes. After that, the excess liquid was shaken off and immersed in another xylene for 5 minutes.
(3) Ethanol and Methanol Treatment Slides were immersed in 100% ethanol for 4 minutes. Excess liquid was shaken off, and immersed in 90% ethanol adjusted with water for 2 minutes. After that, the excess liquid was shaken off, and it was immersed in methanol+hydrogen peroxide solution (4.5%) for 5 minutes.
(4) Washing Excess liquid ethanol was shaken off, and the slide was washed with water (twice for 2 minutes each).

3. Antigen retrieval treatment (microwave method)
The Tris buffer was tris(hydroxymethyl)aminomethane (nacalai tesque, Kyoto, Japan) adjusted to 0.5 M with water and adjusted to pH 10 with sulfuric acid. Further, the slides were immersed in 10-fold diluted Tris buffer and heated in a microwave oven as described below.
(1) 3 minutes at 500 W (2) 6 minutes at 100 W (3) 10 seconds at 500 W (4) 6 minutes at 100 W After that, the container was cooled under running water for 30 minutes and washed 3 times with PBS for 3 minutes. .

4. Permeabilization treatment Saponin (Sigma-Aldrich, St. Louis, Mo.) adjusted to 0.05% with PBS was added dropwise to a glass slide from which excess liquid had been shaken off, and allowed to react for 5 minutes at room temperature in a wet box. Washed with PBS for 3 minutes x 3 times.

5. Blocking A 120-fold diluted normal goat serum with PBS (Vector laboratories, Burlingame, Calif., USA) was added dropwise to the slide glass from which excess liquid had been shaken off, and allowed to react for 30 minutes at room temperature in a wet box.

6. Primary antibody Anti-human CHI3L1 rabbit polyclonal antibody (Bioss Antibody, Boston, MA, USA: Cat# bs-10215R) diluted 50-fold with PBS was added dropwise to a glass slide after shaking off excess liquid, and placed in a humid box. The reaction was carried out at 4°C overnight. Then, it was washed with PBS for 3 minutes×3 times.

7. Secondary Antibody A biotin-labeled anti-rabbit IgG antibody (Vector) diluted 200-fold with PBS was added dropwise to the slide glass from which excess liquid had been shaken off, and reacted in a wet box at room temperature for 30 minutes. Then, it was washed with PBS for 3 minutes×3 times.

8. Avidin-biotin-labeled enzyme conjugate Avidin-biotin-labeled enzyme conjugate (Vector) was diluted 100-fold with PBS 30 minutes before use. An avidin-biotin-labeled enzyme complex (Vector) was added dropwise to the slide glass from which excess liquid had been shaken off, and allowed to react for 45 minutes at room temperature in a wet box. Then, it was washed with PBS for 3 minutes×3 times.

9. Color development detection AEC 3-amino-9-ethylcarbazole (Abcam, Cambridge, MA, USA) was added dropwise to a slide glass from which excess liquid was shaken off, and reacted in an incubator at 37°C for 8 minutes. After that, it was washed with water for 3 minutes×3 times.

10. Hematoxylin Staining Excess liquid was shaken off, and the slide glass was immersed in a hematoxylin solution (Dako, Glostrup, Denmark) for 1 minute for reaction. After that, it was washed with water for 3 minutes×3 times.

11. Excessive liquid was shaken off, and the slide glass was immersed in a lithium carbonate solution for 5 seconds and washed once with water.

12. Encapsulation Glycergel Mounting Medium (Dako) warmed in an incubator at 60° C. was added dropwise to the slide glass from which excess liquid had been shaken off, and the slide was then encapsulated with a cover glass.

13. Evaluation of Staining Intensity Each tissue staining was photographed with an upright microscope (Axio imager2: ZEISS, Berlin, Germany) using a 10x ocular lens and a 40x objective lens (magnification: 400x). For each tissue staining, cytoplasmic and nuclear staining intensity was scored in all 12 fields (4 fields only if the lesion was localized) according to the criteria shown in FIG.

Experimental Results (1) Oral Squamous Cell Carcinoma A histological staining image of oral squamous cell carcinoma is shown in FIG. FIG. 2A is an oral squamous cell carcinoma tissue without infiltration limited to the epithelium, and FIG. 2B is a CHI3L1-stained image of an oral squamous cell carcinoma tissue invading below the basal layer. Table 1 shows the score of the visual field with the highest staining intensity, and Table 2 shows the aggregated results. When the score was the same for cytoplasm and nucleus, it was determined as cytoplasm. The ratio of nuclear to cytoplasmic scores is shown in FIG.

Among oral squamous cell carcinomas, in carcinoma in situ that does not invade (Fig. 2A), a strong CHI3L1 expression is found specifically in the nucleus, and in carcinomas that invade beyond the basal layer (Fig. 2B), CHI3L1 was localized in the cytoplasm and no nuclear expression was observed (Tables 1 and 2).

In oral squamous cell carcinoma, intranuclear expression of CHI3L1 was observed in 5 out of 6 carcinoma in situ. In all of the 4 cases of advanced cancer, diffuse expression was observed in the cytoplasm, and expression in the nucleus was not observed at all. Therefore, the intracellular localization of CHI3L1 in oral squamous cell carcinoma tissue can be used as a marker for the progression of oral squamous cell carcinoma.

(2) Oral leukoplakia A tissue staining image of oral leukoplakia is shown in FIG. FIG. 4A is an oral leukoplakia tissue with epithelial dysplasia, and FIG. 4B is a CHI3L1-stained image of an oral leukoplakia tissue without epithelial dysplasia. Table 3 shows the highest score of staining intensity, and Table 4 shows the aggregated results. The ratio of cytoplasmic and nuclear scores is shown in FIG.

In cases of oral leukoplakia with histopathological dysplasia (Fig. 4A), expression of CHI3L1 in the nucleus was observed, and in cases without dysplasia (Fig. 4B), nuclear expression was not observed. (Tables 3 and 4, Figure 5). In addition, in the process of progressing from the early stage of cancer to invasive cancer, nuclear translocation of CHI3L1 completely disappeared, and it was confirmed that CHI3L1 was diffusely localized in the cytoplasm (Fig. 4A, Fig. 2A, Fig. 2B).

In leukoplakia, expression in the nucleus was observed in 11 out of 13 cases with epithelial dysplasia, and the diagnostic sensitivity of dysplasia based on the presence or absence of CHI3L1 expression in the nucleus was 73.3% and the specificity was 85. .7%, both very high (Table 4). Therefore, the subcellular localization of CHI3L1 in oral leukoplakia tissue can be used as a marker of dysplasia.

Test 2: Immunofluorescence staining
Experimental method 1. Primary antibody In the same manner as the procedure for immunohistochemical staining, the slide glass to which the tissue section was attached was subjected to the steps of deparaffinization, antigen retrieval, permeabilization, and blocking to react with the primary antibody. Anti-human CHI3L1 rabbit polyclonal antibody (Bioss Antibody) diluted 50-fold with PBS (final concentration: 20 µg/ml) was used as the primary antibody in the same manner as in immunohistochemical staining, and reacted at room temperature for 1 hour. Then, it was washed with PBS for 3 minutes×3 times.

2. Secondary Antibody A CF488A anti-rabbit IgG antibody (Biotium Inc, Fremont, Calif., USA) diluted 200-fold with PBS (final concentration: 1 μg/ml) was dropped onto a glass slide after shaking off excess liquid, and placed in a wet box. at room temperature for 1 hour. Then, it was washed with PBS for 3 minutes×3 times.

3. Nuclear Staining An aqueous Hoechst33342 solution (nacalai) diluted 1000-fold with PBS (final concentration: 1 μg/ml) was added dropwise to the slide glass from which excess liquid had been shaken off, and reacted in an incubator at 37° C. for 10 minutes. Then, it was washed with PBS for 3 minutes×3 times.

4. Mounting A mounting medium, Vectashild (Vector laboratories), was dropped onto a slide glass from which excess liquid had been shaken off, with the tissue-attached side facing up, and a cover glass was placed on the slide glass. A Kimwipe was placed on the cover glass and lightly pressed to absorb the protruding mounting medium, and nail polish (manicure) was applied to the four sides of the cover glass to completely shield the cover glass.

5. Histological Observation Each stained slide was observed under a confocal microscope (Digital Eclipse C1; Nikon, Tokyo, Japan) using a 10x eyepiece and a 40x objective lens, and EZ C1 for image processing. Using 3.5 software (Nikon), the image was taken at 2x high magnification (final magnification: 800x).

6. Measurement of Fluorescence Intensity The obtained RGB image was divided into nuclear staining (Blue) and CHI3L1 (Green) using image processing software ImageJ (NIH, Bethesda, MD, USA) and measured.
(1) In the Blue image, the nucleus of each tissue was measured in 5 fields, and the average value was obtained.
(2) In order to keep the fluorescence intensity constant, the ratio was calculated based on the value of the fluorescence intensity of the nucleus of leukoplakia without dysplasia (leukoplakia without dysplasia: 1, invasive cancer: 0.8847, epithelial Intracarcinoma: 0.9278).
(3) In the Green image, the cytoplasmic portion and the nuclear portion were measured in 5 fields each. The obtained value was multiplied by the ratio obtained in (2) to obtain the fluorescence intensity value.

Experimental results The measurement results of fluorescence intensity are shown in FIG. FIG. 7 shows the ratio of cytoplasmic and nuclear fluorescence intensities. In fluorescent staining images, similar to immunohistochemical staining, CHI3L1 expression in the nucleus was strong in carcinoma in situ among oral squamous cell carcinomas, and CHI3L1 was localized in the cytoplasm and the expression in the nucleus was weak in advanced cancers. . In oral leukoplakia, the expression of CHI3L1 in the nucleus was strong in histopathologically confirmed dysplasia, and weak in the nucleus in cases without dysplasia. Therefore, the intracellular localization of CHI3L1 can be used as a marker for the progression of oral squamous cell carcinoma in oral squamous cell carcinoma tissue, and can be used as a marker for dysplasia in oral leukoplakia tissue. confirmed.

According to the present disclosure, it is possible to predict canceration of oral leukoplakia or determine the presence or absence of invasion of oral squamous cell carcinoma, which is useful in the medical field.

Claims (14)

If the level of nuclear-localized chitinase 3-like protein 1 (CHI3L1) is higher than the reference value in cells of diseased tissue obtained from subjects with oral leukoplakia, oral leukoplakia is predicted to become cancerous. , a method of predicting canceration of oral leukoplakia, comprising predicting that oral leukoplakia will not become cancerous when the level of CHI3L1 localized in the nucleus is low compared to a reference value. 2. The method of claim 1, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus. 2. The method of claim 1, wherein the level of CHI3L1 localized in the nucleus is the ratio of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm. 4. The method according to any one of claims 1 to 3, comprising the step of measuring the amount of CHI3L1 localized in the nucleus. 4. The method according to any one of claims 1 to 3, comprising the step of measuring the amount of CHI3L1 localized in the cytoplasm. The method according to any one of claims 1 to 5, wherein the level of CHI3L1 is measured by immunostaining. The method according to any one of claims 1 to 6, wherein the reference value is a cutoff value for the level of CHI3L1. In the cells of the lesion tissue obtained from the oral squamous cell carcinoma subject, if the level of CHI3L1 localized in the nucleus is higher than the reference value, it is determined that the oral squamous cell carcinoma has not invaded. A method for determining the presence or absence of infiltration of oral squamous cell carcinoma, comprising determining that oral squamous cell carcinoma has infiltrated when the level of CHI3L1 localized in the body is low compared to a reference value. 9. The method of claim 8, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus. 9. The method of claim 8, wherein the level of CHI3L1 localized in the nucleus is the ratio of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm. The method according to any one of claims 8 to 10, comprising the step of measuring the amount of CHI3L1 localized in the nucleus. The method according to any one of claims 8 to 10, comprising the step of measuring the amount of CHI3L1 localized in the cytoplasm. The method according to any one of claims 8 to 12, wherein the level of CHI3L1 is measured by immunostaining. The method according to any one of claims 8 to 13, wherein the reference value is a cutoff value for the level of CHI3L1.

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