Method for predicting the progression of oral leukoplakia to cancer and method for determining the presence or absence of invasion of oral squamous cell carcinoma

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

[Technical Field]

[0001] The present disclosure relates to a method for predicting the canceration of oral leukoplakia and a method for determining the presence or absence of invasion of oral squamous cell carcinoma. [Background technology]

[0002] Oral leukoplakia is a keratinized white lesion on the oral mucosa, gingiva, and tongue, and is a typical precancerous lesion of oral cancer. The rate of carcinogenesis is estimated to be 2-18%, while leukoplakia precedes approximately 18% of tongue cancers. Clinically, oral leukoplakia is characterized by white patches that cannot be removed by friction and are not associated with other diagnosable diseases. Histopathologically, the lesions range from squamous epithelial hyperplasia to dysplasia. The epithelial dysplasia of oral leukoplakia is said to frequently transform into cancer.

[0003] In histopathological examinations of oral leukoplakia, the epithelial histology is usually determined by hematoxylin and eosin staining. Anti-keratin antibodies such as CK13 and CK17 are also used to differentiate squamous intraepithelial neoplasia and epithelial dysplasia. However, it is difficult to completely distinguish between normal epithelium and dysplasia using these stains, and there is an urgent need to establish a test method that can detect early signs of cancer from dysplasia.

[0004] Chitinase 3-like protein 1 (CHI3L1) is a mammalian chitinase (chitin hydrolase) that does not have enzymatic activity and is produced by various cells, including epithelial cells, chondrocytes, macrophages, and neutrophils. CHI3L1 has been shown to induce chronic inflammation, epithelial dysplasia, and carcinogenesis, and its expression has also been reported in colon cancer and breast cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide a method for predicting the canceration of oral leukoplakia.An object of the present disclosure is to provide a method for determining the presence or absence of invasion of oral squamous cell carcinoma. [Means for solving the problem]

[0006] The inventors found that in oral leukoplakia cases where dysplasia was found histopathologically, CHI3L1 expression was observed in the nucleus, but not in cases without dysplasia; and that in oral squamous cell carcinoma with no infiltration limited to the epithelium, strong CHI3L1 expression was observed specifically in the nucleus, but in cancers with infiltration below the basal layer, CHI3L1 expression was limited to the cytoplasm and not in the nucleus.

[0007] Therefore, in one aspect, a method for predicting the canceration of oral leukoplakia is provided, which includes predicting that oral leukoplakia will become cancerous if the level of CHI3L1 localized in the nucleus is high compared to a reference value in cells of diseased tissue obtained from a subject with oral leukoplakia, and predicting that oral leukoplakia will not become cancerous if the level of CHI3L1 localized in the nucleus is low compared to a reference value.

[0008] In one aspect, a method for determining whether or not oral squamous cell carcinoma has invaded is provided, comprising determining that oral squamous cell carcinoma has not invaded 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, and determining that oral squamous cell carcinoma has invaded if the level of CHI3L1 localized in the nucleus is lower than a reference value. [Effects of the Invention]

[0009] According to the disclosure of the present application, it is possible to predict the canceration of oral leukoplakia or to determine the presence or absence of invasion of oral squamous cell carcinoma. [Brief explanation of the drawings]

[0010] [Figure 1] The evaluation criteria for CHI3L1 expression in immunohistochemical staining images are shown. [Figure 2] Figure 2A shows CHI3L1 staining images of early-stage oral squamous cell carcinoma tissue, and Figure 2B shows CHI3L1 staining images of advanced oral squamous cell carcinoma tissue. [Figure 3]The ratio of CHI3L1 cytoplasmic to nuclear localization scores in oral squamous cell carcinoma tissues is shown. [Figure 4] Figure 4A shows CHI3L1-stained images of oral leukoplakia tissue with and without epithelial dysplasia, respectively. [Figure 5] The ratio of CHI3L1 cytoplasmic to nuclear localization scores in oral leukoplakia tissues is shown. [Figure 6] Fluorescence intensity of CHI3L1 in the cytoplasm and nucleus of oral squamous cell carcinoma tissues and oral leukoplakia tissues is shown. [Figure 7] The nuclear / cytoplasmic ratio of CHI3L1 fluorescence intensity in oral squamous cell carcinoma tissues and oral leukoplakia tissues is shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0012] I. Methods for predicting the progression of oral leukoplakia to cancer This method uses cells from lesional tissue obtained from a subject with oral leukoplakia. Oral leukoplakia is a white patch-like lesion caused by hyperkeratinization of the oral mucosa, and is defined as "a prominent white lesion of the oral mucosa that cannot be characterized as any other disease." Histopathological findings range from epithelial hyperkeratosis (hyperorthokeratosis, acanthosis, or hyperparakeratosis) to epithelial dysplasia. Cells may be collected from the subject's lesional tissue, or from lesional tissue collected from the subject. Lesioned tissue may also be collected from the subject and the cells contained therein observed. Although multiple cells are usually used, a single cell may also be used.

[0013] Chitinase 3-like protein 1 (CHI3L1), also known as 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. Chitinases are hydrolases that degrade the glycosidic bonds of chitin, but CHI3L1 does not have enzymatic activity. CHI3L1 protects cells from apoptosis and plays an important role in cell proliferation and differentiation, angiogenesis, inflammation, extracellular matrix remodeling, and innate immune responses.

[0014] In the present disclosure, CHI3L1 may contain a sequence in which one or several amino acids are deleted, substituted, or added to the original amino acid sequence, as long as its function is maintained. "Several" preferably means 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.

[0015] Furthermore, as long as its function is maintained, CHI3L1 may contain an amino acid sequence that has an identity with the original amino acid sequence of at least about 60% 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, and most preferably about 97%, about 98%, or about 99% or more when calculated using BLAST or the like (e.g., when using the default i.e., initial condition parameters of BLAST).

[0016] The level of CHI3L1 localized in the nucleus may be the amount of CHI3L1 localized in the nucleus, or may be the relative value of the amount of CHI3L1 localized in the nucleus to the amount of CHI3L1 localized in the cytoplasm. For example, the level of CHI3L1 localized in the nucleus may 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 a label described below.

[0017] The CHI3L1 level may be a score representing 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 may be scored using the following criteria: 0: negative, 1: weakly positive, 2: positive, and 3: strongly positive. Alternatively, for example, the maximum amount of CHI3L1 localized in the nucleus may be scored as 100%, with 0% or more and less than 25% being scored as 1, 25% or more and less than 50% being scored as 2, 50% or more and less than 75% being scored as 3, and 75% or more and less than 100% being scored as 4. In some cases, the amount of CHI3L1 localized in the cytoplasm may also be used for scoring. For example, the ratio of the score of CHI3L1 localized in the nucleus to the score of CHI3L1 localized in the cytoplasm may be used.

[0018] For example, the amount of CHI3L1 can be measured by contacting a sample with a reagent that specifically binds to CHI3L1 and measuring the amount of reagent bound to CHI3L1 in the nucleus and, optionally, the cytoplasm. The sample can be cells from diseased tissue obtained from a subject. For example, the diseased tissue can be sectioned and the cells can be observed, or cells from the diseased tissue can be fixed to a substrate such as a glass slide and observed. The reagent can be, for example, an antibody, RNA, DNA, a polypeptide, or an aptamer. These reagents can also be fragments, derivatives, or analogs that can specifically bind to CHI3L1. General techniques for in vitro detection of substances in a sample, such as sample fixation and permeabilization, are well known in the art.

[0019] The reagent can be labeled with a detectable substance. Examples of detectable substances include radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzyme labels, biotin, and electron-dense substances that are easily visualized by electron microscopy, such as ferritin or gold colloid. A substance that specifically binds to the reagent and has a label, such as a secondary antibody, can also be used.

[0020] The label is visualized under a microscope such as an upright microscope or a fluorescence microscope, and images are taken using a CCD camera to measure the signal intensity of the label in the nucleus and, optionally, in the cytoplasm. Image processing software, such as ImageJ software (NIH, Bethesda, MD, USA), can be used to measure the signal intensity.

[0021] Typically, 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 observed within a certain field of view under a microscope or in cells expressing CHI3L1, and the average value is calculated. The field of view may be one or multiple. The measured CHI3L1 level may be corrected using endogenous controls such as measurements of housekeeping genes or measurements of nuclear staining. Statistical analysis software such as Prism may be used for statistical analysis. Alternatively, the CHI3L1 level may be evaluated visually.

[0022] The level of CHI3L1 may be measured using automated imaging and image analysis techniques, such as high-content analysis (HCA). HCA techniques are known in the art and involve automated imaging of a large number of cells followed by quantitative image analysis. Synonyms for HCA include high-content imaging and high-content screening. Robotic or automated equipment or microfluidic devices may be used to speed up the measurement.

[0023] In the Examples described below, it was found that the level of CHI3L1 localized in the nucleus was high in oral leukoplakia cases in which dysplasia was observed histopathologically, and low in cases in which dysplasia was not observed. Oral leukoplakia with dysplasia is known to have a high frequency of carcinogenesis. Therefore, in this method, if the level of CHI3L1 localized in the nucleus is high compared to the reference value, it is predicted that oral leukoplakia will become carcinomatous, and if the level of CHI3L1 localized in the nucleus is low compared to the reference value, it is predicted that oral leukoplakia will not become carcinomatous.

[0024] When the level of CHI3L1 localized in the nucleus is equivalent to a reference value, it is predicted that oral leukoplakia will or will not become cancerous, and this can be set arbitrarily depending on the purpose of the prediction, etc. Thus, in one embodiment, when the level of CHI3L1 localized in the nucleus is equal to or greater than the reference value, it is predicted that oral leukoplakia will become cancerous, 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, when the level of CHI3L1 localized in the nucleus is higher than the reference value, it is predicted that oral leukoplakia will become cancerous, and when the level of CHI3L1 localized in the nucleus is equal to or less than the reference value, it is predicted that oral leukoplakia will not become cancerous.

[0025] In one embodiment, the reference value is a cutoff value for the CHI3L1 level. The cutoff value is a value that can statistically significantly separate a group of oral leukoplakia patients into a group of patients with and without dysplasia. The cutoff value can be set by known methods using various statistical analysis techniques. For example, the cutoff value can be set by statistically analyzing the levels of CHI3L1 localized in the nucleus in samples obtained from a group of oral leukoplakia patients with dysplasia and the levels of CHI3L1 localized in the nucleus in samples obtained from a group of oral leukoplakia patients without dysplasia. Statistical significance can be analyzed using known testing methods such as the chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, and Cox proportional hazards. Statistical analysis software such as Prism can be used to set the cutoff value.

[0026] The cutoff value can be set based on sensitivity and / or specificity. Preferably, the cutoff value exhibits both high sensitivity and high specificity. Here, sensitivity refers to the true positive rate. Specificity refers to the true negative rate. For example, a CHI3L1 level that exhibits a high positive rate in a patient group with oral leukoplakia with dysplasia and a high negative rate in a patient group with oral leukoplakia without dysplasia can be set as the cutoff value.

[0027] For example, a cutoff value can be determined using receiver operating characteristic (ROC) analysis, a commonly used method for assessing the usefulness of diagnostic tests. In 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. For tests with no diagnostic ability, the ROC curve is a straight diagonal line. As diagnostic ability improves, the curve curve curves upward and to the left. The cutoff value that provides the point on the ROC curve with the smallest distance from the upper left corner is considered to have excellent sensitivity and specificity. Alternatively, the cutoff value can be determined based on the Youden index. Specifically, the sensitivity and specificity are calculated from the CHI3L1 levels of patients with oral leukoplakia with and without dysplasia, and an ROC curve is created using commercially available analysis software based on these values. The values ​​at which the sensitivity and specificity are as close to 100% as possible are then determined and used as the cutoff value.

[0028] Furthermore, for example, the diagnostic efficiency (i.e., the ratio of the total number of cases in which patients with oral leukoplakia with dysplasia were correctly diagnosed as having dysplasia and the total number of cases in which patients with oral leukoplakia without dysplasia were correctly diagnosed as not having dysplasia) can be calculated, and the CHI3L1 level that yields the highest diagnostic efficiency can be used as the cutoff value.

[0029] Cutoff values ​​can also be set for patient subgroups according to characteristics, for example, cutoff values ​​may be set according to gender, age group, or race.

[0030] 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 (weak positive) or higher, oral leukoplakia is predicted to become cancerous, and if the score is less than 1, oral leukoplakia is predicted not to become cancerous.

[0031] In some embodiments, the predictive results of the present methods can provide information for diagnosis. In one embodiment, there is provided a method for predicting the canceration of oral leukoplakia, comprising: (1) Obtaining cells from diseased tissue from a subject with oral leukoplakia; (2) measuring the level of CHI3L1 localized in the nucleus in cells of diseased tissue; and (3) To predict that oral leukoplakia will become cancerous when the level of CHI3L1 localized in the nucleus is high compared to the reference value, and to predict that oral leukoplakia will not become cancerous when the level of CHI3L1 localized in the nucleus is low compared to the reference value; A method is provided which includes:

[0032] In one embodiment, in a subject in whom oral leukoplakia is predicted to develop into cancer by the present method, treatment of oral leukoplakia and / or prevention of cancer development is carried out, for example, surgical removal of the diseased tissue is carried out.

[0033] At least a portion of the above-described method may be executed by a computer. For example, a tester, such as a medical professional, obtains cells from diseased tissue from a subject with oral leukoplakia, processes them appropriately as necessary, and places them in an image analyzer. The computer then causes the image analyzer to measure the level of CHI3L1 localized in the nucleus and obtains the measurement results. The computer then predicts whether the subject's oral leukoplakia will become cancerous based on the obtained level of CHI3L1 localized in the nucleus. The computer then outputs the prediction results thus obtained, allowing the tester to obtain information about the subject. Thus, for example, a program is provided that causes a computer to execute a method utilizing the obtained level of CHI3L1 localized in the nucleus. The program may also cause a computer to execute a method for measuring the level of CHI3L1 localized in the nucleus.

[0034] So, 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) predicting that oral leukoplakia will become cancerous if the level of CHI3L1 localized in the nucleus is higher than the reference value, and predicting that oral leukoplakia will not become cancerous if the level of CHI3L1 localized in the nucleus is lower than the reference value; and (4) Step to output the prediction results A program for causing a computer to execute the above is provided.

[0035] A computer-readable recording medium having the program recorded thereon is also provided, which may be, but is not limited to, a magnetic disk, an optical disk, a flash memory device, or the like.

[0036] In another embodiment, a kit for predicting the canceration of oral leukoplakia is provided, comprising a reagent that specifically binds to CHI3L1. The reagent may be dissolved in water or a suitable buffer, such as phosphate-buffered saline (PBS), or lyophilized and provided in a suitable container. Suitable containers include bottles, vials, syringes, test tubes, plates, membranes, and the like. The container may be made of a variety of materials, such as glass or plastic. The kit may further include other components or reagents necessary for detecting CHI3L1. For example, the kit may further include a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, 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.

[0037] In one embodiment, a reagent that specifically binds to CHI3L1 is provided for predicting the canceration of oral leukoplakia. In one embodiment, there is provided use of a reagent that specifically binds to CHI3L1 for producing a kit for predicting the canceration of oral leukoplakia.

[0038] II. Methods for determining the presence or absence of invasion of oral squamous cell carcinoma This method uses cells from diseased tissue obtained from a subject with oral squamous cell carcinoma. Oral squamous cell carcinoma includes, for example, tongue cancer, floor of mouth cancer, gingival cancer, buccal mucosa cancer, and hard palate cancer. Cells may be collected from the subject's diseased tissue, or from diseased tissue collected from the subject. Diseased tissue may also be collected from the subject and the cells contained therein may be observed. Although multiple cells are usually used, a single cell may also be used. The level of CHI3L1 localized in the nucleus is the same as in I above.

[0039] In the Examples described below, it was found that the level of CHI3L1 localized in the nucleus was high in oral squamous cell carcinoma cases in which the cancer was confined to the epithelium and low in cases in which the cancer had invaded below the basal layer. Therefore, in this method, if the level of CHI3L1 localized in the nucleus is high compared to a reference value, it is determined that the oral squamous cell carcinoma has not invaded, and if the level of CHI3L1 localized in the nucleus is low compared to a reference value, it is determined that the oral squamous cell carcinoma has invaded.

[0040] When the level of CHI3L1 localized in the nucleus is equivalent to a reference value, oral squamous cell carcinoma is determined to have not invaded or to have invaded, which can be arbitrarily set depending on the purpose of the determination. Thus, in one embodiment, when the level of CHI3L1 localized in the nucleus is equal to or greater than the reference value, oral squamous cell carcinoma is determined to have not invaded, whereas when the level of CHI3L1 localized in the nucleus is less than the reference value, oral squamous cell carcinoma is determined to have invaded. In another embodiment, when the level of CHI3L1 localized in the nucleus is higher than the reference value, oral squamous cell carcinoma is determined to have not invaded, whereas when the level of CHI3L1 localized in the nucleus is equal to or less than the reference value, oral squamous cell carcinoma is determined to have invaded.

[0041] In one embodiment, the reference value is a cutoff value for the CHI3L1 level. The cutoff value is a value that can statistically significantly separate a group of oral squamous cell carcinoma patients into a group of patients with non-invasive cancer and a group of patients with invasive cancer. The cutoff value can be set by known methods using various statistical analysis techniques, as in I above. For example, the cutoff value can be set by statistically analyzing the levels of CHI3L1 localized in the nucleus in samples obtained from a group of patients with non-invasive oral squamous cell carcinoma and the levels of CHI3L1 localized in the nucleus in samples obtained from a group of patients with invasive oral squamous cell carcinoma.

[0042] When setting a cutoff value based on sensitivity and / or specificity, for example, a CHI3L1 level that shows a high positive rate in a patient group with non-invasive oral squamous cell carcinoma and a high negative rate in a patient group with invasive oral squamous cell carcinoma can be set as the cutoff value.

[0043] When setting a cutoff value using ROC analysis, for example, sensitivity and specificity can be determined from the CHI3L1 levels of a group of patients with non-invasive oral squamous cell carcinoma and a group of patients with invasive oral squamous cell carcinoma, and based on these values, an ROC curve can be created using commercially available analysis software.The values ​​at which sensitivity and specificity are as close to 100% as possible can be determined, and these values ​​can be used as the cutoff value.

[0044] When setting a cutoff value based on diagnostic efficiency, for example, the ratio of the total number of cases in which patients with non-invasive oral squamous cell carcinoma were correctly judged to be "non-invasive" and the total number of cases in which patients with invasive oral squamous cell carcinoma were correctly judged to be "invasive" can be calculated, and the CHI3L1 level that yields the highest diagnostic efficiency can be used as the cutoff value.

[0045] Cutoff values ​​can also be set for patient subgroups according to characteristics, for example, cutoff values ​​may be set according to gender, age group, or race.

[0046] 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 (weak positive) or higher, it is determined that oral squamous cell carcinoma has not invaded, and if it is less than 1, it is determined that oral squamous cell carcinoma has invaded. In another example, if the score of CHI3L1 localized in the nucleus is 2 (positive) or higher, it is determined that oral squamous cell carcinoma has not invaded, and if it is less than 2, it is determined that oral squamous cell carcinoma has invaded.

[0047] In some embodiments, the results of the present 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 comprises: (1) Obtaining cells from diseased tissue from a subject with oral squamous cell carcinoma; (2) measuring the level of CHI3L1 localized in the nucleus in cells of 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 when the level of CHI3L1 localized in the nucleus is lower than the reference value, it is determined that the oral squamous cell carcinoma has invaded. A method is provided which includes:

[0048] In one embodiment, in a subject in whom the presence or absence of invasion of oral squamous cell carcinoma has been determined by this method, treatment of oral squamous cell carcinoma is carried out according to the presence or absence of invasion. Usually, the lesion tissue and its surrounding area are resected regardless of the presence or absence of invasion, but if invasion is determined to be present, the area around the lesion may be resected widely, and if invasion is determined not to be present, the extent of resection around the lesion may be reduced.

[0049] At least a portion of the above-described method may be executed by a computer. For example, a tester, such as a medical professional, obtains cells from diseased tissue from a subject with oral squamous cell carcinoma, processes them appropriately as necessary, and places them in an image analyzer. The computer then causes the image analyzer to measure the level of CHI3L1 localized in the nucleus and obtains the measurement results. The computer determines whether the subject's oral squamous cell carcinoma is non-invasive or invasive based on the obtained level of CHI3L1 localized in the nucleus. The computer outputs the determination results thus obtained, allowing the tester to obtain information about the subject. Thus, for example, a program is provided that causes a computer to execute a method utilizing the obtained level of CHI3L1 localized in the nucleus. The program may also cause a computer to execute a method for measuring the level of CHI3L1 localized in the nucleus.

[0050] So, 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) determining that oral squamous cell carcinoma has not invaded when the level of CHI3L1 localized in the nucleus is higher than the reference value, and determining that oral squamous cell carcinoma has invaded when the level of CHI3L1 localized in the nucleus is lower than the reference value; and (4) Step to output the prediction results A program for causing a computer to execute the above is provided.

[0051] A computer-readable recording medium having the program recorded thereon is also provided. Details of the recording medium are as described in I above.

[0052] In another embodiment, there is provided a kit for determining the presence or absence of invasion of oral squamous cell carcinoma, comprising a reagent that specifically binds to CHI3L1. Details of the kit are as described in I above.

[0053] In one embodiment, a reagent that specifically binds to CHI3L1 is provided for determining the presence or absence of invasion of oral squamous cell carcinoma. In one embodiment, there is provided use of a reagent that specifically binds to CHI3L1 for producing a kit for determining the presence or absence of invasion of oral squamous cell carcinoma.

[0054] For example, the following embodiments are provided: [1] A method for predicting the canceration of oral leukoplakia, comprising predicting that oral leukoplakia will become cancerous if the level of CHI3L1 localized in the nucleus is high compared to a reference value in cells of diseased tissue obtained from a subject with oral leukoplakia, and predicting that oral leukoplakia will not become cancerous if the level of CHI3L1 localized in the nucleus is low compared to a reference value. [2] The method according to paragraph 1, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus. [3] The method according to paragraph 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, which comprises a step of measuring the amount of CHI3L1 localized in the nucleus. [5] The method according to any one of items 1 to 3, which comprises a 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 cutoff value for the level of CHI3L1.

[0055] [8] A method for determining whether or not oral squamous cell carcinoma has invaded, comprising determining that oral squamous cell carcinoma has not invaded 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, and determining that oral squamous cell carcinoma has invaded if 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 paragraph 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, which comprises a step of measuring the amount of CHI3L1 localized in the nucleus.

[12] The method according to any one of items 8 to 11, which comprises a 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 cutoff value for the level of CHI3L1.

[0056] All documents cited herein are hereby incorporated by reference. The above description is non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea. The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0057] Test 1: Immunohistochemical staining Experimental Method 1. Specimen Slide Preparation Formalin-fixed, paraffin-embedded sections of oral squamous cell carcinoma and oral leukoplakia tissues from the Dental and Oral Medical Center at Kurume University Hospital were used. The tissue sections were cut at 4 μm and attached to glass slides.

[0058] 2. Deparaffinization (1) Heat treatment The slides were heated in a 60°C incubator for 30 minutes. (2) Xylene treatment The slides were immersed in xylene for 5 minutes, then the excess liquid was shaken off and the slides were immersed in another xylene for 5 minutes. (3) Ethanol and methanol treatment The slides were immersed in 100% ethanol for 4 minutes, shaken off excess liquid, and then immersed in 90% ethanol diluted with water for 2 minutes. Then, shaken off excess liquid, the slides were immersed in methanol and hydrogen peroxide (4.5%) for 5 minutes, shaken off excess liquid, and then immersed in 70% ethanol diluted with water for 2 minutes. (4) Cleaning Excess liquid ethanol was shaken off and the slides were washed with water (2 times for 2 minutes each).

[0059] 3. Antigen retrieval treatment (microwave method) Tris buffer was prepared by adjusting tris(hydroxymethyl)aminomethane (Nacalai Tesque, Kyoto, Japan) to 0.5 M with water and then adding sulfuric acid to pH 10. Slides were immersed in a 10-fold diluted Tris buffer and heated in a microwave oven as described below. (1) 500W for 3 minutes (2) 100W for 6 minutes (3) 500W for 10 seconds (4) 100W for 6 minutes Thereafter, the container was cooled under running water for 30 minutes, and then washed with PBS three times for 3 minutes.

[0060] 4. Permeabilization After shaking off excess liquid, saponin (Sigma-Aldrich, St. Louis, MO) adjusted to 0.05% in PBS was added dropwise to the slide, and the slide was incubated at room temperature for 5 minutes in a humidified chamber. The slide was then washed three times with PBS for 3 minutes each.

[0061] 5. Blocking Excess liquid was shaken off from the slide, and normal goat serum (Vector Laboratories, Burlingame, CA, USA) diluted 120-fold with PBS was added dropwise to the slide, followed by reaction in a humidified box at room temperature for 30 minutes.

[0062] 6.Primary antibody After shaking off excess liquid, anti-human CHI3L1 rabbit polyclonal antibody (Bioss Antibody, Boston, MA, USA: Cat# bs-10215R) diluted 50-fold with PBS was added dropwise to the slide and incubated overnight at 4°C in a humidified chamber. The slide was then washed three times for 3 minutes with PBS.

[0063] 7.Secondary antibody After shaking off excess liquid, a biotin-labeled anti-rabbit IgG antibody (Vector) diluted 200-fold with PBS was dropped onto the slide, and the mixture was incubated at room temperature for 30 minutes in a humidified chamber. The slide was then washed three times with PBS for 3 minutes each.

[0064] 8. Avidin-biotin-labeled enzyme complex Avidin-biotin-labeled enzyme complex (Vector) was diluted 100-fold with PBS 30 minutes before use. After shaking off excess liquid, a drop of avidin-biotin-labeled enzyme complex (Vector) was added to a slide and allowed to react for 45 minutes at room temperature in a humidified chamber. The slide was then washed three times with PBS for 3 minutes each.

[0065] 9. Chromogenic Detection After shaking off excess liquid, AEC 3-amino-9-ethylcarbazole (Abcam, Cambridge, MA, USA) was added dropwise to the slide and allowed to react in an incubator at 37°C for 8 minutes, after which the slide was washed with water three times for 3 minutes each.

[0066] 10. Hematoxylin Staining After shaking off excess liquid, the slides were immersed in hematoxylin solution (Dako, Glostrup, Denmark) for 1 minute and then washed with water three times for 3 minutes.

[0067] 11. Color Dashi The slide was shaken off excess liquid, immersed in lithium carbonate solution for 5 seconds, and washed once with water.

[0068] 12. Enclosure After shaking off excess liquid, Glycergel Mounting Medium (Dako) that had been warmed in an incubator at 60°C was added dropwise to the slide, and the slide was then sealed with a cover glass.

[0069] 13. Assessing Staining Intensity Each tissue stain was photographed under an upright microscope (Axio Imager2: ZEISS, Berlin, Germany) using a 10x eyepiece and a 40x objective (magnification: 400x). For each tissue stain, the staining intensity of the cytoplasm and nucleus was scored in all 12 fields (4 fields only when the lesion was localized) according to the criteria shown in Figure 1.

[0070] Experimental results (1) Oral squamous cell carcinoma Figure 2 shows histological staining images of oral squamous cell carcinoma. Figure 2A shows oral squamous cell carcinoma tissue with no invasion limited to the epithelium, and Figure 2B shows a CHI3L1-stained image of oral squamous cell carcinoma tissue with invasion below the basal layer. The scores of the fields with the highest staining intensity are shown in Table 1, and the tabulated results are shown in Table 2. When the scores for the cytoplasm and nucleus were the same, the staining was determined to be cytoplasmic. The ratio of the nuclear score to the cytoplasmic score is shown in Figure 3. [Table 1] [Table 2]

[0071] Among oral squamous cell carcinomas, strong CHI3L1 expression was observed specifically in the nucleus in non-invasive intraepithelial carcinomas (Fig. 2A), whereas in carcinomas that had invaded beyond the basal layer (Fig. 2B), CHI3L1 was localized to the cytoplasm, with no nuclear expression (Tables 1 and 2).

[0072] In oral squamous cell carcinoma, nuclear CHI3L1 expression was observed in 5 of 6 cases of intraepithelial carcinoma. In advanced cancer, diffuse cytoplasmic expression was observed in all 4 cases, with no nuclear expression observed. Therefore, the intracellular localization of CHI3L1 in oral squamous cell carcinoma tissues may be used as a marker for the progression of oral squamous cell carcinoma.

[0073] (2) Oral leukoplakia Figure 4 shows stained images of oral leukoplakia tissue. Figure 4A shows an image of CHI3L1 stained oral leukoplakia tissue with epithelial dysplasia, and Figure 4B shows an image of CHI3L1 stained oral leukoplakia tissue without epithelial dysplasia. The highest staining intensity scores are shown in Table 3, and the tabulated results are shown in Table 4. The ratio of cytoplasmic to nuclear scores is shown in Figure 5. [Table 3]

[0074] [Table 4]

[0075] Among oral leukoplakia cases, nuclear CHI3L1 expression was observed in cases with histopathologically confirmed dysplasia (Fig. 4A), but not in cases without dysplasia (Fig. 4B) (Tables 3 and 4, Fig. 5). Furthermore, as cancer progresses from the early stage to invasive cancer, nuclear translocation of CHI3L1 completely disappears, and the CHI3L1 is diffusely localized in the cytoplasm (Fig. 4A, Fig. 2A, Fig. 2B).

[0076] In leukoplakia, nuclear expression was observed in 11 of 13 cases with epithelial dysplasia, and the sensitivity and specificity of the diagnosis of dysplasia based on the presence or absence of nuclear CHI3L1 expression were both very high (73.3%) and 85.7%, respectively (Table 4). Therefore, the intracellular localization of CHI3L1 in oral leukoplakia tissue can be used as a marker for dysplasia.

[0077] Test 2: Immunofluorescence staining Experimental Method 1.Primary antibody Similar to the immunohistochemical staining procedure, the slides with the tissue sections attached were deparaffinized, antigen retrieval, permeabilization, and blocking were performed, and then the primary antibody was reacted. As with the immunohistochemical staining, the primary antibody used was an anti-human CHI3L1 rabbit polyclonal antibody (Bioss Antibody) diluted 50-fold in PBS (final concentration: 20 μg / ml), and reacted for 1 hour at room temperature. The slides were then washed three times for 3 minutes with PBS.

[0078] 2.Secondary antibody After shaking off excess liquid, a 200-fold diluted anti-rabbit CF488A antibody (Biotium Inc., Fremont, CA, USA) was added dropwise to the slide, which was then incubated at room temperature for 1 hour in a humidified chamber. The slide was then washed three times for 3 minutes with PBS.

[0079] 3. Nuclear Staining After shaking off excess liquid, a 1000-fold diluted Hoechst 33342 solution (Nacalai) with PBS (final concentration: 1 μg / ml) was added dropwise to the slide, and the slide was incubated in an incubator at 37°C for 10 minutes. The slide was then washed three times with PBS for 3 minutes each.

[0080] 4. Encapsulation After shaking off excess liquid, a drop of Vectashild (Vector Laboratories) mounting medium was applied to a slide with the tissue side facing up, and a cover glass was placed on top. A Kimwipe was placed on top of the cover glass and gently pressed to absorb any excess mounting medium, and nail polish was applied to all four edges of the cover glass to completely seal it.

[0081] 5. Tissue Observation Each stained slide was photographed under a confocal microscope (Digital Eclipse C1; Nikon, Tokyo, Japan) using a 10x eyepiece and a 40x objective lens at a high magnification of 2x (final magnification: 800x) with EZ C1 3.5 software (Nikon) for image processing.

[0082] 6. Fluorescence Intensity Measurement The acquired RGB images were divided into nuclear staining (Blue) and CHI3L1 (Green) using the image processing software ImageJ (NIH, Bethesda, MD, USA) and then measured. (1) In the Blue image, the nuclei of each tissue were measured in five fields and the average value was calculated. (2) To keep the fluorescence intensity constant, the ratio was calculated using the fluorescence intensity value of the nucleus of leukoplakia without dysplasia as the standard (leukoplakia without dysplasia: 1, invasive carcinoma: 0.8847, carcinoma in situ: 0.9278). (3) In the Green image, the cytoplasmic and nuclear regions were measured in five fields. The obtained values ​​were multiplied by the ratio obtained in (2) to obtain the fluorescence intensity value.

[0083] Experimental results The results of the fluorescence intensity measurements are shown in Figure 6. The ratio of cytoplasmic to nuclear fluorescence intensity is shown in Figure 7. In the fluorescent staining images, similar to the immunohistochemical staining, CHI3L1 expression was strong in the nucleus of oral squamous cell carcinoma in situ, whereas in advanced carcinoma, CHI3L1 was localized to the cytoplasm and weak in the nucleus. Among oral leukoplakia, CHI3L1 expression was strong in the nucleus in cases with histopathologically confirmed dysplasia, whereas nuclear expression was weak in cases without dysplasia. Therefore, fluorescent staining also confirmed that 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 as a marker for dysplasia in oral leukoplakia tissue. [Industrial Applicability]

[0084] INDUSTRIAL APPLICABILITY According to the present disclosure, it is possible to predict the canceration of oral leukoplakia or to determine the presence or absence of invasion of oral squamous cell carcinoma, which is useful in the medical field.

Claims

1. A method for predicting that oral leukoplakia will become cancerous if the level of CHI3L1 localized in the nucleus is high compared to a reference value, and predicting that oral leukoplakia will not become cancerous if the level of CHI3L1 localized in the nucleus is low compared to the reference value, the method comprising a step of measuring the amount of chitinase 3-like protein 1 (CHI3L1) localized in the nucleus in cells of diseased tissue obtained from a subject with oral leukoplakia.

2. The method of claim 1, wherein the level of CHI3L1 localized in the nucleus is the amount of CHI3L1 localized in the nucleus.

3. 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 of claim 3, comprising measuring the amount of CHI3L1 localized in the cytoplasm.

5. The method of any one of claims 1 to 4, wherein the level of CHI3L1 is measured by immunostaining.

6. The method according to any one of claims 1 to 5, wherein the reference value is a cutoff value for the level of CHI3L1.

7. A kit comprising a reagent that specifically binds to chitinase 3-like protein 1 (CHI3L1), for predicting that oral leukoplakia will become cancerous if the level of chitinase 3-like protein 1 (CHI3L1) localized in the nucleus is higher than the reference value in cells of diseased tissue obtained from a subject with oral leukoplakia, and for predicting that oral leukoplakia will not become cancerous if the level of CHI3L1 localized in the nucleus is lower than the reference value.

8. The kit described in claim 7, wherein the reagent that specifically binds to CHI3L1 is an anti-CHI3L1 antibody.

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