Method for diagnosing HPV-associated oropharyngeal premalignant lesion or HPV-associated oropharyngeal microcarcinoma by using p16 protein immunostaining and optical clearing of tonsil

Abstract

One purpose of the present application is to provide a method that is for efficiently detecting, through visualization, a p16 protein expression site in a tonsil, and that contributes to the diagnosis of an HPV-associated oropharyngeal premalignant lesion or an HPV-associated oropharyngeal microcarcinoma. The present application discloses, as one embodiment of the present invention, a method for detecting a p16 protein expression site in a tonsil, the method comprising (1) (i) a step for subjecting a tonsil removed from a subject to optical clearing and (ii) a step for subjecting the cleared tonsil to immunostaining of the p16 protein, and further comprising (2) a step for observing fluorescence emitted from the immunostained p16 protein in the tonsil subjected to the steps in (1).

Description

Diagnostic method for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas by tonsil clearing and p16 protein immunostaining.

[0001] As one embodiment of this invention, the present invention provides a method for efficiently detecting the expression site of the p16 protein in the tonsils by visualizing it. This detection method is useful in the diagnosis of HPV (human papillomavirus)-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas, and is useful in the medical field and other areas.

[0002] HPV is a circular double-stranded DNA virus with over 200 genotypes. HPV infection can cause cervical cancer, HPV-associated oropharyngeal cancer, anal cancer, penile cancer, vulvar cancer, and vaginal cancer, but the HPV strains that cause these HPV-associated cancers are classified as high-risk types. The most representative HPV-associated cancers are cervical cancer and HPV-associated oropharyngeal cancer. In recent years, HPV-associated oropharyngeal cancer has been gradually increasing in developed countries, and in the United States and the United Kingdom, the number of oropharyngeal cancer cases has already exceeded that of cervical cancer. Approximately 90% of the high-risk HPV strains that cause HPV-associated oropharyngeal cancer are HPV-16.

[0003] In HPV-related cancers (such as cervical cancer, HPV-related oropharyngeal cancer, etc.), the function of Rb is inhibited by the viral protein E7, and p16 protein is diffusely overexpressed. Therefore, the expression of p16 protein in tumor cells serves as an alternative marker for HPV-related cancers. Thus, in medical practice, when a lesion is confirmed in the oropharynx by visual inspection, biopsy of the site and detection of the expression of p16 protein by immunohistochemistry of the tumor tissue are considered necessary examination methods for the diagnosis of HPV-related oropharyngeal cancer (Non-Patent Document 1). As described above, when a lesion is found in the oropharynx by visual inspection, the site is biopsied for histological evaluation, and p16 immunohistochemistry is performed to diagnose HPV-related oropharyngeal cancer. However, when a lesion in the oropharynx cannot be clearly confirmed by visual inspection, for example, when a precancerous lesion described in detail later exists, it is impossible to identify the site by visual inspection. That is, evaluation by biopsy is impossible. Therefore, the diagnosis of HPV-related oropharyngeal precancerous lesions has been possible only by preparing serial sections from the excised tonsils for histological evaluation and p16 immunohistochemistry. That is, the identification of HPV-related oropharyngeal precancerous lesions is not easy, and efficient diagnosis has been difficult with conventional pathological examination methods. In addition, in many cases of HPV-related oropharyngeal cancer, the lesion in the primary focus is so small that it rarely presents like a cancer of unknown origin. In such cases as well, the lesion cannot be clearly confirmed by visual inspection, and there are the same difficulties as in the diagnosis of HPV-related oropharyngeal precancerous lesions. As described above, with conventional pathological examination methods, it has been difficult to efficiently and quickly make a definitive diagnosis of HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas to meet the requirements of medical practice. Therefore, in medical practice, the development of a more convenient and efficient method has been strongly desired.

[0004] Iene, Head and Neck Cancer, 44(3), 253-257, 2018

[0005] An object of the present invention is to contribute to the definitive diagnosis of HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in medical practice by providing a simple and efficient method for detecting the site of p16 protein expression in the tonsils.

[0006] The inventors of the present invention have diligently studied and, in order to solve the above problems, have discovered that by utilizing "tissue clearing technology," which has been researched and developed in recent years as a technology that enables three-dimensional structural analysis of tissues and organs, it is possible to "clearly" the tonsil itself and perform immunostaining for p16 protein on the entire tonsil, without having to prepare pathological tissue specimens which require the preparation of numerous sections from tonsils excised from subjects as required in conventional examinations, and thereby extremely efficiently detect the expression site of p16 protein in the tonsil, thus completing the present invention.

[0007] Incidentally, the diagnosis of HPV-related oropharyngeal cancer is carried out in a series of tests, starting with a series of pharyngeal examinations such as visual inspection and palpation, and ending with a definitive diagnosis by biopsy of the target tissue if HPV-related oropharyngeal cancer is suspected. The diagnostic method provided by the present invention involves testing for the presence or absence of p16 protein expression using tonsils removed from the patient at the final stage of this series of tests. As described later, the existence of HPV-related oropharyngeal precancerous lesions has not been proven, and even less has a method for their early detection been found. However, the inventors have demonstrated their existence and found that the presence of HPV-related oropharyngeal precancerous lesions can be detected by a non-invasive method of detecting the presence of high-risk HPV mRNA in oral samples taken from subjects. In addition, it was confirmed that the p16 protein is expressed in HPV-related oropharyngeal precancerous lesions (a patent application for this finding has been filed as Japanese Patent Application No. 2024-167655 (filing date: September 26, 2024) (the term "precancerous lesion of HPV-related oropharyngeal cancer" in this patent application is synonymous with "HPV-related oropharyngeal precancerous lesion"), and an international patent application has also been filed as PCT / JP2025 / 033940 (filing date: September 25, 2025)). The method for detecting the p16 protein expression site in the tonsils, provided as an embodiment of the present invention, together with the method for detecting HPV-related oropharyngeal precancerous lesions in the subject concerned, is of great significance as it will construct a platform for the early detection of HPV-related oropharyngeal cancer.

[0008] Specific embodiments of the present invention are given below, but the present invention is not limited to these embodiments. [1] A method for detecting the expression site of p16 protein in a tonsil, comprising the steps of (1) (i) subjecting a tonsil extracted from a subject to clearing, and (ii) subjecting the cleared tonsil to immunostaining for p16 protein, and further comprising (2) observing the fluorescence emitted from the immunostained p16 protein in the tonsil subjected to step (1). [2] The detection method according to [1] above, further comprising the step of determining that an HPV-related oropharyngeal precancerous lesion or HPV-related oropharyngeal microcarcinoma is present in the subject when p16 protein expression is detected in the tonsil. [3] The detection method according to [1] or [2] above, wherein the clearing of the tonsil is performed by a tissue clearing technique using a water-soluble reagent. [4] The detection method according to any one of [1] to [3] above, wherein the clearing of the tonsil is performed by the CUBIC method. [5] The detection method according to any one of [1] to [4] above, wherein immunohistochemistry is performed by an indirect method. [6] The detection method according to any one of [1] to [5] above, wherein immunohistochemistry is performed by a fluorescent labeling method. [7] The detection method according to any one of [1] to [6] above, wherein the fluorescence emitted from the p16 protein is observed using a light microscope. [8] The detection method according to any one of [2] to [7] above, which diagnoses the presence of an HPV-associated oropharyngeal precancerous lesion.

[0009] [9] The detection method according to any one of [1] to [8] above, further comprising the step (I) of detecting the presence of high-risk HPV mRNA in an oral sample taken from a subject before step (1) to screen for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas.

[10] The detection method according to [9] above, further comprising the step of determining that the subject has the precancerous lesion or microcarcinoma if the presence of high-risk HPV mRNA is detected in the oral sample in step (I).

[11] The detection method according to [9] or

[10] above, for the early detection of HPV-related oropharyngeal cancer.

[12] The detection method according to any one of [9] to

[11] above, wherein the high-risk HPV mRNA is mRNA of the E6 gene and / or the E7 gene.

[13] The detection method according to any one of [9] to

[12] above, wherein the oral sample is a gargle sample, a saliva sample, or a pharyngeal swab sample.

[14] The detection method according to any one of [9] to

[13] above, wherein the oral sample is a gargle sample.

[0010]

[15] A kit for detecting p16 protein expression in tonsils, for the detection method described in any of [1] to

[14] above, comprising an article for clearing tonsils collected from a subject, and an article for immunostaining p16 protein in the cleared tonsils.

[0011] One embodiment of the present invention provides a simple and efficient method for detecting p16 protein expression sites in the tonsils. This detection method makes it possible to efficiently perform definitive diagnosis of HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas (hereinafter, both may be collectively referred to as "HPV-related oropharyngeal precancerous lesions, etc.") in a medical setting.

[0012] Figure 1 shows the tonsils before and after clearing in Example 1 described below. In the figure, the left image shows the tonsils before clearing, and the right image shows the tonsils after clearing. Figure 2 shows the tonsils during immunostaining in Example 2 (note that the light shielding was temporarily removed for the purpose of taking the photograph). Figure 3 shows the tonsils after refractive index adjustment in Example 4 described below. Figure 4 shows the imaging results of 3D imaging of the tonsils using a light sheet microscope in Example 6 described below. Figure 5 shows the imaging results for the identification of p16-positive lesions in the tonsils in Example 6 described below. Figure 6 shows an overview of each step for identifying HPV-related oropharyngeal precancerous lesions described in the Examples section below. Items A to D in Figure 6 refer to the following: A. The step of collecting "gargle samples" to screen patients with pharyngeal HPV infection, i.e., patients whose HPV DNA is positive in "gargle samples", from patients undergoing tonsillectomy for non-malignant diseases. B. The process involves: C. After formalin fixing tonsils extracted from patients who tested positive for high-risk HPV DNA in a gargle test, dividing them into 2-3 sections and embedding them in paraffin to create serial sections of the entire tonsil. D. After extracting DNA from every 50 slides, HPV E6 / E7 DNA is quantified by digital PCR, and slides that test positive for high-risk HPV DNA of the genotype detected in the gargle test are selected. E. Using slides adjacent to the slides that tested positive for high-risk HPV E6 / E7 DNA, hematoxylin & eosin staining (H&E staining), p16 and Ki-67 immunostaining, and high-risk HPV mRNA in situ hybridization are performed. Figure 7 shows an overview of "Test Results 2" described in the Examples section below. Serial sections were prepared from the entire tonsils of three patients whose gargle samples were positive for HPV16 DNA. DNA was extracted every 50 slides, and HPV16 E6 / E7 DNA was quantified by digital PCR. The vertical axis of the graph shows the number of HPV16 E6 and E7 droplets, and the horizontal axis shows the block number of the divided tonsil (number before the hyphen) and the slide number (number after the hyphen). If HPV16 DNA-positive slides were clustered, the cluster was considered a single HPV16 DNA-positive site.For patient 1's left tonsil, slides 1-068, 1-168, 1-217, and 3-251 were positive. However, because there was a slide with a suspected positive result (1-118) in between, further detailed analysis of every 10 slides revealed the existence of two distinct clusters. Three HPV16 DNA-positive sites were identified in patient 1's left tonsil (Site 1-1, Site 1-2, Site 1-3), three in patient 2's right tonsil (Site 2-1, Site 2-2, Site 2-3), and one in patient 3's left tonsil (Site 3-1). Figure 8 shows details of Lesson 1-2-1, Lesson 1-2-2, and Lesson 2-1, which are HPV16-related precancerous lesions identified in "Test Results 3 (1)" described in the Examples section below. Items A to E in Figure 8 refer to the following: A. H&E staining of Site 1-2 and Site 2-1 (whole tonsil). Two HPV16-related precancerous lesions were identified in Site 1-2 of patient 1 (positive for high-risk HPV mRNA in gargle sample), and one in Site 2-1 of patient 2 (no high-risk HPV mRNA testing performed on gargle sample). Each lesion is enclosed in a rectangle and labeled Lesson 1-2-1, Lesson 1-2-2, and Lesson 2-1, respectively. All lesions were localized in the tonsillar crypts. B. H&E staining (magnified view). Arrowheads indicate significantly enlarged nuclei, arrows indicate mitotic figures, and neovascularization is circled. Abnormal findings such as a high N / C ratio, nuclear enlargement, coarse chromatin, and nuclear inequality were observed in at least the basal two-thirds of the epithelium. C. p16 immunostaining. The lower panel shows a magnified view of the area enclosed by the rectangle in the upper panel. Diffuse overexpression of p16 was observed in the nucleus and cytoplasm of atypical epithelium. D. Ki-67 immunostaining. Ki-67-positive cells were observed throughout all layers, including the outermost layer, of atypical epithelium. E. RNA in situ hybridization performed with a probe that recognizes high-risk HPV. In cells exhibiting atypicality, expression of high-risk HPV mRNA was observed sporadically in Lessons 1-2-1 and 1-2-2, and densely in Lesson 2-1. The scale bar corresponds to 100 μm in B, C, and D, and 20 μm in E.Figure 9 shows details of Lesson 1-1 and Lesson 1-3, which are HPV16-related precancerous lesions identified in "Test Results 3 (1)" described in the Examples section below. Items A to E in Figure 9 refer to the following: A. H&E staining (whole tonsil) of Site 1-1 and Site 1-3 of Patient 1 (positive for high-risk HPV mRNA in gargle sample). One HPV16-related precancerous lesion was identified from Site 1-1 and one from Site 1-3. Each lesion is enclosed in a rectangle and labeled Lesson 1-1 and Lesson 1-3, respectively. B. H&E staining (magnified view). Neovascularization is circled. Abnormal findings such as a high N / C ratio, nuclear enlargement, coarse chromatin, and nuclear inequality were observed in at least the basal two-thirds of the epithelium. C. p16 immunohistochemistry. The lower panel shows a magnified view of the area enclosed by the rectangle in the upper panel. Diffuse overexpression of p16 was observed in the nucleus and cytoplasm of atypical epithelium. D. Ki-67 immunostaining. Ki-67-positive cells were observed throughout all layers, including the outermost layer, of atypical epithelium. E. RNA in situ hybridization performed with a probe that recognizes high-risk HPV. Expression of high-risk HPV mRNA was observed in cells showing atypicality. Figure 10 shows the overall tonsil image of each site identified in "Test Results 2" described in the Examples section below, based on p16 immunostaining. Figure 11 shows the overall tonsils of Site1-1, Site1-2, and Site1-3, identified from Patient 1 (positive for high-risk HPV mRNA in gargle samples), and Site2-1, identified from Patient 2 (no high-risk HPV mRNA testing performed on gargle samples). p16-positive areas are enclosed in rectangles. All lesions were located in the crypts, not on the tonsil surface. Figure 11 shows the results of the examination described in "Test Results 3 (2)" in the Examples section below. A to E in Figure 11 indicate the following: A. H&E staining of Site2-2 and Site3-1 (overall tonsil view). In Site2-2 of Patient 2 (no high-risk HPV mRNA testing performed on gargle samples), no precancerous lesions were observed, but the area enclosed in rectangles showed expression of high-risk HPV mRNA and was designated as Lesson 2-2.In Patient 3 (negative for high-risk HPV mRNA in gargle sample), no precancerous lesions were confirmed in Site 3-1, but there were areas with koilocytosis-like findings in the epithelium, so it was designated as Lesson 3-1. B. Magnified images of H&E staining are shown. Lesson 2-2 did not show clear cellular atypia, but angiogenesis was observed (circled). Lesson 3-1 showed koilocytosis-like findings (arrow), but did not show clear cellular atypia. C. p16 immunostaining. Lessons 2-2 and 3-1 were p16 negative. D. Ki-67 immunostaining. In Lesson 2-2, Ki-67 positive cells were observed throughout all layers, including the outermost layer. In Lesson 3-1, Ki-67 positive cells were localized to the basal and parabasal layers. E. RNA in situ hybridization was performed using a high-risk HPV probe. Epithelial cells weakly expressing high-risk HPV mRNA were sparsely observed in Lesson 2-2, but no epithelial cells expressing high-risk HPV mRNA were observed in Lesson 3-1. The scale bars correspond to 100 μm in B, C, and D, and 20 μm in E. Figure 12 shows the results of the spatial transcriptome analysis in "Test Results 4" described in the Examples section below. After removing non-biological batch effects, data obtained from multiple samples were integrated into one. Next, the proportion of cell types in each spatial region (spot) was estimated, and only spots where the purity of epithelial cells was determined to be 90% or higher were extracted. After extraction, dimensionality reduction was performed using UMAP (Figure left), and clustering analysis was performed after visualization. As a result, cancer (0, red), precancerous lesions (3, green), and normal epithelium (2, yellow-green) each formed distinct clusters, and the precancerous lesions showed an intermediate gene expression profile between cancer and normal epithelium. The right side of the figure shows Lesson 2-1, an HPV16-related precancerous lesion, and the center of the figure shows an HPV16-related invasive cancer lesion. Figure 13 shows details of Lessons 4-1, 5-1, and 6-1, which are high-risk HPV-related precancerous lesions other than HPV16 identified in "Test Results 5" described in the Examples section below. Items A to E in Figure 3 refer to the following: A. H&E staining of the tonsillectomy surface containing the lesions. Each lesion is enclosed in a rectangle. B. H&E staining (magnified view). C. p16 immunohistochemistry.The lower panel shows a magnified view of the area enclosed by the rectangle in the upper panel. D. Ki-67 immunostaining. E. RNA in situ hybridization performed with a probe that recognizes high-risk HPV. Lessons 4-1 and 6-1 showed mild cellular atypia, but p16 expression was observed, and Ki-67 was positive in all layers. Expression of high-risk HPV mRNA was also observed. In Lesson 5-1, severe cellular atypia was observed, p16 was diffusely expressed, and Ki-67 was positive in all layers. High-risk HPV mRNA was also diffusely positive. The scale bar corresponds to 2 mm in A and 100 μm in B, C, D, and E.

[0013] The present invention will be described in detail below based on embodiments, but the present invention is not limited to these. Those skilled in the art can modify embodiments of the present invention in various ways without departing from the meaning of the present invention, and such modifications are also included within the scope of the present invention.

[0014] One embodiment of the present invention is a method for detecting the expression site of p16 protein in the tonsils, comprising: (1) (i) a step of clearing a tonsil extracted from a subject; and (ii) a step of subjecting the cleared tonsils to immunostaining for p16 protein; and further comprising: (2) a step of observing the fluorescence emitted from the immunostained p16 protein in the tonsils subjected to step (1) (Embodiment A). This embodiment relates to an efficient method for detecting the expression site of p16 protein (p16-positive lesions) in the tonsils (hereinafter also referred to as "this detection method"), based on observing the fluorescence emitted from the immunostained p16 protein in the tonsils after subjecting the entire tonsil to immunostaining for p16 protein by "clearing" the tonsils, instead of preparing a pathological tissue specimen by making serial sections from the tonsils extracted from a subject in conventional pathological diagnosis. Furthermore, if the expression site of the p16 protein is detected in the tonsils using this detection method, the subject will be diagnosed with an HPV-related oropharyngeal precancerous lesion or the like. The detection method will be described in detail below.

[0015] [Subjects] The subjects for this detection method are those who are undergoing screening for the early detection of HPV-related oropharyngeal cancer, or those who have been judged to be suspected of having HPV-related oropharyngeal cancer. The examination using this detection method will be performed using tonsils that have been previously removed from such subjects. An embodiment of early detection screening for HPV-related oropharyngeal cancer, in which subjects are identified in advance by detecting the presence of high-risk HPV mRNA in oral samples taken from subjects to determine the presence of HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas, will be described later.

[0016] [Tonsil Clearing Process] (1) Tissue Clearing Technology The "tonsil clearing" in this detection method can be performed by applying histological techniques ("tissue clearing technology") that have been rapidly developing in recent years and make it possible to observe and analyze biological tissues that originally have a three-dimensional structure in three dimensions. Tissue clearing enables optical microscope observation of the inside of the tissue by chemical treatment that allows light to pass through the tissue. Tissue clearing has the advantage of making it possible to observe the entire target tissue in three dimensions without physical cutting or sectioning.

[0017] (2) Procedure for clearing tonsils Tissue clearing is achieved by 1) suppressing the scattering of light within biological tissue, and 2) suppressing the absorption of light within biological tissue (degreasing and decolorization). Specifically, "clearing tonsils" in this detection method can be performed, for example, by "immersing the tonsils in a tissue clearing reagent (tissue clearing reagent)". (i) Tissue clearing reagent The tissue clearing reagent used in this detection method is not particularly limited, and for example, any method that has already been developed and provided as a tissue clearing reagent can be appropriately selected and carried out according to its standard experimental protocol. Examples of tissue clearing reagents that use water-soluble reagents include the Scale method (see Nat Neurosci. 2015; 18: 1518-1529, Nat Neurosci. 2011; 14: 1481-1488), the CUBIC method (see Cell. 2014; 157: 726-739, Cell. 2014; 159: 911-924), and the SeeDB method (see Nat Neurosci. 2013; 16: 1154-1161, Cell Rep. 2016; 14: 2718-2732). Another method is the CLARITY method (see Nature. 2013; 497: 332-337, Nat Protoc. 2014; 9: 1682-1697), which involves forming a hydrogel within the tissue to retain the protein, followed by clearing with a water-soluble reagent. On the other hand, clearing techniques using organic solvents include the BABB method (see Nat Methods. 2007; 4: 331-336) and the 3DISCO method (see Nat Protoc. 2012; 7: 1983-1995). (ii) Immersed tonsils are cleared by immersing them in a tissue clearing reagent (e.g., CUBIC-L) for a predetermined time. The immersion temperature is not particularly limited, but it is preferably 10 to 60°C, and more preferably 20 to 40°C. The immersion time is not particularly limited, but for example, clearing can be achieved by immersing for 1 day to 4 weeks. The immersion conditions can be appropriately set by those skilled in the art depending on the tissue clearing technique applied. (iii) Immobilization In this detection method, before the tonsil clearing procedure described above, fixation may be performed using conventionally known histopathological methods.Furthermore, the fixation procedure may be combined with conventionally known degreasing treatments as needed. For fixation, a method of immersing the tonsils in a formalin solution (10% neutral formalin solution, 4% paraformaldehyde buffer solution, etc.) can be employed. After fixation, it is preferable to wash off the formalin and then perform a tissue clearing procedure. When using tonsils that have been fixed with formalin and then embedded in paraffin, it is preferable to deparaffinize them using xylene or ethanol, and then wash the tonsils before subjecting them to tissue clearing.

[0018] [Immunostaining Process of Tonsils] In this detection method, since the expression of p16 protein is used as a surrogate marker for HPV-related oropharyngeal cancer, the tonsils are subjected to immunostaining for p16 protein to detect the presence or absence of p16 protein expression sites in the tonsils. In this immunostaining, a labeling substance is used to visualize the antigen-antibody reaction with p16 protein. As such labeling methods, "fluorescent labeling" or "enzyme labeling" are known, but in this detection method, p16 protein is preferably fluorescently stained by "fluorescent labeling". Fluorescent staining is performed using a fluorescent dye (e.g., FITC (Fluorescein Isothiocyanate), Alexa Fluor). ＴＭEither dyes (such as Cy dyes) or fluorescent proteins (e.g., PE (Phycoerythrin), APC (Allophycocyanin), etc.) can be used, but the use of fluorescent dyes is preferred. Immunostaining can be performed using either direct or indirect methods. The indirect method is widely used because multiple secondary antibodies bind to the primary antibody, resulting in the binding of numerous labeling substances, which amplifies the immune reaction and makes visualization clearer. It is understood that the indirect method is also preferred in this detection method. When using the indirect method, it is preferable to select an antibody with high specificity and affinity for the p16 protein as the primary antibody. As the secondary antibody, one can be produced by immunizing animals such as goats, sheep, and rabbits with immunoglobulins from the animal species used to produce the primary antibody. As the secondary antibody, one that recognizes the entire primary antibody or one that recognizes a part of the primary antibody (Fab, Fc, etc.) can be used. The introduction of labeling substances into the secondary antibody can be carried out by conventional methods in this art. If primary and secondary antibodies are commercially available, these commercially available products may be used. Examples of the primary antibodies mentioned above include p16 INK4A (JC8) mouse monoclonal antibody (Santa Cruz Biotechnology) and CINTec p16 (E6H4) (VENTANA). Examples of labeled secondary antibodies include Alexa Fluor 647 AffiniPure fab fragment goat anti-mouse IgG2a, Fcγ fragment specific (Jackson Immuno Research). The above immunohistochemical staining can be performed according to the standard methods in this art.

[0019] The above describes the process of clearing the tonsils and the process of immunohistochemical staining of the tonsils. These two processes may be performed consecutively or with a time difference. The people performing both processes may be the same or different.

[0020] [Step for detecting the p16 protein expression site] In this detection method, the tonsils prepared by the [tonsil clearing step] and [tonsil immunostaining step] described above are used to observe the fluorescence emitted from the p16 protein, thereby detecting the p16 protein expression site in the tonsils. The outline of the procedure is as follows: (1) Adjustment of refractive index In order to maintain the transparency of the tonsils and to perform optical detection effectively, it is preferable to adjust and homogenize the refractive index of the tonsils prior to observing the fluorescence. Various water-soluble solvents such as organic solvents such as BABB and dibenzyl ether, sugars, alcohols, and aromatic amides are used to adjust the refractive index. The refractive index may be adjusted according to the standard experimental protocol of the "tissue clearing technique" described above (for example, the use of CUBIC-R). (2) Observation of fluorescence and detection of the p16 protein expression site In this detection method, the tonsils are then gel-embedded using a conventional method as appropriate, and then observed using an optical method to detect the presence or absence of fluorescence emitted from the p16 protein. Observation by optical method is not particularly limited as long as it is a method capable of detecting fluorescence, and can be performed using any type of optical microscope. For example, observation can be performed using a light sheet microscope, a two-photon laser microscope, and / or a confocal microscope. Observation using a light sheet microscope is particularly preferred. The images obtained from the observation are processed on a computer and reconstructed as 3D images for accurate pathological analysis of the presence of the p16 protein. (3) The above-described [step for detecting the p16 protein expression site] (step (2)) may be performed consecutively with step (1) which involves clearing the tonsils and immunostaining of the p16 protein, or it may be performed with a time difference. Also, the person performing each step may be the same person or different people.

[0021] [Diagnosis based on this detection method] (1) HPV-associated oropharyngeal precancerous lesions As mentioned above, the expression of p16 protein is a surrogate marker for HPV-associated cancers (cervical cancer, HPV-associated oropharyngeal cancer, etc.), and the detection of p16 protein expression by immunohistochemistry is positioned as an essential diagnostic method for HPV-associated oropharyngeal cancer. Furthermore, as mentioned above, "precancerous lesions" exist in the process of developing HPV-associated oropharyngeal cancer, and it has been demonstrated that p16 protein is expressed in these precancerous lesions (see the description in "Test Result 3: Verification of HPV-associated tonsillar dysplasia" in the reference example below). Therefore, using this detection method, it is possible to diagnose HPV-associated oropharyngeal precancerous lesions based on the detection of p16 protein expression in the tonsils. (2) HPV-associated oropharyngeal microcarcinomas Even microcarcinomas of HPV-associated oropharyngeal cancer express p16 protein. Although microcarcinomas are often not identified by conventional histopathological examination even after tonsillectomy, this detection method makes it possible to diagnose HPV-related oropharyngeal microcarcinoma based on the detection of p16 protein expression in the tonsils. Therefore, with this detection method, even if a lesion cannot be confirmed by conventional histopathological examination, the presence of p16 protein expression sites in the tonsils can be used as a criterion to diagnose that the subject has HPV-related oropharyngeal cancer. In addition, in diagnoses based on this detection method, it may be possible to diagnose HPV-related oropharyngeal precancerous lesions and HPV-related oropharyngeal microcarcinoma simultaneously, and this case is also included within the scope of the present invention. (3) Early detection of HPV-related oropharyngeal cancer This detection method makes it possible to identify HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinoma, and to perform early detection of HPV-related oropharyngeal cancer.

[0022] Another embodiment of the present invention is "[B] A method for detecting the expression site of the p16 protein in the tonsils, further comprising the step (I) of detecting the presence of high-risk HPV mRNA in an oral sample taken from a subject prior to step (1) for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in the detection method of Embodiment A" (Embodiment B). More specifically, the method is described as: "[B] (I) a step of screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas by detecting the presence of high-risk HPV mRNA in an oral sample taken from a subject; (1) (i) a step of clearing tonsils excised from subjects determined to have HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas by step (I); and (ii) a step of subjecting the cleared tonsils to immunostaining for p16 protein; further comprising (2) a step of detecting the expression site of p16 protein in tonsils by observing fluorescence emitted from p16 protein in the tonsils subjected to step (1)." In this embodiment, prior to implementing the "detection method" of Embodiment A, a step (I) is performed to screen for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas. Based on the determination that a subject has HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas, the "detection method" is performed using tonsils excised from the subject. Based on the expression of p16 protein in the tonsils, a definitive diagnosis is made that the subject has precancerous lesions or microcarcinomas. Combined with the implementation of step (I), this constructs a platform for the early detection of HPV-related oropharyngeal cancer (hereinafter also referred to as the "early detection method"). The early detection method will be described in detail below. Steps (1) and (2) can be described by referring to the explanation of the detection method in Embodiment A, so only step (I) will be described below.

[0023] [High-Risk HPV] Oropharyngeal cancer is a type of head and neck cancer that develops in the oropharynx. Oropharyngeal cancer is divided into two types: HPV-unrelated oropharyngeal cancer, which is caused by smoking and drinking, and HPV-related oropharyngeal cancer, which is caused by infection with high-risk HPV. HPV is a circular double-stranded DNA virus and is a common virus that is transmitted through contact. There are more than 200 types (genotypes) of HPV, which are classified into high-risk and low-risk types. High-risk HPV can induce cervical cancer, anal cancer, vaginal cancer, and HPV-related oropharyngeal cancer. HPV-related oropharyngeal cancer is on the rise globally, mainly in developed countries, and in the United States and the United Kingdom, the incidence of oropharyngeal cancer has already surpassed that of cervical cancer. High-risk HPV genotypes identified include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Approximately 70% of cervical cancers are caused by types 16 (HPV16) and 18 (HPV18). In HPV-associated oropharyngeal cancer, various high-risk HPV types can be the cause, but in approximately 90% of cases, type 16 (HPV16) is the cause. In the [Reference Examples] section below, in addition to "HPV16," which is the most representative cause of the disease, "HPV52, HPV56, and HPV59" are used to specifically explain step (I). However, step (I) is not limited to cases where HPV16 etc. are the cause, but can also be applied when other high-risk HPV genotypes are the cause.

[0024] (Precancerous lesions in HPV-associated oropharyngeal cancer) A "precancerous lesion" refers to a state in which cells are "not currently considered cancerous, but have a high probability of progressing to cancer." More specifically, a lesion is classified as a "precancerous lesion" if 1) a high-grade dysplastic lesion is histologically observed, more specifically, if the atypical cells of the lesion are found to be p16-positive and high-risk HPV mRNA-positive, and / or 2) regardless of the degree of cellular atypia, for example, even if the degree of cellular atypia is slight, if the cells constitute a lesion in which they are found to be p16-positive and high-risk HPV mRNA-positive (see, for example, test result 3(1) below). Dysplasia refers to a state in which cells are on the borderline between malignant and benign (borderline malignancy). High-grade dysplasia is a state in which dysplasia extends to two-thirds to all layers of the epithelium (without rupturing the basement membrane and remaining within the epithelium). In this specification, the term "precancerous lesion" is used to refer collectively to the above 1) and / or 2). In the cervix, a series of processes (natural history) from HPV infection to the development of invasive cancer via precancerous lesions has been clarified, and screenings are conducted to detect cervical cancer early. In cervical cancer screenings, a specimen is taken from the cervix for cytological examination and HPV testing. If the presence of a precancerous lesion is suspected, the cervix is ​​observed using a corticoscope and a biopsy is performed. If a high-grade precancerous lesion is confirmed, a cervical conization is performed to prevent cervical cancer. On the other hand, in the oropharynx, HPV infects numerous depressions in the tonsils called crypts, and the carcinogenic process progresses in the crypts. It is impossible to directly observe the crypts, and it is also impossible to directly collect specimens from the crypts. Therefore, the presence of precancerous lesions has not been proven in HPV-associated oropharyngeal cancer, and its natural history has not been elucidated. Consequently, there is no method for the early detection of HPV-associated oropharyngeal cancer. Under these circumstances, as detailed in the [Reference Examples] section below, the inventors diligently investigated and succeeded in identifying HPV-related oropharyngeal precancerous lesions, thus demonstrating for the first time the existence of "precancerous lesions" in the developmental process of HPV-related oropharyngeal cancer. Based on this new finding, the inventors further investigated and conceived a screening method for HPV-related oropharyngeal precancerous lesions.This screening method makes it possible to recognize HPV-related oropharyngeal cancer at the precancerous lesion stage, leading to early detection and treatment of HPV-related oropharyngeal cancer.

[0025] [Detection of the presence of high-risk HPV mRNA in oral specimens] The inventors focused on high-risk HPV mRNA detected in oral specimens and conducted research, finding a correlation between "the presence of high-risk HPV mRNA in oral specimens" and "the presence of HPV-related oropharyngeal precancerous lesions." As a result, they found that the presence of HPV-related oropharyngeal precancerous lesions in subjects can be screened (detected) by detecting the presence of high-risk HPV mRNA in oral specimens collected from subjects, and thus completed the screening method for step (I).

[0026] The detection process described above will be explained in detail below. The "oral sample" is not particularly limited as long as it is a sample that can detect and / or quantify the presence of high-risk HPV mRNA leaking into the sample from cells in the state of HPV-associated oropharyngeal precancerous lesions or HPV-associated oropharyngeal microcarcinoma, or a sample that contains cells in the state of precancerous lesions or microcarcinomas of HPV-associated oropharyngeal cancer and can detect and / or quantify the presence of high-risk HPV mRNA in those cells. For example, "gargle samples," "saliva samples," or "pharyngeal swab samples" from subjects are preferred samples. The "oral sample" can be a sample that has been collected by being provided in advance by the subject. The timing of the collection of the sample is not particularly limited, and it may be collected multiple times. For the "genotype of high-risk HPV," refer to the explanation above in the (HPV-associated oropharyngeal cancer) section. As shown in the [Reference Examples] section below, the presence of high-risk HPV mRNA in oral samples clearly indicates the presence of advanced precancerous lesions, and therefore can serve as a biomarker that clearly indicates that a subject has precancerous lesions. Accordingly, the screening method in step (I) is performed by detecting high-risk HPV mRNA in oral samples. As described above, the screening method in step (I) is performed by detecting high-risk HPV mRNA, but since the E6 and E7 genes are strongly involved in carcinogenesis in HPV, as one embodiment, the method may be performed by focusing on both and detecting the mRNA of the E6 and / or E7 genes of high-risk HPV. When detecting both, the detection may be performed simultaneously or at different times. As stated above, the scope of application of the screening method related to process (I) is not limited to cases caused by high-risk HPV types such as HPV16, which are specifically demonstrated in the [Reference Examples] section below, but can be broadly applied to screening for HPV-related oropharyngeal precancerous lesions caused by other high-risk HPV types.

[0027] In the screening method related to step (I), the "detection of the presence of high-risk HPV mRNA in the oral sample" and the "detection of the presence of high-risk HPV E6 gene and / or E7 gene mRNA in the oral sample" can be carried out by methods commonly used in the art for mRNA detection and quantification. For example, commercially available detection kits, such as those used in the examples described below, can be used. A person skilled in the art can appropriately select a detection method according to the nature of the object to be detected and carry out the screening method related to step (I) as appropriate. Note that 1) the examination of whether or not the HPV is of the high-risk type and 2) the examination of whether or not the mRNA of the HPV is present in the sample may be carried out in two steps, for example, by evaluating the HPV DNA in the sample to determine whether or not the genotype is of the high-risk type and detecting the presence of HPV mRNA in the sample, or both the examination of the HPV genotype and the detection of mRNA may be carried out simultaneously in one step. A person skilled in the art can appropriately select a detection method according to the nature of the object to be detected and carry out the screening method related to step (I) as appropriate. Furthermore, if the 1) examination of whether or not the HPV is a high-risk type (Step 1) and the 2) examination of whether or not the mRNA of the HPV is present in the sample (Step 2) are performed in two steps, this screening method applies not only when both steps are performed by the same entity, but also when each step is performed by different entities, but both entities are deemed to be working together to perform both steps as a single unit. For example, this could include a case where the entity performing Step 1 performs Step 1 at the request of the entity performing Step 2.

[0028] In the screening method related to step (I), if the presence of mRNA of a high-risk HPV gene is detected as a result of the "step of detecting the presence of high-risk HPV mRNA in an oral sample" (or "step of detecting the presence of mRNA of the E6 and / or E7 genes of high-risk HPV in an oral sample taken from a subject") as detailed above, the subject will be judged to have an HPV-related oropharyngeal precancerous lesion. Here, "if detected" is not particularly limited to the amount of mRNA detected, and refers to cases where an amount sufficient to determine that high-risk HPV mRNA is present in the sample is detected in a medical setting. Therefore, the screening method related to step (I) may further include the step of making such determination. Even if the step of making such determination is performed by a different implementing body than the implementing body that performs the "step of detecting the presence of high-risk HPV mRNA in an oral sample," if the integrated actions of all implementing bodies result in "the subject being judged to have an HPV-related oropharyngeal precancerous lesion," this constitutes the implementation of this screening method. For example, a testing company might undertake a process to "detect the presence of high-risk HPV mRNA in oral samples," and based on the test results, a physician in a medical setting might "determine that the subject has a precancerous lesion of HPV-related oropharyngeal cancer."

[0029] [Application to Screening for HPV-Related Oropharyngeal Microcarcinoma] As described above, the method in step (I) can be used to screen for the presence of HPV-related oropharyngeal precancerous lesions in subjects. HPV-related oropharyngeal cancer often presents as a small primary lesion, resembling cancer of unknown primary origin, and in such cases, it cannot be detected by visual inspection, similar to HPV-related oropharyngeal precancerous lesions. However, since the cells themselves have already become cancerous, mRNA is actively expressed. Therefore, the presence of mRNA of high-risk HPV genes in oral samples can be detected. Consequently, there is a correlation between "the presence of high-risk HPV mRNA in oral samples" and "the presence of HPV-related oropharyngeal microcarcinoma," and therefore, the screening method in step (I) can also be used to screen for HPV-related oropharyngeal microcarcinoma in subjects.

[0030] Another embodiment of the present invention is a kit for detecting p16 protein expression in tonsils for the “detection method” of Embodiment A or the “early detection method” of Embodiment B, comprising [C] an article for clearing tonsils collected from a subject, and an article for immunostaining the cleared tonsils for p16 protein (Embodiment C). Embodiments of the present invention also include the above kit.

[0031] The above kit is for carrying out the "detection method" of Embodiment A or the "early detection method" of Embodiment B, and comprises an article for clearing tonsils collected from a subject, and an article for immunostaining of the cleared tonsils for p16 protein. Articles for detecting other components may be included as needed. These articles are not particularly limited, but examples include articles for detecting high-risk HPV mRNA in oral samples. The kit may also include drugs, instruments, containers, instructions, etc., necessary for using the kit. The meanings and preferred embodiments of various terms such as "clearing tonsils" and "immunostaining of p16 protein" in this embodiment can be understood by referring to the detailed descriptions of the corresponding terms in Embodiment A or Embodiment B.

[0032] The present invention will be described in detail below based on examples and reference examples, but the present invention is not limited to these. Those skilled in the art can modify embodiments of the present invention in various ways without departing from the meaning of the present invention, and such modifications are also included within the scope of the present invention.

[0033] <Examples> Specific examples of this diagnostic method are shown below. Example 1: Clearing of tonsils Below, a specific example using the CUBIC method is described as an example. The CUBIC method uses a combination of two reagents: CUBIC-L and CUBIC-R. CUBIC-L contains N-butyldiethanolamine, a type of amino alcohol, and Triton X-100, and has degreasing and decolorizing activity, and is used for tissue clearing. CUBIC-R is a highly efficient refractive index modifier that combines two types of aromatic amines. Both reagents are commercially available and can be easily obtained by those skilled in the art. [Pretreatment] (In the case of unfixed specimens) The tonsils were immersed in 4% paraformaldehyde and fixed at 4°C for 24 hours. Furthermore, they were washed with PBS (phosphate-buffered saline). For washing, the tonsils were shaken at 37°C for more than 2 hours while immersed in PBS. This procedure was repeated three times. (In the case of formalin-fixed paraffin-embedded specimens) Unnecessary paraffin was removed from the embedded specimen as much as possible with a scalpel. The specimen was placed in an incubator (60°C) for 1 hour to remove further paraffin. The resulting specimen was immersed in xylene, treated with overnight, and then treated with xylene for 3 hours. Next, it was washed with 100%, 90%, 80%, and 70% ethanol for 3 hours each, in that order. Furthermore, it was washed with PBS for 6 hours. [Degreasing and decolorization] For degreasing and decolorization of the tonsils, Tissue clearing reagent CUBIC-L (for animals) (CUBICStars) was used. First, the tonsils were immersed in 50% CUBIC-L solution and shaken at 37°C for 1 day. Next, they were immersed in 100% CUBIC-L solution and shaken at 37°C for 2 weeks. During this process, the solution was replaced with fresh solution every two to three days. Figure 1 shows the tonsils before and after clearing. In the figure, the left image shows the tonsils before clearing, and the right image shows the tonsils after clearing.

[0034] Example 2: Immunostaining of p16 protein (1) The solution in which defatted and destained tonsils were immersed was replaced with a Staining Buffer and shaken at room temperature for 2 hours. In parallel, the primary antibody: p16 INK4A (JC8) mouse monoclonal antibody (Santa Cruz Biotechnology) and the dye-labeled secondary antibody: Alexa Fluor 647 AffiniPure fab fragment goat anti-mouse IgG2a, Fcγ fragment specific (Jackson Immuno Research) were pre-mixed. If the volume of the mixture was less than 120 μL, Staining Buffer was added to adjust the total volume to 120 μL. Then, the mixture was protected from light and the antibody was docked at room temperature for 2 hours. (2) After replacing with Staining Buffer in (1), the solution was discarded, and then the docked antibody solution and nuclear staining reagent: SYTOX green fluorescent dye (Invitrogen) were added, and Staining Buffer was added so that the entire tissue was completely immersed. Then, the entire sample was protected from light and shaken at room temperature for 1 to 3 weeks (depending on the size of the tissue). On the final day, shaking was performed at 4°C. (3) Furthermore, the sample was washed with Wash Buffer for 1 hour twice (4°C, shaking). *The Staining Buffer and Wash Buffer are included in the CUBIC-HV2 3D immunostaining kit (CUBICStars). Figure 2 shows the tonsils during immunostaining (the light shielding was temporarily removed for the purpose of taking the photograph).

[0035] Example 3: Post-fixation The tonsils stained in Example 2 were immersed in a 1% formalin solution prepared by diluting 37% formalin with Wash Buffer and shaken at 4°C for 1 day. Subsequently, they were shaken at 37°C for 1 hour. The fixed tonsils were immersed in ultrapure water and shaken at 37°C for 2 hours.

[0036] Example 4: Refractive Index Adjustment The refractive index was adjusted using the tissue clearing reagent CUBIC-R+(N) (for animals) (CUBICStars). Specifically, the tonsils immobilized in Example 3 were immersed in a 50% R solution and shaken at room temperature for one day. Next, the tonsils were immersed in a 100% R solution and shaken twice for two days. Figure 3 shows the tonsils after refractive index adjustment.

[0037] Example 5: Samples were embedded in 2% agarose gel-CUBIC-R+(N) using a gel-embedded multiwell plate. Specifically, the first layer of gel was solidified at 4°C for 20 minutes, the sample was placed on top, the second layer of gel was poured in, and solidified at room temperature for several hours. Air bubbles in the gel before solidification were removed as much as possible using a micropipette.

[0038] Example 6: Imaging with a light sheet microscope A self-made light sheet microscope (0.63x objective lens) was used to acquire p16 immunostained images at an excitation wavelength of 592 nm and an fluorescence wavelength of 625 nm. Similarly, nuclear stained images were acquired at an excitation wavelength of 488 nm and an fluorescence wavelength of 625 nm. Figure 4 shows the 3D imaging of the tonsils using a light sheet microscope, and Figure 5 shows the imaging for the identification of p16-positive lesions in the tonsils.

[0039] <Example> The following is a specific example of screening (Step (I)) for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in this early detection method.

[0040] Reference Example: Identification of HPV-related oropharyngeal precancerous lesions and verification of the relationship between the presence of such lesions and the E6 / E7 mRNA of high-risk HPV oncogenes detected in gargle samples (hereinafter also referred to as "this study") 1. Overview of "Identification of HPV-related oropharyngeal precancerous lesions" in this study In the oropharynx, HPV infects the tonsillar crypts, which are difficult to observe directly or to collect samples directly. Therefore, there is no way to prove HPV-related oropharyngeal precancerous lesions other than to examine the removed tonsils. Accordingly, the inventors conducted a study targeting patients who underwent tonsillectomy for benign diseases such as habitual tonsillitis and sleep apnea syndrome. Gargle samples were collected during outpatient visits before surgery, and HPV DNA was evaluated to assess the genotype of the detected HPV. Gargle samples were used for evaluation because it is not possible to collect samples directly from the tonsillar crypts. Then, immediately before surgery, gargle samples were collected again from patients in whom high-risk HPV DNA was detected in gargle samples taken during outpatient visits. HPV DNA was evaluated (presence / absence, identification of genotype), and the presence or absence of high-risk HPV mRNA was also evaluated. The excised tonsils were fixed in formalin and embedded in paraffin, and serial sections 6 μm thick were prepared from the entire tonsil. The following studies were conducted to prove the existence of HPV-related oropharyngeal precancerous lesions. Because preparing serial sections requires an enormous amount of effort, in this study, patients in whom HPV16 DNA, which accounts for almost all cases of HPV-related oropharyngeal cancer, was found in gargle samples, i.e., patients with HPV16 pharyngeal infection, were examined regardless of whether high-risk HPV mRNA was detected in the gargle samples. However, in patients in whom DNA of high-risk HPV other than HPV16 was found in gargle samples, i.e., patients with high-risk HPV pharyngeal infection other than HPV16, the study was limited to patients in whom high-risk HPV mRNA was detected in the gargle samples.

[0041] Figure 6 shows an overview of the steps involved in identifying HPV-related oropharyngeal precancerous lesions.

[0042] 2. Details of this study [Methods] The study methods used in this study are described below. Study Method 1: Patients and Sample Collection The patients were those undergoing tonsillectomy for non-malignant diseases at Osaka University Hospital and its affiliated facilities, and patients under 20 years of age were excluded. At the time of outpatient visit, the subjects gargled with 10 mL of phosphate-buffered saline (PBS) or saline for 30 seconds, and the gargle sample was collected in a sterile tube (primary gargle sample). For patients whose primary gargle sample was positive for high-risk HPV DNA, another gargle sample was collected in the same manner on the day of surgery (secondary gargle sample). Of the secondary gargle sample, 2 mL was dispensed into a tube for Aptima (Hologic). In addition, for patients whose primary gargle sample was positive for HPV16 DNA, a blood sample (8.5 mL) was collected in a Cell-Free DNA Collection Tube (Roche) immediately before surgery. Plasma was collected by centrifugation at 3500 rpm for 10 minutes and stored at -80°C until use. The excised tonsils were fixed with 10% formalin, cut into two or three pieces, and embedded in paraffin. This study was approved and registered by the ethics review committee of each hospital (UMIN study ID: UMIN000036583) and conducted with informed consent from all patients.

[0043] Test Method 2: Evaluation of HPV DNA and mRNA in gargle samples, and evaluation of circulating tumor HPV16 DNA (ctHPV16DNA). DNA was extracted from primary and secondary gargle samples and analyzed using the GENOSEARCH HPV31 kit (Medical & Biological Laboratories) to determine the presence or absence of HPV DNA and its genotype. The GENOSEARCH HPV31 kit can detect and determine the genotype of 31 types of HPV, including high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and low-risk types (6, 11, 26, 42, 44, 53, 54, 55, 61, 62, 70, 71, 73, 82, 84, 90, CP6108). The second gargle samples were also evaluated using the Aptima HPV assay kit. The Aptima HPV assay kit can detect E6 / E7 mRNA of 14 high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68), but it cannot determine genotype. Cell-free DNA was extracted from plasma, and the HPV16 E6 / E7 copy number was quantified using droplet digital PCR (ddPCR) to calculate ctHPV16 DNA.

[0044] Test method 3: Identification of high-risk HPV DNA-positive sites in tonsillar tissue. Formalin-fixed paraffin-embedded tonsillar tissue was sectioned at a thickness of 6 µm using a manual rotary microtome (Leica Biosystems), and serial sections of the entire tonsil were prepared. One to three sections were placed on a single slide. The sections were spread on a stretching table (42°C), dried in an incubator (60°C) for several hours, and then stored at 4°C until use. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) every 50 slides. Droplet digital PCR (ddPCR) using the QX200 Droplet Digital PCR System (Bio-Rad) was performed to quantify the E6 / E7 copy number of high-risk HPV genotypes detected from gargle specimens. The absorbance of the droplets was measured using the QX200 Droplet Reader (Bio-Rad), and the results were analyzed using QuantaSoft software v1.7.4.0917 (Bio-Rad). When the slide was E6 and / or E7 positive and the droplet count was 2 or more, the surrounding area including the slide was considered HPV DNA positive.

[0045] Test method 4: Histological evaluation and evaluation of p16, Ki-67, and high-risk HPV mRNA expression. H&E staining, immunostaining of p16 and Ki-67, and in situ hybridization of high-risk HPV mRNA were performed using slides adjacent to those determined to be high-risk HPV DNA positive. The primary antibodies used for immunostaining were anti-p16 INK4A (JC8) mouse monoclonal antibody (Santa Cruz Biotechnology) and anti-Ki-67 (D2H10) rabbit monoclonal antibody (Cell Signaling Technology). For RNA in situ hybridization, an RNAscope cocktail probe (Advanced Cell Diagnostics) that recognizes the E6 / E7 mRNA of 18 high-risk HPV types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) was used.

[0046] [Results] The results of this study are described below. Study Result 1 Between February 2018 and March 2022, 1180 patients were enrolled. Among the underlying diseases for which tonsillectomy was indicated, the most common was habitual tonsillitis (958 cases, 81.2%). In the first gargle test, 49 cases (4.2%) were positive for HPV DNA, of which 32 cases (2.7%) were high-risk types, and 3 cases (0.3%) were limited to HPV16. A second gargle test was planned for 32 cases that were positive for high-risk HPV DNA in the first gargle test. However, in one case the tonsillectomy was canceled and in one case the surgery was performed without collecting a gargle sample, so a second gargle sample was collected from 30 cases. Additionally, one gargle sample intended for HPV DNA testing was discarded, so HPV DNA testing was performed on 29 cases and HPV mRNA testing on 30 cases. The median interval between the first and second gargle tests was 30.5 days (IQR, 24.3–55.3 days). Of the 29 cases that tested positive for high-risk HPV DNA in the first gargle test, 15 cases (51.7%) remained positive for high-risk HPV DNA in the second gargle test. Of the 15 cases that tested positive for high-risk HPV DNA in the second gargle test, high-risk HPV mRNA was detected in 5 cases (33.3%). Of these 5 cases with high-risk HPV mRNA, one case tested positive for HPV16 DNA in the second gargle test, and four cases tested positive for high-risk HPV DNA other than HPV16 in the second gargle test. Specifically, in the second gargle test, one case was considered positive for HPV16 mRNA, and four cases were considered positive for high-risk HPV mRNA other than HPV16. Of the three cases that were positive for HPV16 DNA in the first gargle test, second gargle samples and blood samples were collected from two of them. In both cases, the second gargle test was also positive for HPV16 DNA, but HPV mRNA was positive and negative in one case each. ctHPV16DNA was not detected in either case. In the remaining case, it was not possible to collect a second gargle sample or blood sample. Table 1 shows the patient characteristics and the results of the gargle test. Table 2 shows the genotype of the HPV DNA identified in the gargle test for each patient, and whether or not high-risk HPV mRNA was expressed in the gargle sample.Note that the notation "NT" in Table 2 indicates "not tested".

[0047]

[0048]

[0049] Test Result 2: HPV16 DNA-Positive Sites in Tonsillar Tissue Since most HPV-related oropharyngeal cancers are caused by HPV16 infection, three cases that tested positive for HPV16 DNA in the gargle test were first examined in detail. Subsequently, those who tested positive for high-risk type HPV mRNA in the gargle test are presented as Patient 1, those with unknown high-risk type HPV mRNA as Patient 2, and those with negative high-risk type HPV mRNA as Patient 3. Patient 1 and Patient 3 are men in their late 20s, and Patient 2 is a man in his late 40s. All of them underwent tonsillectomy for habitual tonsillitis. As a result of preparing serial sections from the entire excised tonsils, the number of slides was 1,215 for the right tonsil and 1,275 for the left tonsil of Patient 1, 886 for the right tonsil and 768 for the left tonsil of Patient 2, and 970 for the right tonsil and 948 for the left tonsil of Patient 3. To narrow down the HPV16 DNA-positive sites in the tonsils, DNA was extracted and evaluated every 50 slides. In Patient 1, there were 4 slides (1-068, 1-168, 1-217, 3-251) showing HPV16 DNA positivity in the left tonsil. Since 3 positive slides (1-068, 1-168, 1-217) were adjacent, sandwiching a slide (1-118) that only slightly expressed E6, this area was further evaluated in more detail every 10 slides, and it was found that there were 2 clusters. Thus, the HPV16 DNA-positive sites in Patient 1 were found to be 3, designated as Site1-1, Site1-2, and Site1-3, respectively. Similarly, in Patient 2, there were 3 sites (Site2-1, Site2-2, Site2-3) in the right tonsil, and in Patient 3, there was 1 site (Site3-1) in the left tonsil where HPV16 DNA was positive. The expression level of HPV16 DNA was extremely high at Site2-1 compared to other sites. An overview of the above test results is shown in Figure 7.

[0050] Test Result 3: Verification of HPV16-related precancerous tonsillar lesions (1) Histological evaluation was performed on slides adjacent to each slide that was positive for HPV16 DNA. As a result, one high-grade dysplastic lesion was identified each in Site 1-1, Site 1-3, and Site 2-1, and two in Site 1-2, for a total of five lesions. These were designated as Lesson 1-1, Lesson 1-3, Lesson 2-1, Lesson 1-2-1, and Lesson 1-2-2, respectively. Figure 8 shows details of Lesson 1-2-1, Lesson 1-2-2, and Lesson 2-1, and Figure 9 shows details of Lesson 1-1 and Lesson 1-3. Figure 8A shows the overall tonsils in Site 1-2 and Site 2-1. The tonsil parenchyma consists of non-keratinized stratified squamous epithelium and lymphoid tissue, and has crypt structures that branch from the surface to the deeper parts of the parenchyma. Normal epithelium is composed of a single layer of basal cells and two to three layers of parabasal cells, which gradually flatten, and the nucleus-to-cytoplasm ratio (N / C ratio) decreases towards the surface. In contrast to normal epithelium, abnormal findings such as a high N / C ratio, nuclear enlargement, aggregated chromatin, and nuclear inequality were observed in at least the basal two-thirds of the epithelium in Lesson 1-2-1, Lesson 1-2-2, and Lesson 2-1. However, the polarity of cell maturation from the basal side to the surface was maintained, and the basement membrane was preserved, and no abnormal findings that would lead to a diagnosis of invasive cancer were observed (Figure 8B). Invasion of thin fibrovascular stroma into the epithelium, indicating angiogenesis, was also observed (circled). Lessons 1-2-2 and 2-1 showed particularly high degrees of atypia, with many markedly enlarged nuclei (Figure 8B, arrowheads) and mitotic figures (Figure 8B, arrows). Diffuse and strong expression of p16, a surrogate marker for HPV-related cancer, was observed in the nuclei and cytoplasm of the atypical epithelium (Figure 8C). Ki-67, a marker of cell proliferation, was positive only in basal and parabasal cells in the normal region, but was positive throughout all layers of the atypical epithelium (Figure 8D). Atypical cells showed scattered positive expression of high-risk HPVE6 / E7 mRNA in Lessons 1-2-1 and 1-2-2, and diffuse positive expression in Lesson 2-1 (Figure 8E). Figure 9A shows the overall tonsils in Site 1-1 and Site 1-3.Similar to the lesions in Figure 3, Lesson 1-1 and Lesson 1-3 also showed abnormal findings such as a high N / C ratio and nuclear enlargement, and invasion of thin fibrovascular stroma into the epithelium indicating neovascularization was observed (circled), but no abnormal findings were found that would lead to a diagnosis of invasive carcinoma (Figure 9B). Atypical cells strongly expressed p16 diffusely (Figure 9C), Ki-67 was positive throughout the entire thickness (Figure 4D), and high-risk HPV E6 / E7 mRNA was also positive (Figure 9E). Figure 10 shows the overall tonsil image after p16 immunostaining.

[0051] (2) The results for Site 2-2 and Site 3-1 are shown in Figure 11. Site 3-1 showed a region with koilocytosis-like findings and was designated Lesson 3-1, but no clear cellular atypia was observed, p16 was negative, Ki-67 expression was observed only in basal and parabasal cells, and high-risk HPV E6 / E7 mRNA expression was not observed, so it could not be considered a precancerous lesion. Site 2-2 did not show any clear precancerous lesions, but a region with sporadic expression of high-risk HPV E6 / E7 mRNA was identified (Lesson 2-2). Lesson 2-2 was p16 negative, but Ki-67 was expressed throughout the entire cell layer, and angiogenesis was also observed. From these findings, Lesson 2-2 may be a lesion in the preliminary stage before showing morphological abnormalities. On the other hand, the epithelium of Site 2-3 was similar to normal epithelium both morphologically and in terms of p16 / Ki-67 expression (data not presented), suggesting that it was a state in which only HPV infection was observed.

[0052] As described above, based on a study using HPV16, the most representative high-risk HPV that causes approximately 90% of HPV-related oropharyngeal cancers, a clear correlation was demonstrated between "the presence of high-risk HPV mRNA in gargle samples collected from subjects" and "the presence of HPV-related oropharyngeal precancerous lesions in subjects." This clearly demonstrated that "the presence of high-risk HPV mRNA in gargle samples collected from subjects" can serve as a biomarker for determining the presence of oropharyngeal precancerous lesions. Based on this novel finding, the inventors have completed the present invention. Below, we conducted further investigations to confirm the above. Specifically, we confirmed the presence of precancerous lesions from the perspective of spatial transcriptome analysis, and also confirmed, as expected, that the same results as those obtained with HPV16 could be obtained when using high-risk HPV other than HPV16.

[0053] Test Result 4: Spatial Transcriptome Analysis of HPV16-Associated Tonsil Precancerous Lesions Spatial transcriptome analysis was performed on HPV16-associated tonsil invasive cancer lesions and HPV16-associated tonsil precancerous lesions (Lesion 2-1) using Visium HD (10X Genomics). After removing non-biological batch effects, data from multiple samples were combined. Next, the cell type composition ratio in each spatial region (spot) was estimated, and only spots with an epithelial cell purity of 90% or higher were extracted. After extraction, dimensionality reduction was performed using UMAP (Figure 12 left), followed by visualization and clustering analysis. Cancer (0, red), precancerous lesions (3, green), and normal epithelium (2, yellow-green) formed distinct clusters, revealing that precancerous lesions (Figure 12 right) possessed an intermediate gene profile between cancer (Figure 12 center) and normal epithelium.

[0054] Test Result 5: Verification of High-Risk HPV-Associated Tonsillar Precancerous Lesions Other Than HPV16 Four patients who tested positive for high-risk HPV DNA other than HPV16 and also positive for high-risk HPV mRNA in the second gargle test (Patients 4, 5, 6, and 7) were examined in detail in the same way as the three patients who tested positive for HPV16 DNA in the gargle test (Patients 1, 2, and 3). Patient 4 tested positive for HPV52, Patient 5 for HPV59, Patient 6 for HPV56 and HPV59, and Patient 7 for HPV58 in the second gargle test. Patient 4 was a 42-year-old male, Patient 5 was a 30-year-old male, Patient 6 was a 38-year-old male, and Patient 7 was a 56-year-old male. The underlying disease for all of them was habitual tonsillitis. Serial sections were prepared from the entire excised tonsils, resulting in the following number of slides: 934 for the right tonsil and 1145 for the left tonsil of patient 4; 1345 for the right tonsil and 1223 for the left tonsil of patient 5; 1195 for the right tonsil and 1352 for the left tonsil of patient 6; and 1233 for the right tonsil and 1045 for the left tonsil of patient 7. Slides positive for HPV DNA of the same genotype as the high-risk HPV detected in the second gargle test were identified, and H&E staining, p16 and Ki-67 immunostaining, and in situ hybridization of HPV mRNA were performed using slides adjacent to these identified slides. The lesions of patient 4 are shown as Lesson 4-1, the lesions of patient 5 as Lesson 5-1, and the HPV56-related lesions of patient 6 as Lesson 6-1 in Figure 13. Figure 13A shows an overall view of the tonsil, with the lesion enclosed in a rectangle. Lessons 4-1 and 6-1 showed mild cellular atypia (Figure 13B), but p16 expression was observed (Figure 13C), and Ki-67 was positive throughout all layers (Figure 13D). High-risk HPV E6 / E7 mRNA was also positive (Figure 13E). In Lesson 5-1, severe cellular atypia was observed (Figure 13B), p16 was diffusely expressed (Figure 13C), and Ki-67 was positive throughout all layers (Figure 13D). High-risk HPV E6 / E7 mRNA was also diffusely positive (Figure 13E). Patient 6 also showed HPV59-related lesions with similar findings (data not presented).Since no HPV58-related lesions were found in patient 7, it was considered possible that HPV58-related lesions were latent in the base of the tongue.

[0055] The above test results indicate that patients 1 and 2 have precancerous lesions derived from HPV16, which is p16-positive and positive for high-risk HPV mRNA. Patients 4, 5, and 6 also have precancerous lesions derived from high-risk HPV types other than HPV16, which is p16-positive and positive for high-risk HPV mRNA. In other words, it was demonstrated that patients 1, 2, 4, 5, and 6 have HPV-related oropharyngeal precancerous lesions. These lesions were named Tonsillar Intraepitis Neoplasia (TIN). HPV16 DNA in gargle samples was positive in all three patients (1-3), but high-risk HPV mRNA in gargle samples was positive in patient 1, who had HPV-related precancerous lesions, while it was negative in patient 3, who did not have HPV-related precancerous lesions. Furthermore, while evaluation of high-risk HPV mRNA in the gargle sample was not performed for patient 2, who had HPV-related precancerous lesions, considering that patient 2's lesions expressed high-risk HPV mRNA more strongly than patient 1's lesions, it is likely that if patient 2 had undergone evaluation of high-risk HPV mRNA in the gargle sample, the result would have been positive. In addition, in patients 4, 5, and 6, who also had HPV-related precancerous lesions, high-risk HPV DNA other than HPV16 was positive in the gargle sample in all cases, and high-risk HPV mRNA was also positive. Therefore, it became clear that a positive result for high-risk HPV mRNA in a subject's gargle test, that is, the detection of high-risk HPV E6 and / or E7 gene mRNA in the gargle sample, suggests that the subject has HPV-related oropharyngeal precancerous lesions. As described above, a clear correlation was demonstrated between "the presence of high-risk HPV mRNA in gargle samples collected from subjects" and "the presence of HPV-related oropharyngeal precancerous lesions in subjects." High-risk HPV mRNA is detected in gargle samples from subjects with HPV-related precancerous lesions, but not in gargle samples from subjects without HPV-related precancerous lesions. Therefore, it can be said that it is a highly reliable biomarker for determining the presence of oropharyngeal precancerous lesions through evaluation of gargle samples.

[0056] As described above, the method of process (I) can also be applied to the screening of HPV-related oropharyngeal microcarcinomas. Therefore, high-risk HPV mRNA can be considered a highly reliable biomarker for determining the presence of such microcarcinomas by evaluating gargle samples, similar to the case for determining the presence of oropharyngeal precancerous lesions.

[0057] As one embodiment of the present invention, a method for efficiently detecting the expression of the p16 protein in the tonsils by visualizing it is provided. This detection method is useful in the diagnosis of HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas and is useful in the medical field and other areas. This application is based on Japanese Patent Application No. 2025-018647 (filing date: February 6, 2025), the contents of which are fully incorporated herein.

Claims

(1) (i) A step of clearing the tonsils extracted from the subject, and (ii) The process of subjecting the cleared tonsils to immunostaining for p16 protein, It includes, and further, (2) A step of observing the fluorescence emitted from the immunostained p16 protein in the tonsils subjected to step (1), including, A method for detecting the expression site of the p16 protein in the tonsils. 　 The process further includes the step of determining that a subject has an HPV-related oropharyngeal precancerous lesion or HPV-related oropharyngeal microcarcinoma if p16 protein expression is detected in the tonsils. The detection method according to claim 1. 　 Tonsil clearing is performed using tissue clearing techniques that utilize water-soluble reagents. The detection method according to claim 1 or 2. 　 Tonsil clearing is performed using the CUBIC method. The detection method according to claim 1 or 2. 　 Immunostaining is performed using an indirect method. The detection method according to claim 1 or 2. 　 Immunostaining is performed using the fluorescent labeling method. The detection method according to claim 1 or 2. 　 The fluorescence emitted from the p16 protein is observed using an optical microscope. The detection method according to claim 1 or 2. The presence of an HPV-related oropharyngeal precancerous lesion is diagnosed. The detection method according to claim 2. 　 Before step (1), The method further includes a step (I) of detecting the presence of high-risk HPV mRNA in an oral sample taken from a subject to screen for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas. The detection method according to claim 1 or 2. 　 Step (I) further includes a step in which, if the presence of high-risk HPV mRNA is detected in the oral sample, it is determined that the subject has the said precancerous lesion or HPV-associated oropharyngeal microcarcinoma. The detection method according to claim 9. This is for the early detection of HPV-related oropharyngeal cancer. The detection method according to claim 9 or 10. 　 The mRNA of high-risk HPV is the mRNA of the E6 gene and / or the E7 gene. The detection method according to claim 9 or 10. 　 Oral specimens include gargle specimens, saliva specimens, or pharyngeal swab specimens. The detection method according to claim 9 or 10. 　 Oral sample is a gargle sample. The detection method according to claim 9 or 10. 　 A kit for detecting p16 protein expression in tonsils, comprising an article for clearing tonsils collected from a subject, and an article for immunostaining p16 protein in the cleared tonsils, for carrying out the detection method according to claim 1.

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