Biomarker contributing to detection of human papillomavirus-related oropharyngeal pre-cancerous lesion or human papillomavirus-related oropharyngeal microcarcinoma

Abstract

The present application discloses, as one embodiment thereof, a screening method for a human papillomavirus (HPV)-related oropharyngeal precancerous lesion or an HPV-related oropharyngeal microcarcinoma, the method being useful for early detection of HPV-related oropharyngeal cancer and including a step for detecting the presence of high-risk type HPV mRNA in an oral specimen collected from a subject.

Description

Biomarkers that contribute to the detection of human papillomavirus-associated oropharyngeal precancerous lesions or human papillomavirus-associated oropharyngeal microcarcinomas.

[0001] This invention is based on the discovery of the presence of HPV-related precancerous lesions in the developmental process of human papillomavirus (HPV)-associated oropharyngeal cancer. One embodiment of this invention discloses a screening method for HPV-associated precancerous lesions in subjects by detecting the presence of high-risk HPV mRNA derived from such precancerous lesions in oral samples from subjects as a biomarker for HPV-associated precancerous lesions. This screening method can also be applied to the screening of HPV-associated oropharyngeal microcarcinomas and contributes to the early detection of HPV-associated oropharyngeal cancer, making it useful in the fields of medicine and pharmaceuticals.

[0002] HPV is a circular double-stranded DNA virus that is transmitted through contact. There are over 200 types (genotypes) of HPV, which are classified into high-risk and low-risk types, with high-risk HPV types inducing cancer. The main cancers induced by high-risk HPV infection are cervical cancer and HPV-associated oropharyngeal cancer. Approximately 70% of cervical cancers are caused by HPV16 and HPV18, while approximately 90% of HPV-associated oropharyngeal cancers are caused by HPV16. HPV-associated oropharyngeal cancer is increasing 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. Oropharyngeal cancer is divided into HPV-unassociated oropharyngeal cancer, which is caused by smoking and drinking, and HPV-associated oropharyngeal cancer, which is caused by high-risk HPV infection. On the other hand, in the case of cervical cancer, since almost all cases are caused by high-risk HPV infection, this classification is not made. Most HPV-related oropharyngeal cancers occur in the tonsils or base of the tongue, with the former being more common. Furthermore, both HPV-related and non-HPV-related oropharyngeal cancers are more common in men.

[0003] The cervix can be directly observed, and specimens can be directly collected from it. Because of this, the entire process (natural history) from HPV infection to the development of precancerous lesions and then invasive cancer in the cervix has been elucidated. Furthermore, techniques for screening precancerous lesions have been established, and cervical cancer screening is conducted. On the other hand, in the oropharynx, HPV infects depressions called crypts. While numerous crypts exist, it is impossible to directly observe their interiors, nor is it possible to directly collect specimens from them. For these reasons, HPV-related precancerous lesions have not yet been identified in the oropharynx, and the natural history from HPV infection to the development of invasive cancer remains unknown. Furthermore, techniques for the early detection of HPV-related oropharyngeal cancer have not been established, and no screening methods exist (Non-Patent Literature 1).

[0004] Cancer prevention is classified into primary prevention and secondary prevention. The prevention of cancer is achieved by both primary and secondary prevention working in tandem. Primary prevention aims to reduce the risk of cancer, and typical examples include smoking cessation and moderation of alcohol consumption. In the case of HPV-related cancers such as cervical cancer and HPV-related oropharyngeal cancer, vaccination with the HPV vaccine to prevent HPV infection serves as primary prevention. However, in Japan, while regular vaccination of women with the HPV vaccine is carried out, regular vaccination of men is not. That is, primary prevention of HPV-related oropharyngeal cancer is not being implemented. Secondary prevention is the early detection of cancer, and cancer screening corresponds to this. For cervical cancer, screening methods have been established, and samples are directly collected from the cervix for evaluation by cytology and evaluation by HPV DNA. On the other hand, for HPV-related oropharyngeal cancer, the very existence of pre-cancerous lesions is unknown, making it impossible to perform early detection. For these reasons, in order to prevent HPV-related oropharyngeal cancer, which is on the rise in developed countries including Japan, it is an urgent issue to introduce regular vaccination of men with the HPV vaccine and establish a screening method after clarifying the existence of pre-cancerous lesions. There is a report regarding the detection of HPV-related pre-cancerous lesions or HPV-related cancers located in the cervix, anorectal region, oropharynx / nasopharynx, oral pharynx, penis, or vulva, including the detection of LGR6, LGR5, and / or LGR4 (LGRs) in a sample and the detection of HPV (Patent Document 1). In this report, the detection of HPV may be by DNA, RNA, or protein. However, in this report, the detection of LGRs is merely a necessary condition for the detection of the pre-cancerous lesion, and it does not conclude that the pre-cancerous lesion can be detected solely by the detection of HPV mRNA. Furthermore, the existence of HPV-related oropharyngeal pre-cancerous lesions is not demonstrated at all in this report. According to the only report different from Patent Document 1 that analyzed the expression of LGRs in HPV-related oropharyngeal cancer, approximately 30% of HPV-related oropharyngeal cancers do not express LGRs (Non-Patent Document 2). Similarly, approximately 30% of head and neck cancers other than HPV-related oropharyngeal cancer also do not express LGRs. This indicates that LGRs cannot be a useful biomarker for detecting head and neck cancers including HPV-related oropharyngeal cancer.

[0005] International Publication No. 2016 / 207414

[0006] Oncogene (2024) 43:543-554 Oral Oncology (2020) 105:104657

[0007] In view of the urgent issues in the medical field described above, the present invention aims to clarify the existence of HPV-related precancerous lesions in the developmental process of HPV-related oropharyngeal cancer, and, based on this novel finding, to provide, as one embodiment, a screening method for HPV-related precancerous lesions in subjects for the early detection of HPV-related oropharyngeal cancer.

[0008] Specifically, the following embodiments of the present invention are mentioned, but the present invention is not limited to these embodiments. [1] A screening method for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas, comprising the step of detecting the presence of high-risk HPV (human papillomavirus) mRNA in an oral sample taken from a subject. [2] The screening method according to [1], 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 an oral sample. [3] The screening method according to [1] or [2], for the early detection of HPV-related oropharyngeal cancer.

[0009] [4] The screening method according to any one of [1] to [3] above, wherein the mRNA of the high-risk HPV is the mRNA of the E6 gene and / or the E7 gene. [5] The screening method according to any one of [1] to [4] above, wherein the oral sample is a gargle sample, a saliva sample, or a pharyngeal swab sample. [6] The screening method according to any one of [1] to [5] above, wherein the oral sample is a gargle sample.

[0010] [7] A biomarker containing high-risk HPV mRNA for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects. [8] High-risk HPV mRNA to be used as a biomarker for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects. [9] High-risk HPV mRNA in an oral sample taken from a subject, which is a biomarker for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

[10] Use of high-risk HPV mRNA in an oral sample taken from a subject as a biomarker for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

[0011]

[11] A kit for screening for HPV-associated oropharyngeal precancerous lesions or HPV-associated oropharyngeal microcarcinomas in a subject, comprising an article for detecting mRNA of high-risk HPV types in an oral sample taken from a subject. (*) In this specification, HPV-associated oropharyngeal precancerous lesions are synonymous with precancerous lesions of HPV-associated oropharyngeal cancer, and HPV-associated oropharyngeal microcarcinomas are synonymous with microcarcinomas of HPV-associated oropharyngeal cancer.

[0012] This invention has for the first time revealed the presence of HPV-related precancerous lesions in the developmental process of HPV-related oropharyngeal cancer. Based on this novel finding, one embodiment of this invention is disclosed as a screening method for HPV-related oropharyngeal cancer or HPV-related oropharyngeal microcarcinoma in a subject, which is useful for the early detection of HPV-related oropharyngeal cancer or HPV-related oropharyngeal microcarcinoma by detecting the presence of high-risk HPV mRNA derived from such precancerous lesions or microcarcinomas in the subject's oral sample as a biomarker for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinoma.

[0013] Figure 1 shows an overview of each step for identifying HPV-related oropharyngeal precancerous lesions, as described in the Examples section below. Items A to D in Figure 1 refer to the following: A. A step to collect "gargle samples" from patients undergoing tonsillectomy for non-malignant diseases to screen for patients with pharyngeal HPV infection, i.e., patients whose HPV DNA in the "gargle sample" is positive. B. A step to prepare serial sections of the entire tonsil by fixing the tonsils removed from patients who tested positive for high-risk HPV DNA in the gargle test with formalin, dividing them into 2 to 3 sections, and embedding them in paraffin. C. A step to extract DNA every 50 slides, quantify HPV E6 / E7 DNA by digital PCR, and narrow down the slides to those that are positive for high-risk HPV DNA of the genotype detected in the gargle test. D. The procedure involves performing hematoxylin and eosin staining (H&E staining), p16 and Ki-67 immunostaining, and high-risk HPV mRNA in situ hybridization using slides adjacent to slides that tested positive for high-risk HPV E6 / E7 DNA. Figure 2 shows an overview of "Test Results 2" described in the Examples section below. After preparing serial sections from the entire tonsils of three patients whose gargle samples tested 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 droplets of HPV16 E6 and E7, 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. In 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, a more detailed analysis of each of the 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 3 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 3 refer to the following: A. H&E staining of Site 1-2 and Site 2-1 (overall view of the tonsils). 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 test 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. Overexpression of p16 was diffusely 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 showing 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 4 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 4 refer to the following: A. H&E staining (overall view of the tonsils) 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 Site 1-3. Each lesion is enclosed in a rectangle and labeled as 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 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. Expression of high-risk HPV mRNA was observed in cells showing atypicality. Figure 5 shows the overall tonsils of each site identified in "Test Results 2" described in the Examples section below, based on p16 immunostaining. Figure 6 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 6 shows the results of the examination described in "Test Results 3 (2)" in the Examples section below. A to E in Figure 6 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 confirmed, 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 performed with a high-risk HPV probe.In Lesson 2-2, sparsely expressed epithelial cells with weak expression of high-risk HPV mRNA were observed, but no such cells were found in Lesson 3-1. The scale bars correspond to 100 μm in B, C, and D, and 20 μm in E. Figure 7 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 different clusters, and 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 8 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 lesion. Each lesion is enclosed in a rectangle. B. H&E staining (magnified view). C. p16 immunostaining. The lower panel shows a magnified view of the area enclosed in 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 throughout all layers. High-risk HPV mRNA expression was also observed. In Lesson 5-1, severe cellular atypia was observed, p16 was diffusely expressed, and Ki-67 was positive throughout 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.

[0014] 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.

[0015] [Screening Method for HPV-Related Precancerous Lesions, etc.] One embodiment of the present invention is "[A] A screening method for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinoma, comprising the step of detecting the presence of high-risk HPV mRNA in an oral sample taken from a subject" (Embodiment A). Hereinafter, the above step may be referred to as "Step (I)". This embodiment is a screening method for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinoma (hereinafter also referred to as "this screening method") that aims to evaluate the risk of developing HPV-related oropharyngeal cancer in a subject using a non-invasive and simple method, based on the clarification of the presence of HPV-related precancerous lesions in the developmental process of HPV-related oropharyngeal cancer, and to detect it early (hereinafter also referred to as "this screening method"). The screening method will be described below.

[0016] (HPV-associated oropharyngeal cancer) Oropharyngeal cancer is a type of head and neck cancer that develops in the oropharynx. Oropharyngeal cancer is divided into two types: HPV-unassociated oropharyngeal cancer, which is caused by smoking and drinking, and HPV-associated oropharyngeal cancer, which is caused by high-risk HPV infection. HPV is a circular double-stranded DNA 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 types induce cervical cancer, anal cancer, vaginal cancer, HPV-associated oropharyngeal cancer, etc. HPV-associated 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 approximately 90% of cases are caused by type 16 (HPV16). In the [Examples] section below, the present invention is specifically explained using "HPV52, HPV56, and HPV59" in addition to "HPV16," which is the most representative cause of the disease. However, the present invention is not limited to cases where these high-risk HPV genotypes are the cause, but can also be applied to cases where other high-risk HPV genotypes are the cause. In other words, the present invention can be applied to all genotypes of high-risk HPV.

[0017] (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." In this invention, more specifically, a lesion is considered 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 from two-thirds to all layers of the epithelium (without rupturing the basement membrane and remaining within the epithelium). In this specification, the term "precancerous lesions" is used to refer collectively to items 1) and / or 2) above. As mentioned earlier, the cervix can be directly observed, and specimens can be directly collected from it. For these reasons, the entire process (natural history) from HPV infection to the development of invasive cancer via precancerous lesions has been elucidated in the cervix. Furthermore, techniques for screening precancerous lesions have been established, and cervical cancer screening is conducted. In contrast, in the oropharynx (tonsils, base of the tongue), HPV infects depressions called crypts. Numerous crypts exist, but it is impossible to directly observe their contents, nor is it possible to directly collect specimens from them. For these reasons, HPV-related precancerous lesions have not yet been identified in the oropharynx. Under these circumstances, the inventors, as detailed in the [Examples] section below, 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 the onset of HPV-related oropharyngeal cancer at the precancerous lesion stage, and also enables the recognition of HPV-related oropharyngeal microcarcinomas, thereby leading to early detection and treatment of HPV-related oropharyngeal cancer.

[0018] (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 this screening method.

[0019] 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 HPV-associated oropharyngeal precancerous lesions or HPV-associated oropharyngeal microcarcinoma 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 section on (HPV-associated oropharyngeal cancer). As shown in the [Examples] section below, the presence of high-risk HPV mRNA in oral samples clearly indicates the presence of precancerous lesions, and therefore can serve as a biomarker that clearly indicates that a subject has precancerous lesions. Accordingly, the screening method of the present invention is performed by detecting high-risk HPV mRNA in oral samples. As described above, this screening method 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 present invention is not limited to cases caused by high-risk HPV genotypes specifically demonstrated in the [Examples] section below, but can be broadly applied to the screening of HPV-related oropharyngeal precancerous lesions caused by other high-risk HPV types.

[0020] In this screening method, "detection of the presence of high-risk HPV mRNA in oral samples" and "detection of the presence of high-risk HPV E6 gene and / or E7 gene mRNA in oral samples" can be performed 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. Those skilled in the art can appropriately select the detection method according to the characteristics of the object to be detected and implement this screening method as appropriate. Note that 1) the examination of whether HPV is of the high-risk type and 2) the examination of whether HPV mRNA is present in the sample may be performed in two steps, for example, by evaluating the HPV DNA in the sample to determine whether 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 performed simultaneously in one step. Those skilled in the art can appropriately select the detection method according to the characteristics of the object to be detected and implement this screening method 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.

[0021] In this screening method, if the presence of high-risk HPV mRNA is detected as a result of the "step of detecting the presence of high-risk HPV mRNA in oral samples" (or "step of detecting the presence of high-risk HPV E6 gene and / or E7 gene mRNA in oral samples collected from subjects") 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, but 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, this screening method may further include the step of making such a determination. Even if the step of making such a determination is performed by a different implementing body than the same implementing body that performed the "step of detecting the presence of high-risk HPV mRNA in oral samples," 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."

[0022] [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.

[0023] Furthermore, if necessary, the detection of high-risk HPV mRNA in oral samples collected from subjects using this screening method may be combined with examination for oropharyngeal cancer (e.g., visual inspection or endoscopic examination). Such embodiments are also included within the scope of the present invention.

[0024] [Biomarker for screening HPV-related oropharyngeal precancerous lesions] Another embodiment of the present invention is "[B] A biomarker comprising high-risk HPV mRNA for screening HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in a subject" (Embodiment B). As described above, the detection of high-risk HPV mRNA serves as a "biomarker" for detecting HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas. Therefore, as one embodiment of the present invention, the use of high-risk HPV mRNA (more specifically, high-risk HPV mRNA in an oral sample taken from a subject) as a "biomarker" for screening HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas is provided. One example of such use as a "biomarker" is to examine whether or not high-risk HPV mRNA as a biomarker is detected in an oral sample of a subject. Furthermore, the meanings and preferred embodiments of various terms such as "mRNA of high-risk HPV" in this embodiment can be understood by referring to the detailed descriptions of the corresponding terms in Embodiment A.

[0025] [Detection Kit for High-Risk HPV mRNA] Another embodiment of the present invention is a kit for screening for HPV-associated oropharyngeal precancerous lesions or HPV-associated oropharyngeal microcarcinomas in a subject, comprising an article for detecting high-risk HPV mRNA in an oral sample taken from a subject (Embodiment C). Embodiments of the present invention also include the above kit.

[0026] The above kit comprises articles for detecting high-risk HPV mRNA in an oral sample. Articles for detecting other components may be included as needed. These articles are not particularly limited, but examples include antibodies. 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 "high-risk HPV mRNA" in this embodiment can be understood by referring to the detailed descriptions of corresponding terms in Embodiment A.

[0027] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. For example, the following examples illustrate the cases where the high-risk HPV types are HPV16, HPV52, HPV56, and HPV59. This is an illustration of the present invention using cases of infection with some high-risk HPV types, including HPV16, which is a representative high-risk HPV that causes HPV-associated oropharyngeal cancer. 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.

[0028] 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 the preparation of serial sections requires an enormous amount of effort, in this study, patients in whom HPV16 DNA, which accounts for the majority of HPV-related oropharyngeal cancers, 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.

[0029] Figure 1 shows an overview of the steps involved in identifying HPV-related oropharyngeal precancerous lesions.

[0030] 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) 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.

[0031] 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 and 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.

[0032] Test Method 3: Identification of High-Risk HPV DNA Positive Sites in Tonsil Tissue Formalin-fixed, paraffin-embedded tonsil tissue was sectioned to a thickness of 6 μm using a manual rotary microtome (Leica Biosystems) to prepare serial sections of the entire tonsil. One to three sections were placed on each glass slide. The sections were spread on a spreading table (42°C), dried in an incubator (60°C) for several hours, and then stored at 4°C until use. DNA was extracted from every 50 slides using the QIAamp DNA FFPE Tissue Kit (Qiagen). The E6 / E7 copy numbers of high-risk HPV genotypes detected from gargle samples were quantified by ddPCR using the QX200 Droplet Digital PCR System (Bio-Rad). The absorbance of droplets was measured using a QX200 Droplet Reader (Bio-Rad), and the results were analyzed using QuantaSoft software v1.7.4.0917 (Bio-Rad). If a slide was E6 and / or E7 positive and its droplet count was 2 or greater, the surrounding area including that slide was considered HPV DNA positive.

[0033] Test Method 4: Histological Evaluation and Evaluation of p16, Ki-67, and High-Risk HPV mRNA Expression H&E staining, immunohistochemical staining for p16 and Ki-67, and in situ hybridization of high-risk HPV mRNA were performed on slides adjacent to those determined to be positive for high-risk HPV DNA. The primary antibodies used for immunohistochemical staining 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, we used an RNAscope cocktail probe (Advanced Cell Diagnostics) that recognizes E6 / E7 mRNA of 18 high-risk HPV strains (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82).

[0034] [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 "NT" in Table 2 indicates "not tested".

[0035]

[0036]

[0037] Test Result 2: HPV16 DNA-Positive Sites in Tonsil Tissue Since most HPV-related oropharyngeal cancers are caused by HPV16 infection, we first examined in detail three cases that tested positive for HPV16 DNA in a gargle test. Hereafter, patients who tested positive for high-risk HPV mRNA in the gargle test will be referred to as Patient 1, those whose high-risk HPV mRNA was unknown as Patient 2, and those who tested negative for high-risk HPV mRNA as Patient 3. Patients 1 and 3 were men in their late 20s, and Patient 2 was a man in his late 40s. All three underwent tonsillectomy for habitual tonsillitis. Serial sections were prepared from the entire excised tonsils. The number of slides obtained was 1215 for the right tonsil and 1275 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 within the tonsils, DNA was extracted and evaluated every 50 slides. In patient 1, there were four slides (1-068, 1-168, 1-217, 3-251) showing HPV16 DNA positivity in the left tonsil. Three of the positive slides (1-068, 1-168, 1-217) were located close together, separated by a slide (1-118) that showed only slight expression of E6. Therefore, this area was further evaluated every 10 slides, revealing the presence of two clusters. Thus, there were three HPV16 DNA-positive sites in patient 1, which were designated Site 1-1, Site 1-2, and Site 1-3. Similarly, in patient 2, three HPV16 DNA-positive sites were found in the right tonsil (Site 2-1, Site 2-2, Site 2-3), and in patient 3, one site was found in the left tonsil (Site 3-1). The expression level of HPV16 DNA was extremely high at Site 2-1 compared to the other sites. A summary of these test results is shown in Figure 2.

[0038] 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 3 shows details of Lesson 1-2-1, Lesson 1-2-2, and Lesson 2-1, and Figure 4 shows details of Lesson 1-1 and Lesson 1-3. Figure 3A 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 3B). 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 3B, arrowheads) and mitotic figures (Figure 3B, 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 3C). Ki-67, a marker of cell proliferation, was positive only in basal and parabasal cells in normal tissue, but was positive throughout all thicknesses in atypical epithelium (Figure 3D). Atypical cells showed scattered positivity for high-risk HPV E6 / E7 mRNA in Lessons 1-2-1 and 1-2-2, and diffuse positivity in Lesson 2 precancerous-1 (Figure 3E). Figure 4A shows the overall tonsils in Site 1-1 and Site 1-3.Similar to the lesions in Figure 3, Lessons 1-1 and 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). However, no abnormal findings were observed that would lead to a diagnosis of invasive carcinoma (Figure 4B). Atypical cells strongly expressed p16 diffusely (Figure 4C), were positive for Ki-67 throughout the entire thickness (Figure 4D), and were also positive for high-risk HPV E6 / E7 mRNA (Figure 4E). Figure 5 shows the overall tonsil image after p16 immunostaining.

[0039] (2) The results for Site 2-2 and Site 3-1 are shown in Figure 6. Site 3-1 showed a region with koilocytosis-like findings and was designated Lesson 3-1. However, 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. No obvious precancerous lesions were found in Site 2-2, but regions with sporadic high-risk HPV E6 / E7 mRNA expression were 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. Therefore, 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.

[0040] 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.

[0041] 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 7 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 7 right) possessed an intermediate gene profile between cancer (Figure 7 center) and normal epithelium.

[0042] 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, as shown in Figure 8. Figure 8A shows an overall view of the tonsil, with lesions enclosed in rectangles. Lessons 4-1 and 6-1 showed mild cellular atypia (Figure 8B), but p16 expression was observed (Figure 8C), and Ki-67 was positive throughout all layers (Figure 8D). High-risk HPV E6 / E7 mRNA was also positive (Figure 8E). In Lesson 5-1, severe cellular atypia was observed (Figure 8B), p16 was diffusely expressed (Figure 8C), and Ki-67 was positive throughout all layers (Figure 8D). High-risk HPV E6 / E7 mRNA was also diffusely positive (Figure 8E). Patient 6 also showed HPV 59-related lesions with similar findings (data not presented). Since no HPV 58-related lesions were found in patient 7, it was considered possible that HPV 58-related lesions were latent in the base of the tongue.

[0043] The above test results indicate that Patient 1 and Patient 2 have precancerous lesions derived from HPV16 that are p16 positive and high-risk type HPV mRNA positive. In addition, Patients 4, 5, and 6 have precancerous lesions derived from high-risk type HPVs other than HPV16 that are p16 positive and high-risk type HPV mRNA positive. That is, it was demonstrated that five patients, namely Patient 1, Patient 2, Patient 4, Patient 5, and Patient 6, have HPV-related oropharyngeal precancerous lesions. These lesions were named Tonsillar Intraepithelial Neoplasia (TIN). The HPV16 DNA in the gargle specimens was positive in all three of Patients 1 to 3. However, the high-risk type HPV mRNA in the gargle specimens was positive in Patient 1 with HPV-related precancerous lesions, but negative in Patient 3 without HPV-related precancerous lesions. Also, although the evaluation of the high-risk type HPV mRNA in the gargle specimens was not performed in Patient 2 with HPV-related precancerous lesions, considering that the lesions of Patient 2 express high-risk type HPV mRNA more strongly than those of Patient 1, if Patient 2 had been evaluated for high-risk type HPV mRNA in the gargle specimens, the result would be considered positive. In addition, in Patients 4, 5, and 6 with HPV-related precancerous lesions, high-risk type HPV DNA other than HPV16 was positive in the gargle specimens and high-risk type HPV mRNA was also positive in all of them. Therefore, it became clear that a positive result for high-risk type HPV mRNA in the gargle test of the subject, that is, the detection of the mRNA of the E6 and / or E7 genes of high-risk type HPV in the gargle specimen, suggests that the subject has HPV-related oropharyngeal precancerous lesions.

[0044] 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.

[0045] As stated above, this screening method 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. Although this invention is excellent as described above, verification is currently underway to make it even more robust and practical.

[0046] The present invention discloses, as one embodiment, a method for screening for HPV-related precancerous lesions or HPV-related microcarcinomas in a subject by detecting the presence of high-risk HPV mRNA derived from the precancerous lesion in the subject's oral sample as a biomarker for HPV-related precancerous lesions or HPV-related microcarcinomas, and is useful in the fields of medicine, pharmaceuticals, etc. This application is based on Japanese Patent Application No. 2024-167655 (filing date: September 26, 2024) and Japanese Patent Application No. 2025-017926 (filing date: February 5, 2025), the contents of which are fully incorporated herein.

Claims

1. A screening method for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas, comprising the step of detecting the presence of high-risk HPV (human papillomavirus) mRNA in an oral sample collected from a subject.

2. The screening method according to claim 1, further comprising the step of determining that a subject has a precancerous lesion or a microcarcinoma if the presence of high-risk HPV mRNA is detected in an oral sample.

3. The screening method according to claim 1 or 2, for the early detection of HPV-related oropharyngeal cancer.

4. The screening method according to claim 1 or 2, wherein the mRNA of high-risk HPV is the mRNA of the E6 gene and / or E7 gene of high-risk HPV.

5. The screening method according to claim 1 or 2, wherein the oral sample is a gargle sample, a saliva sample, or a pharyngeal swab sample.

6. The screening method according to claim 1 or 2, wherein the oral sample is a gargle sample.

7. Biomarkers containing high-risk HPV mRNA for screening HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

8. mRNA of high-risk HPV species to be used as a biomarker for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

9. mRNA of high-risk HPV in oral samples collected from subjects, which serves as a biomarker for screening HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

10. Use of high-risk HPV mRNA from oral samples collected from subjects as a biomarker for screening HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects.

11. A kit for screening for HPV-related oropharyngeal precancerous lesions or HPV-related oropharyngeal microcarcinomas in subjects, comprising an article for detecting high-risk HPV mRNA in oral samples taken from subjects.

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