Method and kit for evaluating carcinogenic risk of human papillomavirus-associated cervical cancer

Abstract

Provided are: a method for evaluating that, in an amino acid sequence encoded by E6 gene of human papillomavirus (HPV) to be analyzed, the carcinogenic risk of cervical cancer is high when the amino acid corresponding to the 34th position of the amino acid sequence represented by SEQ ID NO: 1 is a phenylalanine, the amino acid corresponding to the 40th position is an arginine or lysine, the amino acid corresponding to the 102nd position is a tyrosine or leucine, and the amino acid corresponding to the 120th position is a leucine, and the risk is low for all other combinations; and a test kit for evaluating the carcinogenic risk of HPV-associated cervical cancer, the test kit including a reagent for concentrating a nucleic acid or a nucleic acid fragment containing the E6 gene of HPV in a sample or a reagent for identifying the amino acid sequence encoded by the E6 gene of HPV.

Description

Method and kit for assessing the risk of human papillomavirus-induced cervical cancer

[0001] The present invention relates to a method and a kit for assessing the risk of human papillomavirus (HPV)-induced cervical cancer.

[0002] Papillomaviruses (PVs) are non-enveloped viruses with circular double-stranded DNA. PVs infect the epithelia of vertebrates, including humans, non-human mammals, birds, and reptiles. Due to the lack of a strong antibody response in the host and the difficulty of applying classical culture techniques, PVs are classified based on genome sequence rather than serotype. The genome size of PVs is approximately 8 kb and is evolutionarily stable. A typical PV genome consists of an upstream regulatory region (URR), three early (E) regulatory genes (E6, E7, and E1), and two late (L) structural genes (L1 and L2). The L gene encodes the capsid protein, and the L1 gene is known to be the most conserved gene among all PVs. The type of a newly isolated PV is determined by two criteria: cloning of the full-length genome and a nucleotide sequence in the L1 gene that differs by at least 10% from any known PV type (Non-Patent Document 1). The αPV genus is characterized by its detection in skin and / or mucosal lesions of humans and primates.

[0003] Genital HPV, which infects the genital mucosa, is predominantly alpha-HPV and is the leading cause of cervical cancer. Cervical cancer is the fourth most common cancer among women worldwide. In 2020, 600,000 new cases of cervical cancer were diagnosed, and 340,000 people died from the disease (Non-Patent Document 2). Some genital HPVs have been clinically identified as pathogenic and have been subdivided into several groups. HPV16 and 18 have been reported as the HPVs with the highest risk of developing cervical cancer, and these two HPVs are thought to be associated with more than 70% of cervical cancers worldwide (Non-Patent Document 3).

[0004] reported a list of high-risk and low-risk HPV groups in 2003 based on a pooled analysis of epidemiological data from 3,846 women in nine countries (Non-Patent Document 4). In this report, 18 HPV types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) were identified as high-risk or likely high-risk HPVs, and 12 HPV types (HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89 (referred to as CP6108 in the report)) were identified as low-risk HPVs. Munoz et al. also reported discrepancies between the epidemiological risk classification and the phylogenetic classification based on L1 gene sequences for HPV73 and HPV70.

[0005] Approximately half of αHPVs have not been classified for epidemiological risk, and most of these unclassified αHPVs are not subject to clinical testing. Because any αHPV can potentially infect mucous membranes, including the genital mucosa, characterizing unclassified αHPVs will aid in understanding the clinical outcomes associated with single and multiple αHPV infections. There is a need to resolve the discrepancy between epidemiological risk classification and phylogenetic classification based on L1 gene sequences and to develop novel markers to characterize unclassified HPVs, including newly identified HPVs.

[0006] Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM , Virology (2010) 401(1), 70-79.International Agency for Research on Cancer, Cancer Fact Sheets, Cervix uteri (C53) (2020) https: / / gco.iarc.fr / today / data / factsheets / cancers / 23-Cervix-uteri-fact-sheet.pdf.de Sanjose S, et al., Lancet Oncol. (2010) 11(11), 1048-1056.Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJF, Meijer CJLM, N. Engl. J. Med. (2003) 348(6), 518-527.

[0007] An objective of the present invention is to provide a method and kit for evaluating the risk of human papillomavirus (HPV)-induced cervical cancer, and a method for determining the level of risk of developing cervical cancer in a subject.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions: [1] A method for assessing the risk of human papillomavirus (HPV)-induced cervical cancer, wherein the risk is assessed as high if, in the amino acid sequence encoded by the E6 gene of the HPV being analyzed, the amino acid corresponding to position 34 of the amino acid sequence set forth in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine, and the risk is assessed as low if any other combination is observed. [2] A method for determining a subject's risk of developing cervical cancer, wherein, in the amino acid sequence encoded by the E6 gene of at least one HPV detected in a cervical sample from the subject, the risk is assessed as high if the amino acid corresponding to position 34 of the amino acid sequence set forth in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine. [3] The method according to [2] above, comprising the following steps (a) to (d): (a) extracting nucleic acid from a cervical sample from a subject, (b) concentrating a nucleic acid or nucleic acid fragment comprising the HPV E6 gene from the nucleic acid extracted in step (a), (c) identifying the HPV E6 amino acid sequence, and (d) determining whether, in the E6 amino acid sequence, the amino acid corresponding to position 34 of the amino acid sequence set forth in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine. [4] A test kit for assessing the risk of HPV cervical cancer, comprising a reagent for concentrating nucleic acid or nucleic acid fragment comprising the HPV E6 gene in a sample, wherein the enrichment reagent comprises an amplification primer pair for amplifying a full-length or partial sequence of a nucleic acid comprising the HPV E6 gene or a hybridization capture probe having a sequence complementary to a sequence comprising the HPV E6 gene.[5] A test kit for assessing the risk of HPV-induced cervical cancer, comprising a reagent for identifying at least one amino acid selected from the amino acid corresponding to positions 34, 40, 102, and 120 of the amino acid sequence set forth in SEQ ID NO: 1 in an amino acid sequence encoded by the HPV E6 gene in a sample. [6] The kit according to [5] above, wherein the reagent for identifying the amino acid comprises a reagent for determining the base sequence of the HPV E6 gene.

[0009] The present invention can provide a method and a kit for evaluating the risk of human papillomavirus (HPV)-induced cervical cancer.

[0010] 1 shows a phylogenetic tree of HPVs based on the E6 gene. HPVs classified as high-risk types in the epidemiological classification by Munoz et al. (2003) are indicated with an asterisk, and clades containing these high-risk types are indicated with a box. This is an enlarged view of the clades indicated with a box in FIG. 1. The asterisks indicate HPVs classified as high-risk types in the epidemiological classification by Munoz et al. (2003). This is a phylogenetic tree of HPVs based on the L1 gene. HPVs classified as high-risk types in the epidemiological classification by Munoz et al. (2003) are indicated with an asterisk, and clades containing these high-risk types are indicated with a box. This is an enlarged view of the clade indicated with a box in FIG. 3. The asterisks indicate HPVs classified as high-risk types in the epidemiological classification by Munoz et al. (2003). This is a view showing HPV risk type classification based on the E6 amino acid sequence and the positions of four amino acids conserved in E6-RDAA high-risk types.

[0011] [Method for assessing risk of cervical cancer caused by HPV] The present invention provides a method for assessing risk of cervical cancer caused by human papillomavirus (HPV) (hereinafter referred to as "assessment method of the present invention"). The assessment method of the present invention is characterized in that, in the amino acid sequence encoded by the E6 gene of the HPV to be analyzed, if the amino acid corresponding to position 34 of the amino acid sequence set forth in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine, the risk is assessed as high, and if any other combination is present, the risk is assessed as low.

[0012] As used herein, "human papillomavirus" and "HPV" refer to viruses of the genus Papillomavirus that can infect humans. In the evaluation method of the present invention, the HPV to be analyzed is not particularly limited as long as it is a human papillomavirus, and may be an alpha HPV (hereinafter referred to as alphaHPV) or an unclassified HPV. In the evaluation method of the present invention, the HPV to be analyzed may be a known HPV or a newly identified HPV. Examples of known HPV include HPVs listed in the PaVE (Papillomavirus Episteme) database (van Doorslaer K et al., 2017, https: / / pave.niaid.nih.gov / ).

[0013] In the present invention, "cervical cancer" includes carcinoma in situ and invasive carcinoma of the cervix, which may be, for example, squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumor or other cancer types.

[0014] In the evaluation method of the present invention, the amino acid sequence encoded by the E6 gene of the HPV to be analyzed can be identified by methods well known to those skilled in the art. For example, the HPV genome may be extracted from a sample containing the HPV to be analyzed, and the nucleotide sequence of the region containing the E6 gene may be determined by sequence analysis to identify the amino acid sequence encoded by the E6 gene region, or the region containing the E6 gene may be enriched from the extracted HPV genome and subjected to sequence analysis to determine the nucleotide sequence of the region containing the E6 gene to identify the E6 amino acid sequence.

[0015] The amino acid sequence shown in SEQ ID NO: 1 is a consensus sequence of the amino acid sequences encoded by the HPV E6 gene, identified by the inventors using the following procedure. The inventors annotated potential HPV gene information, including nucleotide and amino acid sequences, for all HPV reference genome sequences (182 HPV types) available from the PaVE database at the time of analysis initiation using the PuMa (papillomavirus annotation tool) software package (Pace et al., 2019, https: / / github.com / KVD-lab / puma). Furthermore, for all available αHPV (64 αHPV types) genome reference sequences among the above HPV reference genome sequences, the nucleotide sequences of the E6 gene predicted by PuMA were multiple aligned using Clustal Omega and converted to amino acid sequences, resulting in the discovery of the consensus sequence shown in SEQ ID NO: 1 (see Example 1).

[0016] The present inventors constructed a phylogenetic tree of HPVs based on the E6 gene and found that the clusters of HPVs classified as high-risk and low-risk according to the epidemiological risk classification by Muñoz et al. (2003) were clearly separated. Furthermore, the present inventors found that, in the amino acid sequence encoded by the HPV E6 gene, the amino acid combination of phenylalanine at position 34, arginine or lysine at position 40, tyrosine or leucine at position 102, and leucine at position 120 of the amino acid sequence set forth in SEQ ID NO: 1 is completely conserved only in the HPVs classified as high-risk according to the epidemiological risk classification by Muñoz et al. (2003). The present inventors defined these four E6 amino acids as E6 risk distinguishable amino acids (E6-RDAAs).

[0017] The present inventors classified HPVs into E6 gene sequences encoded by the HPV E6 gene as follows: HPVs in which the amino acid corresponding to position 34 of the amino acid sequence shown in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine; HPVs with other amino acid combinations are classified as E6-RDAA high-risk HPVs; and HPVs with other amino acid combinations are classified as E6-RDAA low-risk HPVs. The results were consistent with the epidemiological risk classification by Munoz et al. (2003). Therefore, the E6-RDAA combination can be used as an index to assess the risk of cervical cancer caused by HPVs, including HPVs with unknown cervical cancer risk and newly identified HPVs.

[0018] [Method for determining the high risk of developing cervical cancer] The present invention provides a method for determining the high risk of developing cervical cancer in a subject (hereinafter referred to as the "determination method of the present invention"). The determination method of the present invention is characterized in that it determines the risk to be high when the amino acid sequence encoded by the E6 gene of at least one HPV detected in a cervical sample from the subject has the following amino acid combination in the amino acid sequence set forth in SEQ ID NO: 1: phenylalanine at the amino acid 34, arginine or lysine at the amino acid 40, tyrosine or leucine at the amino acid 102, and leucine at the amino acid 120.

[0019] In the determination method of the present invention, the subject is not particularly limited as long as the subject is a subject who may develop cervical cancer and who may be infected with HPV. Preferably, the subject is a human. In the determination method of the present invention, the condition of the subject is not particularly limited. Preferably, the subject is a subject whose status as to whether or not he or she has cervical cancer is unknown. When the determination method of the present invention is performed on a human, for example, a cervical sample from the subject is analyzed in a cancer screening or the like, and if the detected HPV is an E6-RDAA high-risk type, it can be predicted that the subject has a high risk of developing cervical cancer.

[0020] The sample used in the determination method of the present invention may be any sample obtained from the cervix, and is not particularly limited as long as it is collected from a subject and contains cervical cells. For example, it may be cervical mucus collected by scraping the subject's cervix with a spatula, brush, cotton swab, etc., or a tissue fragment collected from the subject's cervix by surgical procedure. The sample used in the determination method of the present invention may be optionally pretreated after collection from the subject, may be frozen, or may be prepared as needed. Pretreatment may include, for example, adjusting the concentration of an internal standard in the sample so that it is constant between samples.

[0021] The determination method of the present invention may comprise the following steps (a) to (d): (a) extracting nucleic acid from a cervical sample from a subject, (b) concentrating nucleic acid or nucleic acid fragments comprising the HPV E6 gene from the nucleic acid extracted in step (a), (c) identifying the HPV E6 amino acid sequence, and (d) determining whether, in the E6 amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 34 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine.

[0022] In step (a), the method for extracting nucleic acids from the subject's cervical sample is not particularly limited and can be performed by methods well known to those skilled in the art. For example, extraction can be performed using a commercially available reagent kit according to the instructions included with the kit.

[0023] In step (b), the method for enriching nucleic acids or nucleic acid fragments is not particularly limited and can be performed by methods well known to those skilled in the art. For example, the region containing the HPV E6 gene may be amplified by a nucleic acid amplification method such as PCR, the region containing the HPV E6 gene may be enriched by a hybridization capture method using a probe, or the region containing the HPV E6 gene may be enriched using a commercially available target enrichment kit according to the instructions of the kit.

[0024] In step (c), the amino acid sequence of HPV E6 detected from the subject's cervical sample can be identified by a method well known to those skilled in the art. For example, the nucleotide sequence of the region containing the HPV E6 gene may be determined by sequence analysis of the subject's cervical sample, and the amino acid sequence encoded by the E6 gene region may be identified. Alternatively, the genomic region containing the HPV E6 gene may be enriched from the subject's cervical sample and sequence analyzed, and the nucleotide sequence of the region containing the E6 gene may be determined, and the amino acid sequence encoded by the E6 gene region may be identified.

[0025] In step (d), based on the E6 amino acid sequence identified in step (c), it can be determined whether the amino acid at position 34 in the amino acid sequence shown in SEQ ID NO: 1 is phenylalanine, the amino acid at position 40 is arginine or lysine, the amino acid at position 102 is tyrosine or leucine, and the amino acid at position 120 is leucine. If an HPV having the amino acid combination of phenylalanine at position 34, arginine or lysine at position 40, tyrosine or leucine at position 102, and leucine at position 120 in the amino acid sequence shown in SEQ ID NO: 1 is detected in a cervical sample from a subject, this suggests that the subject has a high risk of developing cervical cancer. In the determination method of the present invention, if an HPV having an amino acid combination in the amino acid sequence encoded by the HPV E6 gene shown in SEQ ID NO: 1, where the amino acid corresponding to the 34th position is phenylalanine, the amino acid corresponding to the 40th position is arginine or lysine, the amino acid corresponding to the 102nd position is tyrosine or leucine, and the amino acid corresponding to the 120th position is leucine, is not detected in the subject's cervical sample, this suggests that the subject has a low risk of developing cervical cancer.

[0026] The present inventors classified HPV detected in cervical samples as E6-RDAA high-risk HPVs having phenylalanine at position 34, arginine or lysine at position 40, tyrosine or leucine at position 102, and leucine at position 120 in the amino acid sequence set forth in SEQ ID NO: 1, and E6-RDAA low-risk HPVs having other amino acid combinations. The inventors found that the E6-RDAA risk type of the detected HPV was significantly associated with the clinical outcome of the subject from whom the sample was derived. Furthermore, the present inventors found that samples from subjects whose outcome was cervical cancer were positive for at least one HPV type classified as E6-RDAA high-risk, and that HPVs with nonsynonymous mutations in at least one of the four amino acids in E6-RDAA were associated with a low risk of cervical cancer. Therefore, the combination of E6-RDAAs can be used as an indicator to determine a subject's risk of developing cervical cancer.

[0027] Because HPV infection occurs prior to the onset of cervical cancer, the E6-RDAAs combination of positive HPV, which is an indicator of cancer risk, can be detected before the onset of cervical cancer. While tumor marker tests generally detect cancer, this invention is revolutionary in that it can detect cancer risk before cancer onset occurs.

[0028] HPV tests currently in practical use target HPV types classified by phylogenetic classification based on L1 DNA sequences. Furthermore, approximately half of αHPVs have not been classified for epidemiological risk, and most of these unclassified αHPVs have not previously been subject to clinical testing. This invention focuses on the HPV E6 gene and uses E6-RDAAs combinations as an indicator to evaluate the cervical cancer risk of HPVs, including those with unknown cervical cancer risk and newly identified HPVs, and is therefore groundbreaking in that it can determine a subject's risk of developing cervical cancer.

[0029] [Test Kit] The present invention provides a test kit for assessing the risk of HPV-induced cervical cancer (hereinafter referred to as the "test kit of the present invention"). A first embodiment of the test kit of the present invention may include a reagent for enriching the HPV E6 gene region. Examples of reagents for enriching the HPV E6 gene region include, but are not limited to, amplification primers for amplifying the full-length or partial sequence of the HPV E6 gene and labeled artificial oligonucleotides (hybridization capture probes) for hybridization capture assays. In the test kit of the present invention, the amplification primers may be primers specific to sequences containing the E6 gene of each HPV type, degenerate primers for degenerate PCR assays, or primers designed to add tags, adapters, or the like to the amplification products. Amplification primers can be designed and synthesized using techniques well known to those skilled in the art. In the test kit of the present invention, the labeled artificial oligonucleotide (hybridization capture probe) is not particularly limited as long as it has a sequence complementary to a sequence containing the HPV E6 gene, and may be RNA or DNA. The labeled artificial oligonucleotide can be designed and synthesized using techniques well known to those skilled in the art.

[0030] A second embodiment of the test kit of the present invention may include a reagent for identifying at least one amino acid selected from the amino acid corresponding to the 34th, 40th, 102nd, and 120th positions of the amino acid sequence set forth in SEQ ID NO: 1 in the amino acid sequence encoded by the HPV E6 gene in a sample. The selected amino acids may be two, three, or all four. Reagents for identifying amino acids include, but are not limited to, reagents for determining the nucleotide sequence of the HPV E6 gene. Reagents for determining the nucleotide sequence may suitably be those used in the Sanger method or MLPA (Multiplex ligation-dependent probe amplification) method.

[0031] The test kit of the present invention may contain, in addition to the reagent for concentrating the HPV E6 gene region and / or the reagent for identifying specific amino acids in the HPV E6 gene, a reagent for extracting DNA, and may also contain other components, such as, but not limited to, DNA polymerase, dNTPs, a reaction buffer, a blocking oligonucleotide, a substrate, beads, a buffer solution for preparing reagents, a spatula, brush, cotton swab, or the like for collecting specimens, various tubes for storing specimens, and an instruction manual.

[0032] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0033] Example 1: Phylogenetic Classification of αHPV by Gene Sequence and Identification of Amino Acids Characteristic for Cervical Cancer Risk 1. Methods 1-1 Reference HPV Genome Sequences and Gene Annotation All available HPV reference genome sequences (182 HPV types) at the time of analysis were downloaded from the PaVE (papillomavirus episteme) database (van Doorslaer K et al., 2017, https: / / pave.niaid.nih.gov / ). Each genome sequence was processed using the PuMa (papillomavirus annotation tool) software package (Pace et al., 2019, https: / / github.com / KVD-lab / puma) to annotate potential HPV gene information, including nucleotide and amino acid sequences. PuMa also redefined the nucleotide following the stop codon of the L1 gene as the first physical position in the HPV circular genome.

[0034] 1-2 Multiple Alignment and Clustering of HPV Reference Genome Sequences. For all available αHPV genomes (64 αHPV types) among the HPV reference genome sequences, multiple alignment (MSA) of the PuMA-predicted genes and regions was performed using the Clustal Omega software package (ver. 1.2.4, Sievers and Higgins, 2018), and PHYLIP-format files were generated for each HPV gene. These PHYLIP-format files were visualized and manually inspected using Jalview (Waterhouse AM et al., 2009). To construct a phylogenetic tree in Newick format, the PHYLIP files were processed in IQ-TREE 2 (ver. 2.2.0, Minh et al., 2020) using the best-hit model "GTR+F+I+I+R6," selected using the ModelFinder subcommand (Kalyaanamoorthy et al., 2017) of IQ-TREE 2. The created Newick file was visualized using the R language library ggtree (Yu, 2020).

[0035] 2. Results 2-1 Phylogenetic Classification by Gene Sequences Among the HPV reference genome sequences available from PaVE at the start of the analysis, multiple alignments were performed using the full-length sequences of each gene and upstream regulatory region (URR) for all available αHPV genome reference sequences (64 αHPV types), and a phylogenetic tree (cladeogram) was constructed. The HPV phylogenetic tree constructed based on the E6 gene is shown in Figure 1. In the HPV phylogenetic tree based on the E6 gene, clades containing HPVs classified as high-risk HPVs according to the epidemiological risk classification by Muñoz et al. (2003) are shown in Figure 2. In the phylogenetic tree based on the E6 gene, clusters containing HPVs classified as high-risk and low-risk HPVs according to the epidemiological risk classification by Muñoz et al. (2003) were clearly separated (Figure 1). The phylogenetic tree constructed based on the L1 gene is shown in Figure 3. In the HPV phylogenetic tree based on the L1 gene, clades containing HPVs classified as high-risk HPVs according to the epidemiological risk classification by Muñoz et al. (2003) are shown in Figure 4. In the phylogenetic tree based on the L1 gene, the clusters of HPVs classified as high-risk and low-risk according to the epidemiological risk classification by Muñoz et al. (2003) were not clearly separated from each other compared with the phylogenetic tree based on the E6 gene (Fig. 3). Similarly, in the phylogenetic tree based on the L2, E2, and E7 genes, the regions encoding the E1^E4 and E8^E2 splice translation products, and URR, the clusters of HPVs classified as high-risk and low-risk according to the epidemiological risk classification by Muñoz et al. (2003) were not clearly separated from each other. Based on these results, we selected the E6 gene-based classification as the phylogenetic tree that best fits the epidemiological risk classification by Muñoz et al. (2003).

[0036] 2-2 E6 Amino Acids Characteristic for Cervical Cancer Risk For the 64 αHPV genome reference sequences, the nucleotide sequences of the E6 gene predicted by PuMA were multiple aligned using Clustal Omega. The aligned nucleotide sequences of the E6 gene were converted to amino acid sequences, and the following consensus sequence was found: MIYKIREGPKTVQPKPVHIKAVVENSYLXMMMMFSNGAERPRNXFLLCKELNISLHDLRLLCVFCKKELTTAEVYAFAFKELKVVWRDGFPYXACAKCLLFYGKVRELRHWDYSVYGETVEEETKKSLYELLIRCYRCHKPLCPEEKERHVDEKRRFHKIAGXWTGRCLHCWRRCTARRQX (SEQ ID NO: 1)

[0037] We compared the E6 amino acid sequences of 14 HPV groups classified as high-risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 82) and 11 HPV groups classified as low-risk (HPV6, 11, 40, 42, 43, 44, 54, 61, 72, 81, and 89) by multiple alignment. As a first step to identify common amino acids, we excluded HPV70 and HPV73 from the analysis, as Munoz et al. (2003) reported discrepancies between their epidemiological risk classification and the L1 gene-based phylogenetic classification. Figure 5 shows the results of the multiple alignment. The four amino acid combinations of phenylalanine at position 34, arginine or lysine at position 40, tyrosine or leucine at position 102, and leucine at position 120 in the amino acid sequence shown in SEQ ID NO: 1 were completely conserved only in the HPV group classified as high-risk in the epidemiological risk classification by Munoz et al. (2003) (Figure 5). Therefore, in the present invention, these four E6 amino acids are defined as E6 risk distinguishable amino acids (E6-RDAAs), and HPVs that have a combination of amino acids conserved in the HPV group classified as high-risk in the epidemiological risk classification by Munoz et al. (2003), i.e., HPVs in which the amino acid corresponding to position 34 of the amino acid sequence shown in SEQ ID NO: 1 is phenylalanine, the amino acid corresponding to position 40 is arginine or lysine, the amino acid corresponding to position 102 is tyrosine or leucine, and the amino acid corresponding to position 120 is leucine, are classified as E6-RDAA high-risk, while HPVs with other amino acid combinations are classified as E6-RDAA low-risk.

[0038] A comparison of the epidemiological classification by Munoz et al. (2003) and the E6-RDAA classification of the present invention is shown in Table 1. HPVs classified as high-risk in the epidemiological classification were classified as E6-RDAA high-risk types, and HPVs classified as low-risk in the epidemiological classification were classified as E6-RDAA low-risk types. In the report by Munoz et al. (2003), HPV70 (** in Table 1) was epidemiologically classified as low-risk but classified as high-risk in the phylogenetic classification based on the L1 gene. Conversely, HPV73 (* in Table 1) was epidemiologically classified as high-risk but classified as low-risk in the phylogenetic classification based on the L1 gene. In the E6-RDAA classification of the present invention, HPV70 and HPV73 were classified as risk types consistent with their respective epidemiological risks. Furthermore, HPV30, 34, 67, 69, and 85 were unclassified in the classification by Munoz et al. (2003) but were classified as E6-RDAA high-risk types. These results demonstrate that the E6-RDAA classification of the present invention can identify risk types of HPVs whose risk of cervical cancer is unknown.

[0039]

[0040] Example 2: Clinical classification and sequencing analysis of cervical swab specimens 1. Methods 1-1 Participants and sample collection The clinical study design was a prospective observational study. Cervical mucus samples were collected from patients who provided written informed consent at Nagasaki University Hospital and its affiliated facilities between 2007 and 2008. Subjects were patients who visited the hospital for further examination of abnormal cervical cytology. After specimen collection, the subjects' progress was prospectively monitored. Subject outcomes were obtained from the medical records of the facilities.

[0041] 1-2 Clinical Classification of Cervical Swab Specimens Samples were classified into Groups 1–3 according to the following criteria. Group 1 included (1) specimens with normal cervical cytology from the start of follow-up to the end of follow-up, and (2) specimens with cervical intraepithelial lesion (CIN) 1 (mild dysplasia), CIN2 (moderate dysplasia), or CIN3 (severe dysplasia or carcinoma in situ) cytology at the start of follow-up that converted to normal during follow-up. Group 2 included (1) specimens with CIN1, CIN2, or CIN3 cytology at the start of follow-up that converted to CIN3, CIS (cervical carcinoma in situ), or invasive carcinoma during follow-up, (2) specimens with CIN1, CIN2, or CIN3 cytology at the start of follow-up that showed no change during follow-up, (3) specimens with CIN3 or CIS at the start of follow-up that were promptly treated, and (4) specimens that showed carcinoma at the start of follow-up. Group 3 consisted of specimens with no clear outcome information.

[0042] 1-3 Sequencing Analysis of Cervical Swab Samples DNA was extracted from cervical swab samples using the QIAamp DNA kit (QIAGEN) according to the manufacturer's instructions. Reference genome sequences for 64 αHPVs were downloaded from PaVE as described in Example 1. SureSelect custom baits (Agilent Technologies) were designed to cover the downloaded reference genome sequences. HPV177, the latest αHPV, was not included in the analysis. Target enrichment and library construction were performed using SureSelect XT low input reagents (Agilent). The constructed libraries were subjected to paired-end multiplex sequencing using a HiSeq 2500 system (Illumina).

[0043] HPV Genome Mapping and Positive HPV Typing. For each read obtained from the clinical sample library, HPV genome mapping was performed using the HPViewer software package (Hao et al., 2018). During the mapping process, PCR duplicates were removed (deduplicated) using a modified version of HPViewer (https: / / github.com / misshie / HPViewer / tree / dedup) with the SAMtools software package (Danecek et al., 2021). To minimize the size of N-masked nucleotides in the genome sequence, the repeat-masked mode of HPViewer was used instead of the hybrid-masked mode. To renumber the first nucleotide position redefined by PuMA, the repeat-masked HPPV genome sequence from HPViewer was inverted using the FASTA-tools toolkit (Brankovics, 2016, 2023), and then mapped using HPViewer. For each library, a mapped BAM (Binary Alignment / Map) file and the RPK (reads per kilobase) of each detected HPV type were generated using HPViewer. To determine the appropriate RPK threshold for calling a positive HPV type, all HPV types detected at a given RPK threshold were counted and the threshold was set to a logarithm between 1 and 10,000 (log ). 10 ) and divided into 25 levels.

[0044] A 2 × 2 table was created containing the clinical outcome groups (Group 1 and Group 2) and E6-RDAA classification (high-risk and low-risk) described in Section 1-2 of Example 2, and a chi-square test was performed. Yates' continuity correction was not performed. To evaluate whether positive HPV types could predict outcome, a random forest method was performed using the R package random forest library (version 4.7.1, Liaw A and Wiener M, 2002) and kernlab library (version 0.9.29, Karatzoglou et al., 2004). The outcome (Group 1 or Group 2) was used as the objective variable for the decision tree, and the positive HPV type was used as the explanatory variable. The number of samples for training and testing was 200 and 76, respectively. The accuracy was 64.4%.

[0045] 1-5 Detection of HPV Genome Diversity To detect HPV genome diversity, the BAM files were generated using the haplotypecaller command in the Clara Parabricks (NVIDIA) pipeline, which is compatible with the HaplotypeCaller command in GATK (genome analysis toolkit) version 4, to generate genomic VCF (g.vcf) files. All g.vcf files were genotyped together using the GLnexus software package (Yun et al., 2020) to generate a single VCF file. Genetic information was then annotated using the SnpEff software package (Cingolani et al., 2012). A modified GFF3 file created by PuMA was used to construct the SnpEff database. The annotated combined VCF file and RPK information were used to extract mutation information for the four E6-RDAAs of all αHPVs.

[0046] 2. Results 2-1 Clinical Classification of Cervical Swab Samples To validate the E6-RDAA classification from the perspective of clinical outcomes, a total of 325 cervical swab samples collected from 252 participants in Japan were analyzed. At the start of case follow-up, these samples were classified by histopathological diagnosis into normal cytology (52 samples), CIN1 (99 samples), CIN2 (35 samples), CIN3 (67 samples), CIS (29 samples), squamous cell carcinoma (SCC) (35 samples), and carcinoma, including adenocarcinoma, adenosquamous carcinoma, and mucinous carcinoma (8 samples). During follow-up, these samples were classified into Group 1 (recovery), Group 2 (non-recovery), or Group 3 (unknown outcome).

[0047] 2-2 Association between E6-RDAA risk types of positive HPV types and clinical outcomes. A massively parallel sequencing library was constructed using targeted hybridization capture (TCAP) for DNA extracted from each cervical swab specimen and all available αHPV reference genome sequences (64 αHPV types, the same as in Example 1) obtained from the PaVE database. The sequenced reads were processed to measure the reads per kilobase of the decoded HPV genome. HPV types with an RPK of 100 or greater, a value approximately 10 times the average depth, were considered positive HPV types for further analysis. A random forest classification model revealed that HPV16, an epidemiologically high-risk HPV type, was the HPV type most affected by clinical outcomes (Groups 1 and 2).

[0048] The number of specimens positive for HPV types categorized as high-risk or low-risk by the E6-RDAA classification, along with their clinical outcomes, are shown in Table 2. The underlined HPV types in Table 2 were unclassified in the report by Muñoz et al. (2003).

[0049]

[0050] For HPV30, 34, 67, or 69, which were unclassified in the report by Munoz et al. (2003) but were classified as high-risk types by the E6-RDAA classification of the present invention, the number of detected reads in those positive specimens, along with the clinical outcomes, are shown in Table 3. For each HPV type, specimens with 100 or more detected reads were considered positive.

[0051]

[0052] A chi-square test of the 2 × 2 table consisting of the clinical outcome groups (Group 1 and Group 2) and the E6-RDAA classification (high-risk and low-risk types) without Yates' continuity correction showed that the E6-RDAA risk type of positive HPV was significantly associated with clinical outcome (Table 4).

[0053]

[0054] All of the HPV types that tested positive in samples from participants with SCC were high-risk HPV types, except for HPV67, which was unclassified by the epidemiological risk classification by Muñoz et al. (2003) but classified as a high-risk type by the E6-RDAA classification of the present invention (Table 5).

[0055]

[0056] 2-3 Association between HPV genomic mutations and clinical outcomes To evaluate the impact of HPV genomic mutations on clinical outcomes, mutation analysis was performed on four E6-RDAAs. One HPV16-positive specimen had a predicted clinical outcome of Group 2, but the actual outcome was Group 1. HPV16 is a high-risk type according to both our E6-RDAA classification and the epidemiological risk classification by Muñoz et al. (2003). However, the HPV16 genome (lib106) from this specimen was found to have an Arg40Gln nonsynonymous mutation (Table 6). These results indicate that HPVs with a nonsynonymous mutation in at least one of the four amino acids in E6-RDAA have a low risk of cervical cancer.

[0057]

[0058] 3. Discussion The present inventors focused on four amino acids identified by multiple alignment of the E6 gene and found that they can distinguish αHPV groups into high-risk and low-risk types. The E6-RDAA classification of the present invention resolves the discrepancy between the epidemiological risk classification and the phylogenetic classification based on the L1 gene reported by Muñoz et al. (2003). Furthermore, the present inventors found that the clinical outcomes of specimens collected in Japan were significantly associated with the E6-RDAA risk type of the positive HPV. Specimens that resulted in cervical cancer contained at least one HPV type classified as high-risk only by the E6-RDAA classification of the present invention. Furthermore, a nonsynonymous mutation (Arg40Gln) in the E6-RDAAs of the high-risk HPV16 was detected in one specimen that resulted in clinical recovery.

[0059] HPV risk classification based solely on epidemiological studies has limitations due to confounding factors and misclassification (Schiffman et al., 2009). Our E6-RDAA classification is consistent with the epidemiological risk classification by Muñoz et al. (2003) and can resolve the discrepancy between the L1 gene-based phylogenetic classification and the epidemiological risk classification for HPV70 and HPV73. Phylogenetic analysis based on the E6 gene showed that HPV70 and HPV73 are close to clades that are mostly high-risk HPVs (Figures 1 and 2). As shown in Figure 5, HPV73 has a high-risk combination of four amino acids in the E6-RDAA (phenylalanine at position 34, arginine or lysine at position 40, tyrosine or leucine at position 102, and leucine at position 120). In HPV70, the 102nd amino acid of the four E6-RDAAs was a low-risk type (histidine) rather than a high-risk type (leucine or tyrosine). Therefore, HPV70 was classified as a low-risk HPV in the E6-RDAA classification. This classification is consistent with the epidemiological risk classification by Muñoz et al. (2003). These results also suggest that the 102nd amino acid of E6-RDAA is important for tumorigenesis.

[0060] Of the specimens that were positive for the E6-RDAA low-risk HPV70, two specimens (lib273 and lib672) were categorized into Group 2. This can be explained by the fact that these specimens were positive not only for HPV70 but also for other E6-RDAA high-risk HPV types (HPV39, HPV68, and / or HPV51).

[0061] The E6-RDAA classification allows for the annotation of cancer risk for previously unclassified αHPVs. For example, HPVs 30, 34, 67, 69, and 85 were reported to be closely related to high-risk HPVs in a phylogenetic tree constructed by full-length genome alignment (Schiffman et al., 2005). HPVs 30, 34, 67, and 69 have been reported to be detected at low frequencies (<1%) in invasive cervical cancer specimens (de Sanjose et al., 2010). As shown in Table 1, these five HPVs are classified as high-risk by the E6-RDAA classification and therefore merit attention in cancer risk assessment.

[0062] HPV testing is crucial for initial screening and post-treatment follow-up for cervical cancer. Currently, commercially available HPV tests target HPV types classified by phylogenetic classification based on L1 DNA sequences and overlook HPV types not classified by Muñoz et al. (2003), including E6-RDAA high-risk HPVs. Targeted capture sequencing targeting all αHPVs for screening tests can reveal the entire HPV genome, including the E6 gene, and reveal mutations in all cervical HPV viruses. As the cost of next-generation sequencing continues to decrease, HPV genome sequencing may become a viable option for HPV classification. Furthermore, screening with HPV genome sequencing may be more convenient than current HPV genotype testing. Furthermore, identifying HPV genome mutations could provide important information.

[0063] The E6-RDAA classification may provide a useful diagnostic tool for HPV classification; however, similar to the processes by which previous risk classifications have been established, it must be validated in terms of cancer outcomes. This invention integrates the clinical knowledge accumulated to date and enables classification based on minimal HPV genome sequencing using computational methods. In this example, the amount of HPV present in the cervix was not used to correlate with clinical outcomes because accurate quantification and comparison of HPV load in cervical swabs was technically difficult. However, viral load should be considered as a predictor of outcome.

[0064] In this study, we demonstrated the importance of E6 in cancer outcomes. Although there is currently no experimental evidence demonstrating the role of E6 protein in cancer development, we believe that analyzing a large number of SCC specimens in terms of E6-RDAA mutations will help clarify the biological function of E6 protein in cancer development.

[0065] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for evaluating the risk of cervical cancer caused by human papillomavirus (HPV), comprising: a method for evaluating the risk as high when, in the amino acid sequence encoded by the E6 gene of the HPV being analyzed, the amino acid corresponding to the 34th amino acid in the amino acid sequence shown in SEQ ID NO:1 is phenylalanine, the amino acid corresponding to the 40th amino acid is arginine or lysine, the amino acid corresponding to the 102nd amino acid is tyrosine or leucine, and the amino acid corresponding to the 120th amino acid is leucine; and a method for evaluating the risk as low when any other combination is observed.

2. A method for determining a subject's risk of developing cervical cancer, comprising determining that the risk is high if, in the amino acid sequence encoded by the E6 gene of at least one HPV among the HPVs detected in the subject's cervical sample, the amino acid corresponding to the 34th amino acid in the amino acid sequence shown in SEQ ID NO:1 is phenylalanine, the amino acid corresponding to the 40th amino acid is arginine or lysine, the amino acid corresponding to the 102nd amino acid is tyrosine or leucine, and the amino acid corresponding to the 120th amino acid is leucine.

3. The method of claim 2, comprising the following steps (a) to (d): (a) extracting nucleic acid from a cervical sample of a subject; (b) concentrating a nucleic acid or a nucleic acid fragment comprising the HPV E6 gene from the nucleic acid extracted in step (a); (c) identifying the HPV E6 amino acid sequence; and (d) determining whether, in the E6 amino acid sequence, the amino acid corresponding to the 34th position in the amino acid sequence shown in SEQ ID NO:1 is phenylalanine, the amino acid corresponding to the 40th position is arginine or lysine, the amino acid corresponding to the 102nd position is tyrosine or leucine, and the amino acid corresponding to the 120th position is leucine.

4. A test kit for assessing the risk of HPV causing cervical cancer, comprising a reagent for concentrating a nucleic acid or a nucleic acid fragment containing the HPV E6 gene in a sample, the reagent for concentrating comprising an amplification primer pair for amplifying the full length or partial sequence of a nucleic acid containing the HPV E6 gene, or a hybridization capture probe having a sequence complementary to a sequence containing the HPV E6 gene.

5. A test kit for assessing the risk of HPV causing cervical cancer, comprising a reagent for identifying at least one amino acid selected from the amino acid corresponding to the 34th, 40th, 102nd, and 120th amino acids in the amino acid sequence shown in SEQ ID NO:1 in an amino acid sequence encoded by the E6 gene of HPV in a sample.

6. The kit of claim 5, wherein the reagents for identifying amino acids include reagents for determining the base sequence of the E6 gene of HPV.

Images