Methods of human papillomavirus screening

Abstract

A method for identifying the presence of HPV in human tissue may comprise the following steps: collecting a cell from oropharyngeal tissue of a subject utilizing a swab and measuring HPV DNA in the cell or in a solution that contains the contents of the cell. The method may include calculating a risk assessment for the development of cancer in the subject, wherein the risk assessment is calculated from data generated by the measuring of the HPV DNA to determine a persistence of the HPV in human tissue.

Description

PATENT APPLICATIONInventors: Mohammad Kamal, M.D.Docket No.: 33394-00007TITLEMETHODS OF HUMAN PAPILLOMAVIRUS SCREENINGCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 736,216 entitled “SYSTEM AND CORRESPONDING METHOD OF ADMINISTRATION FOR OROPHARYNGEAL HUMAN PAPILLOMAVIRUS TEST,” filed on December 19, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present teachings relate generally to methods of screening for papillomavirus infection in humans and more specifically screening for the persistence of papillomavirus infection in humans that may facilitate cancer development.BACKGROUND

[0003] The human papillomavirus (HPV) is, according to the Center for Disease Control (CDC), the most common sexually transmitted infection in the United States. Epidemiologic studies indicate that HPV-positive oropharyngeal cancer occurs commonly in patients who are of younger age and have a higher number of sexual partners, more exposure to oral sexual practices, and lower smoking rates. There are multiple types of HPV, with some types being more difficult to treat and / or producing more severe symptoms. Some of the more severe types of HPV, known as “High-risk HPV types," and thus targeted for testing purposes, include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66. and 68.

[0004] Most HPV infections spontaneously resolve, but in a small percentage of cases, the infection persists. The development of cancer is linked to persistent HPV. Multiple studies haveshown that HPV infection is linked to female cervical cancer, anal cancer, and oropharyngeal (throat) cancer. As a result of increased Pap smear screening, the incidence of cervical cancer is declining. Unfortunately, HPV-related orophary ngeal cancer cases are at an all-time high. HPV is identified in at least 87% of orophary ngeal cancers. Today, there are more men with HPV-related oropharyngeal cancer than women with cervical cancer. As will be appreciated by one skilled in the art, patients may be hesitant to undergo uncomfortable or invasive testing procedures such as cytology7sample collection that includes invasive scraping or brushing of epithelial cells (cervical, anal tissues, etc.), and this may result in a significant drop in the percentage of the relevant population that gets tested. Furthermore, there are no HPV tests on the market to date that are intended for sampling of the oropharyngeal region as a substrate for HPV testing.

[0005] Detection of HPV nucleic acids in saliva has been reported. There are, however, potential downsides to utilizing saliva for such tests. These include, but are not limited to: 1) Saliva is not specific for orophary ngeal HPV infection — it is general for the oral cavity7as a whole 2) Older patients often do not secrete as much saliva as younger patients and therefore sampling may be difficult and / or inaccurate 3) Saliva varies from person to person 4) Saliva complicates the PCR process since it requires a liquification step of the saliva prior to extraction, amplification and analysis of the DNA. These are not an exhaustive list of downsides. There are other potential downsides to trying to utilize saliva such that it can result in a more difficult test and a potentially more inaccurate result.

[0006] Detection of HPV nucleic acids in oral gargle and / or oral rinse samples has been reported. The downside of this method is the lack of standardization between studies and that the subject largely controls the sample collection. For example, the duration of the gargling or rinsing, the volume of the gargle or rinse solution, and the types of solutions used for conservation or preservation of the sample vary greatly between studies. In addition, dependingon the way a gargle and / or rinse sample is collected, the solution may not contain a sufficient sampling of orophary ngeal cells. Also, a gargle and / or rinse solution found to be positive for HPV may not reflect HPV infection of the orophary nx, but of the oral cavity7alone, depending on how the gargle and / or rinse was performed. Moreover, the weakness of the rinse and gargle method is that it is not specific for the oropharynx and can detect HPV that is anywhere in the mouth and oral cavity7. The rinse and gargle method has a positivity7rate of detecting HPV of 13.1% compared to a targeted tonsillar brushing of 3.6%. Low agreement in paired tonsil brushings and gargles suggests that gargle is not representative of HPV prevalence in the tonsil.

[0007] There is a need therefore for a test that overcomes many of the foregoing shortcomings and disadvantages.

[0008] It should be noted that the above background description includes information that may be useful in understanding aspects of the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention.SUMMARY

[0009] The following presents a summary of this disclosure to provide a basic understanding of some aspects. This summary7is intended to neither identify key or critical elements nor define any limitations of embodiments or claims. Furthermore, this summary may provide a simplified overview of some aspects that may be described in greater detail in other portions of this disclosure.

[0010] A method for identifying the presence of HPV in human tissue may comprise the following steps: collecting a cell from oropharyngeal tissue of a subject utilizing a swab and measuring HPV DNA in the cell or in a solution that contains the contents of the cell. The method may include calculating a risk assessment for the development of cancer in the subject, wherein the risk assessment is calculated from data generated by the measuring of the HPV DNA to determine a persistence of the HPV in human tissue.

[0011] The method may also comprise the following in any combination and order:• determining presence of beta globin in the cell.• stabilizing the cell or the contents of the cell using a transport medium.• the transport medium is PreservCyt Solution.• the transport medium contains guanidine-hydrochloride.• measuring HPV DNA comprises quantitative PCR, In Situ Hybridization, nextgeneration sequencing, or CRISPR-based detection.• the subject is a human.• synthesizing an HPV gene expression library derived from the cell; wherein the HPV gene expression library is not found in nature and measuring HPV gene expression.• synthesizing a HPV gene expression library includes RNA isolation and cDNA synthesis or next-generation sequencing library preparation.• measuring HPV gene expression includes quantitative PCR or next-generation sequencing• analyzing the diversity and abundance of HPV transcripts.• calculating a risk assessment comprises determining a persistence of an HPV infection.• the persistence of the HPV infection is linked to development of oropharyngeal squamous cell carcinoma.• determining the persistence of the HPV infection comprises repeating the HPV DNA in the cell or in the solution that contains the contents of the cell every six months and generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test twenty-four months after the first test.• a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test tw enty four months after the first test.• the subject with the score greater than one is referred to a physician including an otolaryngologist or an oncologist.

[0012] A method of determining the persistence of an HPV infection comprising conducting the method as described above every six months for no less than two years.

[0013] The method may also comprise the following in any combination and order:• generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test twenty-four months after the first test.• a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test twenty four months after the first test.• the subject with the score greater than one is referred to a physician including an otolaryngologist or an oncologist.

[0014] A method for identifying the presence of HPV in human tissue may comprise the following steps: collecting a cell from orophary ngeal tissue of a subject, measuring HPV DNA in the cell or in a solution that contains the contents of the cell, and collecting an oral solution from the subject. The method may also comprise measuring HPV nucleic acid in the oral solution or in nucleic acid isolated from the solution, and calculating a risk assessment for the development of cancer in the subject, wherein the risk assessment is calculated from data generated by the measuring of the HPV DNA from the oropharyngeal tissue.

[0015] The method may also comprise the following in any combination and order:• determining presence of beta globin in the cell.• stabilizing the cell or the contents of the cell using a transport medium.• calculating a risk assessment comprises determining a persistence of an HPV infection.• determining the persistence of the HPV infection comprises repeating the measuring HPV DNA in the cell or in the solution that contains the contents of the cell every six months and generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test tw enty-four months after the first test.a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test tw enty four months after the first test.• the subject with the score greater than one is referred to an otolaryngologist or an oncologist.

[0016] A method for identifying the presence of HPV in human tissue may comprise the steps of collecting an oropharyngeal swab specimen comprising human epithelial cells and / or cellular contents, validating specimen adequacy by detecting beta-globin, placing the sw ab into a transport medium comprising guanidine-hydrochloride and storing at 2-8°C for up to 50 days and isolating nucleic acids by magnetic-bead purification. The method may also comprise performing real-time qPCR targeting HPV E6 / E7 and beta-globin under defined cycling conditions, measuring HPV DNA in the cell or in a solution that contains the contents of the cell, electronically calculating a persistence index from measurements at MO, M6, M12, M18, and M24, computing a risk score for the development of cancer in the subject, and automatically generating a report that, when the risk score > two, recommends referral to a physician.

[0017] The method may also comprise the following in any combination and order:• the risk score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: MO, M12 and M24.• the risk score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• the risk score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• the risk score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.• the risk score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at MO and M24.

[0018] A method for identifying the persistence of HPV infection may comprise the steps of: collecting a swab specimen comprising human squamous cells from am oropharynx of a subject, validating specimen adequacy by detecting beta-globin, placing the collected swab specimen into a container filled or partially filled with a transport medium, and testing for a presence of HPV DNA in the swab specimen or in solutions derived from the swab specimen. The method may also comprise measuring the presence of HPV DNA in the swab specimen or in solutions derived from the swab specimen, electronically calculating a persistence index from measurements at MO, M6, M12, Ml 8, and M24, and computing a risk score for the development of cancer in the subject.

[0019] The method may also comprise the following in any combination and order:• the measuring may use PCR amplification and genome tagging to characterize viral genetic material that has entered or integrated into a human genome as a result of natural infection.evaluating a collection site to determine if the collection site is already inflamed and / or otherwise irritated.• the transport medium comprises guanidine-hydrochloride, which is capable of being stored at 2-8°C for up to 50 days.• the oropharynx comprises a base of a tongue, a palatine tonsils, tonsillar fossae, a soft palate and a posterior phary ngeal wall of the subj ect.DESCRIPTION OF THE DRAWINGS

[0020] The present teachings may be better understood by reference to the following detailed description taken in connection with the following illustrations, wherein:

[0021] FIG. 1 illustrates a method for screening and treating HPV in a patient.

[0022] FIG. 2 illustrates a method for screening and treating HPV in a patient

[0023] FIG. 3 illustrates a method for collecting a cell from oropharyngeal tissue of a subject.

[0024] FIG. 4 illustrates a method for screening and treating HPV in a patient.

[0025] FIG. 5 illustrates a method for screening and treating HPV in a patient.

[0026] FIG. 6 illustrates a method for screening and treating HPV in a patient.

[0027] FIG. 7 depicts a timeline of HPV oncogenesis and biomarker development.

[0028] FIG. 8 is an exemplary HPV persistence scoring method.

[0029] The following description discloses various illustrative aspects. Some improvements and novel aspects may be expressly identified, while others may be apparent from the description alone.DETAILED DESCRIPTION

[0030] Reference will be made in detail to exemplary- embodiments of the present technology'. It is to be understood that other embodiments may be utilized, and structural and functional changes may be made without departing from the respective scope of the disclosure. Moreover, features of the various embodiments may be combined or altered without departing from thescope of the invention. As such, the following description is presented by way of illustration only and should not limit in any way the various alternatives and modifications that may be made to the illustrated embodiments and still be within the spirit and scope of the invention.

[0031] As used herein, the words “example” and “exemplary” mean an instance, or illustration. The words “example” or “exemplary ” do not indicate a key or preferred aspect or embodiment. The use of any and all examples, or exemplary7language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0032] Unless expressly stated otherwise, the transitional terms “comprising,” “having,” “including,” and “containing” are open and non-limiting, and the inclusion of one or more elements or steps does not exclude the presence of additional, unrecited elements or steps. Where “consisting essentially of’ or “consisting of’ is used, such usage is purposeful and limited to the specific claim in which it appears.

[0033] The word “or” is intended to be inclusive rather an exclusive unless context suggests otherwise. As an example, the phrase “A employs B or C,” includes any inclusive permutation (e.g., A employs B; A employs C; or A employs both B and C). As another matter, the articles “a” and “an” are generally intended to mean “one or more” unless context suggests otherwise.

[0034] It is noted that the various embodiments descnbed herein may include other components and / or functionality. Aspects of embodiments may be utilized for various other procedures or as a stand-alone procedure. Moreover, the embodiments may be combined or aspects thereof may be combined with each other or with other medical tests and treatments.

[0035] Unless an explicit temporal or logical sequence is required by the claim language itself, the accompanying drawings and the description of the disclosed methods are not intended to mandate that the recited operations be performed in the precise order in which they areenumerated or illustrated. Rather, each step may be executed individually or in any practicable combination, permutation, or concurrency that a person of ordinary skill in the art would recognize as functionally or operationally equivalent for achieving the stated objectives. Method steps may be added, omitted, subdivided, consolidated, repeated, or carried out in parallel, provided that the intended result of the method is obtained. Accordingly, references in the specification to a method step or similar language are used merely for convenient identification and do not by themselves establish a necessary chronological sequence unless the context clearly dictates otherwise.

[0036] Numerical quantities (including percentages, times, temperatures, concentrations, cycle numbers, Ct thresholds, base-pair lengths, and the like) may be expressed as exact values or as ranges; disclosure of a value supports disclosure of ranges centered on or including that value, and vice versa. Unless otherwise specified, “about,” “approximately,” and similar terms encompass variations attributable to measurement error, manufacturing tolerances, and normal biological, chemical, or computational variability as understood by a skilled artisan.

[0037] This disclosure generally relates to methods of HPV testing. The term HPV may generally refer to any type of HPV. Moreover, aspects disclosed herein may be applicable to testing of viruses other than HPV. Further, while embodiments may refer to a technician or healthcare worker performing a particular action(s), other users of the method including automated machines or the like may perform the methods disclosed herein.

[0038] As used herein, a “nucleic acid” or “polynucleotide” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded form or a double-stranded helix. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The termnucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary' and secondary' structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5 '-3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0039] The term “detect” or “measure” as used herein means to discover the existence of a molecule, such as DNA or RNA. For example, in some embodiments, the presently disclosed methods help detect the presence of HPV DNA. In other embodiments, the presently disclosed methods help detect the presence of HPV RNA.

[0040] Human papillomavirus (HPV) is a small, double-stranded DNA virus that infects epithelial cells, particularly those of the skin and mucous membranes. The HPV genome is approximately 8,000 base pairs long and contains several genes, which are classified as early (E) and late (L) genes: Early genes (El, E2, E4, E5, E6, E7) are involved in viral replication, regulation, and cell transformation. Late genes (LI, L2) encode structural proteins for the viral capsid.

[0041] In some embodiments, the custom-designed pool of HPV-specific capture probes is designed to capture most or all of the genomes of high-risk HPV-specific types. In other embodiments, the custom-designed pool of HPV-specific capture probes is designed to capture only some regions of the genotype-specific regions of at least one high-risk HPV genome, such as 1, 2. 3, 4. or 5 or more regions. In still other embodiments, the custom-designed pool of HPV-specific capture probes is designed to capture 2 to 3 regions of the HPV genome that distinguishes high-risk from low-risk HPV types. In further embodiments, the custom-designed pool of HPV-specific capture probes does not capture low-risk HPV types.

[0042] In some embodiments, the method further comprises performing quantitative PCR (qPCR) to amplify the one or more enriched or unenriched segments of HPV DNAs.

[0043] In general, PCR refers to an in vitro method for amplifying or replicating a specific polynucleotide template sequence. The PCR reaction involves a repetitive series of temperature cycles. The reaction mix usually comprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, and target nucleic acid molecule or template. The PCR step can use a variety of thermostable DNA-dependent DNA polymerases, such as Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'- 5' proofreading endonuclease activity. In real-time or quantitative PCR, the DNA is amplified and simultaneously quantified. Variations on the general PCR method are known in the art.

[0044] The term “substantial identity” or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identify, for example, at least about 60% or more identify, such as 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identify compared to a reference sequence using one of the alignment programs described using standard parameters.

[0045] Embodiments of the methods described herein include sample collection from the postenor oropharyngeal wall of the throat and / or the tonsils of a subject. A medical professional and / or technician may identify and perform a visual assessment of a patient’s posterior oropharyngeal wall prior to collecting the specimen. It is important that the medical professional collecting an oropharyngeal swab specimen be specially trained in the manner in which to obtain a quality sample — which means at least a cell from the surface of oropharyngeal tissue — without injuring the patient or compromising the specimen. Suitable specimens comprise one or more cells collected from the test subject. The one or more cellsmay be epithelial. It is critical, however, to obtain squamous cells from the orophary nx — the presence of beta globin is an internal control for the validity' of the PCR test— it is used verify that the sample is 1) from a human source 2) that a valid specimen was actually collected and performed as a valid test (e.g., a swab could be placed in the collection vial and run on the instrument and have a negative result for HPV , but it would not tell if the specimen was valid unless the presence of beta globin was detected as an internal control.).

[0046] FIG. 1 provides a method of screening and treating an HPV risk in a patient. It should be understood that FIG. 1 illustrates an exemplary' method to which steps can be re-ordered or omitted, and additional steps may be added. A step 101 includes collecting a cell from oropharyngeal tissue of a subject. The method further includes a step 102 of determining the presence of beta globin in the cell. The method further includes a step 103 of stabilizing the cell or the contents of the cell using a transport medium. The step 103 of stabilizing the cell may implement a transport medium. The transport medium may be PreservCyt Solution. The transport medium may additionally or alternatively contain guanidine-hydrochloride. The method further includes a step 104 of measuring HPV DNA in the cell or in a solution that contains the contents of the cell. The step 104 of measuring HPV DNA may include methods such as quantitative PCR, In Situ Hybridization, next-generation sequencing, or CRISPR-based detection.

[0047] The method further includes a step 105 of calculating a risk assessment for the development of cancer in the subject. In step 105, the HPV gene expression library may not be found in nature and the risk assessment may’ be calculated from data generated by the measuring of the HPV DNA and the measuring of the HPV gene expression. The risk assessment of step 105 may further include determining a persistence of an HPV infection. Determining the persistence of the HPV infection may further include repeating the measuring HPV gene expression every six months and generating a score, where a score of zero comprisesa negative measurement of HPV gene expression at a first test, a second test twelve months after the first test and a third test twenty -four months after the first test. A score of one may result from one, two or three positive tests for the measurement of HPV gene expression out of five total tests. A score of three may include two positive tests for the measurement of the HPV gene expression on a first test and a test twenty four months after the first test. A score of zero may result from a negative measurement of HPV gene expression at: a first test, a second test twelve months after the first test and a third test twenty -four months after the first test. A score of one may result from one positive test for the measurement of HPV gene expression out of five total tests. A score of two may result from two positive tests for the measurement of HPV gene expression out of five total tests. A score of three may result from three or more positive tests for the measurement of HPV gene expression out of five total tests. A score of three may result from two positive tests for the measurement of HPV gene expression on a first test and a test twenty four months after the first test.[004S] The method may further include a step 106 of referring the subject patient to a physician. The subject patient may be referred to a physician, such as an otolaryngologist or an oncologist, when the subject has a score greater than one. In the method illustrated in FIG. 1, it should be understood that the subject may be human. The method illustrated in FIG. 1 may be conducted every- six months for no less than two years.

[0049] FIG. 2 provides a method of screening and treating an HPV risk in a patient. It should be understood that FIG. 1 illustrates an exemplary method to which steps can be re-ordered or omitted, and additional steps may be added. A step 201 includes collecting a cell from oropharyngeal tissue of a subject. The method further includes a step 202 of determining the presence of beta globin in the cell. The method further includes a step 203 of stabilizing the cell or the contents of the cell using a transport medium. The step 203 of stabilizing the cell may implement a transport medium. The transport medium may be PreservCyt Solution. Thetransport medium may additionally or alternatively contain guanidine-hydrochloride. The method further includes a step 204 of measuring HPV DNA in the cell or in a solution that contains the contents of the cell. The step 204 of measuring HPV DNA may include methods such as quantitative PCR, In Situ Hybridization, next-generation sequencing, or CRISPR-based detection. The method further includes a step 205 of synthesizing an HPV gene expression library derived from the cell. The step 205 of synthesizing an HPV gene expression library may further include methods of RNA isolation and cDNA synthesis or next-generation sequencing library preparation The method further includes a step 206 of measuring the HPV gene expression. The step 206 of measuring HPV gene expression may include quantitative PCR or next-generation sequencing. The method further includes a step 207 of analyzing the diversity and abundance of HPV transcripts.

[0050] The method further includes a step 208 of calculating a risk assessment for the development of cancer in the subject. In step 208, the HPV gene expression library may not be found in nature and the risk assessment may be calculated from data generated by the measuring of the HPV DNA and the measuring of the HPV gene expression. The risk assessment of step 208 may further include determining a persistence of an HPV infection. Determining the persistence of the HPV infection may further include repeating the measuring HPV gene expression every six months and generating a score, where a score of zero comprises a negative measurement of HPV gene expression at a first test, a second test twelve months after the first test and a third test twenty-four months after the first test. A score of one may result from one, two or three positive tests for the measurement of HPV gene expression out of five total tests. A score of three may include two positive tests for the measurement of the HPV gene expression on a first test and a test twenty four months after the first test. A score of zero may result from a negative measurement of HPV gene expression at: a first test, a second test twelve months after the first test and a third test twenty -four months after the first test. A scoreof one may result from one positive test for the measurement of HPV gene expression out of five total tests. A score of two may result from two positive tests for the measurement of HPV gene expression out of five total tests. A score of three may result from three or more positive tests for the measurement of HPV gene expression out of five total tests. A score of three may result from two positive tests for the measurement of HPV gene expression on a first test and a test twenty four months after the first test.

[0051] The method may further include a step 209 of referring the subject patient to a physician. The subject patient may be referred to a physician, such as an otolary ngologist or an oncologist, when the subject has a score greater than one. In the method illustrated in FIG. 2, it should be understood that the subject may be human. The method illustrated in FIG. 2 may be conducted every six months for no less than two years.

[0052] Oropharyngeal refers to anything related to the orophary nx, a region within the human throat. The oropharynx is the middle part of the pharynx (throat), located behind the oral cavity (mouth). It extends from the soft palate (the back of the roof of the mouth) down to the upper edge of the epiglottis. The oropharynx may contain the base of the tongue, the palatine tonsils and tonsillar fossae, the soft palate and the posterior pharyngeal wall.

[0053] Some embodiments of the present disclosure are directed to a method of administering an oropharyngeal swab HPV test to determine the persistence of the HPV infection. The steps of some embodiments of the method of the present disclosure will be discussed below. As will be appreciated by one skilled in the art, in some embodiments the variation of the sequence of steps, or the omission of some steps, is within the scope of the present disclosure.

[0054] In some embodiments, the method may include the step of identifying, by a skilled clinician, a collection site on a patient's posterior oropharyngeal wall or tonsils. In some embodiments, an evaluation of the collection site may be performed by the skilled clinician to determine if the testing site is already inflamed and / or otherwise irritated to an extent thatspecimen collection should be delayed due to a likelihood of severe irritation to the collection site.

[0055] In some embodiments, the method of the present disclosure may include the collection of an orophary ngeal specimen, by the skilled clinician, using a modified oropharyngeal swab, such as described below.

[0056] In some embodiments, the method of the present disclosure may include placing the collected oropharyngeal swab specimen into a container filled or partially filled with a transport medium, such as described below.

[0057] As a non-limiting example, FIG. 3 illustrates a method of collecting an oropharyngeal specimen. The method includes step 301 of unscrewing the cap of the viral transport medium tube. The method further includes step 302 of opening the package containing the swab. The method further includes a step 303 of carefully removing the swab. It is important to hold the swab correctly when taking an oropharyngeal specimen to avoid injuring the patient and to ensure a good collection. The swab should be held like a pen, between the index and second fingers and the thumb. Further, the method includes a step 304 of directing the patient to open their mouth and extend their tongue — the use of a tongue depressor can help increase visualization. Shining a light source into their mouth to further enhance visualization of the posterior oropharyngeal wall. Directing the patient to say ‘"Ahhh.” This will elevate the soft palate for better access to the posterior oropharyngeal wall. The method further includes a step 305 of inserting the swab into the mouth and gently swabbing against the oropharyngeal wall and the tonsils, rotating the swab as one collects the specimen. Avoiding touching the swab against the cheeks, tongue, or gums. Further, the method includes a step 306 of placing the swab head into the viral transport tube, and a step 307 of either cutting off the excess shaft or breaking it off at the indicated line and securing the tube cap for sample processing.

[0058] In some embodiments, the use of the posterior oropharyngeal wall as a sample collection site may reduce patient discomfort. In some other embodiments, the use of the posterior oropharyngeal wall of a patient as the collection site may also facilitate administration of the HPV test in more settings, as the less invasive nature of the specimen collection — relative to cervical brushing specimen collection — may be performed with less effect on the patient’s privacy.

[0059] It is noted that the method or methods may be applied in an office, in a quick clinic setting, in a hospital, or at another location.

[0060] In some embodiments, an oral solution is collected from the subject. The oral solution may be a gargle solution, an oral rinse solution, or saliva. The solution may be saline or another solution that is safe for oral use and that preserves either the cells or nucleic acids from the cells in the solution. The saliva may be spit by the subject into a tube alone or in combination with a transport medium.

[0061] FIG. 4 illustrates another method of screening and treating an HPV risk in a patient. It should be understood that FIG. 4 illustrates an exemplary method to which steps can be reordered or omitted, and additional steps may be added. It should be further understood that certain steps are similar or identical to those shown in FIGS. 1-3, and thus features from the method shown in FIGS. 1-3 may be incorporated into the method shown in FIG. 4. In FIG. 4, a step 401 includes collecting a cell from oropharyngeal tissue of a subject. The method further includes a step 402 of determining the presence of beta globin in the cell. The method further includes a step 403 of stabilizing the cell or the contents of the cell using a transport medium. The method further includes a step 404 of measuring HPV DNA in the cell or in a solution that contains the contents of the cell.

[0062] The method further includes a step 405 of collecting an oral solution, as noted above.The method further includes a step 406 of measuring HPV nucleic acid. The method furtherincludes a step 407 of calculating a risk assessment for the development of cancer in the subject. Calculating a risk assessment may include determining a persistence of an HPV infection. Determining the persistence of the HPV infection may include repeating the measuring HPV gene expression every six months and generating a score.

[0063] FIG. 5 illustrates another method of screening and treating an HPV risk in a patient. It should be understood that FIG. 5 illustrates an exemplary method to which steps can be reordered or omitted, and additional steps may be added. It should be further understood that certain steps are similar or identical to those shown in FIGS. 1-4, and thus features from the method shown in FIGS. 1-4 may be incorporated into the method shown in FIG. 5. In FIG. 5, a step 501 includes collecting a cell from oropharyngeal tissue of a subject. The method further includes a step 502 of determining the presence of beta globin in the cell. The method further includes a step 503 of stabilizing the cell or the contents of the cell using a transport medium. The method further includes a step 504 of measuring HPV DNA in the cell or in a solution that contains the contents of the cell.

[0064] The method further includes a step 505 of synthesizing an HPV gene expression library derived from the cell. The method further includes a step 506 of measuring the HPV gene expression. The method further includes a step 507 of collecting an oral solution, as noted above. The method further includes a step 508 of measuring HPV nucleic acid. The method further includes a step 509 of calculating a risk assessment for the development of cancer in the subject. Calculating a risk assessment may include determining a persistence of an HPV infection. Determining the persistence of the HPV infection may include repeating the measuring HPV gene expression even’ six months and generating a score.

[0065] FIG. 6 illustrates another method of screening and treating an HPV risk in a patient. It should be understood that FIG. 6 illustrates an exemplary method to which steps can be reordered or omitted, and additional steps may be added. It should be further understood thatcertain steps are similar or identical to those shown in FIGS. 1-5, and thus features from the method shown in FIGS. 1-5 may be incorporated into the method shown in FIG. 6. The method may comprise a step 601 of collecting an oropharyngeal swab specimen including human epithelial cells and / or cellular contents. The method further includes a step 602 of validating specimen adequacy by detecting beta-globin. The method further includes a step 603 of placing the swab into a transport medium comprising guanidine-hydrochloride and storing at 2-8°C for up to 50 days. The method further includes a step 604 of isolating nucleic acids by magnetic- bead purification. The method further includes a step 605 of performing real-time qPCR targeting HPV E6 / E7 and beta-globin under defined cycling conditions. The method further includes a step 606 of measuring HPV DNA in the cell or in a solution that contains the contents of the cell. The method further includes a step 607 of electronically calculating a persistence index from measurements at M0, M6, M12, M18, and M24. The method further includes a step 608 of computing a risk score for the development of cancer in the subject. A risk score of zero may result from a negative measurement of HPV gene expression at: M0, Ml 2 and M24. A risk score of three may result from two positive tests for the measurement of HPV gene expression at M0 and M24. The method further includes a step 509 of automatically generating a report and if the risk score is greater than or equal to two, recommending referral to a physician.

[0066] By way of a non-limiting example, the specimen may be collected using a modified swab. The modified swab may, in some embodiments, be modified to include a longer handle length to facilitate collection of the specimen from a patient’s posterior oropharyngeal wall. In some embodiments, the handle length may be between 6 inches and 10 inches. The handle may be modified in stiffness and flexibility to better accommodate access to anatomical sites in the oral cavity that may be difficult to swab. The handle may be modified to allow bending into a position to better accommodate access and collection. The modified swab may be a soft-flocked swab. However, as will be appreciated by one skilled in the art, in some other embodiments, the modified swab may be a nylon, polyester, non-cotton, or non-calcium alginate swab. In some embodiments, the modified swab may have non-wooden handles formed from a sterile plastic, polymer, or metallic material.

[0067] In an aspect, the method may include placing and / or submerging the swab or modified swab within a transport medium during transport and / or possibly storage before the specimen is processed for molecular testing. The transport medium used for a given specimen and the test to be performed will dictate the type of transport medium that may be used. As will be understood by one skilled in the art, Viral Transport Media (VTM) are designed to reduce the growth of non-viral pathogens, such as bacteria and fungi, and stabilize the virus and nucleic acids, which are prone to degradation. It is important that the molecular integrity of the specimen is maintained. The transport medium may be stored in a sterile environment, such as within a sealed vial or tube. The transport medium may, in some embodiments, also include components that protect the specimen. This may include buffers to ensure a proper pH level is maintained and cryoprotectants such as sucrose to optimize viability of organisms through freezing and thawing. This may also include preservative components, chelating components, and components to neutralize or deactivate pathogens contained within the specimen, including antimicrobial agents to inhibit the growth of bacteria and fungi. In some embodiments, the transport medium may be PreservCyt® Solution (Hologic, Inc.; 250 Campus Drive; Marlborough, MA 01752; USA), but the present teachings are not limited to such.

[0068] Aspects of the disclosed method are directed to the use of transport mediums, such as those containing a guanidine-hydrochloride stabilizing agent, for use with oropharyngeal HPV DNA collected with an oropharyngeal swab. Independent verification of the efficacy of such solutions for use with oropharyngeal swab specimens on oropharyngeal HPV testing has been confirmed with modifications to the transport / storage protocols. For example, using Cobas®PCR Media, a swab sample for STI testing of CT / NG may be stored at a temperature range of 2°-30° C for up to 12 months from the date of collection. However, in some embodiments of the present disclosure, an orophary ngeal specimen collected by a swab and placed in a similar transport medium containing guanidine-hydrochloride and used for orophary ngeal HPV testing may be kept in a narrower 2°-80C temperature range for up to 50 days.

[0069] Some transport mediums contain an alcohol as a primary' preservative component. In some instances, the alcohol may be methanol, isopropanol, and / or ethanol at 70- 100 percent by volume. An example of such a transport medium is PreservCyt® Solution. Cervical epithelial cells transported in PreservCyt® Solution may be stored at room temperature for up to 18 weeks. In some embodiments of the present disclosure, a methanol-based transport medium may be used to transport / store a collected orophary ngeal swab specimen under the same conditions as an oropharyngeal swab specimen transported in a guanidine-hydrochloride based transport medium.

[0070] PCR may be used to amplify the presence of viral DNA collected in a specimen. Many such tests are specific to the LI gene of the virus. Similarly, for some HPV tests that use PCR amplification, fluorescent-dye tagged oligonucleotide probes and quenchers may be used to facilitate so-called ‘‘real-time PCR” wherein the tagged probes bind to specific sequences of target DNA, and the cleaving process of each PCR cycle then activates the fluorescent dye, allowing the accumulation of fluorescence within the PCR sample to be measured each cycle.

[0071] For some samples to be used for PCR-based testing, the sample may contain cells that must be lysed before testing. In some such testing procedures, there may also be a washing and purification process to remove unwanted DNA and cell remnants from the sample prior to testing. A pre-testing process for lysing, washing, and purifying a sample may be modified to allow for the use of a different specimen type, i.e., an oropharyngeal swab specimen. For example, in some embodiments, the lysing process may be omitted as the availability of viralDNA within fluids collected by the oropharyngeal swab may not require a lysing step. In some other embodiments, the washing and purification steps may similarly be adapted for use with oropharyngeal swab collected specimens. In some embodiments, the lysing, extraction, washing, and / or purifying steps may be automated. In some embodiments, these steps may be carried out directly using a specimen tube within a device configured to carry7out the PCR steps of the HPV test. In some other embodiments, an aliquot from a specimen-containing tube, a “primary ” specimen tube, may be removed and placed in a secondary container for use with a PCR testing device.

[0072] In some embodiments of the present disclosure, PCR amplification may be used. In some other embodiments, real-time PCR may be used, including the use of fluorescent-dye tagged oligonucleotide probes and quenchers.

[0073] An embodiment of the method for identify ing HPV within the orophary ngeal tissue of a patient, may involve the use of Roche Cobas PCR tests (Roche Molecular Systems, Inc.; 4300 Hacienda Drive; Pleasanton, C A 94588; USA) or any other suitable reagent and / or method for the isolation and quantification of RNA, DNA, and / or protein from the specimen, including commercially available kits for such purposes. An HPV testing assay may use a real-time PCR method targeting the LI region of the HPV genome that detects 14 high-risk HPV genotypes. (3-globin serves as an internal control for monitoring sample preparation, extraction, and PCR amplification. The use of fluorescent probes specific to HPV targets, including HPV 16 and HPV 18, may ensure high specificity' and reliable real-time detection.

[0074] As described herein, the method may include a test for the presence of HPV in the specimen or in solutions derived from the specimen. The method may include measuring the presence of HPV DNA in ahuman cell. The measuring may use PCR amplification and genome tagging to characterize viral genetic material that has entered or integrated into the human genome as a result of natural infection. HPV, especially high-risk types, can integrate its DNAinto the human genome, and this integration is a key event in the development of HPV- associated cancers, but it is not a universal feature of all HPV infections. More specifically, high-risk HPV types (such as HPV- 16 and HPV- 18) can integrate their DNA into the human genome, especially during the progression from infection to cancer. Genome tagging refers to the use of molecular probes, primers, or sequencing-based approaches to specifically identify and track viral DNA within human cells. This allows for the detection, localization, and characterization of viral genetic material, providing critical insights for diagnosis, monitoring, and research into viral diseases. As such, the disclosed methods factor in the degree of viral DNA integration into the calculation of risk assessment of cancer development.

[0075] Measuring the presence of HPV DNA in the human cell may comprise the following methods: 1) In Situ Hybridization (ISH): labeled DNA probes are designed to bind specifically to viral DNA sequences known to be present in naturally occurring infections. When applied to patient samples, these probes "tag" the viral DNA, allowing visualization under a suitable microscope, such as a fluorescent microscope if the DNA probes are labeled with a fluorophore or equivalent; 2) PCR and / or quantitative PCR: Primers targeting unique viral sequences act as molecular tags, enabling the amplification and quantification of viral DNA directly from patient samples; 3) next-generation sequencing (NGS): High-throughput sequencing can identify’ and map viral DNA within the human genome. Bioinformatic tools "tag" reads that match viral sequences, distinguishing them from human DNA and revealing integration sites or viral load; or 4) CRISPR-based Detection: CRISPR / Cas systems can be programmed to recognize and bind to specific viral DNA sequences, effectively "tagging" them for detection, sometimes coupled with a fluorescent or colorimetric readout.

[0076] In some embodiments of the method, there may be a need for a transport medium that includes one or more buffer compounds to maintain a specified pH level. In some otherembodiments, a nucleic-acid stabilizing compound may also be included in the transport medium.

[0077] In some embodiments, the method of the present disclosure may include separating a testing sample from the collected oropharyngeal swab specimen within the transport medium and placing the remaining portion of the specimen into storage.

[0078] In some embodiments, the method of the present disclosure may include a lysing step to lyse any cells present within the testing sample.

[0079] In some embodiments, the method of the present disclosure may include a washing step to remove unwanted cell remnants from the testing sample.

[0080] In some embodiments, the method of the present disclosure may include a purification step to specifically separate target DNA from the testing sample.

[0081] In some embodiments, the method of the present disclosure may include placing the purified testing sample into a real-time PCR test assay including the specified buffers, PCR medium solution, template DNA, primers, DNA polymerase (Taq polymerase), and nucleotides. In some embodiments, one or more fluorescent-dye tagged oligonucleotide probes and quenchers may be added.

[0082] In some embodiments, the method of the present disclosure may include running a realtime PCR testing sequence carried out over a plurality of PCR cycles. In some embodiments, the number of cycles may be between 15 and 30 cycles. In some embodiments the number of cycles may be 30 cycles. In some other embodiments, the number of cycles may be between 30 and 45 cycles.

[0083] In some embodiments, the presence of different HPV types, including those types associated with higher risk of severe symptoms and / or cancer-risk, may be evaluated separately. In some embodiments, these evaluations may be made contemporaneously.

[0084] In another embodiment, HPV mRNA expression may be measured to identify infections that are actively expressing oncogenic proteins, which are more likely to lead to cellular changes and cancer. HPV mRNA expression data may be combined with HPV DNA data in predicting risk and progression to cancer, and in some embodiments may include a risk score. Orophary ngeal squamous cell carcinoma (“OPSCC”) can occur in cases where the HPV DNA has integrated into the host genome and is expressing E6 / E7 mRNA, but there have also been cases reported in the literature of OPSCC where episomal HPV is present.

[0085] The pathobiology of human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC) — including initial infection, viral persistence, epigenetic methylation, host-genome integration, and host immune responses — provides the basis for identifying oral and circulating biomarkers that differentiate transient, high-prevalence early infection from less common pathogenic viral persistence (see FIG. 7). In certain embodiments, such biomarkers include, without limitation, nucleic acid markers (e.g., HPV DNA load and genotype, E6 / E7 mRNA expression, viral-host integration breakpoints, and locus-specific methylation of viral and / or host genes), protein markers (e g., HPV oncoproteins or host proteins induced by viral oncogenesis), serologic markers (e.g., antibodies against HPV capsid and early proteins), and cell-free or exosomal markers in saliva or blood (e.g., circulating tumor DNA bearing HPV sequences, methylation signatures, or integration junctions). In some embodiments, combinations of these markers, optionally assessed longitudinally, are used to distinguish early, typically self-limited infection from persistent, pathogenic infection associated with oncogenic transformation, to stratify risk, and to inform diagnostic, prognostic, or monitoring applications (see FIG. 7).

[0086] In the present system, the above described test may be utilized to determine the persistence of the HPV infection, which may be linked to OPSCC development. The persistent HPV infection can lead to events that cause a deactivation of the body’s cancer preventivemechanisms. Specifically, persistent HPV infection induces oncogenesis through suppression of tumor suppressor proteins p53 and RB. This can then lead to an increased risk of developing OPSCC. To determine what persistent HPV infection is, a risk assessment may be utilized. The risk assessment may utilize a risk score that can help to show or determine the persistence of the HPV infection. If there is a persistence of an HPV infection there is an increased risk of developing OPSCC. Set forth below is a description of a risk assessment utilizing a risk score. Modifications of such are contemplated herein and this is merely an exemplary embodiment of a way of developing such risk score. It should be understood that alternative risk assessments may be utilized without departing from the present teachings.

[0087] The risk score may be calculated according to a risk score process, such as shown in Figure 6. Since most HPV infection spontaneously resolves within 18-24 months, the present disclosure utilizes a risk score to determine individuals with persistent infection, which is an indicator of the potential for cancer risk or more specifically for OPSCC. The risk score considers the first month of testing Month Zero (MO) followed by repeat testing every 6 months (full testing cycle M6, M12, M18 and M24) if the first test is positive and every 12 months (reduced testing cycle at M12 and M24) if the first test is negative. If an individual in the reduced testing cycle tests positive in M12 or M24, the positive test would be considered MO triggering and full cycle.

[0088] An example of the scoring system is set forth below:• Score Zero: Negative testing at MO, M12 and M24• Score 1 : One positive test out of 5• Score 2: Two positive tests out of 5• Score 3: Three or more positive tests out of 5 or 2 positive tests at MO and M24

[0089] Individuals with score 3 are to be referred to ENT doctors for follow up or to any other appropriate physician including an otolaryngologist or an oncologist. It is worth noting that itis also prudent to refer individuals with a 2 score or high-risk patients to ENT doctors or to any other appropriate physician including an otolaryngologist or an oncologist with the first positive test. The logic is that for a newly implemented test, it is impossible to determine if this positive result represents anew onset infection or existing persistent one.

[0090] It should be understood that modifications may be made to the risk assessment set forth above without departing from the present teachings. For example, while specific timing is disclosed, the actual timing may be modified by a month or two either way from the first test without departing from the present teachings. Moreover, the actual scores may be modified in any appropriate manner, e.g., numerical scores may be utilized, alphanumeric scores may be utilized, word scores or a combination of such may be utilized. The scores are utilized to provide perspective of and show the extent of the HPV infection to help provide context to the actual persistence of the HPV infection.

[0091] Also, a risk score may be calculated using the presence of HPV ty pes by HPV DNA and activity by HPV gene expression, comprising at least five prognostic expression values and / or a pathology method using Al - macros pulled from diagnostic database.

[0092] In certain embodiments, droplet digital PCR (ddPCR) is employed to enhance analytical sensitivity and enable precise quantification of circulating tumor-derived viral DNA present in the peripheral blood of cancer patients. Methylation-specific PCR assays can be used to detect epigenetic alterations within the human papillomavirus (HPV) genome as well as in host tumor suppressor genes, including, for example, EBP41L3 and LY6D. In addition, nextgeneration sequencing (NGS) can be applied to characterize HPV integration events, DNA methylation patterns, and fusion transcripts, and to assess correlations between patterns of viral integration and clinical disease stage or progression.

[0093] In certain embodiments, for subjects with a risk score of two or greater, an initial, low- cost, and low-morbidity7evaluation comprises a comprehensive examination of anatomic sitesat risk for HPV-associated malignancy. This evaluation may comprise a head and neck examination with flexible nasopharyngoscopy, with focused assessment of the palatine tonsils and the base of the tongue, and may further include narrow-band imaging to enhance delineation of mucosal abnormalities. If no abnormality is identified on clinical examination, the subject may proceed to ultrasound imaging to evaluate the bilateral upper cervical lymph node basins for early nodal metastases. If neither physical examination nor ultrasound demonstrates localizing abnormalities, ancillary' biomarker-based localization can be performed to identity' an occult primary in the tonsils or base of tongue. Such biomarkers may include, by way of example, site-directed swab detection of oncogenic HPV DNA from a single tonsil with absence of signal from the contralateral tonsil and base of tongue; detection of sitespecific aberrant DNA methylation patterns; or detection of HPV DNA demonstrating human- viral genomic integration confined to one site. Upon identification of site-specific positivity', a targeted biopsy of the implicated site may be indicated, followed by close clinical surveillance and serial swab sampling in cases of negative initial biopsy.

[0094] In certain embodiments, when biomarker findings indicate markedly elevated risk, more invasive interventions can be considered for biomarker-positive subjects. Such interventions may include positron emission tomography-computed tomography (PET-CT) or other advanced imaging modalities; prophylactic tonsillectomy; administration of systemic or topical site-localizing biopharmaceuticals to facilitate intraoperative directed biopsy; and immunopreventive strategies intended to prevent the emergence of oropharyngeal carcinoma.

[0095] The foregoing description provides support for the full breadth of the claimed subject matter without confining the claims to particular examples, contexts, or configurations. The claims that follow particularly point out and distinctly claim the subject matter regarded as the invention and are intended to encompass all variations, modifications, alternatives, and equivalents that a person of ordinary skill in the art would recognize in view of this disclosure.Any characterization in the description should not be interpreted as limiting unless expressly- set forth in a claim. The claims are intended to cover, and the disclosure supports, all structural, chemical, biological, algorithmic, and procedural equivalents, variants, substitutions, permutations, and rearrangements that a person of ordinary- skill would recognize as insubstantially different from the expressly described subject matter. References to specific materials, instruments, parameters, algorithms, targets, biomarkers, genes, loci, media, transport conditions, or platforms are exemplary and encompass known and later-developed equivalents and functionally interchangeable alternatives.

[0096] No characterization in this specification is intended to narrow the plain meaning of the claims or to effect a disclaimer or disavowal. To the extent any statement could be construed as limiting, it is expressly disclaimed unless affirmatively7adopted into a claim by amendment or argument.

[0097] References to specific technologies, platforms, assays, media, molecular targets, computational models, statistical thresholds, machine-learning approaches, or analytical pipelines are intended to encompass reasonable updates, improvements, and successors that provide substantially the same function or result, as understood by a person of ordinary skill in the art.

[0098] What has been described above includes examples of the present specification. It is, of course, not possible to describe every conceivable combination of components or methodologies for purposes of describing the present specification, but one of ordinary skill in the art may recognize that many further combinations and permutations of the present specification are possible. Each of the components described above may be combined or added together in any permutation to define embodiments disclosed herein. Accordingly, the present specification is intended to embrace all such alterations, modifications and variations that fall within the spirit and scope of the appended claims. Furthermore, to the extent that the term“includes” is used in either the detailed description or the claims, such term is intended to be inclusive in a manner similar to the term “comprising” as “comprising” is interpreted when employed as a transitional word in a claim.

Claims

CLAIMS1. A method for identifying presence of HPV in human tissue comprising the steps of: collecting a cell from oropharyngeal tissue of a subject utilizing a swab; measuring HPV DNA in the cell or in a solution that contains contents of the cell; and calculating a risk assessment for development of cancer in the subject, wherein the risk assessment is calculated from data generated by the measuring of the HPV DNA to determine a persistence of the HPV in human tissue.

2. The method of claim 1 further comprising determining presence of beta globin in the cell.

3. The method of claim 1, further comprising stabilizing the cell or the contents of the cell using a transport medium.

4. The method of claim 3, wherein the transport medium is PreservCyt Solution.

5. The method of claim 3, wherein the transport medium contains guanidinehydrochloride.

6. The method of claim 1, wherein measuring HPV DNA comprises quantitative PCR, In Situ Hybridization, next-generation sequencing, or CRISPR-based detection.

7. The method of claim 1, wherein the subject is a human.

8. The method of claim 1 further comprising synthesizing an HPV gene expression library derived from the cell; wherein the HPV gene expression library is not found in nature and measuring HPV gene expression.

9. The method of claim 8, wherein synthesizing a HPV gene expression library includes RNA isolation and cDNA synthesis or next-generation sequencing library preparation.

10. The method of claim 8. wherein measuring HPV gene expression includes quantitative PCR or next-generation sequencing.

11. The method of claim 10, further comprising analyzing diversity and abundance of HPV transcripts.

12. The method of claim 1, wherein calculating a risk assessment comprises determining a persistence of an HPV infection.

13. The method of claim 12, wherein the persistence of the HPV infection is linked to development of orophary ngeal squamous cell carcinoma.

14. The method of claim 12, wherein determining the persistence of the HPV infection comprises repeating the HPV DNA in the cell or in the solution that contains the contents of the cell every7six months and generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test twenty -four months after the first test.

15. The method of claim 14, wherein a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

16. The method of claim 15, wherein a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

17. The method of claim 16, wherein a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

18. The method of claim 16. wherein a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test twenty four months after the first test.

19. The method of claim 17 or claim 18, wherein the subject with the score greater than one is referred to a physician including an otolaryngologist or an oncologist.

20. A method of determining the persistence of an HPV infection comprising conducting the method of claim 1 every six months for no less than two years.

21. The method of claim 20 further comprising generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test twenty -four months after the first test.

22. The method of claim 21, wherein a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

23. The method of claim 22, wherein a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

24. The method of claim 23, wherein a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

25. The method of claim 23, wherein a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test twenty four months after the first test.

26. The method of claim 24 or claim 25 wherein the subj ect with the score greater than one is referred to a physician including an otolaryngologist or an oncologist.

27. A method for identifying presence of HPV in human tissue comprising the steps of: collecting a cell from oropharyngeal tissue of a subject; measuring HPV DNA in the cell or in a solution that contains contents of the cell;collecting an oral solution from the subj ect; measuring HPV nucleic acid in the oral solution or in nucleic acid isolated from the solution; and calculating a risk assessment for development of cancer in the subject, wherein the risk assessment is calculated from data generated by the measuring of the HPV DNA from the oropharyngeal tissue.

28. The method of claim 27 further comprising determining presence of beta globin in the cell.

29. The method of claim 27 further comprising stabilizing the cell or the contents of the cell using a transport medium.

30. The method of claim 27, wherein calculating a risk assessment comprises determining a persistence of an HPV infection.

31. The method of claim 30, wherein determining the persistence of the HPV infection comprises repeating the measuring HPV DNA in the cell or in the solution that contains the contents of the cell every six months and generating a score, wherein a score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: a first test, a second test twelve months after the first test and a third test twenty- four months after the first test.

32. The method of claim 31, wherein a score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

33. The method of claim 32, wherein a score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

34. The method of claim 33, wherein a score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

35. The method of claim 33, wherein a score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell on a first test and a test twenty four months after the first test.

36. The method of claim 34 or claim 35, wherein the subject with the score greater than one is referred to an otolaryngologist or an oncologist.

37. A method for identifying presence of HPV in human tissue comprising the steps of: collecting an oropharyngeal swab specimen comprising human epithelial cells and / or cellular contents; validating specimen adequacy by detecting beta-globin; placing the swab into a transport medium comprising guanidine-hydrochloride and storing at 2-8°C for up to 50 days; isolating nucleic acids by magnetic-bead purification; performing real-time qPCR targeting HPV E6 / E7 and beta-globin under defined cycling conditions; measuring HPV DNA in the cell or in a solution that contains the contents of the cell; electronically calculating a persistence index from measurements at MO, M6, Ml 2, Ml 8. and M24; computing a risk score for development of cancer in a subject; and automatically generating a report that, when the risk score > two, recommends referral to a physician.

38. The method of claim 37, wherein the risk score of zero comprises a negative measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at: MO, M12 and M24.

39. The method of claim 38, wherein the risk score of one comprises one positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

40. The method of claim 39, wherein the risk score of two comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

41. The method of claim 40, wherein the risk score of three comprises three or more positive test for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell out of five total tests.

42. The method of claim 40, wherein the risk score of three comprises two positive tests for the measurement of the HPV DNA in the cell or in the solution that contains the contents of the cell at MO and M24.

43. A method for identifying a persistence of HPV infection comprising the steps of: collecting a swab specimen comprising human squamous cells from am oropharynx of a subject; validating specimen adequacy by detecting beta-globin; placing the collected swab specimen into a container filled or partially filled with a transport medium; testing for a presence of HPV DNA in the swab specimen or in solutions derived from the swab specimen; measuring the presence of HPV DNA in the swab specimen or in solutions derived from the swab specimen;electronically calculating a persistence index from measurements at MO, M6, M12,M18, and M24; and computing a risk score for development of cancer in the subject.

44. The method of claim 43, wherein the measuring may use PCR amplification and genome tagging to characterize viral genetic material that has entered or integrated into a human genome as a result of natural infection.

45. The method of claim 43 further comprising evaluating a collection site to determine if the collection site is already inflamed and / or otherwise irritated.

46. The method of claim 43, wherein the transport medium comprises guanidinehydrochloride, which is capable of being stored at 2-8°C for up to 50 days.

47. The method of claim 43, wherein the oropharynx comprises a base of a tongue, a palatine tonsils, tonsillar fossae, a soft palate and a posterior pharyngeal wall of the subject.

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