Assay for the detection of oropharyngeal cancer

Abstract

An assay comprising a probe panel for detecting the methylation status of 12 genes and one CPG site is disclosed herein. Also disclosed herein is a method for using oral rinsing and the probe panel to detect the presence of oropharyngeal cancer based on the methylation status of these genes and CPG sites.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 449,665 filed on March 3, 2023, U.S. Provisional Patent Application No. 63 / 452,352 filed on March 15, 2023, and U.S. Provisional Patent Application No. 63 / 455,829 filed on March 30, 2023, which are incorporated herein by reference in their entirety.

[0002] Statement of government support This invention was developed with government support under authorization number R21DE024816, granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Sequence listing reference The sequence listing filed on March 1, 2024, titled "10110-440WO1_ST26.XML", prepared in .XML format, with a file size of 18,980 bytes, is incorporated herein by reference in accordance with 1.52(e)(5) of the U.S. Patent Act. [Background technology]

[0004] As human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) increases among middle-aged men in developed countries, the need for early diagnosis is also growing. While the survival rate for HPV-associated OPC is good, metastasis remains a possibility, with 13–25% of patients experiencing recurrence within two years, and often failing to achieve a cure regardless of HPV status. Primary prevention through screening is impossible for OPC due to the lack of observable precancerous lesions. Therefore, detection of early OPC (small tumors with only a single ipsilateral positive lymph node, T1–T2, N0–N1, <3cm) is key to reducing the severity of post-diagnosis treatment and adverse outcomes. Novel assays for OPC detection are needed. [Overview of the Initiative]

[0005] Assays and biomarker panels for the detection of oropharyngeal cancer, as well as methods for using them for the detection and treatment of oropharyngeal cancer, are disclosed. Such assays and biomarker panels are effective tools for the early detection of OPC and for preventing treatment complications associated with later diagnosis.

[0006] In one embodiment, an assay for detecting oropharyngeal cancer in a subject, comprising one or more genes in a tissue sample, the assay using probes that detect methylation at the CpG site of the gene, including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and EPB41L3 (e.g., CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, c An assay is disclosed in which changes in the amount of methylation at three or more CpG sites (e.g., g14500050, cg14705778, cg04025917, cg06459104, and cg00619915, etc.) compared to a normal control (e.g., increased methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or decreased methylation at HMGB2 or EPB41L3) indicate the presence of oropharyngeal cancer. In one embodiment, the assay includes a probe for the detection of EPB41L3 CpG site 438 and / or HPV SPF for the detection of human papillomavirus type 16 infection or human papillomavirus type 18 infection. 10 PCR-DEIA-LiPA 25 Includes line probes.

[0007] Also disclosed herein is a method for diagnosing and / or detecting the presence of oropharyngeal cancer in a subject, comprising obtaining a tissue sample from the subject and applying the tissue sample to an assay described in any preceding embodiment. For example, a method for diagnosing and / or detecting the presence of oropharyngeal cancer in a subject, comprising: i) obtaining a tissue sample from the subject (including, but not limited to, performing oral rinsing, gargling, pharyngeal swab, scrape, or biopsy on the subject, or collecting saliva from the subject); and ii) one or more genes including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3. A method is disclosed herein that includes measuring methylation at CpG sites of a gene, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control (e.g., an increase in methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or a decrease at HMGB2 or EPB41L3) indicates the presence of oropharyngeal cancer. In one embodiment, the method may further include the detection of methylation at EPB41L3 CpG site 438 and / or HPV16 and / or HPV18 infection.

[0008] In one embodiment, a method for diagnosing and / or detecting the presence of oropharyngeal cancer as described in any prior embodiment is disclosed herein, wherein the detection of CpG sites is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915. For example, a methylation change may be an increase in methylation detected with one or more probes cg25191818, cg19367172, cg12176286, cg19487745, cg11125104, cg27131607, cg14500050, cg04025917, and / or cg0061991, and / or a decrease in methylation detected with one or more probes cg20791412, cg14705778, and / or cg06459104.

[0009] HPV16 and / or HPV type 16 infection is HPV SPF 10 PCR-DEIA-LiPA 25 A method for diagnosing and / or detecting the presence of oropharyngeal cancer as described in any prior embodiment, using a line probe, is disclosed herein.

[0010] In one embodiment, a method for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastasis in a subject is disclosed herein, comprising: i) obtaining a tissue sample from the subject; ii) applying the tissue sample to an assay of any preceding embodiment and / or performing a method for diagnosing and / or detecting the presence of oropharyngeal cancer of any preceding embodiment; and iii) treating the oropharyngeal cancer if detected. For example, a method for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastasis in a subject is disclosed herein, comprising: i) obtaining a tissue sample from the subject (including, but not limited to, performing oral washing, gargling, pharyngeal swab, scrape, or biopsy on the subject, or collecting saliva from the subject); and ii) CpG of one or more genes including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3 A method is disclosed herein that includes measuring methylation at a site, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control (e.g., an increase in methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or a decrease at HMGB2 or EPB41L3) indicates the presence of oropharyngeal cancer, and iii) treating the oropharyngeal cancer if detected. In one embodiment, the method may further include the detection of methylation at EPB41L3 CpG site 438 and / or HPV16 and / or HPV18 infection.

[0011] Also disclosed herein are methods for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastases as described in any prior art, wherein detection of CpG sites is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915. For example, a methylation change may be an increase in methylation detected with one or more probes cg25191818, cg19367172, cg12176286, cg19487745, cg11125104, cg27131607, cg14500050, cg04025917, and / or cg0061991, and / or a decrease in methylation detected with one or more probes cg20791412, cg14705778, and / or cg06459104.

[0012] In one embodiment, a method for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastasis as described in any prior embodiment, wherein HPV16 and / or HPV16 type infection is HPV SPF 10 PCR-DEIA-LiPA 25 A method for detection using a line probe is disclosed herein.

[0013] Furthermore, methods for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastases as described in any prior art are disclosed herein, wherein the cancer is treated with the administration of anticancer agents and / or radiation.

[0014] The accompanying drawings incorporated herein and forming part thereof illustrate several embodiments and, together with the description, illustrate the compositions and methods disclosed. [Brief explanation of the drawing]

[0015] [Figure 1]Figure 1 shows the distribution of Area Under the Curve (AUC) values. Four different variable selection methodologies were implemented to determine which one yielded the best predictive ability (i.e., a higher AUC in the test set). Each experiment consisted of 50 rounds with random selection of training and validation sets of 66.67% and 33.33%, respectively. Using the variables selected by the given method, a LASSO model was constructed and then used to predict the probability that a sample belonged to an early case or control. The experiments consisted of: (1) variables selected only from discoveries using an Illumina EPIC array probe, (2) Step 1 and manual addition of results from pyrosequencing of three EPB41L3 CpG sites, (3) Steps 1, 2 and manual addition of HPV16 status, and (4) Steps 1-3 and addition of EPB41L3 CpG sites identified by Illumina. [Figure 2A] Figure 2A shows the ROC curve of the trained model generated from 14 selected markers. A single bootstrap sample was used to train the model, yielding an AUC of 0.978. [Figure 2B] Figure 2B shows the ROC curve of the test model generated from a corresponding set of “excluded” samples, yielding a validation AUC of 0.935 using boosted rat resampling. [Figure 3] Figure 3 shows the number of non-zero coefficients per variable. In the variable selection process for the final model, we were able to assign non-zero coefficients to variables 1 to 50 times. The variables were ranked in descending order according to the number of times each variable received a non-zero coefficient. Using the heuristic that each variable should be assigned a non-zero coefficient at least half a time, we started at the red solid line, which is a soft cutoff of 25. There was some plateau around 25 (red dotted line), so variables that were assigned a non-zero coefficient 23 times were also included in the final model of a total of 14 variables. [Figure 4]Figure 4 shows a detailed schema of the process used to construct a biomarker panel. The actions on the left represent the process that was part of the cross-validation, and the actions on the right represent the individual steps towards the identification of the biomarkers to be included in the panel. Steps 1-5 correspond to those described in detail in this method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Before the compounds, compositions, articles, devices, and / or methods are disclosed and described, they are not limited to a particular synthetic method or a particular recombinant biotechnology method, or to particular reagents, unless otherwise specified, and as such, may of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0017] A. Definitions As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0018] In this specification, a range may be expressed as "approximately" from a certain value and / or "approximately" to another specific value. Where such a range is expressed, another embodiment includes that certain value and / or to another specific value. Similarly, where a value is expressed as an approximation using the antecedent "approximately", it is understood that a particular value forms another embodiment. It will be further understood that the endpoint of each range is important, whether related to other endpoints or independent of other endpoints. Also, where several values ​​are disclosed in this specification, it is understood that each value is disclosed in this specification not only as the value itself but also "approximately" that particular value. For example, if the value "10" is disclosed, "approximately 10" is also disclosed. As will be appropriately understood by those skilled in the art, where a value is disclosed as "less than or equal to that value", it is also understood that "greater than or equal to that value" and possible ranges between values ​​are also disclosed. For example, where the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. Furthermore, throughout this application, the data is provided in several different formats, and it is understood that this data also represents the range of endpoints and starting points, as well as any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that, in addition to the range between 10 and 15, the values ​​greater than 10, 10 or greater, less than 10, 10 or less, and equal to 10, greater than 15, 15 or greater, less than 15, 15 or less, and equal to 15 are disclosed. It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0019] In this specification and the appended claims, several terms are referenced to have the following meanings:

[0020] "Optional" or "optional" means that the event or situation described below may or may not occur, and this description includes examples of when such event or situation occurs and when it does not.

[0021] "Increase" can refer to any change that results in a greater quantity of symptoms, disease, composition, condition, or activity. An increase can be an increase in any individual value, median, or mean in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, as long as the increase is statistically significant.

[0022] "Reduction" can refer to any change that results in a smaller amount of symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene if the genetic output of the gene product containing that substance is less than the genetic output of the gene product without that substance. Also, for example, a reduction can be a change in the symptoms of a disorder, such that the symptoms are less than previously observed. A reduction can be a statistically significant reduction in any individual, median, or mean amount of a condition, symptom, activity, or composition. Thus, a reduction can be a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, insofar as the reduction is statistically significant.

[0023] "Inhibit," "to inhibit," and "inhibition" mean reducing activity, response, state, disease, or other biological parameters. This may include, but is not limited to, the complete elimination of activity, response, state, or disease. It may also include, for example, a 10% reduction of activity, response, state, or disease compared to natural or control levels. Thus, a reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to natural or control levels.

[0024] The words "reduce" or other forms of the word, such as "to reduce" or "reduce," mean a decrease in an event or characteristic (e.g., tumor growth). This is typically related to some standard or expected value, in other words, it is relative, but it is understood that it is not always necessary to refer to a standard or relative value. For example, "to reduce tumor growth" means to reduce the rate of tumor growth compared to a standard or control.

[0025] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, to stabilize or delay the occurrence or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than reduction, for example, and therefore does not require comparison with a contrast. Where used herein, something may be reduced but not preventable, while something that is reduced may be prevented. Similarly, something may be prevented but not reduced, while something that is prevented may be reduced. Where reduction or prevention is used, it should be understood that the use of other words is also expressly disclosed unless specifically designated otherwise.

[0026] The term "subject" refers to any individual that is the target of administration or treatment. A subject can be a vertebrate, for example, a mammal. In one embodiment, a subject can be a human, a non-human primate, a cattle, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject can be a human or a veterinarian's patient. The term "patient" refers to a subject under the treatment of a clinician, such as a physician.

[0027] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement does not necessarily mean elimination; reduction or alteration may suffice.

[0028] The term “treatment” refers to the medical management of a patient with the intention of curing, improving, stabilizing, or preventing a disease, condition, or disorder. This term includes active treatment, i.e., treatment aimed specifically at improving the disease, condition, or disorder, and causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing, or partially or completely suppressing, the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement other specific treatments aimed at improving the associated disease, condition, or disorder.

[0029] "Biocompatible" generally refers to materials and any metabolites or their breakdown products that are generally non-toxic to the recipient and do not cause serious side effects in the subject.

[0030] "Comprising" is intended to mean that a composition, method, etc., includes the enumerated elements but does not exclude others. When used to define a composition and method, "consisting essentially of" means that it includes the enumerated elements but excludes other elements that have any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharmaceutically acceptable carriers, such as phosphate-buffered saline and preservatives, from isolation and purification methods. "Consisting of" means that it excludes more than trace elements of other components for administering the composition provided and / or claimed in this disclosure, and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0031] A "control" is an alternative subject or sample used in an experiment for comparative purposes. A control can be either "positive" or "negative."

[0032] The “effective dose” of a drug refers to the amount of drug sufficient to provide the desired effect. The amount of drug that is “effective” will vary from subject to subject, depending on many factors such as the subject’s age and general condition, and the specific drug. Therefore, it is not always possible to specify a quantifiable “effective dose.” However, the appropriate “effective dose” for any subject may be determined by those skilled in the art using routine experiments. Also, as used herein, unless otherwise specified, the “effective dose” of a drug may also refer to an amount that covers both the therapeutically effective dose and the prophylactically effective dose. The “effective dose” of a drug required to achieve a therapeutic effect may vary depending on factors such as the subject’s age, sex, and weight. The drug regimen may be adjusted to provide the optimal therapeutic response. For example, the dose may be administered in several divided doses per day, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation.

[0033] A “pharmaceutically acceptable” ingredient can refer to an ingredient that is not biological or otherwise undesirable, i.e., an ingredient that can be incorporated into a pharmaceutical formulation provided herein and administered to an subject described herein without causing a significantly undesirable biological effect or interacting in an adverse manner with any other ingredient in the formulation in which it is contained. When used in reference to administration to humans, the term generally means that the ingredient meets the requirements for toxicity and manufacturing testing or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0034] A “pharmaceutically acceptable carrier” (sometimes referred to as “carrier”) means a carrier or excipient that is generally safe and non-toxic and useful in the preparation of pharmaceutical or therapeutic compositions, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” may include, but are not limited to, phosphate-buffered saline solutions, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term “carrier” includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations and further described herein.

[0035] "Pharmacological activity" (or simply "activity") can refer to a derivative or analog of a "pharmacologically active" compound that has the same type of pharmacological activity as the parent compound, and to approximately the same degree (e.g., salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.).

[0036] "Therapeutic agent" refers to any composition having beneficial biological effects. Beneficial biological effects include both therapeutic effects, e.g., treatment of disorders or other undesirable physiological conditions, and preventive effects, e.g., prevention of disorders or other undesirable physiological conditions (e.g., non-immunogenic cancers). The term also encompasses, but is not limited to, pharmaceutically acceptable and pharmacologically active derivatives of beneficial agents specifically referred to herein, including salts, esters, amides, precursors, active metabolites, isomers, fragments, analogs, etc. When the term "therapeutic agent" is used, and subsequently when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharmaceutically acceptable and pharmacologically active salts, esters, amides, precursors, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0037] The “therapeutic effective amount” or “therapeutic effective dose” of a composition (e.g., a composition containing a drug) refers to the amount effective in achieving a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is the control of type 1 diabetes. In some embodiments, the desired therapeutic outcome is the control of obesity. A given therapeutic effective amount of a therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of a therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), that is effective in promoting a desired therapeutic effect, such as pain relief. The exact desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., the potency of the therapeutic agent, the concentration of the drug in the formulation, etc.), as well as various other factors understood by those skilled in the art. In some cases, the desired biological or medical response may be achieved after administration of multiple doses of the composition to a subject over a period of several days, weeks, or years.

[0038] A "primer" is a subset of probes that can support certain types of enzymatic manipulation and can hybridize with a target nucleic acid so that the enzymatic manipulation can occur. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with the enzymatic manipulation.

[0039] A "probe" is typically a molecule capable of interacting with a target nucleic acid in a sequence-specific manner, for example, through hybridization. Nucleic acid hybridization is well understood in the art and is discussed herein. Typically, probes can be prepared from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

[0040] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference in their entirety to more fully describe the cutting edge of the technology relating to this application. The disclosed references are also incorporated herein by reference individually and specifically to the content contained within the references considered in the sentences in which they are cited.

[0041] B. Composition The components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein, are disclosed. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but it is understood that each is specifically intended and described herein. For example, when a particular oropharyngeal detection assay is disclosed and considered, and numerous modifications that can be made into several molecules including the oropharyngeal detection assay are considered, all possible combinations and permutations of the oropharyngeal detection assay and possible modifications are specifically intended unless otherwise indicated. Thus, when class molecules A, B, and C, and classes D, E, and F are disclosed, and A-D, which is an example of a combination molecule, are disclosed, even if each is not individually listed, it is intended that each individually and collectively means a combination, and it is considered that A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are disclosed. Similarly, any subset or combination of these is also disclosed. Therefore, for example, subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in a method for producing and using the disclosed compositions. Thus, where various additional steps can be implemented, it is understood that each of these additional steps can be implemented in any particular embodiment or combination of embodiments of the disclosed method.

[0042] As human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) increases among middle-aged men in developed countries, the need for early diagnosis is also growing. While the survival rate for HPV-associated OPC is good, metastasis remains a possibility, with 13–25% of patients experiencing recurrence within two years, and often failing to achieve a cure regardless of HPV status. Primary prevention through screening is not possible for OPC due to the lack of observable precancerous lesions. Therefore, detection of early OPC (small tumors with only a single ipsilateral positive lymph node, T1–T2, N0–N1, <3cm) is key to reducing the severity and adverse outcomes of treatment after diagnosis.

[0043] Host tumor suppressor gene, EPB41L3, and HPV16 genome methylation, as assessed from pre-treatment oral rinsing, were significantly higher between cases in male OPC cases (n=228) compared to control and predictive cases with an AUC of 0.82 (p<0.0001). However, the prediction observed in early OPC cases alone (T1-T2, N0-N1, small tumor with only a single ipsilateral positive lymph node <3cm) was low (AUC=0.78), indicating a need for additional biomarkers to improve screening performance.

[0044] Genome-wide methylation at 850,000 CpG sites can now be evaluated using standardized arrays. Therefore, to expand site-specific methylation and explore additional biomarkers that can distinguish early cases from later cases and controls, we performed genome-wide methylation across the same test population to identify additional markers for early HPV-associated OPCs and develop methylation assays for more accurate detection of oropharyngeal cancer.

[0045] In one embodiment, an assay for detecting oropharyngeal cancer in a subject, comprising one or more genes in a tissue sample, the assay using probes that detect methylation at the CpG site of the gene, including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and EPB41L3 (e.g., CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, c An assay is disclosed in which changes in the amount of methylation at three or more CpG sites (e.g., increased methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or decreased methylation at HMGB2 or EPB41L3) compared to a normal control, including g14500050, cg14705778, cg04025917, cg06459104, and cg00619915, etc., are indicated by the presence of oropharyngeal cancer. For example, a methylation change may be an increase in methylation detected with one or more probes cg25191818, cg19367172, cg12176286, cg19487745, cg11125104, cg27131607, cg14500050, cg04025917, and / or cg0061991, and / or a decrease in methylation detected with one or more probes cg20791412, cg14705778, and / or cg06459104. In one embodiment, the assay may be a probe for the detection of EPB41L3 CpG site 438 and / or HPV SPF for the detection of human papillomavirus type 16 infection or human papillomavirus type 18 infection. 10 PCR-DEIA-LiPA 25 Includes line probes.

[0046] C. Methods of treating cancer It is understood and intended herein that the disclosed methods can be used for the detection of oropharyngeal cancer. Accordingly, a method for diagnosing and / or detecting the presence of oropharyngeal cancer in a subject is disclosed herein, comprising: obtaining a tissue sample from the subject; and applying the tissue sample to an assay disclosed herein. For example, a method for diagnosing and / or detecting the presence of oropharyngeal cancer in a subject, comprising: i) obtaining a tissue sample from the subject (including, but not limited to, performing oral rinsing, gargling, pharyngeal swab, scrape, or biopsy on the subject, or collecting saliva from the subject); and ii) one or more including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3 A method is disclosed herein that includes measuring methylation at CpG sites of a gene, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control (e.g., increased methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or decreased methylation at HMGB2 or EPB41L3) indicates the presence of oropharyngeal cancer. In one embodiment, the method may further include the detection of methylation at EPB41L3 CpG site 438 and / or HPV16 and / or HPV18 infection.

[0047] Also disclosed herein is a method for diagnosing and / or detecting the presence of oropharyngeal cancer, wherein the detection of CpG sites is achieved using the CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915. For example, the methylation change can be an increase in methylation detected at one or more of the probes cg25191818, cg19367172, cg12176286, cg19487745, cg11125104, cg27131607, cg14500050, cg04025917, and / or cg0061991, and / or a decrease in methylation detected at one or more of the probes cg20791412, cg14705778, and / or cg06459104.

[0048] In one aspect, HPV16 and / or HPV type 16 infection is detected by HPV SPF 10 PCR-DEIA-LiPA 25 Also disclosed herein is a method for diagnosing and / or detecting the presence of oropharyngeal cancer according to any of the preceding aspects, wherein the detection is achieved using line probes.

[0049] The disclosed method can be used for treating any disease in which uncontrolled cell growth occurs, such as cancer, including oropharyngeal cancer, esophageal cancer, and head and neck cancer, including squamous cell carcinoma tumors of the mouth, throat, and larynx.

[0050] In one embodiment, a method is disclosed herein for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastasis in a subject, comprising: i) obtaining a tissue sample from the subject; ii) applying the tissue sample to an assay disclosed herein and / or performing a method for diagnosing and / or detecting the presence of oropharyngeal cancer disclosed herein; and iii) treating the oropharyngeal cancer if detected. For example, a method for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastasis in a subject, comprising: i) obtaining a tissue sample from the subject (including, but not limited to, performing oral washing, gargling, pharyngeal swab, scrape, or biopsy on the subject, or collecting saliva from the subject); and ii) CpG of one or more genes including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3 A method is disclosed herein that includes measuring methylation at a site, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control (e.g., an increase in methylation at one or more CpG sites among PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, and / or ARMC6, and / or a decrease at HMGB2 or EPB41L3) indicates the presence of oropharyngeal cancer, and iii) treating the oropharyngeal cancer if detected. In one embodiment, the method may further include the detection of methylation at EPB41L3 CpG site 438 and / or HPV16 and / or HPV18 infection.

[0051] Also disclosed herein are methods for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastases, wherein detection of CpG sites is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915. For example, a methylation change may be an increase in methylation detected with one or more probes cg25191818, cg19367172, cg12176286, cg19487745, cg11125104, cg27131607, cg14500050, cg04025917, and / or cg0061991, and / or a decrease in methylation detected with one or more probes cg20791412, cg14705778, and / or cg06459104.

[0052] In one embodiment, HPV16 and / or HPV16 type infection is HPV SPF 10 PCR-DEIA-LiPA 25 Methods for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastases, as detected using a line probe, are disclosed herein.

[0053] Also disclosed herein are methods for treating, inhibiting, reducing, mitigating, improving, and / or preventing oropharyngeal cancer or its metastases, wherein the cancer is treated with the administration of anticancer agents and / or radiation. The disclosed treatment regimens include, alone or not, abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ad-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alecinib), Alectinib, Alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochorin (chlorambucil), Ambochorin chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Allezia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Alzera (ofatumumab), Erwinia Chrysanthemi-derived asparaginase, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, babencio (avelumab), BEACOPP, besenam (carmustine), bereodac (bellinostat), bellinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, bexal (tositumomab and iodine I 131 Tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Brincyte (blinatumomab), bortezomib, Boslif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, busulfex (busulfan), cabazitaxel,Cabometicus (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Campus (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil - topical), carboplatin, carboplatin-taxol, carfilzomib, Carmbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, selvidine (daunorubicin hydrochloride), selvarix (recombinant HPV bivalent vaccine), cetuximab, CE V, Chlorambucil, Chlorambucil-Prednisone, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Chloral (Clofarabine), CMF, Cobimetinib, Cometric (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotelic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Syphos (Ifosfamide), Cyramza (Ramucirumab), Sytarabi Cytarabine liposomes, Cytosal-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, dalzarex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomes, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin difutox, denosumab, DepoCyt (cytarabine liposomes), dexamethasone, dexrazoquinone Sun hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efdex (fluorouracil - topical), Elitek (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, eloxatin (oxaliplatin), eltrombopagolamine, Emend (aprepitant), Emplici (elotuzumab), enasidenib mesylate,Enzalutamide, Epirubicin hydrochloride, EPOCH, Elbic (cetuximab), Eribulin mesylate, Elvege (bismodegive), Erlotinib hydrochloride, Erwinase (Erwinia chrysanthemi-derived asparaginase), Etiol (amifostin), Etopophos (etoposide phosphate), Etoposide, Etoposide phosphate, Evaset (doxorubicin hydrochloride liposome), Everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fareston (toremifene), Faridac (panobinostat), Fastlodex (fulvestrant) FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil - Topical), Fluorouracil Injection, Fluorouracil - Topical, Flutamide, Forex (Methotrexate), Forex PFS (Methotrexate), Forfiri, Forfiri-Bevacizumab, Forfiri-Cetuximab, Forfirinox, Forfox, Forotin (Pralatrexate), FU-LV, Fullbest Lant, Gardasil (recombinant HPV tetravalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine), Gaziba (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Girotrif (afatinib dimaleate), Greebeck (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, Goserelin acetate, Halaben (eribulin mesylate), Hemangeol (propanolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonavalent vaccine, recombinant, HPV tetravalent vaccine, recombinant, Hicamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), Ibritumomab tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride),Idarubicin hydrochloride, Ideracilib, Idohifa (enasidenib mesylate), Ifex (ifosfamide), Ifosfamide, Ifosfamidam (ifosfamide), IL-2 (aldesleukin), Imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Imurizik (tarimodine laherpalepbec), Inrita (axitinib), Inotuzumab ozogamicin, Interferon alpha-2b, Recombinant, Interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), Iodine I 131 Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepirone, ixixazomib citrate, ixempra (ixabepirone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Cadsila (adtrastuzumab emtansine), Keoxyfe N (raloxifene hydrochloride), Kepivans (palifermin), Keytruda (pembrolizumab), Kiskari (ribociclib), Kymriah (tisagenlecleucel), Cyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, raltravo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Ikeran (chlorambucil), leuprolide acetate, leustatin (cladribine), Leblanc (aminolevulinic acid), lymphoridine (chlorambucil), Lipodox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lymphal Za (olaparib), Marquivo (vincristine sulfate liposome), Matsuran (procarbazine hydrochloride), mechloretamine hydrochloride, megesterol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, Mesna, Mesnex (mesna), Metazolaston (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide,Mexart (methotrexate), Mexart-AQ (methotrexate), midostaurine, mitomycin C, mitoxantrone hydrochloride, mitoditrex (mitomycin C), MOPP, mozovir (plelixafall), mustargen (mechloretamine hydrochloride), mutamycin (mitomycin C), mirelan (busulfan), mirosal (azacitidine), mirotalg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle preparation), navelbine (vinorelbine tartrate), necitumumab, Nelarabine, Neosal (cyclophosphamide), neratinib maleate, Nerlinx (neratinib maleate), netupitant and palonosetron hydrochloride, Neurasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (niltamide), nilotinib, niltamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, odomzo (sonidedib), OEPA, ofatumumab, OFF, olaparib, olalatumab, omasetaxin mepesuccinate, Oncaspar (pegaspar gauze), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontac (denileukin difutitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, pa Nitumumab, panobinostat, paraplatin (carboplatin), paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, pegylated interferon alfa-2b, PEG-intron (pegylated interferon alfa-2b), pembrolizumab, pemetrexed disodium, perjeta (pertuzumab), pertuzumab, platinol (cisplatin), platinol-AQ (cisplatin), plerixafall, pomalidomide, pomalist (pomalidomide), ponatinib hydrochloride,Portraza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, proleukin (aldesleukin), prolia (denosumab), promacta (eltrombopagolamine), propranolol hydrochloride, Provenge (ciproisel-T), prinetol (mercaptopurine), prixan (mercaptopurine), ra, Dium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) tetravalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Isera (rituximab and hyaluronidase), rituximab, rituximab and hyaluronidase, lorapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), rubraca (rucaparibucan sylate), rucaparibucan sylate, ruxolitinib phosphate, lydapt (midostaurine), sclerosol intrapleural aerosol (talc), siltuximab, ciproisel-T, somatsurin depot (lanreotide acetate), sonidecib, sorafenib tosylate, splicel (dasatinib), STANFORD V, Sterile talc powder (talc), Steritalk (talc), Stivarga (regorafenib), Sunitinib malate, Stent (sunitinib malate), Cilatron (pegylated interferon alpha-2b), Silvant (siltuximab), Synribo (omacetaxin mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, Tarimodine laherparepbec, Tamoxifen citrate, Tarabine PFS (cytarabine ), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, taromid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Trac (fluorouracil - topical), topotecan hydrochloride, toremifene, Tricel (temsirolimus), tositumomab and iodine I 131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib,Trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbit (lorapitant hydrochloride), Vectibix (panitumumab), VeIP, Verban (vinblastine sulfate), Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Benclexta (venetol Clax), Venetoclax, Belzenio (Abemaciclib), Biadur (Leuprolide acetate), Vidaza (Azacitidine), Vinblastine sulfate, Vincazar PFS (Vincristine sulfate), Vincristine sulfate, Vincristine sulfate liposome, Vinorelbine tartrate, VIP, Bismodesib, Vistguard (Uridine triacetate), Boraxase (Glucarpidase), Vorinostat, Botrient (Pazopanib hydrochloride), Vixeos (Daunorubicin hydrochloride and Sitalabi (Liposome), Welcovorin (Leucovorin calcium), Xarukoli (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium-223 dichloride), Xtandi (Enzalutamide), Elboy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Afilbercept), Zalxio (Filgrastim), Zejura (Niraparib tosylate monohydrate), Zerboraf (Vemurafenib), Zevali It is understood and intended herein that these can be used in combination with known anticancer therapies in the art, including, but are not limited to, PD-1 (e.g., nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, MK-3475, etc.), PD-L1 (e.g., atezolizumab, avelumab, durvalumab, etc.).MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C, etc.), PD-L2 (e.g., rHIgM12B7, etc.), CTLA-4 (e.g., ipilimumab (MDX-010), tremelimumab (CP-675, 206), etc.), IDO, B7-H3 (e.g., MGA271, MGD009, omvaltamab, etc.), B7-H4, B7-H3, Ig and T-cell immune receptors with ITIM domains (TIGIT) (e.g., BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO, etc.), CD96, B and T- The present invention may include, or further include, checkpoint inhibitors containing antibodies that block lymphocyte attenuators (BTLAs), V-domain Ig suppressors (VISTAs) of T cell activation (e.g., JNJ-61610588, CA-170, etc.), TIM3 (e.g., TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661, etc.), LAG-3 (e.g., BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep, etc.).

[0054] 1. Immunoassays and fluorescent dyes Steps for various useful immunoassays are described in scientific literature such as Maggio et al., Enzyme-Immunoassay, (1987), and Nakamura et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol.1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference, in whole, particularly with respect to its teachings on immunoassays. Immunosays in the simplest and most direct sense are binding assays, which involve the binding of antibodies to antigens. Many types and forms of immunoassays are known, all suitable for detecting biomarkers of disclosure. Examples of immunoassays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), radioimmunoprecipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel shift assays, flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and post-bleaching fluorescence recovery / localization (FRAP / FLAP).

[0055] Generally, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as a disclosed biomarker) with an antibody against that molecule, or, in some cases, an antibody against the molecule of interest, under conditions effective for immune complex formation, or contacting an antibody against the molecule of interest (such as an antibody against a disclosed biomarker) with a molecule to which that antibody can bind. Contacting a sample with an antibody against the molecule of interest, or a molecule to which an antibody against the molecule of interest can bind, under conditions effective for immune complex formation (primary immune complex) and for a sufficient period of time, generally simply involves contacting the molecule or antibody with the sample and incubating the mixture for a period long enough for the antibody to form an immune complex with any existing molecule to which it can bind (e.g., an antigen), i.e., to bind to that molecule. In many forms of immunoassays, the sample-antibody composition can then be washed, such as tissue sections, ELISA plates, dot blots, or Western blots, to remove any nonspecifically bound antibody species and to detect only antibodies specifically bound within the primary immune complex.

[0056] Immunoassays can include methods for detecting or quantifying the amount of a target molecule (such as a disclosed biomarker or an antibody thereof) in a sample, and these methods generally involve the detection or quantification of any immune complexes formed during the binding process. Generally, the detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches. These methods generally rely on the detection of labels or markers, such as any radioactive, fluorescent, biological, or enzymatic tags, or any other known labels.

[0057] As used herein, labeling can include fluorescent dyes, members of binding pairs such as biotin / streptavidin, metals (e.g., gold), or epitope tags that can specifically interact with molecules that can be detected by producing a colored substrate or fluorescence. Suitable substances for detectably labeling proteins include fluorescent dyes (also known herein as fluorescent dyes and fluorophores), and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the implementation of the present invention because very small amounts of the fluorescent dye can be detected. Furthermore, when multiple antigens are reacted in a single array, each antigen can be labeled with a different fluorescent compound for simultaneous detection. A fluorometer is used to detect the labeled spots on the array, which are the presence of a signal indicating an antigen bound to a particular antibody.

[0058] Fluorophores are luminescent compounds or molecules. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include 1,5-IAEDANS, 1,8-ANS, 4-methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaptofluorescein, 5-carboxynaptofluorescein (5-TAMRA), 5-hydroxytryptamine (5-HAT), 5-ROX (carboxy-X-rhodamine), 6-carboxyrhodamine 6G, 6-CR 6G, 6-JOE, 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxy-4-I-methylcoumarin, 9-amino-6-chloro-2-methoxyacrycidine (ACMA), ABQ, acid fuchsin, acridine orange, acridine red, acridine yellow, acriflavin, acridine foilgen SITSA, aequorin (luminescent protein), and AFPs (autofluorescent proteins). Biotechnologies) (see sgGFP, sgBFP), Alexa Fluor 350 (trademark), Alexa Fluor 430 (trademark), Alexa Fluor 488 (trademark), Alexa Fluor 532 (trademark), Alexa Fluor 546 (trademark), Alexafluol 568 (trademark), Alexafluol 594 (trademark), Alexafluol 633 (trademark), Alexafluol 647 (trademark), Alexafluol 660 (trademark), Alexafluol 680 (trademark), Alizarin Complexone, Alizarin Red, Allophycocyanin (APC), AMC, AMCA-S, Aminomethylcoumarin (AMCA), AMCA-X, Aminoactinomycin D, Aminocoumarin, Aniline Blue, Anthrosil Stealthol, APC-Cy7, APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7GLL, Atabrin, ATTO-TAG(trademark)CBQCA, ATTO-TAG(trademark)FQ, Auramine, Auranofin G, Aurophosphine, BAO9 (bisaminophenyloxadiazole), BCECF (high pH), BCECF (low pH), Berberine sulfate, β-lactamase, BFP blueshift GFP (Y66H), blue fluorescent protein, BFP / GFP FRET, Biman, Bisbenzimide, Bisbenzimide (Hoechst), Bis-BTC, Brancofol FFG, Brancofol SV, BOBO(trademark)-1, BOBO(trademark)-3, Bodypea 492 / 515, Bodypea 493 / 503, Bodypea 500 / 510, Bodypea, 505 / 515, Bodypea 530 / 550, Bodypea 542 / 563, Bodypea 558 / 568, Bodypea 564 / 570, Bodypea 576 / 589, Bodypea 581 / 591, Bodypea 630 / 650-X, Bodypea 650 / 665-X, Bodypea 665 / 676, Bodypea Fl, Bodypea FL ATP, Bodypea Fl-Ceramide, Bodypea R6G SE, Bodypea TMR, Bodypea TMR-X Conjugate, Bodypea TMR-X, SE, Bodypea TR, Bodypea TR ATP, Body Peel TR-X SE, BO-PRO(trademark)-1, BO-PRO(trademark)-3, Brilliant Sulfaflavin FF, BTC, BTC-5N, Calcein, Calcein Blue, Calcium Crimson, Calcium Green, Calcium Green-1 Ca 2+ Dye, Calcium Green-2Ca 2+ Calcium Green-5N Ca 2+ Calcium Green - C18 Ca 2+Calcium Orange, Chalcoflor White, Carboxy-X-Rhodamine (5-ROX), Cascade Blue (trademark), Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, CFP (Cyanide Fluorescent Protein), CFP / YFP FRET, Chlorophyll, Chromomycin A, Chromomycin A, CL-NERF, CMFDA, Coelenterazine, Coelenterazine cp, Coelenterazine f, Coelenterazine fcp, Coelenterazine h, Coelenterazine hcp, Coelenterazine ip, Coelenterazine n, Coelenterazine O, Coumarin Phalloidin, C-Phycocyanin, CPM I Methylcoumarin, CTC, CTC Formazan, Cy2 (trademark), Cy3.1 8, Cy3.5 (trademark), Cy3 (trademark), Cy5.1 8. Cy5.5(trademark), Cy5(trademark), Cy7(trademark), Cyanide GFP, Cyclic AMP Fluorosensor (FiCRhR), Dabsil, Dansil, Dansilamine, Dansilcadaverine, Dansilchloride, DansilDHPE, Dansilfluoride, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3'DCFDA, DCFH (Dichlorodihydrofluorescein diacetate), DDAO, DHR (Dihydrorhodamine 123), Di-4-ANEPPS, Di-8-ANEPPS (non-ratio), DiA(4-Di16-ASP), Dichlorodihydrofluorescein diacetate (DCFH), DiD-lipophilic tracer, DiD(DilC18(5)), DIDS, Dihydrorhodamine 123 (DHR), Dil(DilC18(3)), I-dinitrophenol, DiO(DiOC18(3)), DiR, DiR(DilC18(7)), DM-NERF (high pH), DNP, Dopamine, Ds Red, DTAF, DY-630-NHS, DY-635-NHS, EBFP, ECFP, EGFP, ELF9 7. Eosin, Erythrosine, Erythrosine ITC, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Euchrysin, EukoLight, Europium (111) chloride, EYFP, Fastblue, FDA, Feulgen (pararoseaniline), FIF (formaldehyde-induced fluorescence), FITC, Furazoorange, Fluo-3, Fluo-4, Fluorescein (FITC), Fluorescein diacetate, Fluoroeme Lardo, Fluorogold (hydroxystilbamidin), FluoroRuby, FluoroX, FM1-43 (trademark), FM4-46, Furared (trademark) (high pH), Furared (trademark) / Fluo-3, Fura-2, Fura-2 / BCECF, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Geneblazer (CCF2), GFP (S65T), GFP Redshift (rsGFP), GFP Field Wild-type non-UV excited (wtGFP), GFP wild-type, UV excited (wtGFP), GFPuv, gloxalic acid, granula blue, hematoporphyrin, Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, hydroxycoumarin, hydroxystilvamidine (Fluorogold), hydroxytryptamine, Indo-1, high calcium, Indo-1 low calcium, indodicarbocyanin (DiD), indocarbocyanin (DiR), Intrawhite Cf, JC-1, JOJO-1, JO-PRO-1, LaserPro, Laurodane, LDS751 (DNA), LDS751 (RNA), Leucofor PAF, Leucofor SF, Leucofor WS, Lisamin Rhodamine, Lisamin Rhodamine B, Calcein / Ethidium Homodimer, LOLO-1, LO-PRO-1, Lucifer Yellow, Lithotracker Blue, Lithotracker Blue-White, Lithotracker Green, Lithotracker Red, Lithotracker Yellow, Lithosensor Blue, Lithosensor Green, Lithosensor Yellow / Blue, Mag Green, Magdalena Red (Phloxine B), Magfla Red, Mag-Fla-2, Mag-Fla-5, Mag-Indo-1, Magnesium Green, Magnesium Orange, Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, merocyanine, methoxycoumarin, Mitotracker Green FM, Mitotracker Orange, Mitotracker Red, mitramycin, monobromoviman, monobromoviman (mBBr-GSH), monobromoviman, MPS (methylgreen pyronin stilbene), NBD, NBDamine, Nile Red, nitrobenzoxedidole, norepinephrine, Nuclear Fast Red, iNuclear Yellow, Nylosa Brilliant Iabine E8G, Oregon Green (trademark), Oregon Green (trademark) 488, Oregon Green (trademark) 500, Oregon Green (trademark) 514, Pacific Blue, pararosalinine (Feilgen), PBFI, PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5.5, PE-Texas Red (Red 613), phloxine B (Magdala Red), Holwhite AR, Holwhite BKL, Holwhite Rev, Holwhite RPA, Phosphine 3R, Photoresist, Phycoerythrin B [PE], Phycoerythrin R [PE], PKH26 (Sigma), PKH67, PMIA, Pontochrome Blue Black, POPO-1, POPO-3, PO-PRO-1, PO-IPRO-3, Primurin, Procyon Yellow, Propidium Iodide (PI), PyMPO, Pyrene, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, QSY7, Quinacrine Mustard, Resorfin, RH414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine Green, Rhodamine Faricidine, Rhodamine Phalloidin, Rhodamine Red, Rhodamine WT, Rose Bengal, R-Phycocyanin, R-Phycoerythrin (PE), rsGFP, S65A, S65C, S65L, S65T, Sapphire GFP, SBFI, Serotonin, Sebron Brilliant Red 2B, Sebron Brilliant Red 4G, Sebron I Brilliant Red B, Sebron Orange, Sebron Yellow L, sgBFP (trademark) (Super Glow GFP), sgGFP (trademark) (Super Glow GFP), SITS (Primulin, Stilbene Isothiosulfonic Acid), SNAFL Calcein, SNAFL-1, SNAFL-2, SNARF Calcein, SNARF1, Sodium Green, Spectrum Aqua, Spectrum Green, Spectrum Orange, Spectrum Red, SPQ (6-Methoxy-N-(3-Sulfopropyl)Quinolinium), Stilbene, Sulforodamine B and C, Sulforodamine Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO85, SYTOX Blue, SYTOX Green, SYTOX Orange, Tetracycline, Tetramethylrhodamine (TRITC), Texas Red (trademark), Texas Red-X (trademark) Conjugate, Thiadicarbocyanin (DiSC3), Thiazine Red R, Thiazole Orange, Thioflavin 5, Thioflavin S, Thioflavin TON, Thiolite, Thiazole Orange, Chinopol CBS (Calcoflor White), TIER, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO Examples include, but are not limited to, -1, TOTO-3, Tricolor (PE-Cy5), TRITC tetramethylrhodamine isothiocyanate, True Blue, True Red, Ultra Light, Uranine B, Ubitex SFC, wt GFP, WW781, X-rhodamine, XRITC, Xylene Orange, Y66F, Y66H, Y66W, Yellow GFP, YFP, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, Cyber ​​Green, Thiazole Orange (intercalate dye), semiconductor nanoparticles such as quantum dots, or caged fluorophores (which can be activated by light or other electromagnetic energy sources), or combinations thereof.

[0059] Modification units, such as radionuclides, can be incorporated into or directly bonded to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, and fluorine-18. In another embodiment, the radionuclide can be bonded to a linking group or to a chelating group, which then bonds to the compound directly or via a linker. Examples of radionuclides useful in this embodiment include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Such radiolabeling techniques are routinely used in the radiopharmaceutical industry.

[0060] Radiolabeled compounds are useful as imaging agents for diagnosing neurological diseases (e.g., neurodegenerative diseases) or mental states, or for tracking the progression or treatment of such diseases or conditions in mammals (e.g., humans). The radiolabeled compounds described herein can be readily used in combination with imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0061] Labeling can be either direct or indirect. In direct labeling, the detection antibody (an antibody for the molecule of interest) or the detection molecule (a molecule that can be bound by an antibody against the molecule of interest) contains the label. Detection of the label indicates the presence of the detection antibody or detection molecule, which in turn indicates the presence of the molecule of interest or the presence of an antibody against the molecule of interest, respectively. In indirect labeling, an additional molecule or part is brought into contact with the immune complex or generated at a site on the immune complex. For example, a signal-generating molecule or part, such as an enzyme, can bind to or associate with the detection antibody or detection molecule. The signal-generating molecule can then generate a detectable signal at a site on the immune complex. For example, an enzyme, when supplied with a suitable substrate, can generate a visible or detectable product at a site on the immune complex. ELISA uses this type of indirect labeling.

[0062] Another example of indirect labeling is the contact of an immune complex with an additional molecule (which can be called a conjugate), such as a second antibody against a primary antibody, that can bind to either the molecule of interest or an antibody against the molecule of interest (primary antibody). The additional molecule may have a label or signal-generating molecule or part. The additional molecule may be an antibody, which can therefore be called a secondary antibody. The binding of the secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complex can be contacted with the labeled secondary antibody under conditions effective to allow the formation of the secondary immune complex and for a sufficient period of time. The secondary immune complex can then generally be washed to remove any nonspecifically bound labeled secondary antibody, after which any remaining label in the secondary immune complex can be detected. The additional molecule may also be one of a pair of molecules or parts that can bind to each other, such as a biotin / avadin pair, or may include one such pair. In this form, the detection antibody or detection molecule should contain the other member of the pair.

[0063] Another form of indirect labeling involves the detection of primary immune complexes using a two-step approach. For example, a secondary immune complex can be formed using a molecule such as an antibody (which may be referred to as the first binder) that has a binding affinity to the molecule of interest or the corresponding antibody, as described above. After washing, the secondary immune complex can be contacted with another molecule (which may be referred to as the second binder) that has a binding affinity to the first binder, under conditions effective for further immune complex formation (and thus for the formation of a tertiary immune complex), and for a sufficient period of time. The second binder can be linked to a detectable label or signal-generating molecule or portion to enable detection of the tertiary immune complex thus formed. This system can provide signal amplification.

[0064] Immunosays involving the detection of substances such as proteins or antibodies against specific proteins include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic methods that determine the presence or absence of a specific protein or antibody against a specific protein in a sample. Protein separation methods are further useful for evaluating the physical properties of proteins, such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein or antibody against a specific protein in a sample. Finally, in vivo detection is useful for evaluating the spatial expression pattern of a substance, i.e., where a substance can be found within a subject, tissue, or cell.

[0065] If the concentration is sufficient, the molecular complex ([Ab-Ag]n) produced by antibody-antigen interaction is visible to the naked eye, but due to their ability to scatter light beams, even smaller amounts can be detected and measured. The formation of the complex indicates the presence of both reactants, and in immunoprecipitation assays, a certain concentration of reagent antibody is used to measure the specific antigen ([Ab-Ag]n) and the reagent antigen is used to detect the specific antibody ([Ab-Ag]n). If the reagent species has been previously coated on cells (in the case of hemagglutination assays) or very small particles (in the case of latex agglutination assays), the "clumping" of the coated particles is visible at much lower concentrations. Various assays based on these basic principles are commonly used, and these include Ouchterlony immunodiffusion assays, rocket immunoelectrophoresis, and immunoturbidimetric and turbidimetric assays. The main limitations of such assays are that their sensitivity is limited (low detection limits) compared to assays using labeling, and in some cases, very high concentrations of the analyte can actually inhibit the formation of the complex, requiring precautions that complicate the procedure further. Some of these Group 1 assays date back to the discovery of antibodies, and none of them have actual "labels" (e.g., Ag-enz). Other types of label-free immunoassays rely on immunosensors, and a variety of instruments are now commercially available that can directly detect antibody-antigen interactions. Most rely on generating evanescent waves on a sensor surface with an immobilized ligand, which allows for continuous monitoring of ligand binding. Immunosensors enable easy investigation of dynamic interactions, and with the emergence of low-cost specialized instruments, they may be widely applied to immunoanalysis in the future.

[0066] The use of immunoassays to detect specific proteins can involve the separation of proteins by electrophoresis. Electrophoresis is the movement of charged molecules in solution in response to an electric field. Their movement speed depends on the strength of the electric field, the net charge, size, and shape of the molecules, as well as the ionic strength, viscosity, and temperature of the medium through which the molecules move. As an analytical tool, electrophoresis is simple, rapid, and highly sensitive. It is used analytically to study the characteristics of single charged species and as a separation technique.

[0067] Generally, samples are electrophoresed in a support matrix such as paper, cellulose acetate, starch gel, agarose, or polyacrylamide gel. The matrix suppresses mixing by convection caused by heating and provides an electrophoretic record: at the end of the electrophoresis, the matrix can be stained and used for scanning, autoradiography, or storage. In addition, agarose and polyacrylamide, the most commonly used support matrices, provide a means of separating molecules by size because they are porous gels. Porous gels can function as sieves by delaying or, in some cases completely hindering, the movement of large macromolecules while allowing smaller molecules to move freely. Diluted agarose gels are generally stiffer and easier to handle than polyacrylamide of the same concentration, so agarose is used to separate larger macromolecules such as nucleic acids, large proteins, and protein complexes. Polyacrylamide, which is easier to handle and to prepare at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for delay.

[0068] Proteins are amphoteric compounds, and therefore their net charge is determined by the pH of the medium in which they are suspended. In solutions where the pH is higher than its isoelectric point, proteins have a net negative charge and move toward the anode in an electric field. Below its isoelectric point, proteins are positively charged and move toward the cathode. The net charge carried by a protein is also independent of its size; that is, the charge carried per unit mass of the molecule (or, assuming proteins and nucleic acids are linear macromolecules, the length) varies from protein to protein. Therefore, at a given pH and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both the size and charge of the molecule.

[0069] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "wrapping" the polypeptide backbone, and SDS binds to proteins quite specifically in a mass ratio of 1.4:1. In doing so, SDS imparts a negative charge to the polypeptide in proportion to its length. Furthermore, it is usually necessary to reduce (denature) disulfide crosslinks in the proteins before forming the random coil configuration required for size-based separation, and this is done using 2-mercaptoethanol or dithiothreitol (DTT). Therefore, in denaturation during SDS-PAGE separation, migration is determined by molecular weight, not by the internal charge of the polypeptide.

[0070] Molecular weight determination is performed by SDS-PAGE of proteins of known molecular weight along with the protein being characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide or native nucleic acid and its Rf. Rf is calculated as the ratio of the distance the molecule travels to the distance the marker dye front travels. A simple method for determining relative molecular weight (Mr) by electrophoresis is to plot a standard curve of migration distance versus log10MW for known samples and read out the logMr of the sample after measuring the migration distance on the same gel.

[0071] In two-dimensional electrophoresis, proteins are fractionated based on two physical properties: first, in a first step, and then in a second step, in a second step, based on a different physical property. For example, isoelectric focusing can be used for the first dimension and can be easily performed in a tube gel, while SDS electrophoresis in a slab gel can be used for the second dimension. An example of the procedure is found in O'Farrell, PH, High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), which is incorporated herein by reference for its teachings on two-dimensional electrophoresis. Other examples include, but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977) and Ornstein, L., Disc electrophoresis, L. Ann. NYA Acad. Sci. 121:321349 (1964), each of which contains instructions on electrophoresis that are incorporated herein by reference in their entirety. Laemmli, UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which discloses a discontinuous system for separating proteins denatured by SDS. The leading ion of the Laemmli buffer system is chloride, and the tailing ion is glycine. Therefore, the separation gel and concentration gel consist of Tris-HCl buffer (with different concentrations and pH), and the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.

[0072] An example of an immunoassay using electrophoresis as intended in the present method is Western blot analysis. Western blotting, or immunoblotting, allows for the determination of the molecular mass of a protein and the measurement of the relative amount of protein present in different samples. Detection methods include chemiluminescence and colorimetric detection. Standard methods for Western blot analysis can be found, for example, in DMBollag et al., Protein Methods (2nd edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent No. 4,452,901, each of which, with respect to teachings on Western blotting, are incorporated herein by reference in their entirety. Generally, proteins are separated by gel electrophoresis, usually by SDS-PAGE. Proteins are transferred to special blotting paper, such as a nitrocellulose sheet, although other types of paper or membranes can also be used. Proteins retain the same separation pattern as they were on the gel. The blot is incubated with a common protein (such as milk protein) to bind to any remaining sticky locations on the nitrocellulose. Then, the antibody is added to a solution that can bind to that specific protein.

[0073] The attachment of specific antibodies to specific immobilized antigens can usually be readily visualized by indirect enzyme immunoassay techniques using chromogenic substrates (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other probe possibilities include fluorescent or radioisotope labeling (e.g., fluorescein). 125 I) The use of a probe for detecting antibody binding can be a probe to a conjugated anti-immunoglobulin, a conjugated Staphylococcus protein A (which binds to IgG), or a biotinylated primary antibody (e.g., a conjugated avidin / streptavidin).

[0074] The capability of this technique lies in the simultaneous detection of specific proteins based on their antigenicity and molecular mass. First, proteins are separated by mass using SDS-PAGE, and then specifically detected in the immunoassay step. In this way, protein standards (ladders) can be run simultaneously to approximate the molecular mass of the target protein in a heterogeneous sample.

[0075] Gel shift assays or electrophoretic mobility shift assays (EMSA) can be used to detect interactions between DNA-binding proteins and their homologous DNA recognition sequences in both qualitative and quantitative ways. Exemplary techniques are described in Ornstein L., Disc electrophoresis-I: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudaira, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which, with respect to teachings regarding gel shift assays, is incorporated herein by reference in its entirety.

[0076] In a typical gel shift assay, purified protein or crude cell extract is labeled (for example, 32After incubation with a radiolabeled (P) DNA or RNA probe, the complex can be separated from the free probe via a non-denaturing polyacrylamide gel. The complex moves more slowly through the gel than the unbound probe. Depending on the activity of the binding protein, the labeled probe can be either double-stranded or single-stranded. For the detection of DNA-binding proteins such as transcription factors, purified or partially purified proteins or nuclear cell extracts can be used. For the detection of RNA-binding proteins, purified or partially purified proteins or nuclear or cytoplasmic cell extracts can be used. The specificity of the DNA or RNA-binding protein to the putative binding site is established by competitive experiments using DNA or RNA fragments, oligonucleotides containing the binding site to the protein of interest, or other unrelated sequences. Differences in the properties and strength of the complexes formed in the presence of specific and non-specific competitors allow for the identification of specific interactions.<http: / / www.promega.com / faq / gelshfaq.html> Please refer to the Promega, Gel Shift Assay FAQ, available (last accessed March 25, 2005), which contains teachings on the gel shift method, and its entirety is incorporated herein by reference.

[0077] Gel shift methods may include, for example, detecting proteins in a gel such as a polyacrylamide electrophoresis gel using a colloidal form of COOMASSIE (Imperial Chemicals Industries, Ltd) blue dye. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985) and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is incorporated herein by reference in its entirety with respect to teachings relating to gel shift methods. In addition to the conventional protein assay methods referenced above, combination washing and protein staining compositions are described in U.S. Patent No. 5,424,000, which is incorporated herein by reference in its entirety with respect to teachings relating to gel shift methods. Solutions may include phosphoric acid, sulfuric acid, and nitric acid, as well as acid violet dyes.

[0078] Radioimmunoprecipitation assays (RIPA) are highly sensitive assays that use radiolabeled antigens to detect specific antibodies in serum. The antigen is reacted with serum and then precipitated using a special reagent, such as protein A Sepharose beads. The bound radiolabeled immunoprecipitate is then analyzed by gel electrophoresis, as is standard practice. RIPA is often used as a confirmatory test to diagnose the presence of HIV antibodies. RIPA is also referred to in the art as Farr assay, precipitate assay, radioimmunoprecipitation assay, radioimmunoprecipitation analysis, and radioimmunoprecipitation analysis.

[0079] The immunoassays described above, which utilize electrophoresis to separate and detect specific proteins of interest, allow for the assessment of protein size, but they are not very sensitive to the assessment of protein concentration. However, immunoassays that involve conjugating proteins or protein-specific antibodies to a solid support (e.g., tubes, wells, beads, or cells) to capture the antibody or the protein of interest from the sample, respectively, are also intended to be used in combination with methods for detecting proteins or protein-specific antibodies on the support. Examples of such immunoassays include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplexed bead assays, and magnetic capture.

[0080] Radioimmunoassay (RIA) is a classic quantitative assay for detecting antigen-antibody reactions using radiolabeled substances (radioligands), either directly or indirectly, to measure the binding of unlabeled substances to specific antibodies or other receptor systems. Radioimmunoassays are used, for example, to test hormone levels in the blood without the need for bioassays. Non-immunogenic substances (e.g., haptens) can also be measured when coupled to larger carrier proteins (e.g., bovine gamma globulin or human serum albumin) that can induce antibody formation. RIAs use radioantigens (radioisotopes because iodine atoms are easily introduced into tyrosine residues in proteins). 125 I or 131This involves mixing a radioactive antigen (often using type I) with an antibody against that antigen. The antibody is generally bound to a solid support such as a tube or beads. Next, a known amount of unlabeled antigen or "cold" antigen is added, and the amount of labeling antigen substituted is measured. First, the radioactive antigen is bound to the antibody. When the cold antigen is added, these two compete for the antibody binding site, and a higher concentration of cold antigen results in more binding to the antibody and substituted for the radioactive variant. The bound antigen is separated from the unbound antigen in the solution, and the binding curve is plotted using the radioactivity of each. This technique is extremely sensitive and specific.

[0081] Enzyme-linked immunosorbent assays (ELISAs), or collectively referred to as EIAs (enzyme-linked immunoassays), are immunoassays capable of detecting antibodies specific to proteins. In such assays, the detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. Upon exposure to a substrate, this enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorescence, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0082] Modifications of the ELISA technique are known to those skilled in the art. In one modification, an antibody capable of binding to a protein can be immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected to contain a marker antigen can then be added to the well. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a second antibody specific to the target protein, linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second antibody, followed by a third antibody having binding affinity to the second antibody, which is linked to a detectable label.

[0083] Another variation is competitive ELISA. In competitive ELISA, the test sample competes for binding to a known amount of labeled antigen or antibody. The amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation using coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species that can bind to the wells, thus reducing the final signal.

[0084] Regardless of the format used, ELISA has certain common features, including coating, incubation or binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. Antigens or antibodies can be bound to solid supports such as plates, beads, test strips, membranes, or column matrices, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a plate with either an antigen or antibody, the wells of the plate are generally incubated with the antigen or antibody solution overnight or for a specific period of time. The wells of the plate can then be washed to remove any incompletely adsorbed material. Subsequently, any remaining available surface of the wells can be "coated" with nonspecific proteins that are antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, and powdered milk solutions. Coating allows for the blocking of nonspecific adsorption sites on the immobilized surface, thereby reducing background caused by the nonspecific binding of antiserum to the surface.

[0085] In ELISA, secondary or tertiary detection methods can also be used instead of the direct procedure. Therefore, after binding the protein or antibody to the wells, coating with a non-reactive material to reduce background noise, and washing to remove unbound material, the immobilized surface is brought into contact with a control clinical or biological sample to be tested under conditions effective in enabling immune complex (antigen / antibody) formation. Detection of immune complexes then requires a labeled second binder, or a second binder combined with a labeled third binder.

[0086] Enzyme-conjugated spot immunoassay (ELISPOT) is an immunoassay that can detect antibodies specific to a protein or antigen. In such an assay, the detectable label bound to either the antibody-conjugating or antigen-conjugating reagent is an enzyme. Upon exposure to a substrate, this enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorescence, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In this assay, a nitrocellulose microtiter plate is coated with the antigen. After exposing the test sample to the antigen, it is reacted in the same manner as in an ELISA assay. Detection differs from conventional ELISA in that it is determined by enumerating spots on a nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. By counting the spots, the number of antigen-specific cells in the sample can be determined.

[0087] "Conditions effective for forming immune complexes (antigen / antibody)" means that the conditions include diluting the antigen and antibody with solutions such as BSA, bovine gamma globulin (BGG), and phosphate-buffered saline (PBS) / Tween to reduce nonspecific binding and promote a reasonable signal-to-noise ratio.

[0088] Preferred conditions also mean that the incubation is at a temperature and duration sufficient to allow for effective bonding. The incubation step can typically be about 1 minute to 12 hours at a temperature of about 20°C to 30°C, or it can be incubated overnight at about 0°C to 10°C.

[0089] After all incubation steps in the ELISA, the contacted surfaces can be washed to remove any uncomplexed material. Washing procedures may include washing with a solution such as PBS / Tween or borate buffer. Specific immunocomplexes can form between the test sample and the original binding substance, and after subsequent washing, the formation of these immunocomplexes, even in trace amounts, can be determined.

[0090] To provide a means of detection, the second or third antibody may have an associated label to enable detection, as described above. This can be an enzyme that can produce a color when incubated with a suitable chromogenic substrate. For this reason, for example, the first or second immunocomplex can be brought into contact with the labeled antibody and incubated for a period of time and under conditions favorable for further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0091] After incubation with the labeled antibody, followed by washing to remove unbound substances, the amount of labeling can be quantified by incubation with a chromogenic substrate such as urea and bromocresol purple, or 2,2'-azidose di(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2) in the case of peroxidase as an enzyme label. Then, quantification can be achieved by measuring the degree of color production, for example, using a visible spectrum spectrophotometer.

[0092] Protein arrays are solid-phase ligand-binding assay systems that use proteins immobilized on surfaces such as glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. Assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). These advantages include being rapid and automatable, highly sensitive, economical in terms of reagents, and providing rich data in a single experiment. Bioinformatics support is crucial, and data processing requires advanced software and data comparative analysis. However, the software can be adapted from that used for DNA arrays, as can many hardware and detection systems.

[0093] One of the main forms is the capture array, in which a ligand-binding reagent, usually an antibody but also an alternative protein scaffold, peptide, or nucleic acid aptamer, is used to detect a target molecule in a mixture such as plasma or tissue extract. In diagnostics, capture arrays can be used to run multiple immunoassays in parallel, for example, testing for several analytes in individual serum samples and simultaneously testing many serum samples. In proteomics, capture arrays are used to quantify and compare protein levels in different samples in healthy and diseased states, i.e., for protein expression profiling. Proteins other than specific ligand-binding reagents are used in array form for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened for many proteins, which can also be done in a multiple array form for multiple protein targets.

[0094] For array construction, protein sources include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production using cell-free translation systems, and peptide synthesis methods. Many of these methods can be automated for high-throughput production. For capture arrays and protein function analysis, it is crucial that proteins are correctly folded and functional, although this is not always the case, for example, when recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful for screening antibody cross-reactivity, identifying autoantibodies, and selecting ligand-binding proteins.

[0095] Protein arrays are designed as miniaturizations of familiar immunoassay methods such as ELISA and dot blotting, and are often facilitated by robotics and high-throughput detection systems that utilize fluorescence readout and allow multiple assays to be performed in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, as well as magnetic and other microbeads. While microdroplets of proteins delivered to a planar surface are the most familiar form, alternative architectures include CD centrifuge devices based on advances in microfluidics (Gyros, Monmouth Junction, NJ), specialized chip designs such as manipulated microchannels in plates (e.g., The Living Chip®, Biotrove, Woburn, MA), and microscopic 3D posts on silicon surfaces (Zyomyx, Hayward, CA). Particles in suspension can also be used as the basis for arrays, provided they are coded for identification. Systems include color coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots®, Quantum Dot, Hayward, CA), as well as bar coding for beads (UltraPlex®, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and polymetallic microrods (e.g., Nanobarcodes® particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).

[0096] Protein immobilization involves both the coupling reagent and the properties of the surface being coupled. A good protein array support surface is chemically stable before and after the coupling procedure, allows for good spot morphology, exhibits minimal nonspecific binding, does not contribute to background in the detection system, and is compatible with different detection systems. The immobilization method used is reproducible, applicable to proteins with different properties (size, hydrophilicity, hydrophobicity), suitable for high-throughput and automation, and compatible with the preservation of fully functional protein activity. The orientation of the surface-bound protein is recognized as a crucial factor in presenting it to the ligand or substrate in an active state, and for capture arrays, the most efficient binding results are generally obtained by using oriented capture reagents that require site-specific labeling of the protein.

[0097] Both covalent and non-covalent methods are used for protein immobilization, each with its own advantages and disadvantages. Passive adsorption to a surface is methodologically simple, but offers little quantitative or oriented control, which may or may not alter the functional properties of the protein, resulting in variable reproducibility and efficiency. Covalent coupling methods provide stable linkage, are applicable to a variety of proteins, and have good reproducibility, but orientation can vary. Chemical derivatization can alter protein function and requires a stable interaction surface. Biological capture methods utilizing tags on proteins provide stable linkage and bind to the protein specifically and in a reproducible orientation, but require thorough initial immobilization of the biological reagent, demand special handling of the array, and have variable stability.

[0098] Several immobilization chemicals and tags are described for the construction of protein arrays. Substrates for covalent bonding include glass slides coated with amino or aldehyde-containing silane reagents. In the Versalinx® system (Prolinx, Bothell, WA), reversible covalent bonding is achieved by interaction between a protein derivatized with phenyl diboronic acid and salicylic acid immobilized on a support surface. This also allows for low background binding, low autofluorescence, and the retention of function by the immobilized protein. Non-covalent bonding of unmodified proteins occurs within porous structures such as HydroGel® (PerkinElmer, Wellesley, MA) based on three-dimensional polyacrylamide gels, and this substrate has been reported to confer particularly low background on glass microarrays with high protein function capability and retention. Widely used biological coupling methods involve biotin / streptavidin or hexahistidine / Ni interactions with appropriately modified proteins. Biotin may be conjugated to a polylysine skeleton immobilized on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0099] Array fabrication methods include robotic contact etching, inkjet processing, piezoelectric spotting, and photolithography. A number of commercially available arrays (e.g., Packard Biosciences) and manual instruments (V&P Scientific) are available. Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression at insights.

[0100] The limits of spot size and density are nanoarrays where spots are on a nanometer spatial scale, allowing thousands of reactions to occur on a single chip smaller than 1 mm square. BioForce Laboratories has developed a nanoarray of 1521 protein spots in 85 square microns at the optical detection limit, which corresponds to 25 million spots per square centimeter. The readout method is fluorescence and atomic force microscopy (AFM).

[0101] Fluorescent labeling and detection methods are widely used. The same instrumentation used to read DNA microarrays is applicable to protein arrays. For differential indication, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cellular states, where cell lysates are directly conjugated and mixed with different fluorophores (e.g., Cy-3, Cy-5) so that the color functions as a readout of changes in target abundance. Fluorescence readout sensitivity can be amplified 10 to 100 times by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultra-high sensitivity fluorescence detection with the added advantage of eliminating the need for washing procedures. High sensitivity can also be achieved by suspension beads and particles using phycoerythrin as a label (Luminex) or by utilizing the properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts have been developed, particularly in the field of commercial biotechnology. These include applications of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonant light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy (BioForce Laboratories).

[0102] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They bind to specific target ligands in a high-throughput manner using high-affinity capture reagents such as conventional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers, and then detect them.

[0103] Antibody arrays possess the necessary characteristics of specificity and acceptable background, and several are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are prepared either by conventional immunization (polyclonal serum and hybridoma) or as recombinant fragments typically expressed in E. coli after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, single V domains from camelids, or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays.

[0104] The term "scaffold" refers to the ligand-binding domain of a protein that is modified into multiple variants capable of binding to diverse target molecules with antibody-like properties of specificity and affinity. These variants are produced in gene library form and can be selected for individual targets by phage, bacterial, or ribosome display. Examples of such ligand-binding scaffolds or frameworks include "Affibody" (Affibody, Bromma, Sweden) based on Staph. aureus protein A, "Trinectin" (Phylos, Lexington, MA) based on fibronectin, and "Anticalin" (Pieris Proteolab, Freising-Weihenstephan, Germany) based on the lipocalin structure. These can be used in capture arrays similar to antibodies and offer advantages in robustness and ease of production.

[0105] Non-protein capture molecules, particularly single-stranded nucleic acid aptamers that bind to protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from a library of oligonucleotides by the Selex™ procedure, and their interaction with proteins can be enhanced by covalent bonding through the incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligands reduces the cross-reactivity of aptamers due to specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis, as well as DNA stability and robustness, and binding can be detected in photoaptamer arrays using universal fluorescent protein staining.

[0106] Protein analytes bound to antibody arrays can be detected in sandwich assays directly or via secondary antibodies. Direct labeling is used for comparing different samples of different colors. When pairs of antibodies targeting the same protein ligand are available, sandwich immunoassays offer high specificity and sensitivity, and are therefore a method for selecting proteins in low abundance, such as cytokines, which also offer the possibility of detecting protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance, and atomic force microscopy, avoid ligand modification. Optimal sensitivity and specificity, with low background to impart a high signal to noise, are required from any method. Sensitivity needs to be appropriately adjusted as analyte concentrations cover a wide range, and serial dilution of samples or the use of antibodies with different affinities are solutions to this problem. The target protein is often a protein present in low concentrations in body fluids and extracts, such as cytokines or low-expression products in cells, requiring detection below the pg range.

[0107] The alternative to arrays of capture molecules is achieved through "molecular imprinting" technology, where peptides (e.g., derived from the C-terminal region of a protein) are used as templates to generate structurally complementary sequence-specific cavities in a polymerizable matrix. These cavities can then specifically capture (denature) proteins that have the appropriate primary amino acid sequence (ProteinPrint®, Aspira Biosystems, Burlingame, CA).

[0108] Another methodology that can be used for diagnostic and expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), in which a solid-phase chromatography surface binds to proteins with similar charge or hydrophobic characteristics from a mixture such as plasma or tumor extract, and the retained proteins are detected using SELDI-TOF mass spectrometry.

[0109] Large-scale functional chips are constructed by immobilizing numerous purified proteins and are used to assay a wide range of biochemical functions, including protein-protein interactions with other proteins, drug-target interactions, and enzyme substrates. Generally, these require expression libraries, which are cloned into E. coli, yeast, or similar organisms, from which the expression proteins are purified and immobilized, for example, via His tags. Cell-free protein transcription / translation is a viable alternative for the synthesis of proteins that are not well expressed in bacterial or other in vivo systems.

[0110] To detect protein-protein interactions, protein arrays can be an in vitro alternative to cell-based yeast 2-hybrid systems and may be useful in cases where the latter is deficient, such as interactions involving secreted proteins or proteins with disulfide crosslinks. High-throughput analysis of biochemical activity on arrays has been described for yeast protein kinases and various functions of the yeast proteome (protein-protein and protein-lipid interactions), where the majority of the entire yeast open reading frame was expressed and immobilized on microarrays. Large-scale "proteome chips" promise to be extremely useful in identifying functional interactions, drug screening, and other applications (Proteometrix, Branford, CT).

[0111] Using protein arrays as a two-dimensional display of individual elements, phage or ribosome display libraries can be screened to select specific binding partners, including antibodies, synthetic scaffolds, peptides, and aptamers. In this way, a "library-versus-library" screening can be performed. Another application of this approach is the screening of drug candidates in combinatorial chemical libraries against arrays of protein targets identified from genome projects.

[0112] Multiplexed bead assays, such as BD® Cytometric Bead Array, are used to capture and quantify soluble analytes, with a series of spectra represented by separate particles. The analytes are then measured by fluorescence-based emission and flow cytometry detection. Multiplexed bead assays are equivalent to ELISA-based assays but generate data in a "multiplexed" or simultaneous manner. Unknown concentrations are calculated for the cytometry bead array, similar to any sandwich-type assay, i.e., by using known standards and plotting the unknown against a standard curve. Furthermore, multiplexed bead assays enable the quantification of soluble analytes in samples that had previously been unconsidered due to sample volume limitations. In addition to quantitative data, they can generate powerful visual images revealing unique profiles or signatures that provide users with additional information at a glance. [Examples]

[0113] The following examples are provided to those skilled in the art to provide a complete disclosure and description of methods for preparing and evaluating the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be purely illustrative and not to limit the disclosure. While every effort has been made to ensure accuracy with respect to numerical values ​​(e.g., quantities, temperatures, etc.), some errors and variations should be considered. Unless otherwise indicated, parts are by weight, temperatures are in °C or ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure.

[0114] 1. Example 1: Identification of a biomarker panel from genome-wide methylation for detecting early HPV-associated oropharyngeal cancer. a) Method (1) Test group Males aged 18 years or older diagnosed with newly histologically confirmed OPC (C01.9 base of the tongue; C05.1 soft palate, otherwise unspecified [NOS]; C05.2 uvula; C09.0 tonsillar fossa; C09.1 trabecular wall; C09.8 overlapping lesion of the tonsil; C09.9 tonsil, NOS; C10.0 vallecula epiglottis; C10.2 lateral wall of the epiglottis; C10.3 posterior wall of the epiglottis; C10.8 overlapping area of ​​the oropharynx, and C10.9 oropharynx, NOS) were recruited from the Head and Neck Cancer Radiation Oncology and Senior Adult Oncology clinics at Moffitt Cancer Center between May 2014 and March 2020. Patients were pre-screened for eligibility by reviewing their medical records. Signed and informed consent was obtained for noteworthy and eligible cases. Cases of individuals who had received prior treatment or who had not completed the study were excluded. Approval was obtained from Advarra's Institutional Review Board and Moffitt Cancer Center's Scientific Review Committee. Cases were defined as either early-stage (small tumors with only one ipsilateral positive lymph node, T1–T2, N0–N1, <3cm) or late-stage.

[0115] Healthy, cancer-free controls were selected from US participants in a male HPV infection (HIM) trial who recruited men aged 18–70 years with no history of HPV-related cancer, HPV vaccination, or HIV / AIDS. Controls were frequently matched for age under 5 years and smoking history (never, formerly, or currently).

[0116] In both trials, demographic information, as well as sexual behavior and substance use history, was collected during trial visits via computer-assisted risk assessment. Males then provided oral rinsing by gargling with mouthwash for 30 seconds, followed by dispensing into 50 ml conical tubes. For processing, samples were centrifuged at 2,000 g for 15 minutes, and the cell pellet was subjected to three washing procedures by resuspending in 20 ml of cold phosphate buffer (PBS), repeatedly mixed by inversion to ensure complete homogenization, and then centrifuged at 2,000 g for 15 minutes at 4°C. The remaining cell pellet was resuspended in 1.2 ml of PBS and stored at -80°C until analysis. Oral HPV DNA was extracted from the oral rinsing cell pellet using an automated BioRobot MDx (Qiagen). All participants' oral rinsing samples were HPV SPF 10 PCR-DEIA-LiPA 25 The results were obtained using a line probe assay (DDL Diagnostic Laboratory, Rijswik, the Netherlands).

[0117] (2) Methylation test (a) HPV and host gene methylation All oral gargle samples were tested for methylation of three CpG sites (438, 427, and 425) within the host tumor suppressor gene EPB41L3 using a validated pyrosequencing method (PyroMark). Briefly, 200 ng of DNA was used in a bisulfite conversion reaction, in which unmethylated cytosine was converted to uracil using the EZ DNA methylation kit (Zymo Research, Irvine, CA). The converted DNA was purified and amplified using PCR primers with one biotin per pair. The primers were designed with short amplicons (90–140 base pairs each) using PyroMark Assay Design software (V2.0.1.15 Qiagen). PCR was performed using equivalent amounts of DNA from 1500 converted cells using the Pyro-Mark PCR kit (Qiagen). Next, the PCR products were captured in 96-well plates using streptavidin beads (GE Healthcare, Buckinghamshire, UK) and pyrosequencing was performed using PyroGold reagent along with the signals analyzed using a PyroMark TMQ96 ID (Qiagen) instrument. All runs included standard curves as positive and non-template controls for 0, 50, and 100% methylated human DNA.

[0118] (b) Genome-wide methylation The extracted DNA was also processed for genome-wide methylation using a MethylationEPIC BeadChip (Illumina) array, which evaluates the methylation status of >850,000 CpG sites across the genome. 500 ng of extracted and Qubit-quantified DNA was bisulfite-converted using Zymo EZ DNA Methylation Kits (Zymo Research, Irvine, CA), and the converted DNA was used to process and hybridize Illumina MethylationEPIC BeadChips according to the manufacturer's protocol (Illumina, Inc., San Diego, CA). Single-base extension and staining were performed using a Tecan Evo liquid handling system in a Te-Flow (GenePaint) chamber. The arrays were then scanned with an Illumina iScan scanner, and QC was performed using Illumina GenomeStudio software. The minfi R package was used to later read and analyze the obtained IDAT files, and then the raw intensity values ​​were preprocessed using the preprocess Funnorm function. Quality control consisted of plotting and inspecting histograms of beta values ​​at the sample level and performing principal component analysis (PCA). Based on the beta histogram, one sample failed, which was clearly an outlier in the plot of 3 out of 4 principal components. After removing that sample, a dataset consisting of 89 early OPC cases and 108 control cases was obtained. Beta values ​​were calculated as the ratio of methylation to total signal intensity (methylated and unmethylated signals). Beta values ​​showing a detection p-value > 0.01 were marked as NA, and then probes were filtered from further analysis if (1) more than 20% of the samples had missing values, or (2) all samples had a range of less than 0.1.

[0119] (3) Construction of a biomarker panel By analyzing data from methylation studies, a biomarker panel capable of distinguishing early OPC from controls was constructed. The overall process consisted of five additional steps: 1) identifying candidate Illumina probes from all available probes; 2) finding key candidate probes from EPB41L3, known to be associated with HPV-related precancerous cervical cancer and HPV-OPC, using alternative selection criteria; 3) manually adding methylation data from EPB41L3 pyrosequencing CpGs; 4) manually adding HPV16 and HPV18 status; and 5) constructing a LASSO model using the variables identified in the previous step to determine which variables are assigned non-zero coefficients. The steps toward constructing the biomarker panel are further described herein and in Figure 4.

[0120] Steps 1 and 2 involved independently comparing detection at individual CpG sites using 10-fold cross-validation to identify Illumina probes that were methylated differently between early OPCs and controls. First, t-tests were systematically performed on 9 out of 10 sets, with one set being excluded each time. This resulted in approximately 850,000 t-tests across 10 rounds. The p-values ​​from the t-tests were converted to q-values ​​to control for multiple tests. The top 100 probes (lowest q-values) and EPB41L3 probes with q-values ​​less than 0.01 were saved from each round. After completing all 10 rounds of cross-validation, the top 100 probes from all rounds of cross-validation and EPB41L3 probes selected in at least 5 of the 10 rounds were selected as candidates for further consideration.

[0121] HPV16 and HPV18 status were added as binary categorical variables (Step 3). Next, candidate probes from previous pyrosequencing methylation signals containing three CpG sites (438, 427, and 425) were added to the EPB41L3 gene (Step 4). Note that these CpG sites are not present in the Illumina array. Finally, in Step 5, a logistic regression model with LASSO normalization was constructed using the previously selected candidates in the glmnet (version 4.1-1) R package and saved with non-zero coefficients.

[0122] The above five steps were repeated for a total of 50 iterations. Candidate markers discovered at the end of each iteration were saved. The final panel of 14 variables had non-zero coefficients in at least 23 of the 50 iterations. After 23 iterations, there was a sharp drop in the CpG sites having non-zero coefficients (Figure 3). The final model was constructed as a simple logistic regression model consisting of these 14 selected variables. To estimate the accuracy of this final model on unseen data, a bootstrap resampling method was used, in which the logistic regression model was trained on bootstrap samples with the 14 selected variables and validation samples excluded in each resampling set.

[0123] (4) Statistical analysis The Cochrane-Mantel-Henzel (CMH) test was used to compare sociodemographic characteristics, sexual behavior, oral health, and oral rinsing HPV status in all cases (n=228) compared to a control group (n=142). Models representing various steps in probe selection were compared using box plots, and AUC was compared using the Wilcoxon rank-sum test. ROC curves from the training and test sets (from bootstrap resampling) were generated by varying the threshold over the predicted probability of the area under the curve (AUC) generated by the Wilcoxon test. Cutpoints were identified using Joden's J (J statistic) and selected with the goal of maximizing specificity while maintaining sensitivity above 70%. Steps for constructing the panel were completed in R. Other analyses were performed in SAS 9.3.

[0124] b) Result There were 228 OPC cases, of which 92 were identified as early OPC, and 142 age- and smoking-matched controls were included in this study. Cases differed significantly from controls (p<0.05) by race, ethnicity, marital status, education, smoking pack years, kissing and oral sex history, tonsillectomy history, number of tooth extractions, and HPV infection by any type or HPV16. While matching for smoking status, cases were more likely to have a higher total pack years than controls (21% vs. 13% of controls). Among cases, 68% had HPV detected in oral rinsing compared to 14% of controls, and 54% were positive for HPV16 compared to 3.5% of controls (Table 1). [Table 1-1] [Table 1-2] [Table 1-3]

[0125] The biomarker selection process identified 14 markers significantly associated with early OPC (p<0.05), including one EPB41L3 CpG site from the Illumina array and one EPB41L3 CpG site (438) from pyrosequencing, as well as the oral HPV16 / 18 status. (Tables 2 and 3)

[0126] Compared to a single-pass analysis using only the Illumina probe (AUC=0.883), there was a significant improvement in the ability to predict early OPCs (p=0.009) compared to a control using the five-step process described herein (AUC=0.908) (Figure 1). [Table 2] [Table 3-1] [Table 3-2]

[0127] A panel constructed from a five-step process was used to train the final logistic regression model on all data (Table 4). This model was able to predict early OPCs compared to a control with an AUC of 0.978 on the training data (Figure 2a). Resampling validation achieved a mean AUC of 0.935, demonstrating adequate internal validity (Figure 2b). [Table 4]

[0128] OPC is a rare event and requires high specificity to minimize false positives. For a 14-marker panel modeled across all samples, 99.1% specificity was achieved for early OPC prediction, with a sensitivity of 70.1%. Reducing specificity to 98.1% resulted in a significant increase in sensitivity to 78.0%. To account for differences in smoking, it was investigated as a covariate in models with little change in outcome (AUC=0.964). We also tested the panel separately for smokers and non-smokers and found that smokers (AUC=0.979) performed slightly better than non-smokers (AUC=0.960).

[0129] c) Discussion In this study, aimed at identifying additional biomarkers for detecting early OPC, we were able to improve ROC estimates to an AUC of 0.78–0.978 using training data and to 0.935 when validated using a resampling method in the same population. These results demonstrate that the addition of several novel methylation-variable CpG sites to our biomarker panel significantly improves the detection of early OPC in a clinically applicable manner by using oral rinsing, which is an easily collected sample.

[0130] Until recently, OPC was considered a rare cancer. However, its incidence is now rapidly increasing, particularly among men, and this increase is projected to continue for decades to come. Therefore, early detection is currently a priority in this anatomical region. OPC can present as a neck mass, dysphagia, earache, dysphonia, or sore throat, but there is likely to be no external physical changes indicating a cancerous diagnosis. Due to the abundant lymphatic vessels in the oropharynx, lymph node metastasis at the time of diagnosis is common. However, diagnosis can be delayed due to its similarity to other benign conditions affecting this anatomical region. The American Joint Committee on Cancer (AJCC) staging system was revised in its 8th edition to reflect HPV-positive and HPV-negative OPC as two distinct entities with separate staging parameters, due to recent understanding of differences in molecular characteristics, tumor properties, and prognosis. The dependence of HPV-transformed cells on the p16 tumor suppressor has led to the use of p16 overexpression, measured via immunohistochemistry (IHC), as a surrogate marker to determine whether OPC is HPV-related. Regardless of the cause of HPV, treatment may include radiotherapy, surgery, and chemotherapy. Current standard treatment for late-stage tumors includes 70 Gy of radiotherapy with concurrent chemotherapy, but in most cases, the treatment can have a significant impact on quality of life, including dysgeusia, xerostomia, dysphagia, soft tissue fibrosis, and rarely neuropathy, radiation osteonecrosis, or other serious complications. Therefore, there is great interest in gradual easing of therapy with minimally invasive surgical techniques, reduction of radiotherapy dose, or modification / reduction / elimination of systemic therapy. However, since most cases are diagnosed at a stage where multimodal therapy is necessary, there is a continuing need to identify and validate biomarkers to improve the early detection of OPC.

[0131] Extensive research has been conducted to investigate biomarkers for oral cancer using techniques in the “omics” approach, namely genomics, transcriptomics, proteomics, and metabolomics. The use of “omics” has gained popularity due to its ability to be better implemented in a higher-dimensional biological setting and its ability to explore the details of the condition they are investigating. For example, by using metabolomics, five salivary biomarkers may be successfully isolated for the early diagnosis of OSCC and represent candidate biomarkers for screening. In this study, we investigate epigenomic changes and identify differently methylated CpG sites that can specifically predict early oropharyngeal cancer. DNA methylation (DNAm) has been useful in recent studies for diagnosis, as well as for prognosis and stratification. DNAm is in clinical use and is currently used in diagnostic kits for specific genes such as SHOX2 for lung cancer or GSTP1 for prostate cancer. Similar to our previous studies, we used multiple DNAm biomarkers (GSTP1, APC, RASSF1, PTGS2, and MDR1) as a panel to achieve nearly 100% sensitivity and specificity for prostate cancer. However, these studies rely on prior knowledge for the identification of target biomarkers. A novel technique using genome-wide profiles, including Illumina 850K methylation arrays, provides unbiased screening during the biomarker discovery phase and allows for evaluation of multiple combinations to enhance sensitivity and specificity, which is the approach we adopted in this study.

[0132] The epigenetic process of DNAm is a promising biomarker for the early detection of OPC in lung and colorectal cancer. It has long been known that DNAm alterations are characteristic of cancer, specifically that DNAm of tumor suppressor genes lead to gene suppression and uncontrolled carcinogenesis. Recent studies have also highlighted DNAm alterations in precancerous conditions. In our previous research toward OPC biomarker development, we applied what was known in the cervix using the EPB41L3 tumor suppressor gene and found that (1) levels of EPB41L3 DNAm correlated between OPC tumor specimens and oral gargle specimens, and (2) EPB41L3 DNAm measured in oral gargle could distinguish cases from controls, but could predict later OPC cases better than controls. In this study, we expanded our knowledge and identified additional markers toward creating a biomarker panel for detecting early oropharyngeal cancer.

[0133] Within the framework of the Early Detection Research Network (EDRN), our trial will progress from Phase 1 to Phase 2. Phase 1 is defined as a preclinical exploratory trial aimed at identifying potentially useful biomarker leads and prioritizing those biomarkers. Phase 2, clinical assay development of the clinical disease, aims to differentiate cases from controls using ROC curves with estimated sensitivity and specificity. The results of this trial will achieve both phases by identifying biomarker leads and identifying a panel for differentiating early OPC from controls. Ultimately, this panel can be used in dental practices with a model similar to that of other early-detected cancers (i.e., oral / oral cancer). Using a defined population at risk, individuals requiring screening can be identified. Oral rinsing samples can be readily collected during routine dental cleanings and tested with a targeted methylation panel, rather than using the genome-wide arrays used in this trial. Those identified as being at risk for OPC can be sent for further evaluation.

[0134] In our study population, >90% of cases were HPV-positive, which is slightly above the 80% HPV-attributable disease attribute in the US population. The biomarker panel disclosed herein can be used in OPC with or without HPV to determine whether it can be used to screen for both. External validation in an independent validation cohort was not possible at this time, so it was done in this manner.

[0135] The specimens from the cases were collected at the time of diagnosis and before any treatment for OPC. Therefore, they represent specimens that could be collected in a screening population. However, other interventions, including biopsies, dietary changes, supplement use, or other changes in oral health, may have been performed. The specimens were also obtained only from cases with a primary OPC diagnosis, the findings were specifically related to the OPC diagnosis, and there was no confounding from previous cancer diagnoses or treatments. Finally, perhaps most importantly for biomarker development, this development was carried out using mouthwash, a sample readily available in multiple environments, and is particularly useful for screening early OPC.

[0136] In conclusion, utilizing data from our previous studies, a basic bioinformatics approach, and a simple method for obtaining samples, we identified a potential biomarker panel for distinguishing early OPC that can be implemented in many settings. Unlike other HPV-related cancers, there are no identifiable prodromal lesions in the oropharynx before cancer development. Therefore, relying on primary prevention through HPV vaccination and early cancer detection of OPC is crucial for improving survival and quality of life. Further research is needed to externally validate this panel and expand it to a broader population. By following the biomarker development framework, the biomarker panel described herein can be used to screen individuals at risk of early OPC and prevent the intensive systemic therapy required for late OPC diagnosis.

[0137] Oral gargle sample collection 1) If applicable, instruct the target to remove it: a. When you come to the clinic for your appointment, chew the gum as soon as possible. b. Insert the denture into the mouth before collecting the sample. 2) Label each 50ml conical tube with the participant's ID (the label needs to be printed out from the tracking system). 3) Fill a 50ml conical tube with 15ml of mouthwash (Scope or similar). 4) Instruct the patient to thoroughly rinse their mouth, including their throat, by swishing the mouthwash from the container into their mouth for approximately 15 seconds. Instruct the subject to cover all surfaces of their mouth. Next, instruct the subject to tilt their head back and gargle in their throat for another 15 seconds. If the patient is unable to gargle in their throat, instruct them to vigorously swish the mouthwash in for 30 seconds. 5) Ask the subject to spit the mouthwash back into the tube. 6) Replace the lid and place the sample in the refrigerator until processing (processing within 24 hours).

[0138] Standard Operating Procedure Manual I. Subject: Processing and storage of oral specimens II. Objective: To ensure that all oral specimens are processed appropriately. III. Responsibilities: Clinical Research Coordinator IV. Frequency: At the time of sample collection V. Required equipment: i. 15 ml of oral rinse sample in a 50 ml conical tube. ii. Centrifugal separator (capable of maintaining a temperature of 4 degrees Celsius) iii. Electric pipette filler / dispenser iv. 5 ml serum pipette v. 25 ml serum pipette vi.1000ul pipette vii. 41.3 ml of PBS (without magnesium, without calcium), refrigerated at 4 degrees Celsius. viii. 2.0 ml cryovial ix. 2.0 ml microcentrifuge tube x.9x9 storage box Access to xi.Lab Vantage xii. Freezer at -80 degrees Celsius VI. Procedure:

[0139] Oral samples must be processed within 24 hours of collection. If a sample is not processed within 1 hour of collection, it must be refrigerated at 4 degrees Celsius. i. Centrifuge the mouth rinse in a 50 ml conical tube at 2000 x g for 15 minutes at 4 degrees Celsius. ii. Using Sharpie, write the patient's MRN into three (3) 2.0 ml cryovials, one 2.0 ml microcentrifuge tube, and three (3) 2.0 ml cryovials. iii. Pipette 1.8 ml of supernatant into three 2.0 ml cryovials (3). iv. Discard the remaining supernatant. Resuspend the pellet in 20 ml of cold PBS (4 degrees Celsius). vi. Ensure the sample is completely resuspended by inverting it 10 times or by mixing it using a pipette. vii. Repeat centrifugation at 2000xg for 15 minutes at 4 degrees Celsius. viii. Discard the supernatant. ix. Repeat steps v., vi., vii., and viiii. Resuspend the oral pellet in 1.3 ml of cold PBS (4 degrees Celsius). xi. Alicoat the sample: a. Transfer 500 µl to a 2.0 ml microcentrifuge tube. b. Transfer 300µl to a 2.0ml cryovial tube. Place c.250ul into a 2.0ml cryovial tube (for use in microbiome). d.250ul into a 2.0ml cryovial tube (for use in p16). xii. Enter all aliquot samples into Lab Vantage (see the SOP for Entering Data into Lab Vantage). xiii. Print the labels and place them on the appropriate tubes. xiv. File all samples into appropriate 9x9 storage boxes and place them in the -80°C freezer in Dr. Giuliano's lab. xv. After processing, the discarded supernatant can be poured into the sink. VII. Documentation: i.Lab Vantage ii. Patient audit sheet

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Claims

1. An assay for detecting oropharyngeal cancer in a subject, comprising a probe for detecting methylation at CpG sites in one or more genes in a tissue sample, wherein the genes include PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and EPB41L3, and an assay in which a change in the amount of methylation at three or more CpG sites compared to a normal control indicates the presence of oropharyngeal cancer.

2. The assay according to claim 1, wherein the detection of the CpG site is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915.

3. The assay according to claim 1, further comprising a probe for detecting EPB41L3 CpG site 438.

4. HPV SPF for the detection of human papillomavirus type 16 infection or human papillomavirus type 18 infection １０ PCR-DEIA-LiPA ２５ The assay according to claim 1, further comprising a line probe.

5. A method for detecting oropharyngeal cancer in a subject, comprising: obtaining a tissue sample from the subject; and applying the tissue sample to the assay described in claim 1.

6. A method for detecting oropharyngeal cancer in a subject, comprising: obtaining a tissue sample from the subject; and measuring methylation at CpG sites of one or more genes including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control indicates the presence of oropharyngeal cancer.

7. A method for detecting oropharyngeal cancer according to claim 5 or 6, wherein the detection of CpG sites is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915.

8. A method for detecting oropharyngeal cancer according to any one of claims 5 to 7, further comprising a probe for detecting EPB41L3 CpG site 438.

9. A method for detecting oropharyngeal cancer according to any one of claims 5 to 8, wherein the tissue sample is obtained via oral washing or gargling.

10. A method for detecting oropharyngeal cancer according to any one of claims 5 to 9, further comprising assaying the sample for the presence of human papillomavirus type 16 (HPV16) infection or human papillomavirus type 18 (HPV18) infection.

11. HPV16 and / or HPV16 type infection is HPV SPF １０ PCR-DEIA-LiPA ２５ A method for detecting oropharyngeal cancer according to any one of claims 5 to 10, which is detected using a line probe.

12. A method for treating oropharyngeal cancer in a subject, comprising: obtaining a tissue sample from the subject; applying the tissue sample to the assay described in claim 1; and, if detected, treating the oropharyngeal cancer.

13. A method for treating oropharyngeal cancer in a subject, comprising: i) obtaining a tissue sample from the subject; ii) measuring methylation at CpG sites of one or more genes including PAPD5, ST8SIA5, SNORD115-20, NCRNA00164, MIR663B, DDX42, RASA3, HMGB2, ARMC6, and / or EPB41L3, wherein a change in the amount of methylation at three or more CpG sites compared to a normal control indicates the presence of oropharyngeal cancer; and iii) if detected, treating the oropharyngeal cancer.

14. A method for treating oropharyngeal cancer in a subject according to claim 13, wherein the cancer is treated with the administration of anticancer drugs and / or radiation.

15. A method for treating oropharyngeal cancer according to claim 13 or 14, wherein detection of CpG sites is achieved using CpG probes cg25191818, cg19367172, cg12176286, cg19487745, cg20791412, cg11125104, cg27131607, cg14500050, cg14705778, cg04025917, cg06459104, and cg00619915.

16. A method for treating oropharyngeal cancer according to any one of claims 13 to 15, further comprising a probe for detecting EPB41L3 CpG site 438.

17. The method for treating oropharyngeal cancer according to any one of claims 13 to 16, wherein the tissue sample is obtained by oral washing or gargling.

18. A method for treating oropharyngeal cancer according to any one of claims 13 to 17, further comprising assaying the sample for the presence of human papillomavirus type 16 (HPV16) infection or human papillomavirus type 18 (HPV18) infection.

19. HPV16 and / or HPV16 type infection is HPV SPF １０ PCR-DEIA-LiPA ２５ A method for treating oropharyngeal cancer according to claim 18, detected using a line probe.

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