Compositions and methods for selective detection of tumor-inducing viral DNA

Abstract

The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, that are effective for detecting tumor-derived human papillomavirus (HPV) and tumor-derived Epstein-Barr virus (EBV), and in particular, that are effective for distinguishing tumor-derived viral DNA from viral DNA derived from infectious viral particles.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 990,438, filed March 16, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the detection of viral nucleic acids in bodily fluids for the purpose of cancer or precancer detection. [Background technology]

[0003] Viruses are known to promote cancer development. For example, infection with high-risk strains of human papillomavirus (HPV) is associated with cancer of the cervix, head / neck, anus, vulva, or penis. Other examples of viruses associated with cancer development include hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphotropic virus 1 (HTLV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human endogenous retrovirus type K (HERV-K), Merkel cell virus, human immunodeficiency virus (HIV), and Kaposi's sarcoma herpesvirus (KSHV). Because circulating tumor DNA (ctDNA) is released by dying cancer cells, these viral nucleic acids can be detectable in the blood of patients with the corresponding cancer type.

[0004] There is great interest in detecting viral nucleic acids in bodily fluids for the purpose of cancer detection, but there are two major challenges associated with this concept. First, the amount of circulating tumor-derived DNA (ctDNA) in blood, urine, or saliva is extremely low, often only a few molecules per mL of blood. Second, viral nucleic acids present in circulation are typically not derived from tumor-associated viruses but rather from virions shed from non-tumor tissues. This is established by the finding that DNA sequences from many different types of viruses have been identified in the blood of healthy volunteers (Moustafa et al., PLoS Pathog. 2017 13(3):e1006292). Given this finding, the detection of circulating viral sequences alone is insufficient to conclude that a patient has cancer associated with that virus, because the detected viral sequences may originate from normal, non-tumor tissues within the patient.

[0005] For example, any viral sequence detected in a blood sample may simply be related to the viral infection itself, and not related to the tumor burden in the host. This major confounding factor poses a significant challenge to using circulating viral DNA as a specific marker for cancer detection. Individuals with detectable circulating viral DNA may simply be infected with the virus and may not have cancer or pre-cancer.

[0006] In line with this, PCR-based methods for detecting HPV DNA in the circulation of patients with HPV-associated pre-neoplasia or cancer have been reported, but these methods also detect HPV DNA in the blood of a significant proportion of patients without a diagnosis of HPV+ cancer (Bodaghi et al., J Clin Microbiol. 2005 43(11):5428-5434; Cocuzza et al., PLoS One. 2017 12(11):e0188592; Ferreira et al., Pathol Res Pract. 2017 213(7):759-765; Chen et al., J Med Virol. 2009 81(10):1792-1796). Therefore, the presence of HPV virus in a person's circulation does not necessarily indicate that the person has HPV-associated cancer, as its detection may result from infection of normal tissues. Currently available PCR detection methods cannot distinguish between tumor-derived HPV DNA and HPV DNA from virus-infected normal tissues, so they lack sufficient specificity to allow early detection of HPV-associated cancers.

[0007] For example, PCR-based methods for HPV detection exhibit only approximately 54% sensitivity for detecting patients with HPV-associated cancers, which is insufficient to be clinically useful for distinguishing patients who require further evaluation from those who do not (Jensen et al., Clin Otolaryngol. 2018 May 15; Higginson et al., Int J Radiat Oncol Biol Phys. 2015 93(3):S78-S-79; Cao et al., Int J Radiat Oncol Biol Phys. 2012 82(3):e351-358; Dahlstrom et al., Cancer. 2015 121(19):3455-3464). Summary of the Invention

[0008] The present disclosure provides methods and compositions of DNA oligonucleotide primer and probe combinations structurally modified with locked nucleic acids, quenchers, and dyes that are effective for detecting tumor-derived viral, e.g., HPV and EBV, DNA, and in particular, are effective for distinguishing tumor-derived viral DNA from viral DNA derived from infectious viral particles.

[0009] The DNA amplification methods and structurally modified primer / probe compositions disclosed herein quantitatively detect tumor-derived viral DNA in a sample. The disclosed methods and compositions distinguish between tumor-derived viral DNA and viral DNA from non-tumor sources, such as infectious viral particles. In particular, methods and compositions for detecting or monitoring human papillomavirus (HPV)-associated malignancies or Epstein-Barr virus (EBV)-associated malignancies in a subject are disclosed herein, which involve detecting the presence or absence of at least one circulating tumor-derived HPV or EBV DNA of a specific size range in a sample from the subject. Compositions and kits for carrying out these methods are also provided.

[0010] In one aspect, the disclosure provides a composition for detecting tumor-derived human papillomavirus type 16 (HPV16), HPV18, HPV31, HPV33, HPV35, or HPV45 in a sample from a subject, the composition comprising primer / probe sets numbered 1-18 as shown in Table 14 for HPV16, primer / probe sets numbered 1-18 as shown in Table 15 for HPV18, primer / probe sets numbered 1-17 as shown in Table 16 for HPV31, primer / probe sets numbered 1-15 as shown in Table 17 for HPV33, primer / probe sets numbered 1-16 as shown in Table 18 for HPV35, or primer / probe sets numbered 1-17 as shown in Table 19 for HPV45. The modified oligonucleotide primer / probe set of the triplet may comprise (consist of) at least three modified oligonucleotide primer / probe sets selected from the set numbers of the three modified oligonucleotide primer / probe sets, or may comprise four, five, six, seven, eight, nine, ten or more modified oligonucleotide primer / probe sets, wherein a first primer / probe set of the triplet is configured to generate a first amplification product signal, a second primer / probe set of the triplet is configured to generate a second amplification product signal, and a third primer / probe set of the triplet is configured to generate a third amplification product signal, and wherein the primer / probe set of the triplet with the lowest set number corresponds to the first primer / probe set and the primer / probe set of the triplet with the highest set number corresponds to the third primer / probe set.

[0011] For example, if one selects three primer / probe sets numbered 3, 5, and 7 from a table, e.g., Table 14, set 3 is the "first primer / probe set," set 5 is the "second primer / probe set," and 7 is the "third primer / probe set." The lowest set number always corresponds to the first set, the highest set number always corresponds to the third set, and thus any numbers in between always correspond to the second set.

[0012] In these compositions, the triplet modified oligonucleotide primer / probe sets can be selected from primer / probe sets numbered 1-18 as shown in Table 14 for HPV16, or primer / probe sets numbered 1-18 as shown in Table 15 for HPV18, or primer / probe sets numbered 1-17 as shown in Table 16 for HPV31, or primer / probe sets numbered 1-15 as shown in Table 17 for HPV33, or primer / probe sets numbered 1-16 as shown in Table 18 for HPV35, or primer / probe sets numbered 1-17 as shown in Table 19 for HPV45.

[0013] In some embodiments, the present disclosure provides compositions for detecting tumor-derived human papillomavirus type 16 (HPV16) in a sample from a subject, comprising triplets of modified oligonucleotide primer / probe sets selected from the primer / probe sets numbered 1 to 18 as shown in Table 14 for HPV16, wherein a first primer / probe set of the triplets is configured to generate a first amplification product signal, a second primer / probe set of the triplets is configured to generate a second amplification product signal, and a third primer / probe set of the triplets is configured to generate a third amplification product signal, and wherein the primer / probe set of the triplets with the lowest set number corresponds to the first primer / probe set and the primer / probe set of the triplets with the highest set number corresponds to the third primer / probe set.

[0014] In another aspect, the present disclosure provides a composition for detecting tumor-derived Epstein-Barr virus (EBV) in a sample from a subject, comprising triplets of modified oligonucleotide primer / probe sets selected from the primer / probe sets numbered 1 to 28 as shown in Table 20 for EBV, wherein a first primer / probe set of the triplets is configured to generate a first amplicon signal, a second primer / probe set of the triplets is configured to generate a second amplicon signal, and a third primer / probe set of the triplets is configured to generate a third amplicon signal, and wherein the primer / probe set of the triplets with the lowest set number corresponds to the first primer / probe set and the primer / probe set of the triplets with the highest set number corresponds to the third primer / probe set.

[0015] In these compositions, the triplets can contain three (or more) primer / probe sets in which no two primer / probe sets are contiguous, or the triplets can contain three (or more) non-contiguous primer / probe sets.

[0016] In certain embodiments, the primer / probe sets are numbered 3, 5, and 7, or 4, 7, and 10, as shown in Table 14 for HPV 16, or 4, 7, and 10, as shown in Table 15 for HPV 18, or 4, 7, and 10, as shown in Table 16 for HPV 31, or 4, 7, and 10, as shown in Table 17 for HPV 33, or 4, 7, and 10, as shown in Table 18 for HPV 35, or 4, 7, and 10, as shown in Table 19 for HPV 45. In some embodiments, the primer / probe sets are numbered 4, 5, and 6, as shown in Table 20 for EPV.

[0017] In various embodiments, the compositions described herein can further comprise one or more reporter moieties, such as a reporter dye, e.g., FAM, HEX, VIC, Cy5™, or Cy5.5. For example, detector probe 5 in Table 14 can be conjugated to the reporter moiety HEX, and detector probes 3 and 7 in Table 14 can be conjugated to the reporter moiety FAM. In another example, detector probe 7 in Table 14 can be conjugated to the reporter moiety FAM, and detector probes 4 and 10 in Table 14 can be conjugated to the reporter moiety HEX.

[0018] In certain embodiments, the composition comprises primer / probe sets numbered 1, 3, and 5, or sets numbered 1, 3, and 8, or probe sets numbered 4, 6, and 9, or sets numbered 14, 16, and 18, or sets numbered 1, 2, and 5, or sets numbered 10, 12, and 13 as shown in Table 14 for HPV16.

[0019] In another aspect, the disclosure provides a method for detecting tumor-derived human papillomavirus type 16 (HPV16), HPV18, HPV31, HPV33, HPV35, or HPV45 in a sample from a subject, the method comprising: providing any triplets of modified oligonucleotide primer / probe sets of any of the compositions described herein, listed in Tables 14, 15, 16, 17, 18, and 19; fractionating a plurality of HPV DNA fragments from the sample into droplets at a concentration such that zero or one molecule of the DNA fragment is present in each droplet; amplifying the HPV DNA in each droplet with the triplicate primer / probe sets to generate an amplification product signal; and detecting any amplification product signal in each droplet, wherein detection in the droplet of the second amplification product but not the first or third amplification product indicates that the HPV DNA fragments fractionated in the droplets are tumor-derived HPV DNA fragments.

[0020] In these methods, the triplet may contain three primer / probe sets from Table 14 and the method detects tumor-derived HPV16, or the triplet may contain three primer / probe sets from Table 15 and the method detects tumor-derived HPV18, or the triplet may contain three primer / probe sets from Table 16 and the method detects tumor-derived HPV31, or the triplet may contain three primer / probe sets from Table 17 and the method detects tumor-derived HPV33, or the triplet may contain three primer / probe sets from Table 18 and the method detects tumor-derived HPV35, or the triplet may contain three primer / probe sets from Table 19 and the method detects tumor-derived HPV45.

[0021] In another aspect, the disclosure provides a method for detecting tumor-derived human papillomavirus type 16 (HPV16) in a sample from a subject, the method comprising: providing triplets of modified oligonucleotide primer / probe sets of any of the compositions described herein, as listed in Table 14; fractionating a plurality of HPV DNA fragments from the sample into droplets at a concentration such that zero or one molecule of the DNA fragment is present in each droplet; amplifying the HPV DNA in each droplet with the triplicate primer / probe sets to generate an amplification product signal; and detecting any amplification product signal in each droplet, wherein detection in the droplet of the second amplification product but not the first or third amplification product indicates that the HPV DNA fragments fractionated in the droplets are tumor-derived HPV DNA fragments.

[0022] In another aspect, the disclosure provides a method for detecting tumor-derived Epstein-Barr virus (EBV) in a sample from a subject, the method comprising: providing any triplets of modified oligonucleotide primer / probe sets of any of the compositions described herein, as listed in Table 20; fractionating a plurality of EBV DNA fragments from the sample into droplets at a concentration such that 0 or 1 molecule of the DNA fragment is present in each droplet; amplifying the EBV DNA in each droplet with the triplets of primer / probe sets to generate an amplification product signal; and detecting any amplification product signal in each droplet, wherein detection in the droplet of the second amplification product but not the first or third amplification product indicates that the EBV DNA fragments fractionated in the droplets are tumor-derived EBV DNA fragments.

[0023] In all of the methods described herein, the DNA fragments can be fractionated into microdroplets by emulsification and / or the DNA can be amplified using PCR-based methods.

[0024] In all of the methods described herein, the sample may be a blood, saliva, mouthwash, or urine sample, for example a blood sample.

[0025] In another aspect, the disclosure provides for detecting tumor-derived HPV16, HPV18, HPV31, HPV33, HPV35, or HPV45 in a sample from a subject utilizing a primer and probe oligonucleotide composition comprising any triplicate of primer / probe sets from Tables 14, 15, 16, 17, 18, or 19, respectively, wherein a first primer / probe set consisting of two primers and one probe is configured to generate a first amplification product signal, and a second primer / probe set consisting of two primers and one probe is configured to generate a second amplification product signal. and a third primer / probe set consisting of two primers and one probe configured to generate a third amplification product signal), fractionating DNA fragments from a sample from a subject into droplets at a concentration such that only 0 or 1 DNA fragment is present in each droplet, amplifying the DNA in each droplet with a set of primer / probe sets to generate an amplification product signal, and detecting the number of amplification product signals in each droplet (detection of the second amplification product, but not the first or third amplification product, indicates that the subject has tumor-derived HPV16, HPV18, HPV31, HPV33, HPV35, or HPV45).

[0026] Also disclosed is a method for detecting tumor-derived EBV in a sample from a subject using a composition consisting of non-sequential triplets of primer / probe sets from Table 20 (a first primer / probe set consisting of two primers and one probe configured to generate a first amplification product signal, a second primer / probe set consisting of two primers and one probe configured to generate a second amplification product signal, and a third primer / probe set consisting of two primers and one probe configured to generate a third amplification product signal), fractionating DNA fragments from the sample from the subject into droplets at a concentration where only 0 or 1 DNA fragment is present in each droplet, amplifying the DNA in each droplet with a series of primer / probe sets to generate amplification product signals, and detecting the number of amplification product signals in each droplet (detection of the second amplification product, but not the first or third amplification product, indicates that the subject has tumor-derived EBV).

[0027] The method of DNA fragmentation into droplets for digital PCR is known, and includes fractionation into microdroplets by emulsification.In some embodiments, DNA is fractionated based on size before emulsifying into microdroplets.This can be carried out to isolate a series of DNA fragments, for example, for size quantification.

[0028] Any known method can be used to amplify the DNA fragment, such as PCR or non-PCR methods.

[0029] In some embodiments, the subject has never been diagnosed with or suffered from an HPV-associated or EBV-associated malignancy, while in other embodiments, the subject has previously been treated for an HPV-associated or EBV-associated malignancy.

[0030] The disclosed methods can also be used to monitor treatment. Accordingly, methods for monitoring HPV- or EBV-related cancer or malignant tumors in a subject are also disclosed herein, which involve using the disclosed methods to quantify tumor-derived cell-free HPV or EBV viral DNA in blood samples collected at two or more time points during the treatment of a subject receiving treatment for an HPV- or EBV-related malignant tumor. In some of these embodiments, the presence of tumor-derived cell-free HPV or EBV viral DNA in a sample collected at a later time point may indicate that the subject receiving treatment for an HPV-related cancer or malignant tumor has an increased likelihood of recurrence of the HPV- or EBV-related malignant tumor. Similarly, in some of these embodiments, rapid clearance or absence of tumor-derived cell-free HPV or EBV viral DNA in a sample collected at a later time point may indicate that the subject receiving treatment for an HPV- or EBV-related malignant tumor has a decreased likelihood of recurrence of the HPV- or EBV-related malignant tumor. In these cases, the methods may also further involve treating the subject with reduced radiation therapy and / or chemotherapy if the subject exhibits rapid clearance or absence of HPV or EBV tumor-derived cell-free DNA sequences in samples collected at later time points during the course of treatment.

[0031] According to aspects of the disclosed methods, longitudinal analysis of tumor-derived viral nucleic acids in blood using the disclosed methods can be utilized for early detection of virus-positive cancers in individuals who do not exhibit any symptoms associated with malignancy, or in individuals where such symptoms have not yet been identified by a clinician. As demonstrated herein, the disclosed methods enable previously unattainable levels of sensitivity and specificity for detecting circulating tumor-derived viral nucleic acids applicable to patients with HPV+ or EBV+ cancers.

[0032] In some embodiments, the disclosed methods may be applied to determine the likelihood of an early diagnosis or recurrence of a virus-associated cancer, comprising detecting the presence or absence of at least one circulating tumor nucleic acid marker of the associated virus in a blood sample collected from a subject at a single time point or over time.

[0033] In some embodiments, the disclosed methods can be applied to select a treatment for oropharyngeal squamous cell carcinoma (OPSCC) or other virus-associated cancers, including detecting the presence or absence of at least one circulating tumor-derived human papillomavirus (HPV) DNA in a sample collected from a subject diagnosed with or being treated for OPSCC before initiating treatment and / or at various time points during treatment, wherein the presence and / or amount of the circulating tumor-derived HPV DNA in a sample collected at a later time point indicates that the subject being treated for OPSCC has an increased likelihood of OPSCC recurrence. Alternatively, rapid clearance or absence of the circulating tumor-derived HPV DNA in a sample collected at a later time point indicates that the subject being treated for OPSCC has a decreased likelihood of OPSCC recurrence, and if the subject shows rapid clearance or absence of the circulating tumor nucleic acid markers of HPV at a particular time point after initiating cancer therapy, the subject is treated with reduced radiation therapy and / or chemotherapy.

[0034] Also disclosed herein are methods for determining a treatment regimen for a human papillomavirus (HPV)-associated cancer or malignancy, comprising detecting the presence or absence of at least one circulating tumor-derived HPV DNA in samples collected at different time points during treatment from a subject diagnosed with or undergoing treatment for an HPV-associated cancer or malignancy, wherein the absence or rapid clearance of the circulating tumor-derived HPV DNA in samples collected at later time points during treatment indicates that the subject can be treated with reduced radiation therapy and / or chemotherapy.

[0035] Also disclosed herein are methods for detecting, monitoring, and / or treating HPV-associated or EBV-associated malignancies in a subject, the methods comprising detecting the presence or absence of at least one circulating tumor-derived HPV or EBV DNA in samples collected from the subject at various time points during the course of treatment, wherein the presence of circulating tumor-derived HPV or EBV DNA in a sample collected at a later time point during the course of treatment indicates that the subject has an HPV-associated or EBV-associated malignancy or an increased likelihood of recurrence of the HPV-associated or EBV-associated malignancy, and wherein rapid clearance or absence of circulating tumor-derived HPV or EBV DNA in a sample collected at a later time point during the course of treatment indicates that the subject does not have an HPV-associated or EBV-associated malignancy or has a decreased likelihood of an HPV-associated or EBV-associated malignancy.

[0036] Also disclosed herein are methods for monitoring and / or treating an HPV- or EBV-associated malignancy in a subject, which include detecting levels of circulating tumor-derived HPV or EBV DNA in samples collected at various time points from a subject diagnosed with or undergoing treatment for an HPV- or EBV-associated malignancy, determining the subject's circulating tumor-derived HPV or EBV DNA profile, and adjusting a treatment regimen for the HPV- or EBV-associated malignancy according to the circulating tumor-derived HPV or EBV DNA profile, wherein subjects with a favorable circulating tumor-derived HPV or EBV DNA profile are treated with a de-intensified treatment regimen.

[0037] Also disclosed herein are kits containing the components and compositions described herein and instructions for their use for detecting, monitoring, and / or treating malignancies in subjects described herein. For example, the kits can include the primer / probe sets described in Tables 14-20 and instructions for their use.

[0038] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0039] [Figure 1A-B] Figure 1A) Dual-fragment HPV16 assay for detecting tumor viral DNA. Digital PCR fluorescence detection plots for simultaneous detection of two distinct fragments of the HPV16 genome are shown. Examples of a single-positive control, a dual-fragment positive control, an intact HPV genome control, and two patient plasma DNA samples are shown. The gates used to quantify single- and double-positive droplets, as described in the methods, are shown. Figure 1B) Using this assay, analysis of 12 plasma DNA samples from patients with HPV+ oropharyngeal cancer consistently demonstrates the presence of tumor-derived viral DNA, indicated by the abundance of both fragments in single-positive droplets and the very rare co-occupancy of both targets in the same droplet (in contrast to the intact genome control). [Figure 2] Analysis of a large cohort of patient blood samples using the HPV blood assay described in Example 1. HPV tumor virus DNA was not detected in 30 healthy volunteers and 50 patients with HPV-negative cancers (breast and pancreatic cancer). In contrast, 95 / 102 patients with a diagnosis of HPV-positive oropharyngeal cancer had pretreatment circulating tumor HPV DNA in their plasma using the blood test described herein. The observed copy number of HPV DNA detected by the assay is also shown, highlighting the dynamic range of the test. Based on this data, the estimated assay specificity and sensitivity at the time of initial diagnosis are 100% and 93%, respectively. [Figure 3A-D]Figure 3A) Schematic diagram of blood collection time points in a cohort of oropharyngeal cancer patients before, during, and after chemoradiotherapy (CRT). Figure 3B) Two patterns of plasma ctHPV16 profiles observed after initiating CRT. In some patients (red line), ctHPV DNA levels are highest before treatment and decrease after treatment begins. In other cases (blue line), ctHPV DNA levels increase soon after treatment begins, likely due to a spike in cancer cell death. In all cases, ctHPV DNA levels decrease significantly at the end of CRT, indicating that ctHPV DNA levels correlate with the patient's active cancer volume. Figure 3C) A subset of patients has rapid ctHPV DNA clearance kinetics during CRT, with >95% of ctHPV DNA cleared by week 4 of CRT. Figure 3D) Another subset of patients has delayed ctHPV DNA clearance kinetics after CRT, which may correlate with an inadequate or delayed response to CRT. [Figure 4A-F]A subset of 20 patients with HPV+ OPC underwent next-generation sequencing (NGS) analysis of their primary tumors and ctHPV DNA analysis of their blood to investigate potential correlations between these assays. (Figure 4A) When applied to tumor biopsy specimens, there was a strong correlation between the HPV DNA dPCR assay described in Example 1 and tumor HPV copy number assessed by NGS. This validates both the HPV DNA assay and NGS using orthogonal assays on the same sample. (Figure 4B) There is a statistically significant correlation between tumor HPV copy number per cellular genome and pretreatment ctHPV DNA in blood normalized to tumor volume ("ctHPV DNA density"). This indicates that the level of ctHPV DNA detected in blood correlates with HPV copy number in the associated tumor. (Figure 4C) Using tumor NGS data, we developed a bioinformatics analysis pipeline to distinguish HPV+ HPV cancers with evidence of HPV integration into the human genome from cancers with purely episomal HPV. Figure 4D) Circos plots showing the observed rearrangements in cancers with episomal HPV (left) and integrated HPV (right). The example with integrated HPV shown here has an integration site that maps to a region on chromosome 8 (see dark black line). Figure 4E) Higher tumor HPV copy number correlates with a greater likelihood of non-integrated (episomal-only) HPV. Figure 4F) Higher ctHPV DNA levels in the blood also correlate with a greater likelihood of non-integrated (episomal-only) HPV in the associated cancer. Thus, ctHPV DNA can also provide information about the status of the HPV genome in the associated cancer, i.e., whether it is integrated or episomal. [Figure 5A-B]FIG. 5A) Schematic diagram for stratifying patients based on ctHPV DNA profiles. Patients with abundant pre-treatment ctHPV16 DNA that is rapidly removed (>95% by day 28) are classified as having a favorable ctHPV DNA profile. All other patients are classified as having an unfavorable ctHPV DNA profile. FIG. 5B) The favorable ctHPV DNA profile is observed in approximately 30% of patients with clinically favorable (<T4 and <=10 pack-year smoking history) disease and in approximately 30% of patients with clinically unfavorable (T4 or >10 pack-year smoking history) disease. [Figure 6A-B] FIG. 6A) Proportion of patients in each subgroup with post-treatment neck dissection positivity (i.e., local persistent disease), local recurrence, and distant metastasis. Patients with unfavorable clinical risk factors and an unfavorable ctHPV DNA profile had the highest risk of adverse disease events. FIG. 6B) Kaplan-Meier analysis of local disease-free (persistent or recurrent) survival stratified by clinical risk and ctHPV DNA profile. Patients with a favorable ctHPV16 DNA profile had 100% local disease control regardless of smoking history (5 patients were heavy smokers). In contrast, patients with a high clinical risk and an unfavorable ctHPV DNA profile had a significantly reduced local disease control. P, two-sided log-rank test for trend. [Figure 7]The ctHPVDNA test described herein was applied to a cohort of 73 patients who completed treatment for HPV+ oropharyngeal cancer. These patients were clinically asymptomatic with no evidence of disease. These patients were monitored with ctHPVDNA blood tests at each follow-up visit. 60 of the 73 patients had undetectable ctHPVDNA at all follow-up visits, and none of these patients developed disease recurrence during the follow-up period. In contrast, 13 of the 73 patients developed positive ctHPVDNA blood tests during the clinical follow-up period. Nine of these 13 patients also developed clinically evident disease recurrence. The ctHPVDNA blood tests remained positive for up to 6 months before diagnostic radiology scans identified recurrent disease. The remaining four patients with positive blood tests are being closely monitored for possible disease recurrence. [Figure 8] Case from a ctHPVDNA surveillance study. This patient was clinically asymptomatic and not considered to have cancer. In June 2017, a ctHPVDNA blood test was positive. Shortly thereafter, he was examined by an oncologist and found to have no evidence of disease. Three months later, he was examined again by a clinician, who identified no evidence of recurrence. However, the patient reported some neck / shoulder pain, which was considered musculoskeletal in nature. A neck / shoulder MRI was ordered, and this examination, 4 months after the positive blood test, identified an isolated, abnormally enlarged lymph node, which was subsequently biopsied and consistent with recurrent HPV+ oropharyngeal cancer. [Figure 9] Representative embodiments of methods for detecting tumor-derived HPV viral DNA assays using the triplet primer / probe sets presented in Tables 14, 15, 16, 17, 18, 19, and 20. Figure 1 shows digital PCR fluorescence detection plots of multiplex digital PCR reactions for detecting tumor-derived HPV 16 DNA in patient blood samples comprising modified primer-probe sets 3, 5, and 7 from Table 14, where detection probe 5 is conjugated to HEX and detection probes 3 and 7 are conjugated to FAM. DETAILED DESCRIPTION OF THE INVENTION

[0040] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be limited by the appended claims.

[0041] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, useful methods and materials are now described.

[0043] All publications and patents cited herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials for which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0044] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0045] Embodiments of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of the art.

[0046] The examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the methods disclosed and claimed herein, and how to make and use the compositions and probes. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0047] Before embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, this disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also possible that steps can be executed in different order in the present disclosure, where this is logically possible.

[0048] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0049] As used herein, "human papillomavirus" or "HPV" refers to small, non-enveloped, double-stranded DNA viruses that infect the skin and / or mucosal epithelia. As will be understood by those skilled in the art, over 100 HPV genotypes are known to exist. Sexually transmitted mucotropic HPVs are further subclassified as high-risk (e.g., HPV16 and HPV18) or low-risk (HPV6 and HPV11).

[0050] As used herein, HPV-associated malignancies include malignancies of the head and neck (larynx, oral cavity, oropharynx, tonsils, and esophagus), respiratory tissue, breast, skin, cervix, vulva, penis, and anus. Malignancy and cancer are used interchangeably.

[0051] As used herein, "detecting" or "detection" means testing, screening, or otherwise determining the presence and / or absence of at least one tumor nucleic acid marker for HPV in a sample. Such detecting or detection can be performed by methods described herein, including those known in the art that are applicable to the present technology, such as nucleic acid amplification, hybridization-based detection, microarrays, and next-generation sequencing.

[0052] Also, as used herein, the terms "treat," "treating," or "treatment" may refer to any type of action that provides a modulatory effect, which may be a beneficial and / or therapeutic effect, to a subject suffering from a condition, disorder, disease, or condition, including, for example, an improvement in the subject's condition (e.g., of one or more symptoms), a delay in the progression of the disorder, disease, or condition, a delay in the onset of the disease, disorder, or condition, and / or an alteration in the clinical parameters of the condition, disorder, disease, or condition, as would be well known in the art.

[0053] As used herein, the term "surveillance" refers to evaluating the therapeutic effectiveness of a treatment for a patient with cancer. As used herein, the term "surveillance" refers to the detection of virus-associated malignancies in subjects who may or may not have clinically diagnosed or symptomatic cancer.

[0054] As used herein, a subject has an "increased likelihood" of a certain clinical characteristic or outcome (e.g., recurrence or progression) if the probability of the subject having the characteristic or outcome exceeds a certain reference probability or value. The reference probability may be the probability of the characteristic or outcome across a general relevant subject or patient population. For example, if the probability of recurrence in the general oropharyngeal cancer population is X%, and a particular patient has been determined by a method to have a Y% probability of recurrence, and if Y>X, then the patient has an "increased likelihood" of recurrence. Alternatively, a threshold or reference value may be determined, and the probability of recurrence for a particular patient may be compared to that threshold or reference value.

[0055] As used herein, "sample" refers to a biological sample containing tumor nucleic acid markers for HPV. The sample may be a tissue, a cell, or any fluid collected from the human body, such as blood, plasma, urine, saliva, etc. In certain embodiments, the sample is a blood-based sample. Thus, the sample may be whole blood or its components, such as serum or plasma.

[0056] Also disclosed herein are methods for the detection, treatment, and surveillance of human papillomavirus-associated malignancies, and kits for accomplishing the same.

[0057] Also disclosed herein are methods for quantifying viral nucleic acids in the circulation that are specifically tumor-derived, and for distinguishing these tumor-derived viral nucleic acids from other sources of circulating viral nucleic acids.

[0058] Disclosed herein is a DNA amplification method for quantifying DNA fragments of target DNA by size in a sample. This can be used, for example, to detect tumor-derived viral DNA in a blood sample and distinguish it from larger viral DNA from non-tumor sources. In particular, disclosed herein is a method for detecting, monitoring, or treating human papillomavirus (HPV)-associated malignancies in a subject, which involves detecting the presence or absence of at least one circulating tumor-derived HPV DNA in a sample from the subject. Kits for achieving this are also provided.

[0059] The disclosed methods can be performed in thousands of microdroplets (also referred to herein as droplets) generated, for example, via emulsification and / or water-in-oil droplet splitting, as described, for example, in Hindson et al., Anal Chem. 2011 Nov 15;83(22):8604-8610, Pinheiro et al., Anal Chem. 2012 Jan 17;84(2):1003-1011, and Kanagal-Shamanna, Methods Mol Biol. 2016;1392:33-42, or any other method known to those skilled in the art for separating a sample into distinct and / or volumetrically defined aliquots for analysis of tumor-derived versus non-tumor-derived DNA present in the aliquots / sample. In the case of microdroplets, the droplets can be microscale, nanoscale, picoscale, or femtoscale in size.

[0060] As disclosed herein, the microdroplets are generated so that each contains at most a single targeted viral nucleic acid. In embodiments in which only two distinct regions of the viral nucleic acid are detected, the microdroplets containing the targeted viral nucleic acid are either single-positive, i.e., positive for one of the detection signals, or double-positive, i.e., positive for both detection signals. As disclosed herein, the relative numbers of single-positive and double-positive microdroplets and double-positive droplets provide quantitative information that allows for quantitative determination of the relative amounts of tumor-derived and non-tumor-derived viral DNA in a sample.

[0061] In embodiments where PCR is used to detect two physically distinct regions, forward and reverse primer pairs corresponding to each detected region are included. In some embodiments, detection can be performed using digital PCR, droplet digital PCR, emulsion PCR, or microdroplet PCR, according to procedures that would be understood by one of ordinary skill in the art. As disclosed herein, microdroplets containing tumor-derived circulating viral nucleic acids have fewer positively detected viral nucleic acid regions compared to microdroplets containing non-tumor-derived circulating viral nucleic acids. In the simplest case where only two distinct regions in the viral nucleic acid are targeted for detection, the disclosed method identifies microdroplets containing non-tumor-derived circulating viral DNA as being positive for both detection signals used for the different targeted regions (double positive); in contrast, in this simplest case, microdroplets containing tumor-derived circulating viral nucleic acids are positive for only one detection signal, but not both signals simultaneously.

[0062] As an illustrative example, if the target nucleic acid region for detection contains fragments of approximately 70-100 bp, two or more target regions separated by 100 bp will not be present on the same viral DNA molecule if the molecule is derived from circulating tumor DNA. In this scenario, simultaneous detection of both target fragments within the same microdroplet must result from the co-occupancy within the same microdroplet of two different target fragment molecules, each containing one of the regions targeted for detection. Therefore, if the analyzed sample contains non-tumor-derived viral DNA, the frequency of microdroplets testing positive for two or more target fragments will be higher than would be expected based on the frequency of each target region analyzed individually.

[0063] For example, one quantitative measure of the proportion of non-tumor-derived viral DNA fragments in a sample is [fraction (droplets double-positive for both detection signals) - fraction (droplets positive only for detection signal 1) * fraction (droplets positive only for detection signal 2)]. Thus, the fraction of tumor-derived viral DNA in a sample is [1 - [fraction (droplets double-positive for both detection signals) - fraction (droplets positive only for detection signal 1) * fraction (droplets positive only for detection signal 2)]]. The corresponding quantitative measure of tumor-derived viral DNA is [# (droplets positive only for detection signal 1) + # (droplets positive only for detection signal 2)] * [proportion of tumor-derived viral DNA fragments] = [# (droplets positive only for detection signal 1) + # (droplets positive only for detection signal 2)] [1 - fraction (droplets positive for both detection signals) + fraction (droplets positive only for detection signal 1) * fraction (droplets positive only for detection signal 2)]. These equations are provided as illustrative examples of how raw detection signal data can be converted into measurements of tumor-derived and non-tumor-derived viral DNA in a sample, and it is understood that there are other equations that can be utilized for the same purpose.

[0064] The disclosed method can also be applied when three or more regions, each with its own detection signal, are detected in each microdroplet or other parallelized microreaction chamber. In this context, the formula for quantification of tumor-derived and non-tumor-derived viral DNA can be generalized based on the reasoning provided above for the two amplified regions.

[0065] While the above examples contemplate detected viral DNA regions of about 70-100 bp in size separated by at least about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, or about 100 bp, it should be understood that both the size of the detected DNA fragments and their distance can vary and are not fixed in connection with the disclosed methods.

[0066] Returning to embodiments in which two different regions are targeted for detection, double-positive microdroplets may occasionally arise from the co-occurrence of two smaller fragments of viral nucleic acid within a single microdroplet, each fragment containing only a single region targeted for detection, as opposed to the desired measurement of one larger fragment encompassing both regions targeted for detection. In some embodiments, this possibility can be eliminated, or the extent to which it occurs, can be quantified by comparing the relative frequencies of double-positive and single-positive droplets, and observing that the frequency of double-positive droplets is approximated by the product of the frequencies of single-positive droplets, with prevalence decreasing by the square of the dilution factor when reanalyzed at lower concentrations.

[0067] In some embodiments, nucleic acids isolated from a blood sample are emulsified into microdroplets so that the majority of the microdroplets contain either one or none of the viral nucleic acids targeted for detection. Experimental methods for determining the correct microdroplet volume and blood sample dilution factor are provided herein. After emulsification, nucleic acid detection methods, which may include PCR-based methods, are used to detect two or more regions of the targeted viral nucleic acid that are physically separated from each other. In some embodiments, each targeted viral region is associated with a unique detection signal, for example, a unique fluorescent color.

[0068] Also disclosed herein are methods for detecting ctHPV DNA in HPV-OPSCC patients. The methods generally involve detecting the presence of tumor-derived viral DNA using nucleic acid amplification, such as polymerase chain reaction (PCR), in the context of an emulsification in which at least two distinct regions are amplified to distinguish between tumor-derived viral DNA in which fragmentation has occurred and intact virions of non-tumor origin in which the DNA has not been fragmented. Nucleic acid probes and primers for use in the methods, as well as kits containing them, are also disclosed herein.

[0069] Also disclosed herein are methods for determining the prognosis of individuals with HPV-associated OPSCC that can be successfully treated with reduced-intensity chemoradiotherapy (CRT), methods for identifying tumor-specific biomarkers that predict recurrence or recurrence of HPV-associated OPSCC after treatment, and methods for determining the prognosis of individuals with HPV-associated OPSCC that are at risk of recurrence or recurrence after treatment.

[0070] Suitable subjects to be treated by the disclosed methods include, but are not limited to, mammalian subjects. Mammals include, but are not limited to, dogs, cats, cows, goats, horses, sheep, pigs, rodents (e.g., rats and mice), lagomorphs, primates, humans, and mammals in utero. Any mammalian subject in need of or desiring treatment is suitable. Human subjects of any gender (e.g., male, female, or transgender) and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult, elderly) can be treated. Subjects may be of any race or ethnicity, including, but not limited to, Caucasian, African American, African, Asian, Latino, Indian, and the like, and combinations thereof. Furthermore, it should be noted that the terms subject and patient are used interchangeably.

[0071] In certain embodiments, the subject has never been diagnosed with or has never suffered from a virus-associated malignancy. In other embodiments, the subject may be diagnosed with, suffer from, suffer from, or be at risk for a virus-associated malignancy. In some embodiments, the subject has previously been treated for a virus-associated malignancy. In other embodiments, the subject may be in remission from a virus-associated malignancy. In some embodiments, the subject is a smoker. In some embodiments, the subject is a non-smoker.

[0072] Detection of ctHPV DNA Methods for determining the level of biomarker nucleic acids, e.g., ctHPV DNA, such as ctHPV16 DNA, in a sample may involve a process of nucleic acid amplification by, for example, PCR, ligase chain reaction (LCR), transcription-based amplification systems, (TAS) self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), and branched DNA (bDNA) amplification, Q-beta replicase, rolling circle replication, rolling circle amplification, or any other nucleic acid amplification method, followed by a process of detecting the amplified molecules using any technique that would be understood by one of skill in the art.

[0073] In some embodiments, nucleic acid detection methods can be performed in microdroplets or other microreaction chambers, such that the detection methods can be performed in a highly parallelized manner. In some embodiments, the microdroplets or microreaction chambers can contain either zero or one copy of the viral DNA region targeted for detection.

[0074] In embodiments involving emulsion PCR, the target nucleic acid or polynucleotide sequence is typically dispersed in microdroplets. In some embodiments, it is essential that the target nucleic acid be emulsified at a concentration such that each microdroplet (or droplet) contains either one or zero copies of the target molecule. For plasma or serum DNA isolated from a patient's blood sample, the appropriate dilution level can be determined by assessing the abundance of a control genomic region and ensuring that the frequency of positive microdroplets remains below 10% (<10%). The control genomic region detects human (i.e., non-viral) DNA and serves as a quality control for the sample (since many negative samples do not have any positive signal in the assay), and is also used to establish that the concentration of DNA fragments is appropriate (not too low or too high).

[0075] The principles of conventional PCR are also applied, in which target molecules are amplified within each microdroplet by reaction with at least one oligonucleotide primer or oligonucleotide primer pair. The primers hybridize to complementary regions of the target nucleic acid, and DNA polymerase extends the primers to amplify the target sequence. Under conditions sufficient to provide a polymerase-based nucleic acid amplification product, a single-size nucleic acid fragment dominates the reaction product (the target polynucleotide sequence, which is the amplification product). Repeated amplification cycles increase the concentration of a single target nucleic acid or polynucleotide sequence within each microdroplet. The reaction can be performed in any thermocycler typically used for PCR.

[0076] Methods for setting up PCR reactions are well known to those skilled in the art. As will be appreciated by those skilled in the art, any known DNA polymerase, nucleoside triphosphates, buffers, additives / enhancers, and conditions for amplification (cycles of denaturation, annealing, and polymerization) may be used in a PCR reaction.

[0077] In some embodiments, the reaction includes a sequence-specific hydrolysis probe conjugated to both a fluorescent molecule and a fluorescence quencher molecule for the reaction mixture, allowing for detection of successful amplification of the target molecule within each droplet. The chemical composition of the particular probe may vary, but will follow established methods for detecting synthesis-based nucleic acid amplification by those skilled in the art.

[0078] The preparation of emulsion for PCR reaction can be achieved by various methods recognized by those skilled in the art.One effective methodology utilizes a fabricated microfluidic chip that mixes aqueous PCR reaction mixture with lipid solution under controlled pressure to generate uniformly sized microdroplets.The disclosed method is applicable to any method for achieving a divided PCR reaction mixture that allows simultaneous detection of two or more viral nucleic acid target molecules.

[0079] After preparation of the PCR reaction mixture, appropriately emulsified or divided into droplets, the reaction mixture is subjected to primer extension reaction conditions ("conditions sufficient to provide polymerase-based nucleic acid amplification products"), i.e., conditions that allow polymerase-mediated primer extension by adding nucleotides to the ends of primer molecules using the template strand as a template. Cycles of denaturation, annealing, and polymerization can be carried out according to any conditions (e.g., number of cycles, temperature, and duration of time) that would be understood by one of skill in the art.

[0080] The cycles of denaturation, annealing, and polymerization may be carried out using an automated device, typically known as a thermocycler. Thermocyclers that can be used are described elsewhere herein, as well as in U.S. Patent Nos. 5,612,473, 5,602,756, 5,538,871, and 5,475,610.

[0081] In other embodiments, non-PCR-based applications can be used to detect target nucleic acid sequences, for example, such targets can be immobilized on solid supports.Methods for immobilizing nucleic acid sequences on solid supports are known in the art and are described in Ausubel et al.Current Protocols in Molecular Biology, John Wiley and Sons, Inc., and in the protocols provided by manufacturers of, for example, membranes: Pall Corporation, Schleicher & Schuell, magnetic beads: Dynal, culture plates: Costar, Nalgenunc, and other supports.

[0082] Other nucleic acid amplification procedures, such as, but not limited to, LCR, TAS, 3SR, NASBA, SDA, bDNA, and isothermal amplification, will be understood by those skilled in the art. The disclosed methods are not limited to the use of PCR amplification, but rather include the use of any nucleic acid amplification method or any other procedure that may be useful for amplifying sequences for the detection and / or quantification of the presence or expression of one or more of the specific nucleic acid sequences described herein.

[0083] The exact amounts of various reagents and variations in conditions (e.g., buffer conditions, cycle times, etc.) of PCR or other suitable amplification procedures that yield similar amplification or detection / quantification results are known to those skilled in the art and are considered equivalent.

[0084] Detection of the presence of target molecules in a sample / microdroplet is not particularly limited and can be achieved by any technique recognized by one of skill in the art. In some embodiments, detection can involve hybridization of the target molecule with a target-specific probe, such as a nucleic acid probe linked to a fluorescent marker. In other embodiments, the marker can be a non-fluorescent marker. The nature of the marker, fluorescent or non-fluorescent, is not particularly limited and can be any marker or label that would be recognized by one of skill in the art.

[0085] Detecting aliquots (droplets) containing target molecules and distinguishing them from aliquots containing zero target molecules is a critical step in digital PCR. This can be achieved using a variety of established techniques. In some embodiments, microfluidic channels and fluorescent detectors are used to individually analyze the microdroplets. Alternatively, advanced microscopy techniques may be implemented to count positive and negative droplets. The disclosed methods can be embodied in any nucleic acid detection method.

[0086] While some of the methods disclosed herein have been performed using digital PCR, the disclosed methods can in principle be used with any nucleic acid detection method capable of detecting a single nucleic acid and distinguishing the size of detected fragments. For example, such methods may include hybridization of unamplified target molecules with fluorescent or non-fluorescent probes. In any such embodiment, the disclosed methods can be applied to distinguish circulating tumor-derived viral nucleic acids from circulating viral nucleic acids and other non-malignant sources of intact virions. A particular aspect of the disclosed methods is the simultaneous detection in a cleavage reaction of at least two DNA fragments in the viral genome separated by a predetermined distance, where the presence of both targets in separate cleavages indicates tumor viral nucleic acids in a bodily fluid, such as blood, and an increased frequency of co-occupancy of both fragments in the same cleavage indicates non-malignant tumor viral nucleic acids.

[0087] The present invention will be described in more detail in the following examples, which are intended as illustrative only, since numerous modifications and variations will be apparent and appreciated by those skilled in the art. Provided below are examples of how the application of this specific and sensitive method for detecting tumor virus nucleic acids in body fluids such as blood can be used to predict patient prognosis during cancer treatment and identify patients at highest risk of disease recurrence among a cohort of patients who are clinically asymptomatic and considered to be in disease remission.

[0088] Several embodiments of the present invention have been described. The disclosure is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0089] Example 1: Digital PCR assay for detecting HPV viral DNA in circulating cell-free DNA An embodiment of the disclosed method is provided herein, which uses digital emulsion PCR to detect tumor-derived viral HPV DNA in circulating cell-free DNA isolated from blood. The methodological details described in this example, such as the nucleic acid amplification and detection methods used, are included solely to establish that the invention has been put into practice. The methodological details provided in this example, which relate only to this particular embodiment, should not be construed as limiting the invention, as described in the Claims and Abstract sections of this document.

[0090] material Reagents: Cell-Free DNA BCT Tubes, RUO (Streck Catalog No. #218962); QIAamp™ Circulating Nucleic Acid Kit, Catalog No. #55114; dPCR Supermax® Bio-Rad Catalog No. #186-3024; Eppendorf™ 96-Well twin.tec™ PCR Plates; Fisher Scientific Catalog No. #E951020362; Pipette Tips (Bio-Rad Catalog No. #186-4121 or #186-4120); Cartridges (Bio-Rad Catalog No. #186-4109 or #186-4108); Sealing Foil (Bio-Rad Catalog No. #181-4040); Optional: VacConnectors® (Qiagen Catalog No. / ID: 19407). This additional connector is useful in case of any failure of the connectors available for the QIAamp Circulating Nucleic Acid Kit, catalog number 55114; bovine serum albumin (BSA); Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific catalog number: #Q32851); Qubit™ Assay Tubes (Thermo Fisher Scientific catalog number: #Q32856); Falcon® 15 ml conical centrifuge tubes (Corning catalog number #352096); Falcon™ 50 ml conical centrifuge tubes (Corning catalog number #352098); disposable sterile 5 ml, 10 ml, and 25 ml serological pipettes (any good quality brand); sterile PCR tubes (any good quality brand); sterile filter tips in 2 μl, 10 μl, 200 μl, and 1200 μl volumes (any good quality brand); and primers and probes.

[0091] Equipment: microcentrifuge (suitable for 1.5 ml Eppendorf tubes, e.g., Eppendorf 5424 microcentrifuge), centrifuge (suitable for 15 ml falcon tubes, e.g., Eppendorf centrifuge 5810R), Qubit fluorimeter (Thermo Fisher Scientific catalog number #Q33226), deep 96-well thermocycler (Bio-Rad C1000 Touch™ thermal cycler with 96-deep well reaction module #1851197); heating block (Denville Scientific catalog number #10540) for drying 1.5 ml Eppendorf tubes, water bath (should have enough space to incubate 24 50 ml conical centrifuge tubes so that 24 samples can be processed at once); automated droplet generator (Bio-Rad catalog number #186-4101); Bio-Rad QX200™ droplet reader catalog number #1864003; portable Pipet-Aid® XP pipette control device, Drummond Scientific, catalog number 4-000-101; pipettes (volumes: 2 μl, 10 μl, 20 μl, 200 μl, and 1000 μl; any good quality brand); 8-channel pipette (any good quality brand, e.g., Eppendorf catalog number #3125000010).

[0092] method Blood Collection: Collect blood into cell-free DNA BCT tubes, RUO (Streck catalog number #218962).

[0093] Plasma Extraction: Ideally, blood collected from Step I should be processed for plasma extraction on the same day. The same tube can be centrifuged at 2000 x g for 10 minutes at room temperature (RT). The supernatant is transferred to a new Falcon™ 15 mL conical centrifuge tube. Care must be taken not to remove the whitish layer in the middle below the plasma. The tube is centrifuged again at 2000 x g for 10 minutes at room temperature. The supernatant is then transferred to a new Falcon™ 15 mL conical centrifuge tube. The plasma is stored in a -80°C freezer until further use. Note: Sometimes the 10-minute centrifugation does not result in a clear separation of the plasma layers. In such cases, the sample should be centrifuged for an additional 10 minutes before removing the plasma. Alternatively, the centrifugation in the first step can be performed for 15-20 minutes. If the plasma appears red, record the sample. Due to hemolysis, additional processing of the sample during the PCR step may be necessary. Blood should be disposed of in 10% bleach, autoclaved, or according to any other facility / company approved protocol.

[0094] Plasma cell-free DNA (cfDNA) extraction: Thaw the stored plasma sample in a water bath for approximately 5 minutes at 37°C. DNA is extracted using a Qiagen kit (QIAamp Circulating Nucleic Acid Kit, catalog #55114) according to the manufacturer's protocol with the following modifications. A standard vacuum available in the laboratory can be used with the protocol. cfDNA is eluted in two steps: first with 100 μl elution buffer, and then with 75 μl elution buffer if the plasma volume exceeds 3 ml. If the collected plasma volume is small to avoid excessive dilution of the cfDNA, elution can be performed with a smaller volume. It has been observed that incubating the column for 3 minutes after adding the elution buffer (as suggested in the protocol) does not fully extract the cfDNA. Generally, incubations of 30 minutes to 1 hour at room temperature are generally followed for both elution steps. cfDNA is quantified using a Qubit fluorimeter. (Note: Generally, 2 μl of eluate is sufficient for quantification purposes.) The eluted cfDNA is stored in a -20 °C freezer until further use. Note: The cfDNA recovered in the first elution is used for all experiments and calculations. The cfDNA recovered in the second elution step is used only if the cfDNA is depleted in the first elution. If more concentrated cfDNA is needed for any purpose, such as NGS, the sample can be concentrated using a speed vacuum.

[0095] dPCR: dPCR involves three steps: droplet generation, PCR, and droplet reading.

[0096] Droplet generation: Prepare a 25 µl reaction for each sample customized according to the following composition (Reagents: dPCR Supermax® Bio-Rad catalog number #186-3024; any nuclease-free PCR-grade water can be used; other reagents such as forward primer, reverse primer, and probe are designed by the user). [Table 1]

[0097] The primer and probe sequences for the ctHPV DNA assay described herein are as follows: [Table 2]

[0098] The working solution of primer mix is ​​assembled by combining 22.5 µL of each of the four primers (100 µM concentration) in a tube and adding 10 µL of nuclease-free water to achieve a final concentration of 22.5 µM for each primer.

[0099] The working solution of the probe mixture is assembled by combining 6.25 µL of each primer (100 µM concentration) in a tube and adding 87.5 µL of nuclease-free water to achieve a final concentration of 6.25 µL for each primer.

[0100] Approximately 22-23 µl of the above reaction mixture was loaded into a 96-well plate (Eppendorf™ 96-Well twin.tec™ PCR Plate, Fisher Scientific catalog number #E951020362) using a multichannel pipettor (Note: The instrument uses only 20 µl from each well. Extra volume is added to avoid any pipetting errors). Droplets were generated using an automated droplet generator (Bio-Rad catalog number #186-4101) according to the manufacturer's protocol. The reagents required for this step are pipette tips (Bio-Rad catalog number #186-4121 or #186-4120) and cartridges (Bio-Rad catalog number #186-4109 or #186-4108). The sample plate was sealed with sealing foil (Bio-Rad catalog number #181-4040) according to the manufacturer's protocol. Note: The volume of cfDNA + water should be 4.3 µl. Generally, using 4.3 μl of cfDNA sample per reaction is not problematic. However, excessive allele copy numbers can result in streaking of positive droplets and many double-positive droplets along the axis. In such cases, the run should be repeated using less sample, and the remaining volume can be adjusted with water. Dilution provides better quantification of allele frequency in such cases. A linear relationship spanning five orders of magnitude was observed for HPV16 copy numbers ranging from 5 copies to 50,000 copies per 20 μl reaction. Care must be taken to properly seal the plate. Failure to seal the plate properly can lead to sample evaporation during the PCR step.

[0101] PCR: PCR thermocycling was performed using the protocol described below.

[0102] [Table 3]

[0103] All wells should be visually inspected after PCR and before reading the plate in the droplet reader. The copy number observed during droplet reading may be inaccurate if sample evaporates from some wells due to improper sealing. It is recommended to leave the plate in the thermocycler for approximately 15–20 minutes after PCR is complete. This allows the plate temperature to drop in a more controlled manner and avoids droplet malformation. Droplet malformation due to static currents can sometimes be avoided by touching another metal surface before removing the plate from the thermocycler. This should be a routine practice to improve the reproducibility of results. PCR plates can be stored overnight after PCR and read the following morning if time is limited. However, completing the entire process in one day is recommended.

[0104] Reading droplets: Use a droplet reader (Bio-Rad QX200™ droplet reader catalog number #1864003) to read the signal in the droplets according to the manufacturer's protocol. Note: Disposal of oil waste: The composition of droplet reader oil is proprietary to Bio-Rad. A typical waste profile contains fluorinated oil (95%), water (5%), bleach (less than 0.5%), proteins, nucleic acids, and fluorescent dyes (less than 0.1%). Appropriate disposal should be planned accordingly.

[0105] Data Analysis: Copy number calculations should be performed according to the manufacturer's guidelines. An example of a dPCR ctHPV DNA assay readout is shown in Figure 1A. The following parameters were set for calculation of FAM single-positive, HEX single-positive, and FAM+HEX double-positive droplets: In the FAM channel, a cutoff of 700 is used to separate negative droplets from positive droplets. Similarly, in the HEX channel, a cutoff of 3000 is used to separate negative droplets from positive droplets. FAM+HEX double-positive droplets are those with a fluorescence intensity of >700 in the FAM channel and >3000 in the HEX channel. Double-negative droplets have a FAM fluorescence of less than 700 and a HEX fluorescence of less than 3000.

[0106] Poisson statistics are used to calculate the copies of each fragment individually in the reaction using the following formula:

[0107] #copies = #total droplets * ln(#total droplets / #single negative droplets).

[0108] This is calculated first for fragment 1 (FAM positive) and fragment 2 (HEX positive).

[0109] Extensive control assays were performed to determine the level of experimental noise. Based on these controls, the following criteria were used to determine the copy number of the target fragment in the assay reactions:

[0110] [Table 4]

[0111] These copy numbers can be used to calculate the frequency of droplets containing the target fragment: Pr(frag) = (# positive droplets / # total droplets). The expected frequency of double-positive droplets when a sample consists of fragmented circulating tumor viral DNA is estimated as follows: Pr(double positive) = Pr(frag1) * Pr(frag2). In contrast, if Pr(double positive) > 2 * Pr(frag1) * Pf(frag2), the sample was considered to contain non-fragmented viral DNA that was not tumor-derived. If Pr(double positive) > Pr(frag1) or Pr(double positive) > Pr(frag2), the sample was interpreted as negative for circulating tumor-derived viral nucleic acid. If Pr(frag1) and Pr(frag2) > 2 * Pr(double positive), the sample was considered positive for circulating tumor-derived viral nucleic acid, but there was evidence of coexisting non-tumor-derived viral nucleic acid. The raw data from this assay applied to plasma DNA from experimental controls and a cohort of 12 patients with HPV+ oropharyngeal cancer are shown in Figures 1A-1B.

[0112] For the HPV16F1 assay, a cutoff of 700 was set on the y-axis (FAM channel). Any droplets above 700 were considered positive for HPV16. For the HPV16F2 assay, a cutoff of 3000 was set on the x-axis (Hex channel). Any droplets above 3000 were considered positive for HPV16F2. Occasionally, the pattern of dPCR readout appears abnormal. This may be due to a bad sample (unanalyzable cfDNA) or a poor dPCR run. Such samples may need to be repeated to correct the problem. Data from such samples cannot be used for interpretation. If any available strategies do not improve the sample quality, the sample should be classified as unanalyzable DNA or a bad sample. Some strategies for improving sample quality are as follows: The presence of heparin in blood samples can interfere with the dPCR reaction. Treating samples with Bacteroides Heparinase I (New England Biolabs catalog number #P0735S) using the manufacturer's protocol improved sample quality. Excessive hemolysis can interfere with dPCR. Improvement in assay readout was observed by adding 0.4% bovine serum albumin. Some representative dot plots from failed samples are provided as separate files along with suggested solutions.

[0113] The use of endogenous genomic sequences as positive controls for dPCR reactions is essential to verify sample quality. dPCR-based detection of a target sequence in the ESR1 gene was utilized as a positive control for sample quality. The assay is described below. [Table 5] [Table 6] [Table 7]

[0114] Workflow for analysis of plasma DNA samples: 1) Test samples using the ESR1 Genomic Control dPCR assay to assess amplifiable DNA and sample concentration. 2) Test the sample for the double fragment HPV16 assay (HPV16ZENv2). 4) If the sample is negative for HPV16, perform the HPV multiplexed assay (HPVmultiplexed_v1). 5) Perform a duplex assay to identify the specific HPV variant.

[0115] Example 2: Multiplex dPCR assay for detecting five different HPV substrains (HPVmultiplexed_v1) The disclosed methods were applied to a given virus substrain. Although the embodiments herein relate to HPV virus substrains, the methods can be easily applied to other virus substrains using established techniques known to those skilled in the art. Variants included in the described method embodiments are HPV16, HPV18, HPV31, HPV33, and HPV35.

[0116] The HPV16 probe was tagged with FAM-Zen, and the other probes were tagged with HEX (LNA version). [Table 8]

[0117] Reaction mixture Primer mix: Mix equal volumes of 10 primers at 100 μM each (each primer was diluted 1:10 in the mix). Probe mix: Mix 6.25 μl of each of the 5 probes in a tube and add 68.75 μl of water (final concentration is 6.25 μM each in a volume of 100 μl). [Table 9] [Table 10]

[0118] Note: An LNA™-FAM probe was also designed to amplify a common region in all known HPV16 variants with the same primers (No. 283 and 285), which was not used in the multiplex assay. 413_HPV16_LNA_FAM-CommonProbe:CA(+C)A(+C)(+G)(+T)A(+G)(+A)CAT(SEQ ID NO: 22)

[0119] Example 3: Duplex dPCR assay for detecting specific HPV variants (HPV_18&33_v1 and HPV_31&35_v1) This assay was designed to determine the identity of specific HPV variants in patient samples. [Table 11]

[0120] Reaction mixture Primer mix: Mix 22.5 μl of each of the four primers and add 10 μl of water (22.5 μM of each primer in the mix). Probe mix: Mix 6.25 μl of both probes in a tube and add 87.5 μl of water (final concentration 6.25 μM each in a volume of 100 μl).

[0121] [Table 12] [Table 13]

[0122] Example 4: Application to patient blood samples in a prospective clinical trial Blood samples from healthy volunteers, non-virus-related cancers, and HPV+ cancers were analyzed. Circulating tumor HPV DNA copy numbers measured using the assay technology described in Example 1 are shown in Figure 2. These data demonstrate that the ctHPVDNA blood test has 100% specificity and 93% sensitivity for identifying patients with HPV+ cancers.

[0123] Blood samples from patients enrolled in two prospective Phase II clinical trials were analyzed. The disclosed method for specifically detecting tumor-derived viral HPV DNA in blood was integrated into the design of both of these clinical trials. Findings from these longitudinal clinical studies illustrate the applicability and usefulness of the disclosed method for both individualizing patient therapy and for cancer surveillance.

[0124] Summary of patient cohort and clinical trial design. Two prospective phase II clinical trials evaluating the efficacy of reduced-intensity chemoradiotherapy regimens in low-risk OPSCC have been completed. In LCCC 1120 (NCT01530997), 45 patients were treated with reduced-intensity CRT. Eligible patients had HPV-positive and / or p16-positive OPSCC, T0-T3, N0-N2c, M0, and <10 pack-years of smoking. Patients received 60 Gy of intensity-modified radiation therapy (IMRT) concurrent with intravenous cisplatin (30 mg / m 2) weekly. All patients underwent primary site biopsy and elective neck dissection to encompass the nodal levels that were positive before treatment within 4–14 weeks after CRT. The primary endpoint of LCCC 1120 was the pCR rate (86% (37 / 43)), and the 3-year cancer control and overall survival rates were 100% and 95%, respectively. In addition to excellent cancer control, patients reported less toxicity and superior quality of life and less symptom burden compared with standard-of-care CRT (70 Gy). In a second-generation phase II trial (LCCC 1413, NCT02281955), PET / CT scans 12 weeks after treatment were used to guide the use of biopsy / neck dissection (i.e., surgical evaluation after CRT was not mandatory and was guided by imaging). Of the 113 patients enrolled, 82 patients have had at least one year of follow-up, and the two-year actuarial cancer control and overall survival rates for these 82 patients are 90% and 95%, respectively (again, excellent results). Patients continue to report a good recovery in quality of life at one year, thus supporting the concept that dose reduction can improve the treatment ratio. A third-generation phase II trial (LCCC 1612, NCT03077243) is currently underway, with 53 of the expected 120 patients enrolled. Blood samples from 113 patients were prospectively analyzed to detect tumor-derived plasma viral HPV DNA.

[0125] The levels of tumor-derived HPV DNA in blood, as quantified by the disclosed method, significantly correlated with HPV viral DNA in the primary tumor. In 63 patients with HPV-OPSCC, serial levels of tumor-derived viral HPV DNA (the week before RT and every week during RT) were prospectively quantified in patient blood samples using the disclosed method (Figure 3A). Forty-nine patients had detectable tumor-derived viral HPV DNA in the circulation before treatment. In all 49 patients, circulating HPV DNA levels significantly decreased by week 6 after the initiation of chemoradiotherapy (Figure 3B), and by the end of the treatment regimen, the majority of patients (90%, n=44 / 49) had decreased to undetectable levels. Across patients, peak levels of tumor-derived viral HPV DNA in blood ranged from 10 copies / mL to approximately 30,000 copies / mL. Furthermore, distinct clearance kinetics of tumor-derived viral HPV DNA were observed in the blood during chemoradiotherapy treatment. In one group, there was rapid clearance kinetics (Figure 3C), with >95% of peak tumor-derived viral HPV DNA in the blood being cleared by day 28 of therapy. In the second group, there was delayed clearance of tumor viral HPV DNA in the blood (Figure 3D), which may be associated with an inadequate or delayed response to therapy.

[0126] The disclosed method was further validated by correlating the levels of tumor-derived viral HPV DNA in blood with analysis of matched primary tumors in a cohort of 20 patients (Figure 4). Normalized HPV DNA copies in tumor biopsies strongly correlate with HPV DNA copies, as measured by next-generation sequencing (Figure 4A). Furthermore, the amount of tumor-derived viral HPV DNA in blood significantly correlated with HPV DNA copy number in matched tumor biopsies after normalization to the patient's overall tumor burden (Figure 4B). Next-generation sequencing was used to identify cancers with HPV integration into the cancer cell genome (Figures 4C-4D). Higher copy numbers of HPV DNA in tumor biopsies correlated with a higher likelihood of episomal (non-integrated) HPV DNA in matched primary tumors (Figure 4E). Similarly, higher copy numbers of tumor-derived viral HPV DNA in blood correlated with a higher likelihood of episomal (non-integrated) HPV DNA in matched primary tumors (Figure 4F). These findings validate the disclosed methods by establishing that quantification of viral DNA in blood using the disclosed methods significantly correlates with measurements of viral DNA in matched tumor tissue. These observations also demonstrate that the disclosed methods can monitor the presence and clearance of tumor-derived HPV viral DNA in longitudinal analysis of patient blood samples.

[0127] Predicting clinical risk in cancer patients. The disclosed method was applied to monitor tumor-derived viral HPV DNA in the blood of 63 patients enrolled in the aforementioned clinical trial before and during therapy. A favorable profile was defined as having tumor-derived viral HPV DNA (>200 copies / mL) with abundant peak levels of ctHPV16 DNA in the blood and rapid clearance kinetics (≤2% of peak value by week 4) (Figure 5A). Eighteen of the 63 evaluable patients (29%) had a favorable profile (Figure 5B), and none of these 18 patients relapsed (regardless of smoking status) (Figures 6A-B). An unfavorable profile was defined as (i) undetectable pretreatment tumor-derived viral HPV DNA in the blood, (ii) low peak levels of tumor-derived viral HPV DNA in the blood (≤200 copies / mL), or (iii) >2% of peak value by week 4 (40 Gy) (Figures 5A-B). Notably, patients with favorable profiles demonstrated 100% disease control in non-smokers (60 Gy) and heavy smokers (60-70 Gy). In contrast, heavy smokers (>10 pack-years) with unfavorable profiles had a significantly poorer local disease control rate of 45% 12 months after completing therapy (Figure 6A-B). These observations demonstrate the clinical utility of applying the disclosed method to quantify tumor-derived viral DNA in blood as a biomarker for predicting clinical risk among patients with virus-associated cancers. Furthermore, these findings demonstrate that real-time assessment of circulating tumor-derived viral DNA can be utilized to individualize the intensity of a patient's therapy based on their estimated risk.

[0128] Early detection of HPV-positive cancer recurrence in healthy patients considered asymptomatic and disease-free using existing clinical procedures. A typical surveillance schedule after chemoradiotherapy involves clinical examinations (physical examination and fiberoptic nasopharyngoscopy) every 2–6 months for the first 5 years. Most head and neck oncologists also obtain PET / CT scans every 6–12 months. Currently, there are no available surveillance blood tests for patients with HPV-associated OPSCC. The availability of a highly sensitive blood-based surveillance test would improve the value of cancer surveillance in this population by aiding in the early detection of cancer recurrence (before clinical or radiological findings) and reducing the frequency of clinic visits and the use of expensive radiological imaging tests. To date, 73 patients have been prospectively observed with HPV-associated OPSCC (Figure 7). Blood samples were obtained at each follow-up visit, regardless of clinical findings. Plasma ctHPV16 DNA became detectable at post-treatment follow-up in 13 patients (median copies / mL and range), nine of whom had clinical / radiological evidence of cancer recurrence. These patients with detectable ctHPV16 DNA after treatment were asymptomatic and underwent radiographic examinations confirming very early, low-dose cancer recurrence. An illustrative case is shown in Figure 8. Here, the patient had already completed curative therapy and was completely asymptomatic with no evidence of disease. However, he developed a positive result for tumor-derived HPV viral DNA in his blood in June 2017. Soon after, he was examined by an oncologist and determined to have no evidence of disease. Three months later, he returned for a follow-up visit, but the oncologist did not identify any evidence of disease recurrence on physical examination. However, the patient reported some neck / shoulder pain, which was considered musculoskeletal in nature. A neck / shoulder MRI was ordered, and this examination, 4 months after the initial positive blood test, identified an isolated, abnormally enlarged lymph node in the neck. This unusual mass was subsequently biopsied and proved to be recurrent HPV+ oropharyngeal cancer.

[0129] This study includes four patients with detectable HPV DNA in their blood using the disclosed method, but without clinical / radiographic evidence of disease, who are being closely followed for recurrence. No cancer recurrences were detected in patients with undetectable ctHPV16 DNA. In summary, the negative and positive predictive values ​​of the plasma ctHPV16 DNA assay for detecting cancer recurrence are 100% and 70%, respectively. These observations suggest that plasma ctHPV DNA is highly specific and sensitive for the early detection of HPV-associated cancers. Application of the disclosed method as part of clinical care may improve the effectiveness and reduce the costs of cancer surveillance for patients with HPV-associated oropharyngeal cancer.

[0130] Example 5: Plasma circulating tumor HPV16 DNA as a biomarker for the treatment of HPV-associated OPSCC The RTOG0129 study showed that exposure influences clinical risk of oropharyngeal cancer. HPV-positive patients tend to fare better, while HPV-negative patients with extensive smoking histories tend to fare worse. Most studies, including RTOG0129, also show an intermediate prognosis for HPV+ cancers that develop in heavy smokers. This is shown in Figure 8.

[0131] Genetic biomarkers can improve clinical risk stratification. For example, a subset of tumors in HPV+ non-smokers may be exceptionally sensitive to therapy, while there may be smokers with HPV+ cancers that have more HPV-like biology, and other smokers with more tobacco-like biology. Biomarkers can better identify these subgroups than clinical parameters alone.

[0132] Plasma ctHPV DNA is detected in the majority of HPV-OPSCC patients. Therefore, ctHPV DNA may be a biomarker of tumor burden and, importantly, response kinetics. Plasma circulating tumor HPV DNA may also inform decisions regarding who is appropriate for depletion therapy for HPV-associated OPSCC.

[0133] The digital PCR assay was developed for HPV16 DNA, i.e., this assay does not cross-detect HPV-18, -31, -33, or 35, has a very low background signal, is linear over five orders of magnitude of copy number (5 to 50,000 copies), has accurate and excellent reproducibility, is ultrasensitive, and is very specific in that it can detect as few as 6 copies of HPV16 with a sensitivity of approximately 80%. This is shown in FIGS. 1A and 1B. FIG. 1A shows the readout of an example of this assay, showing the presence of individual HPV16 DNA molecules where the positive droplets are well separated from the negative droplets in the reaction. FIG. 1B shows the 95% confidence interval of the linear regression, showing surprising linearity and accuracy, and assay variability is only a problem within the 10 target copy range. This assay can detect as few as 6 target molecules of HPV16 DNA using an assay threshold that gives no false positives.

[0134] A study cohort including 64 patients with biopsy-proven HPV-positive OPSCC overexpressed p16 (IHC) and / or was HPV ISH positive. All patients received definitive chemoradiation (without induction chemotherapy). Fifty-four patients (84%) were enrolled in a prospective CRT intensity reduction trial (60 Gy IMRT + cisplatin 30 mg / m 2 )). Of those patients, 46 patients were clinically favorable (<T4 and ≤10 pack-years of tobacco), and 18 patients were clinically unfavorable (T4 or >10 pack-years of tobacco). There were no patients with N3 or M1 disease. Study blood was collected before treatment, weekly during CRT, and at the post-treatment clinic visit (approximately 450 blood samples were analyzed).

[0135] Plasma ctHPV16DNA levels were measured during chemoradiation. Plasma ctHPV16DNA responds to CRT and is, in most cases, "removed" after treatment. The potential as a biomarker of treatment efficacy, and the correlation of peak-ctHPV16DNA levels with smoking status, disease burden, and tumor HPV copy number were evaluated. This regimen is shown in FIG. and normalized levels of ctHPV16DNA are shown in FIG. 3A.

[0136] Plasma ctHPV16 DNA was detected in 77% of patients, and Figure 11 shows the peak values ​​for each patient. As shown in Figure 11, a wide range of values ​​was observed, ranging from as few as 10 copies per mL to a maximum of 30,000 copies per mL. Also, 23% of patients did not have any detectable HPV16 DNA in their plasma. Furthermore, no significant correlation was found between the amount of HPV16 in plasma and smoking status.

[0137] However, plasma ctHPV16 levels did not show a significant correlation with disease burden, as shown in Figure 3C. We investigated many different methods for correlation between disease burden and peak HPV16 DNA levels, but were unable to find one. However, by analyzing a subset of 25 patients matched for tumor sequencing and plasma HPV16 DNA, we identified a modest but statistically significant correlation between plasma circulating tumor HPV16 and tumor HPV16 copy number, as shown in Figure 3D. Thus, the number of HPV16 copies in a particular patient's plasma may reflect the number of HPV copies in the tumor, with prognostic implications beyond disease stage and smoking history.

[0138] Next, we focused on HPV16-negative patients and developed digital PCR assays for the four most common alternative HPV strains. This analysis is shown in Figure 12. Indeed, among non-heavy smokers, all HPV16-negative patients had detectable HPV18 / 31 / 33 or 35 in their plasma. A strikingly different pattern was observed among heavy smokers. HPV16-positive patients were somewhat fewer. However, among HPV16-negative patients, only about one-third had detectable variant HPV, while the remainder were negative for all five HPV strains. Perhaps even more concerning, two of these four patients had positive neck dissections after treatment, and one patient also had tumors that tested positive for p53 mutations. Among non-smokers, we did not observe an increase in disease events with alternative HPV strains, whereas among smokers, only two patients, one of whom developed a local recurrence soon after completing CRT, appear to have an interactive effect between HPV16 negativity and smoking status.

[0139] Different clearance kinetics were also observed among patients who were high HPV16 expressers. As shown in Figure 13, some patients had exponential clearance of HPV16 DNA, while others had a more complex kinetic pattern with delayed clearance from plasma. This was quantified by measuring how much plasma HPV DNA was present at week 4 compared to the peak HPV value. For the top patient, this value was 0%, and for the bottom patient, it was 39%. A median value of 5% was used to stratify patients into those with either rapid or delayed clearance kinetics.

[0140] Collectively, these defined plasma-circulating tumor HPV16-based risk groups, as shown in Figure 14. Favorable-risk patients had abundant HPV16 and rapid clearance kinetics. All other patients had unfavorable circulating tumor HPV16 profiles due to either slow kinetics, low copy number, positivity for variant HPV, or undetectable HPV. For both non-smokers and smokers, approximately 30% of patients had favorable circulating tumor HPV16 profiles, and 70% had unfavorable profiles. Again, note the 20% subset of smokers in whom none of the five most common HPV strains were detected.

[0141] As shown in Figure 4, patients with favorable circulating tumor HPV16 profiles did not experience any local disease events, regardless of smoking status. However, for patients with unfavorable circulating tumor HPV profiles, smoking status had a significant impact. Non-heavy smokers still had favorable outcomes, with 90% having local disease control. However, a minority of heavy smokers or those with T4 disease had poor local disease control if they also had unfavorable plasma circulating tumor HPV16 profiles.

[0142] In summary, plasma ctHPV16 DNA revealed genetic heterogeneity among 4 / 18 (22%) HPV-associated OPSCC patients with >10 pack-years of tobacco use or T4 disease, and p16+ OPSCC were negative for HPV-16 / 18 / 31 / 33 / 35. Approximately 30% of HPV-associated OPSCC have a favorable ctHPV16 profile (i.e., ≥200 copies / mL and rapid clearance kinetics), whereas an unfavorable ctHPV16 profile (i.e., low / undetectable or delayed clearance) is strongly associated with poor local disease in heavy smokers. Assessment of ctHPV16 profiles can help guide treatment intensity in nonsmokers and smokers undergoing treatment for HPV-associated OPSCC.

[0143] Example 6: Application to cancer surveillance in longitudinal clinical trials of patients without any clinical evidence of cancer after therapy A prospective study was conducted on 73 patients who had previously been diagnosed with HPV+ malignancies but were deemed "free of evidence of disease" after curative-intent therapy. HPV blood testing was administered to these patients during routine follow-up. None of the patients with negative HPV blood tests developed new HPV+ cancers or recurrences of previous HPV+ cancers. In contrast, 9 of the 13 patients with positive HPV blood tests were diagnosed with HPV+ cancers.

[0144] Figure 7 shows the results of an HPV blood test. Negative samples can be clearly distinguished from positive samples. Figure 8 shows a summary case of a patient who tested positive by HPV blood test, but showed no abnormalities or evidence of disease at a follow-up visit to an oncologist, and whose disease recurred several months after follow-up.

[0145] A summary of the clinical results is shown in Figure 7. These results demonstrate the predictive validity of the HPV blood test. Of the 73 patients in this study, 60 were negative by HPV blood testing, and none of them had recurrent disease. In contrast, of the 13 patients who were positive by HPV blood testing, 9 had recurrent disease, and the remaining 4 are being closely monitored for recurrence.

[0146] Example 7: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV16 Table 14 below provides primers and probes for the detection of tumor-derived HPV16, where "+" denotes a locked nucleic acid. Tumor-derived HPV16 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 14 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0147] FIG. 9 shows the results of a multiplex digital PCR reaction for detecting tumor-derived HPV16 DNA in a patient blood sample comprising modified primer-probe sets 3, 5, and 7 from Table 14, where detection probe 5 is conjugated to HEX and detection probes 3 and 7 are conjugated to FAM.

[0148] As another example, modified primer probe sets 4, 7, and 10 from Table 14 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV16 DNA, with detector probe 7 conjugated to FAM and detector probes 4 and 10 conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0149] In some embodiments, the primer / probe sets may be selected such that no two sets are contiguous in Table 14. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ... {14, 16, 18}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0150] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0151] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes.

[0152] [Table 14] TIFF0007791583000015.tif249170TIFF0007791583000016.tif236170TIFF0007791583000017.tif254170

[0153] Example 8: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV18 Table 15 below provides primers and probes for the detection of tumor-derived HPV18, where "+" denotes a locked nucleic acid. Tumor-derived HPV18 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 15 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0154] For example, modified primer probe sets 4, 7, and 10 from Table 15 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV18 DNA, with detector probe 7 conjugated to FAM and detector probes 4 and 10 conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0155] In some embodiments, primer / probe sets may be selected such that no two sets are contiguous in Table 15. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ...{14, 16, 18}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0156] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0157] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes. [Table 15] TIFF0007791583000019.tif217170TIFF0007791583000020.tif219170TIFF0007791583000021.tif185170

[0158] Example 9: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV31 Table 16 below provides primers and probes for the detection of tumor-derived HPV31, where "+" denotes a locked nucleic acid. Tumor-derived HPV31 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 16 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0159] For example, modified primer probe sets 4, 7, and 10 from Table 16 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV31 DNA, with detector probe 7 conjugated to FAM and detector probes 4 and 10 conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0160] In some embodiments, the primer / probe sets may be selected such that no two sets are contiguous in Table 16. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ...{13, 15, 17}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0161] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0162] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes. [Table 16] TIFF0007791583000023.tif219170TIFF0007791583000024.tif226170TIFF0007791583000025.tif180170

[0163] Example 10: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV33 Table 17 below provides primers and probes for the detection of tumor-derived HPV33, where "+" denotes a locked nucleic acid. Tumor-derived HPV33 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 17 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0164] For example, modified primer probe sets 4, 7, and 10 from Table 17 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV33 DNA, with detector probe 7 conjugated to FAM and detector probes 4 and 10 conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0165] In some embodiments, the primer / probe sets may be selected such that no two sets are contiguous in Table 17. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ...{11, 13, 15}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0166] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0167] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes.

[0168] [Table 17] TIFF0007791583000027.tif246170TIFF0007791583000028.tif228170TIFF0007791583000029.tif151170

[0169] Example 11: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV35 Table 18 below provides primers and probes for the detection of tumor-derived HPV35, where "+" denotes a locked nucleic acid. Tumor-derived HPV35 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 18 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0170] For example, modified primer probe sets 4, 7, and 10 from Table 18 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV35 DNA, where detector probe 7 is conjugated to FAM and detector probes 4 and 10 are conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0171] In some embodiments, the primer / probe sets may be selected such that no two sets are contiguous in Table 18. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ...{12, 14, 16}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0172] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0173] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes.

[0174] [Table 18] TIFF0007791583000031.tif249170TIFF0007791583000032.tif230170TIFF0007791583000033.tif133170

[0175] Example 12: Modified oligonucleotide compositions and methods for the detection of tumor-derived HPV45 Table 19 below provides primers and probes for the detection of tumor-derived HPV45, where "+" denotes a locked nucleic acid. Tumor-derived HPV45 DNA can be detected and distinguished from non-tumor sources using any three primer / probe sets from Table 19 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. The DNA of the primer / probe set is modified to include a quencher, a dye moiety, and a locked nucleic acid. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0176] For example, modified primer probe sets 4, 7, and 10 from Table 19 can be used in a multiplex digital PCR reaction to detect tumor-derived HPV45 DNA, with detector probe 7 conjugated to FAM and detector probes 4 and 10 conjugated to HEX. Alternatively, detector probe 7 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 10 can be conjugated to a different detector moiety.

[0177] In some embodiments, the primer / probe sets may be selected such that no two sets are contiguous in Table 19. For illustrative purposes, examples of triplets in this embodiment that do not have contiguous primer-probe sets include {1, 3, 5}, {1, 3, 8}, {4, 6, 9}, ...{13, 15, 17}. Examples of triplets that have contiguous primer-probe sets include {1, 2, 5} and {10, 12, 13}.

[0178] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0179] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes.

[0180] [Table 19] TIFF0007791583000035.tif226170TIFF0007791583000036.tif245170TIFF0007791583000037.tif231170

[0181] Example 13: Modified oligonucleotide compositions and methods for the detection of tumor-derived EBV Table 20 below provides primers and probes for the detection of tumor-derived EBV, where "+" denotes locked nucleic acid. Tumor-derived EBV DNA can be detected and distinguished from non-tumor sources using any three consecutive primer / probe sets from Table 20 in a single multiplex digital PCR reaction, with the central probe having a different detection color from both of the other (border) probes. The two border probes may or may not be the same color. In this example, droplets that are positive for the detection label of the central probe and negative for the detection label of the border / distal probes contain tumor-derived viral DNA.

[0182] For example, modified primer probe sets 4, 5, and 6 from Table 20 can be used in a multiplex digital PCR reaction to detect tumor-derived EBV DNA, with detector probe 5 conjugated to FAM and detector probes 4 and 6 conjugated to HEX. Alternatively, detector probe 5 can be conjugated to FAM, detector probe 4 can be conjugated to HEX, and detector probe 6 can be conjugated to a different detector moiety.

[0183] For illustrative purposes, an example of three consecutive primer-probe sets includes {1, 2, 3}, {2, 3, 4}, {3, 4, 5}, ... {15, 16, 17}.

[0184] For purposes of this application, digital PCR refers to any method understood by those skilled in the art in which a PCR reaction is divided into many sub-reactions for analysis. The divisions may be droplets or microwells, or any other division used in digital PCR.

[0185] It should be understood that HEX and FAM are used in all examples merely for clarity and illustrative purposes.

[0186] [Table 20] TIFF0007791583000039.tif222170TIFF0007791583000040.tif254170TIFF00077915830 00041.tif254170TIFF0007791583000042.tif254170TIFF0007791583000043.tif100170

[0187] Other embodiments While the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative, not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Set 1: SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; Set 2: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; Set 3: SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; Set 4: SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34; Set 5: SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37; Set 6: SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; Set 7: SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43; Set 8: SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46; Set 9: SEQ ID NO: 47, SEQ ID NO: 48, and (SEQ ID NO: 49, 50, or 51); Set 10: SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54; Set 11: SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57; Set 12: SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60; Set 13: SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63; Set 14: SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66; Set 15: SEQ ID NO: 67, SEQ ID NO: 68, and (SEQ ID NO: 69 or 70); Set 16: SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; Set 17: SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; and Set 18: SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79; at least three modified oligonucleotide primer / probe sets selected from the group consisting of: a first primer / probe set of the triplet configured to generate a first amplification product signal; a second primer / probe set of the triplet configured to generate a second amplification product signal; a third primer / probe set of the triplet configured to generate a third amplification product signal; and A composition for detecting tumor-derived human papillomavirus type 16 (HPV16) in a sample from a subject, wherein the primer / probe set of the triplet with the lowest set number corresponds to the first primer / probe set, and the primer / probe set of the triplet with the highest set number corresponds to the third primer / probe set.

2. 2. The composition of claim 1, wherein the triplet contains three primer / probe sets, no two of which are contiguous.

3. The composition of claim 1 , wherein the triplet contains three non-contiguous primer / probe sets.

4. The composition described in claim 1, comprising primer / probe sets of sets 3, 5, and 7.

5. providing at least triplet modified oligonucleotide primer / probe sets of the composition of any one of claims 1 to 4; fractionating a plurality of HPV DNA fragments from said sample into droplets at a concentration such that zero or only one molecule of said DNA fragments is present in each droplet; amplifying the HPV DNA in each droplet with the triplet primer / probe set to generate an amplification product signal; detecting any amplification product signal in each droplet; A method for detecting tumor-derived human papillomavirus type 16 (HPV16) in a sample from a subject, wherein detection in the droplet of the second amplification product but not the first or third amplification product indicates that the HPV DNA fragments fractionated in the droplet are tumor-derived HPV DNA fragments.

6. The method of claim 5, wherein the DNA fragments are fractionated into microdroplets by emulsification.

7. 7. The method of claim 5, wherein the DNA is amplified using a PCR-based method.

8. The method according to any one of claims 5 to 7, wherein the sample is a blood, saliva, gargle, or urine sample.

9. The method of claim 8 , wherein the sample is a blood sample.

Images