Compositions and methods for the detection of HPV biomarkers in urine

Abstract

The present disclosure provides compositions, methods, and kits related to detecting and measuring human papillomavirus (HPV) (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), trans-renal cell-free tumor DNA (TR-ctDNA), etc.) associated with an HPV infection or with an HPV-associated cancer. In particular, the present disclosure provides novel compositions, methods, and kits for diagnosing, treating and / or preventing human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) or other HPV-associated cancers.

Description

COMPOSITIONS AND METHODS FOR THE DETECTION OF HPV BIOMARKERS IN URINESEQUENCE LISTING

[0001] The text of the computer readable sequence listing filed herewith, titled “42774- 601_SEQUENCE_LISTING”, created January 9, 2025, having a file size of 10,809 bytes, is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure provides compositions, methods, and kits related to detecting and measuring human papillomavirus (HPV) (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), trans-renal cell-free tumor DNA (TR-ctDNA), etc.) associated with an HPV infection or with an HPV-associated cancer. In particular, the present disclosure provides novel compositions, methods, and kits for diagnosing, treating and / or preventing human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) or other HPV-associated cancers.BACKGROUND

[0003] HPV infection is associated with several cancers, including cancers of the cervix, vulva, vagina, penis, anus, and oropharynx. Further, HPV-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is a growing epidemic that, despite availability of an HPV vaccine, will continue to be a worsening public health problem for decades because of relatively low adoption of the vaccine in the US. Patients who present with high-risk HPV+ OPSCC are likely to have disease recurrence and, when this occurs, patients have a dismal 5-year survival of only 3-26%.

[0004] Data from other solid tumors indicate that during post-treatment surveillance (PTS), recurrences detected by biofluid-based biomarkers (e.g., “biochemical recurrence”) prior to the appearance of CT scan-detectable disease, have the potential to be cured with salvage therapy. Consistent with this, there is improved survival in locally recurrent OPSCC when disease volume is smaller, suggesting that earlier detection of HPV+ OPSCC recurrence would improve outcomes. However, the current standard-of-care for HPV+ OPSCC PTS is periodic clinical examination, which requires recurrent tumors to be relatively large for detection. Unfortunately, CT scan-based PTS has been tried but had low sensitivity, high false positive rate, and did not increase survival.

[0005] Improved HPV-related cancer detection strategies are needed.SUMMARY

[0006] Provided herein are compositions, methods, and kits related to detecting and measuring human papillomavirus (HPV) (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), trans-renal cell-free tumor DNA (TR-ctDNA), etc.) associated with an HPV infection or with an HPV-associated cancer. In particular, the present disclosure provides novel compositions, methods, and kits for diagnosing, treating and / or preventing human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) or other HPV-associated cancers.

[0007] The description in each section of this patent disclosure, regardless of any heading or sub-heading titles, is intended to be read in conjunction with all other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0008] Embodiments of the present disclosure include compositions for use in a HPV ctDNA and / or TR-ctDNA assay. In some embodiments, the composition includes one or more oligonucleotides that detect one or more HPV target regions. In some embodiments, the oligonucleotides are designed to amplify and / or detect one or more target regions of a high- risk HVP type (e.g., HPV16, HPV18, HPV31, HPV33, and / or HPV39). In some embodiments, the HPV target region is single-or double-stranded. In some embodiments, the oligonucleotides are used in the amplification and quantification of single-or double-stranded DNA. In some embodiments, the single-or double-stranded DNA is from a subject that has an HPV-related disease or condition (e.g., cancer (e.g., HPV+ OPSCC)).

[0009] In some embodiments, the single-or double-stranded region of HPV-associated nucleic acid comprises from about 10 nucleotides (or basepairs / bp) to about 100 bp. In some embodiments, the single-or double-stranded region comprising the HPV-associated nucleic acid sequence is about 5 nucleotides (or basepairs / bp), about 10 nucleotides (or basepairs / bp), about 15 nucleotides (or basepairs / bp), about 20 nucleotides (or basepairs / bp), about 25 nucleotides (or basepairs / bp), about 30 nucleotides, (or basepairs / bp) about 35 nucleotides (or basepairs / bp), about 40 nucleotides(or basepairs / bp), about 45 nucleotides (or basepairs / bp), about 50 nucleotides (or basepairs / bp), about 55 nucleotides (or basepairs / bp), about 60 nucleotides (or basepairs / bp), about 65 nucleotides (or basepairs / bp), about 70 nucleotides (or basepairs / bp), about 75 nucleotides (or basepairs / bp), about 80 nucleotides (or basepairs / bp), about 85 nucleotides (or basepairs / bp), about 90 nucleotides (or basepairs / bp), about 95nucleotides (or basepairs / bp), about 100 nucleotides (or basepairs / bp), about 110 nucleotides (or basepairs / bp), about 120 nucleotides (or basepairs / bp), about 130 nucleotides (or basepairs / bp), about 140 nucleotides (or basepairs / bp), about 150 nucleotides (or basepairs / bp), about 160 nucleotides (or basepairs / bp), about 170 nucleotides (or basepairs / bp), about 180 nucleotides (or basepairs / bp), about 190 nucleotides (or basepairs / bp), about 200 nucleotides (or basepairs / bp), about 220 nucleotides (or basepairs / bp), about 240 nucleotides (or basepairs / bp), about 260 nucleotides (or basepairs / bp), about 280 nucleotides (or basepairs / bp), about 300 nucleotides(or basepairs / bp), about 320 nucleotides(or basepairs / bp), about 340 nucleotides (or basepairs / bp), about 360 nucleotides (or basepairs / bp), about 380 nucleotides (or basepairs / bp), about 400 nucleotides (or basepairs / bp), about 420 nucleotides (or basepairs / bp), about 440 nucleotides (or basepairs / bp), about 460 nucleotides (or basepairs / bp), about nucleotides (or basepairs / bp), about 480 nucleotides (or basepairs / bp), about 500 nucleotides (or basepairs / bp), about 520 nucleotides (or basepairs / bp), about 540 nucleotides (or basepairs / bp), about 560 nucleotides (or basepairs / bp), about 580 nucleotides (or basepairs / bp), about 600 nucleotides (or basepairs / bp), about 620 nucleotides (or basepairs / bp), about 640 nucleotides (or basepairs / bp), about 660 nucleotides (or basepairs / bp), about 680 nucleotides (or basepairs / bp), about 700 nucleotides (or basepairs / bp), about 720 nucleotides (or basepairs / bp), about 740 nucleotides (or basepairs / bp), about 760 nucleotides (or basepairs / bp), about 780 nucleotides (or basepairs / bp), about 800 nucleotides (or basepairs / bp), about 820 nucleotides (or basepairs / bp), about 840 nucleotides (or basepairs / bp), about 860 nucleotides (or basepairs / bp), about 880 nucleotides (or basepairs / bp), about 900 nucleotides (or basepairs / bp), about 920 nucleotides (or basepairs / bp), about 940 nucleotides (or basepairs / bp), about 960 nucleotides (or basepairs / bp), about 980 nucleotides (or basepairs / bp), about 1000 nucleotides (or basepairs / bp).

[0010] In some embodiments, the oligonucleotides are comprised of one or more of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 10, or sequences having at least 90% sequence identity thereto.

[0011] In some embodiments, the one or more oligonucleotides is a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, 10. In some embodiments, the forward primer further comprises an iso-base (e.g., a 5’ methyl isodeoxycytosine (Me-isodC) or an iso-dG).

[0012] In some embodiments, the one or more oligonucleotides is a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5.

[0013] In some embodiments, the one or more oligonucleotides is a second reverse primer at least 90% sequence identity to SEQ ID NO: 6.

[0014] In some embodiments, the one or more oligonucleotides is a probe having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the one or more oligonucleotides is a probe having at least 40% sequence identity to SEQ ID NO: 7, at least 45% sequence identity to SEQ ID NO: 7, at least 50% sequence identity to SEQ ID NO: 7, at least 55% sequence identity to SEQ ID NO: 7, at least 60% sequence identity to SEQ ID NO: 7, at least 65% sequence identity to SEQ ID NO: 7, at least 70% sequence identity to SEQ ID NO: 7, at least 75% sequence identity to SEQ ID NO: 7, at least 80% sequence identity to SEQ ID NO: 7, at least 85% sequence identity to SEQ ID NO: 7. In some embodiments, the probe is biotinylated. In some embodiments, the probe is immobilized on a streptavidin-coated surface. In some embodiments, the probe comprises a detectable label. In some embodiments, the probe comprises a Minor Groove Binder (MGB) moiety. In some embodiments, the probe comprises a nonfluorescent quencher (e.g., 3' Eclipse Dark Quencher (EDQ)). In some embodiments, the one or more oligonucleotides is a probe having at least 90% sequence identity to SEQ ID NO: 7, a detectable label, a MGB moiety and a nonfluorescent quencher.

[0015] In some embodiments, the one or more oligonucleotides is a biotinylated probe with a detectable label having at least 40% sequence identity to SEQ ID NO: 7 and is immobilized on a streptavidin-coated surface.

[0016] In some embodiments, the one or more oligonucleotides comprises: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, 10 and comprising a 5’ methyl iso-deoxy cytosine (Me-isodC); a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5; a second reverse primer at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

[0017] In some embodiments, the one or more oligonucleotides comprise two or more of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, 10 and comprising a 5’ methyl i o-deoxy cytosine (Me-isodC); a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5; a second reverse primer having at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

[0018] In some embodiments, the one or more oligonucleotides comprise each of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4 and comprising a 5’ methyl iso-deoxycytosine (Me-isodC); a first reverse primer having atleast 90% sequence identity to SEQ ID NO: 5; a second reverse primer having at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

[0019] In some embodiments, the forward primer and the first reverse primer anneal to a target HPV sequence at a first low annealing temperature and wherein the second reverse primer anneals to the loop region of the forward primer at a second low annealing temperature.

[0020] In some embodiments, the first low annealing temperature is about 30 degrees Celsius, 31 degrees Celsius, 32 degrees Celsius, 33 degrees Celsius, 34 degrees Celsius, 35 degrees Celsius, 36 degrees Celsius, 37 degrees Celsius, 38 degrees Celsius, 39 degrees Celsius, 40 degrees Celsius, 41 degrees Celsius, 42 degrees Celsius, 43 degrees Celsius, 44 degrees Celsius, 45 degrees Celsius, 46 degrees Celsius, 47 degrees Celsius, 48 degrees Celsius, 49 degrees Celsius, 50 degrees Celsius, 51 degrees Celsius, 52 degrees Celsius, 53 degrees Celsius, 54 degrees Celsius, 55 degrees Celsius, 56 degrees Celsius, 57 degrees Celsius, 58 degrees Celsius, 59 degrees Celsius, 60 degrees Celsius.

[0021] In some embodiments, the second low annealing temperature is about 40 degrees Celsius, 41 degrees Celsius, 42 degrees Celsius, 43 degrees Celsius, 44 degrees Celsius, 45 degrees Celsius, 46 degrees Celsius, 47 degrees Celsius, 48 degrees Celsius, 49 degrees Celsius, 50 degrees Celsius, 51 degrees Celsius, 52 degrees Celsius, 53 degrees Celsius, 54 degrees Celsius, 55 degrees Celsius, 56 degrees Celsius, 57 degrees Celsius, 58 degrees Celsius, 59 degrees Celsius, 60 degrees Celsius, 61 degrees Celsius, 62 degrees Celsius, 63 degrees Celsius, 64 degrees Celsius, 65 degrees Celsius, 66 degrees Celsius, 67 degrees Celsius, 68 degrees Celsius, 69 degrees Celsius, 70 degrees Celsius.

[0022] In some embodiments, the one or more target regions is selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 11.

[0023] In some embodiments, the one or more target regions is the HPV16 E6 gene.

[0024] Embodiments of the present disclosure also include methods of detecting and / or quantifying HPV-associated nucleic acid (e.g., viral nucleic acid and / or ctDNA and / or TR- ctDNA) in a sample (e.g., amniotic fluid, ascites, bile, breast milk, breast milk colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysate, eye aqueous humor, eye vitreous humor, feces, paracentesis, pericardial fluid, peritoneal, blood plasma, pleural, semen, blood serum, synovial fluid, tears, thoracentesis, blood, saliva, gargle, or urine) from a subject.

[0025] In some embodiments, provided herein are methods for detecting and / or quantifying HPV subtype 16 (HPV16) nucleic acid, HPV subtype 18 (HP VI 8) nucleic acid, HPV subtype 31 (HPV31) nucleic acid, and / or HPV subtype 39 (HPV39) nucleic acid (e.g., viral nucleic acidand / or ctDNA and / or TR-ctDNA). In some embodiments the HPV nucleic acid is analyzed in a urine sample.

[0026] In some embodiments, the methods comprise detecting and / or quantifying HPV- associated nucleic acid having less than fifty (50) nucleotides in a sample from a subject. In some embodiments, the HPV-associated nucleic acid comprises from about 10 nucleotides (or basepairs / bp) to about 100 bp. In some embodiments, the HPV-associated nucleic acid has less than 100 nucleotides of base pairs (e.g., less than 90, less than 80, less than 70, less than 60, less than 50, less than 50, less than 40, less than 30, less than 20).

[0027] In some embodiments, the detecting and / or quantifying comprises nucleic acid amplification using at least one hairpin primer (e.g., a primer comprising any one or more of SEQ ID NOs: 1, 2, 3, 4, 10). In some embodiments, the detecting and / or quantifying comprises digital amplification. In some embodiments, the digital amplification comprises digital PCR. In some embodiments, the amplification comprises contacting the sample with the hairpin primer and a second primer (e.g., SEQ ID NOs: 5, 6).

[0028] In some embodiments, the detecting and / or quantifying comprises contacting amplicons generated by the hairpin primer and the second primer with one or more probes (e.g., SEQ ID NO: 7).

[0029] In some embodiments, the subject has previously been diagnosed as having an HPV infection or HPV-associated disease or condition. In some embodiments, the subject has been treated with a vaccine, an anti-viral drug, or an anticancer therapy. In some embodiments, the HPV nucleic acid is detected before and / or after administration of therapy. In some embodiments, the results of the assay are employed to modify a therapy (e.g., change dose, change drug, stop treatment, etc.).

[0030] Embodiments of the present disclosure also include methods comprising contacting a sample (e.g., urine) from a subject with one or more of the oligonucleotides. In some embodiments, the sample comprises HPV-associated nucleic acid (e.g., viral nucleic acid and / or ctDNA and / or TR-ctDNA). In some embodiments, the sample comprises HPV subtype 16 (HPV16) nucleic acid, HPV subtype 18 (HPV18) nucleic acid, HPV subtype 31 (HPV31) nucleic acid, and / or HPV subtype 39 (HPV39) nucleic acid.

[0031] In some embodiments, contacting comprises: (i) providing one or more of the oligonucleotides; (ii) fractionating a plurality of HPV DNA from the sample into droplets at a concentration wherein, on average, only 0 or 1 molecule of the DNA is present in each droplet; (iii) amplifying HPV DNA in each droplet with the oligonucleotides to produce amplicon signals; and (iv) detecting in each droplet any amplicon signals. In some embodiments, theDNA is fractionated into micro-droplets by emulsification. In some embodiments, the DNA is amplified using a nucleic acid amplification method.

[0032] In some embodiments, the nucleic acid amplification method comprises polymerase chain reaction (PCR), loop mediated isothermal amplification (LAMP), nucleic acid sequencebased amplification (NASBA), strand displacement amplification (SDA), or multiple displacement amplification (MDA). In some embodiments, the contacting comprises conducting a quantitative PCR (qPCR) assay. In some embodiments, the qPCR assay comprises a digital PCR assay.

[0033] In some embodiments, the digital PCR assay comprises a droplet digital PCR (ddPCR) assay.

[0034] In some embodiments, the sample comprises a ctDNA and / or a TR-ctDNA HPV target nucleic acid and the oligonucleotides hybridize to a HPV target nucleic acid. In some embodiments, the HPV target nucleic acid or a product derived therefrom (e.g., an amplicon) is sequenced. In some embodiments, the sequencing technique is a next generation sequencing technique. The term “next generation sequencing” refers to highly parallelized methods of performing nucleic acid sequencing and comprises the sequencing-by-synthesis or sequencing- by-ligation platforms (e.g., employed by Illumina, Life Technologies, Pacific Biosciences and Roche, etc.). Next generation sequencing methods may also include, but not be limited to, nanopore sequencing methods such as offered by Oxford Nanopore or electronic detectionbased methods such as the Ion Torrent technology commercialized by Life Technologies. In some embodiments, one or more of the primers described herein further comprises an additional sequence (e.g., barcode, adapter, etc.) that finds use in sequencing library preparation, sequencing, and analysis. Suitable nucleic acid sequencing techniques include, but are not limited to, sequencing by synthesis (see e.g., Meyer and Kircher, "Illumina sequencing library preparation for highly multiplexed target capture and sequencing," Cold Spring Harbor Protocols 2010 (6)); single-molecule real-time sequencing (see e.g., Levene et al., "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science. 299(5607): 682-6 (2003)); ion semiconductor sequencing (see e.g., Rusk, “Torrents of sequence,” Nat. Methods 8, 44 (2011)); pyrosequencing (see e.g., Wicker et al., “454 sequencing put to the test using the complex genome of barley,” BMC Genomics, 7:275, 2006); sequencing by ligation (SOLiD sequencing) (see e.g., Margulies et al., “Genome sequencing in microfabricated high-density picolitre reactors,” Nature, 437:376-80 (2005)); nanopore sequencing (see e.g., Goodwin et al., “Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome,” Genome Res., 25(11): 1750-6 (2015)); chaintermination sequencing (Sanger sequencing) (see e.g., Sanger et al., "DNA sequencing with chain-terminating inhibitors, "Proceedings of the National Academy of Sciences of the United States of America, 74 (12): 5463-5467 (1977)); and sequencing with mass spectrometry (see e.g., Edwards et al., "Mass-spectrometry DNA sequencing," Mutation Research, 573(1-2): 3- 12 (2005)).

[0035] In some embodiments, the contacting comprises: conducting a quantitative Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) assay. The term “Single Molecule Recognition through Equilibrium Poisson Sampling” refers to an amplification-free method (e.g., a direct detection approach) for detection and / or quantification of single, unlabeled molecules (e.g., ctDNA and / or TR-ctDNA). Suitable SiMREPS techniques include, but are not limited to, single-molecule kinetic fingerprinting (see e.g., Stephen L. Hayward et al., Ultraspecific and Amplification-Free Quantification ofMutantDNAby SingleMolecule Kinetic Fingerprinting, J Am Chem Soc. 2018 Sep 19; 140(37): 11755-11762.; Alexander Johnson Buck et al., Kinetic Fingerprinting to Identify and Count Single Nucleic Acids, Nat Biotechnol. 2015 Jul;33(7):730-2; and Kunal Khanna et al., Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET -based Dynamic Nanosensor, Biosens Bioelectron. 2021 Oct 15: 190: 113433).

[0036] In some embodiments, the contacting comprises: (i) providing one or more of the oligonucleotides; (ii) immobilizing the one or more of the oligonucleotides onto a surface; (iii) repeatedly transient binding HPV DNA in the sample to the one or more oligonucleotides bound to the surface to produce a kinetic fingerprint; and (iv) detecting any kinetic fingerprints.

[0037] In some embodiments, the HPV DNA is immobilized with a probe. In some embodiments, the one or more oligonucleotides is a probe as described herein.

[0038] In some embodiments, provided herein are methods of detecting HPV nucleic acid and / or ctDNA and / or TR-ctDNA and, in some embodiments, further comprising the step of treating a subject with cancer surveillance, therapy, or other intervention if HPV is detected in a sample. In some embodiments, the cancer treatment comprises an imaging technique, a clinical examination, excision treatment, cryotherapy, thermal ablation, radiotherapy, chemotherapy, and / or salvage therapy. In some embodiments, the treatment is provided prior to appearance of CT scan-detectable cancer.

[0039] In some embodiments, provided herein are methods of detecting HPV nucleic acid and / or ctDNA and, in some embodiments, further comprising the step of treating a subject with an HPV vaccine if HPV is not detected in a sample. In some embodiments, the method further comprises sequencing HPV nucleic acid present in said sample.

[0040] Embodiments of the present disclosure also include kits. In some embodiments, the kits may comprise one or more oligonucleotides and / or sets of oligonucleotides, as described herein. In some embodiments, the kits may further comprise reagents necessary, useful, or sufficient to purify, isolate, detect and / or quantify HPV nucleic acid and / or ctDNA and / or TR- ctDNA. For example, the kits may further comprise amplification reagents, including buffers and enzymes. In some embodiments, the kit may further comprise control samples, if needed or desired. In some embodiments, the kit may comprise solid surfaces (e.g., beads) comprising capture reagents (e.g., oligonucleotides) specific for target ctDNA and / or TR-ctDNA. In some embodiments, the kit can further include containers for holding or storing a sample, reagents, or reaction mixtures (e.g., a container or cartridge for a plasma sample, a container for a cell free DNA sample, etc.). In some embodiments, the kit can also include one or more instrument for assisting with obtaining or manipulating a test sample, such as a syringe. Where appropriate, the kit can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents (e.g., a container for mixing reagents for PCR). In some embodiments, the kit may further comprise instructions for use of the kit. Instructions included in kits can be affixed to packaging material or can be included as a package insert or can be viewed or downloaded from a particular website that is recited as part of the kit packaging or inserted materials. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIGS 1A-1C depict fragment size analysis of urine cfDNA WGS from patients with non-urologic cancers which shows TR-ctDNA enrichment in ultra-short fragments. FIG. 1A includes plots which show log2(CopyNumberRatio) calls on the y-axis for plasma cfDNA or urine cfDNA for three patients, with the x-axis showing the genomic position of the mapped DNA fragments across the indicated chromosomes. Low-pass coverage urine cfDNA WGS data, unfiltered for fragment length, shows CNA patterns qualitatively concordant with those from matched plasma cfDNA WGS (also unfiltered for length) in some but not all patients, where cancer-associated CNA are visible in patients AML14 and AML13, but not in patient ST5. After stratification of analysis by fragment length into 20 bp- wide bins, CNA plotsshowed that restriction to ultra-short bins (<50 bp; i.e., 30-50 bp and 20-40 bp) revealed tumor- associated CNA more robustly than unfiltered data. FIG. IB is a heatmap of estimated percent of tumor DNA content from WGS data. An increased enrichment for tumor DNA with ultra- short fragments (e.g., <50 bp) relative to larger fragment size bins was observed, consistent with the qualitative differences evident in Panel A. FIG. 1C are composite curves of urine cfDNA WGS data from 3 solid tumor and 3 AML patients. The curves labeled as focally amplified, gain, and loss are implicitly derived from tumor- enriched DNA (fragment length of mapped reads emanating from regions with known tumor-associated CNA), while the unaltered curves reflect inferred fragment lengths for reads mapping to the remainder of the genome. As shown, the majority of DNA fragments, and importantly those mapping to genomic regions of tumor-associated CNA, are ultra-short (e.g., <50 bp) in length.

[0042] FIGS. 2A-2D depict ultra-short amplicon HP VI 6 stem -loop ddPCR assay detects TR-ctDNA fragments in urine from HPV+OPSCC patients and shows concordance with results from plasma ctDNA analysis. FIG. 2A is a schematic of the short-amplicon (42 bp) stem-loop two stage PCR approach used to detect and quantify ultra-short HP VI 6 TR-ctDNA present in urine. During Stage 1 of the PCR, a forward primer (Primer 1) forming a stem-loop and a tailed reverse primer (Primer 2), both with short regions of complementarity to the template, anneal at a low temperature to amplify the ultra-short fragments. Stage 2 unlocks the stem-loop, leading to a longer version of the template derived from the amplicons in step 1, which can be amplified with higher annealing temperatures using a third reverse primer (Primer 3) complementary to the loop region of the stem-loop primer (sequence details in FIG. 6). FIG. 2B is an analytical validation of the stem-loop ddPCR assay showing a high correlation between expected and observed copies. Data from a 2-fold dilution series is shown, to define the detectable range of HPV16 ultra-short DNA using the stem-loop ddPCR assay. Synthetic ultra-short HP VI 6 target DNA spiked into the ddPCR reaction (top left plot) or Hindlll digested genomic DNA (gDNA) from HPV16+ head and neck cancer cell line UM-SCC-104 (top right plot) were tested in a background of Hindlll digested non- HPV human gDNA matrix of 200,000 haploid genome equivalents (GEs) per well. The expected copies of spiked in synthetic DNA (x-axis; left plots) or the number of GEs of UM-SCC-104 tested (x-axis; right plots) were plotted against the measured copies (y-axis; top plots) of HPV16 E6 gene DNA (cumulative of 3 replicates). Plots at bottom-left and bottom-right show the observed percent coefficient of variation (% CV) corresponding to measurement each of the serial dilutions of synthetic HPV16 DNA or HPV16- positive cancer cell line DNA, respectively. LoD of the stem-loop assay was determined to be 4.2 copies cumulative for ddPCR triplicates as describedin the Methods section. FIG. 2C is a stem-loop, 42 bp amplicon urine ctDNA assay was used to detect HPV16 E6 ctDNA in matched urine and plasma from 32 patients (HPV+ OPSCC) and 12 negative controls (1 HPV18+ OPSCC patient, 6 HPV-Negative head and neck squamous cell cancer (HNSCC) patients and 5 non-cancer healthy controls). Cancer patients represented both early stage (E) and metastatic (M) cases, as indicated in the panel, and all blood and urine samples from patients were collected pre-treatment. As shown, the assay detected HPV16 ctDNA in both urine and plasma in 27 of the 32 cases of HPV+ OPSCC. HP VI 6 ctDNA was not detected in all the negative controls (12 of 12 cases), showing high concordance between the results for plasma and urine based detection of ctDNA. FIG. 2D is a plot of results comparing loglO HPV16 ctDNA absolute quantification values in urine and matched plasma collected from 31 HPV+ OPSCC patients. A significant correlation was found between HPV16 copies detected in urine TR-ctDNA and plasma ctDNA; Pearson r = 0.5483, *p (two-tailed) = .0014. HPV16 TR-ctDNA values for urine correspond to the mean value of 30 ml samples from two bottles; for plasma they represent values from ~0.9 ml of sample. The values are plotted as the cumulative sum of HPV16 TR- ctDNA copies detected from ddPCR triplicates.

[0043] FIG. 3 depicts a proof-of-concept for earlier detection of cancer recurrence via serial urine HPV16 TR-ctDNA measurements in four patients with HPV+ OPSCC. Shown are ddPCR results from testing of longitudinal urine collections from four patients, using the stemloop 42 bp urine TR-ctDNA assay. Number of HPV16 copies detected (y-axis) were plotted on a loglO scale (patient 1 and patient 3) or linear scale (patient 2 and patient 4) as a cumulative of HPV16 ctDNA values from urine cfDNA samples tested in ddPCR triplicates, collected at different timepoints over several months (x-axis). There were seven timepoints for Patient 1, four timepoints for Patient 2, four timepoints for Patient 3, and three timepoints for Patient 4. Closed symbols represent HPV16 molecules detected above LoD (magenta dotted line), and open symbols represent values that were below the LoD, with serially collected samples at baseline prior to treatment in black, during treatment in green, or during post- treatment surveillance for recurrence in red. The day of treatment is marked as 0 on the x-axis. Patients 1 through 3 underwent chemo-RT treatment (7 weeks) and showed detectable HP VI 6 TR- ctDNA prior to initiation of treatment. Patient 4 underwent surgical resection, but notably, had baseline urine DNA of poor quality and therefore that time point could not be accurately analyzed for HP VI 6 TR-ctDNA. In three subjects, HP VI 6 TR-ctDNA was detected in urine during the surveillance period prior to clinically detected recurrence.

[0044] FIGS. 4A-4C depict addition of EDTA to urine stabilizes spiked-in synthetic DNA fragments for at least 7 days when stored at room temperature. FIG. 4A is a GeneRuler Ultra Low Range DNA (ULDNA) ladder (Fisher #SM1212) which was spiked into urine samples from two healthy controls which were then stored at room temperature and preserved with 1) H2O at pH 6.0, 2) 10 mM EDTA at pH 6.0, 3) 40 mM EDTA at pH 7.0, 4) 30 mM Tris-HCl at pH 7.5, 5) 10 mM EDTA / 50 mM Tris-HCl at pH 7.5, and 6) 40 mM EDTA / 50 mM Tris-HCl at pH 7.5. pH strips were used to measure the preserved urine. * indicates a lane with error during DNA ladder spike-in extraction. FIG. 4B shows the stability of ULDNA ladder which was tested after incubation for 24 hours in urine preserved with 40 mM and 100 mM EDTA. Ladder was spiked into the urine collected from two healthy donors in duplicates stored at room temperature and preserved with 40 mM or 100 mM EDTA. FIG. 4C shows a schematic of the experiment to investigate stability of ULDNA ladder spiked into urine from healthy controls, preserved with 100 mM EDTA and stored at room temperature for up to 7 days. FIG. 4D shows the average concentration of individual ULDNA ladder bands as determined by Bioanalyzer for urine left at room temperature for 0, 3, 5, and 7 days.

[0045] FIGS. 5A-5C depict the assessment of efficiency of urine DNA extraction and low MW / high MW DNA separation. FIG. 5 A shows the efficiency of DNA extraction of ULDNA ladder from total urine cfDNA. Error bars represent standard deviations of duplicate extractions. FIG. 5B shows the efficiency of DNA extraction of Invitrogen Ikb DNA ladder (Ikb DNA) from total urine cfDNA, high molecular weight (HMW) fractions of total urine cfDNA, and low molecular weight (LMW) fractions of total urine cfDNA. Error bars represent standard deviations of quadrupole extractions. FIG. 5C shows the efficiency of DNA extraction of Invitrogen Ikb DNA ladder (Ikb DNA) from total urine cfDNA, HMW fractions of total urine cfDNA, and LMW fractions of total urine cfDNA.

[0046] FIG. 6 depicts the assay design and primer / probe sequences for the HP VI 6 TR- ctDNA stem-loop ddPCR assay. Stem-loop ddPCR assay design to detect and quantify ultra- short TR-ctDNA fragments present in urine, targeting a short-amplicon (42 bp) region in the HPV16 E6.DETAILED DESCRIPTION

[0047] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

[0048] Embodiments of the present disclosure provide compositions, methods, and kits related to detecting and measuring human papillomavirus (HPV) (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), trans-renal cell-free tumor DNA (TR-ctDNA), etc.) associated with an HPV infection or with an HPV-associated cancer. In particular, the present disclosure provides novel compositions, methods, and kits for diagnosing, treating and / or preventing human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) or other HPV-associated cancers, infections or other HPV-related diseases or conditions.

[0049] Solid tumors are known to shed cell-free tumor-derived DNA (ctDNA) that might be detectable bodily fluids including saliva, urine, and plasma. The presence of ctDNA in biofluids has provided opportunities for cancer diagnostics. Although most research in this area has focused on ctDNA present in plasma, ctDNA from the bloodstream can be filtered through the kidney into the urine, as trans-renal ctDNA (TR-ctDNA). Whereas urine-based tests for cancer have traditionally been utilized with cancers that directly interface with the urinary tract (e.g., bladder cancer, prostate cancer), TR-ctDNA holds the promise of urine-based access to a broad variety of cancer types arising in organs throughout the body, although successful development of such technologies has been limited.

[0050] The length of TR-ctDNA fragments present in urine impacts assay design for optimal sensitivity in TR-ctDNA detection. To date, there have been contrasting reports regarding the length of TR-ctDNA fragments. Whereas PCR-based studies of TR-ctDNA have shown greater success in detection when using amplicons shorter than 60 bp, next-generation sequencing (NGS) studies that focused specifically on TR-ctDNA suggested a median length of 112 bp or 101 bp, with additional investigation showing a higher proportion of ultra-short fragments in patients vs. controls. A limitation of reported NGS results is that the specific library preparation methods used (e.g., double-stranded DNA library preparation protocols, hybridization-based capture of ctDNA fragments) are prone to bias against recovery of shorter fragments, especially ultra-short fragments (e.g., <50 bp).

[0051] Single-stranded DNA (ssDNA) NGS methods were developed herein to profile TR- ctDNA size. Resulting data indicated that TR-ctDNA is ultra-short (e.g., <50 bp) and detectable across multiple non-urologic cancer types. In addition to the ssDNA NGS methods, a droplet digital PCR (ddPCR)-based assay was developed to measure TR-ctDNA in urine, which offers absolute quantification, higher precision, and higher throughput compared to NGS. The assay was designed to analyze patients with HPV+OPSCC. In such patients, the HPV DNA sequences are present in the blood circulation as ctDNA and it was hypothesized that such ctDNA fragments that transited the glomerular barrier of the kidney could be detected in urine by ddPCR. It is worth noting that, unlike the setting of HPV+ cervical cancer where shed tumorDNA could be deposited directly into urine, HPV16 signal in the urine of HPV+ OPSCC patients would necessarily be trans-renal.

[0052] A ddPCR assay capable of detecting ultra-short HP VI 6 TR-ctDNA fragments in urine from HPV+ OPSCC patients was developed. The assay (42 bp amplicon) was compared to a conventional -length assay (77 bp amplicon) and it was found that targeting ultra-short fragments was useful for robust urine TR-ctDNA detection. Utilizing the ultra-short amplicon assay, TR-ctDNA detection results were achieved in urine from HPV+ OPSCC patients that were concordant with results from matched plasma ctDNA. Furthermore, using longitudinal urine samples from a small case series, proof-of-concept was shown for early detection of cancer recurrence. The results indicate that by targeting ultra-short DNA fragments, TR-ctDNA is a viable approach for HPV+ OPSCC detection, and for cancer recurrence monitoring after treatment.

[0053] Described herein is the design, development, and validation of compositions, methods, and systems that offers a precise assay test for detection of HPV-related cancers including cancers of the cervix, vulva, vagina, penis, anus, and oropharynx (e.g., oropharyngeal squamous cell carcinoma (HPV+ OPSCC)) or any other HPV-associated cancer, disease, or condition. Moreover, the assay described herein finds use to predict progressive disease prior to radiographic imaging in patients undergoing treatment (e.g., cancer surveillance, therapy, and / or intervention).

[0054] In some embodiments, a highly sensitive and specific droplet digital PCR (ddPCR) assay for absolute quantification of HPV TR-ctDNA from urine specimens is provided herein. However, any number of different detection methodologies may be used, including semi -digital methodologies (e.g., dilution-based approaches where individual reaction chambers may be zero, one, or a small number (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.; less than 100; less than 50; less than 20; less than 10) of target nucleic acid molecules).

[0055] In some embodiments, the compositions, methods, and / or systems may employ any one or more or all of the oligonucleotide primers / probes described below. These primers / probes may be used in combination with any other diagnostic assays, for example, to detect other HPV types / strains, other cancer or pre-cancer biomarkers, inflammation, etc. Likewise alternative primers and probes that target the same or similar regions of HPV target nucleic acid sequences can be employed. The same design approaches may be used to analyze other HPV strains as well, either alone or in combination with HPV16 targets.

[0056] In some embodiments, any suitable sample type may be used. The sample may be obtained from the subject and subsequently used for any of the methods described herein. Insome embodiments, the sample is obtained from the subject and ctDNA is isolated from the sample for use in the methods described herein. Suitable samples include fluids (e.g. amniotic fluid, ascites, bile, breast milk, breast milk colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysate, eye aqueous humor, eye vitreous humor, feces, paracentesis, pericardial fluid, peritoneal, blood plasma, pleural, semen, blood serum, synovial fluid, tears, thoracentesis, blood, saliva, gargle, or urine, etc.), solids, tissues, and gases. In some embodiments, the sample is blood (e.g., whole blood) or a blood product such as plasma, serum, and the like. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a sample obtained from the mouth of the subject. For example, the sample may be a saliva sample. The term “saliva” or “saliva sample” is meant to include any sample containing saliva from the subject, including spit, an oral swab or sponge sample, a mouthwash rinse sample, etc.1. Definitions

[0057] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular

[0058] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0059] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting

[0060] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0061] As used herein, the term “and / or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and / or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C ”

[0062] “ Correlated to” as used herein refers to compared to.

[0063] As used herein, the terms “providing,” and “introducing,” are used interchangeably herein and refer to the placement into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. For example, the compositions disclosed herein can be provided by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.

[0064] The terms “administration of’ and “administering” a composition as used herein refers to administering a composition of the present disclosure to a subject in need of treatment (e.g., antiviral treatment, anticancer treatment, etc.). The compositions of the present disclosure may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, nebulization, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.

[0065] A “subject” or “patient” are used interchangeably herein and refer to both human and nonhuman animals and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the analysis by the methods, compositions, and systems described herein. In some embodiments, the subject may be a human or a non-human. In one embodiment, the subject is a human. The subject or patient may be undergoing various forms of treatment.

[0066] As used herein, “treat”, “treating”, “treatment”, and variations thereof refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includesthe alleviation or prevention of symptoms, preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection), slowing, stopping, or reversing the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder, or condition. A positive response to treatment may indicate a complete response to treatment, a partial response to treatment, or a stable disease state in the subject. A negative response to treatment may indicate disease progression in the subject. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. For example, treating a cancer may include the management and care of the subject for combating and reducing one or more symptoms of the cancer. For example, treating cancer may reduce tumor burden (e.g., reduce the size of one or more tumors in the subject afflicted with cancer and / or reduce the overall number of tumors in the subject afflicted with cancer). Treating a cancer may reduce or completely eliminate the cancer (e.g., completely eliminate the tumor) in the subject.

[0067] As used herein, the term “immunotherapy” refers to any type of cancer treatment that helps the immune system fight cancer. For example, “immunotherapy” may include treatment with an immune checkpoint inhibitor, T-cell transfer therapy, monoclonal antibodies, treatment vaccines, and / or immune modulators. The cancer may be any cancer type.

[0068] As used herein, a method of predicting a response to treatment in a subject may include measuring a baseline level of cancer associated nucleic acid (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA) etc.), and / or trans-renal cell-free tumor DNA (TR- ctDNA) in the subject. The baseline level of said nucleic acid may be measured prior to any treatment in the subject. Alternatively, the baseline level of said nucleic acid may be measured following one or more treatment sessions in the subject.

[0069] The method may further include measuring a follow-on level of cancer associated nucleic acid (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), and / or trans-renal cell-free tumor DNA (TR-ctDNA), etc.) following one or more treatment sessions in the subject. For example, a baseline level of said nucleic acid may be measured prior to any therapy and a follow-on level may be measured following a therapy cycle. Alternatively, a baseline level of said nucleic acid may be measured after a first therapy cycle and a follow-on level may be measured following a second therapy cycle.

[0070] The baseline level and / or follow on level of said nucleic acid may be measured using a suitable method described herein. For example, measuring a baseline level and measuring a follow-on level may include obtaining a sample comprising nucleic acid from the subject.

[0071] The nucleic acid may be isolated from urine obtained from the subject. For example, urine may be obtained and nucleic acid may be isolated using any suitable method. In some embodiments, nucleic acid is isolated using a commercially available kit.

[0072] In some embodiments, the method further involves predicting response to therapy in the subject based upon analysis of HPV (e.g., HPV16) nucleic acid (e.g., viral nucleic acid, cell-free tumor-derived DNA (ctDNA), and / or trans-renal cell-free tumor DNA (TR-ctDNA), etc.) or nucleic acid levels. In some embodiments, the method comprises predicting a positive response to treatment in the subject when a change from the baseline level to the follow-on level is below a threshold value. In some embodiments, the method comprises predicting a negative response to treatment in the subject when a change from the baseline level to the follow-on level is above a threshold value. In some embodiments, the threshold value is 50%. In some embodiments, the threshold value is 60%. For example, the method may comprise predicting a positive response to treatment when there is less than a 60% increase in HPV nucleic acid levels from the baseline level to the follow-on level. Alternatively, the method may comprise predicting a negative response to treatment when there is a 60% or higher increase in HPV nucleic acid levels from the baseline level to the follow-on level. A positive response to treatment may indicate a complete response to treatment, a partial response to treatment, or a stable disease state in the subject. For example, a positive response may indicate no metastasis, no increase in tumor size, and / or no increase in total number of tumors observed in the subject. A negative response may indicate progression of the disease, such as increased total number of tumors and / or increased tumor size.

[0073] An “amplicon” refers to a nucleic acid fragment formed as a product of natural or artificial amplification events or techniques. For example, an amplicon can be produced by PCR.

[0074] As used herein, “amplify”, “amplifying”, “amplification”, and variations thereof refer to method of increasing the number of copies of a target sequence, if present in a sample. In some embodiments, the method further comprises detecting a signal from a detectable label, which is indicative of the presence of the target sequence, if present in the sample. For example, amplification may be performed by polymerase chain reaction (PCR).

[0075] As used herein, “polymerase chain reaction” (PCR) refers to amplification and “quantitative PCR” (qPCR) refers to a method of quantifying the number of copies of theamplified sequence. For example, amplification and quantification of a target sequence may be performed using a digital PCR technique, such as a dPCR technique selected from droplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification, and magnetic), and microfluidic chips.

[0076] As used herein, “digital PCR” refers to an assay that provides an end-point measurement that provides the ability to quantify nucleic acids without the use of standard curves, as is used in real-time PCR. In a typical digital PCR experiment, the sample is randomly distributed into discrete partitions, such that some contain no nucleic acid template and others contain one or more template copies. The partitions are amplified to the terminal plateau phase of PCR (or end-point) and then read to determine the fraction of positive partitions. If the partitions are of uniform volume, the number of target DNA molecules present may be calculated from the fraction of positive end-point reactions using, for example, Poisson statistics, according to the following equation: =-l«(l- ) (1) wherein X is the average number of target DNA molecules per replicate reaction and p is the fraction of positive end-point reactions. From k, together with the volume of each replicate PCR and the total number of replicates analyzed, an estimate of the absolute target DNA concentration is calculated. Digital PCR includes a variety of formats, including droplet digital PCR, BEAMing (beads, emulsion, amplification, and magnetic), and microfluidic chips.

[0077] As used herein, “droplet digital PCR” (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011). A single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well. A “droplet” or “water-in-oil droplet” refers to an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011).

[0078] Droplet digital PCR may be performed using any platform that performs a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. The strategy for droplet digital PCR may be summarized as follows: a sample is diluted and partitioned into thousands to millions (or more) of separate reaction chambers (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule ofinterest. The number of “positive” droplets detected, which contain the target amplicon (e.g., nucleic acid molecule of interest), versus the number of “negative” droplets, which do not contain the target amplicon (e.g., nucleic acid molecule of interest), may be used to determine the number of copies of the nucleic acid molecule of interest that were in the original sample. Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanolitersized droplets; the QX200™ Droplet Digital PCR System by Bio-Rad; and the RainDrop™ digital PCR system by RainDance, which partitions samples containing nucleic acid template into 1,000,000 to 10,000,000 picoliter-sized droplets.

[0079] As used herein, “detectable label” refers to a fluorescent label affixed to an oligonucleotide. For example, suitable fluorescent labels include, FAM (5- or 6- carboxyfluorescein), VIC, NED, Fluorescein, FITC, IRD-700 / 800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor PET, Biosearch Blue™, Marina Blue®, Bothell Blue®, Alexa Fluor®, 350 FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488 JOE™, VIC™ HEX™ TET™, CAL Fluor® Gold 540, Yakima Yellow®, ROX™, CAL Fluor® Red 610, Cy3.5™, Texas Red®, Alexa Fluor® 0.568 Cy5™, Quasar™ 670, LightCycler Red640®, Alexa Fluor 633 Quasar™ 705, LightCycler Red705®, Alexa Fluor® 680, SYTO® 9, LC Green®, LC Green® Plus+, and EvaGreen™. Further, a detectable label is used to detect the presence of a specific molecule in a sample wherein detection of the signal indicates the presence of the target sequence. Quantification of the target sequence may be performed, for example, by determining the number of target molecules present. The number of target molecules present may be calculated from the fraction of positive end-point reactions using Poisson statistics, as described above. The methods of detecting and / or quantifying the target sequence may be used in a variety of diagnostic or predictive methods. For example, the methods may be used to determine whether a subject has cancer and / or for predicting a subject’s response to therapy in a cancer.

[0080] As used herein, “at least 90% sequence identity” refers to an oligonucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the referenced sequence.

[0081] As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s),element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated.

[0082] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.

[0083] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0084] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleicacid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The term “oligonucleotide,” or “oligos,” as used herein, generally refers to a short nucleic acid sequence comprising from about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values) Any of the oligonucleotide sequences described herein may comprise, consist essentially of, or consist of a complement of any of the sequences disclosed herein.

[0085] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, engineered, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.2. Examples

[0086] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0087] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.Example 1

[0088] Materials and Methods.

[0089] Sample collection and cfDNA isolation. Blood was collected from patients and healthy controls into either K2EDTA tubes or Streck Cell-Free DNA BCT. Blood was processed into plasma by double centrifugation at 1,600 x g for 10 minutes, followed by 16,000 x g for 10 minutes, all at room temperature and stored at -80 °C until DNA isolation. Plasma cfDNA was isolated with QIAamp Circulating Nucleic Acid Kit (Qiagen #55114).

[0090] Urine was collected from patients and healthy controls into 500 mL bottles containing 100 mL 0.5 M EDTA (pH 8.0) as the preservative. Urine supernatant was separated from cellular debris by either double centrifugation at 1,600 x g for 10 minutes, followed by 3,000 x g for 10 minutes, or a single centrifugation at 3,000 x g for 10 minutes, followed by filtering through a 0.45 pm polyethersulfone membrane (e.g., Fisher #1690045). The resultant supernatant was either frozen in 50 mL aliquots at -80 °C or progressed for urine cfDNA isolation, and frozen at an intermediate step when bound to Q Sepharose resin (Cytiva #17051001). Urine cfDNA was isolated from a 50 mL aliquot using a Q Sepharose resin based binding strategy as described below in more detail. A 100 bp synthetic, plant-derived nonhuman DNA duplex referred to as “plant spike-in”, was spiked into each 50 ml urine aliquot toassess urine cfDNA extraction efficiency. High molecular weight DNA (> 500 bp) was removed using AMPure XP beads, based on a previously reported protocol.

[0091] Longitudinal stabilization of cfDNA in urine using EDTA. Urine samples were collected from healthy control participants and 0.5M EDTA (pH 8.0) was added at the time of collection to a final concentration of 100 mM. A GeneRuler Ultra Low Range DNA ladder was added to the urine sample, homogenized by inversion, and urine aliquoted into 250 mL tubes stored at room temperature for 0, 3, 5, or 7 days. Day 0 samples had cfDNA isolated immediately. Urine samples were processed and cfDNA isolated using a resin based binding strategy described above. DNA ladder bands were measured using a DI 000 Bioanalyzer chip.

[0092] Whole genome sequencing. Low-pass coverage WGS of urine cfDNA was performed using a ssDNA library preparation protocol. Corresponding plasma cfDNA low- pass WGS libraries were prepared from matched plasma cfDNA using methods reported previously. Data was analyzed using a previously reported pipeline for detecting somatic tumor-associated copy number alterations (CNA) and estimation of % tumor content (also referred to as % tumor DNA in the main text), which utilized a copy number calling algorithm based on comparison to reference control DNA samples. Low-pass coverage WGS and computational data analysis methods were used to analyze cfDNA isolated from n = 15 urine specimens collected from patients with diverse cancer types (multiple samples were collected from some patients, on different days), in whom plasma ctDNA and CNA were known to be detectable by low-pass WGS. Urine WGS results were stratified by inferred cfDNA fragment length into four sliding 20 bp-wide bins (50-70 bp, 40-60 bp, 30-50 bp, 20-40 bp) and the amplitude of tumor-associated CNA signal in each of them plotted. For 6 patients where there was sufficient read depth and cancer-associated CNA (detected via paired tissue and / or plasma WGS), mapped single-stranded urine cfDNA WGS reads were analyzed by stratifying the inferred cfDNA fragment lengths based on the local CNA status (gain, loss, partially amplified, unaltered).

[0093] Urine cfDNA isolation. Urine cfDNA was extracted from a 50 mL aliquot using a protocol adapted from a previously described method. Briefly, 10 pl of plant spike-in synthetic DNA (at 5000 copies / pl) serving as urine cfDNA extraction control, and 0.5 ml Q Sepharose resin slurry (Cytiva #17051001), were added to each 50 ml urine aliquot and rotated at room temperature for 30 minutes. The Q Sepharose resin bound DNA was collected by centrifugation at 1,800 x g for 5 minutes at room temperature, supernatant removed, and washed with 4 ml Tris-EDTA (TE) buffer (Fisher #BP24731). The resin was collected by centrifugation asdescribed above, and excess TE buffer was carefully aspirated out. The resin was then suspended in 0.5 ml of 95% ethanol and frozen at -80 °C until DNA isolation.

[0094] For DNA isolation, the resin was transferred to a Micro Bio-Spin column (Bio-Rad #7326204) and the ethanol removed by centrifugation at 1,000 x g for 30 seconds. The resin was then washed three times with 600 pl of resin wash buffer (10 mM sodium acetate (pH 5.2) + 0.3M lithium chloride) by centrifugation for 1 minute at 800 x g. DNA was eluted twice by adding 450 pl of resin elution buffer (10 mM sodium acetate (pH 5.2) + 2M lithium chloride) to the column each time and centrifugation for 3 minutes at 800 x g. Each 450 pl elute was precipitated by adding 3 volumes of 95% ethanol (1.35 ml) and incubating at room temperature for a minimum of 5 minutes. The mixture (700 pl maximum per spin) was applied onto a QIAquick spin column (Qiagen #28115) for binding of cfDNAby centrifugation at 10,000 rpm for 1 minute, flow-through discarded and repeated until all the mixture was applied. The column was washed twice with 500 pl of column wash buffer 1 ((10 mM sodium acetate (pH 5.2) + 2M lithium chloride) in 70% ethanol final) and twice with 500 pl of column wash buffer 2 (75 mM potassium acetate (pH 5.0) in 80% ethanol final), by centrifugation at 10,000 rpm for 30 seconds each time. Residual ethanol was removed by centrifugation at 10,000 rpm for 3 minutes in a fresh collection tube. cfDNA was eluted by transferring the column to a fresh 1.5 ml tube and adding 53 pl buffer EB (Qiagen), followed by centrifugation at 10,000 rpm for 2 minutes. cfDNA was stored at -20 °C or processed for isolation of low molecular weight (LMW) fraction (< 500 bp) as described below.

[0095] For separation of the high molecular weight (HMW) DNA fraction (> 500 bp) from LMW fraction, 50 pl of cfDNA elute was incubated with an equal volume of suspension buffer (0.6 M sodium chloride + 16% polyethylene glycol 8000) and 10 pl of AMPure XP bead suspension (Beckman Coulter #A63880) for 2 hours at room temperature in a ThermoMixer (Eppendorf), which was programmed to perform agitation at 1,400 rpm for 3 seconds of each one minute segment. The AMPure XP beads bound to HMW cfDNA fraction were then pelleted by incubation on a magnetic rack (Fisher #12321D). With the tubes still on the rack, a 105 pl suspension containing LMW cfDNA fraction was carefully transferred to a 1.5 ml tube without disturbing the pellet, and kept on ice for further processing. The AMPure XP beads were washed with 150 pl of 75% ethanol and HMW cfDNA fraction eluted in 50 pl TE buffer.

[0096] To the 105 pl LMW cfDNA suspension kept on ice, following were added: 20 pl of 1.5 M Sodium Chloride, 30 pl of 50 mM Magnesium Chloride, 1.5 pl of 15 mg / ml GlycoBlue, and finally 300 pl of 100% ethanol, and incubated on ice overnight to precipitate the cfDNA. LMW cfDNA pellet was recovered by centrifugation at 16,100 x g for 30 min at 4 °C andwashed with 80% ethanol by centrifugation at the same speed for 15 min at 4 °C. The pellet was dried to remove residual ethanol by incubating the tube with the lid open at room temperature for 10 minutes. 25-50 pl buffer EB was added to the pellet (and more specifically for ddPCR, samples were always eluted using 25 pl buffer EB) and it was resuspended by incubation at room temperature. cfDNA was quantified using the Qubit dsDNA HS Assay kit (Fisher #Q32854). 19 pl of the 25 pl LMW cfDNA elute was used as template for ddPCR reaction set up in triplicates.

[0097] Droplet digital PCR. All ddPCR experiments were performed using the QX200 Droplet Digital PCR System (Bio-Rad). Each 20 ul reaction mix was partitioned into droplets using the QX200 droplet generator (Bio-Rad), transferred into a 96-well plate, sealed, and cycled in a Cl 000 Thermal Cycler (Bio-Rad). Droplets were read using QuantaSoft Software in the QX200 reader (Bio-Rad). A short-amplicon two stage stem-loop ddPCR reaction was designed as previously described with sequences and PCR cycling conditions provided in FIG. 6. For the PCR set up, a 21.5 pl of sample reaction mix was prepared containing primer 3 and primer 2 at a final concentration of 900 nM each, stem-loop primer 1 and FAM-MGBNFQ probe at a concentration of 250 nM each and the IX ddPCR supermix (Bio-Rad #1863024), of which 20 pl was used for droplet generation. After PCR cycling, the plate was removed from the thermocycler only after the lid temperature fell below 50°C, followed by incubation at room temperature for 5 minutes before proceeding with the droplet reading. The 77 bp HPV ddPCR assay (Table 1) was performed as described below. Primers and synthetic DNA templates were procured from Integrated DNA Technologies and TaqMan probes were procured from ThermoFisher Scientific.

[0098] In Table 1, the 42 bp amplicon, ultra-short TR-ctDNA stem-loop ddPCR assay was compared to a published, conventional ctDNA assay of 77 bp amplicon length for the detection of HPV16 TR- ctDNA in urine specimens. DNA extracted from 60 mL urine per patient was analyzed, corresponding to n=l l patients with early stage (E) or metastatic (M) HP VI 6+ OPSCC. The table shows the number of total human genome equivalents of urine cfDNA analyzed for each patient, along with the number of copies of HP VI 6 TR-ctDNA detected using either the 42 bp amplicon or 77 bp amplicon assay. The loss of signal associated with the 77 bp assay when compared to the 42 bp ultra-short amplicon stem-loop assay is evident from the difference in copies of HP VI 6 TR-ctDNA detected. Control samples are water, normal human genomic DNA (which should have no HPV16) and genomic DNA from the HPV16+ HNSCC cell line UM-SCC-104. Both assays robustly detected HPV16 from the positive control cell line (which represents long DNA), with the 77 bp assay even showing higher copies(884 vs. 582) of HPV16 measured in this positive control sample compared to the ultra-short amplicon assay

[0099] Table 1: Comparison of urine HPV16 TR-ctDNA detection using an ultra-short amplicon stem-loop ddPCR assay vs. a conventional ctDNA ddPCR assay.

[0100] Stem-loop HPV16 TR-ctDNA assay validation and data analysis. The stem-loop ddPCR assay was validated using a synthetic ultra-short HP VI 6 DNA duplex with the strand sequence: 5’AATGC GTTTC AGGAC CCACA GGAGC GACCC AGAAA GTTAC CACAG TTCACT 3’ (SEQ ID NO: 11) which includes five (5) bp non-HPV flanking sequences on each end (indicated in Bold typeface), or using genomic DNA (gDNA) from the HPV+ head and neck cancer cell line UM-SCC-104 digested with Hindlll restriction enzyme (FIG. 2B). LoD of the stem-loop assay was determined to be 1.4 copies per 20 pl reaction (i.e.,4.2 copies cumulative for ddPCR triplicates) and was calculated using the formula LoD = Limit of Blank (LoB) + 1 ,645(Standard Deviation iowconcentration sample) based on the linearity plots. LoB was determined using 200,000 haploid genomes of Hindlll digested non-HPV hgen DNA template (blank) background matrix per 20 pl ddPCR reaction set up in triplicates (n = 40) using the formula LoB = mean blank + 1.645 (Standard Deviation blank).

[0101] For urine samples, low molecular weight cfDNA (equivalent to a 30 ml urine aliquot) from two different urine collection bottles (for results shown in FIG. 2), or from 2-4 bottles per time point (for results shown in FIG. 3) was tested in triplicates. Different bottles in this case represent separate urine collections done on the same day, or within 3 days of each other for a given time point. Data from each aliquot (representing each bottle) was averaged to determine the HPV16 TR-ctDNA values. There was one exception (patient #32E), for which a sample from only one urine bottle was tested (in triplicate ddPCR) (FIG. 2C and FIG. 2D). For plasma samples, cfDNA equivalent to ~0.9 ml aliquot was tested in triplicates and the results were used to compare the HP V 16 values in plasma to urine samples (FIG. 2C and FIG. 2D) In one case (patient #25E), although both urine and plasma were positive for HPV16 ctDNA, the plasma total cfDNA extracted showed an anomaly: its abundance was 41 standard deviations higher than the median plasma total cfDNA values from all other patients in the same batch (n=18), and the HPV ctDNA quantification was inaccurate due to signal saturation, and hence this sample was not included in the correlation analysis (FIG. 2D). Of the 5 patient samples with HPV16 ctDNA values below the LoD in urine and / or plasma (FIG. 2C), for 4 patients no signal was detected in urine and / or plasma, and a nominal value of 1 was assigned, to be able to plot the correlation on a logic scale (FIG. 2D).

[0102] Statistics. To determine the correlation between HP VI 6 copies detected in urine TR-ctDNA and plasma ctDNA, statistical analysis was performed using GraphPad Prism 9.0, and a two-tailed p<0.05 was considered significant.Example 2

[0103] TR-ctDNA is readily stabilized at the point-of-collection with EDTA and effectively isolated using a Q Sepharose resin based strategy. While some work has been done on urine DNA stabilization and isolation methods, there is no accepted consensus yet as to optimal methods, especially with respect to TR-ctDNA. For urine DNA stabilization at the point of collection, the ability of EDTA to effectively preserve spiked-in DNA fragments of varying size in healthy donor human urine was tested. It was found that EDTA at 100 mM final concentration was able to prevent degradation of all size fragments, compared to the rapiddegradation seen within 1 hour when no preservative is added (FIG. 4A and FIG. 4B). Additionally, the ability of EDTA to stabilize DNA fragments in urine samples for up to 7 days, which is important for being able to use urine samples collected at home and mailed to the lab, was tested. It was found that the immediate addition of EDTA (final concentration 100 mM) to urine samples at the point of collection preserved DNA fragments of diverse sizes for at least 7 days (FIG. 4C and FIG. 4D).

[0104] For urine DNA extraction, we tested a previously developed Q Sepharose resinbased TR-ctDNA isolation strategy using an ultra-low range DNA ladder spiked into healthy control urine samples was tested. A magnetic bead-based approach to remove high molecular weight DNA (>500 bp), while retaining lower molecular weight DNA, was further tested. It was found that these strategies were able to effectively isolate and separate short DNA fragments from urine samples (FIG. 5).Example 3

[0105] TR-ctDNA is ultra-short and detectable in multiple cancer types. To test the hypothesis that TR-ctDNA is ultra-short, a single- stranded DNA (ssDNA) NGS library preparation protocol that has been used for sequencing of highly degraded, “ancient DNA” from fossils was adopted. This library preparation method captures ultra-short DNA fragments that are missed by the more commonly used, double-stranded DNA library preparation approaches. To determine the size profile of TR-ctDNA in patients with non-urologic cancers, a ssDNA library preparation coupled with low-pass whole genome sequencing (WGS) to characterize cfDNA isolated from 22 urine specimens, representing urine collections from cancer patients (n=15) with a variety of solid tumors (breast, lung, colon and rectal) or leukemias (AML) was employed. These patients were all known to have ctDNA in the blood, based on detection of tumor-associated copy number alterations (CNA) from sequencing of contemporaneously collected plasma cfDNA.

[0106] The urine WGS data, either unfiltered for DNA fragment length or by restricting fragment length to select size bins, by using a peak-calling algorithm to infer genome-wide tumor CAN was analyzed. The mapping of fragments to regions of CNA served as indication of their tumor-derived origin. Restricting analysis to ultra-short fragments (i.e., 30-50 bp, 20- 40 bp length) enhanced the detection of CNA profiles that were concordant with the CNA patterns observed from plasma cfDNA WGS sequencing data (example CNA plots from three patients are shown in FIG. 1A). Based on the peak-calling algorithm, the percentage of DNA fragments corresponding to tumor DNA (based on their mapping to regions of CNA), as hasbeen reported for plasma ctDNA analysis was also quantified. Performing this analysis on the range of fragment size bins showed a progressive enrichment in % tumor DNA in most cases, with ultra-short bins (i.e., <50 bp) showing the highest fractional representation of tumor DNA (FIG. IB)

[0107] For six of the patients, there were sufficient copy number alterations and read depth to perform a direct fragment length analysis of the genomic regions of cancer-associated CNA, which represent regions that are preferentially derived from tumor DNA in the sample, as compared to non-altered regions. Through this analysis, it was confirmed that the DNA corresponding to cancer-associated CNA regions is ultra-short (<50 bp, with median length ~30 bp) (FIG. 1C).Example 4

[0108] Ultra-short amplicon ddPCR-based analysis of urine TR-ctDNA detects HPV+ OPSCC and shows strong concordance between urine-based and plasma-based ctDNA detection. The results suggested that sensitive detection of TR-ctDNA by ddPCR, which offers the benefits of absolute quantification, high precision, and speed, would require assays designed to target ultra-short fragments (e.g., <50 bp). Conventional ddPCR assays for ctDNA target amplicons >50 bp in length, which, it was hypothesized, may miss the majority of TR- ctDNA due to its ultra-short nature. To test this hypothesis, the effectiveness of HPV16 TR- ctDNA detection in urine from HP VI 6+ OPSCC patients using a custom-designed ultra-short amplicon ddPCR assay vs. a conventional length ctDNA ddPCR assay was compared.

[0109] For the ultra-short amplicon assay, a stem-loop 2-stage PCR approach that allowed for the development of a 42 bp amplicon ddPCR assay (FIG. 2A) was used. It was designed to target the HPV16 E6 gene (FIG. 6), and measure TR-ctDNA in HPV+ OPSCC patients. The HPV 16 E6 gene represents a highly recurrent ctDNA target in the population of HPV+ OPSCC patients, and HPV16 in general provides a high signal -to-noise ratio because of its low sequence similarity to endogenous human genomic sequences that could be the source of “background” signal.

[0110] Table 2: Sequences selected for the ultra-short amplicon assay

[0111] Analytical validation of this assay was performed and was found to have exceedingly low background (i.e., high specificity), providing a Limit of Detection (LoD) of ~4 molecules in a sample, and the sensitivity to detect the presence of HPV16 ctDNA at a representation of <0.01% tumor DNA (i.e., one copy of HPV16 ctDNA in a background of over 10,000 wildtype human genomes) (FIG. 2B). In a series of matched plasma and urine samples (n=32) obtained from patients with either locally advanced (e.g.., early stage) or metastatic disease, prior to the start of treatment and known to be pl6 positive in tissue immunohistochemistry (a surrogate for any HPV subtype-associated OPSCC), we found that HPV16 ctDNA could be consistently detected in both urine and plasma using the 42 bp amplicon assay in 27 of 32 patients (FIG. 2C). In two patients, HPV16 ctDNA was detected only in plasma, and in one patient it was detected only in urine (FIG. 2C). In the remaining two patients, HPV16 ctDNA was undetectable in both urine and plasma (FIG. 2C). HPV16 ctDNA was undetectable in urine from 11 of 11 control individuals who did not have HPV+ cancer and in 1 control (patient #75E) with OPSCC related to the HPV18 subtype rather than HPV16 (FIG. 2C). For HPV+ OPSCC patients, HPV16 ctDNA abundance values in urine and plasma were found to be positively correlated (FIG. 2D).

[0112] As an additional confirmation of our conclusion from urine cfDNA sequencing studies that TR-ctDNA is ultra-short, results from the ultra-short amplicon assay in urine with a validated, conventional length plasma HPV16 ctDNA assay, targeting a 77 bp amplicon was compared. The ultra-short 42 bp assay detected TR-ctDNA in all patient samples studied, whereas the longer conventional 77 bp assay yielded undetectable or extremely low values of HPV16 TR-ctDNA in all the urine samples (Table 1).Example 5

[0113] Proof-of-concept for urine TR-ctDNA based post-treatment surveillance for early detection of HPV+ OPSCC recurrence. After clinically validating the HPV16 urine assay in the specimens described above, the dynamics of urine TR-ctDNA in a small pilot cohort of patients treated for HPV+ OPSCC with curative therapy, who had biopsy-proven recurrence within the first year after treatment was assessed. To do so, ddPCR was performed on urine samples from four patients for whom longitudinal urine samples were available, including at least one sample within 3 months prior to the date of clinical recurrence diagnosis by imaging and / or tissue biopsy (FIG. 3). Three of these patients had been treated with chemoradiation for locally advanced disease, whereas one had been treated by surgical resection. In three of the four patients (Patients 1, 3 and 4), HPV16 TR-ctDNA was detected earlier than clinical diagnosis of recurrence (median of 3.2 months earlier). In the case of Patient 2, HPV16 TR- ctDNA was not detected during the surveillance period. However, it is worth noting that urine samples were only available until 2.8 months before clinical recurrence for this patient.Example 6

[0114] The Examples included herein report that TR-ctDNA is ultra-short and is readily detectable using both NGS and ddPCR approaches when utilizing methods capable of evaluating ultra-short fragments. A ssDNA library preparation and NGS protocol capable of assessing genome-wide somatic copy-number profdes from TR-ctDNA in routine urine samples from patients with hematological and solid tumor malignancies was demonstrated. The TR-ctDNA signal is enriched when restricting analysis to the ultra-short (20-40 bp) fragments was determined. A custom ultra-short stem-loop ddPCR assay was then developed and optimized that allowed for absolute quantification of tumor-derived ctDNA copies in urine from patients with HPV16+ OPSCC with a simpler and faster assay. Furthermore, proof-of- concept was shown that TR-ctDNA can be used to monitor for and detect cancer recurrence through serial non-invasive urine sampling. This points toward how a urine-based TR-ctDNAassay can be paired with traditional imaging and clinical workflows, opening up the potential to provide earlier detection of recurrence and improved outcomes.

[0115] This work highlights the importance of utilizing detection methods compatible with the ultra-short nature of TR-ctDNA. Compared to dsDNA library preparation approaches that only capture longer DNA fragments, this ssDNA library preparation approach allowed capture of TR-ctDNA fragments that would otherwise have been missed. Indeed, previous NGS and PCR investigations into TR-ctDNA that reported different results than these appear to have utilized approaches that did not capture ultra-short fragments. Herein, it is proposed that the lack of knowledge about the ultra-short nature of cancer-derived trans-renal DNA fragments in urine may contribute to the relatively limited study of TR-ctDNA in the literature to date.

[0116] This ddPCR approach demonstrated strong concordance of detection overall between urine and plasma samples for measuring HPV16 ctDNA in OPSCC patients. Based on these results, a model is proposed in which circulating tumor DNA in blood, which at steadystate levels is predominantly in the 150-160 bp size range, is also being processed to smaller fragments (e.g., <50 bp) that are small enough to pass through the glomerular barrier of the kidney into the urine. It is expected that this urine cell-free DNA extraction process will avoid detection of HPV DNA from viral particles during active HPV infection, since it removes DNA fragments >500 bp.

[0117] Urine has many appealing features as a biospecimen type for cancer detection and monitoring, including that it is a fully non-invasive biofluid that is easy to collect at home and can be shipped to a lab, which could help enable healthcare access to populations of people who face challenges with being able to access healthcare facilities (e.g., phlebotomy). Furthermore, urine can be collected more frequently and at a large volume compared to blood, and generally confers a lower biohazard risk. Frequent collection (e.g., daily) allows for TR- ctDNA kinetics to be used as a high time-resolution biomarker for treatment-response monitoring, while collecting larger volumes of urine could be especially important for increasing sensitivity of cancer detection in low disease-burden applications such as early detection of cancer, detection of minimal residual disease (MRD) after therapy, as well as early detection of disease recurrence.

[0118] In some embodiments, assays are employed that detect even shorter fragments (e.g., adapting principles used in approaches for microRNA analysis, where the target nucleic acid is typically ~22 nt long) demonstrating that detecting ultra-short cancer-derived DNA fragments facilitates the full potential of TR-ctDNA analysis for non-invasive, sensitive, and broadly accessible cancer detection and monitoring for a variety of clinical applications.Example 7

[0119] It was found that trans-renal urine HPV16 ctDNA shows relatively low sample-to- sample variability (on average <0.5-fold variation) across multiple urine samples collected from the same patient over a few days. An analysis of sample-to-sample variability of trans- renal urine HP VI 6 ctDNA detection was performed by asking 8 patients with HPV+ OPSCC to each self-collect 400 ml of urine at up to 6 different time points, over a span of 4 days or less. Table 3, below, shows HPV16 ctDNA analysis from up to 6 different collection bottles, sampling cfDNA corresponding to 30 ml urine from each bottle. Values in the table depict deviation from the mean of HPV16 ctDNA abundance calculated across the 6 bottles, without any normalization. It was found that the sample-to-sample variation in trans-renal urine HPV 16 ctDNA levels is relatively low, with an average deviation of <0.5-fold of the mean value across all 5 to 6 samples from a given patient. This average deviation was not further enhanced by normalization of the HPV16 ctDNA copies to measured creatinine and / or total number of cfDNA genome equivalents tested in ddPCR assay. The observed low degree of biological variability supports the use of trans-renal HPV ctDNA in urine for early cancer detection and longitudinal surveillance.Table 3: Day-to-Day Variability of HPV16 ctDNA Copies In Urine From Patients WithHPV+ OPSCC.

Claims

CLAIMSWhat is claimed is:

1. A method comprising: detecting HPV-associated nucleic acid having less than 50 nucleotides in a urine sample from a subject.

2. The method of claim 1, wherein the detecting comprises nucleic acid amplification using at least one hairpin primer.

3. The method of claim 2, wherein the detecting comprises digital amplification.

4. The method of claim 3, wherein the digital amplification comprises digital PCR.

5. The method of claim 2, wherein the amplification comprises contacting the sample with the hairpin primer and a second primer.

6. The method of claim 5, wherein the detecting comprises contacting amplicons generated by the hairpin primer and the second primer with one or more probes.

7. The method of claim 1, wherein the HPV-associated nucleic acid is a HPV16- associated nucleic acid.

8. The method of claim 1, wherein the subject has previously been diagnosed as having an HPV infection or HPV-associated disease or condition.

9. The method of claim 1, wherein the subject has been treated with a vaccine, an antiviral drug, or an anticancer therapy.

10. A composition comprising one or more oligonucleotides that detect one or more HPV target regions, wherein the oligonucleotides comprise a sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 10, or sequences having at least 90% sequence identity thereto.

11. The composition of claim 10, wherein the one or more oligonucleotide primer / probe sets is selected from the group consisting of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, and 10 and comprising a 5’ methyl iso-deoxy cytosine (Me-isodC); a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5; a second reverse primer at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

12. The composition of claim 10, wherein the one or more oligonucleotides comprise two or more of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, and 10 and comprising a 5’ methyl iso-deoxy cytosine (Me-isodC); a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5; a second reverse primer having at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

13. The composition of claim 10, wherein the one or more oligonucleotides comprise each of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NOs: 1, 2, 3, 4, and 10 and comprising a 5’ methyl iso-deoxy cytosine (Me-isodC); a first reverse primer having at least 90% sequence identity to SEQ ID NO: 5; a second reverse primer having at least 90% sequence identity to SEQ ID NO: 6; and a probe having at least 90% sequence identity to SEQ ID NO: 7 and comprising a detectable label.

14. The composition of any one of claims 11-13, wherein the probe further comprises a MGB moiety and a nonfluorescent quencher.

15. The composition of any one of claims 11-13, wherein the forward primer and the first reverse primer anneal to a target HPV sequence at a first low annealing temperature and wherein the second reverse primer anneals to the loop region of the forward primer at a second low annealing temperature.

16. The composition of claim 15, wherein the first low annealing temperature is about 49 degrees Celsius.

17. The composition claim of any one of claims 15-16, wherein the second low annealing temperature is about 60 degrees Celsius.

18. The composition of any one of claims 10-17, wherein the one or more target regions is selected from the group consisting of: SEQ ID NO: 8 and SEQ ID NO: 9.

19. The composition of any one of claims 10-18, wherein the one or more target regions is the HPV16 E6 gene.

20. A method comprising contacting a sample with a composition of any one of claims 10-18.

21. The method of claim 20, wherein the sample is amniotic fluid, ascites, bile, breast milk, breast milk colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysate, eye aqueous humor, eye vitreous humor, feces, paracentesis, pericardial fluid, peritoneal, blood plasma, pleural, semen, blood serum, synovial fluid, tears, thoracentesis, blood, saliva, gargle, or urine.

22. The method of any one of claims 20-21, wherein the contacting comprises: (i) providing one or more of the oligonucleotide primer / probe sets; (ii) fractionating a plurality of HPV DNA from the sample into droplets at a concentration wherein, on average, only 0 or 1 molecule of the DNA is present in each droplet; (iii) amplifying HPV DNA in each droplet with the primer / probe sets to produce amplicon signals; and (iv) detecting in each droplet any amplicon signals.

23. The method of any one of claims 20-22, wherein DNA is fractionated into microdroplets by emulsification.

24. The method of any one of claims 20-23, wherein DNA is amplified using a nucleic acid amplification method.

25. The method of claim 24, wherein the nucleic acid amplification method comprises (i) polymerase chain reaction; (ii) loop mediated isothermal amplification; (iii) nucleic acid sequence based amplification, (iv) strand displacement, amplification; (v) multiple di sp 1 acement amplificati on.

26. The method of claim 20, wherein the contacting comprises conducting a quantitative PCR (qPCR) assay.

27. The method of claim 26, wherein the qPCR assay comprises a digital PCR assay.

28. The method of claim 27, wherein the digital PCR assay comprises a droplet digital PCR (ddPCR) assay.

29. The method of any one of claims 20-21, wherein the contacting comprises: (i) providing one or more of the oligonucleotides; (ii) immobilizing one or more of the oligonucleotides onto a surface; (iii) repeatedly transient binding HPV DNA in the sample to the one or more oligonucleotides bound to the surface to produce a kinetic fingerprint; and (iv) detecting any kinetic fingerprints.

30. The method of claim 29, wherein the one or more of the oligonucleotides is a biotinylated probe.

31. The method of claim 29, wherein the one or more of the oligonucleotides is a biotinylated probe with a detectable label having at least 40% sequence identity to SEQ ID NO: 7 and is immobilized on a streptavidin-coated surface.

32. The method of claim 29, wherein the one or more of the oligonucleotides is a biotinylated probe with a detectable label having at least 60% sequence identity to SEQ ID NO: 7 and is immobilized on a streptavidin-coated surface.

33. The method of claim 29, wherein the one or more of the oligonucleotides is a biotinylated probe with a detectable label having at least 90% sequence identity to SEQ ID NO: 7 and is immobilized on a streptavidin-coated surface.

34. The method of any one of claims 20-33, wherein the sample comprises a ctDNA or a TR-ctDNA HPV target nucleic acid and wherein one or more of the oligonucleotides hybridize to the HPV target nucleic acid.

35. The method of any one of claims 20-34, further comprising a step of treating a subject, if HPV is detected in the sample, with cancer surveillance, therapy, or intervention.

36. The method of claim 35, wherein the step of treating a subject is provided prior to the appearance of CT scan-detectable disease.

37. The method of any one of claims 20-34, further comprising a step of treating a subject, if HPV is not detected in the sample, with an HPV vaccine.

38. The method of any one of claims 20-37, further comprising sequencing HPV nucleic acid present in the sample.

39. A kit comprising a composition of any one of claims 10-19 and reagents to purify, isolate, detect, and / or quantify HPV ctDNA.

40. The kit of claim 39, wherein the reagents comprise: (i) detection reagents; (ii) control samples; (iii) solid surfaces comprising capture reagents specific for target ctDNA; (iv) containers for holding or storing a sample; (v) one or more instruments for assisting with obtaining a test sample; (vi) reaction vessels, mixing vessels, and (vii) instructions for use of the kit.

41. The kit of claim 39 or 40, further comprising a microscope.

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