Compositions and methods for HPV analysis

Abstract

Provided herein are compositions, methods, and kits for detecting, amplifying, and / or quantifying analytes in various samples. For example, the present disclosure provides a composition, method, and kit capable of detecting, amplifying, and / or quantifying human papilloma virus (HPV)-associated cancer circulating tumor DNA (ctDNA).

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 500,979, filed May 9, 2023, which is incorporated herein by reference in its entirety and for all purposes.

[0003] sequence list The text of the computer-readable sequence list, titled "41733-601_SEQUENCE_LISTING" and 163,578 bytes in size, created on May 9, 2024 and submitted with this document, is hereby incorporated in its entirety by reference. Technical Field

[0004] This document provides compositions, methods, and kits for detecting, amplifying, and / or quantifying analytes in a variety of samples. For example, this disclosure provides a composition, method, and kit capable of detecting, amplifying, and / or quantifying human papillomavirus (HPV)-associated cancer circulating tumor DNA (ctDNA). Background Technology

[0005] HPV infection is associated with a variety of cancers, including cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancers, and there remains a significant unmet need in detection, surveillance, and treatment. For example, HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is a growing epidemic and will remain a worsening public health problem for decades to come, despite the availability of HPV vaccines, due to relatively low vaccination rates in the US [1-9]. While patients diagnosed with early-stage disease have excellent prognoses, more than a quarter of HPV+ OPSCC patients present with high-risk disease and are likely to experience recurrence

[10] . The 5-year survival rate for patients with recurrent HPV+ OPSCC is low, ranging from 3-26% [11-13].

[0006] Data from other solid tumors suggest that relapse detected by biomarker testing of biological fluids (i.e., “biochemical relapse”) has the potential to be cured by salvage therapy before the appearance of CT scan detectable disease during post-treatment surveillance (PTS) [14-19]. Consistent with this, survival is improved for locally recurrent OPSCC when the disease volume is small

[20] , suggesting that early detection of relapse in HPV+ OPSCC 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 to detect. Unfortunately, while attempts have been made at CT scan based PTS, it has been low sensitivity, high false positive rate, and has not improved survival [21, 22]. Thus, no biomarker testing approach has shown the potential to improve treatment outcomes in PTS for HPV+ OPSCC, and improved HPV related cancer detection strategies are needed. SUMMARY

[0007] Provided herein are compositions, methods, and kits for detecting, amplifying, and / or quantifying analytes in various samples. For example, the present disclosure provides a composition, method, and kit capable of detecting, amplifying, and / or quantifying circulating tumor DNA (ctDNA) in human papillomavirus (HPV) related cancers.

[0008] Some of the principal aspects provided herein are summarized below. Additional aspects are described in the Description of the Drawings, Definitions, Details, Experiments, and Claims sections of the present disclosure. The description of each section of the present patent disclosure, whether titled or sub-titled, should be read in conjunction with all other sections. Furthermore, the various embodiments described in each section of the present disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0009] In some aspects, provided herein are compositions for multi-probe HPV ctDNA assays. In some embodiments, the compositions comprise one or more sets of modified oligonucleotide primers / probes. In some embodiments, the primer / probe sets are used for amplification and quantification of DNA. In some embodiments, the DNA is HPV-associated cancer (e.g., HPV+ OPSCC). In some embodiments, the primer / probe sets are intended to amplify and / or detect one or more target regions of high-risk HVP types (e.g., HPV16, HPV18, HPV31, HPV33, and / or HPV39). In some embodiments, the target set is selected from the group consisting of: SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 56. In some embodiments, the primer / probe sets comprise one or more oligonucleotides selected from the group consisting of: SEQ ID NO: 1 - 27 and SEQ ID NO: 37 - 51.In some embodiments, at least three of the primer / probe sets are selected from the group consisting of: Group 1 : SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; Group 2: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; Group 3: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; Group 4: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12; Group 5: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; Group 6: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18; Group 7: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21; Group 8: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; Group 9: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27; Group 10: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; Group 11: SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; Group 12: SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; Group 13: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; Group 14: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51. In some embodiments, the primer / probe set is selected from the group consisting of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49; a reverse primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51; a probe comprising a detectable label, wherein the forward primer and the reverse primer anneal to any one or more of: SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 38, 41, 44, 47, 50. In some embodiments, the first set of oligonucleotides comprises SEQ ID NO: 1 - 15 or SEQ ID NO: 37 - 45.In some embodiments, the second set of oligonucleotides comprises SEQ ID NO: 16 - 27 or SEQ ID NO: 46 - 51. In some embodiments, the probes used with any primer set comprise a sequence that hybridizes to a target sequence between or overlapping with the primer sequences. In some embodiments, the primer / probe sets do not have a sequence stretch of greater than eight base pairs that anneal to form a homodimer, hairpin structure, or heterodimer. In some embodiments, the first set of oligonucleotides comprises a first detectable label (e.g., FAM) and the second set of oligonucleotides comprises a second detectable label (e.g., VIC). In some embodiments, the oligonucleotides of the second label set target the viral E6 / E7 junction, where the junction is highly present in HPV16 ctDNA. In some embodiments, the oligonucleotides of the first label set meet the two-fold standard and do not change the background in the second label channel compared to a single set of probe oligonucleotides. In some embodiments, the oligonucleotides of the second label set are selected from the group of SEQ ID No: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51 and the oligonucleotides of the first label set are selected from the group of SEQ ID No: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49. In some embodiments, the oligonucleotides of the second label set target the viral E6 / E7 junction, where the junction is highly present in HPV16 ctDNA, and the oligonucleotides of the first label set meet the two-fold standard and do not change the background in the second label channel compared to a single set of probe oligonucleotides.

[0010] In some embodiments, provided herein are methods of detecting and / or quantifying HPV-associated nucleic acids (e.g., viral nucleic acids and / or ctDNA). In some embodiments, provided herein are methods of detecting HPV subtype 16 (HPV16) ctDNA, HPV subtype 18 (HPV18) ctDNA, HPV subtype 31 (HPV31) ctDNA, and / or HPV subtype 39 (HPV39). In some embodiments, the methods comprise contacting a sample containing viral nucleic acids or ctDNA with a set of oligonucleotides. In some embodiments, the set of oligonucleotides comprises a forward primer, a reverse primer, and a probe. In some embodiments, the methods comprise contacting the sample with a combination of any one or more of the compositions listed above. In some embodiments, the sample is amniotic fluid, ascites fluid, bile, breast milk, colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysis fluid, aqueous humor, vitreous humor, fecal matter, aspirate, pericardial fluid, peritoneal fluid, plasma, pleural fluid, semen, serum, synovial fluid, tear fluid, thoracentesis fluid, blood, saliva, mouthwash, or urine, derived from any such sample, although any other type of sample can also be used. In some embodiments, contacting the sample comprises: providing one or more sets of modified oligonucleotide primers / probes; partitioning a plurality of HPV DNA in the sample into droplets at a concentration where only 0 or 1 DNA molecules exist in each droplet; amplifying the HPV DNA in each droplet with the one or more sets of primers / probes to generate amplicon signals; and detecting any amplicon signals in each droplet. In some embodiments, the DNA is partitioned into microdroplets by emulsification. In some embodiments, the DNA is amplified using a nucleic acid amplification method. In some embodiments, the nucleic acid amplification method comprises polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), or multiple displacement amplification (MDA). In some embodiments, the contacting comprises performing a quantitative PCR (qPCR) assay. In some embodiments, the qPCR assay comprises a digital PCR assay. In some embodiments, the digital PCR assay comprises a droplet digital PCR (ddPCR) assay. In some embodiments, the sample comprises ctDNA HPV target nucleic acids, and wherein one or more of the oligonucleotides hybridize to the HPV target nucleic acids. In some embodiments, the assay has a limit of detection of < 1 genome equivalent of HPV DNA.

[0011] In some embodiments, the HPV nucleic acid or derivative thereof (e.g., amplicon) is sequenced. In some embodiments, the sequencing technology is a next generation sequencing technology. The term "next generation sequencing" refers to a highly parallelized approach to performing nucleic acid sequencing, and includes sequencing-by-synthesis or sequencing-by-ligation platforms (e.g., employed by companies such as Illumina, Life Technologies, Pacific Biosciences, and Roche). Next generation sequencing methods can also include, but are not limited to, nanopore sequencing methods (such as provided by Oxford Nanopore) or electronic detection-based methods (such as the Ion Torrent technology commercialized by Life Technologies). In some embodiments, one or more primers described herein further include additional sequences (e.g., barcodes, adapters, etc.) that can be used for sequencing library preparation, sequencing, and analysis. Suitable nucleic acid sequencing technologies 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(1): 7-9 (2011)); and the like.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); ligation sequencing (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)); chain termination 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 mass spectrometry sequencing (see, e.g., Edwards et al., "Mass-spectrometry DNA sequencing," Mutation Research, 573(1-2): 3-12 (2005)).

[0012] In some embodiments, provided herein are methods of detecting HPV nucleic acid and / or ctDNA, and in some embodiments, further comprise the step of treating the subject with cancer monitoring, treatment, or other intervention if HPV is detected in the sample. In some embodiments, the cancer treatment comprises imaging techniques, clinical examination, resection therapy, cryotherapy, thermal ablation, radiation therapy, chemotherapy, and / or salvage therapy. In some embodiments, the treatment is provided prior to the appearance of disease detectable by CT scan.

[0013] In some embodiments, provided herein are methods of detecting HPV nucleic acids and / or ctDNA, and in some embodiments, further comprising the step of treating the subject with an HPV vaccine if HPV is not detected in the sample.

[0014] In some embodiments, provided herein are kits. In some embodiments, the kits can include one or more sets of oligonucleotides described herein. In some embodiments, the kits can further include reagents necessary, useful, or sufficient for purifying, isolating, detecting, and / or quantifying HPV nucleic acids and / or ctDNA. For example, the kits can further include amplification reagents, including buffers and enzymes. In some embodiments, the kits can further include control samples, if desired or warranted. In some embodiments, the kits can include a solid surface (e.g., magnetic beads) comprising capture reagents (e.g., oligonucleotides) specific for target ctDNA. In some embodiments, the kits can further include containers for placing or storing samples, reagents, or reaction mixtures (e.g., containers or boxes for plasma samples, containers for cell-free DNA samples, etc.). In some embodiments, the kits can further include one or more tools for assisting in obtaining or manipulating test samples, such as a syringe. Where appropriate, the kits can contain reaction vessels, mixing vessels, and other components that facilitate reagent preparation (e.g., containers for mixing reagents for PCR). In some embodiments, the kits can further include instructions for use of the kit. Instructions included in the kits can be affixed to the packaging material, provided as a package insert, or made available from a particular website listed in the kit packaging or insert material. While instructions are typically included in written or printed materials, they are not limited to such. The present disclosure encompasses any medium that is capable of storing such instructions and communicating them to an end user. Such media include, without limitation, electronic storage media (e.g., magnetic disks, magnetic tapes, cartridges, chips), optical media (e.g., CD-ROMS) and the like. As used herein, the term “instructions” can include addresses of internet websites that provide the instructions. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figures 1A-1B Design, development, and experimental testing of multiple primer / probe sets targeting the HPV 16 genome are shown. Figure 1AThe flowchart shown outlines the design and development steps of the HPV16 multiplex assay. Briefly, computational analysis was used to scan the entire HPV16 genome for primer and probe combination regions that met primer / probe design criteria to select a combination predicted to specifically bind the HPV16 genome. A schematic based on the Papillomavirus Epigenome (PaVE) coordinates depicts the regions of the HPV16 genome targeted by the primer / probe combinations at various stages of selection. From the forty-nine (49) primer / probe combinations selected from the computer screen (forward primer = green, reverse primer = red), eighteen (18) non-overlapping combinations were selected, of which examples are circled, and one (1) primer / probe combination that overlapped with another primer combination at the E6 / E7 junction was selected based on higher GC content (see primer / probe combination #4 in Figure 1B Figure 1B The ddPCR droplet plot shown demonstrates FAM signal (blue) for the nineteen (19) selected primer / probe candidates tested in duplicate using 400 genome equivalents (GE) of Hindlll-digested HPV16 cell line UM-SCC-104 genomic DNA (gDNA) or 200,000 genome equivalents (GE) of Hindlll-digested non-HPV human genomic DNA (hgen DNA) as template at a final primer / probe concentration of 900 nM / 250 nM. The threshold (pink line) was kept constant for all primer / probe combinations except primer / probe combination #17 and primer / probe combination #18, where the signal intensity was not high compared to background (gray). The bottom table lists the HPV16 copy number (mean of duplicates) detected with each primer / probe combination compared to the SP assay (orange). In the case of primer / probe combination #14, the background could not be distinguished from the FAM signal. No signal was observed with any primer / probe combination using the negative control non-HPV hgen DNA as template.

[0016] Figures 2A-2C The primer / probe pool selection for multiplex assay development is shown. Figure 2A The pooling scheme for multiplex assay development using primer / probe combinations targeting different regions of HPV16 selected based on high signal intensity compared to background during screening is shown. The schematic shows combinations of 3, 5, 10, or 14 primer / probe combinations, referred to as 3-pool, 5-pool, 10-pool, and 14-pool, respectively.

[0017] ​Figure 2B ddPCR droplet plots showing FAM signal (blue) in singleplex assays for each of the 5 primer / probe sets (#4, #10, #13, #15, and #16) and 3-pool, 5-pool, 10-pool, and 14-pool at 450 nM for each primer and 125 nM for each probe. In the case of 3-pool and 5-pool, the separation of signal from background (gray) allowed for the calculation of HPV16 copy number, but in the case of 10-pool and 14-pool, the signal overlapped with the background and HPV16 copy number could not be calculated. The table on the right lists the HPV16 copy number obtained for individual primer / probe combinations, 3-pool, and 5-pool using sheared UM-SCC-104 gDNA (500 GE) or water control as template. The data shown represent different primer / probe concentrations tested to improve the signal-to-background ratio of the pool (Figure 7). Figure 2C VIC-labeled probes #2, #4, #13, #15, and #16 tested in various combinations of the four (4) primer / probe sets to screen 4-pools at a final primer / probe concentration of 450 nM / 125 nM using sheared UM-SCC-104 gDNA template (100 GE) are shown. Droplet plots show signal intensity (green) and background (gray). HPV16 copy number is listed and 4-pool v20 (highlighted in light green) was selected for further experimental evaluation.

[0018] Figure 3 Screening and selection of dual-color multiplex pools is shown. 2-D ddPCR plots demonstrate droplet plots of single probe FAM assay #6 and different 5-pool combinations containing FAM-labeled probes (blue) each screened at a final primer / probe concentration of 450 nM / 125 nM using sheared UM-SCC-104 gDNA as template (50 GE) with 4-pool v20 (#2+#4+#13+#15) containing VIC-labeled probes (green). HPV16 copy number and fold enhancement of signal measured using 5-pool FAM combinations relative to single probe (#6) assay are listed in the table at the bottom. When comparing the FAM-labeled single probe (#6) assay to different 5-pool FAM combinations, the best separation of background (gray) from signal (blue) was observed in 5-pool v39 FAM (#5+#6+#7+#16+#19) (panel G; highlighted in light red). The threshold of 6200 for 4-pool v20 VIC was kept constant to ensure that the FAM pool did not change the background intensity of 4-pool v20 VIC in the case of dual-channel measurement. In cases where the FAM pool changed the background (e.g., 5-pool v35 and 5-pool v41), HPV16 copy number could not be determined and is listed as ‘nd’.

[0019] Figure 4 Analysis validation of the two-color multiplex CHAMP-16 assay is shown. Panel A shows a linear plot of ddPCR results (average of 3 replicate assays) from a 2-fold dilution series to determine the limit of detection (LoD) and reportable range for HPV16 circulating tumor DNA (ctDNA) using a template comprising 9 synthetic HPV16 dsDNA duplexes corresponding to the genomic regions targeted by the 9 primer / probe combinations pooled in the CHAMP-16 assay. Synthetic HPV16 target DNA pools and sheared UM-SCC-104 genomic DNA were tested in the context of sheared hgen background (approximately 44,000 diploid GE per well) and the expected copy number ( x-axis ) was plotted against the measured copy number (y-axis). Panel B shows a linear plot of ddPCR results (average of 3 replicate assays) from a 2-fold dilution series to determine the limit of detection (LoD) and reportable range for HPV16 ctDNA using sheared UM-SCC-104 gDNA as template. Synthetic HPV16 target DNA pools and sheared UM-SCC-104 gDNA were tested in the context of sheared hgen background (approximately 44,000 diploid GE per well) and the number of GE tested ( x-axis ) was plotted against the measured copy number (y-axis). The LoD was analytically and computationally determined to be 4.1 copies per 20 μΐ reaction. No non-specific signal was observed when using only sheared hgen DNA (approximately 44,000 diploid GE per 20 μΐ reaction) as non-HPV template (n = 61). Panel C shows the % CV of the measured copy number of HPV16 ( y-axis ) at different dilutions compared to the expected copy number of the synthetic pool of 9 DNA targets (converted to log 10 )( x-axis ) at different dilutions. Panel D shows the % CV of the measured copy number of HPV16 ( y-axis ) at different dilutions compared to the number of GE of the sheared UM-SCC-104 gDNA (converted to log 10 )( x-axis ) at different dilutions. An arbitrary 20% threshold is indicated with a dashed line.

[0020] Figure 5 A case study comparing the difference in early detection of recurrence between the CHAMP-16 assay and the conventional SP assay is shown. Plasma samples collected from a biopsy-confirmed HPV+ OPSCC patient who received chemoradiation therapy (CRT) were analyzed for HPV16 ctDNA using the SP assay (orange triangles) and the CHAMP-16 assay (green circles). The detected HPV16 copy number ( y-axis) Cumulative values of paired plasma cfDNA samples tested over months of blood draws x-axis ) plotted. With respect to detection of the presence of cancer, PET (+) indicates a positive PET scan, while PET (-ve) indicates a negative PET scan. Filled symbols represent detection of HPV16 molecules above the LoD (black dotted line), while open symbols represent copy numbers below the LoD. Notably, the CHAMP-16 assay was able to detect HPV16 ctDNA signal approximately 20 months prior to clinical relapse and signal detection by SP.

[0021] Figure 6 Computational screening of primer / probe sets targeting the HPV16 genome is shown. A schematic based on the PaVE coordinates depicts the regions of the HPV16 genome targeted by 292 primer / probe combinations (green for forward oligo sequence, red for reverse oligo sequence) selected by computational analysis using melting temperature, amplicon length, and %GC criteria. Various other criteria (e.g., self-dimer formation, potential non-specific amplification, etc.) were also employed for further screening, resulting in the selection of 49 primer / probe combinations.

[0022] Figures 7A-7C Determination of optimal primer / probe combinations and pool concentrations is shown. Droplet plots display FAM signal (blue) in singleplex assays using UM-SCC-104 gDNA (500 GE) as template for 5 primer / probe sets (#4, #10, #13, #15, and #16) and 3-pool, 5-pool, 10-pool, and 14-pool (pooled according to Figure 2A Note that after 50 or 60 PCR cycles, background (gray) increases significantly with increasing number of pooled primer / probe sets tested. Figure 7A Primer / probe sets tested at concentrations of 900 nM each primer and 250 nM each probe are shown. Figure 7B Primer / probe sets tested at concentrations of 450 nM each primer and 125 nM each probe are shown. Figure 7C Primer / probe sets tested at concentrations of 180 nM each primer and 50 nM each probe are shown. In the case of 3-pool and 5-pool, signal intensity over background allowed for calculation of HPV16 copy number, but in the case of 10-pool and 14-pool, signal overlapped with background and HPV16 copy number could not be calculated. The decrease in signal was not due to Figure 7B and Figure 7C Droplet counts for primer / probe concentrations shown in Figure 7ADroplet counts for 5-pool, 10-pool, and 14-pool were negatively affected at the highest primer / probe concentrations shown (i.e., 900 nm / 450 nm).

[0023] Figure 8 Representative droplet plots are shown demonstrating the criteria used to select 5-pool combinations. Representative data for 5-pool combinations show excluded pools (R1, R2, and R3) that did not undergo further assays and accepted pools (A1, A2, and A3) that underwent further testing based on a 2-fold criteria of signal intensity (blue) to background (gray) ratio. #SP assays refer to primer / probe sets corresponding to previously published single probe HPV16 assays.

[0024] Figure 9 Screening and selection of 5-pool combinations containing FAM-labeled probes are shown. Droplet plots for 8 representative 5-pool combinations were tested, two of which (v18 and v20) were excluded because they contained primer / probe set #10 targeting the E2 gene. 5-pool v2 highlighted in light blue was selected based on 2-fold higher signal intensity (dark blue) compared to background (gray) and the highest number of HPV16 copy numbers detected. Sheared UM-SCC-104 gDNA or water were used as templates for ddPCR at a final primer / probe concentration of 180 nM / 50 nM each.

[0025] Figure 10 Comparison of signal intensity for FAM-labeled probes and VIC-labeled probes in single probe assays is shown. Droplet plots demonstrate signal intensity for each primer / probe set (#2 or #4 or #13 or #15 or #16) at a final primer / probe concentration of 900 nM / 250 nM, respectively, in single probe assays using FAM (blue) or VIC (green) labeled probes when sheared UM-SCC-104 gDNA (100 GE) was used as template for ddPCR. The table below lists the signal amplitude for each channel, as well as the approximate signal intensity fold over background calculated from the amplitude. A significant decrease in signal intensity was observed with VIC-labeled probes compared to FAM-labeled probes.

[0026] Figure 11Signal intensity comparison of FAM-labeled probes and VIC-labeled probes as multiplexed pools is shown. Droplet plots display signal intensity for primer / probe pools containing primer / probe sets #2, #4, #13, #15, and #16 (5-pool v2) with probes labeled with FAM (blue) or VIC (green) fluorophores. 20 or 100 GE of sheared UM-SCC-104 gDNA template was used for ddPCR at a final primer / probe concentration of 180 nM / 50 nM each. The table below lists the signal amplitude for each channel, as well as the approximate signal intensity fold over background calculated from the amplitude. A significant reduction in signal-to-background separation is observed for the VIC-labeled version of the pool when compared to the FAM-labeled version.

[0027] Figure 12 Determination of optimal annealing temperature for the CHAMP-16 assay is shown. The assay was tested over a range of annealing temperatures (64°C to 56°C) using sheared UM-SCC-104 gDNA (50 GE) as template. Representative droplet plots (FAM (blue) and VIC (green)) under different test annealing conditions are shown, and HPV16 copy numbers are listed in the table below. A significant reduction in FAM and VIC positive droplets is recorded at temperatures above 60°C, indicating incomplete amplification, as shown by the representative droplet plot (in parentheses) and listed HPV16 copy numbers at 63°C. From the droplet plots shown, amplification is optimal at 59°C, with no further improvement observed at lower temperatures tested, including the lowest temperature tested, 56°C, as indicated by the HPV16 copy numbers. Therefore, 59°C was chosen as the annealing temperature. Furthermore, no signal was observed at this annealing temperature when tested with non-HPV hgen DNA as template (44,000 diploid GE).

[0028] Figure 13 Comparison of analytical sensitivity for the SP assay and the multiplexed CHAMP-16 assay is shown. HPV16 genomic region, depicting the location of primer / probe combinations relative to the SP assay, 9 primer combinations combined in the CHAMP-16 assay (forward primers in green, reverse primers in red). The table lists HPV16 copy numbers detected by the CHAMP-16 assay and the SP assay using sheared UM-SCC-104 gDNA as template. CV = coefficient of variation.

[0029] Figure 14Each primer / probe set is shown compared to amplification of the respective target as a single probe assay and as a multiplexed pool assay. In the CHAMP-16 assay using ddPCR, HPV16 copy numbers measured for 9 primer / probe combinations were compared when tested as individual primer / probe assays or as a pool. Synthetic DNA corresponding to each of the 9 target regions of HPV16 was spiked into a matrix of 44,000 diploid GE of hgen DNA and used as template (top table). The 9 synthetic DNA targets were combined and tested as an artificial sample at two different dilutions (bottom table) with similar analysis.

[0030] Figure 15 Comprehensive patient demographics and HPV16 ctDNA test results are shown. Demographics for 41 HPV+ OPSCC patients were tested with the SP assay and the CHAMP-16 assay. HPV16 copy numbers (average of duplicates) of the plasma cfDNA analyzed for each assay are listed along with the corresponding plasma volume tested for each replicate assay. Patient samples #1 - #21 were known to be positive for HPV16 ctDNA according to previous analysis using the SP assay. Samples from patients #22 - #41 correspond to residual biospecimen samples collected at baseline (i.e., prior to initiation of treatment) from a historical clinical trial. Notably, these samples were collected in a previous study and were handled differently than the samples from patients #1 - #21, including having undergone one or more freeze-thaw cycles. Furthermore, while tumor tissue from patients #22 - #41 was confirmed to be pl6(+) it was not individually determined to be HPV16 positive. For four patients, HPV16 ctDNA copy numbers were detected with the CHAMP-16 assay but not with the SP assay (in red). Samples that tested negative for the SP or CHAMP-16 assay are shown in dark gray shading.

[0031] Figure 16 Clinical specificity control groups are shown. Eleven HPV-negative cancer patients (patients #1 - #11) and 3 non-cancer control subjects (patients #12 - #14) were tested using the SP and CHAMP-16 assays and no signal was detected. This is consistent with the analytical data for the CHAMP-16 assay, which was negative when tested with 44,000 diploid GE of non-HPV human reference DNA.

[0032] Figure 17Patient demographics and SP assay and CHAMP-16 assay results compared to reported HPV16 ctDNA NavDx assay values. Plasma samples collected from 8 HPV+ OPSCC patients near the time sent for commercial NavDx analysis were tested using the CHAMP-16 assay and the SP assay. For the 3 patients (patients #42-#44) that were positive for the NavDx assay, a significant signal boost was observed with the CHAMP-16 assay compared to the commercial assay. The samples collected from 5 patients (patients #45-#49) 3 months post-CRT showed no HPV16 signal with the NavDx assay, and were also negative for HPV16 testing with the SP and CHAMP-16 assays.

[0033] Figure 18 Longitudinal HPV16 ctDNA analysis showing recurrence - patient #19. Plasma samples collected from a biopsy-confirmed HPV+ OPSCC patient were tested using the CHAMP-16 assay. HPV16 and reference gene RPP30 copy numbers are listed here and presented as a timeline in Figure 5 .

[0034] Figure 19 Longitudinal HPV16 ctDNA analysis showing patient #20. Plasma samples collected from a biopsy-confirmed HPV+ OPSCC patient were tested using the CHAMP-16 assay. HPV16 and reference gene RPP30 copy numbers are listed here. Despite very high HPV16 copy numbers at baseline, all subsequent time points measured post-surgical resection were negative for HPV16, in contrast to patient #19 who experienced cancer recurrence Figure 5 and Figure 18 .

[0035] Figure 20 Clinical sample testing using the CHAMP-16 assay is shown. Plasma samples from 21 p16+ HNSCC patients known to be positive for HPV16 ctDNA using the conventional SP assay were analyzed using both the SP assay and the CHAMP-16 assay. The HPV16 copy numbers (average of duplicates) of the plasma cfDNA analyzed for each assay are listed. All patients tested positive in both assays; the CHAMP-16 assay signal boost was on average 6.6-fold higher than the SP assay and highlighted in green.

[0036] Figure 21The performance of CHAMP-16 was compared with that of the SP assay and the commercially available NavDx assay. Plasma samples from three HPV+ OPSCC patients who reported positive HPV16 ctDNA values ​​using the NavDx assay were tested using both the SP and CHAMP-16 assays. The HPV16 copy number detected in plasma cfDNA (mean of duplicates) is listed, extrapolated to 1 mL of plasma for comparison with values ​​reported by the NavDx assay. Values ​​observed with the SP assay were similar to those reported by the NavDx assay, while a significant signal enhancement was observed using the CHAMP-16 assay. Patient #42's NavDx test blood draw was performed seven days earlier than the pretreatment blood draw for the SP and CHAMP-16 assays; otherwise, all assays were performed concurrently. Notably, five samples collected from the patient three months after receiving chemotherapy-radiotherapy (CRT) showed no signal using the NavDx assay and were also negative for HPV16 ctDNA using both the SP and CHAMP-16 assays. Figure 16 This supports the conclusion that the signal enhancement observed in the CHAMP-16 assay was not due to nonspecific signals.

[0037] Figure 22 This section presents the analytical validation – limit of detection (LOD) for the CHAMP-hr assay used to quantify other high-risk HPV types. Data from a 2-fold dilution series of ultrashort synthetic targets (45–55 bp) of the E6 gene targeting five known high-risk HPV types (HPV18, HPV31, HPV33, HPV35, and HPV39) are shown to determine the detectable range. The expected copy number (cumulative value of three replicate assays) of the synthetic ultrashort HPV DNA corresponding to the high-risk type is shown in the ddPCR reaction. The LOD for the high-performance assay for each targeted HPV type is also shown.

[0038] Figure 23 The development of dual assays for the RPP-30 reference gene and plant spike protein was demonstrated: a comparison of the plant spike protein assay and the RPP-30 reference gene assay using ddPCR showed that they performed equally well as single and dual assays.

[0039] Figure 24 The computational analysis of HPV16 is shown. This image is a schematic diagram of the selection process. Of the 49 primer / probe sets that met the computer screening criteria, 19 non-overlapping sets were experimentally tested, and 9 sets were selected for the CHAMP-16 assay. Of the 40 primer / probe sets not selected for the CHAMP-16 assay, 10 sets were excluded after experimental testing, while the remaining 30 sets were not tested. Detailed Implementation

[0040] It is known that solid tumors release ctDNA, which can be detectable in bodily fluids. Bodily fluid ctDNA analysis can allow for non-invasive longitudinal monitoring of tumor-specific genomic alterations. To date, no comprehensive prospective analysis of ctDNA method characteristics and predictive efficacy has been performed for certain HPV-associated cancers, such as head and neck cancer.

[0041] Provided herein are compositions, methods, and kits for detecting, amplifying, and / or quantifying analytes in various samples. For example, the present disclosure provides a composition, method, and kit capable of detecting, amplifying, and / or quantifying human papillomavirus (HPV) and associated circulating tumor DNA (ctDNA) of cancer.

[0042] The compositions, methods, and kits described herein can employ any one or more or all of the oligonucleotides (e.g., primer / probe sets) described herein. These oligonucleotides can be used in conjunction with any other diagnostic assay (e.g., for detecting other HPV types / strains, other cancer or precancer biomarkers, inflammation, etc.).

[0043] Any suitable sample type can be used for any of the compositions, methods, and kits described herein. A sample can be obtained from a subject and subsequently used in any of the methods described herein. In some embodiments, a sample is obtained from a subject, and the analyte is isolated, purified, and / or concentrated from the sample for use in the methods described herein.

[0044] The section headings and all the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0045] 1. Definitions Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the application is described, it is to be understood that this application is not limited to particular molecules, compositions, methodologies or protocols described herein, as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms or embodiments only and is not intended to limit the scope of the embodiments described herein.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. However, in the event that there is a differing or conflicting definition of a term then that which appears in this specification, including annexed drawings, prevails. In addition, unless otherwise specified, singular terms shall include plural forms and plural terms shall include singular forms. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0047] For recitations of numerical ranges by endpoints, each intervening number by the same degree of precision contained in the base number is explicitly included. For example, for the range of 6 to 9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 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 included.

[0048] As used herein, and throughout the claims that follow, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a domain" is a reference to one or more domains and equivalents thereof known to those skilled in the art.

[0049] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. For example, "A and / or B" is intended to cover A alone, B alone, as well as any and all combinations of A and B.

[0050] As used herein, the terms "comprising," "having," "has," "can," "including," and variants thereof are open-ended transitional phrases, terms, or words that are intended to be synonymous with each other.

[0051] For any composition, method, and kit described herein, any suitable sample type can be used. The term "sample" sample means a fluid (e.g., amniotic fluid, ascites fluid, bile, breast milk, colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysis fluid, aqueous humor, vitreous humor, fecal, puncture fluid, pericardial fluid, peritoneal fluid, blood (e.g., whole blood), blood product (e.g., plasma, serum), pleural fluid, semen, synovial fluid, tear fluid, thoracentesis fluid, saliva, mouthwash, or urine, etc.), solid, tissue, and gas.

[0052] The term "saliva" or "saliva sample" sample means any sample containing saliva of a subject, including a spit, oral swab or sponge sample, mouthwash sample, etc.

[0053] The term "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have consecutive sets of identical monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only in conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated as follows: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions at which the identical (or similar) monomers occur (e.g., the same amino acid occurs in both sequences, a similar amino acid occurs in both sequences) to arrive at the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, the specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptide A and peptide B are each 20 amino acids in length and have identical amino acids at all but one position, then peptide A and peptide B have 95% sequence identity. If the amino acid at the non-identical position has the same biophysical characteristics (e.g., both are acidic), then peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 of the 15 amino acids in peptide D have identity with a portion of peptide C, then peptide C and peptide D have 70% sequence identity, but the best comparison window of peptide D has 93.3% sequence identity to peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gaps in the aligned sequences are treated as mismatches at that position.

[0054] Any peptide described herein having a particular percent sequence identity or similarity (e.g., at least 70%) to a reference sequence ID number can also be expressed as having a maximum number of substitutions (or terminal deletions) relative to the reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) to SEQ ID NO:Z (e.g., 20 amino acids) can have at most X substitutions (e.g., 2) relative to SEQ ID NO:Z, and thus can also be expressed as "having X (e.g., 2) or fewer substitutions relative to SEQ ID NO:Z."

[0055] As used herein, "at least 90% sequence identity" means that an oligonucleotide has 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 a reference sequence.

[0056] As used herein, the term “comprising” and its grammatical variants are used inclusively of the recited features, elements, method steps, etc, that they introduce such that determinative issues are not presented by whether the features, elements, method steps, etc, are present or not. In contrast, the term “consisting” and its grammatical variants indicate the presence of the recited features, elements, method steps, etc, and the absence of any element not recited, except for impurities ordinarily associated with the composition, system or method. The phrase “consisting essentially of’ indicates the recited features, elements, method steps, etc, and any additional features, elements, method steps etc, that do not materially affect the basic nature of the composition, system or method. Many of the embodiments herein are described using open-ended “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of’ embodiments, which can alternatively be claimed or described using such language. As used herein, to include a sequence or a SEQ ID NO generally means that at least one copy of the recited sequence is present in the peptide or polynucleotide. However, two or more copies are also contemplated.

[0057] As used herein, the term “contacting” means to bring into contact or place in contact, to contact or to become in contact. The term “contacting” as used herein refers to a state or condition of touching or being in direct or close proximity. The composition can be brought into contact with the target destination such as, but not limited to, an organ, tissue, cell, or tumor, by any means of administration known to one of skill in the art.

[0058] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate involvement of human hand. These terms, when referring to a nucleic acid molecule or polypeptide, indicate that the nucleic acid molecule or polypeptide is at least substantially isolated from at least another component with which it is associated in nature or as found in nature.

[0059] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases (preferably cytosine, thymine, and uridine, and adenine and guanine, respectively) (see Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982)). The 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, among others. The polymer or oligomer can be heterogenous or homogenous in composition and can be isolated from naturally occurring sources or can be artificially or synthetically produced. Furthermore, the nucleic acid can be DNA or RNA or a mixture thereof, and can exist permanently or transitorily in single- or double-stranded forms, and, in the latter case, in either a homo- or heteroduplex state. In some embodiments, the nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures, such as, for example, DNA / RNA hybrids, peptide nucleic acids (PNA), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Patent No. 5,034,506), locked nucleic acids (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 ribozymes. Thus, the term “nucleic acid” or “nucleic acid sequence” can also encompass a strand comprising non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (“nucleotide analogs”); furthermore, as used herein, the term “nucleic acid sequence” refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, as well as DNA or RNA of genomic or synthetic origin, which can be single- or double-stranded, and represent the sense or anti-sense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence” are used interchangeably. They refer to polymeric forms of nucleotides of any length (deoxyribonucleotides or ribonucleotides, or their analogs). The term “oligonucleotide” or “oligos” as used herein generally refers to short nucleic acid sequences consisting of 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 can comprise, consist essentially of, or consist of the complement of any of the sequences disclosed herein.

[0060] The terms "protein" and "peptide" and "polypeptide" are used interchangeably herein to refer to polymers of amino acid residues linked together by peptide bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide can refer to a single protein, or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, e.g., by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnyl group, an isofarnyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide can also be a single molecule, or can be a multimeric complex. A protein, peptide, or polypeptide can be only a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, engineered, or synthetic, or any combination thereof. Any of the proteins provided herein can be produced using any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification methods, which are particularly suitable for fusion proteins comprising peptide linkers. Methods of recombinant protein expression and purification are well known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0061] As used herein, the terms "providing," "administering," and "introducing" are used interchangeably herein and refer to placing into a cell, organism, or subject by a method or route that results in at least partial localization to the desired site. For example, the compositions disclosed herein can be administered by any appropriate route that results in delivery to the desired site in the cell, organism, or subject.

[0062] A“subject” or“patient” are used interchangeably herein to refer to a human or non- human animal, and can include, for example, an animal strain or species used as a“model system” for research purposes, such as a mouse model as described herein. Likewise, a patient can include an adult or a juvenile (e.g., a child). Further, a patient can refer to any living organism, preferably a mammal (e.g., human or non-human), that can benefit from administration of a protein, nucleic acid, or composition encompassed herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like.

[0063] As used herein,“treat,”“treating,”“treatment,” and variations thereof, refer to clinical intervention by a patient exhibiting or who can be predisposed to a disease, disorder, or physiological malady. The objectives of treatment include alleviating or preventing symptoms, slowing or arresting disease, disorder, or malady progression or worsening, and / or relieving the disease, disorder, or malady. A positive response to treatment can indicate that the treatment is completely effective, partially effective, or that the patient’s condition is stable. A negative response to treatment can indicate that the subject’s disease is progressing. For example, treating cancer can include managing and caring for a patient to combat and alleviate one or more symptoms of cancer. For example, treating cancer can reduce tumor burden (e.g., reduce the size of one or more tumors in a subject having cancer and / or reduce the total number of tumors in a subject having cancer). Treating cancer can reduce or completely eliminate cancer in a patient (e.g., completely eliminate a tumor).

[0064] As used herein, the term“immunotherapy” refers to any type of cancer treatment that can help the immune system fight cancer. For example, an“immunotherapy” can include treatment with an immune checkpoint inhibitor, a T-cell transfer therapy, a monoclonal antibody, a therapeutic vaccine, and / or an immunomodulator. The cancer can be any cancer type.

[0065] As used herein, a method of predicting a subject’s response to treatment can include measuring a baseline level of cancer-associated ctDNA in a subject. The baseline level of the ctDNA can be measured prior to any treatment of the subject. Alternatively, the baseline level of the ctDNA can be measured after one or more treatment periods in the subject.

[0066] The method can further comprise measuring a subsequent level of the cancer- related ctDNA in the subject after one or more treatment periods. For example, a baseline level of the ctDNA can be measured prior to any treatment, and a subsequent level can be measured after the end of a treatment period. Alternatively, a baseline level of the ctDNA can be measured after a first treatment period, and a subsequent level can be measured after a second treatment period.

[0067] The baseline level and / or the subsequent level of the ctDNA can be measured using a suitable method described herein. For example, measuring the baseline level and measuring the subsequent level can comprise obtaining a sample comprising the ctDNA from the subject.

[0068] Cell-free DNA can be isolated from plasma obtained from the subject. For example, plasma can be obtained, and cell-free DNA can be isolated using any suitable method. In some embodiments, cell-free DNA is isolated using a commercially available kit.

[0069] In some embodiments, the method further involves predicting a response of the subject to the treatment based on the HPV (e.g., HPV16) ctDNA level. In some embodiments, the method comprises predicting a positive response of the subject to the treatment when the change from the baseline level to the subsequent level is below a threshold. In some embodiments, the method comprises predicting a negative response of the subject to the treatment when the change from the baseline level to the subsequent level is above a threshold. In some embodiments, the threshold is 50%. In some embodiments, the threshold is 60%. For example, the method can comprise predicting a positive response to the treatment when the HPV ctDNA level increases by less than 60% from the baseline level to the subsequent level. Alternatively, the method can comprise predicting a negative response to the treatment when the HPV ctDNA level increases by 60% or more from the baseline level to the subsequent level. A positive response to the treatment can indicate that the treatment is completely effective, the treatment is partially effective, or the patient’s condition is stable. For example, a positive response can indicate no metastasis, no increase in tumor size, and / or no increase in the total number of tumors observed in the subject. A negative result can indicate disease progression, such as an increase in the total number of tumors and / or an increase in tumor size.

[0070] “Amplification product” refers to a nucleic acid fragment that is formed as a product of an amplification event or technique. For example, an amplification product can be produced by PCR.

[0071] As used herein, “amplify,” “amplifying,” “amplification,” and variants thereof refer to a method that increases the copy number of a target sequence, if present in a sample. In some embodiments, the method further comprises detecting a signal from a detectable label, indicating the presence of the target sequence, if present in the sample. For example, amplification can be performed by polymerase chain reaction (PCR).

[0072] As used herein, "polymerase chain reaction" (PCR) refers to amplification, and "quantitative PCR" (qPCR) refers to a method of quantifying the copy number of an amplified sequence. For example, a target sequence can be amplified and quantified using a digital PCR technique, such as a dPCR technique selected from the group consisting of droplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification, and magnetism), and microfluidic chip.

[0073] As used herein, "digital PCR" refers to an assay that provides an end-point measurement that enables quantification of nucleic acids without the use of a standard curve, as used in real-time PCR. In a typical digital PCR experiment, a sample is randomly distributed into discrete partitions, such that some partitions contain no nucleic acid template, while other partitions contain one or more copies of the template. The partitions are amplified to the terminal plateau (or end-point) of PCR, and then read to determine the proportion of positive partitions. If the volume of each compartment is uniform, then the number of target DNA molecules can be calculated from the proportion of positive end-point reactions using, for example, Poisson statistics, according to the following equation:

[0074] where λ is the average number of target DNA molecules per replicate assay reaction, and p is the proportion of positive end-point reactions. From the value of k, in combination with the volume of each replicate assay PCR and the total number of replicate assays analyzed, an estimate of the absolute target DNA concentration is calculated. Digital PCR includes various formats, including droplet digital PCR, BEAMing (beads, emulsion, amplification, and magnetism), and microfluidic chip.

[0075] 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, volume-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 can consist of at least 20,000 partitioned droplets per well. "Droplet" or "water-in-oil droplet" refers to the individual partitions of a droplet digital PCR assay. Droplets support PCR amplification of template molecules using chemistry 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).

[0076] Droplet digital PCR can be performed using any platform that performs a digital PCR assay by counting nucleic acid molecules encapsulated in discrete, volume-defined, water-in-oil droplet partitions that support PCR amplification to measure absolute quantities. The strategy for droplet digital PCR can be summarized as follows: a sample is diluted and partitioned into thousands to millions of individual reaction chambers (water-in-oil droplets) such that each reaction chamber contains one or zero copies of the nucleic acid molecule of interest. The ratio of the number of "positive" droplets detected containing a target amplicon (e.g., nucleic acid molecule of interest) to the number of "negative" droplets that do not contain a target amplicon (e.g., nucleic acid molecule of interest) can be used to determine the copy number of the nucleic acid molecule of interest in the original sample. Examples of droplet digital PCR systems include Bio-Rad's QX100™ droplet digital PCR system, which partitions a sample containing nucleic acid templates into 20,000 nanoliter-sized droplets; Bio-Rad's QX200™ droplet digital PCR system; and RainDance's RainDrop™ digital PCR system, which partitions a sample containing nucleic acid templates into 1,000,000 to 10,000,000 picoliter-sized droplets.

[0077] As used herein, a "detectable label" refers to a fluorescent label attached 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, the detectable label is used to detect the presence of a particular molecule in a sample, where detection of the signal indicates the presence of the target sequence. For example, the target sequence can be quantified by determining the number of target molecules present. As described above, the number of target molecules can be calculated from the proportion of positive endpoint reactions using Poisson statistics. Methods of detecting, amplifying, and / or quantifying target sequences can be used in a variety of diagnostic or predictive methods. For example, these methods can be used to determine whether a subject has cancer, and / or to predict a subject's response to cancer treatment.

[0078] 2. Compositions Embodiments of the present disclosure include compositions comprising one or more oligonucleotides (e.g., primer / probe sets suitable for use in a multiprobe HPV ctDNA assay).

[0079] In some embodiments, the present disclosure provides the design, development, and validation of multiprobe HPV compositions that can provide accurate assay testing for the detection of HPV-associated cancers (e.g., posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and oropharyngeal cancer (e.g., HPV+ OPSCC)).

[0080] In some embodiments, one or more oligonucleotides (e.g., primer / probe sets) are designed to detect, amplify, and / or quantify one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA)) from various samples.

[0081] In some embodiments, detection and / or amplification of one or more analytes is achieved by target amplification (e.g., polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), strand displacement amplification, transcription amplification), signal amplification (e.g., branched DNA assay, hybrid capture), probe amplification (e.g., ligase chain reaction, cleavase-invader, cycling probe), or post-amplification analysis (e.g., sequencing of amplification products, microarray analysis, and melting curve analysis, as done in real-time PCR).

[0082] In some embodiments, quantification of one or more analytes is by absorbance (e.g., UV spectroscopy), fluorescence (e.g., using fluorescent dyes (e.g., specific dyes used only to stain specific types of nucleic acids (e.g., ssDNA, miRNA, dsDNA, and / or RNA))), electrophoresis (e.g., if the DNA to be quantified is a plasmid, a fluorescent dye (e.g., ethidium bromide or SYBR Green) is added to the gel or sample, then the sample is run in parallel with a DNA ladder), PCR (e.g., quantitative real-time PCR (qPCR), digital PCR, or droplet digital PCR (ddPCR)).

[0083] In some embodiments, one or more analytes is HPV-associated cancer (e.g., posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and oropharyngeal cancer (e.g., HPV+ OPSCC) DNA.

[0084] In some embodiments, oligonucleotides (e.g., primer / probe sets) are designed to detect, amplify, and / or quantify one or more target regions of HPV types (e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0085] In some embodiments, the oligonucleotides (e.g., primer / probe sets) comprise: SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 56.

[0086] In some embodiments, the oligonucleotides (e.g., primer / probe sets) comprise: SEQ ID NO: 1 - 27 and SEQ ID NO: 37 - 51.

[0087] In some embodiments, at least a triad of oligonucleotides (e.g., primer / probe sets) comprises: Group 1: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; Group 2: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; Group 3: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; Group 4: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12; Group 5: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; Group 6: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18; Group 7: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21; Group 8: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; Group 9: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27; Group 10: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; Group 11: SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; Group 12: SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; Group 13: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; Group 14: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.

[0088] In some embodiments, the oligonucleotides (e.g., primer / probe sets) comprise: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49; a reverse primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51; a probe comprising a detectable label, wherein the forward primer and the reverse primer anneal to a target sequence.

[0089] In some embodiments, the probe has at least 90% sequence identity to any one or more of: SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 38, 41, 44, 47, 50.

[0090] In some embodiments, the first set of oligonucleotides comprises SEQ ID NO: 1 - 15 or SEQ ID NO: 37 - 45.

[0091] In some embodiments, the second set of oligonucleotides comprises SEQ ID NO: 16 - 27 or SEQ ID NO: 46 - 51.

[0092] In some embodiments, the probe used with any oligonucleotide (e.g., primer / probe set) comprises a sequence that hybridizes to a target sequence between or overlapping with a primer sequence.

[0093] In some embodiments, the oligonucleotides (e.g., primer / probe sets) do not have a base annealing to form a homodimer, hairpin structure, or heterodimer of more than eight base pair sequence segments.

[0094] In some embodiments, the first set of oligonucleotides comprises a first detectable label and the second set of oligonucleotides comprises a second detectable label.

[0095] In some embodiments, the second set of oligonucleotides (comprising the second detectable label) targets a viral E6 / E7 junction.

[0096] In some embodiments, the viral E6 / E7 junction is highly present in HPV16 ctDNA.

[0097] In some embodiments, the oligonucleotides of the first label set meet the two-fold standard and do not change the background in the second label channel compared to a single set of probe oligonucleotides.

[0098] In some embodiments, the second set of oligonucleotides comprising a second detectable label is selected from the group of SEQ ID No: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51.

[0099] In some embodiments, the first set of oligonucleotides comprising a first detectable label is selected from the group of SEQ ID No: SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49.

[0100] In some embodiments, the second set of oligonucleotides (comprising a second detectable label) targets the viral E6 / E7 junction.

[0101] 3. Methods Embodiments of the present disclosure include methods of detecting, amplifying, and / or quantifying one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))) from various samples.

[0102] In some embodiments, any number of different detection, amplification, and / or quantification methods can be used.

[0103] In some embodiments, the detection, amplification, and / or quantification occurs prior to disease onset (e.g., prior to awareness of the disease state of the subject (e.g., prior to the appearance of disease detectable by CT scan)).

[0104] In some embodiments, the methods are designed to detect, amplify, and / or quantify one or more target regions of HPV types (e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0105] In some embodiments, the detection, amplification, and / or quantification is performed by using an assay method that labels an antigen.

[0106] In some embodiments, the detection, amplification, and / or quantification is performed by contacting a sample comprising one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))) with one or more oligonucleotides.

[0107] In some embodiments, the one or more oligonucleotides comprise a forward primer, a reverse primer, and a probe.

[0108] In some embodiments, detecting, amplifying, and / or quantifying comprises isolating, purifying, and / or concentrating analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))) from various samples.

[0109] In some embodiments, purifying is used to concentrate one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))) from various samples so that they can be used directly in downstream applications, such as subsequent analysis methods (e.g., to amplify, identify, quantify, and / or detect the presence or absence of certain target nucleic acids in a sample).

[0110] In some embodiments, the one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA)) in the sample are HPV types (e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68).

[0111] In some embodiments, the one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))) are detected and / or quantified by contacting the sample with one or more oligonucleotides (e.g., a primer / probe set).

[0112] In some embodiments, the one or more oligonucleotides (e.g., a primer / probe set) comprise a forward primer, a reverse primer, and a probe.

[0113] In some embodiments, the sample is amniotic fluid, ascites fluid, bile, breast milk, colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysis fluid, aqueous humor, vitreous humor, fecal matter, aspirate, pericardial fluid, peritoneal fluid, plasma, pleural fluid, semen, serum, synovial fluid, tear fluid, thoracentesis fluid, blood, saliva, mouthwash, or urine, derived from any such sample, although any other type of sample can also be used.

[0114] In some embodiments, contacting the sample comprises: providing one or more oligonucleotides (e.g., primer / probe sets); fractionating a plurality of analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and HPV+OPSCC) DNA))) from the sample into droplets (e.g., fractionating by emulsification).

[0115] In some embodiments, the concentration of droplets is wherein there is only 0 or 1 analyte molecule (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and HPV+OPSCC) DNA))) present in each droplet; amplifying the analyte in each droplet with one or more oligonucleotides (e.g., primer / probe sets) to generate amplicon signals; and detecting any amplicon signals in each droplet.

[0116] In some embodiments, one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+OPSCC) DNA))) are fractionated into microdroplets by emulsification.

[0117] In some embodiments, one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+OPSCC) DNA))) are amplified using a detection and / or amplification method.

[0118] In some embodiments, detection and / or amplification of one or more analytes is achieved by target amplification (e.g., polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), strand displacement amplification, transcription amplification), signal amplification (e.g., branched DNA assay, hybrid capture), probe amplification (e.g., ligase chain reaction, lyase-invader, cycling probe), or post-amplification analysis (e.g., sequencing of amplification products, microarray analysis, and melting curve analysis, as done in real-time PCR).

[0119] In some embodiments, quantification of one or more analytes is by absorbance (e.g., UV spectroscopy), fluorescence (e.g., using a fluorescent dye (e.g., a specific dye used only to stain a particular type of nucleic acid (e.g., ssDNA, miRNA, dsDNA, and / or RNA))), electrophoresis (e.g., if the DNA to be quantified is a plasmid, a fluorescent dye (e.g., ethidium bromide or SYBR Green) is added to the gel or sample, then the sample is electrophoresed in parallel with a DNA ladder), PCR (e.g., quantitative real-time PCR (qPCR), digital PCR, or droplet digital PCR (ddPCR)). In some embodiments, the contacting comprises performing a quantitative PCR (qPCR) assay. In some embodiments, the qPCR assay comprises a digital PCR assay. In some embodiments, the digital PCR assay comprises a droplet digital PCR (ddPCR) assay.

[0120] In some embodiments, the sample comprises a target nucleic acid (e.g., an HPV target nucleic acid). In some embodiments, one or more oligonucleotides hybridize to the target nucleic acid.

[0121] In some embodiments, the assay, amplification, and / or quantification method (e.g., assay) has a limit of detection (LOD) of < 1 genome equivalent of the target nucleic acid (e.g., HPV target nucleic acid).

[0122] In some embodiments, the target nucleic acid (e.g., HPV nucleic acid) or a derivative product thereof (e.g., amplicon) is sequenced.

[0123] In some embodiments, the sequencing technology is a next-generation sequencing technology. The term “next-generation sequencing” refers to highly parallelized methods of performing nucleic acid sequencing and includes sequencing-by-synthesis or sequencing-by-ligation platforms (e.g., employed by companies such as Illumina, Life Technologies, Pacific Biosciences, and Roche). Next-generation sequencing methods can also include, but are not limited to, nanopore sequencing methods (such as provided by Oxford Nanopore) or electronic detection-based methods (such as the Ion Torrent technology commercialized by Life Technologies).

[0124] In some embodiments, one or more oligonucleotides (e.g., primers) described herein further comprise additional sequences (e.g., barcodes, adapters, etc.) that can be used for sequencing library preparation, sequencing, and analysis.

[0125] In some embodiments, the sequencing technology is a next generation sequencing technology. The term "next generation sequencing" refers to highly parallelized methods of performing nucleic acid sequencing and includes sequencing-by-synthesis or sequencing-by-ligation platforms (e.g., employed by companies such as Illumina, Life Technologies, Pacific Biosciences, and Roche). Next generation sequencing methods can also include, but are not limited to, nanopore sequencing methods (such as provided by Oxford Nanopore) or electronic detection-based methods (such as the Ion Torrent technology commercialized by Life Technologies). In some embodiments, one or more primers described herein further include additional sequences (e.g., barcodes, adapters, etc.) that can be used for sequencing library preparation, sequencing, and analysis.

[0126] 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.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 mass spectrometry sequencing (see, e.g., Edwards et al., "Mass-spectrometry DNA sequencing," Mutation Research, 573(1-2): 3-12 (2005)).

[0127] Embodiments of the present disclosure also include methods of detecting one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and HPV+ OPSCC) DNA)) in a sample provided by a subject, and treating the subject if HPV-associated cancer DNA is detected in the sample.

[0128] In some embodiments, treating a subject means treating the subject with cancer monitoring (e.g., closely observing the subject for the condition, but not administering treatment unless test results change to indicate that the condition is worsening), with a cancer therapeutic, or with another intervention.

[0129] In some embodiments, treating a subject means treating with an imaging technique (e.g., computed tomography (CT), magnetic resonance imaging (MRI) scan, x-ray and other radiological examination, mammography, nuclear medicine scan (e.g., bone scan, PET scan, thyroid scan, MUGA scan, gallium scan), and ultrasound examination), clinical examination, resection therapy, thermal ablation (e.g., cryoablation (e.g., PVP laser surgery, cryosurgery, or cryotherapy), radiofrequency ablation), radiation therapy (e.g., intraoperative radiotherapy, stereotactic surgery, proton therapy, three-dimensional conformal radiation therapy, brachytherapy, proton, total body irradiation, electron, internal irradiation, short-range radiation therapy, volumetric modulated arc therapy, linear accelerator, photon), chemotherapy (e.g., alkylating antineoplastic drugs, local chemotherapy, peritoneal chemotherapy, cyclophosphamide, etoposide, doxorubicin, irinotecan, antibiotic), topoisomerase inhibitor, plant alkaloid, carboplatin, daunorubicin, oral, gemcitabine), hormone therapy (e.g., hormone therapy, hormonal therapy, or endocrine therapy), and / or salvage therapy (e.g., a second therapy performed after a first therapy was ineffective or the patient was intolerant).

[0130] In some embodiments, treating a subject means treating with a method such as palliative care, participation in a clinical trial, precision medicine or personalized medicine, off-label use, a biosimilar, a tumor-agnostic drug, and / or pharmacogenomic testing.

[0131] In some embodiments, the treatment is provided prior to disease onset (e.g., prior to awareness of the subject’s disease state (e.g., prior to the appearance of disease detectable by CT scan)).

[0132] Embodiments of the present disclosure also include methods of detecting HPV-associated cancer (e.g., oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA in a sample provided by a subject, and methods of treating the subject with an HPV vaccine (e.g., 9-valent HPV vaccine (e.g., Gardasil 9, 9vHPV), quadrivalent HPV vaccine (e.g., Gardasil, 4vHPV), and bivalent HPV vaccine (e.g., Cervarix, 2vHPV)) if HPV is not detected in the sample.

[0133] 4. Kits As described herein, embodiments of the present disclosure include kits comprising one or more oligonucleotides (e.g., primer / probe sets).

[0134] In some embodiments, the kits can also include reagents necessary, available, or sufficient for purifying, isolating, detecting, and / or quantifying one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA)))). For example, the kits can also include amplification reagents, including buffers and enzymes.

[0135] In some embodiments, the kits can also include control samples, if desired or

[0136] In some embodiments, the kits can include a solid surface (e.g., magnetic beads) comprising a capture reagent (e.g., an oligonucleotide) specific for one or more analytes (e.g., nucleic acids (e.g., prognostic or predictive DNA (e.g., viral nucleic acids and / or ctDNA (e.g., HPV-associated cancer (e.g., a posterior oropharyngeal cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, HPV+ OPSCC) DNA))).

[0137] In some embodiments, the kits can also include containers for placement or storage of samples, reagents, or reaction mixtures (e.g., containers or boxes for plasma samples, containers for samples (e.g., ctDNA samples), etc.).

[0138] In some embodiments, the kits can also include one or more tools (e.g., syringes) for assisting in the procurement or manipulation of test samples. Where appropriate, the kits can include reaction vessels, mixing vessels, and other components that facilitate reagent preparation (e.g., containers for mixing reagents for analysis).

[0139] In some embodiments, the kits can also include instructions for use of the kit. Instructions included in the kits can be affixed to the packaging material, provided as a package insert, or can be viewed or downloaded from a particular website listed on the kit packaging or insert material. While instructions are typically included as written or printed material, they are not limited to these. Any medium capable of storing such instructions and communicating them to an end user is encompassed by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., diskette, tape, cartridges, chips), optical media (e.g., CD-ROM) and the like. As used herein, the term "instructions" can include addresses of internet websites that provide the instructions.

[0140] 5. Sequences Table 1: Sequences of primer and probe sets that were also experimentally tested, in addition to the sequences of the primer and probe sets selected for use in the CHAMP-16 assay pool.

[0141]

[0142] Table 2: Sequences of primer and probe sets selected for computer screening.

[0143]

[0144]

[0145] Table 3: Sequences of primer and probe sets selected for CHAMP-16 assay pool. Synthetic primers corresponding to the 9 target regions were purchased from Integrated DNA Technologies with custom formulation at 500 uM in buffer IDTE, pH 8.0. FAM - MGB NFQ and VIC - MGB NFQ labeled probes were purchased from ThermoFisher Scientific. Sequences are shown below:

[0146] Table 4: Sequences of primer and probe sets selected for CHAMP-hr assay pool.

[0147]

[0148] Table 5: Sequences of primer and probe sets selected for reference gene RPP30.

[0149]

[0150] Table 6: Sequences of primer and probe sets selected for plant spike protein.

[0151]

[0152] Table 7: Synthetic target DNA sequences corresponding to CHAMP-16 assay. Synthetic dsDNA templates corresponding to the 9 target regions of HPV-16 used in the CHAMP-16 assay were purchased from Integrated DNA Technologies (Coralville, IA). The positive strand sequences are shown below, with the underlined sequences being specific to HPV16, and the flanking sequences (AATGC and TCACT) not underlined to distinguish the synthetic fragments from the HPV16 genomic DNA.

[0153]

[0154] Table 8: Synthetic target DNA sequences corresponding to CHAMP-hr assays. Synthetic dsDNA templates for testing high-risk HPV subtype-specific primer / probe set performance were purchased from Integrated DNA Technologies (Coralville, IA). The positive strand sequence is shown below, with the underlined sequence specific to HPV16 and the flanking sequences (AATGC and TCACT) not underlined used to distinguish the synthetic fragment from HPV16 genomic DNA.

[0155]

[0156] Table 9: Synthetic target DNA sequences for testing plant spike protein assays.

[0157]

[0158] 6. Examples It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein of the present disclosure are readily applicable and obvious in light of the teachings herein and 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 intended for purposes of illustration only and are not intended to be limiting of the scope of the present disclosure. All journal references, U.S. patents and published patent applications cited herein are hereby fully incorporated by reference in their entirety.

[0159] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0160] Example 1 Subjects were enrolled, and specimens were collected and processed. Subjects > 18 years of age with HPV+ OPSCC were enrolled, and their informed consent was obtained. p16 was determined by tumor immunohistochemistry and used as a surrogate marker for the HPV status of the subject’s cancer. Enrolled subjects had baseline collection of clinical data on demographics, disease characteristics, treatment plan, and prior biomarker testing. Blood samples were collected prior to initiation of definitive treatment (radiation + / - chemotherapy or surgery) and every 3 months after treatment completion. Plasma was collected in Streck or PaxGene tubes designed for ctDNA preservation and isolated by two sequential centrifugation steps as recommended by the manufacturer, as previously described

[29] . Collection continued for 3 years until the subject withdrew informed consent or at the discretion of the investigator. The frequency of monitoring imaging and clinic visits was determined at the discretion of the treating physician. Imaging times and corresponding results were recorded in the database. gDNA extraction and gDNA fragmentation from cell lines are described below.

[0161] Genomic DNA extraction and fragmentation. Genomic DNA of HPV16-positive cell line UM-SCC-104 and HPV18-positive cell line UM-SCC-105 was extracted using Wizard DNA Purification Kit protocol (Promega # A1120). UM-SCC-104 gDNA and non-HPV human genomic DNA (hgen DNA) (Promega # G3041) were restriction digested using Hindlll (New England Biolabs # R0104S) by incubating at 37°C overnight at the recommended concentration by the vendor, followed by incubation at 80°C for 20 minutes to inactivate the enzyme. Shearing of UM-SCC-104 gDNA, UM-SCC-105 gDNA, and hgen DNA (Promega # G3041) was performed at the Advanced Genomics Core (University of Michigan) using a Covaris S2 Focused-ultrasonicator. The sample volume was kept constant at 130 µl, and the DNA concentration ranged from 10 ng / µl to 50 ng / µl. The sheared DNA size ranged from 50 bp - 500 bp, with the majority between 150 bp - 300 bp. Upon initial screening with un-sheared UM-SCC-104 gDNA, 16 out of 17 selected primer / probe sets (except primer / probe set #8) Figure 1B ) met the screening with sheared gDNA, and no detectable cross-reaction with sheared non-HPV hgen DNA was observed (data not shown). Based on these results, sheared UM-SCC-104 gDNA was used as the template for all further screening experiments to develop the multi-probe assay.

[0162] CHAMP-16 assay development and droplet digital PCR. The assay was developed by combining bioinformatics-based probe selection (Table 10) [35-39], empirical validation and optimization of PCR conditions (Table 1-Table 9, Table 11-Table 19) [40-42]. The specific parameters for primer / probe screening using ddPCR

[43] and calculations for LoD

[44] are described below.

[0163] Droplet digital PCR. Each ddPCR reaction contained template DNA, 2X ddPCR Supermix for Probes No dUTP (Bio-Rad # 186-3024), HPV16 primer / probe assay mix, and nuclease-free water (see supplemental table for final concentrations and volumes). Reaction mixtures were dispensed using a QX200 Droplet Generator (Bio-Rad), transferred to a 96-well plate, sealed, and cycled in a C1000 Thermal Cycler (Bio-Rad). Droplets were read using QuantaSoft software in a QX200 reader (Bio-Rad). For single primer / probe assay tests set up during the screening process, the annealing temperature was set to 58°C according to the lowest primer / probe melting temperature of the selected 19 combinations, and PCR was performed for 40 cycles according to the standard ddPCR protocol with a ramp rate of 2°C / second. To develop the multi-probe pool, the ramp rate was set to 1°C / second for each step, the annealing temperature was set to 60°C, and the number of cycles was set to 50. After finalizing the pool, the PCR conditions for the pool were optimized for analysis and clinical validation, with the annealing temperature set to 59°C Figure 12 ). The sheared UM-SCC-104 (HPV16+) cell line gDNA was included as a positive control. UM-SCC-105 (HPV18+) cell line gDNA and 44,000 sheared non-HPV hgen DNA diploid GE (Promega # G3041) were used as negative controls. For each assay, the threshold used to distinguish between positive and negative droplets was standardized. If 10 or more primer / probe combinations were combined together, the background could not be distinguished from the signal Figure 2B 、 Figure 7A 、 Figure 7B and Figure 7C ), positive droplets could not be determined.

[0164] Limit of detection and coefficient of variation calculation for CHAMP-16 assay. The limit of blank (LoB) for the CHAMP-16 assay was calculated using the following formula: LoB = mean 空白 + 1.645 (standard deviation 空白 ) and was determined to be 0.48 copies per 20 ul reaction using approximately 44,000 sheared non-HPV hgen DNA diploid GE as blank (n = 61). The limit of detection (LoD) for the CHAMP-16 assay was calculated using the following formula: LoD = LoB + 1.645 (standard deviation 低浓度样本 ).

[0165] To determine the LoD using UM-SCC-104 cell line gDNA, 12 gDNA serial dilution samples starting from 140 haploid GE were analyzed Figure 3K). For the measurement of the lowest concentration sample, the standard deviation was determined to be 1.3 copies. From this value and the LoB determined above, the LoD was determined to be 2.6 copies per 20 μΐ reaction using the UM-SCC-104 cell line gDNA.

[0166] To determine the LoD using the synthetic HPV16 DNA pool of 9 targets, 12 serially diluted samples of this DNA pool were analyzed starting from 4000 total copies Figure 3 J). For the measurement of the lowest concentration sample, the standard deviation was determined to be 2.2 copies. From this value and the LoB determined above, the LoD was determined to be 4.1 copies per 20 μΐ reaction using the synthetic HPV16 DNA pool of 9 targets. This value was used as the LoD for the analysis of patient samples. The coefficient of variation (CV) was calculated using the following formula: % CV = (standard deviation / mean) * 100.

[0167] Example 2 Assay design: HPV16 genome used for computational analysis and screening of candidate primer / probe sets. To design a multi-target assay covering the entire HPV16 genome, the commonly referenced variant (NC_001526.4) was chosen and the genomic sequence was computationally screened for candidate primers and probes Figure 1A ). Briefly, the HPV16 genomic sequence was scanned using overlapping 0.8 to 1 kb windows to identify primers and probes of variable length and melting temperature range adaptable to ddPCR conditions that are ideal for plasma cfDNA assays [35, 36]. From this, 292 primer / probe combinations Figure 6 ) were determined, checked for homology to the human genome using the BLAST algorithm

[37] , and then aligned to eliminate those combinations with high risk of homodimerization, heterodimerization, and hairpin formation [38, 39]. From this, 49 primer / probe combinations Figure 1A and Figure 6 ) of which 19 candidates with the best design for multiplex assays were selected for further screening and development Figure 1A ).

[0168] Example 3 Assay development: experimental screening of candidate primer / probe combinations using ddPCR. A SP ddPCR assay for the detection of HPV16 ctDNA targeting a 77 bp region in the E6 gene and the upper regulatory region was previously reported [29-31]. The SP ddPCR assay has a LoD of 4.2 GE for HPV16 and was analytically validated for clinical sensitivity and specificity in plasma samples from HPV+ and HPV- OPSCC patients

[29] . Since the SP ddPCR primer / probe combination met the criteria for computational screening, it was included in the development screening of the multiplexed assay. To test the performance of 20 primer / probe candidates (19 new primer / probe sets and the published SP assay) in ddPCR, Hindlll digested gDNA from the low copy number HPV16 positive cell line UM-SCC-104

[45] was used as template and the candidate sets were tested as individual assays Figure 1B ). Out of the 20 primer / probe candidates, seventeen had a signal intensity 3-fold or more above background and were thus selected for further screening; whereas 2 sets (#17 and #18) with lower signal intensity and 1 set (#14) with suboptimal amplification and presence of a large raindrop pattern were excluded. None of the tested combinations cross-reacted with a saturated amount (200,000 GE) of Hindlll digested non-HPV hgen DNA control Figure 1B ), indicating that the selected candidates displayed specificity for HPV16 DNA detection.

[0169] Determination of the upper limit of multiplexing. To mimic plasma cfDNA derived from the nuclear genome with fragment sizes of approximately 166-167 bp [35, 46, 47], 17 primer / probe candidates were screened using sonicated UM-SCC-104 gDNA and non-HPV hgen DNA (median size 300 bp; see Supplementary Methods). Sonication of the gDNA was also performed to avoid potential generation of amplicons longer than the intended target length (<150 bp), which could occur during development of the multiplexed assay with primers from different pairs. Furthermore, to determine the feasibility of multiplexing the selected primer / probes and to determine the maximum number that could be included in a single reaction

[40] , different numbers of pools of FAM-labeled primer / probe sets were tested Figure 2A and 2B ). Multiplexing resulted in an increase of background that was proportional to the number of multiplexed probes, whereas the signal from positive droplets (theoretically representing only a single amplification target per droplet) did not increase proportionally Figure 2B). Therefore, a signal intensity at least 2-fold higher than background was set as the selection criterion (referred to as the "2-fold criterion") to evaluate the number of probes that can be combined to reliably measure HPV16 copy number.

[0170] To determine the effect of changing primer / probe concentrations and PCR cycling conditions on signal intensity and background

[41] , each pool was tested at the vendor-recommended primer / probe concentrations (900 nM / 250 nM final concentration) ( Figure 7A ) or 2-fold and 5-fold lower primer / probe concentrations ( Figure 2B , 7B and 7C). The combination containing the combination of 5 primer sets and probes (referred to as the "5-pool") met the 2-fold criterion at all different parameters tested ( Figure 2B , 7A , 7B and 7C). In the case of the 10-pool and 14-pool versions, the background signal was significantly elevated, resulting in signal-to-signal overlap as determined by the no-template water blank ( Figure 2B , 7A , 7B and 7C). These results indicated that at most 5 primer / probe combinations can be combined to obtain an acceptable signal intensity relative to background to detect HPV16 copy number.

[0171] Fifty different 5-pool combinations were then screened using FAM-labeled probes. Many combinations did not meet the 2-fold criterion and were therefore excluded ( Figure 8 ). Six-pool versions of the selected primer / probe combinations that met the 5-pool test were also attempted, but they did not meet the 2-fold criterion (data not shown). In addition, pools containing one or more of the five primer / probe sets #3, #9, #10, #11 and #12 were not selected for further analysis because the target region was partially or entirely located in the viral E2 gene ( Figure 1A ) which is frequently lost during integration of viral DNA into the host genome

[29] . Among the combinations that met the 2-fold criterion, the combination that measured the highest HPV16 copy number (5-pool v2) was selected for further analysis and development ( Figure 9 ).

[0172] Selection of dual-color multi-probe pools. To expand the coverage of HPV16 genome detection, a 5-pool method using VIC-labeled probes compatible with the FAM-labeled probes was developed, and the dual-color detection capability of ddPCR that can potentially target up to 10 different regions of the HPV16 genome was utilized. VIC-labeled probes tend to have weaker signal intensity compared to FAM [41, 42]. Therefore, the strong signal intensity when compared to background in the candidate screening ( Figure 9), the VIC labeled version of the FAM pool (5-pool v2) was selected for testing. When tested as a standalone assay using sheared UM-SCC-104 gDNA, all 5 probes VIC labeled versions showed lower signal compared to the FAM labeled versions, assessed by signal intensity over background ( Figure 10 ). Thus, a significant reduction in signal intensity was observed for the VIC labeled 5-pool v2 version, below 2-fold standard ( Figure 11 ). Therefore, the 4-pool combination was tested with the VIC labeled version of the 5 selected probes comprising 5-pool v2, and all 4-pools tested with VIC labeled probes met the 2-fold standard ( Figure 2C ). In the two 4-pools with the highest HPV16 copy number measured, 4-pool v20VIC was selected for further development of the dual color assay because it contains primer / probe #4, which targets the highly present viral E6 / E7 junction in HPV16 ctDNA [29, 48, 49].

[0173] To find a compatible 5-pool containing FAM labeled probes, eight combinations were tested (not including the 4 primer / probe set used in 4-pool v20VIC) for HPV-16 detection in dual color format ( Figure 3 ). To check for any impact on VIC pool background due to FAM pool, the individual FAM labeled primer / probe set (#6) was also tested in combination with 4-pool v20VIC ( Figure 3 A). Out of the eight FAM 5-pools screened, the background of the VIC labeled pool changed for four pools, thus excluded ( Figure 3 C, 3F, 3H, and 31). According to the two-dimensional droplet plot, 5-pool v39FAM was selected as the pool compatible with 4-pool v20VIC ( Figure 3 G) because it met the 2-fold standard and did not change the background in the VIC channel compared to the single probe set ( Figure 3 A). The selected 9-pool (5-pool v39FAM + 4-pool v20VIC) assay was named “CtDNA HPV16 evaluation using multiple probes” (CHAMP-16) assay. The CHAMP-16 assay did not show any signal when tested with sheared non-HPV hgen DNA (about 44,000 GE) or sheared genomic DNA from HPV18+ cell line UM-SCC-105, indicating that it is specific for HPV16 ( Figure 16 ).

[0174] Example 4 Assay validation: Analytical validation of the CHAMP-16 assay and the CHAMP-hr assay. Prior to analytical validation, the optimal PCR conditions for the CHAMP-16 assay were determined (Table 4). Figure 12 ) The CHAMP-16 assay showed an average 9.5-fold higher analytical sensitivity compared to the SP assay when tested with sheared UM-SCC-104 gDNA Figure 13 ). To calculate the LoD, the CHAMP-16 assay was tested in a range of concentrations of a pool of chemically synthesized HPV16 DNA fragments corresponding to the 9 target sites (Table 6, Figure 13 ) and in serial dilutions of sheared UM-SCC-104 gDNA as template at different concentrations. Each of the 9 synthetic DNA templates was individually validated by testing in a sheared non-HPV human genomic DNA background matrix, using the corresponding primer / probe as a single probe assay, or using the CHAMP-16 multi-probe assay as a primer / probe pool. The copy numbers detected with a single target as template were comparable when tested with the corresponding single probe assay or the CHAMP-16 multi-probe assay (Table 5). As expected, the HPV16 copy numbers detected by the CHAMP-16 assay using the pool of all 9 synthetic targets as template were on average about 9-fold higher than the copy numbers detected with a single target as template (Table 5).

[0175] To determine the LoD of the CHAMP-16 assay, the synthetic pooled DNA template containing the 9 targets or the sheared UM-SCC-104 cell line gDNA template was spiked into hgen DNA matrix (about 44,000 diploid GE) and tested at different concentrations. From these serial dilution experiments, using the synthetic DNA library and the sheared UM-SCC-104 cell line gDNA template, the LoD was determined to be <5 copies of HPV16 Figure 4 A and Figure 4 B). As the CHAMP-16 assay targets 9 different regions of HPV16, these results indicate that the LoD of the assay is <1 GE of HPV16. Using the synthetic HPV16 DNA pool or the UM-SCC-104 gDNA, the coefficient of variation (CV) was calculated at different template concentrations, determining results of less than 20% for detection of 1 GE to 5 GE; and results of less than 10% for detection of >5 GE Figure 4 C and Figure 4 D).

[0176] Using the analytical testing strategy of the CHAMP-16 assay as described above, the analytical characteristics of the high-risk HPV pool were determined, including the LoD for the targeted high-risk HPV types. This indicates an analytical LoD of 2.4 to 5 copies per mL of plasma, depending on the HPV type Figure 22). Preliminary analysis showed that the high-risk multiprobe pool had excellent signal-to-noise ratio and did not amplify non-HPV human genomic DNA, and these primer / probe sets have been combined for development of the CHAMP high-risk (CHAMP-hr) HPV assay.

[0177] To control the processing of the raw samples (patient specimens) and the molecular analysis of the analytes (processed samples), quality control measures were incorporated. To control sample processing, synthetic 150 bp plant (galium) DNA was spiked as a "technical control" to normalize the recovery of plasma cfDNA extraction as described in

[54] . In addition, to control analytes with signal below the LoD and to assess biological variation between samples, the widely used cfDNA analysis reference gene RPP30 assay was included in the technical control as a dual-color combined assay to allow independent quantification of the two quality control targets per sample. Thus, a dual-color assay was developed and its performance was analytically tested ( Figure 23 ).

[0178] Testing of clinical plasma samples using the CHAMP-16 assay. Plasma cfDNA from 21 patients diagnosed with locally advanced HPV+ OPSCC that were detectable for HPV16 ctDNA with the SP assay were used to compare the CHAMP-16 assay and the SP assay for detection of HPV16 ctDNA. Figure 20 and Figure 15 ). The CHAMP-16 assay detected significantly higher HPV16 copy numbers compared to the SP assay (mean 6.6-fold) ( Figure 20 ). The CHAMP-16 assay was also highly specific for HPV as samples from HPV-negative cancer patients (n=l l) and non-cancer control subjects (n=3) did not show any signal upon testing ( Figure 16 ).

[0179] In addition to comparing the CHAMP-16 assay to the SP assay for testing of clinical samples, it was also compared to the commercially available NavDx assay. HPV16 ctDNA copy numbers were determined in plasma samples from 3 patients in which detectable HPV16 ctDNA levels were reported at the same or similar time points using the NavDx assay, and in 5 patients in which HPV16 ctDNA was reported negative with the NavDx assay. Consistent with the signal enhancement observed when compared to the SP assay, the CHAMP-16 assay exhibited higher signal intensity than the NavDx test vendor reported values in patients with detectable HPV16 ctDNA (mean 6.9-fold) ( Figure 21 and Figure 17 ), while no signal was observed in the 5 patients that were previously negative with the NavDx test ( Figure 17). Overall, the CHAMP-16 assay showed significantly higher HPV16 ctDNA signal detection from plasma compared to tumor tissue modification virus (TTMV)-HPV DNA quantification reported by the SP assay or the NavDx assay.

[0180] To gain insight into the clinical benefits of enhanced signal detection using this assay, samples from one patient who experienced recurrent disease (confirmed by PET / CT and subsequent biopsy) after completing definitive chemoradiation and with scheduled blood draws every three months were analyzed. It was found that the CHAMP-16 assay detected HPV16 ctDNA 20 months prior to clinical diagnosis, while the SP assay only detected HPV16 ctDNA at a time point 1.5 weeks prior to clinical diagnosis (out of available blood draw time points) Figure 5 and Figure 18 ). For patients with higher baseline ctDNA levels but without clinical recurrence, longitudinally collected plasma samples were further tested and it was found that the CHAMP-16 assay did not show any detectable signal in samples collected after the baseline sample Figure 19 ), in contrast to patients who experienced cancer recurrence Figure 5 and Figure 18 ). This highlights the value of the CHAMP-16 assay to provide increased signal strength and detect HPV16 ctDNA significantly earlier than the SP assay.

[0181] Finally, the assay was applied in an exploratory manner to residual biobanked samples from a historical clinical trial Figure 15 ; patients 22 to 41) to evaluate its performance in detecting HPV16 ctDNA at baseline (i.e., prior to initiation of treatment). Notably, these samples were collected and processed in a different manner than the aforementioned 21 patient serum samples Figure 20 ) in a previous study, including having undergone one or more freeze-thaw cycles. Furthermore, although the tumor tissues of these patients were confirmed to be pl6(+) it was not well understood whether these tissues were positive for HPV16. Focusing on samples that were positive using both the SP and CHAMP-16 assays Figure 15 ; patients 22 to 25), the CHAMP-16 assay showed an average 8.7-fold increase in signal compared to the SP assay. An additional 4 samples in this cohort had detectable HPV16 ctDNA signal with the CHAMP-16 assay, but not with the SP assay Figure 15 ; patients 26 to 41). These data collectively demonstrate that the CHAMP-16 assay can improve the sensitivity of HPV16 detection and improve baseline detection compared to conventional single probe assays.

[0182] HPV ctDNA analysis has had a rapid impact on the field of HPV-associated malignancies. It is clear that HPV ctDNA testing can have substantial long-term impact on the paradigm of clinical decision making for oropharyngeal cancer. Past experience in the development of tumor biomarker assays supports the importance of the availability of multiple assay options, such as the multiple gene expression profile tests for women with breast cancer (MammaPrint, Oncotype Dx) [51, 52]. The development of multiple assays for an important target such as HPV is critical because different clinical application cases can benefit from various optimization parameters. Given the current sensitivity limitations of HPV ctDNA LDTs, there is a clear clinical need to develop higher sensitivity tests with specific settings for the detection of minimal residual disease

[53] .

[0183] Accordingly, recognizing the need for other HPV ctDNA ddPCR LDTs with fully published assay details and analytical characteristics for benchmark testing, and the need for future rigorous validation clinical studies to advance the use of HPV ctDNA as a biomarker, a multi-probe HPV ctDNA assay was developed that has higher analytical sensitivity relative to the SP assay, and has a 6.9x higher signal strength than the commercial assay (NavDx) when tested directly using a limited number of available time-matched samples. Accordingly, this enhancement in signal allows the assay to have an LOD < 1 GE, which is comparable to what has been observed in ultra-sensitive HPV-seq studies using next-generation sequencing

[53] . It is also noted that the reporting metrics for the NavDx assay have recently changed. Accordingly, there is a limited number of samples currently available for performance benchmarking comparisons with other assays, which underscores the urgent need to establish a shared library of HPV+ plasma samples, especially given the multiple HPV ctDNA assays currently being developed by both academic and commercial entities.

[0184] Overall, this high performance ddPCR approach is still significantly more inexpensive, rapid, and simple than NGS approaches. Accordingly, the low-cost ddPCR-based approach developed herein has advantages for early detection and / or monitoring screening, where more frequent testing is beneficial to the patient.

[0185] Example 5 Table 10: Calculation screen. Primers and probes were designed using HPV16 sequence (NC_001562.4 - 7906 bp). Using the PrimerQuest tool from Integrated DNA Technologies, using the qPCR (2 primers + probe) option, scan the HPV16 genomic sequence with the following parameters: (a) Reaction conditions

[0186] The concentration in ddPCR Supermix (#186-3024, Bio-Rad) is proprietary information and the values reported are estimates based on personal communication.

[0187] (b) Primer standards

[0188] (c) Probe standards

[0189] (d) Amplicon standards

[0190] Each search using the PrimerQuest tool returned a maximum of 50 results. To avoid saturation, a 0.8 - 1 kb window of the 7906 bp HPV16 sequence was scanned with a 200 bp overlap at the 5' and 3' ends. Using the above criteria in the PrimerQuest tool, a total of 321 primer and probe (primer / probe) combinations (963 oligonucleotide sequences) were identified across the HPV16 genome. After removing redundant primer / probe combinations in every 200 bp overlapping sequence between different 0.8-1 kb batches, a total of 292 unique combinations (876 oligonucleotide sequences) were identified as candidates for further screening.

[0191] The selected 292 combinations were checked for homology to the human genome (taxonomy ID: 9606) using the NCBI BLASTn megablast program. Any primer or probe with more than 15 bp of homology to an annotated region was excluded. This criterion led to the selection of 67 combinations (201 oligonucleotide sequences).

[0192] Since the primers and probes were designed to be combined in a single reaction and to detect multiple regions of HPV16 from cfDNA samples, the selected 67 primer / probe combinations were checked for heterodimerization with other primers or probes using the Primer Tools program from the National Institute of Standards and Technology. Up to 100 batches of sequences can be screened using this tool. Therefore, the primer / probe combinations were screened in batches of 33 combinations (99 oligonucleotide sequences) in multiple iterations and heterodimers with homologous regions with more than 8 bp (contiguous) and melting temperatures more than 20 °C were excluded. After heterodimer screening, the primer and probe self- annealing to hairpin structures or homodimer formation were screened using the same Primer Tools program and the PCR Primer Stats program from Genscript, respectively. Primers or probes that were predicted to not have more than 8 bp base sequence segments annealing to form homodimers, hairpin structures, or heterodimers were selected, resulting in a total of 49 primer / probe combinations (147 oligonucleotide sequences).

[0193] Table 11: Final PCR conditions for CHAMP-16 assay, CHAMP-hr assay, and RPP-30 / Plant Spike Dual assay.

[0194]

[0195] Table 12: 38x CHAMP-16 assay mix.

[0196]

[0197] Table 13: 38x CHAMP-hr assay mix.

[0198]

[0199] Table 14: 38x (SP assay + RPP30 reference gene assay) mix.

[0200]

[0201] Table 15: 38x (RPP30 reference gene assay + Plant Spike assay) mix.

[0202]

[0203] Table 16: Droplet generation settings for CHAMP-16 assay.

[0204]

[0205] Table 17: Droplet generation settings for CHAMP-hr assay.

[0206]

[0207] Table 18: Droplet generation settings for SP assay + RPP30 reference gene assay.

[0208]

[0209] Table 19: Droplet generation settings for RPP30 reference gene assay + plant spike protein assay.

[0210]

[0211] References:

[0212]

[0213]

[0214]

[0215]

[0216]

Claims

1. A composition comprising one or more oligonucleotides that detect one or more target regions selected from the group consisting of: SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO:

56.

2. The composition of claim 1, wherein the one or more oligonucleotides are selected from the group consisting of: SEQ ID NO: 1 - 27 and SEQ ID NO: 37 - 51.

3. The composition of claim 1 or 2, wherein at least a triad of the one or more oligonucleotides is selected from the group consisting of: Group 1: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; Group 2: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; Group 3: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; Group 4: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12; Group 5: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; Group 6: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18; Group 7: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21; Group 8: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; Group 9: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27; Group 10: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; Group 11: SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; Group 12: SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; Group 13: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; Group 14: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:

51.

4. The composition of claim 1 or 2, wherein the one or more oligonucleotides are selected from the group consisting of: a forward primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49; a reverse primer having at least 90% sequence identity to any one or more of: SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51; a probe comprising a detectable label, wherein the forward primer and the reverse primer anneal to a target HPV sequence.

5. The composition of claim 4, wherein the probe has at least 90% sequence identity to any one or more of: SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 38, 41, 44, 47, 50.

6. The composition of any one of claims 1-5, wherein the first set of oligonucleotides comprises SEQ ID NO: 1 - 15 or SEQ ID NO: 37 - 45.

7. The composition of any one of claims 1-6, wherein the second set of oligonucleotides comprises SEQ ID NO: 16 - 27 or SEQ ID NO: 46 - 51.

8. The composition of any one of claims 1-7, wherein the one or more oligonucleotides do not have a base sequence of more than eight base pairs that anneal to form a homodimer, a hairpin structure, or a heterodimer.

9. The composition of any one of claims 1-8, wherein the first set of oligonucleotides comprises a first detectable label and the second set of oligonucleotides comprises a second detectable label.

10. The composition of claim 9, wherein the second set of oligonucleotides target a viral E6 / E7 junction, and wherein the junction is highly present in HPV ctDNA.

11. The composition of any one of claims 9-10, wherein the second set of oligonucleotides is selected from the group of SEQ ID No: 2, 5, 8, 11, 14, 17, 20, 23, 26, 39, 42, 45, 48, 51 and the first set of oligonucleotides is selected from the group of SEQ ID No: 1, 4, 7, 10, 13, 16, 19, 22, 25, 37, 40, 43, 46, 49.

12. A method comprising contacting a sample with the composition of any one of claims 1-11.

13. The method of claim 12, wherein the sample is blood.

14. The method of claim 12, wherein the sample is derived from blood.

15. The method of claim 12, wherein the sample is amniotic fluid, ascites fluid, bile, breast milk, colostrum, bronchoalveolar lavage fluid, cerebrospinal fluid, dialysis fluid, aqueous humor, vitreous humor, fecal matter, aspirate fluid, pericardial fluid, peritoneal fluid, plasma, pleural fluid, semen, serum, synovial fluid, tear fluid, thoracentesis fluid, blood, saliva, mouthwash, or urine.

16. The method of claim 12, wherein the contacting comprises: (i) providing one or more oligonucleotides; (ii) fractionating a plurality of HPV DNA in the sample into droplets at a concentration wherein there is an average of only 0 or 1 of the DNA molecules per droplet; (iii) amplifying the HPV DNA in each droplet with the one or more oligonucleotides to generate amplicon signals; and (iv) detecting any amplicon signals in each droplet.

17. The method of claim 16, wherein the DNA is fractionated into microdroplets by emulsification.

18. The method of claim 16 or 17, wherein the DNA is amplified using a nucleic acid amplification method.

19. The method of claim 12, wherein, The contacting comprises performing a quantitative PCR (qPCR) assay.

20. The method of claim 19, wherein the qPCR assay comprises a digital PCR assay.

21. The method of claim 20, wherein the digital PCR assay comprises a droplet digital PCR (ddPCR) assay.

22. The method of claim 12, wherein the sample comprises ctDNA HPV target nucleic acids and wherein one or more of the oligonucleotides hybridize to HPV target nucleic acids.

23. The method of claim 12, further comprising the steps of: If HPV is detected in the sample, the subject is treated with cancer monitoring, treatment, or intervention.

24. The method of claim 23, wherein the step of treating the subject is provided prior to the appearance of disease detectable by CT scan.

25. The method of claim 12, further comprising the steps of: If HPV is not detected in the sample, the subject is treated with an HPV vaccine.

26. The method of claim 12, further comprising sequencing HPV nucleic acids present in the sample.

27. A kit comprising the composition of any one of claims 1-12 and reagents for purifying, isolating, detecting, and / or quantifying HPV ctDNA.

28. The kit of claim 26; wherein the reagents comprise: (i) amplification reagents; (ii) control samples; (iii) a solid surface comprising capture reagents specific for target ctDNA; (iv) containers for placing or storing samples; (v) one or more instruments for assisting in obtaining test samples; (vi) reaction vessels, mixing vessels, and (vii) instructions for use of the kit.

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