[go: up one dir, main page]

WO2016049559A1 - Procédés de diagnostic et de pronostic pour des virus et des maladies et affections associées - Google Patents

Procédés de diagnostic et de pronostic pour des virus et des maladies et affections associées Download PDF

Info

Publication number
WO2016049559A1
WO2016049559A1 PCT/US2015/052414 US2015052414W WO2016049559A1 WO 2016049559 A1 WO2016049559 A1 WO 2016049559A1 US 2015052414 W US2015052414 W US 2015052414W WO 2016049559 A1 WO2016049559 A1 WO 2016049559A1
Authority
WO
WIPO (PCT)
Prior art keywords
hpv
amplicon
sample
virus
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2015/052414
Other languages
English (en)
Inventor
Thuraiayah Vinayagamoorthy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2016049559A1 publication Critical patent/WO2016049559A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to the detection, diagnosis, prediction, prognosis, and staging of viruses, such as human papilloma virus (HPV), and in particular to the detection of viral nucleic acids and viral-associated diseases and conditions, including cancer, such as cervical cancer.
  • viruses such as human papilloma virus (HPV)
  • HPV human papilloma virus
  • the disclosure relates in some aspects to methods for discerning between different forms of viruses, such as free, episomal, and integrated forms.
  • Various methods are available for detecting the presence of virus and viral nucleic acids and for prognosis, diagnosis, prediction, and staging of diseases, such as cancer. For example, methods are available for the detection, isolation, and sequencing of viral nucleic acids, and for screening and diagnosis of cancer and other diseases and conditions. Available methods have not been entirely satisfactory. Improved methods are needed. For example, assays are needed for the detection of viruses and the diagnosis, prognosis, and monitoring of associated diseases and conditions, such as cancer. For example, assays are needed with improved sensitivity, specificity, accuracy, efficiency, and/or prognostic or predictive capabilities. Among the provided embodiments are methods, compositions, and systems that meet such needs.
  • kits for assessing samples such as biological samples obtained from a subject, for example, to detect, diagnose, and/or provide prognostic or predictive information about a virus capable of infecting the subject and/or a disease or condition associated with the virus.
  • the virus typically is an oncogenic virus, such as HPV, EBV, or a poxvirus.
  • the disease or condition in some embodiments is a malignancy, neoplasm, tumor, cancer and/or other proliferative disorder, such as cervical cancer.
  • the methods involve (a) detecting the presence or absence of an episomal form of the virus, such as a virus associated with cancer, in the biological sample or a composition derived therefrom; (b) detecting the presence or absence of a nucleic acid of the virus (e.g., genome or partial genome) integrated into a host genome, e.g., into a host gene, in the sample or a composition derived therefrom; and (c) detecting the presence or absence of a host gene or alteration therein, such as a cancer-associated genetic alteration, in the sample or composition derived therefrom.
  • an episomal form of the virus such as a virus associated with cancer
  • detection in (b) involves incubating the sample or a nucleic acid derived therefrom under conditions designed to produce a second amplicon only in the presence of a nucleic acid of the virus integrated into a host genome, or only in the presence of the nucleic acid integrated into a coding region of a host gene, said second amplicon comprising a junction between the integrated nucleic acid and the host gene, thereby, where viral nucleic acid integrated into a coding region of a host gene is present in the sample, producing the second amplicon.
  • the incubation in (b) is carried out in the presence of a primer complementary to a nucleotide sequence of the virus, such as an annealing site in an HPV E7 gene, and a primer comprising a poly-T sequence.
  • the sample is a biological sample, such as one from a subject, e.g., a human subject.
  • the methods involve (a) incubating a composition derived from the sample, which is a cellular fraction, under conditions designed to produce a first amplicon only in the presence of closed circular DNA of a virus associated with cancer, thereby producing the first amplicon where an episomal form of the virus is present in the sample, and optionally, where the first amplicon is produced, determining the sequence of at least a portion of it; and (b) incubating the sample or a nucleic acid derived therefrom under conditions designed to produce a second amplicon only in the presence of a nucleic acid of the virus integrated into a host genome, or only in the presence of the nucleic acid integrated into a coding region of a host gene, said second amplicon comprising a junction between the integrated nucleic acid and the host gene, thereby, where viral nucleic acid integrated into a coding region of a host gene is present in the sample, producing the second amplicon, and optionally, where the second amplicon is produced
  • the incubation in (b) is carried out in the presence of a primer complementary to a nucleotide sequence of the virus, such as an annealing site in an HPV E7 gene, and a primer comprising a poly-T sequence.
  • the amplicon in (a) comprises a nucleotide sequence of a junction between the head and tail portion of a closed circular viral genome, such as a closed circular HPV viral genome.
  • (a) comprises incubating the sample or composition with a primer complementary to a primer annealing site in an HPV L2 gene and a primer
  • the methods involve incubating the sample or a nucleic acid derived therefrom in the presence of a pair of primers designed to produce an amplicon only in the presence of a viral nucleic acid (e.g., HPV nucleic acid) integrated into a coding region of a host gene.
  • a viral nucleic acid e.g., HPV nucleic acid
  • the amplicon in some aspects includes a junction between the integrated viral nucleic acid and the host gene, such that, where viral nucleic acid integrated into a coding region of a host gene is present in the sample, the amplicon is produced.
  • the amplicon comprises a nucleotide sequence of a junction between the head and tail portion of a closed circular viral genome, such as a closed circular HPV viral genome.
  • the methods further include detecting the presence of the virus in the sample or another sample derived from the subject, prior to the detection in (a) or (b) or prior to the incubation with the primers.
  • the detection in (a) is carried out by incubating the sample or composition under conditions designed to produce a first amplicon only in the presence of a closed circular DNA of the virus and, where produced, detecting the presence of the first amplicon, wherein the sample or composition is the composition, which is a cellular fraction derived from the sample; and/or the detection in (b) is carried out by incubating the sample or composition under conditions designed to produce a second amplicon only in the presence of nucleic acid of the virus integrated into a coding region of a host gene and, where produced, detecting the presence of the second amplicon.
  • the detection in (b) is carried out in the presence of a primer complementary to a nucleotide sequence of the virus, such as an annealing site in an HPV E7 gene, and a primer comprising a poly-T sequence.
  • the methods further include detecting the presence of the amplicon(s), e.g., first and second amplicons, where produced. In some aspects, this detecting includes determining the nucleotide sequence of at least a portion of the respective amplicon(s), where produced.
  • Such determining in some embodiments is carried out by combining: the amplicon or amplicons (.e.g., produced in step (a) and/or (b)); and sequencing primer(s) complementary thereto, such as a sequencing primer complementary to a sequencing primer annealing site in the first amplicon; and a sequencing primer complementary to a sequencing primer annealing site in the second amplicon.
  • the sequencing primer annealing site in the second amplicon comprises a portion of a human papilloma virus (HPV) E4 gene.
  • the pair of primers comprises a primer complementary to a nucleic acid sequence, such as an HPV nucleotide sequence, and a primer comprising a poly-T sequence.
  • the virus is an HPV, an Epstein Barr virus (EBV), or a poxvirus.
  • various reactions are carried out in the same reaction and/or are carried out simultaneously.
  • the method includes amplifications in (a) and (b)
  • the amplifications in (a) and (b) may be carried out in the same reaction mixture and/or are carried out simultaneously.
  • the method further includes determining a subtype or category of subtype of the virus in the sample.
  • the sequence so- determined indicates the presence or absence of a particular subtype or category of subtypes of the virus.
  • the subtype is a high-risk HPV subtype or the category is a category of high-risk HPV subtypes.
  • the high-risk subtype or category is a subtype or category indicates cervical cancer or a high risk of developing cervical cancer, such as a high- risk subtype or category that is or includes HPV-16, HPV-18, HPV-31 or HPV-33; is or includes HPV-16, HPV-18, HPV-31, HPV-33HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-55, HPV-56, HPV-58, HPV-59, and/or HPV-68; or is or includes HPV-16 or HPV-18.
  • a high- risk subtype or category that is or includes HPV-16, HPV-18, HPV-31 or HPV-33; is or includes HPV-16, HPV-18, HPV-31, HPV-33HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-55, HPV-56, HPV-58, HPV-59, and/or HPV-68; or is
  • the virus is HPV and the detection of the virus in the sample or another sample derived from the subject includes carrying out an amplification reaction in the presence of a primer complementary to a primer annealing site in an HPV L2 gene and a primer complementary to a primer annealing site in an HPV E2, E4, or E5 gene.
  • the sample is a cervical sample.
  • the sample is from a patient with a diagnosis of atypical squamous cells of undetermined significance (ASCUS).
  • ASCUS atypical squamous cells of undetermined significance
  • the methods further include detecting the presence or absence of a genetic alteration in a host gene, such as an oncogene or tumor suppressor.
  • a host gene such as an oncogene or tumor suppressor.
  • the host gene is selected from the group consisting of p53, Rb, PTEN, pl6INK4a, cyclins, e.g., Cyclin Dl, MYC, RAS genes, EGFR, HST-l, HST-l, and LKB 1.
  • the methods further include detecting the presence or absence of a pathogen other than the virus.
  • the presence of the pathogen indicates a risk of development of the viral- associated disease or condition such as cancer.
  • the pathogen is associated with a sexually transmitted disease, vaginitis, and/or inflammation.
  • kits including kits for carrying out any of the provided methods, including kits comprising the various primers and any other reagents for any of the above embodiments of the methods.
  • the kits include (a) a first pair of primers designed to produce a first amplicon in the presence of closed circular viral DNA, such as closed circular HPV DNA.
  • the first amplicon includes a junction between head and tail portions of the closed circular DNA.
  • the kits further include (b) a second pair of primers designed to produce a second amplicon in the presence of viral nucleic acid, e.g., HPV nucleic acid, integrated into a coding region of a host gene.
  • the second amplicon includes a junction between an integrated HPV nucleic acid and a host gene into which the HPV is integrated.
  • the kits further include (c) a third pair of primers designed to produce an amplicon comprising at least a portion of a host genome, e.g., gene, e.g., one comprising a locus of a genetic alteration in a host gene.
  • the kit includes (a), (b), and (c)
  • it further includes sequencing primer(s).
  • it further includes (d) a first sequencing primer complementary to a sequencing primer annealing site in the first amplicon; (e) a second sequencing primer complementary to a sequencing primer annealing site in the second amplicon; and/or (f) a third sequencing primer complementary to a sequencing primer annealing site in the third amplicon.
  • kits for carrying out the methods.
  • the methods detect the presence or absence of a virus and/or one or more particular form(s) or subtype(s) of the virus in a sample or subject.
  • the methods provide diagnostic, staging, prognostic, monitoring, and/or predictive information regarding a disease or condition associated with the virus, and/or a risk factor, symptom, outcome, or progression thereof.
  • the provided embodiments can offer various advantages compared to available methods.
  • the methods may in some embodiments be carried out in a single test or assay that is comprehensive and/or capable of definitively identifying a subject who has or is at high risk of developing a virus-associated disease, such as a virally-induced cancer, e.g. HPV-induced cancer of the cervix.
  • a virus-associated disease such as a virally-induced cancer, e.g. HPV-induced cancer of the cervix.
  • the methods detect the presence or absence, in a sample or subject, of (a) virus and/or high-risk subtype or group of subtypes thereof; (b) episomal stage(s) of the virus; (c) integrated form(s) of the virus in a host genome, gene, or coding region; and/or (d) disease-associated genetic alteration(s) in one or more host genes, e.g. housekeeping genes or tumor suppressor genes, e.g. p53. Any of a number of known housekeeping genes may be used. Exemplary housekeeping genes are ACTB, GAPDH, GUSB, LDHA, NONO, PGK1, PPIH, RPLP0, ⁇ -globulin and TFRC. Exemplary primers for amplifying ⁇ -globulin include PC03 ( AC AC A ACTGTGTTC ACT AGC) , PC04
  • viruses include those that are associated with, cause, or predispose subjects to neoplasia, malignancy, cancer, and/or proliferative diseases and disorders.
  • the methods are carried out by assessing a single sample taken from the subject, or one or more compositions derived therefrom, in contrast to methods involving the testing of multiple samples, separately obtained from a subject to assess various parameters.
  • the sample and/or compositions are placed under various reaction conditions for the detection of viral and host nucleic acids.
  • the detection or identification of a plurality of nucleic acids, biomarkers, markers, and/or genetic alterations is carried out simultaneously and/or are performed in the same reaction vessel.
  • the presence or absence of two or more of a nucleic acid indicating a virus's presence, a nucleic acid indicating a virus's subtype or category thereof, a nucleic acid indicating a particular form of the virus, and/or a host genetic alteration are detected or determined simultaneously or in the same reaction vessel.
  • the provided methods in some embodiments reveal diagnostic or prognostic information for a subject who does not yet have the disease, or in whom the disease has not progressed, to assess risk of developing the disease or progressing.
  • the provided methods are carried out on a sample that is not cancerous or is not yet at a post-transformed state.
  • the provided embodiments allow for early disease detection, prediction, and/or prognosis, and permit the continued monitoring and/or prevention of diseases and associated symptoms.
  • the methods are useful in prognosis, diagnosis, staging, prediction, risk assessment, and monitoring, of diseases and conditions, such as cancer, and/or in the context of personalized medicine, for example, by identifying the presence of a virus or particular form(s) thereof that is known to be associated with the disease, disease state, progression, or condition and/or genetic alterations in the host known to be associated with the disease or condition.
  • the presence of a viral nucleic acid, e.g., HPV genome, integrated into a coding region of a host gene and/or the detection of one or more tumor-associated mutations in a host gene indicates that the subject is at high risk for developing cancer, e.g., cervical cancer.
  • the methods are capable of detecting the presence or absence of multiple specific viral subtypes in a single reaction, simultaneously, or in compositions derived from the same sample from the subject.
  • the methods include one or more steps for amplifying and/or detecting the presence of or identifying one or more nucleic acids, such as DNA, cDNA, RNA, and/or mRNA.
  • the nucleic acid(s) in some aspects are detected in the sample or composition derived therefrom.
  • the nucleic acids include viral and/or host nucleic acids.
  • the amplification and/or detection is specific for episomal viral nucleic acids and/or for viral nucleic acid integrated into a host genome. In some aspects, it is specific for integration into a gene and/or coding region thereof, e.g., into a particular host gene or coding region thereof.
  • the assay identifies the presence of an integrated viral nucleic acid and also identifies the gene or other sequence into which the virus has integrated.
  • the provided methods are those in which an amplicon is generated or sequence detected in the presence of an episomal or a closed circular viral form but not merely in the presence of other forms of the virus, and/or in the presence of an integrated viral forms, but not merely in the presence of other forms of the virus.
  • the provided assays are advantageous in that they definitively and directly identify viral DNA integrated into a host genome, as opposed to indirectly measuring the levels of products known to be associated with integration, which may not be entirely accurate.
  • high levels of oncogenic genes associated with integrated virus e.g. HPV E6/E7, may be present at the episomal or non- integrated stage and thus methods that use these as markers may result in false positives.
  • the methods specifically amplify and/or detect a nucleic acid that is particular to a type, subtype, or group of subtypes of the virus, such as a subtype or group known to be associated with a high risk of developing a disease or condition such as cancer.
  • the methods include steps in which an amplicon is generated or nucleic acid or sequence detected only in the presence of the relevant subtype or a member of the group of subtypes, but not merely in the presence of other subtypes or members of another group of subtypes of the virus.
  • the subtypes are high-risk HPV subtypes, including HPV-16, HPV-18, HPV-31, and HPV-33.
  • the methods detect or determine the amount or relative amount of virus or viral load in the sample.
  • the methods further detect the presence or absence of one or more nucleic acids containing a genetic alteration, e.g., mutation, in a host gene, for example, in a housekeeping gene or tumor suppressor gene, e.g. p53, such as one associated with the progression of cancer associated with the virus. In some embodiments, they detect the presence of additional pathogenic nucleic acids.
  • the methods further detect the presence or absence of co- infection with other pathogens, such as sexually transmitted pathogens, e.g., those associated with a higher risk of HPV-associated cancer progression.
  • pathogens include herpes simplex virus (HSV) strains such as HSV-6 and HSV-2, bacteria such as Chlamydia trachomatis and Neisseria gonorrhoeae, cytomegaloviruses (CMV) and EBV.
  • HSV herpes simplex virus
  • CMV cytomegaloviruses
  • the identification or detection methods include steps for amplifying and detecting sequences of the nucleic acids, such as amplification, e.g., by polymerase chain reaction (PCR), followed by sequencing.
  • amplification e.g., by polymerase chain reaction (PCR)
  • sequencing e.g., sequencing of various nucleic acids and/or amplicons is carried out simultaneously or in the same reaction vessel.
  • the isolation, amplification, detection, and/or sequencing is carried out using the same apparatus or equipment and/or simultaneously.
  • the methods reduce the amount of time, steps, cost, complexity, and/or number of required resources compared to available assays.
  • the methods provide diagnostic or prognostic information, such as determination of the presence of or risk of developing the disease or condition.
  • HPV e.g., a high-risk subtype
  • the methods indicate or indicate a likelihood of an epithelial, non-cancerous, lesion, such as a low grade cervical squamous intraepithelial lesion (LSIL).
  • LSIL low grade cervical squamous intraepithelial lesion
  • a subject is determined to have a more progressed lesion, such as a high grade cervical squamous intraepithelial lesion (HSIL).
  • HSIL cervical squamous intraepithelial lesion
  • an additional risk factor or indicator such as elevated oncogene expression and/or tumor suppressor mutation in a host gene
  • a subject is determined to have or be likely to have or progress to a malignant or invasive cancer.
  • the viruses detected by the provided assays generally include viruses associated with and/or that cause or predispose subjects to a disease or condition, such as a neoplasia, malignancy, cancer, and/or other proliferative disease or disorder.
  • the viruses may include but are not limited to, human papilloma virus (HPV), including high-risk HPV subtypes, including those associated with particularly high risk of developing one or more particular cancers, such as cervical and/or ovarian cancer.
  • HPV human papilloma virus
  • the viruses also may include Epstein Barr virus (EBV), Marek's disease virus, herpesviruses, such as human herpesviruses, e.g., human herpesvirus 6 (HHV-6) and HHV-7, poxviruses, and adeno-associated viruses (AAV).
  • EBV Epstein Barr virus
  • HHV-6 human herpesvirus 6
  • HHV-7 herpesvirus 6
  • poxviruses e.g., poxviruses
  • AAV adeno-associated viruses
  • the virus-associated and/or induced diseases and conditions include, but are not limited to, proliferative diseases and disorders, including neoplasms, malignancies, tumors, and cancers such as cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, vaginal cancer, head/neck cancer, and oropharyngeal cancer (in some aspects associated with HPV); Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinomas (in some aspects associated with EBV); and lymphomas, leukemias, cervical cancers, and brain tumors (in some aspects associated with HHV-6).
  • proliferative diseases and disorders including neoplasms, malignancies, tumors, and cancers such as cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, vaginal cancer, head/neck cancer, and oropharyngeal cancer (in some aspects associated with HPV); Hodgkin's lymphoma, Burkitt
  • HPV represents a group of over 100 related viruses, including different subtypes, several of which are sexually transmitted.
  • HPV is the causative organism in most cases of cervical cancer, which affects thousands of women in the United States and millions worldwide.
  • HPV subtypes also are causative in most anal cancers and some vaginal, vulvar, penile and oropharyngeal cancers.
  • Sexually transmitted HPV subtypes are generally grouped into one of two categories: high-risk and low-risk. While low-risk subtypes generally do not cause cancer, high-risk or oncogenic subtypes generally can cause cancer.
  • Low-risk HPV subtypes include HPV-6, HPV-11, HPV- 13, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, HPV-61, HPV-70, HPV-72, and HPV-81.
  • High-risk subtypes include HPV- 16, HPV- 18, HPV-31, HPV-33, HPV-34, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-55, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, and HP-70 [0039] HPV 16 and HPV 18 are the most common high-risk subtypes associated with cervical cancer (approximately 70 % of cases), with HPV-16 being the most prevalent. One or both of these subtypes is also associated with most cases of anal cancer, vaginal cancer, vulvar cancer, and penile cancer, with HPV-16 being the most common subtype associated with anal cancer.
  • HPV-16, HPV-18, HPV-31 and HPV-33 are the most common high-risk subtypes associated cervical cancer (approximately 90 % of cases). HPV subtypes in some aspects may be identified based on their nucleotide sequences. See Williams et al., Future Virol. 2011 January 1, 6(1): 45-57; Castillo, Carcinogenesis, Kathryn Tonissen, January 23, 2013, Chapter 3, "Human Papillomavirus and Carcinogenesis in the Upper Aero-Digestive Tract.”
  • HPVs have a circular, double- stranded DNA genome, one strand of which contains open reading frames (ORFs) that are transcribed. It includes eight ORFs and an upstream regulatory region called the long control region (LCR), including an origin of replication and cis-acting transcriptional regulatory elements.
  • LCR long control region
  • the genome is divided into the early region (including six ORFs corresponding to El, E2, E4, E5, E6, and E7, encoding proteins involved in replication and cell transformation) and the late region (including two ORFs, corresponding to LI, and L2, encoding two proteins of the viral capsid). See Castillo, 2013, Figure 1.
  • E6 and E7 are HPV oncogenes. Both gene products interfere with cell cycle regulation and can contribute to malignant transformation. For example, the E6 gene product promotes p53 degradation; E7 promotes and retinoblastoma (Rb) inactivation.
  • the HPV genome exists in an extra-chromosomal (episomal) form in basal epithelial host cells.
  • E2 generally tightly controls expression of E6 and E7, which are typically expressed at high levels in terminally differentiated cells within the intermediate and/or upper layers of the epithelium. See Evans et al., FLOS One. 2014 March 13, 9(3):e91142.
  • Cancer progression generally involves expression of these oncogenes throughout the epithelium. Progression to high-grade intraepithelial lesions and invasive carcinomas is generally associated with a persistent high-risk HPV infection and upregulation of E6/E7 expression. Upregulation of E6 and E7 in many cases results from integration of viral nucleic acids, e.g., of truncated viral genomes, into host genomes. Generally, such integration events are associated with reduced regulation of E6 and/or E7 expression, e.g., due to loss of all or part of the E2 gene. [0042] In a subject in whom HPV is present in the episomal stage, one or more outcomes may result. For example, more than 40% of HPV infections are transient and are cleared with time. Alternatively, the subject may remain a carrier of the virus, which persists in an episomal stage without progression to a cancerous lesion. In some instances, cancer progression occurs.
  • Neoplastic progression often results from, integration of all or part of the HPV genome into the genome of the host cell.
  • the controlling E2 gene is frequently lost, which can result in uncontrolled expression of E6 and/or E7 proteins, which may provide a selective growth advantage for infected cells.
  • E6 and/or E7 proteins which may provide a selective growth advantage for infected cells.
  • integration occurs within a functional cellular gene such as a tumor suppressor gene such as p53, it may destabilize the cell, making it vulnerable for transformation.
  • HPV-associated cancers including some cervical cancers
  • an elevation in episome copy number is accompanied by an increase in viral oncogene expression, which, as with increased expression due to HPV integration, can lead to cell transformation.
  • Host genetic alterations such as mutations in tumor suppressors, e.g., p53, Rb, PTEN, and/or LKBl, or oncogenes, can also promote transformation.
  • Exemplary genes are p53, Rb, PTEN, pl6INK4a, cyclins, e.g., Cyclin Dl, MYC, RAS genes, EGFR, HST-1, HST-1, and LKBl.
  • cyclins e.g., Cyclin Dl, MYC, RAS genes, EGFR, HST-1, HST-1, and LKBl.
  • the provided assays are those that provide diagnostic and prognostic information about HPV-associated transformation events even in the absence of integration.
  • the provided assays in some aspects detect additional markers, such as the presence and/or expression of or alterations in host gene(s) involved in transformation and progression to cancer and/or a host housekeeping gene. Any of a number of known housekeeping genes may be used. Exemplary housekeeping genes are ACTB, GAPDH, GUSB, LDHA, NONO, PGK1, PPIH, RPLP0, and TFRC.
  • such detection provides additional information regarding susceptibility to progression of virus-associated cancer, e.g., cancer of the cervix.
  • Infection with other pathogens such as viral and bacterial pathogens that cause sexually transmitted diseases (STDs) or sexually transmitted infections (STIs) also can be associated with a higher risk of developing cancer, e.g., HPV-associated cancers. See Williams et al., Future Virol. 2011 January 1, 6(1): 45-57.
  • STDs sexually transmitted diseases
  • STIs sexually transmitted infections
  • HPV-associated cancers See Williams et al., Future Virol. 2011 January 1, 6(1): 45-57.
  • Such infections can cause inflammation; in HPV-infected women, cervical inflammation is associated with cervical neoplasia. Since HPV infection of the cervix by itself is not highly inflammatory, viral, e.g.
  • herpes simplex virus HSV
  • bacterial e.g. Chlamydia trachomatis
  • Co-infection with either C. trachomatis or HSV can be associated with a greater risk of developing cervical cancer.
  • HPV integration e.g. via increased HPV integration
  • other STDs such as Neisseria gonorrhoeae.
  • co-infection of HPV 16 with herpesviruses, such as HSV6 or HSV2, and/or cytomegalovirus or EBV can increase the frequency of HPV 16 integration.
  • trachomatis infection favors the entry and persistence of multiple high-risk HPV types in cervical epithelium.
  • Infection-induced inflammation also increases the risk of other HPV-induced cancers, such as penile cancer, where infection with genital lichen sclerosis increases the risk of neoplasia in HPV-infected men.
  • the provided assays detect the presence or absence of virus such as HPV and/or particular subtype(s) thereof, the presence or absence of particular forms or states of the virus, such as whether it is present in episomal and/or integrated forms, the presence or absence of host genetic alterations, e.g. in housekeeping genes or tumor suppressor genes, and/or the presence or absence of an additional pathogen, such as a non-HPV pathogen.
  • virus such as HPV and/or particular subtype(s) thereof
  • particular forms or states of the virus such as whether it is present in episomal and/or integrated forms
  • host genetic alterations e.g. in housekeeping genes or tumor suppressor genes
  • an additional pathogen such as a non-HPV pathogen.
  • one or more or all of these detection features is carried out
  • the provided methods offer various advantages compared with available methods for detection, prediction, and prognosis of viruses and virus-associated cancer progression.
  • the provided assays offer advantages compared to other cervical cancer and HPV screening assays.
  • Available testing protocols for prediction and detection of cervical cancer include the Papanicolau- stained (Pap) smear or Pap test, based on cytological grading of a biological sample obtained from a subject. The detection is based on the identification of transformed cells using the nucleus-to-cell ratio. The effectiveness of Pap smear screening can vary, depending on the quality of the sample being used and/or subjective parameters in the analysis.
  • Such assays in some aspects are unable to reliably predict the behavior of some pre-invasive lesions.
  • the Pap test is limited in that it results in 20-40% atypical squamous cells of undetermined significance (ASCUS).
  • ASCUS atypical squamous cells of undetermined significance
  • the provided methods and systems are useful for further resolving such samples.
  • pre-malignant stages include cervical intraepithelial neoplasia (CIN)-l, CIN2, and CIN3, representing increasing degrees of dysplasia; or low grade cervical squamous intraepithelial lesion (LSIL), and high grade cervical squamous intraepithelial lesion (HSIL).
  • CIN cervical intraepithelial neoplasia
  • LSIL low grade cervical squamous intraepithelial lesion
  • HSIL high grade cervical squamous intraepithelial lesion
  • ASC atypical squamous cells
  • ASCUS atypical squamous cells of undetermined significance
  • Certain available pathological methods may not be predictive of risk for developing cervical cancer. For example, it may not be determined whether a CINl score or LSIL score will progress into a CIN2+/CIN3 or HSIL score and/or to an invasive or malignant lesion. In some aspects, the provided assays resolve the status of CIN1+ or LSIL score.
  • Certain methods detect viral nucleic acids in biological samples.
  • the most commonly used assays of this type for HPV are based on direct hybridization or DNA-based amplification techniques.
  • hybridization microplate-based assay provides a semiquantitative measure of viral load. Such tests can have relatively low analytical sensitivity, lack appropriate internal controls, e.g., for the amount of input DNA, and/or be associated with cross-reactivity with HPV types not included in the probe mix, which can result in false-negative and/or false-positive results.
  • In situ hybridization (ISH) by chromogenic or fluorescence techniques based on the pairing of a labeled probe to HPV nucleic acids can be used to demonstrate the localization of viral genomes in individual cells, where punctate signals indicate integrated virus and diffuse signals indicate episomal virus.
  • ISH Diffuse staining arising from the episomal genome can mask the punctate staining when both episomal and integrated forms are present, as is commonly the case in cervical cancers.
  • ISH also generally is labor-intensive and can lack sufficient clinical sensitivity to be used in screening.
  • Such assays generally do not identify specific HPV subtypes; additional genotyping techniques, such as polymerase chain reaction (PCR), often are used.
  • PCR polymerase chain reaction
  • Some methods detect the presence of virus by measuring an immune response to the virus or component thereof.
  • a serological survey is performed to detect the presence of antibodies against the virus.
  • antibody responses to the virus e.g. HPV
  • ELISA enzyme-linked immunosorbent assay
  • PCR tests are used for genotyping HPV subtype, generally based on consensus primers and/or type specific assays, the most common of which targets the highly conserved LI region of HPV. Such assays can in some cases give false-negative results, for example, where HPV has integrated into the human genome, which frequently results in the loss of the LI region. Thus, certain available methods may result in false-negative misdiagnosis, in particular, of women with more advanced lesions and/or greater and more immediate need for treatment. [0056] Certain available assays assess HPV integration status indirectly, e.g., by measuring the ratio of E2 to E6 (given that E2 is frequently lost upon integration).
  • Detection using primers to E6, which generally varies in sequence among different HPV subtypes can involve a separate PCR and separate reaction vessel for assessing each HPV subtype.
  • the provided assays offer advantages as compared to such available assays, for example, by offering more definitive prediction and prognosis, reducing the likelihood of false positive/false negative results, providing for direct detection of viral nucleic acid integration and location within the host genome, and/or by allowing multiple detections simultaneously, in the same reaction vessel, and/or based on a single sample obtained from a subject.
  • the provided methods generally are carried out on or assess samples, such as biological samples, and/or subjects from which the samples are derived.
  • samples such as biological samples, and/or subjects from which the samples are derived.
  • the sample is obtained from a subject suspected of having a particular disease or condition and/or having or suspected of having been infected with a virus associated with such disease or condition.
  • the subject has received a histological or pathology- based evaluation indicative of a lesion, such as a pre-cancerous and/or low-grade lesion, which is associated with a risk of progression to cancer.
  • the sample may be any sample or material obtained from a living or viral source or other source of macromolecules and biomolecules, and can include any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • isolated nucleic acids that are amplified constitute a biological sample.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.
  • Exemplary tissues are cervical and other epithelial-cell containing tissues.
  • the sample is obtained from a subject suspected of having cervical cancer and/or suspected of being infected with HPV.
  • the subject has been screened, e.g., by another method or test, for example, with a Pap test and/or the assignment of a pre-cancerous (e.g., CIN, ASCUS, or LSIL) score as determined by cytological grading.
  • a pre-cancerous e.g., CIN, ASCUS, or LSIL
  • the methods resolve samples where a Pap test resulted in a score of ASC or ASCUS, or where a CINl score has been assigned based on pathology.
  • the provided assays allow for the prediction, diagnosis, and/or prognosis of cancer, such as cervical cancer, at an early stage of disease progression. For example, in some cases, the provided assays predict progression to invasive or malignant stage to a greater degree than an available method, such as a Pap or other cytology-based screening test. In some embodiments, the methods do not require one or more waiting periods that is or are necessary with other methods. In some aspects, the methods detect viral nucleic acids before any change in morphology is observable by pathology or by a particular pathology- based test, such as by a Pap test. In some aspects, the sample is from a subject who does not display an abnormal morphology based on cytology or pathology.
  • the sample is derived from a subject who has tested positive for HPV.
  • the provided assays are predictive of the risk for disease
  • the subject has been diagnosed with a low grade cervical squamous intraepithelial lesion (LSIL).
  • LSIL low grade cervical squamous intraepithelial lesion
  • the methods detect or predict cancer for patients having received a score of LSIL for HPV based on pathology-based assessment.
  • the methods are carried out within at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks of the assessment resulting in the pre-cancerous lesion score, e.g., the LSIL score.
  • the subject has been classified as having a high grade cervical squamous intraepithelial lesion (HSIL) and has been referred for a tissue biopsy.
  • HSIL cervical squamous intraepithelial lesion
  • the sample is from a subject who has already been diagnosed with cervical cancer.
  • the provided methods monitor disease states in early and/or late stages.
  • the sample is or is obtained from a tissue biopsy, such as an epithelial cell sample, e.g., a sample obtained from a cervical biopsy.
  • the sample is an extracellular sample, for example, a sample that comprises or is derived from an extracellular fluid, such as plasma.
  • the biological sample may comprise, in some aspects, a mixture of cellular and acellular materials. Samples for use with the provided methods include those that are derived from such biological samples, such as those prepared as a result of nucleic acid purification from such a biological sample.
  • the sample is a body fluid, such as blood, plasma, serum, tissue, saliva, urine, or semen.
  • the cells may be found in a cervical smear collected, for example, by a cervical brush.
  • the biological sample is obtained from a Pap smear or tissue biopsy.
  • the sample may include, in some embodiments, epithelial cells of the cervix.
  • the patient sample is a cervical-associated body fluid.
  • fluids include, for example, blood fluids, lymph, ascitic fluids, gynecological fluids, urine, and fluids collected by peritoneal rinsing.
  • the sample or composition derived therefrom is an acellular composition, which contains no or substantially no live cells or intact cells or cells or no or substantially no live cells or intact cells or cells derived from the subject.
  • the sample or composition derived therefrom is a cellular composition, which contains cells, e.g., derived from the subject.
  • the cellular sample contains cells and does not contain or contains substantially no extracellular material from the subject, such as serum or other bodily fluid and/or extracellular matrix components.
  • the methods include steps for isolating or separating one or more fractions from the sample.
  • the steps include separating acellular and cellular fractions of the sample.
  • nuclear and non-nuclear fractions are separated.
  • acellular and cellular fractions are separated by gradient centrifugation or membrane filtration.
  • affinity-based methods are used, such as immunoaffinity-based methods for separation of material containing host proteins and material containing viral proteins, such as using antibodies specific for host, e.g., human proteins, and antibodies specific for viral proteins generally. Separation may be carried out, for example, as described in Fleissner, or J. Virol, Nov 1971; 8(5): 778-785; Ikeda et al., J Virol Jul 1975; 16(1): 53-61.
  • the same assay specific for closed circular viral nucleic assay may be used to identify free virus and episomal viral genome.
  • the methods include analyzing the acellular and cellular fractions separately.
  • the methods include performing steps on a plurality of compositions derived from a sample, which plurality include an acellular fraction and a cellular fraction. Such analysis can be useful, for example, to identify the presence of viral particles or free virus or viral nucleic acid, on the one hand, and separately to identify viral nucleic acids within a host cell, such as integrated or episomal nucleic acids.
  • the methods include carrying out amplification and/or detection steps separately on the acellular and cellular fractions or compositions derived therefrom.
  • separation can be useful to distinguish, in the assays, free virus from virus within a host cell that is in the episomal state.
  • the acellular fraction comprises virus present as a free particle and the cellular fraction comprises virus in an episomal and/or integrated form.
  • the methods include steps for isolating one or more nucleic acids from a sample.
  • the sample may include both viral and host nucleic acids.
  • the nucleic acids can include DNA, RNA, for example, mRNA, and/or derivatives of DNA such as amplicons, cDNA, and DNA analogs.
  • the provided methods include isolating DNA, RNA, or other nucleic acids from the sample, for example, using any of a number of known methods.
  • Methods for isolation of nucleic acids include, for example, separation using affinity and/or physical properties, such as those employing particles, such as magnetic particles, e.g., beads, membrane filtration, solid-phase separation, e.g., using sephadex columns and/or beads.
  • particles such as magnetic particles, e.g., beads, membrane filtration, solid-phase separation, e.g., using sephadex columns and/or beads.
  • Total nucleic acids for example DNA
  • DNA can be extracted from the sample using known methods and kits, e.g. a QIAmp DNA Minikit (Qiagen).
  • mRNA is isolated from the sample or composition and is converted to cDNA by reverse transcriptase (RT)-PCR.
  • RT reverse transcriptase
  • the purity and yield of the nucleic acid can be determined, for example, with the use of a standard spectrophotometer.
  • the methods detect the presence or absence of a virus.
  • the presence of the virus or subtype is carried out by detecting the presence or absence of a viral nucleic acid, such as one that is specific for the virus or the subtype.
  • a viral nucleic acid such as one that is specific for the virus or the subtype.
  • the sample, or composition derived therefrom (such as an isolated fraction such as the acellular or cellular fraction), is incubated under conditions designed to produce an amplicon only in the presence of a viral nucleic acid, and/or in the presence of a nucleic acid from a particular virus or subtype thereof.
  • the sample is incubated with primer pairs for amplification that anneal to a portion of the viral nucleic acid.
  • primers are used that anneal to a consensus sequence in a conserved, e.g., highly conserved, region of the virus.
  • detection of an amplicon indicates that the sample contains viral nucleic acid.
  • the detection of viral nucleic acids indicates that the virus is present as a free particle.
  • the sample is incubated under conditions designed to produce an amplicon only where HPV DNA is present.
  • the sample is incubated with primer pairs that anneal to a portion of the HPV DNA.
  • the primers are consensus primers and/or are capable of annealing to a consensus sequence in the LI region of HPV subtypes.
  • exemplary consensus primers target the HPV LI ORF.
  • primers include GP5+ (TTTGTTACTGTGGTAGATACTAC) and GP6+ (GAAAAATAAACTGTAAATCATATTC) primers that target a fragment within the region targeted by MY09 and MY11.
  • primer sequences include 5'- TTTGTTACTGTGGTAGATACTAC- 3 ' and/or 5'-GAAAAATAAACTGTAAATCATATTC- 3'. See Siddiqa et al., Viruses 2014 July 17, 6(7):2762-2777.
  • the primers include a primer set that amplifies a segment of the LI region of the HPV genome, such as a 65-base pair segment, e.g., SPF 10 . See also Kleter et al., Am. J. Pathol. Dec 1998; 153(6): 1731-39.
  • the amplicons are detected by sequencing.
  • an amplicon containing all or part of the viral nucleic acid sequence e.g. the region of interest, for example the LI region of HPV, indicates that the viral nucleic acid is present.
  • one or more particular types, subtypes, or groups of subtypes of viruses are detected, such as the presence of a high-risk or low-risk subtype and/or a particular high or low-risk or other subtype.
  • primer pairs are used that are capable of annealing specifically to particular sites within the viral genome that are particular to the subtype or category of interest, e.g. sites that allow differentiation of the different viral subtypes.
  • the sample is incubated with primers specific for the various high- risk and low-risk subtypes.
  • the detection of a particular subtype is carried out by determining the sequence of the amplicon.
  • the amplification primers are designed to produce an amplicon in the presence of any virus, in the presence of any type of virus, such as any HPV, and sequencing is used to detect the presence or absence of a particular subtype or subtypes or to determine the identity of the subtype.
  • the amplification primers are designed such that they anneal to regions within the viral genome that are common among all or most subtypes, but to produce an amplicon covering a region that is variable among the different subtypes of interest, such that production and/or detection of the amplicon indicates the presence of the virus generally and sequencing the amplicon reveals or can reveal information about the presence or absence of particular subtypes.
  • the primers are capable of annealing to a region of HPV within the E6 gene, such as a region that is highly variable across the various HPV subtypes.
  • the primers are specific for one or more high-risk and/or low-risk subtypes such as HPV-16 and HPV-18, and/or HPV-6 and HPV-11, respectively.
  • the primers are designed to anneal to portions of the genome that are less variable, but that produce an amplicon covering a region within the highly variable region, e.g. within E6 or the variable region of E6.
  • primers include 5'-CGG TCG GGA CCG AAA ACG G- 3'and 5'-AGC ATG CGG TAT ACT GTC TC-3'.
  • primers include WD72, WD76, WD66, WD67, and/or WD154. See Resnick RM et al., Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J. Natl. Cancer Inst. 821477-1484 (1990).
  • primer sequences include 5'-GGG GAA TTC GCA TGG AGA TAC ACC TAC ATT C-3', 5'-GGG CTC GAG TGG TTT CTG AGA ACA GAT GG -3', 5'-GGA TCC GCA TGG ACC TAA GGC AAC ATT-3', and/or 5'- GAA TTC GCT GCT GGG ATG CAC ACC A-3'.
  • primers include C16E7F (GGGGAATTCGCATGGAGATACACCTACATTC), C16E7R
  • the provided methods are advantageous in that they allow detection of specific viral subtype(s) by analysis of a single sample or compositions derived therefrom, and/or test for the presence of more than one subtype simultaneously.
  • the detection of a high-risk viral subtype may indicate that the patient from whom the biological sample was derived is at high-risk for developing the disease or condition associated with the viral infection.
  • the patient may be considered to have an increased risk for developing cervical cancer.
  • the methods detect the presence or absence of an episomal form of the virus.
  • the biological sample comprises both episomal and integrated forms of virus.
  • the methods are capable of distinguishing between episomal and integrated forms.
  • the provided methods detect whether the virus is present in any episomal stages.
  • episomal forms are detected separately in a cellular fraction derived from the sample.
  • the cellular fraction is incubated under conditions designed to produce an amplicon only in the presence of closed circular viral DNA, for example, where the virus is a DNA virus.
  • separation of the cellular fraction permits the distinction, using this method, between episomal viral nucleic acid and viral nucleic acid contained within free virus, as may be present in the acellular fraction.
  • the methods conclusively determine the presence of episomal DNA, as opposed to free virus, by using closed circular- specific primers on compositions which contain only cellular fractions as opposed to acellular fractions.
  • the same assay e.g., production of an amplicon only in the presence of closed circular viral DNA, can be conducted separately on the acellular fraction to identify the presence of free virus.
  • the primers in some embodiments are head-to-tail primers, which produce an amplicon, e.g., a particular amplicon, e.g., across the junction of the head and tail regions of the viral particle, when the virus is present in a closed circular form, which junction is generally disrupted, for example, upon integration into the host genome.
  • the cellular fraction is incubated with forward primers capable of annealing within the viral nucleic acid, e.g., within the LI region, beginning a certain number of nucleotides, e.g., within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or within 20, 50, or 100 nucleotides from the head or from the end of an LI HPV exon or from the end of an L2 HPV exon, and reverse primers capable of annealing viral nucleic acid, e.g., within the E2 or E5 region beginning a certain number of nucleotides, e.g., within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or within 10, 20, 50, or 100 nucleotides from the tail or from the end of an E2 or an E5 exon.
  • forward primers capable of annealing within the viral nucleic acid, e.g
  • the generated amplicon is sequenced and where the sequence spans the junction between the head and tail regions of the virus, HPV is determined to be present in an episomal form.
  • no amplicon, or an amplicon that does not comprise the sequence of the junction of the head and tail regions of the virus, or does not comprise the sequence of the natural junction formed in the circular form of the virus when intact (as opposed to junctions that may be generated upon head-to-tail tandem integration) will be produced when incubated with these primers where the viral particle is not present or is only present in an integrated form, thus distinguishing episomal from integrated states.
  • the biological sample, composition derived therefrom and/or cellular fraction is incubated under conditions that produce an amplicon, e.g., a particular amplicon, only when a portion of the viral genome that is typically lost upon integration is present.
  • amplicon e.g., a particular amplicon
  • nucleic acids e.g. DNA and/or RNA isolated from the sample, and/or cDNA produced by RT-PCR
  • primers are capable of annealing to a region of the viral nucleic acid that is frequently not present in integrated forms of virus, e.g.
  • the amplicon is sequenced, and the detection of the viral nucleic acid sequence from a region typically absent from integrated forms of virus indicates that the virus is present in episomal forms.
  • the sample is incubated with primers designed to produce an amplicon when the E2 region of HPV is present. Since E2 is frequently lost upon viral integration, in some aspects, the detection of an amplicon that is produced only when E2 is present, and/or that comprises all or a fraction of the E2 gene, indicates that HPV in present in an episomal form.
  • the methods directly detect the presence or absence of viral nucleic acid, e.g., viral DNA, which has been integrated into the host genome.
  • the provided assays are advantageous over other methods that indirectly assess viral integration, for example, in the case of HPV, by measuring the ratio of E2 to E6 gene product expression.
  • the sample is incubated under conditions designed to produce an amplicon only in the presence of viral nucleic acid that has been integrated into the host genome.
  • the amplicon is sequenced to definitively detect viral integration.
  • the sample is incubated under conditions designed to produce an amplicon, e.g., a particular amplicon, only when all or part of the viral genome has integrated into a host gene, or has integrated into a coding region of a host gene.
  • the conditions include incubation with primers that anneal to viral and/or host nucleic acids.
  • a first primer is capable of annealing to viral nucleic acid
  • a second primer is capable of annealing to host nucleic acid.
  • the second primer is a primer capable of annealing to a poly-A or poly-T region of a host mRNA or cDNA, such as a poly-A or poly-T primer.
  • the primer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more consecutive A or T residues.
  • the production or detection of an amplicon using such primers indicates or determines that the viral nucleic acid has integrated into the host genome, and that it has done so into a coding region of a gene, such that an mRNA including the integrated nucleic acid has been produced.
  • Such information can in some aspects, predict a greater risk of progression or development of the associated disease or condition, e.g., cancer, as compared with lack of integration and/or as compared with integration into a non-gene or non-coding region portion of the host genome.
  • the first primer is designed such that it is capable of annealing to a portion of the viral nucleic acid, such as one that is typically maintained or maintained in tact upon integration into the host genome.
  • the amplicon that is produced following amplification includes a junction between viral and host nucleic acids.
  • sequencing of the amplicon reveals the presence of both the viral and host nucleic acid, for example, adjacent to one another, directly confirming that the virus has integrated into the host genome.
  • the first primer is designed such that it is capable of annealing to a specific region of the HPV genome, such as the El, E2, E4 or E6 region, and a second primer designed such that it is capable of annealing to a poly-A region of the host nucleic acid.
  • the methods determine where, i.e., into which gene in particular, in the host genome a virus has integrated.
  • such information is determined by sequencing of the resulting amplicon.
  • sequencing of the amplicon containing both viral and host nucleic acid reveals the location within the host genome in which the virus has integrated.
  • the detection of the location of viral integration provides for the prediction of the level of risk for disease progression, for example, by determining whether the virus has integrated into the coding region of a functional cellular gene, e.g. a tumor suppressor gene.
  • the detection of integrated viral nucleic acid and the detection of the location of viral integration are carried out simultaneously, from the same starting material, and/or in the same reaction vessel.
  • the methods include detecting whether a genetic alteration, such as a mutation, is present in the host cells, genome, or gene, within the subject or sample, for example, in cervical cells included in a sample obtained from a patient, e.g. from a Pap smear or tissue biopsy.
  • the provided methods detect whether the host cells carry a mutation in one or more genes, for example, tumor suppressor genes or oncogenes.
  • mutations in tumor suppressor genes and/or oncogenes such as p53, Rb, PTEN, and/or liver kinase Bl (LKB1), and/or pi 6INK4a, cyclins, e.g., Cyclin Dl, MYC, RAS genes, EGFR, HST-1, and/or HST-1, are detected by the present methods.
  • the presence of a mutation in a host gene in the biological sample indicates that the patient is at high risk for developing a condition or disease, e.g. cervical cancer.
  • such detection is carried out by incubating the sample with primers and/or probes, e.g., amplification primers that are capable of specifically annealing to the host nucleic acid, e.g., the host gene or portion thereof of interest.
  • primers and/or probes e.g., amplification primers that are capable of specifically annealing to the host nucleic acid, e.g., the host gene or portion thereof of interest.
  • the primers anneal to specific regions of host genes such that an amplicon produced following amplification will contain a region of the nucleic acid sequence in which mutations are known to occur.
  • the provided methods detect whether a mutation is present in the nucleic acid sequence of the host gene.
  • ThT fluorescence was determined (emission -450 nM and excitation - 485 nM). Standard curves were generated by measuring ThT fluorescence with different concentrations of amyloid beta fibrils.
  • the primers are capable of annealing only if a particular genetic alteration, such as a single nucleotide polymorphism (SNP) is present.
  • SNP single nucleotide polymorphism
  • an amplicon is only produced when the particular alteration of interest is present in the host gene.
  • the amplicon when generated, is detected by sequencing to confirm that the nucleic acid sequence is that of the host gene in question.
  • multiple host mutations are detected simultaneously, using the same starting material, sample, or composition derived therefrom, and/or in the same reaction vessel.
  • the step of detecting host mutations is performed simultaneously, on the same starting material, and/or in the same reaction vessel as the detecting of the viral nucleic acid.
  • the methods are advantageous because they reduce the amount of time, steps, cost, complexity, and/or number of required resources compared to other methods.
  • the methods include detecting the presence or absence of and/or identifying one or more additional pathogens in the sample or subject.
  • the additional pathogen may be viral or bacterial in nature.
  • the additional pathogen is associated with an STD and/or vaginitis and/or other disease or condition, risk of progression to cancer and/or inflammation.
  • the additional pathogen is, for example, herpes simplex virus (HSV), Chlamydia trachomatis, and/or Neisseria gonorrhoeae.
  • the pathogen is or includes HHV-6, HHV-7, and/or cytomegalovirus (CMV).
  • the methods further detect whether inflammatory mediators, or high levels of inflammatory mediators, are present, e.g. resulting from co-infection with the additional pathogen.
  • the inflammatory mediators are cyclooxygenase (COX)- 2 and/or NF-KB.
  • COX cyclooxygenase
  • the presence of an additional pathogen and/or inflammatory mediators indicates that the patient is at high risk for developing a disease or condition.
  • the methods further include detection of the expression or amount or level thereof of one or more viral genes, such as oncogenic viral genes, e.g., E6 and/or E7.
  • viral genes such as oncogenic viral genes, e.g., E6 and/or E7.
  • the presence of such genes and/or elevated or high levels thereof indicates a risk of developing a disease or condition and/or that the subject has or is likely to have such disease or condition.
  • Exemplary assays include PCR, RT-PCR, QPCR, northern or western blotting, and other methods for determining gene expression.
  • the detection, identification, and/or determination by the methods provides diagnostic, prognostic, and/or predictive information.
  • the presence or absence of the nucleic acid(s), forms of virus, subtype of virus, and/or presence of virus provides diagnostic and/or prognostic information.
  • such information in some embodiments provides such information about a disease, disorder, condition, or stage or state thereof that is associated with or caused by the virus, for example, in a subject from which the sample, e.g., biological sample is taken.
  • the detection, determination, or identification of the nucleic acid in the sample determines the presence, absence, severity, prognosis, stage, or other information about a disease, condition, or other event, such as cancer, tumor, metastasis, malignancy, proliferative disease or disorder, genetic predisposition, or likelihood of success or treatment outcome using a particular therapy or treatment, such as drug, biologic, surgery, intervention, or other treatment, in or by the subject from which the biological sample is taken.
  • the condition is a healthy state.
  • the prognostic, diagnostic, and/or predictive information informs treatment and/or further monitoring decisions.
  • the result indicates that a subject should be subjected to a further test and/or should be monitored by administering the same or different test at a later date and/or regularly.
  • the results indicate that a patient should be treated, e.g., with a particular therapeutic regimen.
  • the methods resolve a previous diagnosis, e.g., based on pathology, which was inconclusive, such as one reporting ASCUS.
  • the methods reveal that such a subject has or is likely to have a high grade cervical squamous epithelial lesion (HSIL), or has or is likely to have a low grade cervical squamous epithelial lesion (LSIL), or has an invasive or metastatic cancer, such as an invasive carcinoma.
  • HSIL high grade cervical squamous epithelial lesion
  • LSIL low grade cervical squamous epithelial lesion
  • an invasive or metastatic cancer such as an invasive carcinoma.
  • the detection of the presence of the virus indicates that the subject has or is likely to have a LSIL.
  • the subject in some cases is further assessed, such as by colposcopy or further monitoring, and/or such further assessment is recommended.
  • the detection of the presence of the virus indicates that the subject has or is likely to have a HSIL.
  • the subject in some cases is further assessed, such as by colposcopy or further monitoring, and/or such further assessment is recommended.
  • the detection of the presence of the virus, even in the absence of integration, coupled with elevated oncogenic viral genes and/or host mutations, such as p53 or Rb mutations, indicates that the subject has or is likely to have an invasive carcinoma.
  • the subject in some cases is further assessed, such as by colposcopy, and/or such further assessment is recommended.
  • the presence of an additional risk factor increases the likelihood of HSIL or invasive cancer, e.g., carcinoma.
  • the present methods are able to produce the diagnosis with a greater degree of certainty than available methods.
  • the methods determine the presence of HSIL, ISIL, and/or invasive cancer, in a subject that has been or would be determined to be negative for such a diagnosis, and/or for which the diagnosis was not conclusive, based on another method, such as a pathology or cytological method.
  • the methods determine that the patient is not at high risk for or does not have HSIL, ISIL, and/or cancer or invasive cancer, where the patient would have been determined to have such a diagnosis or risk thereof by another method.
  • the provided molecular methods provide improved specificity and/or sensitivity as compared with available methods.
  • the methods provide earlier detection of cancer or risk thereof or progression thereto, such as providing a diagnosis for a patient that has not progressed beyond and early stage in cancer progression.
  • the detection and assaying of nucleic acids by the provided methods are used in some embodiments to extract clinical information for use in early diagnosis, prevention, and management of diseases and other conditions. Identification and testing of nucleic acids are particularly useful in some embodiments in non-invasive diagnosis and prognosis, such as noninvasive oncology testing, screening for genetic alterations, and personalized medicine.
  • the detected presence of viral nucleic acid(s), e.g., a particular form or forms or subtypes, and/or the presence of a genetic alteration indicates the presence of a disease or condition or predicts risk for developing a disease or condition, such as a disease or condition associated with or caused by the virus, such as a sexually transmitted disease or cancer.
  • the cancer associated with viral infection is cervical cancer, ovarian cancer, endometrial cancer, vulvar squamous cell carcinoma, vaginal squamous cell carcinoma, penile cancer, anal cancer, head and neck squamous cell carcinoma, or
  • the cancer is cervical cancer.
  • the methods provide diagnostic or prognostic information for a subject who does not yet have the disease, or who has not progressed, to assess risk of developing the disease or progressing. For example, compared to certain available methods, in some embodiments the provided methods are carried out on a sample that is not at a post- transformed state. Thus, in some aspects, the provided embodiments allow for early disease detection, prediction, and/or prognosis, and permit the continued monitoring and/or prevention of diseases and associated symptoms.
  • the methods are useful in prognosis, diagnosis, staging, prediction, risk assessment, and monitoring, of diseases and conditions, such as cancer, and/or in the context of personalized medicine, for example, by identifying the presence of a virus or particular form(s) thereof that is known to be associated with the disease or condition and/or genetic alterations in the host known to be associated with the disease or condition.
  • the methods include determining whether the subject has or is at risk for developing the disease or condition or stage or prognosis thereof.
  • the detected presence of a high-risk virus, episomal stages, virus integrated into a host gene, one or more host genetic alterations, and/or co-infection with an additional pathogen indicates that the subject is at high risk for developing the disease or condition, e.g., the cancer.
  • the methods include monitoring and/or treating the subject determined to be at or have a particular risk, stage, prognosis for or of the disease or condition or having the disease or condition or symptom thereof. Monitoring can include periodic testing by the provided or other diagnostic or prognostic methods. Treatment can include administering a particular therapy or altering the therapy with which a patient currently is being treated.
  • the methods provide information regarding whether treatment is or has been effective in the subject, such as for monitoring treatment.
  • a patient is determined to be at high risk for developing cervical cancer if one or more of the following are detected in the biological sample: episomal HPV, integrated HPV, HPV integrated into a coding region of a host gene, a mutation in a host gene, co-infection with an additional pathogen, for example, one associated with a sexually transmitted disease or infection, and/or elevated levels or E6 and/or E7 expression.
  • a patient from whom a biological sample is derived that tests positive for a combination of any two or more of the presence of episomal HPV, integrated HPV, HPV integrated into a host gene coding region, a mutation in a host gene, such as a tumor suppressor gene, and/or co-infection with an additional pathogen, is determined to be at high risk for developing cervical cancer.
  • the methods are useful as part of a treatment or therapeutic method for the disease, disorder, or condition.
  • detection of the presence or absence of the nucleic acid in the sample or subject indicates the success or likelihood of success of a particular treatment, e.g., drug or surgical intervention.
  • a particular treatment e.g., drug or surgical intervention.
  • the method may indicate that a treatment specifically targeting that gene is likely to treat the subject in which the viral nucleic acid has been identified.
  • the methods are carried out by detecting the presence or absence one or more nucleic acids, viruses, and/or forms thereof, and/or information regarding host genes or other infectious agents or risk factors, in a sample derived from a subject as described herein.
  • the information determined indicates that treatment is likely to be successful or is not likely to be successful in treating the subject.
  • the methods further include administering, discontinuing, and/or altering or adjusting a treatment to the subject, such as the treatment which it is determined is likely to be successful in treating the subject.
  • the methods further include discontinuing or altering the treatment of the subject, for example, discontinuing treatment that it is deemed is unlikely to treat the subject, for example, in favor of a more appropriate treatment.
  • One or more steps of the methods generally involve amplification of one or more nucleic acid of interest, such as viral nucleic acids, host nucleic acids, and combinations thereof.
  • amplification is followed by detection of the resulting amplicon, e.g., by detecting its presence and/or by determining its sequence, such as by sequencing methods.
  • Amplification may be carried out by polymerase chain reaction (PCR).
  • the agent for amplification of the nucleic acid may be any compound or system that will function to accomplish the synthesis of amplicons, including enzymes.
  • Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat- stable enzymes ⁇ i.e., those enzymes which produce amplicons after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase.
  • the suitable enzyme will facilitate combination of the nucleotides in the proper manner to form an amplicon that contains the nucleic acid sequence of the region between the forward and reverse amplification primers.
  • the amplification will be initiated at the 3' end of each nucleic acid and proceed in the 5' direction, until amplification terminates, producing amplicons.
  • a sample following amplification of the nucleic acid and/or amplicon is cleaned, or enriched for the amplicon, to facilitate identification of the nucleic acid sequence.
  • Methods for cleaning and/or enriching for the amplicon include column purification and membrane-based size exclusion, among others. Exemplary methods of nucleic acid purification are disclosed in U.S. Patent No. 4,923,978 (purification through a hydroxylated support), U.S. Patent No. 4,935,342 (anion exchange columns), U.S. Patent No. 4,946,952 (DNA isolation by precipitation with water-soluble ketones), and U.S. Patent No. 4,900,677 (DNA purification using chao tropes).
  • the methods generally include a detection or identification step, for example, whereby the presence or absence of the nucleic acid is detected in the sample or biological sample or subject from which the sample is derived.
  • the provided methods in some embodiments include the detection of nucleic acids, such as the amplicons produced in the amplification reaction.
  • sequencing methods which can be used in conjunction with others of the provided methods, e.g., the amplification methods.
  • the determining includes performing nucleotide sequencing on the amplicon.
  • the nucleotide sequence of all or a portion of the amplicon is determined, for example, by sequencing technologies known to one of skill in the art, thereby detecting the nucleic acid in the sample.
  • Suitable sequencing or sequence determination techniques include but are not limited to mass spectroscopy (e.g., matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF), LC-MS/MS, and TOF/TOFTM LC/MS/MS), nuclear magnetic resonance imaging, and nucleic acid sequencing. Exemplary sequencing techniques can be found in U.S. Patent Application Publication No.
  • a sequencing primer hybridizable to the 3' end of an amplicon can be used to sequence all or a portion of the amplicon, such that the nucleic acid sequence including any mutation therein can be determined.
  • the amplicon since the nucleic acid sequence is known, and the amplicon includes a sequence of a portion of the nucleic acid, sequencing primers hybridizable to various regions of the nucleic acid and/or amplicon can be designed and used.
  • the AMPure system is used to purify the amplicons and sequencing of the amplicons is performed.
  • the sequencing primers are designed such that each subsequent primer is larger than the previous primer plus the product allowing for the identification of each of the amplicons that are generated simultaneously.
  • the amplicon being sequenced comprises both viral and host nucleic acid sequences, such that viral and host sequences are determined or identified in a single sequencing reaction using a single primer.
  • sequencing of the amplicon provides for determination of the location of viral integration into the host genome.
  • the determining of sequence is carried out using a detectable probe, for example, a detectable probe containing a label or an antibody, a fluorescent probe, or a radiolabeled probe.
  • the determining of sequence includes contacting the sample with a restriction enzyme, carrying out fragment analysis, and/or conducting mass spectrometry or nuclear magnetic resonance imaging.
  • fragment analysis e.g., mass spectroscopy or nuclear magnetic resonance imaging, is used.
  • the determination of sequencing is carried out using a sequencing method described herein.
  • the methods are capable of sequencing multiple different nucleic acids, such as multiple products from the various amplifications, e.g., multiple amplicons, either based on different target genes or regions and/or based on different alleles or genetic variations at a single locus, simultaneously or in the same reaction vessel.
  • the provided methods include multiplexing (e.g., detecting or sequence multiple loci simultaneously), including multiplex or multi-loci sequencing methods.
  • this is carried out by employing multiple (first, second, etc.) respective sequencing primers, where a second sequencing primer is designed to be larger in size and/or molecular weight than any of the products generated by extension using the first sequencing primer.
  • the incubation is further in the presence of chain-terminating nucleotides, where, for example, Sanger sequencing is used to determine sequence.
  • products generated by the different sequencing primers thus are distinguished, e.g., separated, based on size or molecular weight.
  • the provided methods are capable of simultaneously sequencing multiple nucleic acids.
  • the sequences of a plurality of nucleic acids are determined.
  • the methods in some aspects include performing Sanger-based sequencing on each of those nucleic acids, using first and second sequencing primers, designed such that the second sequencing primer is larger than the largest of the products generated by extension of the first sequencing primer.
  • the products of such extension can be differentiated, e.g., separated, from each other, such that two different nucleic acids may be assessed simultaneously.
  • the provided methods include:
  • the determining is by multi-loci or multiplex sequencing as described herein.
  • the first nucleic acid is or corresponds to a mutant allele or portion of a gene or locus
  • the second nucleic acid corresponds to a wild-type or normal allele or portion.
  • the first and second nucleic acids each correspond to different mutations or genetic or epigenetic variations, e.g., in the same or different genes.
  • the multiplexing sequencing includes combining in a reaction mixture the first and second (and optionally, third, fourth, etc.) nucleic acids, first and second (and optionally third, fourth, etc.) labeled sequencing primers hybridizable respectively to the first and second (and optionally third, fourth, etc.) nucleic acids, a polymerase, nucleotides, and a chain-terminating nucleotide, under conditions that permit hybridization of the first and second (optionally more) sequencing primers to the first and second (optionally more) nucleic acids, respectively, and extension of the first and second (optionally more) sequencing primers, wherein the periodic incorporation of the chain-terminating nucleotide by the polymerase terminates polymerization, thereby producing a pool of first target products of a plurality of lengths having
  • the second sequencing primer has a molecular weight at least as large as or larger than the largest of the first target products, whereby each of the second target products has a molecular weight greater than each of the first target products, or wherein the second sequencing primer has a molecular weight at least as large as or larger than the first nucleic acid.
  • the products then generally are differentiated, e.g., separated, based on differences in size or molecular weight.
  • the first target products are separated from the second target products, and optionally, third, fourth, etc. target products.
  • this sequencing method can be used in connection with any of the embodiments disclosed herein.
  • the multiplexing sequencing methods are methods for
  • sequencing methods are methods for allele- specific sequencing.
  • the multiplexing sequencing methods are allele-specific multiplex sequencing (ASMS) methods.
  • ASMS allele-specific multiplex sequencing
  • the ASMS methods are modifications of the Sanger sequencing technology, in which one or more nucleic acids are sequenced and detected simultaneously.
  • the methods are useful for detection of a nucleic acid present at low copy numbers among a corresponding but different nucleic acid, such as a mutant allele present at low copy number among wild-type alleles.
  • the ASMS methods achieve this by designing extension primers, e.g., sequencing primers, which prime extension only from a particular allele or variant, such as a mutant variant, and not from a corresponding other variant, such as a wild-type variant, and using multiple such primers in the same reaction.
  • the primers are also typically designed such that products of their extension may be separated, generally by size or molecular weight, such that the two (or more) sequencing reactions may be carried out in the same reaction volume simultaneously and the products distinguished and identified.
  • the methods use a plurality of different sequencing primers (e.g., first, second, etc. sequencing primers), each containing at its 3' terminus a nucleotide that pairs with, i.e., is complementary to, the nucleotide present at the corresponding position in either the variant sequence or the normal sequence when hybridized to a nucleic acid containing the variant or wild-type sequence.
  • different sequencing primers e.g., first, second, etc. sequencing primers
  • primers are otherwise 100 % identical, or at least 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 % identical, except for this 3' terminal nucleotide.
  • the first primer primes extension only when paired to a nucleic acid containing the mutant or varied sequence
  • the second primer primes extension only when paired to a nucleic acid containing the wild-type or normal position.
  • the primers are designed such that one primer is larger in size or molecular weight than all products generated by extension with the other primer, such that products of extension using the two primers can be separated from one another based on size.
  • the methods include: (a) combining a first extension primer (e.g., first sequencing primer) a second extension primer (e.g., sequencing primer), a polymerase, and a sample containing a first nucleic acid, a second nucleic acid, or both the first and second nucleic acids, under conditions whereby the first primer is extended by the polymerase when hybridized to the first nucleic acid, thereby producing a pool of first extension products and the second primer is extended by the polymerase when hybridized to the second nucleic acid, thereby producing a pool of second extension products; and (b) differentiating the extension products based on differences in molecular weight.
  • the extension primers typically are sequencing primers.
  • the first extension primer contains sequence identity to the second extension primer.
  • each of the first and second extension primers is hybridizable to each of the first and second nucleic acids.
  • the 3'-terminal residue of the first extension primer is a first nucleotide, which is complementary to a paired nucleotide in the first nucleic acid when the first extension primer is hybridized to the first nucleic acid.
  • the 3 '-terminal residue of the second extension primer is a second nucleotide, which typically is different from the first nucleotide and generally complementary to a paired nucleotide in the second nucleic acid when the second extension primer is hybridized to the second nucleic acid.
  • the second extension primer has a molecular weight that is greater than that of each of the first extension products. In some aspects, the second extension primer has a molecular weight that is greater than that of the first nucleic acid. In some aspects, the second extension primer has a molecular weight that is at least 1.5, 2, 3, 4, 5, or more times the molecular weight of the first extension primer. In some aspects, the second extension primer is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides in length. In particular embodiments, the first extension products are separated from the second extension products in step (b) based on molecular weight.
  • a third, fourth, and so-forth, extension primer are used in the method in connection with a third, fourth, and so-forth nucleic acid, whereby third, fourth, etc., extension product pools are produced, with the first, second, third, fourth, etc., extension products separated by molecular weight.
  • the third extension primer has a molecular weight that is greater than that of each of the second extension products; and the fourth extension primer has a molecular weight that is greater than that of each of the third extension products.
  • the third extension primer has a molecular weight that is greater than that of the second nucleic acid; and the fourth extension primer has a molecular weight that is greater than that of the third nucleic acid. In some aspects, the third extension primer has a molecular weight that is at least 1.5, 2, 3, 4, 5, or more times the molecular weight of the second extension primer; and the fourth extension primer has a molecular weight that is at least 1.5, 2, 3, 4, 5, or more times the molecular weight of the third extension primer. In some aspects, the third extension primer is at least 40, 45, 50, 55, typically at least 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides in length.
  • the allele- specific sequencing method is used in connection with any of the embodiments herein.
  • the target nucleic acid can include an allelic difference or point mutation compared to a corresponding wild-type sequence
  • ASMS allele-specific multiplex sequencing
  • the method detects the target nucleic acid at a frequency as low as one copy of target nucleic acid per one million copies of a corresponding wild-type sequence.
  • the allele-specific multiplex sequencing (ASMS) method is used to detect a plurality of genetic or genomic alterations or variations
  • this process is based on use of a first sequencing primer with a nucleotide complementary to a nucleotide within a nucleic acid at its 3' terminus, and typically a second sequencing primer that contains at a corresponding position a nucleotide that does not pair with the nucleic acid, i.e., is not complementary to the nucleotide within the nucleic acid.
  • the second primer is complementary to a wild-type nucleotide.
  • the primers are designed such that the first or second primer has a molecular weight greater than all products generated by the second or first primer, respectively, such that the products can be separated by molecular weight.
  • ASMS generates a verifiable result format.
  • ASMS achieves a sensitivity of as low as about 1 in 1,000, as low as aboutl in 10,000, as low as about 1 in 100,000, or as low as about 1 in
  • identification includes or is carried out by labeling the target nucleic acid with a detectable probe, e.g., fluorescent probes, antibody-based probes, or radiolabeled probes.
  • a detectable probe e.g., fluorescent probes, antibody-based probes, or radiolabeled probes.
  • the target nucleic acid or nucleic acid can be identified based on digestion patterns, for example, by restriction enzymes.
  • fragment analysis e.g., mass spectroscopy or nuclear magnetic resonance imaging, is used.
  • compositions including reagents such as primers, primer pairs, buffers, polymerase, nucleotides, thermocycling conditions buffers, and solutions, and combinations thereof, and systems, such as kits, assays, and tests, for carrying out the methods.
  • primers and probes for use in connection with the provided methods, including amplification primers and sequencing primers, and compositions containing the same.
  • primers designed to produce the various amplicons discussed herein and/or sequencing primers for sequencing one or more of the amplicons.
  • primers produce an amplicon spanning a junction, e.g. a head-to-tail region of a virus.
  • primers are provided that are designed to produce an amplicon containing both viral and host nucleic acid.
  • the primers produce an amplicon containing a host mutation, e.g. a mutation in a tumor suppressor gene or oncogene.
  • the primers produce an amplicon in the presence of an additional pathogen, e.g. in the presence of co-infection by the virus and an additional pathogen.
  • kits are provided for carrying out the methods.
  • the kits include primers or probes designed to detect the presence or absence of an episomal or otherwise closed circular viral nucleic acid, e.g., genome.
  • the kit includes primers designed to produce an amplicon in the presence of viral nucleic acid, such as HPV.
  • the primers comprise a first pair of primers designed to produce an amplicon in the presence of episomal viral nucleic acid and/or closed circular viral nucleic acid.
  • the amplicon contains or spans a junction between head and tail portions of the closed circular or episomal nucleic acid, e.g., genome.
  • the kit further includes primers and/or probe(s) designed to detect the presence or absence of integrated viral nucleic acid, e.g., genome or partial genome.
  • the kit includes a pair of primers designed to produce a second amplicon in the presence of an integrated viral (e.g., HPV) nucleic acid, such as one that has integrated into a host gene, e.g., into a coding region of a host gene.
  • the amplicon comprises a junction between an integrated viral nucleic acid and a host gene into which the virus is integrated.
  • the kit further includes a probe and/or primers to detect a genetic alteration in the host, such as another pair of primers designed to reveal the presence or absence of a host genetic alteration, such as one that implicates cancer or risk of progression thereof.
  • the primers are designed to produce an amplicon comprising a locus of a genetic alteration in a host gene, such as p53, Rb, PTEN, pl6INK4a, cyclins, e.g., Cyclin Dl, MYC, RAS genes, EGFR, HST-1, HST-1, or LKB1.
  • the kit further includes a primer(s) or probe(s) designed to detect an additional risk factor, such as a pathogen or inflammatory indicator, such as one associated with an STD, cancer risk, inflammation, and/or vaginitis.
  • an additional risk factor such as a pathogen or inflammatory indicator, such as one associated with an STD, cancer risk, inflammation, and/or vaginitis.
  • a pair of primers designed to produce an amplicon in the presence of the additional pathogen or risk factor.
  • the kit further includes compounds for determining sequence, such as of the amplicons so generated.
  • it includes a sequencing primer complementary to a sequencing primer annealing site in one or more, e.g., all, amplicons designed to be generated by the various primer pairs included in the kit.
  • the primers are designed such that the multiplexing sequencing methods as described herein may be used to sequence the products in the same vessel or mixture, e.g., simultaneously.
  • kits comprising primers designed to produce an amplicon where episomal viral nucleic acids are present.
  • primers are provided that are designed to produce an amplicon comprising a sequence of the viral genome that is indicative of virus present in an episomal form, such as within the E2 gene of HPV.
  • the kit further comprises primers that bind to one or more of the amplicons described herein, e.g. sequencing primers that are capable of sequencing one or more of the amplicons produced.
  • primers are provided that are designed to produce an amplicon where a specific viral subtype is present.
  • primers are provided that are designed to produce an amplicon where HPV 16 and/or HPV 18 is present.
  • primers that are designed to detect viral subtype may be included in a kit in combination with primers designed to detect episomal forms of virus, integrated forms of virus, host mutations, e.g. in genes that are indicative of risk of developing associated diseases or conditions, primers that detect the presence of an additional pathogen, and/or primers for sequencing the amplicons produced by one or more of the primer pairs described herein.
  • nucleic acid can refer to one or more nucleic acids
  • method includes reference to equivalent steps and methods disclosed herein and/or known to those skilled in the art, and so forth.
  • an individual includes any living organism, such as humans and other mammals.
  • a subject as used herein refers to a mammal, such as a human or other mammal. In some aspects, the subject is a primate.
  • a composition refers to any mixture of two or more products, substances, or compounds. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • nucleic acid oligonucleotide
  • nucleic acid and “nucleic acid molecule” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and can include ribonucleotides (e.g., containing D-ribose), deoxyribonucleotides (e.g., containing 2-deoxy-D-ribose), analogs thereof, or mixtures thereof.
  • nucleic acid examples include any suitable length, such as at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides. Nucleic acids can also form stable complex with themselves or others, for example, DNA- protein complex, DNA-DNA complex, DNA-RNA complex.
  • nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.
  • a primer refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a nucleic acid, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed.
  • the sequence of nucleotides added during the extension process is determined by the sequence of the template nucleic acid.
  • Primers usually are extended by a polymerase, e.g., a DNA polymerase.
  • complementary and substantially complementary refer to the ability to hybridize or base pair or form of a duplex between nucleotides or nucleic acids, for instance, between the two strands of a double- stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single- stranded nucleic acid.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single-stranded nucleic acids, e.g., RNA or DNA, or regions thereof, are said to be complementary or substantially
  • nucleotides of one strand or region optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the other strand or region, usually at least about 90% to about 95%, and even about 98% to about 100%.
  • Sequence identity or complementarity can be measured along the full length of a nucleic acid or along a region of the molecule.
  • two complementary sequences of nucleotides are capable of hybridizing, for example, with less than 25%, for example, with less than 15%, for example, with less than 5%, for example, with no mismatches between opposed
  • the two molecules will hybridize under conditions of high stringency.
  • genetic or genomic variations include those in genomic DNA, mitochondrial DNA, episomal DNA, and/or derivatives of DNA such as amplicons, RNA transcripts, cDNA, DNA analogs, etc.
  • Genetic or genomic changes that can be detected by a method of the present disclosure can be any types of DNA alterations including base change, deletion, duplication, amplification, polymorphism, microsatellite instability, loss of heterozygosity (LOH), epigenetic modification, and any combination thereof.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés, des compositions et des systèmes de détection, de diagnostic, de prévision, de pronostic et de stadification de virus, tels que le papillomavirus humain (HPV) et des acides nucléiques viraux et des maladies et affections associées telles que le cancer chez des sujets et dans des échantillons biologiques dérivés de ceux-ci. Dans certains aspects, ces procédés permettent de détecter et/ou de différencier des formes de virus, comme des formes libre, épisomiques et intégrées et de fournir des informations pronostiques et prédictives sur les maladies ou les affections. Parmi les virus, il y a les papillomavirus humain (HPV), le virus d'Epstein Barr (EBV) et les poxvirus. Parmi les maladies et affections, il y a des maladies, des troubles et des affections néoplasiques, cancéreux, pré-malins et prolifératifs.
PCT/US2015/052414 2014-09-25 2015-09-25 Procédés de diagnostic et de pronostic pour des virus et des maladies et affections associées Ceased WO2016049559A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462055558P 2014-09-25 2014-09-25
US62/055,558 2014-09-25

Publications (1)

Publication Number Publication Date
WO2016049559A1 true WO2016049559A1 (fr) 2016-03-31

Family

ID=55582110

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/052414 Ceased WO2016049559A1 (fr) 2014-09-25 2015-09-25 Procédés de diagnostic et de pronostic pour des virus et des maladies et affections associées

Country Status (1)

Country Link
WO (1) WO2016049559A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018207022A1 (fr) * 2017-05-10 2018-11-15 Genomic Vision Association entre l'intégration de génomes de vph à haut risque détectée par peignage moléculaire et la gravité et/ou le résultat clinique de lésions du col de l'utérus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020132227A1 (en) * 2000-03-28 2002-09-19 Biosearch International Pty. Ltd. Approaches for HPV detection and staging by targeting the E6 gene region of the viral genome
US20090111090A1 (en) * 2007-10-31 2009-04-30 Sysmex Corporation Method for detecting integrated hpv dna
US20110275059A1 (en) * 2010-05-05 2011-11-10 Deutsches Krebsforschungszentrum Diagnostic Transcript and Splice Patterns of HR-HPV in Different Cervical Lesions
US20120322049A1 (en) * 2011-02-24 2012-12-20 Qiagen Gaithersburg Inc. Materials and methods for detection of hpv nucleic acids

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020132227A1 (en) * 2000-03-28 2002-09-19 Biosearch International Pty. Ltd. Approaches for HPV detection and staging by targeting the E6 gene region of the viral genome
US20090111090A1 (en) * 2007-10-31 2009-04-30 Sysmex Corporation Method for detecting integrated hpv dna
US20110275059A1 (en) * 2010-05-05 2011-11-10 Deutsches Krebsforschungszentrum Diagnostic Transcript and Splice Patterns of HR-HPV in Different Cervical Lesions
US20120322049A1 (en) * 2011-02-24 2012-12-20 Qiagen Gaithersburg Inc. Materials and methods for detection of hpv nucleic acids

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COLLINS ET AL.: "Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study", CANCER RESEARCH, vol. 69, 1 May 2009 (2009-05-01), pages 3828 - 3832 *
KLIMOV ET AL.: "Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences", BMC CANCER., vol. 2, 13 October 2002 (2002-10-13), pages 1 - 8 *
LU ET AL.: "Multiple-Integrations of HPV16 Genome and Altered Transcription of Viral Oncogenes and Cellular Genes Are Associated with the Development of Cervical Cancer", PLOS ONE, vol. 9, no. e97588, 3 July 2013 (2013-07-03), pages 1 - 9 *
SNIJDERS ET AL.: "Human Papillomavirus Type 33 in a Tonsillar Carcinoma Generates Its Putative E7 mRNA via Two E6* Transcript Species Which Are Terminated at Different Early Region Poly(A) Sites", JOURNAL OF VIROLOGY, vol. 66, no. 5, 1 May 1992 (1992-05-01), pages 3172 - 3178, XP008029424 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018207022A1 (fr) * 2017-05-10 2018-11-15 Genomic Vision Association entre l'intégration de génomes de vph à haut risque détectée par peignage moléculaire et la gravité et/ou le résultat clinique de lésions du col de l'utérus

Similar Documents

Publication Publication Date Title
EP2352845B1 (fr) Transcription de diagnostic et motifs d'épissage du HPV16 dans différentes lésions cervicales
Huang et al. Multiple HPV genotypes in cervical carcinomas: improved DNA detection and typing in archival tissues
Brink et al. HPV testing in cervical screening
WO2006075245A2 (fr) Systemes, methodes et compositions pour la detection de papillomavirus humain dans des echantillons biologiques
Tezcan et al. Human papillomavirus genotype distribution and E6/E7 oncogene expression in Turkish women with cervical cytological findings
CN103725792A (zh) 人类乳头瘤病毒（24型）检测（荧光pcr法）试剂盒及其检测方法
Wang et al. Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions
EP2195468A1 (fr) Composition comprenant un mélange oligonucléotidique pour une meilleure détection de génotypes de papillomavirus humains
KR101761701B1 (ko) Hpv 특이적 프로브 및 이를 포함하는 hpv 유전자형 검사용 dna 칩
Wang et al. Diagnostic performance of the E6/E7 mRNA-based Optimygene HR-HPV RT-qDx assay for cervical cancer screening
Choi et al. The clinical performance of primary HPV screening, primary HPV screening plus cytology cotesting, and cytology alone at a tertiary care hospital
Lee et al. Comparison of human papillomavirus detection and typing by hybrid capture 2, linear array, DNA chip, and cycle sequencing in cervical swab samples
US10947603B2 (en) HPV detection method
WO2016049559A1 (fr) Procédés de diagnostic et de pronostic pour des virus et des maladies et affections associées
Theelen et al. Increase in viral load, viral integration, and gain of telomerase genes during uterine cervical carcinogenesis can be simultaneously assessed by the HPV 16/18 MLPA-assay
US8741568B2 (en) Detection of human papillomavirus
Chranioti et al. Performance evaluation of manual and automated (MagNA pure) nucleic acid isolation in HPV detection and genotyping using Roche Linear Array HPV Test
JP3600616B2 (ja) ヒトパピローマウイルスを検出するためのプライマーセット、検出方法および検出用ｄｎａアレイ
Melo et al. Potential diagnostic techniques for cervical cancer prevention-Review
Wang et al. Analytical performance evaluation of the HPV OncoCheck assay for detection of high-risk HPV infection in liquid-based cervical samples
EP2508620A1 (fr) Évaluation de la charge virale dans des infections multiples par HPV en tant que prédicteur pour la présence de lésions cervicales
WO2005100610A2 (fr) Procede de detection de virus, amorces correspondantes et necessaire de criblage
Ursu et al. Detection of HPV 16 and HPV 18 viral loads by real time PCR in women with cervical dysplasia
Chranioti et al. Research Article Performance Evaluation of Manual and Automated (MagNA Pure) Nucleic Acid Isolation in HPV Detection and Genotyping Using Roche Linear Array HPV Test
da Silva Reis et al. Molecular Tools for Detection Human Papillomavirus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15844727

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15844727

Country of ref document: EP

Kind code of ref document: A1