WO2010060103A1 - Biomarqueurs pour la détection de tumeurs de la tête et du cou - Google Patents

Biomarqueurs pour la détection de tumeurs de la tête et du cou Download PDF

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WO2010060103A1
WO2010060103A1 PCT/US2009/065796 US2009065796W WO2010060103A1 WO 2010060103 A1 WO2010060103 A1 WO 2010060103A1 US 2009065796 W US2009065796 W US 2009065796W WO 2010060103 A1 WO2010060103 A1 WO 2010060103A1
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hpv
host cell
biomarker
biological sample
dna
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PCT/US2009/065796
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English (en)
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Penelope J. Duerksen-Hughes
Maria Filippova
Valeri Filippov
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Loma Linda University
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Priority to EP09828392A priority Critical patent/EP2364371A4/fr
Priority to US13/130,934 priority patent/US8715926B2/en
Publication of WO2010060103A1 publication Critical patent/WO2010060103A1/fr
Priority to US14/162,628 priority patent/US20140194317A1/en

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    • 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
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • C12Q2600/154—Methylation markers
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • HPV Human Papillomavirus
  • HNC Head and Neck Cancer
  • Head and neck cancers arise in the mucosal epithelia that line the throat, oropharynx and mouth. Together, they represent the sixth most common cancer in the US; once diagnosed, patients have a survival rate of approximately 50% (1). It was estimated that 34,360 men and women (24,180 men and 10,180 women) would be diagnosed with and 7,550 men and women would die of cancer of the oral cavity and pharynx in 2007 (2). Approximately 20-30% of the HNC cases are linked to HPV; the remainder are thought to be linked to other risk factors such as tobacco and alcohol.
  • HPV Human papillomavirus
  • HPV sequences were detected in oral cells from 23% of patients in one study, these sequences were also detected in 11% of control subjects (14). This is consistent with findings in cervical cancer, where many more people are infected than actually develop cancer. For example, it is estimated that 10 million women in the US have cervical human papillomavirus infections, while only 15 thousand develop cancer.
  • HPV During the normal HPV life cycle, HPV enters the tissue through a cut or wound and thus comes in contact with the basal keratinocytes of the squamous epithelia. After entering the cells, it remains as a circular episome within this layer, expressing low levels of early viral proteins (including E6 and E7) and replicating its genome in concert with replication of the cellular genome. Typically, at this stage, viral RNA and DNA are found at very low levels, with 50 - 100 episomal copies per cell. As the cells move upward, they become increasingly differentiated into keratinocytes, and these changes in turn trigger changes in HPV activities. The virus enters the vegetative state, and begins to produce the Ll and L2 proteins that will provide the outer coat of the virus.
  • early viral proteins including E6 and E7
  • HPV 16 and HPV 18 High-risk strains of human papillomaviruses code for two oncogenes, E6 and E7. Under normal, episomal conditions, E6 and E7 are expressed at low levels, and are thought to function by creating conditions in the infected keratinocytes that will favor replication of the virus and prevent apoptosis of the host cells. Their expression is negatively regulated, at least in part, by the E2 protein (19-23).
  • E6 is best known for its ability to bind to and mediate the degradation of the tumor suppressor p53 (24). This is not the only activity of E6, however, E6 actually binds to many additional cellular proteins and can affect their biological activities (reviewed in (25, 26)). Several of these proteins, including p53, myc, bak, TNF Rl, FADD and procaspase 8, are involved in cellular apoptotic pathways.
  • E7 is best known for its ability to bind to and inactivate the tumor suppressor Rb protein (27, 28). However, like E6, E7 also has multiple cellular activities (29).
  • the tumor suppressors p53 and Rb are degraded or inactivated, other biological events modulated by E6 and E7 occur, and the chances that the infected cell will divide inappropriately and will fail to undergo apoptosis increase (see (5, 30-33) for reviews). This clearly sets the stage for the development of cancer. It is likely that that the full development of the cancerous phenotype normally takes years to decades to develop, as most women are infected with the high-risk strains of HPV in their late teens and early twenties, and present with cancer in their late forties and early fifties.
  • HNC and/or HPV infection can influence the methylation and therefore the expression of a number of cellular genes.
  • Feng and coworkers (29) examined hypermethylation of 20 genes in patients with increasingly severe CIN and ICC. They found that the best panel of hypermethylated genes included DAPKl, RARB and TWISTl.
  • Henken et al (43) examined promoter methylation that occurred sequentially with progression of CIN and cervical cancer by looking at cells and cell lines that represented the various stages. This group found that a number of genes, many known to be involved in regulation of cell cycle, apoptosis and malignancy, appear to become sequentially methylated with progression of the disease.
  • IGF-II Insulin-like Growth Factor-II
  • IGF-BP3 IGF-Binding Protein 3
  • IMPOD Integrated Microfluidic Platform for Oral Diagnostics
  • MMP-8 matrix metalloproteinase-8
  • this lab was able to identify four genes, interleukin 1-beta (ILlB), ornithine decarboxylase antizyme 1 (OAZ), spermidine/spermine Nl -acetyl transferase (SAT) and interleukin 8 (IL-8) that together, could identify saliva from cancer patients in nine out of ten samples from a group of 32 patients.
  • IL-8 interleukin 1-beta
  • OAZ ornithine decarboxylase antizyme 1
  • SAT spermidine/spermine Nl -acetyl transferase
  • IL-8 interleukin 8
  • one object of the present invention is to provide a rapid accurate and cost-effective diagnostic tool for the early identification of pre-cancerous and cancerous lesions in the head and neck area using saliva as a sample source.
  • one embodiment of the present invention provides a method that detects a series of biomarkers that can distinguish between a non-diseased condition and a situation where the patient is likely to develop or has HPV-associated head and neck cancer.
  • the method detects increases/decreases in biomarker gene expression.
  • the method detects changes in specific DNA methylation patterns.
  • the technology can be adapted to high-throughput, clinically compatible applications where a plurality of HPV and HNC-associated host cell biomarkers are simultaneously detected from a single sample.
  • the method comprises, first, obtaining a biological sample from a patient, such as for example, a tissue, plasma and/or saliva sample.
  • a biological sample from a patient, such as for example, a tissue, plasma and/or saliva sample.
  • the samples are processed to isolate DNA and/or RNA.
  • the biological sample(s) are then subjected to screening for the presence or absence of the biomarker.
  • One embodiment of the present invention provides a method of detecting biomarkers associated with head and neck tumors in a subject.
  • the first step of the method comprises contacting a first biological sample from the subject, wherein the first biological sample is selected from the group consisting of saliva, whole blood, white blood cells, serum, plasma and biopsy tissue from the throat, oropharynx or mouth, with: (1) a first reagent that specifically binds to one or more than one human papillomavirus (HPV) biomarker; and (2) a second reagent that specifically binds to one or more than one host cell biomarker, wherein the host cell biomarker is differentially expressed in head and neck tumor cells as compared to normal cells.
  • HPV human papillomavirus
  • the next steps of the method comprise detecting the presence or absence of the HPV marker; and determining whether or not the host cell marker is differentially expressed in the biological sample.
  • differential expression of the host cell marker is accomplished by comparing the expression level of the host cell marker in the biological sample to the expression level of the same host cell marker for at least one reference sample, where the reference sample is a comparable biological sample obtained from a disease-free subject.
  • the first reagent is an oligonucleotide and the
  • HPV biomarker is a HPV- specific nucleic acid.
  • the oligonucleotide reagent can comprise at least 15 nucleotides.
  • each oligonucleotide can comprise at least 20, 25, 50, 75, 100, 125, 150, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides.
  • the HPV biomarker is a HPV mRNA or a complement thereof.
  • the biomarker is an mRNA encoded by an HPV gene, such as E2, E5, E6, E6* or E7. The preferred method may further entail identifying splice variants of the HPV gene(s).
  • the HPV mRNA is selected from the group consisting of E2 mRNA, E6 mRNA and E7 mRNA.
  • the first reagent is an antibody and the HPV biomarker is a HPV polypeptide.
  • the first reagent can be an HPV antigen and the HPV biomarker can be an anti-HPV antibody.
  • the first reagent specifically binds to a plurality of
  • HPV biomarkers and/or the second reagent specifically binds to a plurality of host cell biomarkers.
  • one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or more host cell biomarkers can be selected from a group of biomarkers differentially expressed in HNC cells, such as H3F3A, TPTl, FTHl, NC0A4, ARCR, IGF-II, IGF-BP3, soluble ⁇ chain of the IL-15 receptor.
  • one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty, forty, fifty or more host cell biomarkers can be selected from a group of biomarkers differentially expressed in HPV positive HNC cells, such as AL833646, BF055370, BUBlB, CCDC5, CCNAl, CCNBl, CCNDl, CCND2, CCNE2, CDC2, CDC7, CDK2, CDKN2A, CDKN2B, CDKN2C, CENPF, CHEKl, E2F2, E2F3, E2F7, EHHADH, EREG, FKSG14, 10 FLJ31952.
  • biomarkers differentially expressed in HPV positive HNC cells such as AL833646, BF055370, BUBlB, CCDC5, CCNAl, CCNBl, CCNDl, CCND2, CCNE
  • the second reagent is an oligonucleotide and the host cell biomarker is a nucleic acid.
  • the oligonucleotide reagent can comprise at least 15 nucleotides.
  • each oligonucleotide can comprise at least 20, 25, 50, 75, 100. 125, 150, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides.
  • the host cell biomarker is a host cell mRNA or a complement thereof.
  • the HPV biomarker or the host cell biomarker is DNA.
  • the DNA is a CpG containing promoter and the method further comprises determining whether or not the CpG-containing promoter is aberrantly methylated.
  • the differential methylation of one, two, three, four or more HPV genes e.g. the E2, E5, E6 or E7 promoter, is determined.
  • whether or not the CpG-containing promoter is aberrantly methylated is determined by comparing the methylation of the CpG-containing promoter in the biological sample to the methylation of the CpG-containing promoter for at least one reference sample, where the reference sample is a comparable biological sample obtained from a disease-free subject.
  • one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or more CpG containing promoters such as the host cell promoters for DAPKl, RARB, TWISTl, TIMP3, APC, KLKlO, TP73, CDH13, IGSF4, FHIT, ESRl, CHFR, N0L4, LHFPL4, SOXl, PAXl, LMXlA, NKX6-1, WT-I and ONECUTl, are selected for analysis.
  • the HPV biomarker is DNA and the method further comprises distinguishing between a high risk strain of HPV and a low risk strain of HPV. In a preferred embodiment, the method further comprises identifying HPVl 6 DNA or HPV 18 DNA. In another embodiment, the integration of HPV DNA in the host genome is determined.
  • the method further comprises comparing the expression level of the host cell marker in the biological sample to the expression level of the same host cell marker for one or more than one additional reference sample, where the reference sample is a comparable biological sample obtained from a patient with an HPV positive head and neck tumor or a patient with an HPV negative head and neck tumor.
  • the first biological sample is compared to a second biological sample from the subject.
  • the first biological sample is saliva and the second biological sample is whole blood, blood cells, serum, plasma, or a tissue sample from the throat, oropharynx or mouth.
  • the method further comprises the additional steps of contacting the second biological sample from the subject with; (1) a reagent that specifically binds to a HPV biomarker, and (2) a reagent that specifically binds to a host cell marker differentially expressed or in head and neck tumor cells as compared to normal cells.
  • the next steps are detecting the presence or absence of the HPV marker; and determining whether or not the host cell marker is differentially expressed in the first sample and/or the second sample.
  • Figure 1 shows PCR amplification with DNA-specific and RNA-specific primers.
  • Figure IA shows PCR amplification results when nucleic acids from 0.280 ml of plasma and saliva were isolated using the QIAamp kit (Qiagen) and eluted into 40 ⁇ l of water. One ⁇ l of purified sample was used for PCR amplification of SRF6 for 39 cycles using genomic DNA specific primers.
  • Figure IB shows PCR amplification results when the DNA/RNA samples were further purified with RNeasy for RNA cleanup, eluted in 10 ⁇ l of water, and 5 ⁇ l was used for cDNA synthesis by ImProm-II reverse transcriptase (Promega) in a 20 ⁇ l reaction volume.
  • Figure 1C shows expression levels of selected genes in plasma and saliva RNA samples.
  • Real-time PCR was performed using the Absolute QPCR SYBR Green kit (ABgene) using 1 ⁇ l of standard cDNA synthesis reaction for saliva samples and 2 ⁇ l for plasma samples.
  • Ct values for blank PCR probes were subtracted from the Ct values obtained for plasma and saliva probes. In some cases the subtracted values were negative and were considered to be equal to zero.
  • Figure 2 shows analysis of the differentially methylated pTOPO plasmid containing the CDK2B promoter region.
  • Figure 2A shows the results following preparation and analysis of the methylated form of pTOPO-CDKN2.
  • Unmethylated pTOPO-CDKN2 was methylated with Hhal methylase, then incubated with Hhal. The methylated, but not the unmethylated version, displays resistance to Hhal-mediated digestion.
  • Figures 2B. 2C, 2D and 2E are graphs that display the MS-MPLA analyses of methylated and unmethylated forms of the plasmid. Black arrows point to the peaks corresponding to the generated PCR products, which are indicative of an intact DNA sequence at the restriction site.
  • Figures 2B and 2C show methylated DNA, either undigested (Figure 2B) or digested with Hhal ( Figure 2C); Figures 2D and 2E show unmethylated DNA, either undigested ( Figure 2D) or digested with Hhal ( Figure 2E). The peaks remaining in Figure 2E show the DNA ladder.
  • Figure 3 shows analysis of the methylation status of the CDKN2B and TP73 promoters in two cervical cancer cell lines: CaSki and SiHa. The two panels to the left show undigested samples and the two panels to the right show samples digested with Hhal. The peaks of the MS-MLPA products for each of the two cell lines are shown as arrows.
  • Figure 4 shows analysis of gene expression when normal control saliva was mixed 1: 1 with lysis reagent, then processed for qNPA. The data for replicates is shown for saliva versus negative control (no saliva). The level of genes with measured expression are detailed in Table 6. Note the repeatability of measurement.
  • the methods of this invention find particular use in diagnosing or providing a prognosis for head and neck cancer (HNC) by detecting human papilloma virus (HPV) markers and host cell markers, which are differentially expressed (down or upregulated) in HNC tumor cells. These markers can thus be used diagnostically to distinguish HPV+ HNC from HPV- HNC or normal cells.
  • HPV human papilloma virus
  • the markers can be used alone or in combination.
  • a method for the detection of changes in expression levels of selected viral and cellular genes or biomarkers there is provided a method for the detection of changes in expression levels of selected viral and cellular genes or biomarkers.
  • Another embodiment of the present invention provides a method for detecting changes in the methylation status of their promoters in tissues, blood/serum and saliva.
  • Several sets of matched samples can be used to identify biomarkers that can be found in saliva and have the ability to distinguish between HPV-associated head and neck cancer and controls.
  • the methods provide a rapid, accurate and cost-effective diagnostic tool for the early identification of pre-cancerous and cancerous lesions in the head and neck area, using saliva as a sample source.
  • head and neck cancer refers to a group of biologically similar cancers originating from the upper aerodigestive tract, including the lip, oral cavity (mouth), nasal cavity, paranasal sinuses, pharynx, and larynx. Most head and neck cancers are squamous cell carcinomas, originating from the mucosal lining (epithelium) of these regions.
  • HPV Human papillomavirus
  • HPV16 is a suggested causal factor for head and neck squamous cell carcinoma (HNSCC).
  • HNSCC head and neck squamous cell carcinoma
  • Approximately 15 to 25% of HNSCC contain genomic DNA from HPV, and the association varies based on the site of the tumor, especially in the oropharynx, with highest distribution in the tonsils, where HPV DNA is found in (45 to 67%) of the cases, less often in the hypopharynx (13%— 25%), and least often in the oral cavity (12%-18%) and larynx (3%-7%).
  • the term "marker” or “biomarker” refers to a molecule (typically protein, nucleic acid, carbohydrate, or lipid) that is expressed in the cell, expressed on the surface of a cancer cell, secreted by a cancer cell or modified in a cancer cell in comparison to a normal cell, and which is useful for the diagnosis of cancer, for providing a prognosis, and for preferential targeting of a pharmacological agent to the cancer cell.
  • markers are molecules that are differentially expressed, e.g., overexpressed or underexpressed in a HPV + HNC cell in comparison to a normal cell, for instance, 1-fold over/under expression, 2- fold over/under expression, 3-fold over/under expression or more in comparison to a normal cell, a HPV HNC cell or a HPV + cervical cancer cell.
  • a marker can be a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell.
  • Accession numbers for nucleic acid and protein sequences of representative biomarkers, which may be differentially expressed in HNC cells include the following:
  • CD44 CD44 molecule Indian NM_000610, NM_001001389, NM_001001390, blood group) NM_001001391, NM_001001392, M59040
  • Accession numbers for nucleic acid and protein sequences of representative biomarkers, which may be differentially expressed in HPV + HNC cells include the following:
  • markers may be used singly or in combination with other markers for any of the uses, e.g., the diagnosis or prognosis of HPV + HNC, disclosed herein.
  • Bio sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include whole blood and blood fractions or products (e.g., serum, plasma, white blood cells, and the like), sputum, saliva, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, etc.
  • the biological sample is typically obtained from a eukaryotic organism, preferably a mammal, most preferably a primate, e.g., a human subject.
  • a "biopsy” refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the location of the tissue to be evaluated (e.g the lip, oral cavity, nasal cavity, paranasal sinuses, pharynx, larynx, etc.) and the size of the tumor, among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, and surgical biopsy. An “excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • a diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy” of the tumor mass, or a “fine-needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V.
  • overexpress refers to a protein or nucleic acid (RNA) that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a normal cell.
  • the term includes overexpression due to transcription, post transcriptional processing, translation, post- translational processing, cellular localization ⁇ e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell.
  • Overexpression can be detected using conventional techniques for detecting inRNA ⁇ i.e., RT-PCR, PCR, hybridization) or proteins ⁇ i.e., ELISA, immunohistochemical techniques).
  • Overexpression can be 10%, 20%, 30%, 40%. 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold or more higher levels of transcription or translation in comparison to a normal cell.
  • underexpress refers to a protein or nucleic acid (RNA) that is transcribed or translated at a detectably lower level, usually in a cancer cell, in comparison to a normal cell.
  • the term includes underxpression due to transcription, post transcriptional processing, translation, post- translational processing, cellular localization ⁇ e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell.
  • Underexpression can be detected using conventional techniques for detecting mRNA ⁇ i.e., RT-PCR, PCR, hybridization) or proteins ⁇ i.e., ELISA, immunohistochemical techniques).
  • Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% etc. in comparison to a normal cell. In certain instances, underexpression is 1-fold, 2-fold, 3-fold, 4-fold or more lower levels of transcription or translation in comparison to a normal cell.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).
  • sequences are then said to be "substantially identical," This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, /. MoI. Biol.
  • a preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. MoI. Biol. 215:403-410 (1990), respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • Nucleic acid refers to deoxynbonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.. MoI. Cell. Probes 8:91-98 (1994)).
  • nucleic acid is used interchangeably with gene. cDNA, mRNA, oligonucleotide, and polynucleotide.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants" and nucleic acid sequences encoding truncated forms of cancer antigens.
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant or truncated form of that nucleic acid.
  • "Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons.
  • Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Nucleic acids can be truncated at the 5' end or at the 3' end. Polypeptides can be truncated at the N-terminal end or the C-terminal end. Truncated versions of nucleic acid or polypeptide sequences can be naturally occurring or recombinantly created.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes,
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). See, e.g., Creighton, Proteins (1984).
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T 1n ) for the specific sequence at a defined ionic strength pH.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T 1n , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e,g,, and Current Protocols in Molecular Biology, ed. Ausubel, et al. , supra.
  • a temperature of about 36 0 C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length.
  • a temperature of about 62 0 C is typical, although high stringency annealing temperatures can range from about 5O 0 C to about 65 0 C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9O 0 C - 95 0 C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72 0 C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y.).
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • Antibodies can be polyclonal or monoclonal, derived from serum, a hybridoma or recombinantly cloned, and can also be chimeric, pnmatized, or humanized.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 a dimer of Fab which itself is a light chain joined to V H -C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab) 5 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al. Nature 348:552-554 (1990)).
  • Certain embodiments of the present invention provide methods to qualitatively and/or quantitatively analyze a panel of candidate biomarkers.
  • matched sample sets of HPV+ and HPV- tonsillar tissue, blood and saliva can be analyzed for the presence of HPV DNA sequences, the presence and levels of HPV-encoded mRNA and proteins, changes in the expression of host-encoded biomarkers, and the methylation status of selected host and viral promoters.
  • the most suitable biomarkers will correlate with HPV and cancer status.
  • matched sets of samples that include cancer tissue, adjacent normal tissue, blood samples and saliva samples are obtained from the same patient, which may contain both HPV positive and HPV negative lesions. Screening the panel for HPV biomarkers will specifically and sensitively identify HPV-positive malignancies. Saliva collected from individuals known to have HPV+ head and neck cancer can serve as a positive control.
  • Various embodiments of the present invention detect and compare one or more of three sets of phenomena - the presence of HPV DNA, the presence of altered levels of cellular and viral messages, and alterations in methylation patterns for cellular and viral genes.
  • Another embodiment of the present invention detects and compares these biomarkers in one or more of three types of material - tissue, blood and saliva.
  • a panel of biomarkers are selected, which are measureable in saliva.
  • biomarkers will be selected from previously published global analyses, while for the viral mRNA expression levels and methylation, the biomarkers are selected on the basis of what is known regarding the molecular activities of the virus and its proteins.
  • nucleic acids encoding biomarkers or their encoded polypeptides refer to all forms of nucleic acids (e.g., gene, pre-mRNA, mRNA) or proteins, their polymorphic variants, alleles, mutants, and interspecies homologs that (as applicable to nucleic acid or protein): (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%.
  • amino acid sequence identity preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence, immunogenic fragments thereof, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides
  • a polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal.
  • the nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules. Truncated and alternatively spliced forms of these antigens are included in the definition.
  • the phrase "specifically (or selectively) binds" when referring to a protein, nucleic acid, antibody, or small molecule compound refers to a binding reaction that is determinative of the presence of the protein or nucleic acid, often in a heterogeneous population of proteins or nucleic acids and other biologies.
  • nucleic acids an oligonucleotide, polynucleotide or nucleic acid specifically binds to a particular nucleic acid biomarker under stringent hybridization conditions.
  • a specified antibody may bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.
  • polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • DIAGNOSTIC AND PROGNOSTIC METHODS are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • the present invention provides methods of diagnosing or providing prognosis of head and neck cancer (HNC) by detecting the expression of markers differentially expressed in HNC cells.
  • Diagnosis involves determining the level of a HPV or host cell polypeptide or nucleic acid in a patient or patient sample and then comparing the level to a baseline or range.
  • the baseline value is representative of levels of the polypeptide or nucleic acid in a healthy person not suffering from HNC, as measured using a biological sample, such as a tissue biopsy, blood or saliva.
  • Variation of levels of a polypeptide or nucleic acid of the invention from the baseline range indicates that the patient has a cancer or is at risk of developing a cancer, depending on the marker used.
  • the term "providing a prognosis” refers to providing a prediction of the probable course and outcome of HNC.
  • the methods can also be used to devise a suitable therapy for HNC treatment, e.g., by indicating whether or not the HNC tumor is still at a benign stage or if the HNC tumor had advanced to a stage where aggressive therapy would be ineffective.
  • Nucleic acid binding molecules such as probes, oligonucleotides, oligonucleotide arrays, and primers can be used in assays to detect differential RNA expression in patient samples, e.g., RT-PCR.
  • RT-PCR is used according to standard methods known in the art.
  • PCR assays such as Taqman ® assays available from, e.g., Applied Biosystems, can be used to detect nucleic acids and variants thereof.
  • qPCR and nucleic acid microarrays can be used to detect nucleic acids.
  • Reagents that bind to selected cancer biomarkers can be prepared according to methods known to those of skill in the art or purchased commercially.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • Applicable PCR amplification techniques are described in, e.g., Ausubel et al. and Innis et al, supra.
  • General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization,” BIOS Scientific Publishers, 1999.
  • Amplification or hybridization of a plurality of nucleic acid sequences can also be performed from mRNA or cDNA sequences arranged in a microarray.
  • Microarray methods are generally described in Hardiman, “Microarrays Methods and Applications: Nuts & Bolts,” DNA Press, 2003; and Baldi et al, “DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge University Press, 2002.
  • PCR-based analysis includes a Taqman ® allelic discrimination assay available from Applied Biosystems.
  • sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods MoI.
  • sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat. BiotechnoL, 16:381-384 (1998)), and sequencing by hybridization.
  • MALDI-TOF/MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Non- limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.
  • Other methods for detecting nucleic acid variants include, e.g., the INVADER ® assay from Third Wave Technologies, Inc., restriction fragment length polymorphism (RFLP) analysis, allele-specific oligonucleotide hybridization, a heteroduplex mobility assay, single strand conformational polymorphism (SSCP) analysis, single - nucleotide primer extension (SNUPE) and pyrosequencing.
  • RFLP restriction fragment length polymorphism
  • SSCP single strand conformational polymorphism
  • SNUPE single - nucleotide primer extension
  • antibody reagents can be used in assays to detect expression levels of HPV of host cell polypeptides in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, “Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach,” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al, Curr. Opin. Biotechnol, 7:60-65 (1996).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence.
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEIA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassay
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al, J. Immunol. Methods, 204:105-133 (1997).
  • nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al, J. Clin. Chem, Clin. Biochem., 27:261-276 (1989)).
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labeled with iodine- 125 ( 123 I) can be used.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the nucleic acid is suitable for sensitive, non-radioactive detection of protein levels.
  • An antibody labeled with fluorochrome is also suitable.
  • fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ - galactosidase, urease, and the like.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB chromogenic substrate tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, MO).
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125 I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • a detectable moiety can be used in the assays described herein.
  • a wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions.
  • Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green , rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.
  • fluorescent dyes e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green , rhodamine, Texas red, tetrarhodimine isothiocynate (TRI
  • Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different markers.
  • Such formats include microarrays and certain capillary devices. See, e.g., Ng et al, J. Cell MoI. Med., 6:329-340 (2002); U.S. Pat. No. 6,019,944.
  • each discrete surface location may comprise antibodies to immobilize one or more markers for detection at each location.
  • Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection.
  • Analysis can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion.
  • the antibodies or nucleic acid probes of the invention can be applied to sections of patient biopsies immobilized on microscope slides.
  • the resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.
  • the panel is used to test several matched sets of patient samples - tissue, blood and saliva. This screen will confirm which of the biomarkers are most meaningful, consistent, and not due to random chance.
  • This further streamlined panel may then be utilized on a population of saliva samples, using saliva from known HPV+ HNC patients as a positive control.
  • Our overall objective is to provide a panel of measurements that can be made using an easily-obtained material such as saliva that will accurately predict the development of HNC in human patients.
  • Tumor tissues can be obtained as surgical specimens. Matching sets of adjacent normal tissue, blood and saliva from the same patient may also provided. These samples can be processed for the analysis of DNA sequences, methylation status, the presence of viral and cellular RNA, and the presence of specific proteins using standard procedures. The basic methods regarding the extraction and processing of DNA and RNA from tissues and blood are well-established. In addition, formalin-fixed, paraffin-embedded tissues may be employed by using commercially available kits to extract the genomic DNA and RNA (43). Protocols regarding the extraction and processing of DNA and RNA from saliva are described in further detail in the "Examples" section and shown in Figures 1 and 4, which demonstrate that one can relatively easily obtain these materials from both blood and saliva.
  • blood samples can be obtained from the same patients that provided the tissue samples and stored in the frozen state. After thawing the samples, they can be processed as described in the "Examples" section of the present disclosure.
  • PCR can then be used to look for the presence of HPV DNA
  • RT PCR or qNPA can be used to look for the presence and level of the selected viral and cellular transcripts
  • MS-MLPA can be used to look for the methylation of selected promoter sequences, as described above and in the "Examples' " section.
  • the presence of anti-HPV antibodies can be detected in serum samples, which could provide evidence of a recent infection.
  • Saliva samples can be obtained, preferably from the same patients that provided the tissue and blood samples, and stored in the frozen state. After thawing, the samples are then processed as described in further detail in the "Examples" section. PCR can be used to look for the presence of HPV DNA, RT PCR or qNPA can be used to look for the presence and level of the selected viral and cellular transcripts, and MS-MLPA can be used to look for the methylation of selected promoter sequences, as described above and in the "Examples' " section. Saliva samples can be collected using the collection and preservation kits produced by Oragene. These kits enable the collection and preservation of DNA and RNA from human saliva until processing.
  • matched sample sets each of which will include cancer tissue, non-cancerous adjacent tissues, blood/serum and saliva, are obtained from the same individual.
  • negative controls no diagnosed HPV-related, cervical, or HNCs
  • positive controls e.g., from patients with diagnosed HPV+ tonsillar cancer (early stage and late stage), from patients with HPV- head/neck cancer, and from patients with HPV+ cervical cancer, may be obtained.
  • Using this group of matched sets will permit calibration of the panel based on the detection of biomarkers important to head/neck cancer in general, to HPV-associated tonsillar cancer specifically, to HPV-associated cancer in general (both head/neck and cervical), and to HPV-associated cervical cancer.
  • each of the samples will be processed as appropriate and each measurement should be made in triplicate.
  • the expression levels of each target message can be compared to expression of the GAPDH gene in the same samples.
  • Changes in expression levels in cancer tissue vs. adjacent normal tissue can be compared to the observed levels in saliva and plasma/serum to see if changes indicative of the presence of cancer can also be detected in saliva and/or plasma/serum.
  • DNA samples isolated from different biological samples from the same donor can be analyzed to see how the methylation measurements from the different materials do or do not correlate.
  • DNA can be extracted from tissues using the DNeasy Blood and Tissue Kit
  • HPV sequences themselves, as by definition, they are expected to be present in HPV-associated tumors. Once is it confirmed that HPV sequences are present, the HPV type can be determined by cloning and sequencing the PCR product. It has previously been shown that PCR followed by DNA sequencing can be used to detect HPV sequences from oral exfoliated cells (14).
  • MS-MLPA methylation-specific multiplex ligation-dependent probe amplification kits
  • This kit has been used successfully in previous studies of cervical cancer (43).
  • the same MS-MLPA technique can be used for the analysis of other promoters that are not included in the kit; these include both viral and cellular promoters.
  • the LCR and early promoter region of the HPV region can be methylated, and it has been suggested that the DNA methylation state of HPV can vary depending on the viral life cycle and on the presence or absence of integration (62).
  • RNA extracted from the tissues using Trizol Reagent can be quantitatively analyzed for the presence and level of both viral and cellular messages.
  • Viral messages to be analyzed include those for the E2, E5, E6 and E7 genes, as their levels will provide valuable information regarding the current status of the virus and the possible progression of transformation.
  • our understanding of the biological events that occur as HPV-associated malignancies develop leads us to predict that in cases where transformation may be occurring, the levels of E6 and E7 expression should be higher than in cases where no transformation is occurring.
  • E6 one may further analyze for the presence of messages coding for both the large and small isoforms.
  • the levels of E2 and E5 expression may provide further information regarding viral integration (37).
  • a preferred set of candidates includes CDKN2B, TP73, CD44, pl6, TCAMl, SYCP2, STAG3, CDC2, CEC7, E2Fs, several of the MCMs, cytokeratin 17 and p63.
  • CDKN2B has been shown to be upregulated in HPV-associated cancers (38, 42).
  • CD44 and specifically, certain splice variants of CD44 is of interest, as evidence exists that the presence of specific variants may be associated with the presence of HNC (63-69).
  • pl6 CDKN2A is another gene which has been associated with tonsillar carcinomas and papillomavirus status (36, 70-72), as well as with HPV-associated cervical cancer (72). TCAMl, SYCP2 and STAG3 are normally testis-specific but were found to be expressed in HPV positive cancer cells (38). Expression of these genes should be negligible in normal cells found in the oral cavity, so detection of them in saliva will be a good indication that transformation is occurring in the head and neck region.
  • the Pyeon group identified a number of additional genes that regulate proliferation; any tumor cells found may display an up-regulation of genes that control the cell cycle by enhancing proliferation.
  • This list includes PCNA (proliferating cell nuclear antigen), CDC2, CDC7, ElFs, and MCM.
  • Cytokeratin 17 and p63 are also included in the list, as they may be markers for cervical stem cells (73), which have been suggested to serve as target cells for HPV.
  • immunohistochemistry can be employed to look for evidence of expression of viral proteins, such as E6 and E7, in tissues.
  • viral proteins such as E6 and E7
  • Some studies have shown an increase in protein expression of certain cellular proteins, such as pl6 (36, 70) following HPV infection, Therefore, tissue sections can be examined for up-regulation of these proteins as well.
  • PCR polymerase chain reaction
  • RT-PCR real time RT-PCR
  • both positive and negative controls are used, because negative results don't necessarily indicate that the HPV DNA, mRNA or host cell mRNA is not present or expressed in a subject (false negative).
  • negative controls include controls for the extraction procedure and water control for the PCR run. It is also desirable to confirm positive results to avoid “false positives” in which the presence of HPV or host cell biomarkers is indicated in error.
  • Positive controls include a control for extraction and PCR.
  • the sample can be "spiked” with a weak positive control in order to detect any PCR inhibitory substances that would interfere with the test.
  • high throughput genomic methods are used to detect multiple target nucleic acids that may be present in a biological sample from a subject. These procedures typically use a multiple-well microtiter plate, containing multiple different oligonucleotide probes specific for multiple target agents (nucleic acid: DNA or RNA, or protein) in each well, that may or may not be present in the biological sample, where the probes are attached to the surface of each well.
  • target agents nucleic acid: DNA or RNA, or protein
  • the ability to test several targets simultaneously is known as "multiplexing.”
  • the assays are performed using reagents and conditions effective for reaction of the probe with its respective target molecule. High Throughput methods are known in the art, for example, as described in issued U.S. Pat. Nos.
  • a high throughput assay can be run using multiple (e.g. 100) plates with "wells" for containing the reactions, such as 96-well microplates, simultaneously.
  • Each well of a plate can have multiple, different tests performed in it, by using an array of corresponding probes.
  • 100 plates, with 96 wells per plate, and each with 16 tests per well can be used. In this case, each of 9,600 different biological samples can be tested simultaneously, for 16 different parameters or assays.
  • High throughput assays provide much more information for each biological sample, than do assays which test only one target nucleic acid or protein at a time. Thus, it is possible in a single initial high throughput screening assay to determine whether a sample from a subject contains any of several target nucleic acids or proteins.
  • a high throughput method that detects messenger RNA (mRNA) or DNA corresponding to HPV or host cell biomarkers, and does not involve any RNA extraction, amplification, purification or biosynthetic steps.
  • This method is known as the "quantitative nuclease protection assay or "qNPA,” (High Throughput Genomics, Inc., Arlington, Ariz.), that can quantitatively measure mRNA, from samples of fewer than 1,000 cells, without extraction or amplification (U.S. Pat. No. 6,238,869, incorporated by reference herein).
  • the qNPA produces a stoichiometric amount of the specific nuclease protection probe for each gene, or a quantitative amount of a chemical mirror image. All the reagents that bind to the plate are synthetic and structurally unaltered by the assay. Assays can be conducted using a microplate washer, incubator and standard pipetting station. Standard automation and workstations perform all assay steps. Assay results are detected using known imaging devices, such as the Omix Imager.TM. (HTG, Arlington, Ariz.).
  • RNA isolation was obtained from fresh blood samples by centrifugation at 2600 x g for 15 min at 4°C, and then processed in the same way as the saliva samples.
  • Nucleic acids from both saliva and blood samples were isolated using the QIAamp Viral RNA kit, using a modified version of the manufacturer's protocol. In this modified protocol, glycogen was used rather than carrier RNA to increase the yield of DNA and RNA. Isolated samples were either used directly for PCR analysis with the indicated DNA probes, or used for purification of RNA.
  • Saliva may therefore offer a significant advantage over plasma samples as a potential source for biomarkers in HNC. RT-PCR analysis of RNA samples.
  • the primers were designed to contain intron-intron junctions or to be located within different introns in order to prevent amplification of genomic DNA sequences.
  • To perform real time PCR we used the Absolute QRCR SYBR Green kit.
  • For each reaction we used 1 ⁇ l of saliva cDNA (l/20 th of total cDNA synthesis reaction mixture) and 2 ⁇ l of the plasma samples.
  • Real-time PCR detected the presence of GAPDH, DAPKl, Fas and NFKB transcripts in plasma.
  • saliva from the same donor contained transcripts of all the genes tested except for CFLl, though the SESN3 and GSTPl gene transcripts were present at marginal levels (Figure 1C).
  • mRNA was considered to be present in a particular blood or saliva sample if the difference between its Ct value and the Ct value of background exceeded 2, and to be marginally present when the Ct value difference was lower than 2 but greater than 0.5.
  • saliva samples contain DNA and mRNA at considerably higher levels than plasma. Therefore, saliva can be regarded as a useful source in the search for biomarkers, especially with regards to HNC.
  • this kit does not include HPV-specific probes, and two of the sequences we wish to assess are HPV sequences. For this reason, we developed a modified protocol, using the CDKN2B gene as a model. Modifications were necessary due to our use of a variety of different enzymes that required buffers and temperature conditions that were often incompatible.
  • the CDKN2B gene was selected as our test gene. The first step was to clone the Hhal-containing CDKN2B promoter region of 0.8 Kb into pTOPO.
  • the plasmid DNA as initially isolated is unmethylated, since E.coli does not possess a CpG methylation system.
  • To methylate Hhal sites in the pTOPO-CDKN2B plasmid we incubated plasmid DNA with Hhal methylase according to the manufacturer's protocol (New England Biolabs). and monitored methylation by resistance to digestion with Hhal restrictase (Figure 2).
  • the unmethylated plasmid DNA digest consists of a group of short fragments with molecular weights of less than 500 bp, while DNA treated with Hhal methylase prior to digestion remains intact, showing almost complete methylation of the Hhal sites.
  • the modified MS-MLPA protocol was then applied to both the methylated and unmethylated samples. Using this protocol, we were able to distinguish between methylated and unmethylated variants of the CDKN2B plasmid ( Figure 2). To show that this protocol is compatible with other probes, we also used it to monitor methylation of the TP73 promoter region, Using both the CDKN2B and TP73 probe sets, we analyzed methylation of DNA isolated from Siha and Caski cell lines, both of which are derived from HPV16-mediated cervical carcinomas ( Figure 3). These results show that the CDKN2B site is not fully methylated (though it may be hemi-methylated) and that TP73 is methylated in both cell lines, consistent with data obtained previously.
  • the following example shows the use of qNPA assays for quantitative detection of HPV and/or host cell nucleic acids from HPV-infected cells, cervical clinical samples and saliva.
  • the reagents include a nuclease protection probe that is specific for a specific target, programming linker, detection linker and detection probe.
  • a Universal Array is manufactured by printing 16 different DNA "anchor” sequences, 25 bases each, onto polystyrene microplates. To program this Universal Array, a cocktail of 16 different "programming linker” capture probes (each 50 bases long of synthetic DNA) is added, each in large excess, incubated at 5O 0 C for 30 min, and then washed.
  • One species of programming linker will hybridize (across 25 bases) to only one anchor, or specific element, of the array.
  • the other 25 base half of the programming linker is designed to hybridize specifically to 25 bases of one specific nuclease protection probe.
  • the specificity of hybridization of each element is converted to capture a specific set of 16 different nuclease protection probes.
  • the sample processing and assay protocol includes the following steps.
  • a lysis reagent is added to the sample, e.g., cells, tissue, blood or saliva, incubated at 95 0 C for 10 min then cooled and frozen or tested immediately. If not already in the lysis solution, a cocktail of nuclease protection probes is added (each 50 bases long, synthetic DNA, each designed to hybridize to a different target gene), and incubation is carried out for 6 hr at 6O 0 C.
  • Sl nuclease is then added and incubated for 60 min at 5O 0 C, during which time all the nonspecific RNA and DNA is destroyed, all the excess single stranded nuclease protection probes and non-hybridized target RNA are destroyed and only the specific probe/target hybrid duplexes remain, thereby providing the quantitative stoichiometry of the assay.
  • Base is added to dissociate the probes from the target RNA and destroy the released target RNA.
  • the solution is transferred onto a previously programmed Array Plate (described above), and the probes captured during an overnight incubation at 5O 0 C.
  • Detection linker can be added at the time the sample is transferred, or added separately and incubated for 60 min.
  • Luminescence substrate is added and the entire microplate is imaged to measure the level of each gene. The amount of luminescence indicates how much of each target gene was present in the sample; the position in the array identifies which gene is being measured.
  • Table 4 depicts the sensitivity and specificity of measurement of HPV 16 viral
  • Table 5 presents the qNPA data from cervical PAP smears collected in
  • Laimins LA The biology of human papillomaviruses: from warts to cancer. Infect Agents Dis 1993;2(2):74-86.
  • Soluble CD44 is a potential marker for the early detection of head and neck cancer. Cancer Epidemiol Biomarkers Prev

Abstract

L’invention concerne un procédé de détection de la présence d’un papillomavirus humain spécifique et de biomarqueurs de cellules hôtes associés à des tumeurs de la tête et du cou dans des échantillons biologiques, tels que la salive, le sang ou un tissu de biopsie obtenu d'un sujet.
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US9315854B2 (en) 2010-10-21 2016-04-19 Advanced Cell Diagnostics, Inc. Ultra sensitive method for in situ detection of nucleic acids
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WO2012103414A3 (fr) * 2011-01-28 2012-10-11 Advanced Cell Diagnostics, Inc. Dosage pvh rnascope® pour la détermination du statut pvh dans les cancers de la tête et du cou et les lésions cervicales
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WO2013074793A1 (fr) * 2011-11-15 2013-05-23 University Of Miami Procédés pour détecter des papillomavirus humains et obtenir un pronostic pour un carcinome malpighien de la tête et du cou
US10180431B2 (en) 2011-11-15 2019-01-15 University Of Miami Methods for detecting human papillomavirus and providing prognosis for head and neck squamous cell carcinoma
US10180430B2 (en) 2011-11-15 2019-01-15 University Of Miami Methods for detecting human papillomavirus and providing prognosis for head and neck squamous cell carcinoma
AU2018203361B2 (en) * 2011-11-15 2020-08-27 University Of Miami Methods for detecting human papillomavirus and providing prognosis for head and neck squamous cell carcinoma
WO2015017692A1 (fr) * 2013-07-31 2015-02-05 University Of Miami Compositions et procédés d'identification d'un risque de développement d'un cancer chez un patient
CN104360070A (zh) * 2014-11-28 2015-02-18 山东创新药物研发有限公司 肽基精氨酸脱亚胺酶2在制备肿瘤临床诊断试剂中的应用
WO2016082444A1 (fr) * 2014-11-28 2016-06-02 山东创新药物研发有限公司 Application de peptidylarginine déiminase 1 dans la préparation d'un réactif pour le diagnostic clinique de tumeurs
US11078528B2 (en) 2015-10-12 2021-08-03 Advanced Cell Diagnostics, Inc. In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto
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