WO2018031545A1 - Compositions et méthodes de détection de carcinomes à cellules squameuses de la cavité buccale - Google Patents

Compositions et méthodes de détection de carcinomes à cellules squameuses de la cavité buccale Download PDF

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WO2018031545A1
WO2018031545A1 PCT/US2017/045898 US2017045898W WO2018031545A1 WO 2018031545 A1 WO2018031545 A1 WO 2018031545A1 US 2017045898 W US2017045898 W US 2017045898W WO 2018031545 A1 WO2018031545 A1 WO 2018031545A1
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nucleic acid
hybridization
probes
virus
detected
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PCT/US2017/045898
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English (en)
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Erle S. Robertson
James C. ALWINE
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The Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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

  • Oral cancer remains the second most common cause of death in the US preceded by heart disease, accounting for nearly 1 of every 4 deaths.
  • Oral cancer is one of the most common cancers worldwide, and incidence rates are higher in men compared to women.
  • the predicted new oral cancer cases in 2016 will be 48,250 in the US, with predicted new cases annually exceeding 450,000, worldwide.
  • Oral cancer is newly diagnosed in about 115 new individuals each day in the US alone, and 1 person dies from it every hour.
  • Oral squamous cell carcinoma (OSCC) is the most common oral cancer, comprising about 90% of all the oral cancers. In the US, 3% of cancers in men and 2% in women are OSCC, most of which occur after age 50.
  • the present invention relates to compositions and methods for detecting oral squamous cell carcinoma.
  • the invention includes a method of detecting oral squamous cell carcinoma in a tumor tissue sample from a subject.
  • the method comprises hybridizing a detectably-labeled nucleic acid from the tumor tissue sample to a PathoChip array to generate a first hybridization pattern and hybridizing a detectably-labeled nucleic acid from a reference sample to a PathoChip array to generate a second hybridization pattern.
  • the reference sample is from an otherwise identical non- tumor tissue from a subject.
  • the first and second hybridization patterns are compared. When the first hybridization pattern is substantially a microbial hybridization signature and the second hybridization pattern is substantially not a microbial hybridization signature, oral squamous cell carcinoma is detected in the tumor tissue sample.
  • the invention includes a method of detecting oral squamous cell carcinoma in a tumor tissue sample from a subject, comprising hybridizing a detectably- labeled nucleic acid from the tumor tissue sample to a first microarray comprising at least three nucleic acid probes from microbes selected from the group consisting of Human papillomavirus 16 (HPV16), Eshcherichia, Rothia, Peptoniphilus, Brevundimonas, Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus,
  • the reference sample is from an otherwise identical non-tumor tissue from a subject.
  • the first and second hybridization patterns are compared.
  • oral squamous cell carcinoma is detected in the tumor tissue sample.
  • Another aspect of the invention includes a composition comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • Yet another aspect of the invention includes a microarray comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • Yet another aspect of the invention includes a microarray comprising at least three nucleic acid probes selected from the group of microbes consisting of Human
  • papillomavirus 16 HPV16
  • Eshcherichia, Rothia Peptoniphilus
  • Brevundimonas Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus, Veillonella, Fonsecaea, Mobiluncus, Propionibacterium, Mycobacterium, Malassezia, Pleistophora, Prosthodendrium, Contracaecum, Toxocara, Parvovirus 2, Human herpesvirus 6A, Adenovirus 1, Swine fever virus, JC polyo
  • kits comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76, and instructional material for use thereof.
  • kit comprising a microarray comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76, and instructional material for use thereof.
  • kit comprising a microarray comprising at least three nucleic acid probes selected from the group of microbes consisting of Human
  • papillomavirus 16 HPV16
  • Eshcherichia, Rothia Peptoniphilus
  • Brevundimonas Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus, Veillonella, Fonsecaea, Mobiluncus, Propionibacterium, Mycobacterium, Malassezia, Pleistophora, Prosthodendrium, Contracaecum, Toxocara, Parvovirus 2, Human herpesvirus 6A, Adenovirus 1, Swine fever virus, JC polyo
  • the microbial hybridization signature is generated by hybridization of the detectably-labeled nucleic acid from the tumor tissue sample to at least three nucleic acid probes on the PathoChip, wherein the probes are from microbes selected from the group consisting of Human papillomavirus 16 (HPV16), Eshcherichia, Rothia,
  • the tumor tissue sample is selected from the group consisting of a biopsy, formalin-fixed, paraffin-embedded (FFPE) sample, or non-solid tumor.
  • the subject is human.
  • the subject when oral squamous cell carcinoma is detected in the tumor tissue sample from a subject, the subject is provided with a treatment for oral squamous cell carcinoma.
  • the treatment comprises surgery, chemotherapy, or radiotherapy.
  • the detectably-labeled nucleic acid is labeled with a fluorophore, radioactive phosphate, biotin, or enzyme.
  • the fluorophore is Cy3 or Cy5.
  • the nucleic acid probes are selected from about 10 to about 30 microbes and comprise about 3 to about 5 probes per microbe.
  • the microarray is a biochip, glass slide, bead, or paper.
  • FIGs.1A-1F illustrate viral signatures detected in oral cancer and control samples.
  • FIG.1A shows the viral signatures that are detected with hybridization signal (g-r>30) by PathoChip screen of 100 oral cancer samples ranked according to decreasing
  • FIGs.1B-1C show the hybridization signals and prevalence for the viral signatures detected in matched (MC) and non-matched (NC) controls respectively, ranked in descending order.
  • FIG.1D shows the association of different molecular signatures of viral families with cancer and controls, represented as a Venn diagram, and a bar graph.
  • FIG. 1E shows a heat map of hybridization signals detected by PathoChip screen of the HPV probes (Y-axis) with the oral cancer and control samples (x-axis). The hybridization signals of the cancer samples to each of these probes were compared to MCs and NCs. Samples were screened individually or in pools (marked with a ⁇ ).
  • FIG.1F shows the percentage of HPV16 probes detected with low (g-r>30-300), medium (g-r>300-3000) and high (g-r>3000) hybridization signal in 100 oral cancer samples screened individually and in pools ( ⁇ ) and 20 each of MCs and NCs screened in pools of 5.
  • FIGs.2A-2F illustrate bacterial signatures detected in oral cancer samples.
  • FIG. 2A is a series of pie charts showing the percentage of different groups and phyla of bacteria detected in oral cancer, matched (MC) and non-matched controls (NC).
  • FIGs. 2B-2D show the bacterial signatures that are detected with hybridization signal (g-r>30) by PathoChip screen of 100 oral cancer samples and in MCs and NCs ranked according to decreasing hybridization signal (weighted score sum of all the probes per accession) and prevalence.
  • FIG.2E shows a heat map of the hybridization signal for the bacterial probes of bacterial genera a-xyz, labeled in FIGs.2B-2D, detected by PathoChip screen with the cancer, matched (MC) and non-matched control (NC) samples. Samples were screened individually and in pools (marked ⁇ ).
  • FIG.2F shows the association of molecular signatures of different bacterial genera with oral cancer and/or controls, represented as a Venn diagram as well as a bar graph.
  • FIGs.3A-3J illustrate fungal (FIGs.3A-3E) and parasitic (FIGs.3F-3J) signatures detected in oral cancer samples.
  • FIG.3A shows the fungal signatures that are detected with hybridization signal (g-r>30) by PathoChip screen of 100 oral cancer samples ranked according to decreasing hybridization signal (weighted score sum of all the probes per accession) and prevalence.
  • FIGs.3B-3C show the fungal signatures detected in the matched (MC) and non-matched controls (NC) respectively, ranked according to decreasing hybridization signal and prevalence.
  • FIG.3D shows a heat map of the hybridization signal for the fungal probes of fungi a-g, labeled in FIG.3A, detected by PathoChip screen with the cancer, matched (MC) and non-matched control (NC) samples. Samples were screened individually and in pools (marked ⁇ ).
  • FIG.3E shows the association of molecular signatures of different fungal genera with oral cancer and/or controls, represented as a Venn diagram and bar graph.
  • FIG.3F shows the parasitic signatures that are detected with hybridization signal (g-r>30) by PathoChip screen of 100 oral cancer samples ranked according to decreasing hybridization signal (weighted score sum of all the probes per accession) and prevalence.
  • FIGs.3G-3H show the parasitic signatures detected in the matched and non-matched controls (MC and NC) respectively, ranked according to decreasing hybridization signals and prevalence.
  • FIG.3I shows a heat map of the hybridization signal for the parasitic probes of parasites a-f, labeled in FIG.3F, detected by PathoChip screen with the cancer, matched (MC) and non-matched control (NC) samples. Samples were screened individually and in pools (marked ⁇ ).
  • FIG. 3J shows the association of molecular signatures of different parasitic genera with oral cancer and/or controls, represented as a Venn diagram and a bar graph.
  • FIGs.4A-4C illustrate hierarachial clustering of 100 oral cancer samples.
  • FIG.4A shows hierarchial clustering by R program using Euclidean distance, complete linkage and non-adjusted values. Samples marked ( ⁇ ) were the samples that were screened in pools, the rest were screened individually.
  • FIG.4B shows clustering of the OSCC samples using NBClust software [CH (Calinski and Harabasz) index, Euclidean distance, complete linkage].
  • FIG.4C shows topological analysis using Ayasdi software, using Euclidean (L2) metric and L-infinity centrality lenses.
  • the OSCC samples that had similar detection for viral and microbial signatures formed the nodes, and those nodes are connected by an edge if the corresponding nodes have detection pattern in common with the first node. Nodes are color coded according to the detection of HPV 16.
  • FIGs.5A-5D illustrate probe capture sequencing alignment for individual capture pools (HPV16, O, B, F and P).
  • HPV16 capture probes were comprised of a set of HPV16 specific probes
  • O capture probes consisted of certain viral and bacterial probes
  • B pool was comprised of bacterial probes
  • F consisted of fungal probes
  • P was comprised of parasitic probes that are mentioned in Table 1.
  • the hybridization signals of the HPV probes used for capture are shown as a heat map in FIG.5A.
  • FIGs.5A-5D show the Miseq reads from individual capture when aligned with the metagenome of PathoChip (Chip probes), which cluster mostly at the capture probe regions. The genomic location along with the number of MiSeq reads are shown in the figure for each organism.
  • FIGs.6A-6F illustrate microbial genomic integrations in the host chromosome.
  • FIG.6A is a series of bar graphs showing the number of viral (HPV16 and JC Polyoma viral) integration sites in host human chromosomes and the percentage of viral genomic sites for integration into host chromosomes.
  • FIG.6C is a karyogram plot of bacterial insertion sites (lines above
  • FIG. 6E is a schematic representation of viral and microbial genomic insertional sites in human chromosome 17. The genomic co-ordinates of the pathogens integrated and that of the host chromosome integration sites are mentioned.
  • FIG. 6F shows the association of host genes affected by viral/microbial genomic integrations to neoplasia of epithelial cells, analyzed by the Ingenuity Pathway Analysis (IP A) program that showed a />value of 7.17E 10 for such association.
  • IP A Ingenuity Pathway Analysis
  • FIGs. 7A-7G illustrate capture probe sequencing results.
  • FIG. 7A is a series of histograms showing the percentage of reads >0 in each capture pool reaction (B for bacterial, F for fungal, HPV for papillomaviral, O for others, that included certain viral and bacterial capture probes and P for parasitic capture probes). The number of reads captured by individual capture pools co-related with the type of capture probes used. For example, 100 percent of the reads from B library aligned with the bacterial (B) capture probes.
  • FIGs. 7B-7G show probe capture sequencing alignments post MiSeq. The MiSeq reads from individual capture when aligned with the metagenome of PathoChip (Chip probes) was found to cluster mostly at the capture probe regions.
  • the genomic location along with the number of MiSeq reads are shown in the figures.
  • the alignment for O capture pool library is shown in FIG. 7B; that of B library is shown Figure FIG. 7C-7D; that of F library is shown FIG. 7E; that of P library is shown in FIG. 7F-7G.
  • FIG. 8 is a schematic representation of viral, bacterial, fungal and parasitic genomic insertional sites in human chromosomes. The genomic co-ordinates of the pathogens integrated and that of the host chromosome integration sites are mentioned.
  • the co-ordinates for human chromosomes are from GRCh37/hfl9 Assembly.
  • FIGs. 9A-9B are a set of tables illustrating microbial genomic integration sites in the OCSCC host somatic chromosomes. DETAILED DESCRIPTION OF THE INVENTION
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • A“biomarker” or“marker” as used herein generally refers to a nucleic acid molecule, clinical indicator, protein, or other analyte that is associated with a disease.
  • a nucleic acid biomarker is indicative of the presence in a sample of a pathogenic organism, including but not limited to, viruses, viroids, bacteria, fungi, helminths, and protozoa.
  • a marker is differentially present in a biological sample obtained from a subject having or at risk of developing a disease (e.g., an infectious disease) relative to a reference.
  • a marker is differentially present if the mean or median level of the biomarker present in the sample is statistically different from the level present in a reference.
  • a reference level may be, for example, the level present in an environmental sample obtained from a clean or uncontaminated source.
  • a reference level may be, for example, the level present in a sample obtained from a healthy control subject or the level obtained from the subject at an earlier timepoint, i.e., prior to treatment.
  • Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio.
  • Biomarkers alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest.
  • the differential presence of a marker of the invention in a subject sample can be useful in characterizing the subject as having or at risk of developing a disease (e.g., an infectious disease), for determining the prognosis of the subject, for evaluating therapeutic efficacy, or for selecting a treatment regimen.
  • a disease e.g., an infectious disease
  • agent any nucleic acid molecule, small molecule chemical compound, antibody, or polypeptide, or fragments thereof.
  • alteration or“change” is meant an increase or decrease. An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.
  • biological sample any tissue, cell, fluid, or other material derived from an organism.
  • capture reagent is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
  • the terms“determining”,“assessing”,“assaying”,“measuring” and“detecting” refer to both quantitative and qualitative determinations, and as such, the term“determining” is used interchangeably herein with“assaying,”“measuring,” and the like. Where a quantitative determination is intended, the phrase“determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase“determining a level” of an analyte or“detecting” an analyte is used.
  • detectable moiety is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron- dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • fragment is meant a portion of a nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.
  • “Homologous” as used herein refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • Hybridization means hydrogen bonding, which may be Watson-Crick,
  • Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases.
  • adenine and thymine are complementary nucleotides that pair through the formation of hydrogen bonds.
  • Identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • an“instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • isolated denotes a degree of separation from original source or surroundings.
  • a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • purified can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • marker profile is meant a characterization of the signal, level, expression or expression level of two or more markers (e.g., polynucleotides).
  • microbe any and all organisms classed within the commonly used term“microbiology,” including but not limited to, bacteria, viruses, fungi and parasites.
  • nucleic acid refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double- stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that specifically binds a target nucleic acid (e.g., a nucleic acid biomarker). Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • “A” refers to adenosine
  • “C” refers to cytosine
  • “G” refers to guanosine
  • “T” refers to thymidine
  • “U” refers to uridine.
  • “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • polypeptide As used herein, the terms“peptide,”“polypeptide,” and“protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
  • polypeptides derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • the level of a target nucleic acid molecule present in a sample may be compared to the level of the target nucleic acid molecule present in a clean or uncontaminated sample.
  • the level of a target nucleic acid molecule present in a sample may be compared to the level of the target nucleic acid molecule present in a corresponding healthy cell or tissue or in a diseased cell or tissue (e.g., a cell or tissue derived from a subject having a disease, disorder, or condition).
  • sample includes a biologic sample such as any tissue, cell, fluid, or other material derived from an organism.
  • nucleic acid probe or primer that recognizes and binds a molecule (e.g., a nucleic acid biomarker), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e -3 and e -100 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Bio
  • substantially microbial hybridization signature is a relative term and means a hybridization signature that indicates the presence of more microbes in a tumor sample than in a reference sample.
  • substantially not a microbial hybridization signature is a relative term and means a hybridization signature that indicates the presence of less microbes in a reference sample than in a tumor sample.
  • subject is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, feline, mouse, or monkey.
  • the term“subject” may refer to an animal, which is the object of treatment, observation, or experiment (e.g., a patient).
  • target nucleic acid molecule is meant a polynucleotide to be analyzed. Such polynucleotide may be a sense or antisense strand of the target sequence.
  • target nucleic acid molecule also refers to amplicons of the original target sequence.
  • the target nucleic acid molecule is one or more nucleic acid biomarkers.
  • A“target site” or“target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • therapeutic means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • treat refers to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • tumor tissue sample any sample from a tumor in a subject including any solid and non-solid tumor in the subject.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the present invention features compositions and methods for the detection or diagnosis of oral squamous cell carcinoma (OSCC) in a tissue sample from a subject.
  • Oral squamous cell carcinoma is meant to include, but is not limited to, oropharyngeal squamous cell carcinoma (OPSCC) and oral cavity squamous cell carcinoma (OCSCC).
  • OPSCC oropharyngeal squamous cell carcinoma
  • OCSCC oral cavity squamous cell carcinoma
  • Metagenomic signatures comprising detecting genetic material from a number of viral, bacterial, fungal, and parasitic microbes were identified that indicate that a subject has oral squamous cell carcinoma.
  • the microbiome is fundamentally one of the most critical organs in the human body. Dysbiosis can result in critical inflammatory responses and can lead to neoplastic events. The dysbiotic oral microbiome and the pathobionts associated with cancers can provide clues as to the major contributors to oral squamous cell carcinomas (OSCCs).
  • OSCCs oral squamous cell carcinomas
  • a pan-pathogen array technology (PathoChip) coupled with next- generation sequencing was used to establish the microbial signatures in human OSCCs. Signatures for DNA and RNA viruses including oncogenic viruses, gram positive and negative bacteria, fungi and parasites were detected. Cluster and topological analyses identified 2 distinct groups of microbial signatures related to OSCCs.
  • PathoChip a pan-pathogen array technology referred to as PathoChip was used to detect metagenomics signatures of oral squamous cell carcinomas (OSCCs) including oropharyngeal squamous cell carcinoma (OPSCC) and oral cavity squamous cell carcinoma (OCSCC).
  • OSCCs oral squamous cell carcinomas
  • OPSCC oropharyngeal squamous cell carcinoma
  • OCSCC oral cavity squamous cell carcinoma
  • PathoChip is comprised of oligonucleotide probes that can detect all sequenced viruses as well as known pathogenic bacteria, fungi and parasites, and family-specific conserved probes, providing a means for detecting previously uncharacterized members of a family (Baldwin et al., (2014) mBio 5, e01714-01714; Banerjee et al., (2015) Sci Rep 5, 15162).
  • OSCC OSCC
  • Results presented herein showed a slight decrease in the abundance of Firmicutes and Actinobacteria in oral cancer samples compared to matched controls, whereas, little or no difference in the abundance of these members was observed when comparing the cancer with the matched and non-matched controls.
  • an increase in the detection of the members of Proteobacteria was observed in the cancers compared to the matched and non-matched controls.
  • 11/13 belong to Proteobacteria.
  • the actinobacteria genus Rothia was detected only in the OSCC cancer samples in the present study.
  • yeasts such as Rhodotorula, Geotrichum and
  • Rhodotorula and Pneumocystis are well-known opportunistic pathogens, and without wishing to be by specific theory, may find the cancer microenvironment amiable for survival. This might also transform harmless commensals to pathogenic oral mucosal micro-organisms, leading to increased morbidity and mortality in cancer patients.
  • Fonsecaea was detected in both OSCC cancer and the adjacent normal matched control tissues, but not from non-matched controls. Without wishing to be bound by any specific theory, this is could be due to the spread of the infection to the adjacent non-cancerous tissues from the tumor site.
  • microsporidia Pleistophora was detected in cancer as well as in controls, the detection being much more significant in the cancer group compared to the controls.
  • Fungi of low pathogenicity like Malassezia and Absidia, along with dermatatious aetiologic agents of chromoblastomycosis Phialophora and Cladophialophora associated significantly with the oral cancer patients as compared to both controls.
  • the fungi that were detected only in the controls and not in the cancer samples were common dermatatious, low pathogenic fungi.
  • Some parasitic worms of the human body, or parasites acquired by ingesting raw fish and meat can also increase the risk of developing certain cancers.
  • molecular signatures of the intestinal parasites, Hymenolepis, Centrocestus and Trichinella were detected in almost all the OSCC samples screened but not in the control samples. Without wishing to be bound by any specific theory, these organisms may find the cancer microenvironment favorable for their growth. Probes of both Hymenolepis, Centrocestus showed very high signals (g-r>3000) when hybridized to the whole genome amplified products of the cancer samples described herein. In the present study, molecular signatures of Toxocara were found in cancer as well as in both the controls, although, the hybridization signal was significantly less in the controls than cancer.
  • results of the present study showed an association of certain viral and microbial signatures with cancer that can be used as microbial biomarkers for oral cancer (Table 3).
  • the microbial signatures that were associated with cancer as well as adjacent matched control tissues also should not be ignored for their potential as microbial biomarkers (Table 3), given there is a possibility of the spread of infection from cancer cells to the adjacent non-cancer cells.
  • the present invention includes a method of detecting oral squamous cell carcinoma in a tumor tissue sample from a subject.
  • the method comprises hybridizing a detectably-labeled nucleic acid from the tumor tissue sample to a PathoChip array to generate a first hybridization pattern, then hybridizing a detectably-labeled nucleic acid from a reference sample to a PathoChip array to generate a second hybridization pattern, wherein the reference sample is from an otherwise identical non-tumor tissue from a subject.
  • the first and second hybridization patterns are compared, wherein when the first hybridization pattern is substantially a microbial hybridization signature and the second hybridization pattern is substantially not a microbial hybridization signature, oral squamous cell carcinoma is detected in the tumor tissue sample.
  • the method comprises wherein the microbial hybridization signature is generated by hybridization of the detectably-labeled nucleic acid from the tumor tissue sample to at least three nucleic acid probes on the PathoChip, wherein the probes are from microbes selected from the group consisting of Human papillomavirus 16 (HPV16), Eshcherichia, Rothia, Peptoniphilus, Brevundimonas, Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus, Veillonella, and Fonsec
  • Another aspect of the invention includes a method wherein the first hybridization pattern is generated by hybridization of the detectably-labeled nucleic acid from the tumor tissue sample to at least three nucleic acid probes on the PathoChip, wherein the probes are selected from the group consisting of SEQ ID NOS: 1-76.
  • the tumor tissue sample can be a biopsy, formalin-fixed, paraffin-embedded (FFPE) sample, or non-solid tumor.
  • the detectably- labeled nucleic acid can be labeled with a fluorophore, radioactive phosphate, biotin, or enzyme and the fluorophore can be Cy3 or Cy5.
  • the methods can also include providing the subject with a treatment for oral squamous cell carcinoma when oral squamous cell carcinoma is detected in the tumor tissue sample from the subject.
  • treatments include, but are not limited to, surgery, chemotherapy, or radiotherapy.
  • compositions and methods of the invention are useful for the identification of a target nucleic acid molecule in a biological sample to be analyzed.
  • Target sequences are amplified from any biological sample that comprises a target nucleic acid molecule.
  • Such samples may comprise fungi, spores, viruses, or cells (e.g., prokaryotes, eukaryotes, including human).
  • Such samples may comprise viral, bacterial, fungal, and parasitic nucleic acid molecules.
  • compositions and methods of the invention detect one or more nucleic acid sequences from one or more pathogenic organisms, including viruses, viroids, bacteria, fungi, helminths, and/or protozoa.
  • a sample is a biological sample, such as a tissue or tumor sample.
  • the level of one or more polynucleotide biomarkers e.g., to detect or identify viruses, viroids, bacteria, fungi, helminths, and/or protozoa
  • the biological sample is a tissue sample that includes a tumor cell, for example, from a biopsy or formalin-fixed, paraffin-embedded (FFPE) sample.
  • FFPE formalin-fixed, paraffin-embedded
  • Exemplary test samples also include body fluids (e.g.
  • a target nucleic acid of a pathogen is amplified by primer
  • oligonucleotides to detect the presence of the nucleic acid sequence of an infectious agent in the sample.
  • nucleic acid sequences may derive from pathogens including fungi, bacteria, viruses and yeast.
  • Target nucleic acid molecules include double-stranded and single- stranded nucleic acid molecules (e.g., DNA, RNA, and other nucleobase polymers known in the art capable of hybridizing with a nucleic acid molecule described herein).
  • primer/template oligonucleotide of the invention include, but are not limited to, double- stranded and single-stranded RNA molecules that comprise a target sequence (e.g., messenger RNA, viral RNA, ribosomal RNA, transfer RNA, microRNA and microRNA precursors, and siRNAs or other RNAs described herein or known in the art).
  • a target sequence e.g., messenger RNA, viral RNA, ribosomal RNA, transfer RNA, microRNA and microRNA precursors, and siRNAs or other RNAs described herein or known in the art.
  • primer/template oligonucleotide of the invention include, but are not limited to, double stranded DNA (e.g., genomic DNA, plasmid DNA, mitochondrial DNA, viral DNA, and synthetic double stranded DNA).
  • Single-stranded DNA target nucleic acid molecules include, for example, viral DNA, cDNA, and synthetic single- stranded DNA, or other types of DNA known in the art.
  • a target sequence for detection is between about 30 and about 300 nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 nucleotides).
  • the target sequence is about 60 nucleotides in length.
  • a target sequence for detection may also have at least about 70, 80, 90, 95, 96, 97, 98, 99, or even 100% identity to a probe sequence. Probe sequences may be longer or shorter than the target sequence.
  • a 60- nucleotide probe may hybridize to at least about 44 nucleotides of a target sequence.
  • a biomarker is a biomolecule (e.g., nucleic acid molecule) that is differentially present in a biological sample.
  • a biomarker is taken from a subject of one phenotypic status (e.g., having oral squamous cell carcinoma) as compared with another phenotypic status (e.g., not having oral squamous cell carcinoma).
  • a biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio.
  • Biomarkers, alone or in combination provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for characterizing a disease (e.g., having triple negative breast cancer).
  • the sets of probes used herein are based on the construction of a metagenome and its use to select probes that identify target nucleic acid molecules associated with an infectious agent.
  • metagenome refers to genetic material from more than one organism, e.g., in an environmental sample. The metagenome is used to select the sets of probes and/or to validate probe sets.
  • the metagenome comprises the sequences or genomes of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000 or more organisms.
  • the nucleic acid sequences of thousands of organisms were linked to generate a metagenome comprising 58 chromosomes.
  • low complexity sequences are masked using mdust (http://doc.bioperl.org/bioperl- run/lib/Bio/Tools/Run/Mdust.html) followed by BLASTN 2.0MP-WashU31
  • N nonspecific nucleotides
  • the invention includes at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • the invention includes a kit comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76, and instructional material for use thereof.
  • the nucleic acid probes can be selected from between about 10 to about 30 microbes and comprise about 3 to about 5 probes per microbe.
  • sample preparation involves extracting a mixture of nucleic acid molecules (e.g., DNA and RNA).
  • sample preparation involves extracting a mixture of nucleic acids from multiple organisms, cell types, infectious agents, or any combination thereof.
  • sample preparation involves the workflow below.
  • microarray e.g., PathoChip
  • microarrays are washed at various stringencies.
  • Microarrays are scanned for detection of
  • Target nucleic acid sequences are optionally amplified before being detected.
  • the term“amplified” defines the process of making multiple copies of the nucleic acid from a single or lower copy number of nucleic acid sequence molecule.
  • the amplification of nucleic acid sequences is carried out in vitro by biochemical processes known to those of skill in the art.
  • the viral sample Prior to or concurrent with identification, the viral sample may be amplified by a variety of mechanisms, some of which may employ PCR.
  • primers for PCR may be designed to amplify regions of the sequence.
  • RNA viruses a first reverse transcriptase step may be used to generate double stranded DNA from the single stranded RNA. See, for example, PCR Technology: Principles and Applications for DNA Amplification (Ed. H.A. Erlich, Freeman Press, NY, N.Y., 1992); PCR
  • LCR ligase chain reaction
  • LCR ligase chain reaction
  • DNA for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)
  • transcription amplification Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315
  • self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and
  • WO90/06995 selective amplification of target polynucleotide sequences
  • CP-PCR consensus sequence primed PCR
  • AP-PCR arbitrarily primed PCR
  • NABSA nucleic acid based sequence amplification
  • Other amplification methods that may be used are described in, US Patent Nos 5,242,794, 5,494,810, 4,988,617 and in US Ser No 09/854,317.
  • the biomarkers of this invention can be detected by any suitable method.
  • the methods described herein can be used individually or in combination for a more accurate detection of the biomarkers.
  • Methods for conducting polynucleotide hybridization assays have been developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3 rd Ed. Cold Spring Harbor, N.Y, 2001); Berger and Kimmel Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).
  • the hybridized nucleic acids are detected by detecting one or more labels attached to, or incorporated within, the sample nucleic acids.
  • the labels may be attached or incorporated by any of a number of means well known to those of skill in the art.
  • the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids.
  • PCR with labeled primers or labeled nucleotides will provide a labeled amplification product.
  • transcription amplification as described above, using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.
  • a labeled nucleotide e.g. fluorescein-labeled UTP and/or CTP
  • PCR amplification products are fragmented and labeled by terminal deoxytransferase and labeled dNTPs.
  • a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed.
  • Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example, nick translation or end-labeling (e.g.
  • label is added to the end of fragments using terminal deoxytransferase.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include, but are not limited to: biotin for staining with labeled streptavidin conjugate; anti-biotin antibodies, magnetic beads (e.g., Dynabeads TM .); fluorescent dyes (e.g., Cy3, Cy5, fluorescein, texas red, rhodamine, green fluorescent protein, and the like); radiolabels (e.g., 3 H, 125 I, 35 S, 4 C, or 32 P); phosphorescent labels; enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA); and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • radiolabels may be detected using photographic film or scintillation counters; fluorescent markers may be detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
  • a sample is analyzed by means of a microarray.
  • the nucleic acid molecules of the invention are useful as hybridizable array elements in a microarray.
  • Microarrays generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached.
  • a capture reagent also called an adsorbent or affinity reagent
  • the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.
  • the array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate.
  • Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins.
  • Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No.5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci.93:10614-10619, 1996), herein incorporated by reference.
  • US Patent Nos 5,800,992 and 6,040,138 describe methods for making arrays of nucleic acid probes that can be used to detect the presence of a nucleic acid containing a specific nucleotide sequence. Methods of forming high- density arrays of nucleic acids, peptides and other polymer sequences with a minimal number of synthetic steps are known.
  • the nucleic acid array can be synthesized on a solid substrate by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling.
  • light-directed chemical coupling and mechanically directed coupling.
  • hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringency See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977);
  • One embodiment of the invention includes a microarray comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • the nucleic acid probes can be selected from about 10 to about 30 microbes and comprise about 3 to about 5 probes per microbe.
  • the microarray comprises at least three nucleic acid probes selected from the group of microbes consisting of Human papillomavirus 16 (HPV16), Eshcherichia, Rothia, Peptoniphilus, Brevundimonas, Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus, Veillonella, and Fonsecaea.
  • the microarray can be a biochip, or on a glass slide, bead, or paper. Detection by Nucleic Acid Biochip
  • a sample is analyzed by means of a nucleic acid biochip (also known as a nucleic acid microarray).
  • a nucleic acid biochip also known as a nucleic acid microarray.
  • oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.).
  • a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
  • nucleic acid molecules useful in the invention include polynucleotides that specifically bind nucleic acid biomarkers to one or more pathogenic organisms, and fragments thereof.
  • a nucleic acid molecule derived from a biological sample may be used to produce a hybridization probe as described herein.
  • the biological samples are generally derived from a patient, e.g., as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell or population of cells isolated from a patient sample. For some applications, cultured cells or other tissue preparations may be used.
  • the mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are well known in the art.
  • the RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the biochip.
  • Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30 ⁇ C, of at least about 37 ⁇ C, or of at least about 42 ⁇ C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 ⁇ C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 37 ⁇ C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA). In other embodiments, hybridization will occur at 42 ⁇ C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 ⁇ C, of at least about 42 ⁇ C, or of at least about 68 ⁇ C.
  • wash steps will occur at 25 ⁇ C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In other embodiments, wash steps will occur at 68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Detection systems for measuring the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences are well known in the art. For example, simultaneous detection is described in Heller et al., Proc. Natl. Acad. Sci.
  • a scanner is used to determine the levels and patterns of fluorescence. Diagnostic assays
  • the present invention provides a number of diagnostic assays that are useful for the identification or characterization of a disease or disorder (e.g., oral squamous cell carcinoma), or a propensity to develop such a condition.
  • oral squamous cell carcinoma is characterized by quantifying the level of one or more biomarkers from one or more pathogenic organisms, including viruses, viroids, bacteria, fungi, helminths, and protozoa. While the examples provided below describe specific methods of detecting levels of these markers, the skilled artisan appreciates that the invention is not limited to such methods.
  • Marker levels are quantifiable by any standard method, such methods include, but are not limited to real-time PCR, Southern blot, PCR, and/or mass spectroscopy.
  • the level of any two or more of the markers described herein defines the marker profile of a disease, disorder, or condition.
  • the level of marker is compared to a reference.
  • the reference is the level of marker present in a control sample obtained from a patient that does not have oral squamous cell carcinoma.
  • the reference is a healthy tissue or cell (i.e., that is negative for oral squamous cell carcinoma).
  • the reference is a baseline level of marker present in a biologic sample derived from a patient prior to, during, or after treatment for oral squamous cell carcinoma.
  • the reference is a standardized curve.
  • the level of any one or more of the markers described herein e.g., a combination of viral, bacterial, fungal, helminth, and/or protozoan biomarkers
  • one or more organisms described herein may be isolated or extracted from a sample using a capture reagent (e.g., an antibody) and/or detected using ELISA.
  • a capture reagent e.g., an antibody
  • reagents for capturing the pathogenic organism include streptavidin bound magnetic beads and biotin labelled probes. Such techniques can be further used to obtain nucleic acids pathogenic organism detection using nucleic acid based probes or for direct sequencing (e.g., MiSeq; Illumina). Kits
  • kits for the detection of a biomarker which is indicative of the presence of one or more biological sequences or agents associated with oral squamous cell carcinoma.
  • the kits may be used for detecting the presence of multiple biological agents associated with triple negative breast cancer.
  • the kits may be used for the diagnosis or detection of oral squamous cell carcinoma.
  • the kit comprises a panel or collection of probes to nucleic acid biomarkers (e.g., PathoChip) delineated herein as specific for detection of oral squamous cell carcinoma.
  • the kit comprises an antibody specific for a pathogenic organism associated with oral squamous cell carcinoma. Such antibodies may be used for ELISA detection or for extraction of a pathogenic organism associated with oral squamous cell carcinoma (e.g., a biotin labelled antibody in conjunction with streptavidin bound magnetic beads).
  • the kit comprises one or more sterile containers which contain the panel of probes, nucleic acid biomarkers, or microarray chip.
  • sterile containers which contain the panel of probes, nucleic acid biomarkers, or microarray chip.
  • Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • the instructions will generally include information about the use of the composition for the detection or diagnosis of triple negative breast cancer.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of oral squamous cell carcinoma or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • kits comprising at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • the kit can include probes from about 10-30 organisms with about 3-5 probes per organism.
  • Another embodiment of the invention is a kit comprising a microarray with at least three nucleic acid probes selected from the group consisting of SEQ ID NOS: 1-76.
  • the kit comprises a microarray comprising at least three nucleic acid probes selected from the group of microbes consisting of Human papillomavirus 16 (HPV16), Eshcherichia, Rothia, Peptoniphilus, Brevundimonas, Comamonas, Alcaligenes, Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Caulobacter, Cardiobacterium, Plesiomonas, Serratia, Edwardsiella, Haemophilus, Frateuria, Rhodotorula, Geotrichum, Pneumocystis, Hymenolepis, Centrocestus, Trichinella, Acinetobacter, Actinobacillus, Veillonella, and Fonsecaea.
  • the kits contain instructional materials for use thereof. Microbial Integrations into Host Genomes
  • NGS data was used to identify sites of integration of the identified pathogens within the host genome. Numerous open reading frames were detected within the host genome in which integration had occurred. Integration hotspots for HPV16 was identified as well as other identified integration sites for a number of viruses, including the JC polyomavirus, as well as other pathogenic and tumorigenic bacteria, fungi and parasites in these OSCC samples. Data shown herein strongly suggest greater molecular intimacy between the host genome and the genetic elements of associated microbial agents in the tumor microenvironment.
  • JC Polyomavirus Large T antigen sequence insertions in the host chromosomes were detected. Without wishing to be bound by specific theory, these insertions may lead to transformations by inducing mutations of the target genes. Also detected herein were VP1, VP2 and VP3 viral genomic sequence insertion sites in multiple regions
  • Some of the host genes that had microbial genomic insertions supported by high sequence reads showed significant association with neoplasia of epithelial tissue, and thus these microbial insertions at or near cancer associated genes can be a contributing factor for OSCC development.
  • the PathoChip Array design has been previously described (Baldwin et al., (2014) mBio 5, e01714-01714; Banerjee et al., (2015) Sci Rep 5, 15162). Briefly, the array was generated from a metagenome of 58 chromosomes in silico. It is comprised of 60,000 probe sets of sequenced microorganisms from Genbank, which are manufactured as SurePrint glass slide microarrays (Agilent Technologies Inc.), containing 8 replicate arrays per slide (Baldwin et al., (2014) mBio 5, e01714-01714). Each probe is a 60-nt DNA oligomer that targets multiple genomic regions of pathogenic viruses, prokaryotic, and eukaryotic microorganisms.
  • PathoChip screening utilized both DNA and RNA extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues.100 de-identified FFPE oral squamous cell carcinoma (OSCC) samples were received as 10 ⁇ m sections on non-charged glass slides, and 20 each of matched and non-matched control samples were provided as paraffin rolls. Matched controls were obtained from the adjacent non-cancerous oral tissue of the same patient from which the cancer tissues were obtained, while non-matched controls were oral tissues obtained from otherwise healthy individuals. DNA and RNA were extracted in parallel from rolls or mounted sections of each FFPE sample. The quality of extracted nucleic acids was determined by agarose gel electrophoresis and the A 260/280 ratio.
  • OSCC formalin-fixed paraffin-embedded
  • RNA and DNA samples were subjected to whole transcriptome amplification (WTA) using 50 ng each of RNA and DNA as input.
  • WTA whole transcriptome amplification
  • a total of 60 arrays were used to screen the 100 OSCC samples, with 48 individual and the rest pooled in groups of 4-5 samples.
  • the 20 matched and 20 non-matched control samples were pooled for screening using 4 arrays for each se of controls.
  • the WTA products were analyzed by agarose gel electrophoresis and showed a range of 200-400bp amplicon sizes.
  • Human reference RNA and DNA were also extracted from the human B cell line, BJAB and 15ng of each were used for WTA.
  • the WTA products were purified, (PCR purification kit, Qiagen, Germantown, MD, USA), and 2 ⁇ g of the amplified products from the OSCC tissues was labelled with Cy3 and that from the human reference was labelled with Cy5 (SureTag labeling kit, Agilent Technologies, Santa Clara, CA). Human reference DNA and RNA was used to determine cross-hybridization of probes to human DNA.
  • the labelled DNAs were purified and the efficiencies of labeling were determined by measuring absorbance at 550nm (for Cy3) and 650nm (for Cy5).
  • the labelled samples (Cy3 plus Cy5) were hybridized to the PathoChip as described previously (Baldwin et al., (2014) mBio 5, e01714-01714; Banerjee et al., (2015) Sci Rep 5, 15162).
  • the hybridization cocktail (CGH blocking agent and hybridization buffer), was added to each of the labeled test sample (Cy3) mixed with reference (Cy5), denatured and hybridized to the arrays in 8- chamber gasket slides. The slides were incubated at 65°C with rotation and washed, then scanned for visualization using an Agilent SureScan G4900DA array scanner.
  • Raw data from the microarray images were extracted using Agilent Feature Extraction software; normalization and data analyses were done in the Partek Genomics Suite (Partek Inc., St. Louis, MO, USA).
  • Model-based analysis of tiling arrays (MAT) which utilized a sliding window to scroll through the entire metagenome of the array to detect positive hybridization signal, was used to detect positive regions in the metagenome for each tumor.
  • Analysis at the individual probe level both for specific and conserved probes), and at the accession level (taking into account all the probes per accession), were performed as previously described (Baldwin et al., (2014) mBio 5, e01714-01714; Banerjee et al., (2015) Sci Rep 5, 15162).
  • Probes of the microorganisms were detected in the samples by both outlier analyses (detecting probes in few samples) and paired t-tests with False Discovery Rate (FDR) multiple correction (detecting probes of significance in the majority of the tumor samples analyzed).
  • FDR False Discovery Rate
  • One sided t-tests were performed to determine if cancer samples have significant detection of the candidate signature of organisms compared to the control (both matched and non-matched) samples.
  • the cancer samples were also subjected to hierarchical clustering, based on the detection of microbial signatures in the samples, using the R program (Euclidean distance, complete linkage, non-adjusted values), and NBClust software [CH (Calinski and Harabasz) index, Euclidean distance, complete linkage] (Charrad et al., (2014) Journal of Statistical Software 61, 1-36).
  • the significant differences between the clusters observed by these methods were determined using t-test.
  • Additional topological-based data analyses were conducted using the Ayasdi software (Ayasdi, Inc.), (Correlation metric, and L-infinity centrality lenses) where statistical significance between different groups was determined using two-sided t- test.
  • WTA products of the oral cancer samples were pooled together for hybridization with selected biotinylated probes that were identified for microbial signatures in the oral cancer samples by the PathoChip screen.
  • the targeted sequences were then captured by Streptavidin coated magnetic beads and libraries were generated for NGS.
  • the selected probes were synthesized as 5′-biotinylated DNA oligomers (Integrated DNA
  • Capture probe pool 1 contained 19 selected probes associated with bacteria (B capture), pool 2 contained 12 selected probes associated with the fungi (F capture), pool 3 contained 14 selected probes associated with parasitic signatures (P capture), pool 4 contains 36 other probes associated with viral and some bacterial signatures (O capture), pool 5 contains 6 HPV16 probes (HPV16 capture) (Table 1).
  • Each of the 5 capture probe pools was added separately to the pooled WTA of the oral cancer samples (150ng/ul) in 5 separate reaction mixtures containing 3 M tetra-methyl ammonium chloride, 0.1% Sarkosyl, 50 mMTris- HCl, 4 mM EDTA, pH 8.0 (1XTMAC buffer).5 target capture reactions were done (Table 1). The reaction mixtures were denatured (1000C for 10 mins) followed by a hybridization step (600C for 3 hours).
  • Streptavidin Dynabeads (Life Technologies, Carlsbad, CA, USA) were added with continuous mixing at room temperature for 2 hours, followed by three washes of the captured bead-probe-target complexes in 0.30 M NaCl plus 0.030 M sodium citrate buffer (2XSSC) and three washes with 0.1 ⁇ SSC.
  • Captured single-stranded target DNA was eluted in Tris-EDTA and used for library preparation using Nextera XT sample preparation kit (Illumina, San Diego, CA, USA) followed by NGS.
  • the 5 libraries were examined for quality control and submitted for NGS using an Illumina MiSeq instrument with paired-end 250-nt reads. Adapters and low-quality fragments of raw reads were first removed using the Trim Galore software
  • Virus-Clip (Ho et al. (2015) Oncotarget 6, 20959-20963) was used to identify the virus fusion sites in the human genome. Specifically, the virus genome was used as the primary read alignment target, and first aligned reads to the PathoChip genome. Some mapped reads may contain soft-clipped segments. Soft-clipped reads were then extracted from the alignment and mapped (containing sequences of potential pathogen-integrated human loci) to the human genome. Utilizing this mapping information, the exact human and pathogen integration breakpoints at single-base resolution can be identified. All the integration sites were then automatically annotated with the affected human genes and their corresponding gene regions.
  • IPA Ingenuity Pathway Analysis
  • Example 1 Microbial signatures detected in OSCCs
  • the PathoChip technology was used to screen 100 FFPE pathologically defined OSCC patient samples as well as 20 matched and 20 non-matched oral tissue control samples for distinct viral and microbial signatures associated with the tumor tissue.
  • Samples analyzed in this study were carcinomas taken from tongue, base of tongue, tonsil, floor of mouth, cheek and predominantly oropharynx which are collectively referred to herein as OSCC (Table 2).
  • OSCC carcinomas taken from tongue, base of tongue, tonsil, floor of mouth, cheek and predominantly oropharynx which are collectively referred to herein as OSCC (Table 2).
  • OSCC carcinomas taken from tongue, base of tongue, tonsil, floor of mouth, cheek and predominantly oropharynx which are collectively referred to herein as OSCC (Table 2).
  • OSCC carcinomas taken from tongue, base of tongue, tonsil, floor of mouth, cheek and predominantly oropharynx which are collectively referred to herein as OSCC (Table 2).
  • OSCC DNA and RNA were extracted from the samples, subjecte
  • RNA and DNA viruses associated with the cancer and control samples were identified (FIGs.1A-1F, Table 3). Viral sequences belonging to Papillomaviridae showed the highest hybridization signal in the OSCC samples screened, followed by that of Herpesviridae, Poxviridae, Retroviridae and Polyomaviridae (FIG.1A). Viral signatures belonging to these families were seen to be >75% prevalent among the 100 OSCC samples screened. Interestingly, Papillomaviridae was detected in 98% of the cases (FIG.1A).
  • FIG.1F shows the detection of almost all of the HPV16 specific probes in the PathoChip across the majority of the OSCC samples with medium to high hybridization signal, while the HPV16 probes were detected with significantly lower hybridization signals in both matched and non-matched controls (FIGs.1B-1C). Signatures of Reoviridae,
  • Herpesviridae, Poxviridae, Orthomyxoviridae, Retroviridae and Polyomaviridae were detected in OSCC samples with high prevalence and at hybridization signals that were 2- 3 logs higher than in controls (FIG.1A & Table 4). Notably, viral signatures of
  • Coronoviridae, Picornaviridae, Adenoviridae, Anelloviridae, Hepadnaviridae and Flaviviridae were significantly detected in the controls along with signatures of non- HPV16 papillomaviridae (FIGs.1B-1C). These data show that viral signature is significantly changed when compared specifically to the OSCC tissue. Table 3. Microbial signatures detected in OSCC and control samples. The putative microbial biomarkers are in bold.
  • FIGs.2A-2F and Table 3 show the variety of bacterial signatures found in OSCC, matched and non-matched control samples. These include Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Fusobacteria. There were differences in gram-positive and gram-negative microbiota in OSCCs compared to control samples. In the non-matched controls about 55% of the organisms were gram-negative compared to 40% in the matched controls and 49% in the OSCC samples.43%, 50% and 36% of the bacterial agents were gram-positive in the OSCCs, matched and non-matched controls, respectively (FIG.2A).
  • Proteobacteria one of the major gram negative phylum (includes Esherichia, Vibrio and Salmonella) was much more pronounced in OSCCs at 41% compared to matched and non-matched control at 25% and 18%, respectively (FIG.2A).
  • the Bacteroides were more pronounced in the non-matched controls at 27% compared to 4% and 5% in the OSCC and matched controls, respectively (FIG.2A).
  • the gram-positive phylum Actinobacteria were similar across all samples at 31%, 30% and 36% (FIG.2A).
  • the Firmicutes phylum of gram-positive bacteria was more pronounced in the matched controls at 35% compared to 24% and 18% in OSCCs and non-matched controls, respectively (FIG.2A).
  • Proteobacteria that were detected in the OSCC samples were the genera of Escherichia, Brevundimonas, Aeromonas, Bordetella, Comamonas, Alcaligenes, Caulobacter, Acinetobacter, Citrobacter, Sphingomonas, Plesiomonas, Actinobacillus, Serratia, Edwardsiella, Haemophilus, Frateuria and Cardiobacterium.
  • Proteobacteria generas detected in cancer cases showed low to moderate hybridization signals, but interestingly, they were highly prevalent (>75%), except for the generas Serratia, Plesiomonas, Edwardsiella, Citrobacter (46-62%) (FIGs.2B-2E).
  • the matched control samples shared some of the bacterial signatures that were detected in the cancer samples along with other bacterial signatures of normal oral flora (FIGs.2B-2D).
  • Table 3 shows the list of bacterial genera detected and shared among the cancer, matched and non-matched control samples.
  • Bacterial signatures of the genera Actinomyces were detected with the highest prevalence (100%) and hybridization signal intensity in the matched controls (FIGs.2B-2D).8 of the 14 bacterial genera detected in matched controls were also detected in the OSCC samples (Table 3). They represented the genera of Arcanobaterium, Actinomyces, Aeromonas, Bordetella, Aerococcus, Pediococcus, Acinetobacter, and Veillonella (Table 3).
  • the Venn diagram (FIG.2F) summarizes findings showing that bacterial signatures representing 13 genera are found to be specifically associated with OSCC samples and not with the matched or non-matched controls. These are the Proteobacteria including Escherichia, Brevundimonas, Comamonas, Alcaligenes, Caulobacter,
  • the bacterial microbial signatures showed a significant divergence in the OSCC when compared to the normal signatures and were more robust.
  • Table 4 Significant detection of the probes of micro-organisms in cancer compared to the matched (MC) and non-matched control (NC) samples. Weighted score sum of the hybridization signals of all the probes of an organism was calculated in cancer and controls, and significance (p- value ⁇ 0.05) was calculated using one sided t-tests.
  • the matched controls detected some of the common oral flora along with some fungal signatures that were detected in the cancer samples. All the matched control samples significantly detected probes of Phialophora, Cladosporium, Fonsecaea, Alternaria and Cladophialophora (FIG.3B). Except for the probes of Alternaria, all others mentioned above were detected in the matched control samples with high hybridization signal intensity (FIG.3B). Probes of Absidia were detected with low hybridization signal intensity in 75% of the matched control samples screened.
  • the Venn diagram shows that three fungal signatures. Rhodotorula, Geotrichum and Pneumocystis, were associated only with OSCCs (Table 3, FIG.3E). Again, a significant change in the fungal biome of OSCC was observed when compared to control oral samples. iv.) Parasitic signatures associated with OSCC
  • Distinct molecular signatures for parasites were detected in OSCCs (FIGs.3F and 3I, Table 4). Probes from 28S and/or 18S rRNA of Hymenolepis, Centrocestus and Prosthodendrium were detected in all the OSCC samples with very high hybridization signal (FIGs.3F and 3I, Table 4). Probes of Contracaecum, Dipylidium, Trichinella and Toxocara were detected in >95% of the cancer samples with moderate hybridization signal intensity (FIGs.3F and 3I, Table 4).
  • FIG.3J The Venn diagram in FIG.3J summarizes the findings of parasitic signature associations with cancer and control samples. Molecular signatures of Hymenolepis, Centrocestus and Trichinella were found to be associated only with OSCC and not with the controls. Signatures of Echinococcus was found to be associated only with matched control samples and that of Anisakis and Echinostoma was found to be associated only with non-matched control samples. Thus distinct signatures differentiate cancer, matched controls and non-matched controls.
  • Example 2 Hierarchial clustering of OSCC samples based on detection of microbial signatures
  • Hierarchial clustering was done based on the detection of the microbial signatures in the 100 OSCC samples. Signature of Cladosporium was ignored as it was not significantly detected in the cancer samples compared to the controls. Using hierarchial clustering analysis R program, the OSCC samples fell into 2 major groups (A and B) based on specific microbiome (FIG.4A). Molecular signatures for HPV16 were detected in the 2 major groups identified (FIG.4A). Apart from HPV16 probes, group A OSCC samples also showed signatures of other viral probes, primarily belonging to
  • Each of the sub-group is again sub-clustered based on the detection of certain probes belonging to Retroviridae, Poxviridae and Polyomaviridae.
  • group B some samples (Sub-group B1) had a lower level of detection of the molecular signatures compared to the majority of group B samples (Sub-group B2).
  • Samples in Sub-group B2 are further clustered based on the low detection of bacterial signatures in some of them.
  • Clustering of the OSCC samples was visualized using NBClust software (FIG. 4B). Two distinct clusters, clusters 1 and 2, were observed similar to the one described above. While there were no significant differences between the two clusters for the signatures of HPV 6b, HPV 16, HPV 26, HHV 8, HHV 6B, HHV 5, retroviral signatures, certain pox viral signatures, parapox viral signatures and polyoma viral signatures, there were significant differences in the detection of some of the viral and all the bacterial, fungal and parasitic signatures between the two clusters, cluster 1 having higher detection than 2.
  • viral signatures were signatures of Orthomyxoviridae, Reoviridae, HPV 34, HHV 6A, Mouse mammary tumor virus-like (MMTV-like) and certain poxvirus that were detected significantly higher in cluster 1 than in cluster 2.
  • Additional analyses using a topological approach represented data by grouping cases with similar detection for viral and microbial signatures into nodes, and connecting those nodes by an edge if the corresponding node have detection pattern in common to the first node (FIG.4C).
  • Topological analysis visualized all the OSCC cases into two clusters,‘Group a’ and‘Group b’, along with certain cases that did not have common detection pattern (ungrouped or singletons) (FIG.4C).
  • the clusters were characterized by detection of microbial signature patterns that may provide clues as to the stage of the disease.
  • the nodes were colored based on HPV16 detection.
  • the two major groups a and b showed significant differences in detection of certain micro-organisms between them, comprising significant higher detection of certain bacterial signatures (Actinobacillus, Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Alcaligenes, Arcanobacterium, Bordetella, Brevundimonas, Cardiobacterium, Citrobacter, Comamonas, Edwardsiella, Escherichia, Frateuria, Haemophilus, Mobiluncus, Mycobacterium, Pediococcus, Peptoniphilus,Peptostreptococcus, Plesiomonas, Prevotella, Propionibacterium, Rothia, Serratia, Sphingobacterium, Sphingomonas, Streptococcus, Veillonella), parasitic signatures of Trichinella, Contracaecum, Prosthodendrium and Toxocara, fungal signature of Pleistophora, Malassezia
  • Rhodotorula, Pneumocystis and Phialophora and viral signatures of Orthomyxoviridae and Reoviridae in‘Group b’ than‘Group a’.
  • significantly higher detection of HPV16 was found in‘Group a’ compared to‘Group b’.
  • the samples within‘Group b’ ranged from having no to very high HPV16 signals.
  • the ungrouped samples had significantly lower detection of the majority of bacterial signatures along with fungal signatures of Malassezia, Geotrichum, Pneumocystis, Fonsecaea, Absidia, Cladophialophora, Phialophora, Rhodotorula, and Pleistophora, viral signatures of Reoviridae,
  • Orthomyxoviridae Herpesviridae, Retroviridae ( MMTV-like), Poxviridae,
  • Polyomavirus were used to enrich genomic regions of late mRNA and VP2/VP3 and VP1 of the virus, respectively from the cancer samples. Importantly, the captured sequences were found to align at the capture probe regions as expected (FIGs.5A-5D).
  • Capture probe designed from 16S rRNA region of the bacteria Rothia captured most of the genomic sequence of the bacteria. Thus the sequence reads aligned not only with the capture probe region, but also extended across the genome of the bacteria (FIGs.5A-5D). Other bacterial sequence reads aligned with their respective capture probe regions, further validating the PathoChip screen results (FIGs.7C-7D).
  • Sequence reads of fungi were also found to align with sequences at or adjacent to their respective capture probe regions (FIGs.5A-5D, and FIG.7E). For example, 1432 sequence reads of Pneumocystis, aligned at the capture probe location in their genome as well as outside of it (FIGs.5A-5D). 2057 sequence reads of Pleistophora, aligned at the capture probe location of its genome (FIG. 7E). High sequence reads (>4000) were obtained for the skin fungus Malassezia (FIG. 7E).
  • FIG.6B represents the data in a Circos plot highlighting the insertions.
  • the number of viral insertions were lower compared to the other microbial insertions, the 79 insertional sites for JC and HPV16 were represented on the Circos plot.
  • the Circos plot shows insertions going from the inner concentric circle to outer circle in the order of fungus, JC Polyomavirus, HPV16, parasites and bacteria. This is then comprehensively shown with its represented colors in the outermost circle with all insertions (FIG.6B).
  • a karyotype plot also shows the representative bacterial and fungal, parasitic and viral insertional sites in each chromosome (FIGs.6C-6D).
  • the sites with >20 reads for bacterial, fungal and parasitic genomic insertions were considered, and all the viral integration sites were included.
  • Bacterial insertions are shown for all chromosomes in FIG.6C.
  • the number of insertions for each chromosomes are shown to the left of each chromosome number.
  • chromosomes 1, 2, 3, 6 and 8 showed over 50 insertions each, and the Y chromosome having the least insertions (FIG.6C).
  • the mitochondrial chromosome also showed 4 insertions in this analysis (FIG. 6C).
  • Genomic elements of HPV16 and JC Polyomavirus were found to be integrated in the human chromosomes of OSCC cells.
  • 7 insertion sites were detected in chromosome 17 (chr17), 6 in chromosome 5, and between 1 and 4 in other the chromosomes except for chromosomess 13, X and Y (FIG.6A).
  • the genomic fragment of HPV16 that was identified most frequently integrated in the human genome was at the genomic co-ordinates 4,172 (based on accession NC_001526.2), which is located around the polyA sequence of E5 gene.
  • HPV16 integrations included HPV genomic co-ordinates 3,393-3,425 in coding sequence of the E1 gene; 10% at co-ordinates 7,206-7,627 near the polyA sequence of the L1 gene), 9% in the coding region of L1 from co-ordinates 6,030-6,715; as well as lower percentage integrations in the coding sequences of the E4 gene (3,358-3,394), the E2 gene (3,393-3,425), the E7 gene (674-693) and the L2 gene (5,201-5,221) (FIG.6A).
  • JC polyoma (JC) viral genomic integration was observed in human chromosomes 1, 2, 5, 6, 10, 13, 15, 16, 17, 19, X and Y (FIG.6A).
  • the JC viral co-ordinates (accession NC_001699), that were integrated in the host chromosomes were mostly at the region of large T antigen (co-ordinates 2,623-2,653) (Frisque et al., (1984) J Virol 51, 458-469) that accounted for 62% of the JC polyomaviral genomic insertions detected in the study.
  • Viral integrations were detected at many genomic regions. Examples of these insertions are represented in FIG.6E, FIG.8, and FIGs.9A-9B. Viral integration sites were mostly intronic, followed by intergenic sites for integration. Viral genomic integrations were also observed upstream or downstream and at 3’UTR of genes as well as at the ncRNA intronic regions (FIGs.6A-6F, FIG.8).
  • HPV16 genomic hotspots for integrations at co-ordinates 4,188-4,243 (Seedorf et al., (1985) Virology 145, 181-185) detected in this study was integrated mostly at the intronic regions (53%) of genes LAMA3, ATXN10, INADL, ABCA10, EVC2, WDR89, CADPS2, HAUS6, EPHA6, FAM179B, COL14A1, MRPS27, FUCA2, ADAMTS12, TRIOBP, CSMD1, KCNQ1, and at the intronic ncRNA gene (6%) of the FAM35BP gene (FIG.6E and FIG.8).
  • LOC102724957 while 2 other integration sites for E4, and 1 site for E7 fragments were intergenic (FIG.8).
  • Human genomic integration sites for L1 fragments were also detected at intronic regions of genes PAFAH1B1 (2/5) and ncRNA LOC100506207 (2/5), and an intergenic region (1/5).
  • L2 fragments were found integrated within the intronic region of gene SSH2.
  • the genomic DNA located at the PolyA sequence of HPV16 L1 were integrated at the intronic regions of gene DEPDC4, and within the intergenic regions and at the 3’UTR region of the MKLN1 gene.
  • the JC Polyoma virus integration sites for large T antigenic regions were found to be within the intronic regions of host genes CMTR1 and ME1 on chromosome 6; gene CPO of chromosome 2, and within intergenic regions of chromosomes 1, 2 and 3.
  • Elements of the VP1 ORF were also found to be integrated in the intergenic regions of human chromosome 10, about 41Kb downstream of the lncRNA gene SFTA1P which is known to be upregulated in carcinoma (Zhao et al., (2014) Sci Rep 4, 6591.) (FIG.8). It was also seen integrated upstream of the ABCA9 gene in chromosome 17, known to be associated with melanoma (Hedditch et al., (2014) J Natl Cancer Inst 106(7). pii, dju149), and in the 3’UTR of the epigenetic regulator gene MECP2 located in chromosome X ( Figure 6E).
  • Genomic elements of VP2 and VP3 integration sites in the intronic region of the FAM13B gene on chromosome 5 and the PCCA gene on chromosome 13, respectively were also detected.
  • Agnoprotein Jvgp1 DNA elements were detected in the intronic regions of the MSH3 gene on chromosome 5, and the PHLSB3 gene on chr19.
  • Late mRNA transcripts 191-253 (NC_001699.1) integration sites were seen at the intergenic regions of chromosome 16, 97Kb downstream of the NPIPA7 gene, also at 99Kb upstream of the NPIPA5 gene, and at intronic region of the SSG5 gene on chromosome 15.
  • FIGs.9A-9B show the various integration sites for JCV in the human genome, again these insertions could affect gene expression in ways that would promote oncogenesis.
  • FIGs.6A-6F Numerous insertional sites were observed for bacterial genomic fragments in exonic, intronic, intergenic, 3’ and 5’ UTR region, upstream and downstream regions of numerous genes of human chromosomes.
  • FIGs.9A-9B Several particularly interesting inserts within human gene related to cancer are shown in FIGs.9A-9B. For example, detected were:
  • NC_008595.1 genomic elements 24065-24105 insertions at the exonic regions of the tumor suppressor ADAMTSL1 gene on chromosome 9; Aeromonas (NC_008570.1) genomic elements insertion sites in the exon of the RASSF5 (1q32.1), a member of Ras association domain family that functions as a tumor suppressor and shown to be inactivated in a variety of cancers (van der Weyden and Adams, (2007) Biochim Biophys Acta 1776, 58-85); Sphingomonas (NC_009511.1) genomic elements insertions in exonic regions of chromatin re-modelling gene SRCAP in chromosome 16; Bordetella (NC_002929.2) genomic insertional site within the exon of the proto-oncogene WNT3 on chromosome 17 (FIGs.6A-6F); Escherichia coli (NC_013008.1) genomic insertional site at the end of the SMURF2 gene, a
  • Genomic fragments of the fungal OSCC flora were also detected at 125 insertion sites in the intergenic (46%), intronic (42%), upstream or downstream of genes or, ncRNA but not exonic regions in the human chromosomes (FIGs.6B-6E).25 insertional sites were observed for the genomic fragments of Malassezia in the host genome, 24 each for Rhodotorula and Pleistophora, 19 for Absidia, 15 for Geotrichum, 7 for
  • Sequences of a parasite were found to have multiple insertional sites within the host chromosomes. A large number of sequence reads were obtained for the 28S rRNA genomic fragment of Prosthodendrium (AF151921.1)-human genomic fusion at the intergenic region of chromosome 8, 37Kb upstream of the proto-oncogene Lyn. Trichinella (AY851263.1) sequence insertion sites were detected on chromosome 17 at the intronic region of the AKAP1 gene which is known to be associated with epithelial cancers (Sotgia et al., (2012) Cell Cycle 11, 4390- 4401) and also within the intergenic region of chromosome 10, 353KB upstream of the NRG3 gene.
  • ANKRD30BL Diphyllobothrium sequences at the ncRNA ANKRD30BL gene and in the intergenic region of chromosome 9 about 106Kb upstream of the TRIM49B gene. Mutations in ANKRD30BL are known to be associated with cancer (Weinhold et al., (2014) Nat Genet 46, 1160-1165); as well as that of TRIM49B, one of the RING type E3 ubiquitin ligase involved in deregulation of tumor suppressors (Hatakeyama, (2011) Nat Rev Cancer 11, 792-804).
  • Echinococcus (EGU27015) sequence insertion sites were observed at the intronic region of the chromatin re-modelling gene ATRX on the X chromosome, and mutation of which was shown to be associated with cancer (Lovejoy et al., (2012) PLoS Genet 8, e1002772). Besides the higher number of reads for parasite-host fusion regions, there were also lower reads for a number of other parasitic insertions. Nevertheless, the insertion sites are important as they might contribute to cancer.
  • NC_009460.1 DNA elements detected 21Kbp upstream of tumor suppressor FGFR2 gene (FIG.8), and mutation or abnormal expression is known to lead to cancer development.
  • Strongyloides (NC_005143.1) 28s rRNA genomic fragment insertion sites were noted on chromosome 17 and at the intronic region of the tumor suppressor SPECC1 gene (FIG.6E).
  • FIGs.9A-9B highlights some of the integrations that may affect human genes involved in cancer.

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Abstract

La présente invention concerne des compositions et des méthodes pour la détection d'un carcinome à cellules squameuses de la cavité buccale. L'invention concerne également des compositions et des méthodes qui permettent de détecter une signature métagénomique dans un échantillon de tissu d'un sujet qui indique que le sujet est atteint d'un carcinome à cellules squameuses de la cavité buccale.
PCT/US2017/045898 2016-08-11 2017-08-08 Compositions et méthodes de détection de carcinomes à cellules squameuses de la cavité buccale WO2018031545A1 (fr)

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US201662494527P 2016-08-11 2016-08-11
US62/494,527 2016-08-11
US201762602529P 2017-04-26 2017-04-26
US62/602,529 2017-04-26

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WO2020093040A1 (fr) * 2018-11-02 2020-05-07 The Regents Of The University Of California Procédés de diagnostic et de traitement du cancer à l'aide d'acides nucléiques non humains
CN111118187A (zh) * 2020-02-25 2020-05-08 福建医科大学 一种检测食管鳞癌癌组织与癌旁组织差异菌群的引物组、试剂盒和检测方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020093040A1 (fr) * 2018-11-02 2020-05-07 The Regents Of The University Of California Procédés de diagnostic et de traitement du cancer à l'aide d'acides nucléiques non humains
EP3874068A4 (fr) * 2018-11-02 2022-08-17 The Regents of the University of California Procédés de diagnostic et de traitement du cancer à l'aide d'acides nucléiques non humains
CN111118187A (zh) * 2020-02-25 2020-05-08 福建医科大学 一种检测食管鳞癌癌组织与癌旁组织差异菌群的引物组、试剂盒和检测方法

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