WO2008076077A1 - Méthodes de diagnostic précoce de cancers associés au virus d'epstein-barr, et réactifs et trousses associés - Google Patents

Méthodes de diagnostic précoce de cancers associés au virus d'epstein-barr, et réactifs et trousses associés Download PDF

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WO2008076077A1
WO2008076077A1 PCT/SG2006/000401 SG2006000401W WO2008076077A1 WO 2008076077 A1 WO2008076077 A1 WO 2008076077A1 SG 2006000401 W SG2006000401 W SG 2006000401W WO 2008076077 A1 WO2008076077 A1 WO 2008076077A1
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gene
seq
determining
expression level
set forth
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Li-Fu Hu
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Innogene Kalbiotech Pte Ltd
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Priority to CN200680056919A priority patent/CN101617056A/zh
Priority to TW096147160A priority patent/TW200839015A/zh
Publication of WO2008076077A1 publication Critical patent/WO2008076077A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/05—Epstein-Barr virus

Definitions

  • the invention relates to methods of diagnosing cancer associated with Epstein- Barr virus infection in subjects.
  • the invention utilizes the determination of the gene load and the gene expression profiling of certain molecular biomarkers related to Epstein-Barr virus related cancers.
  • the invention further relates to reagents and kits for performing methods of the invention.
  • Epstein-Barr virus is a lymphotropic human Herpes virus that establishes lifelong latency. In childhood an EBV infection often is asymptomatic. However, in adolescents and adults a primary EBV infection causes the self-limiting lymphoproliferative disorder, infectious mononucleosis (IM). After the primary EBV infection the virus persists in the organism and is lifelong excreted via the saliva. Li addition, the virus is latently present in about 1 in 10,000 human periphery B cells. In rare cases, an EBV infection can lead to the so-called X-linked lymphoproliferative syndrome (XLP -syndrome) or Duncan syndrome, which is correlated with a gene defect on the X chromosome.
  • XLP -syndrome X-linked lymphoproliferative syndrome
  • Duncan syndrome which is correlated with a gene defect on the X chromosome.
  • the EBV is prevalent worldwide and about 95% of the adult population is seropositive for EBV.
  • the B-cells in which the virus latently persists have the capability to develop into immortalized cells in cell culture, whereas the unregulated proliferation of these cells is normally suppressed by a functional cellular immunity in vivo.
  • the EBV-infected B cells may proliferate uncontrolled and thus cause a B cell lymphoma.
  • the virus Due to this property to stimulate cell proliferation and immortalize an infected cell, the virus is strongly associated with a number of human malignant disorders, characterized by high titers of anti-EBV antibody profiles and an increased circulating EBV DNA level.
  • the EBV is very strongly associated with Burkitt lymphoma, nasopharyngeal carcinoma (NPC) and salivary gland cancer and to a lower extent also associated to Hodgkin lymphomas, T-cell lymphomas and gastric adenocarcinomas.
  • NPC nasopharyngeal carcinoma
  • salivary gland cancer to a lower extent also associated to Hodgkin lymphomas, T-cell lymphomas and gastric adenocarcinomas.
  • the specific role of EBV in the etiology of the above malignancies has not been completely elucidated yet.
  • NPC Nasopharyngeal carcinoma
  • Exemplary etiology factors may include the life style, such as the consumption of nitrosamines in salt fish and certain herbal medicines, and genetic factors, including inheritable, i.e. somatic, and epigenetic changes.
  • NPC is the most common ear-nose-throat (ENT) tumor with high prevalence among native populations and a yearly overall incidence estimated at 6.2 per 100.000 inhabitants. Extremely high incidence was recently documented in native populations living on the island of Sulawesi.
  • Yogyakarta central Java, NPC is the most prevalent tumor among man and 4 th most prevalent among females, with a male/female ration of 2.4, constituting respectively 22 and 8% of all diagnosed malignancies.
  • the Burkitt lymphoma has a high endemic incidence in about seven to nine year old children in equatorial Africa and New Guinea. About 98% of these endemic Burkitt lymphomas show Epstein-Barr viruses in the transformed cells, hi the sporadic form of the Burkitt lymphoma, which is found predominantly in adults, about 25% of the cases are associated with the EBV.
  • EBNAl has been found to be a DNA binding protein important for the long-term maintenance of the viral genome in the cell nucleus.
  • the tumorigenesis is thought to be a multi-step process so that even in cases where EBV seems to be an essential factor in the events leading to malignancy, it is clear that the virus-related tumor development and progression is a multistep process also involving secondary cellular events.
  • both genes gene and epigenetic
  • environmental factors including EBV
  • Epigenetic change happens to the modifications of genetic material instead of the genetic material itself. Epigenetic changes are also inheritable via the cell division, and play an important role in many physiological and pathophysiological conditions. Research has shown that epigenetic mechanisms provide an "extra" layer of transcriptional control that regulates how genes are expressed. These mechanisms are critical components in the normal development and growth of cells. Epigenetic abnormalities have been found to be causative factors in cancer, genetic disorders and pediatric syndromes as well as contributing factors in autoimmune diseases and aging.
  • promoter hypermethylation has been the most detailed studied and could be a main mechanism for the inactivation of tumor suppressor genes in cancer.
  • Cytosine DNA methylation is a covalent modification of DNA, in which a methyl group is transferred from S-adenosylmethionine to the C-5 position of cytosine by a family of cytosine (DNA-5)-methyltransferases. DNA methylation occurs almost exclusively at CpG nucleotides and has an important contributing role in the regulation of gene expression and the silencing of repeat elements in the genome.
  • NPC patients have higher anti-EBV EA/VCA IgA antibody titers than healthy EBV carriers and elevated antibody titers can be found prior to onset of clinical symptoms in NPC patients with primary or recurrent carcinoma, and this method has been used for risk prediction for the population at high-incidence area, the low detection rate and high rate of false positive results make this method disadvantageous.
  • EBV DNA value may be useful for prognostic monitoring, but the EBV DNA level in the blood is even in patients with NPC low or negative due to tumor apoptosis or necrosis.
  • EBV is essential for the pathogenesis of EBV-related cancers, it is not sufficient, because the initiation of tumors involves multiple other etiological factors with multiple processes and steps.
  • the present invention provides a method of diagnosing Epstein-Barr virus (EBV) associated cancer by determining the EBV gene load and gene expression level of certain molecular biomarkers of, for example, viral as well as cellular origin that are related to cancers.
  • EBV Epstein-Barr virus
  • the invention relates to a method of diagnosing Epstein-Barr virus-related cancer in a subject, comprising collecting a biological sample from said subject, determining in said biological sample the amount of the Epstein-Barr virus gene load by determining the EBNAl (Epstein-Barr nuclear antigen 1) gene load and the expression level of at least one gene selected from the group consisting of the Epstein-Barr virus gene LMPl (latent membrane protein 1) and the cellular genes RASSFl (ras association domain family IA), CHFR (Checkpoint with fork-head associated and ring finger) and DAPK (death associated protein kinase), and comparing the determined expression levels of said genes in said biological sample with a reference.
  • EBNAl Epstein-Barr nuclear antigen 1
  • LMPl latent membrane protein 1
  • RASSFl ras association domain family IA
  • CHFR Checkpoint with fork-head associated and ring finger
  • DAPK death associated protein kinase
  • the gene load of EBNAl and the expression levels of at least two genes, or at least three genes selected from the group consisting of the Epstein-Barr virus gene LMPl and the cellular genes RASSFl, CHFR and DAPK are determined and compared to a reference.
  • the expression levels of LMPl and RASSFlA are determined.
  • the expression levels of CHFR and/or DAPK may also be determined.
  • the expression level of the above indicated marker genes can be determined via the epigenetic status of said genes.
  • the promoter hypermethylation of the tumor suppressor genes RASSFlA, CHFR and DAPK is of particular interest, because a hypermethylated promoter region of these genes leads to a decreased or completely abolished expression of the gene products and thus to a loss of the tumor suppressor function of these gene products.
  • the methylation status of the EBV gene LMPl is also of major interest, because LMPl codes for a protein that activates transcription factors and blocks apoptosis and has been associated with tumor development and progression.
  • the methylation status of the above genes can, for example, be determined via methylation specific PCR (MSP).
  • MSP methylation specific PCR
  • the MSP can be carried out in form of multiple methylation specific PCR (MMSP).
  • MMSP multiple methylation specific PCR
  • the primers used to determine the EBV gene load may be designed to take into account the changes of nucleotides after bisulfate treatment (for the conversion of unmethylated cytosines to uraciles) so that the EBV gene load is also determined by means of methylation specific PCR (MSP).
  • oligonucleotide primers suited for this purpose may be used.
  • Suitable upstream (sense) primers for carrying out the method of the invention may comprise, consists essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, whereas suitable downstream (antisense) primers may comprise, consists essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
  • each of LMPl, RASSFlA, CHFR and DAPK which can occur together or individually
  • one set of primers i.e. for the methylated or the unmethylated gene, depending on the expected methylation status of the gene in question in EBV-related cancers, may be used.
  • a primer set for the unmethylated variant of LMPl and/or a primer set for the methylated variant of at least one of RASSFl A, CHFR and DAPK may, for example, be utilized.
  • two sets of oligonucleotide primers may be employed. Both sets of oligonucleotide primers (sense and antisense primer) may be directed to the methylated or unmethylated variant of the target gene. The additional primer for each gene may cover the rare event of a gene variation of the gene to be detected.
  • two sets of oligonucleotide primers one set of primer for the methylated variant of the gene and the other primer set for the unmethylated variant, may be employed. Such an approach allows, for example, the quantification of gene methylation in a sample by correlating the detected signals for the unmethylated and the methylated gene variant.
  • Exemplary upstream (sense) primers for unmethylated LMPl may comprise, consist essentially of or consist of the nucleotide sequences set forth in SEQ ID NO: 5 and 7.
  • Exemplary upstream (sense) primers for methylated LMPl may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 23.
  • Exemplary downstream (antisense) primers for unmethylated LMPl may comprise, consist essentially of or consist of the nucleotide sequences set forth in SEQ ID NO: 6 and 8.
  • Exemplary downstream (antisense) primers for methylated LMPl may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 24.
  • Exemplary upstream (sense) primers for methylated RASSFlA, CHFR and DAPK may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 11, SEQ ID No.: 13 or SEQ ID NO: 15, and SEQ ID NO: 17 or SEQ ID NO: 19, respectively.
  • Exemplary upstream (sense) primers for unmethylated RASSFlA, CHFR and DAPK may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively.
  • Exemplary downstream (antisense) primers for methylated RASSFlA, CHFR and DAPK may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, and SEQ ID NO: 18 or SEQ ID NO: 20, respectively.
  • Exemplary downstream (antisense) primers for unmethylated RASSFlA, CHFR and DAPK may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30, respectively.
  • the reference to which the determined expression levels or the epigenetic status of the selected genes is compared, may be normal (healthy) tissue, such as normal epithelial cells, or any other reference tissue.
  • said reference tissue can be non- cancerous tissue composed of non-EBV-infected cells, such as non-EBV infected epithelial cells.
  • the reference tissue can either originate from the subject the biological sample is collected from or from any other adequate source, for example, a subject not suffering from an EBV infection or an EBV-related cancer or a non-EBV infected cell line, e.g. a non-EBV infected epithelial cell line.
  • an EBV-related tumor tissue or cell line for example the Namalwa BL cell line, may provide a positive control.
  • the biological sample can be either assayed directly, or the DNA contained in the sample can be isolated and purified and subsequently subjected to the invented method.
  • the isolated and purified DNA is genomic DNA.
  • control gene may be any suitable housekeeping gene, for example actin or glyceraldehyde-3-phosphat dehydrogenase (GAPDH).
  • GPDH glyceraldehyde-3-phosphat dehydrogenase
  • suitable primers may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 21 (sense) and SEQ ID NO: 22 (antisense).
  • EBV-related cancers that can diagnosed using the method of the present invention include, but are not limited, to B-cell lymphomas, for example in immune-suppressed patients, Burkitt lymphomas, including the endemic and sporadic form, nasopharyngeal carcinomas (NPC), salivary gland cancer, Hodgkin lymphoma, posttransplant lymphomas, AIDS-associated lymphomas, T-cell lymphomas, such as lethal midline granuloma, gastric adenocarcinoma and leiomyosarcoma in immune-suppressed patients, as well as certain benign tumors, such as oral hairy leukoplakia.
  • EBV-related cancers may express all marker genes disclosed in the present invention, they may be diagnosed by using a suitable subset of the markers. Burkitt lymphomas, for example, do not express LMPl. However, they may still be diagnosed according to the method of the invention by utilizing the other marker genes disclosed herein.
  • the present invention relates to oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID Nos: 1-30.
  • the present invention relates to a method of modifying a DNA molecule, wherein the DNA molecule is denatured, mixed with liquid agarose, pipetted in a chilled liquid to form agarose beads, with the agarose beads containing the denatured DNA molecules, wherein the resulting agarose beads are subsequently subjected to the following modification and purification steps (cf., paragraph 0119 below).
  • Figure 1 demonstrates the feasibility of Multiplex MSP by directly comparing the obtained results with those of single MSP.
  • the biological sample used is the Namalwa cell line, an EBV positive Burkitt lymphoma cell line expressing EBNAl and LMPl that contains one copy of EBV genome DNA per cell (and which can be obtained from American Type Culture Collection (ATCC), for example). These cells were subjected to MMSP or single MSP with primers specific for the indicated marker genes. The obtained PCR fragments were then analyzed on an agarose gel. The density of all bands from MMSP are corresponding to one from single MSP, indicating that reaction condition of MMSP is optimal for each gene and maximally reduced the competition among primers.
  • ATC American Type Culture Collection
  • Figure 2 shows the results of an agarose gel electrophoresis of a number of samples that demonstrate the sensitivity of MMSP.
  • Namalwa cells were used as a reference by semi-quantitative evaluation for EBV DNA load and by evaluation of the housekeeping gene actin for sample DNA input.
  • the DNA quantity corresponding to the number of analyzed Namalwa cells, was serially diluted and applied to MMSP.
  • the sensitivity of this assay is equivalent to up to 2 cells from 10 5 EBV negative cells.
  • Figure 3 illustrates results obtained by MMSP from known NPC cell lines.
  • Cl 5 designates a nude mouse bearing an LMPl expressing NPC.
  • C666-1 is an EBV positive, but LMPl non-expressing cell line as confirmed by Western Blot and immunostaining analysis. The rest are EBV negative NPC cell lines. EBNAl and LMPl could be detected in Cl 5 and EBNAl in C666-1.
  • Figure 4 shows MMSP result from biopsy and paraffin NPCs. 5FB, 9FB and 18FB indicate biopsy samples from NPCs, whereas 56 par., 58 par., 60 par., 63 par. indicate paraffin NPC samples. C15 indicates sample from a nude mice bearing an LMPl expressing NPC and H 2 O indicates a negative control.
  • Figure 5 shows a comparison of MMSP results between paraffin NPC samples (par.) and brush NPC samples (brush).
  • Cl 5 indicates sample from a nude mice bearing an LMPl expressing NPC and Namalwa indicates a Namalwa cell sample (EBV positive Burkitt lymphoma cell line expressing EBNAl and LMPl). H2O indicates a water control.
  • One object of the present invention is to provide an early diagnostic and prognostic tool for various EBV-related tumors.
  • the invention provides a method of determining the tumor-specific methylation patterns of a sample.
  • the sample may be collected from body fluid that is close to the primary tumor, e.g. sputum from lung cancer patients, urine from bladder or prostate cancer patients, and nasopharyngeal swab and mouth washing fluid from nasopharyngeal carcinoma (NPC) patients.
  • NPC nasopharyngeal carcinoma
  • detection using a blood sample is also possible even though use of a blood sample may lead to a rather non- specific signal.
  • the method allows the semi-quantitative evaluation of the expression level and pattern of different markers from materials that can be obtained by non-invasive methods and that are simple and fast to collect.
  • the present invention is directed to a method of diagnosing Epstein-Barr virus-related cancer in a subject, comprising collecting a biological sample from said subject; determining in said biological sample the amount of the Epstein-Barr virus gene load by determining the EBNAl gene load and the expression level of at least one gene selected from the group consisting of the Epstein-Barr virus gene LMPl and the cellular genes RASSFlA, CHFR and DAPK, and comparing the determined expression levels of said genes in said biological sample with a reference.
  • the method of the invention is typically carried out in a non-invasive manner, for example, by using as a biological sample a bodily fluid, such as mouth washing fluid or a nasopharyngeal swab/brush.
  • a biological sample may as well be blood or a tissue sample.
  • the EBV-related cancer may be any malignancy associated with Epstein-Barr virus infection.
  • NPC nasopharyngeal carcinoma
  • salivary gland carcinoma nasopharyngeal carcinoma
  • T cell lymphomas such as lethal midline granuloma, gastric adenocarcinomas, leiomyosarcoma in immune-suppressed subjects.
  • benign tumors associated with EBV for example oral hairy leukoplakia.
  • the method of the invention is used for the diagnosis of NPC in a subject.
  • the EBV gene load is determined by selection of a suitable marker gene, for example EBNAl, and determining the copy number of said gene per cell, for example by semi-quantitative PCR. To calculate the gene load the obtained result may be compared to a reference, for example a cell line with a defined copy number of EBV genomes per cell.
  • a reference for example a cell line with a defined copy number of EBV genomes per cell.
  • a specific example for such cells is the Namalwa cell line, which is derived from a human Burkitt lymphoma and has a single copy of the EBV genome per cell.
  • EBNAl is the abbreviation for Epstein-Barr nuclear antigen 1 and is used in the context of the present invention to designate the gene encoding said protein.
  • the gene product of EBNAl is essential for the virus maintenance and also may be important for growth transformation of human primary B lymphocytes, because it is involved in the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes and avoids proteasomal processing and cell-surface presentation.
  • the EBNAl protein in implicated to influence cellular events by sequestering key regulatory proteins.
  • EBNAl is a gene which is expressed in essentially every EBV infected cell and as such is suitable to determine the EBV gene load, which is correlated to the clinical course and prognosis of EBV-related cancers as well as their tendency to metastasize and hence may be monitored during therapy.
  • the oligonucleotide primers set forth in the present invention may be used.
  • the oligonucleotide primers disclosed by the present invention to determine the EBNAl gene load in a sample comprise, consist essentially of or consist of the nucleotide sequences set forth in SEQ ID NOs.: 1-4, with SEQ ID NOs.: 1 and 3 representing upstream, i.e. sense, primers, and SEQ ID NOs.: 2 and 4 representing downstream, i.e. antisense, primers.
  • complements, variants and fragments of the nucleotide sequences set forth in SEQ ID NOs.: 1-4 are also contemplated by the present invention.
  • the expression levels of the marker genes LMPl, RASSFlA, CHFR and DAPK may be determined by any suitable method known by skilled person. Such methods may include Northern Blot, Western Blot and nucleic acid amplification techniques, for example TMA (transcription mediated amplification) and PCR. In one embodiment of the invention, the epigenetic status, for example the methylation status of the marker genes is determined.
  • the methylation status can be determined by use of any suitable method, such as for example, but not limited to, methylation-sensitive DNA restriction enzymes, methylation specific nucleic acid probes and methylation specific polymerase chain reaction (MSP). Particularly useful for carrying out the present invention is the MSP technique.
  • Methylation specific PCR is a bisulfite conversion based PCR technique for the study of DNA CpG methylation. For an MSP experiment, oligonucleotide primer pairs specific for methylated DNA (M, M primer) and/or for unmethylated DNA (U) are necessary.
  • each primer (or at least one of the pair) sequence one or more CpG sites are included.
  • DNA is modified with sodium bisulfite and purified.
  • the treatment of DNA with bisulfite converts all unmethylated cytosines (C) to uraciles, so that instead of Watson-Crick base- pairing with guanosines (G) they now pair with adenosines (A).
  • the utilized primer pairs are designed to detect the target region with methylated Cs by having a G at the respective complementary position (M primer) or the target region with unmethylated Cs that have been converted to U's by having an A at the respective complementary position (U primer).
  • PCR reactions are performed using M primer pair and/or U primer pair. Successful amplification from M pair and/or U pair indicates methylation and unmethylation, respectively.
  • MMSP multiple methylation specific PCR
  • the MMSP technique has the advantage the methylation status of several target genes can be evaluated in a single reaction. Therefore, this approach reduces DNA requirement from samples and primer competition during PCR amplification.
  • the EBV protein LMPl (latent membrane protein 1) is absolutely essential for the immortalization of B lymphocytes by EBV and is expressed in most of the NPCs and in several EBV-associated tumors. It is a direct target of EBNA2 and crucial for the survival and proliferation of latently infected cells. Investigation of deletion mutants has shown that the transmembrane as well as the signaling domain of the LMPl protein are necessary for its transforming properties.
  • the LMPl protein has a molecular weight of approximately 62 kDa and is 386 amino acids long, with the N-terminal 186 amino acids forming the transmembrane domain and the C-terminal 200 amino acids forming the cytoplasmic signaling domain.
  • the transmembrane domain consists of six transmembrane regions connected via short loop regions that mediate the multimerization.
  • the multimerization of the LMPl proteins leads independently from ligand binding to a constitutively activated signaling complex.
  • TNF-R tumor necrosis factor receptor
  • LMPl shares many functional similarities with these proteins. So does LMPl interact via its C-terminal activating regions 1-3 (CTAR 1-3) with many of the proteins the TNF-R family proteins interact with, including TRAF 1, 2, 3 and 5, TRADD and RIP. Upon binding of these proteins the canonical and non-canonical NFKB signaling pathway is activated.
  • LMPl induces the expression of activation and adhesion markers, such as C54, MHC class II, CD 23, CD95, and CD86, and of anti-apoptotic protein, such as Bcl2, BcIX and A20.
  • activation and adhesion markers such as C54, MHC class II, CD 23, CD95, and CD86
  • anti-apoptotic protein such as Bcl2, BcIX and A20.
  • LMPl is the only viral protein of EBV for which a direct oncogenic potential has been demonstrated. Accordingly, LMPl expression has been found in several EBV-related malignancies. The level of LMPl protein expression depends strongly from the methylation status of LMPl. In the unmethylated form found in many EBV-related cancers, the LMPl protein is constitutively expressed.
  • the presence or amount of unmethylated LMPl gene can be determined by methylation specific PCR (MSP) employing oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence set forth in SEQ ID NO: 5 or 7 (upstream, sense) and SEQ ID NO:6 or 8 (downstream, antisense), as well as complements, variants and fragments thereof.
  • MSP methylation specific PCR
  • the RASSFlA (Ras association domain family IA) protein is a cellular tumor suppressor encoded by RASSFlA, which acts through inhibition of the Gl/S phase progression of the cell cycle.
  • the RASSFlA protein has been shown to bind to the Ras- GTP binding protein Nore-1, consistent with its role as a negative regulator of the Ras proto-oncogene (Ortiz-Vega et al. (2002) Oncogene 21(9):1381-1390). From the high frequency with which RASSFlA is inactivated by promoter methylation it has been deduced that it plays a key role in the development of many primary human tumors. However, the mechanism of RASSFlA action remains yet unknown.
  • RASSFlA also associates with microtubules and it has been shown that this association is essential for RASSFlA to mediate its growth inhibitory effects. Overexpression of RASSFlA promotes the formation of stable microtubules, whereas a dominant-negative fragment of RASSFlA destabilizes microtubule networks. Because RASSFlA localizes to the mitotic spindle and blocks activated Ras-induced genomic instability, it has also been implicated to effect genomic stability. Thus, it is presently assumed that RASSFlA plays a role in the control of microtubule polymerization and potentially in the maintenance of genomic stability (Vos et al. (2004) Cancer Research 64:4244-4250).
  • promoter hypermethylation of the RASSFlA gene can be determined via methylation specific PCR utilizing oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID NOs. :9 or 11 (upstream, sense) and SEQ ID NO: 10 or 12 (downstream antisense), as well as complements, variants and fragments thereof.
  • oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID NOs. :9 or 11 (upstream, sense) and SEQ ID NO: 10 or 12 (downstream antisense), as well as complements, variants and fragments thereof.
  • CHFR Checkpoint with fork-head associated and ring finger
  • CHFR is an ubiquitin ligase and the key component in a new mitotic checkpoint that acts at prophase and delays chromosome condensation in response to mitotic stress (Scolnick and Halazonetis (2000) Nature 406(6794):430-5).
  • the cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome and the fidelity of sister chromatid separation. Failure of such checkpoint functions results in genomic instability, a condition that predisposes cells to neoplastic transformation and tumor progression.
  • CHFR seems to be required for delaying prophase in human cells.
  • CHFR is ubiquitously expressed in normal tissues, it is frequently downregulated in human cancers, mostly owing to hypermethylation of its promoter region.
  • CHFR gene A significant proportion of human esophageal cancer has loss of expression of the CHFR gene (Shibata et al. (2002) Carcinogenesis 23(10): 1695-1700; Toyota et al. (2003) PNAS 100(13):7818-7123; Yu et al. (2005) Nature Genetics 37(4) :401-406).
  • promoter hypermethylation of the CHFR gene can be determined via methylation specific PCR utilizing oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID Nos.:13 or 15 (upstream, sense) and SEQ ID Nos.:14 or 16 (downstream antisense), as well as complements, variants and fragments thereof.
  • oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID Nos.:13 or 15 (upstream, sense) and SEQ ID Nos.:14 or 16 (downstream antisense), as well as complements, variants and fragments thereof.
  • DAPK death associated protein kinase
  • DAPK is a Ca2+/Calmodulin-dependent, cytoskeletal associated protein kinase whose expression is implicated in the sensitivity of cells to apoptotic effects of TNF ⁇ and Interferon- ⁇ . Deletion of the Calmodulin-binding domain results in a constitutively active mutant with a more potent cytotoxic effect. In contrast, the catalytically inactive mutant exerts a dominant negative effect, reducing cytotoxicity and protecting cells from Interferon- ⁇ induced cell death. DAPK expression is frequently lost in human carcinomas and B -cell leukemia, and lower levels of expression correlate with high rates of metastasis.
  • DAPK death-associated protein kinase
  • promoter hypermethylation of the DAPK gene can be determined via methylation specific PCR utilizing oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO:17 or SEQ ID NO:19 (upstream, sense) and SEQ ID NOs.:18 or 20 (downstream, antisense), as well as complements, variants and fragments thereof.
  • oligonucleotide primers comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO:17 or SEQ ID NO:19 (upstream, sense) and SEQ ID NOs.:18 or 20 (downstream, antisense), as well as complements, variants and fragments thereof.
  • the methylated form of the LMPl gene and the unmethylated forms of the RASSFlA, CHFR and/or DAPK genes may be determined separately or in addition to the above.
  • Suitable primers to determine methylated LMPl comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NO: 23 (upstream, sense) and SEQ ID NO: 24 (downstream, antisense).
  • SEQ ID NO: 23 upstream, sense
  • SEQ ID NO: 24 downstream, antisense
  • unmethylated RASSFlA, CHFR and/or DAPK primers that comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID NOs: 25, 27, and 29, respectively (upstream, sense) and SEQ ID NOs: 26, 28, and 30, respectively (downstream, antisense), may be utilized.
  • the method of the invention is directed to determining the EBNAl gene load and the methylation status of the EBV oncogene LMPl.
  • the methylation status of the cellular tumor suppressor gene RASSFlA may be determined.
  • the methylation status of the cellular tumor suppressor genes CHFR and DAPK may be also determined.
  • the determination of the gene load and the expression level is carried out by PCR, for example MSP or MMSP.
  • the DNA Prior to the determination step, the DNA may be isolated and purified from the biological sample, and then subjected to the respective determination technique.
  • the isolated and purified DNA may contain both viral and cellular DNA and may be genomic DNA. Suitable methods and protocols to isolate DNA or genomic DNA from a sample are known to the skilled person and kits for performing DNA isolation and purification are commercially available.
  • a control gene may be concomitantly determined.
  • Suitable control genes are all housekeeping genes (a housekeeping gene is typically a constitutive gene that is transcribed at a relatively constant level) of a cell, for example actin, ubiqitin or GAPDH. The absence of such a house-keeping gene in a DNA preparation indicates that the DNA preparation is of a quality (purity) that is not suitable for carrying out the diagnostic method described here.
  • suitable oligonucleotide primers for the detection of actin may comprise, consist essentially of or consist of the nucleotide sequences set forth in SEQ ID NO:21 (upstream, sense) and SEQ ID NO:22 (downstream, antisense), as well as complements, variants and fragments thereof.
  • the expression level of the selected marker genes may be compared to a reference.
  • This reference may be normal tissue, i.e. non-cancerous tissue, which is preferably of the same tissue type as the biological sample.
  • the reference may originate from the subject the biological sample was collected from or any other suitable source, such as healthy tissue form another donor or cultured cells. If the expression level of a gene of interest exceeds a certain, for example, predetermined threshold value compared to the reference sample, the expression level of the gene is considered to be altered.
  • the expression level can either by increased or reduced compared to the reference sample, depending on the biological function of the selected gene.
  • the level of LMPl protein expression depends strongly from the methylation status of the LMPl gene.
  • the LMPl protein In the unmethylated form found in several EBV-related cancers, the LMPl protein is constitutively expressed. In contrast, no LMPl Protein expression is found in normal (healthy) tissues, which are normally EBV genome negative or wherein the LMPl gene promoter is typically methylated.
  • the cellular tumor suppressor genes RASSFlA, CHFR and DAPK are usually constitutively expressed in normal (healthy) cells.
  • the expression of these genes is decreased or abolished, and the gene products can no longer be found in the affected cells. Accordingly, missing or reduced RASSFlA, CHFR and/or DAPK protein expression is found in many cancers.
  • the gene load may be determined by comparison of the obtained results with a reference with a defined EBV gene load.
  • a reference may be a cell from a cell line with a defined EBV gene load, such as Namalwa cells.
  • the subject the biological sample is collected from may be mammal, in particular a human b eing.
  • NPC samples are obtained from brush or mouth-washing, being from elapsed tumor cells, and tumor DNAs are modified by chemicals and subsequently amplified by methylation and unmethylation specific PCR through a single reaction.
  • This design is able to reduce DNA amount from precious samples and primer competition during PCR amplification.
  • an actin marker can be used as an internal DNA quality control.
  • Namalwa cells can be used as a control for systematic working conditions and a reference for semi-quantity control for EBV DNA load.
  • the "expression" pattern of NPC related genes can then be evaluated for early detection /prognosis. This method is a simple and feasible way to be used in a clinical laboratory.
  • the method described here may not only have a value for the diagnosis, but also may for monitoring the treatment of cancer. As such the method may generate a new- criteria for tumor classification from a molecular basis, opening the way to a host of innovative diagnostic, preventive and therapeutic strategies. Accordingly, the results obtained from the method of the invention may be used in developing a therapeutic or preventive strategy for Epstein-Barr virus-associated cancers.
  • the present invention is directed to reagents for carrying out the method of the invention.
  • Such reagents allow the determination of the gene load of EBNAl and/or the expression level of any one of LMPl, RASSFlA, CHFR and DAPK.
  • Exemplary reagents suitable for determining the gene load of EBNAl and/or the expression level of a gene selected from the group of LMPl, RASSFlA, CHFR and DAPK by means of a nucleic acid amplification technique may be oligonucleotide primers. These primers may comprise, consist essentially of or consist of the nucleotide sequences set forth in SEQ ID Nos. 1-20 and 23-30 as well as complements, variants and fragments thereof.
  • the present invention is directed to a kit for carrying out the method of the invention.
  • the present invention also features a kit for the diagnosis of EBV-related cancer in a subject, comprising at least one reagent for determining the EBV gene load, for example by determining the EBNAl gene load, and at least one reagent for determining the expression level of a marker gene, said marker gene being selected from the EBV gene LMPl and the cellular genes RASSFlA, CHFR and DAPK.
  • the kit of the invention may further include reagents for the determination of the expression of a reference gene.
  • the reagents comprised in the kit are oligonucleotides, preferably oligonucleotide primer pairs.
  • Said oligonucleotides may comprise, consists essentially of or consist of any one of the nucleotide sequences set forth in SEQ ID NOs.: 1-30 as well as complements, variants and fragments thereof.
  • the invented kit comprises a oligonucleotide primer pair for the determination of the EBNAl gene load and at least one further primer pair for the determination of the expression of a marker gene selected from LMPl, RASSFlA, CHFR and DAPK.
  • the kit may comprise further reagents, for example buffers, solvents and compounds for detection of the expression level such as nucleic acid polymerase or antibodies for Western Blots.
  • reagents for example buffers, solvents and compounds for detection of the expression level such as nucleic acid polymerase or antibodies for Western Blots.
  • fragments with respect to nucleic acids relates to 3' and/or 5' shortened versions of a given nucleotide sequence.
  • Such fragments include for example nucleic acids which are shortened by one or more nucleotides at their 3' and/or 5' end(s) compared to their full length counterpart. It is preferred that these fragments have a sufficient length to retain the functionality of the full length version, i.e. in case of oligonucleotide primers for nucleic acid amplification, such fragments preferably have a length of at least 9, more preferably at least 12, most preferably at least 15 nucleotides.
  • variant refers to chemically modified nucleic acids, for example backbone-modified nucleic acids, or mutated nucleic acids, derived from a given nucleotide sequence.
  • the mutants may comprise one or more base substitutions, however it is preferred that the functionality of the original nucleic acid molecule is retained. Thus, it is preferred that a variant shares at least 70, 75, 80, more preferably 90, most preferably 95% nucleotide sequence identity with the original nucleic acid.
  • the base substitution may include any of the naturally occurring standard bases A, G, T, C and U but may also include other bases.
  • the chemical modification may occur at the base, the sugar moiety or the phosphate backbone and may alter the properties of the nucleic acid molecule, e.g. with respect to its stability.
  • "Complementary” relates to the property of a nucleic acid molecule to base-pair with another nucleic acid molecule via canonical Watson-Crick base pairs. Accordingly, a “complement” is a nucleic acid that can hybridize with a given nucleotide sequence via Watson-Crick base pairs.
  • a "gene” is a part of the genomic DNA, encoding for a polypeptide.
  • An “expression product” is a product of gene expression and comprises the transcription product, i.e.
  • “Expression level” as used herein relates to the extent a gene is transcribed.
  • the expression level can be determined in the present invention by any suitable methodology, for example, via Northern Blotting, transcription-mediated amplification or other known techniques to determine the presence or amount of a specific mRNA.
  • the expression level may also be determined by determining the presence of the encoded protein, for example by use of Western Blotting, immunostaining, immunoprecipitation or other known techniques.
  • the expression level can also be directly determined by evaluating the epigenetic status of a gene.
  • Promoter hypermethylation indicates that the expression level is low or virtually non-existent, whereas an unmethylated promoter region indicates that the gene of interest is expressed.
  • the degree of methylation in a promoter of a specific gene is correlated with the gene expression level, shown by RT-PCR (mRNA) or Western blotting (protein) or by methylation specific PCR (DNA fragment).
  • Epigenetic status as used herein relates to modifications of genetic material that are inheritable via the cell division, and play an important role in many physiological and pathophysiological conditions.
  • Epigenetic mechanisms provide an "extra" layer of transcriptional control that regulates how genes are expressed. These mechanisms are critical components in the normal development and growth of cells.
  • epigenetic events such as methylation, deacetylation and chromatin re-modeling, promoter hypermethylation has been the most detailed studied.
  • Methods relate predominantly to the methylation of the 5-position of cytosines in CpG dinucleotides that may be clustered in groups called CpG islands.
  • a methylated gene thus includes 5-methylated cytosines, primarily in the promoter region or in exon 1 of this gene, whereas an unmethylated gene lacks these extra methyl islands at cytosine.
  • a "tumor suppressor” is a gene or gene product that is involved in the control of cellular proliferation or apoptosis. In case such a gene is mutated to lose its function or is deleted the cell loses a fundamental control mechanism that would normally help to regulate cell growth, cell proliferation or apoptosis and thus contribute to prevent cellular transformation. Accordingly, loss-of-function mutations of tumor suppressor genes are frequently found in tumors.
  • An "oncogene” is a gene that promotes cellular transformation. Such an oncogene may be of viral origin or may originate from a constitutively activated cellular proto-oncogene, for example by an activating mutation. Numerous viral oncogenes and cellular proto-oncogenes have been implicated to play a major role in the development and progression of cancer.
  • Gene load as used in the present invention, relates to the copy number of a certain gene or genome, in particular the EBV genome, in a cell or cell-containing sample.
  • the expression “gene load” refers to the amount of a certain gene, for example the EBV gene EBNAl, in a cell or biological sample. This amount can be determined qualitatively, i.e. by determining if it is higher, equal to or lower than that of a reference sample, or quantitatively, e.g. by calculating the copy number of the gene of interest by comparison with a reference with a defined copy number. Determination of the gene load as used herein also includes detecting the presence or absence of a gene such as EBNAl. Over the last years viral DNA load was found to be a good prognosticator for subsequent clinical events (Tan et al. (2006) BMC Cancer 6:227).
  • CNEl, CNE2, TW03, C666-1 and HONEl NPC cell lines were used in this study. Cells were grown at 37°C in IMDM medium with 10% fetal calf serum and antibiotics, except for C666-1, which was cultured according to conditions specified by Cheung et al. (Cheung et al. (1999) Int. J. Cancer 83(1):121 -126).
  • Namalwa cells are an EBV positive lymphoma cell line expressing EBNAl and LMPl, and containing one copy of EBV genome DNA per cell.
  • Xenograft Cl 5 mice (nude mice with a human NPC) were obtained from Dr. P. Busson, Institute Gustave Roussy, Paris.
  • 133 NPC samples from pathology-verified NPC patients, 35 unmatched NPC and 24 normal nasopharyngeal epithelia were obtained from GMU, GMU and HMU (ethical approval: no.00-312 Swedish, Sweden and local committee in China) for this study.
  • 98 cases are paired samples with biopsy/paraffin NPC and brush/swab samples.
  • NPC brush/swabs were collected under surface anesthesia with 1% cocaine solution.
  • cotton/brush topped sticks were inserted into the left and right nasal cavity and moved until they reached the nasopharyngeal wall.
  • the cotton swab was then rotated several times against the posterior and lateral nasopharyngeal walls.
  • the cotton stick with the absorbed material was transferred in a Falcon® tube containing 3 ml saline and immediately processed by centrifugation at 3000x g for lOmin.
  • Cell pellets were stored in 400ul TE buffer (10 ⁇ M Tris 1 ⁇ M EDTA, pH 7.0) at -80 0 C until further use.
  • the absorbed material was soaked and washed in TE buffer containing 0.5%SDS/50 ⁇ g/ml proteinase K (Invitrogen, Carlsbad, CA) and the DNA extracted via the phenol/chloroform (CH 3 Cl) method.
  • Mouth washing was collected at morning with 20ml of 0.9% NaCl, twice.
  • NPC biopsy tissues were snap frozen in liquid nitrogen until further use. Upon thawing the biopsies were homogenized and treated with TE buffer containing 50 ⁇ g/ml proteinase K (Invitrogen, Carlsbad, CA) for 3 hours at 56 0 C. High molecular weight genomic DNA was obtained by conventional phenol/chloroform and ethanol extraction. [00117] Genomic DNA from the swab cell pellets was also extracted by conventional phenol/chloroform method. [00118] DNA from paraffin section was adapted by silicon method. Four sections of a formalin fixed and paraffin embedded biopsy with a thickness of 5 ⁇ m were subjected to DNA extraction.
  • genomic DNA was denatured by incubation in 0.3 M NaOH for 15 minutes at 37 0 C, then mixed with two volumes of 2% low melting point agarose.
  • the agarose/DNA mixture was pipetted into chilled mineral oil to form agarose beads. Each bead was placed in an individual tube to which we added aliquots of 200 ⁇ l 5M bisulfite solution (2.5M sodium metabisulfite, Sigma; 10OmM hydroquinone, Sigma; pH 5.0).
  • the reaction mixture was then incubated in darkness for 16 hours at 5O 0 C. Treatments were stopped by equilibration against ImI of TE buffer followed by a desulfonation step in 500 ⁇ l of 0.2 M NaOH.
  • Twenty-five ⁇ l of bisulfite sequencing PCR reaction contained 20 ng of bisulfite treated DNA as template, 10 pmol of each primer, 100 pmol of each deoxynucleoside triphosphate, 10 ⁇ PCR buffer, and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA). Cycle conditions were as follows: 95°C for 10 minutes, followed by 34 cycles at 94°C for 45 seconds, 55°C for 45 seconds, and 72 0 C for 90 seconds. PCR products were purified using QIA quick Gel Extraction Kit (Qiagen, Chatsworth, CA).
  • PCR primer sequences which are specific for the genes EBNAl, unmethylated LMPl, methylated RASSFlA, methylated CHFR, methylated DAPK and Actin, and the expected PCR product sizes are summarized in Table 1.
  • PCR reaction 2 ⁇ l of bisulfite-modified DNA were added in a final volume of 25 ⁇ l of PCR mixture containing Ix PCR buffer, 1.5 mM MgCl 2 , 100 pmol of each deoxynucleotide triphosphate, 100 pmol of each utilized primer and one unit of AmpiTaq Gold (Applied Biosystems, Branchburg, NJ).
  • PCR amplification was performed at 95°C for 10 minutes, followed by 34 cycles at 94 0 C for 30 seconds, the specific annealing temperature 55°C for 45 seconds, and 72°C for 90 seconds.
  • Each reaction included a positive methylation control (Namalwa cell DNA, one EBV genome per cell), a negative methylation control (normal nasopharyngeal epithelial tissue), and a water blank control.
  • MMSP products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. AU the experiments were repeated to evaluate the reproducibility of the results and semi-quantitatively evaluated for EBV DNA load with comparison to Namalwa cells as a reference for one copy of EBV genome per cell.
  • the evaluation of the samples yielded the results depicted in Table 2.
  • EBV EBNAl gene exists in all NPC, but not in normal epithelial cells. Therefore it appears to be essential for diagnosing or predicting an NPC.
  • LMPl is expressed in more than 60% of NPC.
  • a sample positive for both EBNAl and LMPl leads to the diagnosis of an NPC with 100% certainty.
  • To diagnose NPC in a LMPl negative sample it requires positive EBNAl plus positive from either of methylated RASSFlA or both methylated CHFR/D APK. Accordingly, the criterion for the diagnosis of NPC is the detectability of EBNAl plus either of unmethylated LMPl, methylated PvASSFlA or methylated CHFR/D APK.
  • the present invention provides for the first time a highly specific, highly sensitive, non-invasive method for the early diagnosis of EBV-related cancers, such as nasopharyngeal carcinoma.

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Abstract

L'invention porte sur des méthodes de diagnostic de types de cancers associés aux infections par le virus Epstein-Barr (EBV) et utilisant la détermination de la charge en gènes d'EBV et le profil de l'expression génique de certains biomarqueurs moléculaires d'origine virale ou cellulaire liés aux cancers associés à l'EBV.
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