CN1790021A - Screening of precancerous, carcinoma in situ, and carcinomatous lesions using circulating Ibovirus DNA - Google Patents

Abstract

The present invention relates to methods of screening for different types and stages of cancer, whether or not such cancer is associated with ibovirus. The present invention is based on the following findings: analysis of the level of ibo virus DNA in the plasma or serum of an individual, once or in successive times, can predict different types of cancer at different stages of development.

Description

Screening of precancerous, carcinoma in situ, and carcinomatous lesions using circulating Ibovirus DNA

technical field

The present invention relates to the discovery that Epstein-barr virus (EBV) can be found in the cell-free fluid of a patient's blood, and when the virus is found, the patient may already have precancer, carcinoma in situ, or other Any form of, but not limited to, Ebovirus-associated cancer. The present invention is based on the theory that we found that circulating CD25+CD4+regulatory T (T _reg ) lymphocytes were elevated in normal individuals and cancer patients with positive EBV DNA in their plasma. We also found positive titers of circulating EBV DNA in the serum or plasma of patients with certain cancers of unknown relationship to Ibovirus. Combining the above observations, the findings demonstrate that EBV reactivation can lead to suppression of cytotoxic T cells, leading to a loss of cancer immune surveillance. The present invention proposes a new universal screening method, which uses circulating EBV DNA in serum or plasma to screen precancer, carcinoma in situ and cancer lesions.

technical background

Cancer cells of various tumors are known to release tumor-derived DNA (Anker et al., Cancer Metastasis Rev. 18:65-73 (1999)). Examples include oncogene mutations arising from pancreatic cancer (Anker et al., Gastroenterology. 112:4-1120 (1997)), microsatellite alterations arising from lung cancer (Chen et al., Cancer Res. 59:3 (1999) ) and epigenetic changes produced by liver cancer (Wong et al., Cancer Res. 59:3 (1999)). In addition, in many cancers known to be associated with viral infection, viral DNA can be found in circulation. Examples include nasopharyngeal carcinoma (Mutirangura et al., Clin Cancer Res. 4:665-9 (1998)); Lo et al., Cancer Res. 59:1188-91 (1999)) and certain lymphomas (Lei et al., Br JHaematol.111:239-46(2000); Gallagher et al., Int J Cancer.84:442-8(1999); Drouet et al., J Med Virol.57:383-9(1999 )) and human papillary carcinoma virus DNA from head and neck cancers (Capone et al., Clin Cancer Res. 6:4171-5 (2000)).

Currently, investigators are very concerned about the presence of tumor-derived DNA in the plasma and serum of cancer patients (Chen, X.Q. et al., Nat. Med., 2: 1033-1035 (1996); Nawroz, H. et al., Nat. Med ., 2:1035-1037 (1996)). In virus-associated cancers, cell-free tumor-associated viral DNA has been detected in the plasma and serum of patients (Mutirangura, A. et al., Cancer Res., 4:665-669 (1998); Lo, Y.M.D. et al ., Clin. Cancer Res. 59: 1188-1191 (1999); Capone, R.B. Clin. Cancer Res., 6: 4171-4175 (2000)). An important virus associated with various types of malignancies is EBV (Cohen, J.I.N. Engl. J. Med., 343:481-492 (2000)). EBV is a human herpes virus that infects most people. EBV is usually transmitted through saliva and forms a latent infection in B lymphocytes that can accompany the host for life. For this reason, in nasopharyngeal carcinoma (NPC) (Mutirangura, A. et al., Cancer Res., 4: 665-669 (1998); Lo, Y.M.D. et al., Clin. Cancer Res., 59: 1188 -1191(1999)) patients and certain malignant lymphoid tumors (Lei, K.I.et al., Br.J.Haematol., 111:239-246(2000); Drouet, E.et al., J.Med.Virol , 57: 383-389 (1999); Gallagher, A. et al., Int. J. Cancer, 84: 442-228 (1999)) circulating EBV DNA has been detected in the plasma and serum of patients.

It has also been reported that some gastric cancers are also related to EBV infection (Shibata, D. et al., Am. J. Pathol., 140:769-774 (1992)). In Hong Kong, about 10% of gastric cancer cases were found to be associated with EBV infection (Yuen, S.T. et al., Am. J. Surg. Pathol., 18: 1158-1163 (1994)).

Currently, suppressor T cells with a CD3+CD4+CD25+ phenotype have been found to play a role in the development of cancer. Although these T cells were originally found to protect the host against the occurrence of autoimmunity, they also simultaneously prevent effective immune surveillance of the host against tumor antigens. Thus, the rise of cells with this phenotype can lead to cancer formation and progression from precancerous, carcinoma in situ to full-fledged cancer.

In addition, we can also find EBV DNA in the plasma of patients with some cancers not related to EBV, which further proves that EBV does play a role in cancer (except nasopharyngeal carcinoma, gastric cancer and lymphoma caused by different pathogens). effect.

Contents of the invention

A first aspect of the present invention provides a method for routine screening for cancer by determining the amount of Ebovirus DNA (EBV DNA) present in a patient's serum or plasma. Therefore, the present invention has wide applications in clinical medicine, especially in latent EBV infection (existing in more than 90% of certain populations).

Method of the present invention generally comprises the steps:

obtaining a blood sample from a patient;

Extract DNA from the blood sample;

analyzing the amount of circulating EBV DNA present in the extracted DNA;

Repeat the above steps; and

comparing the amount of circulating EBV DNA present in said blood sample with a control,

Wherein the constant or rising trend of the content of the EBV DNA indicates that the patient suffers from cancer or precancer.

The cancer described in the present invention is preferably selected from nasopharyngeal cancer, gastric cancer and lymphoma; or from lung cancer, colon cancer, parotid gland cancer, breast cancer and liver cancer. Cancers other than those described above may also be used in the present invention.

Preferably, the blood sample of the present invention is an acellular fluid sample. The cell-free liquid fraction means that the sample is serum or plasma after extraction of cells, wherein the extraction of serum is obtained by coagulation and separation of cells from the remaining liquid, or the liquid fraction (plasma) is achieved by inhibiting coagulation and centrifuging extraction. EBV DNA was analyzed in this liquid fraction.

Preferably, repeated steps in the methods of the invention are performed within 4-12 weeks of the first assay. It can also be done at a time other than 4-12 weeks.

In a certain embodiment of the present invention, the method for analyzing the content of EBV DNA is carried out by hybridization, amplification, and quantitative methods, and any sequence or sequence fragment of the EBV genome is selected as the binding site of any primer and probe or a combination thereof point.

A second aspect of the present invention provides a diagnostic kit comprising suitable reagents for detecting EBV DNA in a patient's serum or plasma. Kits of the invention may also comprise one or more devices for obtaining a blood sample from a patient, for isolating EBV DNA from the blood sample, and for quantifying the amount of EBV DNA present in the blood sample. The kit can be used for routine screening of cancer, and the screening is realized by measuring the content of Epstein-Barr virus DNA (EBV DNA) existing in serum or blood of patients.

Brief description of the drawings

Figure 1 shows the results of a series of analyzes of titers in a cohort of cancer patients with positive plasma EBV DNA titers, all untreated patients exhibiting level up.

Figure 2 shows the results of a series of second or third analyzes performed at intervals of 4-12 weeks after the first and second determinations, respectively, of a group of normal individuals with positive plasma EBV DNA titers, Results showed zero EBV DNA titer readings for all but one individual.

Figure 3 shows the results of EBV DNA titers also positive in patients with cancers not directly related to EBV in a cohort of different patients suspected of having different types of cancer. Treatment of a patient with colon cancer who had a complete response was also accompanied by disappearance of her EBV DNA titer.

Detailed description of the invention

The present invention relates to a method for routine screening for cancer by determining the amount of Ebovirus DNA (EBV DNA) present in a patient's serum or plasma. The method involves detecting and measuring the amount of EBV DNA present in the plasma fluid of these patients. The method of the present invention has wide application in clinical medicine.

Clinically, circulating EBV DNA has long been used in the diagnosis and control of nasopharyngeal carcinoma (Lo, Y.M.D. et al., Cancer Res., 60:6878-6881).

Similarly, circulating EBV DNA can also be used in the diagnosis of some documented gastric cancers and lymphomas that have been associated with Ibovirus. However, these assays have never been applied to screen for EBV-associated cancers in the general population.

Any conventional DNA amplification or signal amplification method can be applied to detect EBV DNA. In most cases, the target sequence will need to be amplified using any of the nucleic acid amplification procedures known in the art. In particular, nucleic acid amplification refers to the enzymatic synthesis of nucleic acid amplicons (copies) comprising the complement of the nucleic acid sequence being amplified. Examples of nucleic acid amplification procedures employed in the art include polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR) and transcription associated amplification (TAA). Nucleic acid amplification is especially effective when the target sequence is present in very small amounts in the sample. In order to better detect the nucleic acid of the target organism or virus in the sample, fewer target sequences are required at the beginning of the analysis, so by amplifying the target sequence and detecting the synthesized amplicon, the sensitivity of the analysis can be greatly improved.

Methods of nucleic acid amplification are well described in the literature. For example, Mullis et al. describe PCR amplification methods in US Pat. Examples of SDA can be found in Walker, PCR Methods and Applications, 3: 25-30 (1993), Walker et al., Nucleic Acids Res., 20: 1691-1996 (1992) and Proc. Natl. Acad. Sci., 89: 392-396 (1991). The LCR method is described in US Patent Nos. 5,427,930 and 5,686,272. Moreover, different forms of TAA are provided in various publications, such as Burg et al. in U.S. Patent No. 5,437,990; Kacian et al. in U.S. Patent Nos. 5,339,491 and 5,554,516; 01302, international application number PCT/US87/01966 and publication number WO88/01315, international application number PCT/US88/02108.

Real-time quantitative PCR is the preferred way to monitor EBV DNA, which is based on continuous light monitoring of the progress of the fluorescent PCR reaction (Heide et al. Genome Res. 6: 986-694, 1996 and Lo et al. Am J. Hum. Genet. 62: 768-775, 1998). In this system, in addition to the two amplification primers used in conventional PCR, a double-labeled fluorescent hybridization probe (Livak, et al. PCRMethods Appl., 4357-362, 1995) is also included. One fluorochrome is used as a reporter molecule (FAM) and its emission spectrum is suppressed by a second fluorochrome (TAMRA). During the extension phase of PCR, the 5' to 3' exonuclease activity of Taq DNA polymerase (9) strips the reporter molecule from the probe, thereby releasing it from the inhibitor, resulting in fluorescent emission at 518nm enhanced.

Method of the present invention generally comprises the steps:

obtaining a blood sample from a patient;

extracting DNA from said blood sample;

analyzing the amount of circulating EBV DNA present in the extracted DNA;

Repeat the above steps; and

The amount of circulating EBV DNA present in the blood sample is compared to a control, wherein a constant or increasing trend in the amount of EBV DNA indicates that the patient has cancer or precancer.

Generally, in the present invention, the liquid fraction can be obtained by centrifuging the blood sample, and EBV DNA is analyzed in the liquid fraction.

Those skilled in the art should understand that the step of obtaining a blood sample from a patient and comparing the circulating EBV DNA content in the blood sample with the control is not an essential technical feature of the present invention. Therefore, in the embodiment of the present invention, none or one of them may be included.

Those skilled in the art can extract DNA from blood samples by many methods known in the art. A preferred method is to use the QIAamp Blood Kit. Meanwhile, circulating EBV DNA can also be analyzed by using a method known or new in the art. Especially preferred are methods involving real-time PCR amplification systems. Standard procedures for comparing the levels of EBV DNA detected by the methods described above to controls can be readily devised to assess the significance of the resulting values statistically.

The number of copies of EBV DNA can be analyzed over a period of time, and as demonstrated in our clinical findings, this series of analyzes can separate patients who screen positive for precancer, cancer, or malignancy from those who do not have cancer but because of EBV viral relapse. Individuals whose activation caused sporadic spikes in their EBV DNA titers were distinguished. Thus, the present invention (used alone or for serial analysis) provides a non-invasive method for the routine screening of cancer by determining the amount of EBV DNA present in the serum or plasma of such patients.

A second aspect of the invention relates to a diagnostic kit comprising appropriate reagents for the detection of EBV DNA in a patient's serum or plasma. Kits of the invention may also comprise one or more devices for obtaining a blood sample from a patient, for isolating EBV DNA from the blood sample, and for quantifying the amount of EBV DNA present in the blood sample. The kit can be used for routine screening of cancer, which is achieved by measuring the content of EBV DNA present in the patient's serum or blood.

All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be apparent to those skilled in the art that, without departing from the spirit and scope of the appended claims, Various changes and modifications may be made to the teachings of the invention.

Example

The following examples are offered by way of illustration only, not limitation. Those skilled in the art should readily identify various non-critical parameters by which substantially similar results can be obtained by changing or modifying them.

Example 1

Materials and methods

Blood samples from 132 high-risk cancer patients (Group A) and 3987 approximately normal individuals (Group B) have been sent to our laboratory in Hong Kong for the determination of circulating EBV DNA.

DNA Extraction from Plasma Samples Peripheral blood (5 ml) was collected from each subject and injected into EDTA tubes for separation of plasma. Blood samples were centrifuged at 1600 x g, and the plasma was carefully separated from the EDTA-containing tubes and transferred into blank polypropylene tubes. This sample was stored at -20°C until further manipulation. DNA was extracted from plasma samples using the QIAamp Blood Kit (Qiagen, Hilden, Germany) using the blood and body fluids procedure according to the manufacturer's recommendations (2). DNA extraction was performed using plasma samples (130-800 μl/column). Record the exact amount used to calculate the target DNA concentration. The volume of final eluate obtained from the extraction column used was 50 [mu]l.

The concentration of circulating EBV DNA was analyzed using a real-time quantitative PCR system targeting the BamHI-W fragment region of the EBV genome (Lo, Y.M.D et al., Cancer Res., 59:1181-1191 (1999)). The principals of the real-time quantitative PCR and reaction preparation procedures were described previously (Lo, Y.M.D et al., Cancer Res., 59:1188-1191 (1999)). Data were collected using ABI Prism 7700 Sequence Detector and analyzed with Sequence Detection System software (version 1.6.3) developed by Applied Biosystems. Results are expressed as the number of copies of the EBV genome contained per milliliter of serum.

All serum DNA samples were also analyzed for β-globin gene by real-time PCR (Lo, Y.M.D. et al., Cancer Res., 59:1188-1191 (1999)), which showed positive signals in all tested samples, thus demonstrating the extraction the quality of the DNA. Multiple negative water blanks were included in each analysis.

More specifically, two real-time quantitative PCR systems for EBV DNA detection were developed: (a) one targeting the BamHI-W region; and (b) the other targeting the EBNA-I region (Baer, et alNature, 310: 207-211, 1984). The BamHI-W system consists of amplification primers (SEQ ID NO: 1) W-44F (5'-CCCAACACTCCACCACACC-3') and (SEQ ID NO: 2) W-119R (5'-TCTTAGGAGCTGTCCGAGGG-3') and double The labeled fluorescent probe (SEQ ID NO: 3) W-67T[5'-(FAM)CACACACTACACACACCCAC-CCGTCTC(TAMRA)-3'] constituted. The EBNA-1 system consists of amplification primers (SEQ ID NO: 4) EBNA-1162F (5'-TCATCATCATCCGGGTCTCC-3') and (SEQ ID NO: 5) EBNA-1229R (5'-CCTACAGGGT-GGAAAAATGGC-3') and a double-labeled fluorescent probe (SEQ IDNO: 6) EBNA-1186T[5'-(FAM)CGCAGGCCCCCTCCAGGTA-GAA(TAMRA)-3']. These fluorescent probes contain a 3'-blocking phosphate group to prevent probe extension during PCR. Primer/probe combinations were designed using Primer Express software (Perkin-Elmer Corp., Foster City, CA). The sequence data of the EBV genome (accession number V01555) was obtained from the GenBank Sequence Database. As a control for plasma DNA amplification, PCR amplification was performed using primers and probes for real-time quantitative PCR of the beta globin gene as previously described in Lo, et al. Am J. Hum Genet 62:768-775, 1998.

A fluorescent PCR reaction with a reaction volume of 50 µl was performed using the components provided by TaqMan PCR Core Reagent Kit (Perkin-Elmer Corp.) except the fluorescent probe and amplification primers. Fluorescent probes were custom made at Perkin-Elmer Applied Biosystems. PCR primers were synthesized by Life Technologies, Inc. (Gaithersburg, MD). Each reaction contained 5 μl of 10× buffer A; 300 nM of each amplification primer; 25 nM (referring to EBV probe) or 100 nM (referring to beta globulin probe) of the corresponding fluorescent probe; 4 mM MgCl ₂ ; dATP, dCTP 400 μM dUTP; 1.25 units of AmpliTaq Gold; and 0.5 units of AmpErase uracil N-glycosylase.

DNA amplification was performed in an Applied Biosystems 7900 Sequence Detector in 384-well reaction plates. Each sample was analyzed twice. Multiple negative water blanks were used for each analysis.

DNA extracted from the EBV-positive cell line Namalwa (American Type Culture Collection CRL-1432; See Klein et al., Int J. Cancer, 10:44-57, 1972) was used as a standard in parallel and repeated in each analysis detection to draw a calibration curve. Namalwa is a diploid cell strain containing two sets of combined viral genomes/cells. Copy number was calculated using a conversion factor of 6.6 pg for DNA/diploid cells (Saiki et al., Science, 239:487-491, 1988). Concentrations of circulating cell-free EBV DNA were expressed as EBV genome copies/ml plasma.

The temperature characteristics of the EBV BamHI-W PCR and EBNA-I PCR systems are the same. Thermal cycling consists of an initial incubation at 50°C for 2 minutes for uracil N-glycosylase, followed by a denaturation step at 95°C for 10 minutes, followed by cycles of denaturation at 95°C for 15 seconds and denaturation at 56°C for 1 minute 40 times.

The amplification data was collected using Sequence Detector, stored in a Dell computer, and then analyzed with the Sequence Detection System software developed by Perkin-Elmer Applied Biosystems. Further concentration calculations were performed using the average of each replicate. The plasma concentration (expressed in copy number/ml) of EBV DNA or β-globin gene was calculated by the following equation.

$\mathrm{<span\; class="notranslate">\; C</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Q</span>}<span\; class="notranslate">\; \&times;</span>\frac{{\mathrm{<span\; class="notranslate">\; V</span>}}_{\mathrm{<span\; class="notranslate">\; dna</span>}}}{{\mathrm{<span\; class="notranslate">\; V</span>}}_{\mathrm{<span\; class="notranslate">\; PCR</span>}}}<span\; class="notranslate">\; \&times;</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{{\mathrm{<span\; class="notranslate">\; V</span>}}_{\mathrm{<span\; class="notranslate">\; ext</span>}}}$

Wherein: C represents the concentration (copy number/ml) of the target substance in blood plasma, Q represents the quantity (copy number) of the target substance determined with sequence detector in PCR, V _DNA represents the total volume of the DNA that obtains after extracting ( Typically 50 μl/Qiagen extract), V _PCR represents the volume of DNA solution used for PCR (typically 5 μl), and V _ext represents the volume of extracted plasma/serum (typically 0.13-0.80 ml).

For, and only those individuals with positive EBV DNA titers, they are asked to return for a second identical test 4-12 weeks after the first test for reconfirmation. In some cases, a second series of measurements may need to be performed after 12 weeks. More often than not, a third series of measurements was performed 12 weeks or more after the second measurement.

result

In the high-risk group A, a total of 42 cases were positive for EBV DNA. Most of these were patients with EBV-associated cancers, such as nasopharyngeal carcinoma and anaplastic lymphoepithelioma of the head and neck region. In this population, serial analysis of EBV DNA was performed in 3 patients 4-12 weeks after the first assay. As shown in Figure 1, all patients with no known history of cancer treatment showed an upward trend in EBV DNA titers.

A total of 74 cases of EBV DNA were positive in normal group B. In this cohort, 27 patients underwent serial testing for EBV DNA between 4-12 weeks after the first analysis. As shown in Figure 2, all but one of these individuals had EBV DNA titers that dropped to zero by the second analysis. The third test results (if done) were all negative.

Few cancer patients with tumor types for which no direct genomic association with Ibovirus were identified never exhibited positive EBV DNA titers in their plasma (Figure 3). A patient with colon cancer had the above positive titer until his tumor had a complete response to treatment, at which time he also became negative for EBV DNA.

Example II

Materials and methods

For 20 patients with positive EBV DNA titer (Group C), the percentage of CD4+CD25+ T cells in peripheral blood was compared with the EBV DNA-negative control group.

Peripheral blood from these 20 patients was obtained at the time of blood collection, injected into a Vacutainer tube containing heparin, diluted 2:1 with 1×Dulbecco's phosphate buffer (Mediatech) without calcium and magnesium, and then incubated at room temperature on poly Glycosomes (ficoll) were separated by centrifugation at 1000×g for 20 minutes on a density gradient. Peripheral blood mononuclear cells were collected, washed and cryopreserved in RPMI1640 containing 20% fetal bovine serum and 10% DMSO for future use.

Cell types were determined using a four-color hemocytometer and flow cytometric analysis using activity markers of anti-CD3-PerCP, anti-CD4-APC or anti-CD8-FIT, anti-CD25-PE. Briefly, cells were incubated for 30 min at room temperature in the dark, washed once with FACS buffer (PBS 0.05%, FCS 2 mM, EDTA and 0.01% sodium azide), and fixed with 2% formaldehyde. Flow cytometry analysis was then performed.

result

Compared with EBV DNA-negative individuals, CD4+CD25+ T cells were significantly increased in EBV DNA-positive individuals.

discuss

These data demonstrate that circulating cell-free EBV DNA testing of plasma samples obtained from ordinary individuals can be used to screen for the development of various forms or types of cancer.

Also, for the first time, it was shown that the percentage of regulatory T cells with a CD3+CD4+CD25+ phenotype and a role in cancer development was increased in circulating EBV DNA-positive individuals. Although these T cells were originally discovered to prevent host autoimmunity, they also hinder the immune surveillance of the host against different tumor antigens. Thus, the rise of cells with this phenotype leads to the development of cancer, from precancerous, carcinoma in situ, to full-fledged cancer.

This increase in cancer incidence is not limited to cancers directly related to EBV such as nasopharyngeal cancer, some gastric cancers, and various lymphomas, but has also been demonstrated in other common cancers such as breast, lung, liver, and colon cancers. As explained further below, there are indeed several reasons why the present invention may be useful as a routine cancer screening tool.

One reason is that EBV is the most prevalent virus that spends almost its entire life dormant in the human body. Furthermore, the virus is one of the most proto-oncoviruses not only in the oncogenic potential of EBV but also in a variety of cancers in which cancer genome changes are directly associated with the virus. When this highly efficient proto-oncogene is bound to the regulatory T cell population, it is more likely to lead to the development of cancer cells, so the theoretical hypothesis is that the presence or absence of this EBV characteristic can cause cancer in the absence of genomic integration.

In addition to using a single determination of EBV DNA titer as a routine cancer screening tool, we show for the first time that serial Tests that can distinguish those cancer patients from non-cancer patients (with one exception).

We also show that cancer patients with no genomic association with EBV also screen positive by the present invention. In one case, the patient's EBV viral titer became negative after full recovery.

The above discussion concludes that the present invention is a very valuable tool for routine screening of cancers including precancerous and carcinoma in situ lesions and other cancers that have not been shown to be directly or genomically associated with EBV. Of course, it can be used as a screening tool for EBV-related or concomitant cancers.

Sequence Listing

SEQ ID NO: 1: W-44F (5'-CCCAACACTCCACCACACC-3')

SEQ ID NO: 2: W-119R (5'-TCTTAGGAGCTGTCCGAGGG-3')

SEQ ID NO: 3: W-67T[5'-(FAM)CACACACTACACACACCCAC-CCGTCTC(TAMRA)-3']

SEQ ID NO: 4: EBNA-1162F (5'-TCATCATCATCCGGGTCTCC-3')

SEQ ID NO: 5: EBNA-1229R (5'-CCTACAGGGT-GGAAAAATGGC-3')

SEQ ID NO: 6: EBNA-1186T[5'-(FAM)CGCAGGCCCCCTCCAGGTA-GAA(TAMRA)-3']

Claims (12)

1. A method for screening whether a patient has cancer or precancer, said method comprising the steps of: (a) obtaining the fluid portion from a patient blood sample; (b) extracting DNA from said liquid portion; (c) analyzing the amount of Ebovirus (EBV) DNA present in said DNA; and (d) repeat steps (a)-(c), Wherein the constant or rising trend of the content of the EBV DNA indicates that the patient suffers from cancer or precancer. 2. The method of claim 1, wherein said cancer is selected from nasopharyngeal carcinoma, gastric cancer and lymphoma. 3. The method of claim 1, wherein said cancer is selected from the group consisting of lung cancer, colon cancer, parotid gland cancer, breast cancer and liver cancer. 4. The method of claim 1, wherein said cancer is selected from cancers other than nasopharyngeal cancer, gastric cancer, lymphoma, lung cancer, colon cancer, parotid gland cancer, breast cancer and liver cancer. 5. The method of claim 1 or 2 or 3 or 4, wherein said liquid fraction is serum or plasma. 6. The method of claim 1 or 2 or 3 or 4, wherein said repeating steps are performed within 4-12 weeks after the first determination. 7. The method of claim 5, wherein said repeating steps are performed within 4-12 weeks after the first determination. 8. The method of claim 1 or 2 or 3 or 4, wherein said repeating step is performed outside of 4-12 weeks after the first measurement. 9. methods as claimed in claim 1 or 2 or 3 or 4, wherein the method for analyzing the content of EBV DNA is to carry out by hybridization, amplification, quantitative methods, and select any sequence or sequence fragment of the EBV genome as Binding sites for any primer and probe or combination thereof. 10. methods as described in claim 1 or 2 or 3 or 4, also comprise the steps: A blood sample is obtained from the patient. 11. methods as described in claim 1 or 2 or 3 or 4, also comprise the steps: The amount of EBV DNA present in the liquid fraction is compared to a control. 12. A screening kit for detecting the presence of EBV DNA in patient serum or plasma, wherein said kit comprises reagents suitable for said analysis.