WO2012072515A2

WO2012072515A2 - Lmp1 a diagnostic and prognostic marker for epstein-barr virus (ebv)-associated lymphoma

Info

Publication number: WO2012072515A2
Application number: PCT/EP2011/071055
Authority: WO
Inventors: Tadamasa Ooka; Karim Houali; Sylvie Fiorini
Original assignee: Centre National De La Recherche Scientifique (Cnrs); Universite Claude Bernard Lyon 1
Priority date: 2010-11-29
Filing date: 2011-11-25
Publication date: 2012-06-07
Also published as: WO2012072515A3

Abstract

Method for detecting an Epstein-Barr Virus (EBV)-associated lymphoma comprising measuring the amount of LMP1 antigen of EBV in a blood sample.

Description

LMPl a diagnostic and prognostic marker for Epstein-Barr virus (EBV)-associated lymphoma

The present invention relates to methods for diagnostic and prognostic of EBV- associated lymphoma based on the detection of the LMPl antigen in a blood sample.

The Epstein-Barr Virus (EBV) is associated with various human cancers. These cancers include cancers of the epithelial cells such as Nasopharyngeal carcinoma (NPC) and Gastric carcinoma (GC) but also lymphomas such as Burkitt lymphoma (BL), Hodigkin lymphoma (HL) and Non Hodgkin lymphoma (NHL). Worldwide these cancers are an important problem of public health. There are no diagnostic and predictive tests to detect the development of these tumors. Classic diagnostic tests for detection of EBV are generally based on the detection of immunoglobulines against viral proteins of EBV. However, infection by the Epstein-Barr virus is ubiquitous in humans and antibodies directed to EBV are therefore not sufficient for detecting EBV associated lymphomas. Diagnosis of EBV-associated lymphomas is mainly based on clinical manifestions and may be confirmed with a test detecting anti-EBV antibodies.

Burkitt's lymphoma (BL) associated with EBV has a high incidence in Africa (more than 95% of BL is associated with EBV) while only 20-30% of BL is associated with EBV in European populations. More than 60% of Non-Hodgkin lymhoma (NHL) are also associated with EBV. High incidence of NHL and primitive brain lymphoma (PBL) in HIV-infected patients are all EBV positive. Further, cancerous B cells infected by EBV are generally more virulant than EBV negative cancerous B cells.

Consequently, there is a need to develop a new generation of specific and feasible diagnostic tests for EBV-related lymphomas.

EBV encodes two main oncoproteins : LMPl (Latent Membrane Protein 1) and

BARF 1 (BamHl A right frame 1 ). Both oncoproteins play a crucial role for the development of EBV-associated tumor. LMPl (Latent membrane protein- 1) belongs to a family of latent antigens expressed on the surface of cells infected by EBV and indispensable for B cell immortalization. LMPl possesses six transmembrane domains and an intracellular C-terminal domain. The C-terminal region includes two major functional domains, CTARl, and CTAR2. The extracellular domains called « short loops » of LMPl protein are present on the surface of EBV-infected cells. LMP l is essential for B cell immortalisation activating several cellular genes, like NFkB, A20 and EGF-R which can inhibit cell differentiation when transfected into epithelial cells (Ooka T : 2005. In. Epstein-Barr Virus. Horizon Press, Annette Griffin: Edited by Erie S. Robertson. Chapter 28 : p.p 613-630.). Classically, LMP l proteins have been localized on the cellular membrane. However recent data showed that LMPl could be secreted and localized in exosomal components in the culture medium of PC-derived c666-l cell line (Houali K, X. Wang, Y. Shimizu, D. Djennaoui, J. Nicholls, S. Fiorini, A. Bougermouh and T. Ooka. Clin. Cancer Res. 2007 13 : 4993-5000). The essential oncogenic role of LMPl is determined by its activation of NFkB. The inhibition of LMPl expression resulted in cell apoptosis linked to the diminution of NFkB expression (Kieff and Rickinson, 2007, Fields Virology 5th Edition-Fields BN, Knipe DM,Howley PM (ed.) Lippincott-Williams & Wilkins Publishers : Philadelphia, 2007, pp.2603-2654).

The specific secretion of two oncoproteins (LMPl and BARFl) in serum and saliva of NPC patients was recently demonstrated (Houali K, X. Wang, Y. Shimizu, D. Djennaoui, J.Nicholls, S. Fiorini, A. Bougermouh and T. Ooka. Clin. Cancer Res. 2007. 13 :4993-5000). LMPl found in serum is complexed with an exosomal component (LMPl/exosome). This complex is able to activate the cell cycle by an autocrine mechanism, while free LMPl (without exosome) is unable to activate the cell cycle (Houali K, X. Wang, Y. Shimizu, D. Djennaoui, J. Nicholls, S. Fiorini, A. Bougermouh and T. Ooka. Clin. Cancer Res. 2007. 13 : 4993-5000). However, the presence of both BARF l and LMPl in the serum of lymphoma patients has never been investigated. Moreover, NPC is a cancer of the epithelial cells whereas lymphomas are cancers of the lymphocytes.

EP 1 229 043 describes peptides derived from Epstein Barr Virus (EBV) proteins LMPl, LMP2 and BARFl and use of said peptides for immunization and therapeutic vaccination. EBV+-cells or circulating EBV-infected cells in blood may be visualized in situ using anti-LMPl . EP 1 229 043 does not describe detection of secreted LMPl in blood samples.

WO 03/048337 is related to antibodies and antibody fragments directed against extracellular domains of EBV LMP proteins. These antibodies may be used to detect cells expressing LMP proteins to quantify the level of expression. WO 03/048337 does not describe detection of secreted LMPl in blood samples.

These documents describe detection of LMP l in circulating cells or in tumor biopsies. However, EBV infected lymphocytes are found in the healthy population and infectious mononucleosis (IM) is associated with high levels of EBV-infected lymphocytes in the blood circulation. Detection of EBV in lymphocytes or in blood was therefore not considered as a useful diagnostic tool to detect EBV-associated lymphomas.

The present invention now surprisingly shows that LMPl is detected in the serum of patients with EBV-associated lymphoma whereas LMPl is not detected in the serum of patients with infectious mononucleosis (IM) or in the serum of a healthy population comprising individuals infected with EBV.

LMPl oncoprotein was searched in the serum of Algerian patients with BL (98% positive for EBV) and also in the serum of Algerian patients with infectious mononucleosis (IM). The results were compared with those of healthy individuals living in this high risk area (North Africa). The detection of LMPl oncoprotein in serum was carried out by ELISA test. Unexepctedly, the LMPl antigen was found specifically in the serum of BL patients. Detection of LMPl oncoprotein in serum therefore constitutes a specific tumor marker to detect the development of EBV-associated lymphoma. Summary of the invention

A first object of the present invention is a method for detecting an Epstein-Barr Virus (EBV)-associated lymphoma comprising detecting the presence of the LMPl antigen of EBV in a blood sample previously taken from a patient.

Preferably, secreted LMPl or secreted LMPl complexed with exosomes is detected and/or quantified in the blood sample.

In a preferred embodiment, the present invention encompasses methods for detecting an Epstein-Barr Virus (EBV)-associated lymphoma in a patient comprising the following steps:

a) Measurement of the amount of LMPl antigen of EBV in a blood sample previously taken from said patient;

b) Comparing the amount of LMPl antigen of EBV in the blood sample with a reference value selected from the median level of LMP l antigen in a healthy population, the median level of LMPl antigen in patients suffering from infectious mononucleosis and at least lOOng/ml;

wherein an amount of EBV LMPl in the blood sample greater than the reference value is indicative of an EBV-associated lymphoma.

A second object of the present invention is a method for determining the prognosis of an EBV-associated lymphoma comprising detecting the presence of the LMPl antigen of EBV in a blood sample previously taken from a patient wherein the presence of the LMP1 antigen is associated with an aggressive cancer and/or with a poor prognosis.

Preferably, secreted LMP1 or secreted LMP1 complexed with exosomes is detected and/or quantified in the blood sample.

In a preferred embodiment, the present invention encompasses methods for determining the prognosis of an EBV-associated lymphoma in a patient comprising the following steps:

a) Measurement of the amount of LMP1 antigen of EBV in a blood sample previously taken from said patient;

b) Comparing the amount of EBV LMP1 in the blood sample with a reference value selected from the median level of LMP 1 antigen in a healthy population, the median level of LMP1 antigen in patients suffering from infectious mononucleosis and at least lOOng/ml;

wherein an amount of EBV LMP1 in the blood sample greater than the reference value is is associated with an aggressive cancer and/or with a poor prognosis.

In preferred embodiments, the reference value is lOOng/ml, 200 ng/ml, 500 ng/ml or 1000 ng/ml.

In preferred embodiments, the EBV-associated lymphoma is selected from Burkitt's lymphoma, Hodgkin lymphoma, Non Hodgkin lymphoma and Primitive brain lymphoma.

Preferably, the EBV-associated lymphoma is Burkitt's lymphoma.

Preferably, the EBV-associated lymphoma is Primitive brain lymphoma in HIV- infected patients.

In preferred embodiments, the blood sample is a plasma sample.

More preferably, the blood sample is a serum sample.

In preferred embodiments, measurement of the amount of LMPl antigen and/or detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody.

Preferably, measurement of the amount of LMPl antigen and/or detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody specifically binding the LMPl antigen of SEQ ID No. 1.

More preferably, measurement of the amount of LMPl antigen and/or detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMPl antigen fragment from position 188 to position 386 of SEQ ID No. 1.

Even more preferably, measurement of the amount of LMP l antigen and/or detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMPl antigen fragment from position 306 to position 318 of SEQ ID No. 1.

In preferred embodiments, measurement of the amount of LMPl antigen and/or detecting the presence of the LMPl antigen of EBV is performed by ELISA.

Preferably, the amount of LMPl antigen in the sample is > lOOng/ml.

In preferred embodiments, the methods of the present invention further comprise detecting the presence of the BARF 1 antigen of EBV in said blood sample previously taken from said patient.

Sequence listing

SEQ ID No. 1 : Amino acid sequence of LMPl(Latent Membrane Protein-1) from human Herpesvirus 4 type 1 (Genbank YP_401722.1).

Description of the invention

The present invention is related to the use of the LMPl antigen from the Epstein- Barr virus (EBV) as a marker for the diagnosis and/or prognosis of EBV-associated lymphomas. LMPl is advantageously detected in blood samples of patients suffering from EBV-associated lymphomas allowing for a quick, cheap and simple diagnosis of these lymphomas. Detection of the LMPl antigen in the blood also provides an early diagnosis of these cancers whereas other methods only provide for detection at later stages. The presence of EBV has been associated with more aggressive lymphomas and detection of the LMPl antigen in the blood of lymphoma patients also provides a tool for determining the prognosis of these cancers.

The LMPl (Latent Membrane Protein-1) antigen of EBV is a well-known oncoprotein of EBV. LMPl has been extensively described in the scientific literature. The amino acid sequence of LMPl (Latent Membrane Protein-1) from human Herpesvirus 4 type 1 is for example described as Genbank YP_401722.1. The sequence of LMPl from EBV is also shown in SEQ ID No. 1.

Detection of LMPl in blood is correlated and/or associated with the presence of EBV-associated lymphoma.

Preferably, the methods of the present invention are in vitro methods. The methods of the present invention are carried out on a biological sample previously taken from a patient. The term "sample" refers to any biological sample obtained/taken from a patient including a blood sample, a plasma sample, a tissue sample, a cell sample or a tumor sample.

Advantageously, the biological sample is a blood sample or a saliva sample.

The terms "blood sample" refer to a whole blood sample, a plasma sample or a serum sample. In preferred embodiments, the biological sample is a plasma or serum sample.

In the methods of the present invention, the patient may be or may not be a patient suspected of suffering from an EBV-associated lymphoma. Advantageously, the methods of the present invention provide for an early diagnosis of EBV-associated lymphomas.

A second object of the present invention is a method for determining the prognosis of an EBV-associated lymphoma comprising detecting the presence of the LMPl antigen of EBV in a blood sample previously taken from a patient wherein the presence of the LMPl antigen is associated with an aggressive cancer and/or with a poor prognosis.

Cancer prognosis is often determined based on histological and clinical data such as tumor size, invasion, spread to lymph nodes or metastasis. However, this determination is often difficult to make at early stages when clinical symptoms and histological data are not available or do not provide sufficient information.

In the methods of the present invention, the presence of the LMPl antigen of EBV is statistically significantly correlated with aggressive lymphoma, increased disease recurrence and worse prognosis.

The terms "invasive" or "aggressive" refer to a lymphoma or to a quickly growing tumor having a tendency to extend beyond its boundaries into adjacent tissues.

A "prognosis" is the likely course and outcome of a disease. The prognosis may include the likelihood of complications of the cancer, of metastasis, of spread, probable outcome of the cancer, likelihood of recovery, overall survival rate and /or overall death rate. Preferably, it is the probability that a patient will recover or have a recurrence/relapse of the cancer. This information is useful to the patient but also to the physician and/or clinician in determining the most effective course of treatment. A determination of the likelihood for a cancer relapse or of the likelihood of metastasis can assist the physician and/or clinician in determining whether a more conservative or a more radical approach to therapy should be taken. Prognosis provides for selection and classification of patients who are predicted to benefit from a given therapeutic regimen.

In some embodiments, the methods of the present invention provide prognosis for a EBV-associated lymphoma after it has been diagnosed.

The terms "EBV-associated lymphoma" refer to Burkitt' s lymphoma, Hodgkin lymphoma, Non Hodgkin lymphoma and Primitive brain lymphoma.

The methods of the present invention are preferably directed to the diagnosis and/or prognosis of Burkitt's lymphoma.

In other embodiments, the methods of the present invention are related to the diagnosis and/or prognosis of Primitive brain lymphoma in HIV-infected patients.

The LMPl antigen of EBV may be detected and/or quantified in the biological sample taken from the patient by any appropriate method.

In preferred embodiments, detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody. The anti-LMPl antibody may be a polyclonal antibody or a monoclonal antibody.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies of any isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies, chimeric antibodies, humanized antibodies and antibody fragments. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid.

The terms "anti-LMPl antibody" refers to antibodies that are reactive with the LMPl antigen of EBV and/or antibodies that bind specifically to the LMPl antigen of EBV.

A typical IgG antibody is comprised of two identical heavy chains and two identical light chains that are j oined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions", which are primarily responsible for binding an epitope of an antigen. They are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N- terminus. The more highly conserved portions of the variable regions are called the "framework regions".

As used herein, " VH" or " VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab')2 fragment. Reference to "VL" or "VL" refers to the variable region of the immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab')2 fragment.

A "polyclonal antibody" is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

A "monoclonal antibody", as used herein, is an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies forming this population are essentially identical except for possible naturally occurring mutations which might be present in minor amounts. These antibodies are directed against a single epitope and are therefore highly specific.

An "epitope" is the site on the antigen to which an antibody binds.

As used herein, a "chimeric antibody" is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass.

Preferably, detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody specifically binding the LMPl antigen of SEQ ID No. 1.

More preferably, detecting the presence of the LMPl antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMPl antigen fragment from position 188 to position 386 of SEQ ID No. 1.

Even more preferably, detecting the presence of the LMP l antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMPl antigen fragment from position 306 to position 318 of SEQ ID No. 1.

As used herein the term "binding" refers to an antibody or antibody fragment that reacts with an epitope of the intracellular domain of LMP l corresponding to the polypeptide from position 188 to position 386 of SEQ ID No. l or that was raised against the intracellular domain of LMPl corresponding to the polypeptide from position 188 to position 386 of SEQ ID No. l . Preferably, the antibody reacts with an epitope from the peptide from position 306 to 318 of SEQ ID No. 1 or was raised against the peptide from position 306 to 318 of SEQ ID No. 1. Preferably, the antibody binds specifically to the intracellular domain of LMPl and does not crossreact with other antigens. Thus, the antibody reacts with one specific antigen.

Antibodies binding specifically to the intracellular domain of LMPl are available commercially such as for example antibody S 12 available from BD Sciences (France). Alternatively, antibodies binding specifically to the intracellular domain of LMPl or to fragments thereof may be produced by standard techniques. Preferred antibodies are antibodies binding to the peptide having the sequence from position 306 to 318 of SEQ ID No. 1 which is also specifically bound by monoclonal antibody S 12. Preferably, the antibodies bind to the same epitope as antibody SI 2.

The methods of the present invention comprise the detection of the LMPl antigen in a biological sample taken from a patient using any appropriate test. These test methods include for example western blots or immunoblotting or any methods derived thereof. In preferred embodiments, detection of the LMPl antigen is performed by ELISA (Enzyme- linked immunosorbent assay), a sandwich assay or any appropriate immunoassay based on anti-LMPl antibodies to specifically detect this antigen.

The methods of the present invention comprise the detection of he LMPl antigen of EBV for the diagnosis and/or prognosis of EBV-associated lymphomas. The methods of the present invention may further comprise a quantification of LMPl in the sample taken from the patient or a measurement of the amount of LMPl antigen present in the sample taken from the patient.

In preferred embodiments, an amount of LMPl antigen in the sample greater than lOOng/ml, 200 ng/ml, 500ng/ml or lOOOng/ml is indicative of an EBV-associated lymphoma. The sample is preferably a serum sample.

The amount of LMPl antigen may be compared to a control sample.

In some embodiments, the control sample is the median level of LMPl antigen observed in a healthy population or in patients suffering from infectious mononucleosis. The methods of the present invention may further comprise the detection of another oncoprotein from EBV and more preferably may further comprise the detection of the BARFl antigen from EBV.

The BARFl (BamHI A right fragment) antigen of EBV is a well-known oncoprotein of EBV. BARF l has been extensively described in the scientific literature. The amino acid sequence of BARFl from human Herpesvirus 4 type 1 (Epstein-Barr virus) is for example described as Genbank No. YP 401719.1.

The BARFl antigen is preferably detected in a blood sample and more preferably in a plasma or serum sample.

The BARFl antigen may also be detected in saliva.

LMP 1 and BARF l may be detected in the same biological sample or may be detected in two separate biological samples taken from the same patient.

Another object of the present invention is a method for detecting an Epstein-Barr Virus (EBV) associated lymphoma comprising the following steps:

- obtaining a blood sample from a patient,

- testing said sample for the presence of the LMP1 antigen.

A further object of the present invention is a method for detecting an Epstein-Barr Virus (EBV) associated lymphoma comprising the following steps:

obtaining a blood sample from a patient,

- testing said sample for the presence of the LMP1 antigen wherein the presence of the LMP1 antigen is a marker for an Epstein-Barr Virus (EBV) associated lymphoma.

The present invention is also directed to a method for determining the prognosis of an Epstein-Barr Virus (EBV) associated lymphoma comprising the following steps:

- obtaining a blood sample from a patient,

- testing said sample for the presence of the LMP1 antigen wherein the presence of the LMP1 antigen in the sample is associated with a poor prognosis.

Figures

Figure 1 : Analysis of LMP1 oncoprotein in serum of BL and IM patients and healthy donors Examples

[I] Samples and preliminary testing

For Burkitt's lymphoma (BL), there are two kinds of clinical manifestations of BL: abdominal and maxillary mass. In Algeria, the presence of an abdominal mass represents the majority of clinical cases with about 90% and the presence of a maxillary mass represents about 10%. The sera samples used here come from young patients, between 5 and 7 years old, exhibiting an abdominal mass. To confirm the clinical manifestation a diagnostic test to detect anti-EBV antibodies was used (commercial ELISA kit from EUROIMMUN® A.G., Germany). This test is based on detection of anti-Ig anti-EA-R (early viral protein).

For Infectious mononucleosis (IM), the sera used here come from young patients, age between 9 and 24 years old. IM was identified by ELISA test with anti-IgM anti-VCA (for fresh infection) and with anti-IgG anti-EBNA (for old infection). These kits are for example commercially available from EUROIMMUN® A.G. (Germany). For the serological diagnosis of fresh infections has also been established by investigating the antibody avidity (EUROIMMUN® A.G., Germany).

[II] Detection of LMP1 with anti-LMPl by ELISA test

1) Coating ΙΟΟμΙ containing 50μ1 of human serum and 50μ1 PBS in a well of 96 wells, incubated over night et 4°C.

2) Remove the serum, then wash 4 times with PBS containing 0.005%) Tween 20 at room temperature under agitation

3) Add ΙΟΟμΙ of 2% BSA (Bovin serum albumin) and incubate 2-4 hours at 4°C

4) Remove BSA and 4 times washing with PBS-0.005%) Tween 20 at room temperature under agitation

5) Add ΙΟΟμΙ of monoclonal anti-LMPl antibody (described in WO2010/015704) diluted to 1 : 1000 and incubated 30 mins at 37°C

6) Remove anti-LMPl antibody and 4 times washing with PBS-0.005%> Tween 20 at room temperature under agitation

7) Add anti-mouse antibody coupled with peroxydase diluted to 1 : 1000 and incubated 30 mins at 37°C under agitation

8) Remove anti-mouse, then wash 4 times with PBS-0.005%> Tween 20 under agitation 9) Add ΙΟΟμΙ of ODP solution and incubate 30 mins at room temperature under agitation, then stop the reaction by addition of 25 μΐ of 5 mM sulfuric acid

10) Read the samples by absorbance at 405nm. [II] Results

More than 98 % of sera from BL patients contained high levels of LMP1 oncoprotein. While LMP1 was not detected or at very low levels in sera from healthy individuals. Sera from IM patient (Infectious Mononucleosis) in Algeria showed values similar to those of healthy donors. In conclusion, LMP1 oncoprotein was detected significantly and specifically in BL patients. Anti-LMPl antibody used here are a useful tool for predictive diagnosis of lymphoma development.

REFERENCES

Ooka T : 2005. In. Epstein-Barr Virus . Horizon Press, Annette Griffin: Edited by Erie S. Robertson. Chapter 28 : p.p 613-630

- Houali K, X. Wang, Y. Shimizu, D . Dj ennaoui, J. Nicholls, S . Fiorini, A.

Bougermouh and T. Ooka. Clin. Cancer Res. 2007 13 : 4993-5000

Kieff and Rickinson, 2007, Fields Virology 5th Edition-Fields BN, Knipe DM,Howley PM (ed.) Lippincott-Williams & Wilkins Publishers : Philadelphia, 2007, pp.2603-2654

PATENT REFERENCES WO2010/015704

Claims

1. Method for detecting an Epstein-Barr Virus (EBV)-associated lymphoma in a patient comprising the following steps:

b) Comparing the amount of LMP1 antigen of EBV in the blood sample with a reference value selected from the median level of LMP 1 antigen in a healthy population, the median level of LMP1 antigen in patients suffering from infectious mononucleosis and at least lOOng/ml;

wherein an amount of EBV LMP1 in the blood sample greater than the reference value is indicative of an EBV-associated lymphoma.

2. Method for determining the prognosis of an EBV-associated lymphoma in a patient comprising the following steps:

wherein an amount of EBV LMPl in the blood sample greater than the reference value is associated with an aggressive cancer and/or with a poor prognosis.

3. Method according to anyone of claims 1-2, wherein the reference value is at least lOOng/ml, 200 ng/ml, 500 ng/ml or at least 1000 ng/ml.

4. Method according to anyone of claims 1-3, wherein the EBV-associated lymphoma is selected from Burkitt's lymphoma, Hodgkin lymphoma, Non Hodgkin lymphoma and Primitive brain lymphoma.

5. Method according to anyone of claims 1-4 wherein the EBV-associated lymphoma is Burkitt's lymphoma.

6. Method according to anyone of claims 1-4 wherein the EBV-associated lymphoma is Primitive brain lymphoma in HIV-infected patients.

7. Method according to anyone of claims 1-6 wherein the blood sample is a plasma sample.

8. Method according to anyone of claims 1-6 wherein the blood sample is a serum sample.

9. Method according to anyone of claims 1-8 wherein measurement of the amount of LMP1 antigen of EBV is performed with an anti-LMPl antibody.

10. Method according to claim 9 wherein measurement of the amount of LMP1 antigen of EBV is performed with an anti-LMPl antibody specifically binding the LMP1 antigen of SEQ ID No. 1.

11. Method according to claim 10 wherein measurement of the amount of LMP1 antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMP1 antigen fragment from position 188 to position 386 of SEQ ID No. 1.

12. Method according to claim 11 wherein measurement of the amount of LMP1 antigen of EBV is performed with an anti-LMPl antibody specifically binding to the LMP1 antigen fragment from position 306 to position 318 of SEQ ID No. 1.

13. Method according to anyone of claims 1-12 wherein measurement of the amount of LMP1 antigen of EBV is performed by ELISA.

14. Method according to anyone of claims 1 to 13, further comprising detecting the presence of the BARF1 antigen of EBV in said blood sample previously taken from said patient.