The diagnosis of Epstein-Barr virus-associated polymorphic B cell lymphoma in immunocompromised patients : Review of methods

P BEAUPARLANT, C ALFIERI, J-H JoNCAS. The diagnosis of Epstein-Barr virus-associated polymorphic B cell lymphoma in immunocompromised patients: Review of methods. Can J Infect Dis 1991 ;2(3) :109-115. Polymorphic B cell lymphoma and diffuse B cell lymphoproliferation associated with Epstein-Barr virus infection is increasingly reported in immunodeficient patients. Accurate diagnosis of these pathologies is essential because the appropriate treatment regimens for the patients in question differ from those for patients with other lymphoproliferative diseases. Two complementary techniques are' currently used in the d iagnosis and characterization of Epstein-Barr virus-associated B cell lymphomas and diffuse B celllymphoproliferalion . Immunofluorescence a llows specific detection of Epstein-Barr nuclear antigens in lymphomatous tissue. Molecular hybridization with the Bam Hl-W and/or Bam Hl -NJ probes confirms the presence of the Epstein-Barr vi rus genome in tumour cells. The Bam H 1-NJ probe is also usefu l in determining the clonality of the tumour and the replication mode, episoma l or linear. of the viral genome. The polymerase chain reaction method allows detection of the Epstein-Barr virus o-enome within 24 h in these tumours and is more sensitive.

permet de deceler les antigenes nucleaires codes par le virus d 'Epstein -Barr dans les Ussus lymphomateux.tandis que l'hybridation molecula ire utilisanl les sondes Bam-Hl -W ou Bam H l -NJ con fi rme Ia presence du genom e d u virus d'Epstein -Barr dans les cell u les tumora les.La sond e Bam H l -NJ per met egalement de determiner Ia clon a lite de Ia tume ur et le mode de replication .episomique ou lin eaire .du genome viral.L'amplification de I'ADN vi ra l par Ia reaction en chaine a Ia polymerase permel la detection du genome du virus Epslein -Barr dans ces tu m eurs en 24 h et cette meth ode est plus sensible.
E PSTEIN -BARR VlRUS.THE CAUSATIVE AGENT OF' infectious mononucleosis (1), is a DNA tumour virus associated with Burkitt's lymphoma and nasopharyngeal carcinoma (2).More recently, polymorphic B cell lymphomas and diffuse B cell lymphoproliferations associated with Epstein-Barr virus infection have been reported in patients with congenital (3) and acquired (4-9) immunodeficiencies .These pathological conditions have been reported in transplant patients (4)(5)(6)(7), in patients receiving chemotherapy for leukemia (8) and.more frequently, in patients suffering from the acquired immune deficiency syndrome (AIDS) (9).These tumours may be monoclonal or polyclonal , and monoclonal tu mours may be associated with chromosomal abnormalities ( 1 0).The term 'polymorphic' refers to the histological appearance of these tumou rs. which may be described as consisting of a mixture of undifferentiated B cell lymphoblasts, immunoblasts and fully differentiated plasma cells (11) .Because this virus has the property of immortalizing B cells in vitro, it is thou ght that th e same phenomenon might occur in vivo, leading to B cell lymphomatous proliferations in th e absence of adequate immune function.An accu rate d iagnosis of these tumours is essential becau se the treatment regimens appropriate fo r th ese patients differ from those for patients with oth er lymphoproliferative diseases.It was reported th at two leukemic patients who developed polymorp hic B cell lymphomas recovered when im munosuppressive drugs were discontinued, and th eir leukemia has not recurred to date , after periods of four and six years, respectively (12).
In order to facilitate th e diagnosis of Epstein-Barr virus-associated lymphomatous proliferations, the authors report two case studies in which the lymphomas were Epstein-Barr virus positive and monoclonal as assessed by immunofluorescence and molecular hybridization.

MATERIALS AND METHODS
Immunofluorescence for detection of viral antigens in tissue: Detection of the Epstein-Barr nuclear antigen on imprints and frozen sections of biopsy specimens was performed by anticomplement immunofluorescence with positive and nega-110 tive control antisera (1 3 ).Th ese reference sera contained no antinuclear antibodies as determined by anticomplement immunofluorescence on the Epstein-Barr virus negative Molt-4 cell line.Determination of t umour clonality by molecular hybridi zation: Molecular hybridization with a specific Epstein-Barr virus probe (Bam Hl-NJ) was used to assess whether the tumou r was polyclonal or monoclonal ( 14).Briefly, the 7 kilobasepair cloned Epstein-Barr virus restr iction fragment , Bam Hl-NJ, which was constructed by fusion of the outermost 3' and 5' Bam H 1 fragments of the linear genome while deleting the terminal repeat segments, was labelled with phosphorus-32 by the random priming meth od (15) with modifications (16).Ten micrograms of tumour DNA was digested with Bam HI, electrophoresed and blotted onto n itrocellu lose by the method of Southern (17).After drying for 2 h in an 80°C oven , blots were prehybridized for 3 hat 42°C in 50% formamide, 6x sse.0.2% polyvinylpyrrolidone, 0.2% fico ll , 50 mM sodium p h osphate (pH 7.0) and 100 11g/ mL of sheared, freshly denatured salmon spenn DNA.The radioactive probe (Epstein-Barr virus Bam Hl-NJ) was then added to the prehybridization solution, and the blots were incubated in a 42°C waterbath overnight.After a series of washes ( 1 0 mins in 2x SSC at 20°C: 60 m ins in 0.1x SSC p lus 0.1% sodium dodecylsulphate at 60°C; 3 mins in 0.1x SSC at 20°C), blots were dried in a 60°C oven for 30 mins .Autoradiography was perfom1ed with Kodak XAR film placed between two Cronex screens.

RESULTS
The following case studies, used here to illustrate the authors' diagnostic approach, have been discu ssed in detail elsewhere (12).and will only be briefly described below.Case 1: A girl suffering from congenital combined immune deficiency associated with adenosine deaminase deficiency d ied at the age of three years from a monoclonal (IgM and gamma-light chain positive) primruy B cell lymphoma of the brain (Figure 1).Case 2 : A 4-year-old boy, on maintenance chemotherapy for acute lymphoblastic leukemia in remission, subsequently developed primruy

Diagnosis of EBV in immunocompromised patients
Epstein -Barr virus infection and intestina l tumours.These tumours were polymorphic in appearance: one was positive for IgA-kappa and another for IgM-gamma , suggesting multiclonality.
Immunofluorescence studies using a human Epstein ~Barr n u clear antigen positive antiseru m revealed Epstein-Barr nuclear antigens in the fresh tumour cells (imprints and frozen sections) from both cases (Figure 2).No fluorescence was observed with the negative control serum.
Molecular hybridization using the Bam H1 -NJ probe was performed on DNA extracted from control laboratory cell lines P3HR-1.B95-8 , Raji and Molt-4, as well as from tumour biopsies obtained from cases 1 and 2 (Figure 3).Molt -4 is aT cell leukemia line which is negative for Epstein-Barr virus.The Raji cell line, which is nonproductive for virus r eplication, contains approximately 50 copies of episomal Epstein-Barr virus DNA per cell but no linear Epstein-Barr virus DNA (18).The virus-producing P3HR-1 and B95-8 cell lines contain linear Epstein-Barr virus genomes in addition to episomal copies (19)(20)(21) .Hyb1idization results reveal the presence of one high molecular weight band for both Raji and the tumour biopsies (Figure 3) .Thus. one may deduce that the BamH1 -NJ probe identified the large fused terminal regions of

4.4-
Figure 3) Left Molecular hybridization using Bam Hl-NJ probe performed on DNA ext.raciedfrom the laboratory cell line B95-8 (1).Raji (2).Molt-4 (3) and P3HR1 (4).Right Molecular hybridization using the Bam H 1-NJ probe performed on DNA e>tl.ract.edfromtumour biopsies obtainedjrom cases 1 (1) and 2 (2).MW Molecular weight in Jcilobasepairs the Epstein-Barr virus episome consisting of the Bam H1-N fragment plus the Bam H1-J fragment linked by the presence of a clonally defined number of terminal repeat segments (each consisting of 500 basepairs) (14).These findings confirmed the monoclonality of the tumours.Molecular hybridization on B95-8 and P3HR-1 viral DNA demonstrated multiple DNA fragments of molecular weights ranging from 4.4 to 9.4 kilobasepairs (Figure 3).The high molecular weight fragment corr esponds to the fused region of the Epstein-Barr virus episome, as described above.Fragments of low molecular weight correspond to the individual unfused BamH1-N and Bam H 1-J segments of the linear genome, each attached to one or more terminal repeat units ( 14).

DISCUSSION
Epstein-Barr virus infects and transforms B lymphocytes both in vitro and in vivo .The resulting polyclonal lymphoblastoid cell lines generally carry multiple episomal (circular) copies of the genome and express only latent Epstein-Barr virus antigens.In the occasional cell.Epstein-Barr virus is spontaneously reactivated leading to lytic antigen expression.In cases in which lytic antigen expression culminates in the production of virions, the presence of linear genomes can be demonstrated (22).In vitro studies have shown that acyclovir is effective only in reducing the number of linear replicative Epstein-Barr virus genomes (19,23).Acyclovir, when used therapeutically in the treatment of polymorphic B cell lymphoma associated with Epstein-Barr virus, is believed to prevent viral replication and thus to block the spread of infection to other B lymphocytes (24).thereby limiting extension of the lymphoproliferative process.Acyclovir has been reported to influence the outcome of polyclonal.but not monoclonal, tumours (11}.However. the major factor influencing the outcome of this tumour appears to be the relief of an immunosuppressive state (25).
A monoclonal tumour is one that originates from a single infected cell, whereas a polyclonal tumour evolves from multiple infected cells.Mul- Epstein-Barr virus replication is associated with the linearization of the viral genome at the terminal repeat segment.Virions differ in the number of terminal repeat segments contained in their genomes (14).Thus, if each clone of cells results from the proliferation of one cell infected by one virus particle, then the number of terminal repeat segments in all of the episomes in the clone will be identical, yielding only one upper band in a Southern blot of the DNA when probed with Bam H1-NJ.This upper band is composed of the terminal BamH1-N and BamH1-J fragments joined, in a given episome, by means of a determined number of terminal repeat segments.A polyclonal population will, on the other hand, give rise lo multiple upper bands, because the number of terminal repeat elements varies among the clonal populations comprising the polyclonal tumour.This occurs as a result of the mode of replication of the virus (26).Based on this information, one may conclude that the Raji cell line is monoclonal, as well as the tumours in cases 1 and 2 in which no virus replication can be demonstrated by Southern blot analysis.BecaLlSe of virus replication in the B95-8 and P3HR-1 cell lines, these appear polyclonal by Southern blot.although they Diagnosis of EBV in immunocompromised patients are monoclonal by immunoglobulin gene rearrangement.; in these lines the mode of replication of the virus by the rolling circle model gives rise lo additional longer fragments which have been shown to disappear fo ll owing acyclovir inhibition of viral replication (26).Therefore, in the absence of viral replication within a tumour, as judged by lhe absence of short Bam Hl-N and Bam H1-J bands, the presence of one longer Bam Hl-NJ band confirms the monoclonality of the tumour.The significance of clonality in this type of tumour is presently unclear (12).Another point to be highlighted is lhe capacity of this hybridization technique lo reveal whether Epstein-Barr virus is replicating in tumour cells.Because acyclovir acts only on the replicative form ( 19,23).the degree of replication is believed lo correlate with the expectation of a clinical response to acyclovir treatment (24).The genome of replicating Epstein-Barr virus is linear, implying that the Bam H1-N and -J fragments are separated from each other.Thus, the molecular hybridization results should reveal two or more fragments of low molecular weight.(Figure 4).Molecular hybridization of lhe three laboratory cell lines indicated that P3HR-1 and B95-8, but not.Raji, harboured replicative linear forms of Epstein-Barr virus.The tumour biopsies from the present two patients were like Raji in that they only contained latent.episomal copies of the Epstein-Barr virus genome.However, a percentage of these tumours has been shown to contain replicative copies of the Epstein-Barr virus genome (27), theoretically justifying the use of acyclovir in such cases to prevent infection of additional cells.
Other Epstein-Barr virus restriction fragments may also be used as probes for lhe presence of the Epstein-Barr virus genome in lymphoma tissue.The Epstein-Barr virus Bam H1-W segment.(termed Bam H1-V in some publications) is perhaps the best choice where sensitivity is concerned, because it is repeated several times within the IR1 region of the genome (28); however, it gives no information concerning the slate of the Epstein-Barr virus genome (linear or episomal) or the clonality of the tumour.
In cases in which it is only necessary to determine the presence (or absence) of Epstein-Barr virus DNA in tissue specimens , dot blot hybridization is a fast and simple alternative (12).Whole cells or DNA extracted from these cells may be spotted directly onto nitrocellulose filters.which are then hybridized with the probe of choice.
Recently, at the National Centre for Epstein-Barr Virus at the Sainte-Justine Hospital Pediatric Research Centre, the polymerase chain reaction method of DNA amplification allowed detection of the Epstein-Barr virus genome within 24 h by ethidium bromide staining of an amplified 110 basepair viral DNA segment in th e same number of tumours that were found to b e viral DNA positive by conventiona l hybridization (12).These results were subsequently confirmed within three days using a phosphorus-32-labelled oligoprobe spanning the amplified segment located within the Bam H1-W fragment.An additional tumour sample which was doubtful by the conventiona l method was definitely positive by the labelled oligoprobe confirmatory test.All other tumour samples which were negative by the conventional method a lso gave negative results by polymerase chain reaction (29).
The mechanism of B cell transformation induced by Epstein-Barr virus h as yet to be determined.However, it is presently b elieved that three Epstein-Barr virus proteins are involved in this process.Two are nuclear proteins, namely Epstein-Barr nuclear a ntigens 1 and 2 (30)(31)(32)(33), and the other, referred to as latent membrane protein 1 (34,35).is associated with the cytoplasm a nd plasma m embrane.The authors have previously reported the presence of Epstein-Barr nuclear antigen in Epstein-Barr virus-associated polymorphic B cell lymphoma (12) .and emphasize the diagnostic value of the sensitive Epstein-Barr nuclear antigen anticomplem ent immunofluorescence technique for d etecting the presence of Epstein-Barr virus antigens in the tumour cells.Other laboratories have used immunofluorescence with monoclonal a ntibodies to Epstein-Barr nuclear antigen 2 and la tent membrane protein 1 to demonstrate expression of th ese a ntigens in tumour tissu e (36).Because immunofluorescen ce can be rather subj ective a nd, therefore, under-standably dependent on the experience of the observer, molecular hybridization is useful to confirm the diagnosis .This technique also allows detection of the Epstein-Barr virus genome in tumour tissue since the use of the restriction fragment Bam H 1-NJ as a probe indicates tumour clonality.In addition, the presence of linear copies of the genome, which are associated with Epstein-Barr virus replication , may warrant the use of acyclovir to limit further replication of the virus and recurrent lymphoproliferation (24) .
Polymorphic B cell lymphomas have been reported to occur in 1 to 3% of organ transplant patients (11 ,25,37) .Since the majority of these lymphomas are associated with primary Epstein-Barr virus infection or reactivation, the authors have monitored Epstein-Barr virus infection in transplant patients at th eir institution .Primary Epstein-Barr virus infection in young children was frequent , reaching an incidence of over 50% (in a total of 29 cases) during the first six to 12 months following transplantation (unpublished data) .These patients were followed between 1984 and 1990 with serial serological tests for Epstein-Barr virus IgG and IgM a ntibodies to viral capsid antigens and IgG antibodies to early antigens, in a ddition to viru s isolation from saliva.The diagnosis of primary Epstein-Barr virus infection was confirmed by the appearance ofEpstein-Barrviral capsid antigen IgM antibody and s table seroconversion after the disappearance of passively transferred antibodies from blood a nd/ or other biological products.None of the children has yet developed lymphoma .These results may b e explained by the small population sample, the len gth of follow-up, and the fact that immunosuppressive protocols were relatively mild compared to those u sed in other centres.

Figure 1 )
Figure 1) Computed tomography scan of a primary B cell lymphoma of ihe brain (case 1)

Figure 4 )
Figure 4) Schematic representation of hybridization resulis with Bam H 1-NJ probe La d ete rmine tumour clonality and the form of Epste in-Barr virus DNA (episomal or linear) contained in cetl lines or lymphoma tissue.TR Terminal repeat s egment