Background. Endemic Burkitt ‘s lymphoma ( eBL ) is a B cell tumor that is thought to originate from arrested ripening of originative centre B cells. However, information is presently missing on the peripheral B cell compartment and whether alterations in B cell subsets mirror elevated Epstein-Barr virus ( EBV ) loads in eBL patients. Methods. We used flow cytometry to analyse peripheral B cell subset distribution and real-time quantitative polymerase concatenation reaction ( Q-PCR ) to analyse EBV viral tonss in 32 kids showing with eBL and 25 age-matched controls from western Kenya. Results. No cells with a BL tumour phenotype ( CD19+CD10+CD77+CD38+ ) were found in the peripheral circulation of either eBL patients or controls. The frequences of naif ( CD19+IgD+CD27- ) and classical memory ( CD19+IgD-CD27+ ) B cells were comparable in the two groups, while there were significantly higher frequences of B cell receptor ( BCR ) -deficient ( CD19+?-?- ) and CD19+CD27-CD5-CD10- memory B cells in eBL patients as comparative to controls ( p=0.0038 and p=0.0001, severally ) . Importantly, EBV viral burden was more elevated in eBL patients as compared to controls ( P & A ; lt ; 0.0001 ) , and was positively correlated with the frequence of CD19+CD27-CD5-CD10- B cells ( r=0.4952, p=0.0191 ) . Conclusions. Take together, these informations suggest that there is profound disturbance of B cell homeostasis in eBL patients with attendant lift of EBV viral tonss being associated with CD19+CD27-CD5-CD10- B cells, and non with peripheral circulation of tumour cells.
Endemic Burkitt ‘s lymphoma ( eBL ) is a high class B cell lymphoma, and is the most common paediatric malignance in malaria-endemic parts of sub-Saharan Africa and Papua New Guinea [ 1 ] . Epstein Barr virus ( EBV ) can be found in up to 95 % of eBL tumours, implicating the virus as critical in the etiology of this malignant neoplastic disease [ 2,3 ] . In add-on, there is a profound dysregulation of EBV continuity and unsusceptibility in kids [ 4-6 ] in malaria-endemic countries, proposing that changes in EBV continuity due to repeated malaria infection precede the outgrowth of a malignant ringer.
Germinal centre ( GC ) reactions play an of import function in the coevals of effectual humoral immune responses [ 7 ] . Bodily hypermutation and class-switch recombination are important stairss in originative centre reactions for coevals of functional B cell receptors ( BCR ) , which, in bend, are of import in the endurance of B cells in peripheral circulation [ 8,9 ] . Ideally, all B cells that fail to develop functional BCR through acquisition of hurtful mutants during bodily hypermutation are efficaciously removed through programmed cell death [ 9,10 ] . However, there are surveies showing that EBV can deliver BCR-deficient B cells from programmed cell death [ 11-14 ] . Defects in BCR look in mouse theoretical accounts have been implicated in arrested ripening of B cells and in B cell lymphomagenesis [ 15,16 ] . Interestingly, several surveies have reported that BL tumour cells may be derived from BCR-deficient originative centre B cells, with the EBV viral proteins, latent membrane protein ( LMP ) -1 and LMP-2a playing critical functions in the endurance of these pre-malignant B cell subsets [ 13,14,17 ] . BL tumours express authoritative markers of originative centre B cells, such as CD10, CD38 and CD77 [ 16-18 ] and molecular profiling indicates that many cistrons expressed in originative centres are besides expressed in BL tumours [ 7,19 ] . However, the cell type is controversial and some have argued that they have post-germinal centre memory-cell beginning [ 20 ] .
We and others have found elevated EBV viral tonss in DNA extracted from whole blood of eBL patients [ 4,21,22 ] , but it is unknown whether this elevated viral burden is due to presence of a leukemic stage of the disease or merely release of EBV viral DNA into circulation from necrotic tumour cells. In healthy EBV sero-positive persons, EBV is purely cell-associated and found in the CD19+IgD-CD5-CD27+ memory B cell compartment [ 12,23,24 ] , but in patients with EBV-associated post-transplant lympho-proliferative disease, inveterate elevated viral tonss are declarative of an addition in circulation of Ig ( Ig ) -null B cells that merely show CD19 and no other authoritative B cell markers such as CD10 [ 11,12,25 ] . In this survey, we examined the peripheral B cell phenotype and EBV viral burden in eBL patients and controls from a malaria-endemic part with high incidence of eBL. Our consequences revealed that the EBV viral burden does non correlate with an addition in go arounding BL tumour cells, but instead with lift of CD19+CD5-CD27-CD10- B cell population.
Information about the survey population and design has been reported elsewhere [ 21 ] . Briefly, following informed consent by the survey participant ‘s parents/guardians, we enrolled 34 kids showing with eBL at the Nyanza Provincial General Hospital ( NPGH ) , Kisumu, Kenya. Two of these patients were excluded from farther analysis ; one patient had acute leukaemia and another was diagnosed to be HIV+ . We besides enrolled 25 age-matched controls ( herein referred to as ‘controls ‘ ) from Kanyawegi small town along the shores of Lake Victoria in Kisumu West District, Nyanza Province. Kanyawegi is a rural territory that has a high incidence of eBL and experiences relentless P. falciparum transmittal [ 4 ] . This protocol was approved by the Kenya Medical Research Institute ( KEMRI ) Ethical Review Committee, the Institutional Review Board for Human Studies at the University Hospitals of Cleveland and Case Western Reserve University ( Dr. Moormann ‘s institutional association at the clip of sample aggregation ) and at SUNY Upstate Medical University, USA.
Blood aggregation and processing
Finger-prick blood was collected in EDTA tubings and measurings of haemoglobin ( Hb ) degrees were determined utilizing a portable B-hemoglobin photometer ( Hemocue AB Angelholm, Sweden ) . Complete blood counts were performed with a Beckman colter AcT diff2 ( Beckman-coulter corporations, Miami, FL, USA ) on finger-prick blood, collected in the EDTA-containing phials. An aliquot of finger-prick blood was stored at -80oC for subsequent isolation of DNA. In add-on, 2-5 milliliter of venous blood was drawn from both eBL patients and controls. Ficoll-hypaque denseness gradient centrifugation of blood was carried out within 1 hr of blood aggregation. Peripheral blood mononuclear cells ( PBMC ) were instantly analyzed for B cell phenotype by flow cytometry.
Preparation of blood and flow cytometric analysis
Cell viability was determined utilizing the Trypan Blue dye exclusion check and all cells were routinely greater than 98 % feasible. Following PBMCs isolation, immunofluorescence labeling was done as antecedently described [ 26 ] . The cell surface staining was done utilizing the undermentioned antibodies to lymphocyte surface receptors ; CD45-APC, CD19-PerCP, CD3-APC, CD8-PE, CD4-FITC, CD3-FITC, CD27-FITC, CD10-PE, CD5-APC, Kappa ( ? ) -FITC, Lambda ( ? ) -PE, CD10-APC, CD77-FITC and IgD-PE. Isotypic controls were IgG2a, K-FITC ( mouse ) , IgG1, K-PE ( mouse ) , IgG1, K-PerCP ( mouse ) and IgG1, K-APC ( mouse ) ( BD Pharmigen, San Diego, CA, USA ) . Data was collected utilizing a FACsCalibur flow cytometer ( Becton Dickinson Immunocytometry Systems, San Jose, USA ) and analysed utilizing FlowJo package ( Tree Star Inc. San Carlos, CA, USA ) .
Human Immunoglobulin Free Light Chains ? and ? ELISA
Degrees of serum free ? and ? visible radiation ironss checks were determined utilizing Human Immunoglobulin Free Light Chains ? and ? ELISA kit ( BioVendor Laboratorni Medicina, Modrice, Czech Republic ) , harmonizing to the makers ‘ protocol.
Quantitative PCR to quantify EBV DNA
Deoxyribonucleic acid was extracted from 200 i?l of finger-prick whole blood utilizing Qiagen DNAeasy kit ( Qiagen, Valencia, CA, USA ) harmonizing to the makers ‘ protocol. Deoxyribonucleic acid was eluted from the column in 200 i?l of H20 and stored at -20oC. Primers and investigations to observe a 70 bp part of the EBV BALF5 cistron and the i??-actin cistron were antecedently described [ 4 ] . The viral burden was normalized to the i??-actin transcript figure, log-transformed and so calculated based on transcripts of EBV genome/µg of whole blood. The EBV viral burden was reported as log EBV copies/µg DNA.
Differences in non-parametric values between two defined groups were determined utilizing Mann-Whitney U trial. Correlations were examined utilizing Spearman ‘s rank correlativity trial. A P & A ; lt ; 0.05 was considered statistically important. Data was analyzed utilizing GraphPad prism version 5 ( GraphPad Software, Inc, La Jolla, CA, USA ) .
General features of the survey population
We enrolled 32 eBL patients ( 21 males and 11 females ) and 25 healthy age-matched controls ( 16 males and 9 females ) . The demographic, parasitological and haematological features of the survey populations are summarized in Table 1. Age ( p=0.7584 ) and distribution of gender ( p=0.8822 ) were comparable between the two groups. The proportions of kids who were parastemic was significantly higher in controls comparative to eBL patients ( p & A ; lt ; 0.0001 ) . However, most kids showing with eBL at NPGH have been referred from another infirmary, and in most instances it was reported that these patients have been treated with anti-malarial drugs prior to admittance at NPGH. Leukocyte counts were comparable in eBL patients and controls, as there were no important differences in white blood cell counts ( WBC ) ( p=0.1356 ) , lymphocyte counts ( LY ) ( p=0.3220 ) , monocytes counts ( MO ) ( p=0.2458 ) and granulocyte counts ( GR ) ( p=0.0569 ) . Red blood cell counts ( RBC ) were besides comparable ( p=0.4661 ) . In contrast, Hb degrees were significantly lower in the eBL patients compared to controls ( p=0.0009 ) and a higher frequence of eBL patients were anaemic compared to controls ( P & A ; lt ; 0.0001 ) . Lactate dehydrogenase ( LDH ) degrees, which were used as a clinical marker of tumour load were significantly higher in eBL patients compared to controls ( P & A ; lt ; 0.0001 ) .
Elevated EBV viral tonss in eBL patients are non associated with go arounding tumour cells in peripheral blood
Elevated EBV viral tonss in BL patients are thought to ensue from increased circulation of BL tumour cells in peripheral circulation [ 4 ] . To prove this possibility, we quantified EBV viral tonss from whole blood utilizing Q-PCR. Consistent with earlier observations [ 4,22 ] , we found significantly higher EBV viral tonss in eBL patients compared to controls ( average 4.14 log EBV copies/µg DNA [ range 0.00-4.11 ] versus 0.90 log EBV copies/ µg DNA [ range 0.00-5.23 ] , P & A ; lt ; 0.0001 ) ( Fig. 1 ) .
To find if the elevated EBV viral tonss observed in peripheral blood from eBL patients were due to the presence of go arounding tumour cells, we analyzed peripheral B cells for grounds of tumours cells. PBMC were stained with a panel of mAbs particular for CD19, CD10, CD38 and CD77. The CD19 is a pan-B cell marker found on both normal and malignant B cells. The CD10, CD38 and CD77 are expressed by BL cells [ 19,27 ] such that if there were go arounding BL cells, we would expect high proportions of cells showing these markers. As these markers are besides found in normal peripheral B cells albeit at low degrees, we besides analyzed the peripheral B cell phenotype of the controls. Consequences demonstrated that there were no cells with untypical BL phenotype ( CD19+CD10+CD38+CD77+ ) go arounding in peripheral blood of either eBL patients or controls ( Fig A ) or eBL patients ( Fig. 2B ) . It is possible that EBV down-regulation of the CD10 and CD38 germinal centre B cell markers, besides used as BL tumour cell marker [ 11,16,17 ] , can take to miss of sensing of these cell types utilizing flow cytometry. To look into for the presence of these cell types in peripheral circulation, we analyzed PBMCs for surface look of these markers. As shown in Table 2 and Fig.2 C and D, severally, we were able to observe ( CD19+CD38+CD10+77- ) B cells showing these originative centre markers in peripheral circulation, although at a lower frequence in eBL patients compared to controls ( average 8.37 % [ scope 1.81-18.24 ] versus 21.93 % [ 13.65-28.78 ] ; p=0.0005 ) , proposing that down-regulation or loss of BL tumour markers may non explicate the deficiency of sensing of BL tumour cells in peripheral circulation.
As a 2nd marker, to look into for clonality of go arounding cells declarative of tumour cells, we besides tested PBMC for the surface look of the kappa ( i?« ) or lambda ( i?¬ ) concatenation on B cells. For the presence of monoclonal cell populations, we would detect skewing of distribution of surface Ig visible radiation concatenation towards either ? or ? light ironss. Typically, in healthy grownup blood, about 46.0 % of B cells are i?«-positive and 39.0 % are i?¬-positive [ 28 ] . In conformity with this, in the samples of control survey population, we observed a average frequence of 47.7 % of B cells were ?-positive, 32.3 % were ?-positive while 2.48 % expressed both i?«- and i?¬-light ironss. In add-on, 17.76 % neither uttered i?«- or i?¬- visible radiation ironss ( Fig.3 A and C ) . In contrast, in eBL patients, there was a average frequence of 4.36 % of B cells showing i?«-positive, 17.32 % were i?¬-positive while 1.01 % expressed both i?«- and i?¬-light ironss. A higher proportion 73.9 % neither uttered i?«- or i?¬-light ironss ( BCR-deficient ) ( Fig. 3 B and D ) iˆ®iˆ iˆ Remarkably, eBL patients had significantly higher frequence of CD19+?-?- ( BCR-deficient ) B cells comparative to controls ( average 73.39 % [ scope 11.18-95.09 ] versus 17.76 % [ scope 10.82-19.76 ] ; p=0.0038 ) , while the controls had normal distribution of i?«- or i?¬-light ironss ( Table 2 ) . These informations suggest that the elevated EBV viral tonss reported in eBL patients are non associated with go arounding BL tumour cells.
Endemic Burkitt ‘s lymphoma patients have normal degrees of serum free ? and ? visible radiation ironss in their plasma
We next explored whether low frequences of B cells showing a functional B cell receptor ( BCR ) on B cells was due to cytogenic aberrances that impair heavy and light ironss partner offing, or as a consequence of down-regulation of heavy concatenation but non light concatenation written text [ 29 ] . Therefore, we assayed for serum free ?- and ?- visible radiation ironss in plasma of both eBL patients and controls. There was no important difference in the degrees of serum free ? visible radiation ironss in controls comparative to eBL patients ( average 13.03mg/L [ scope 5.31- 28.83 ] versus 9.11 mg/L [ scope 1.23-40.07 ] ; p=0.0678 ) ( Fig.3E ) . In add-on, the degrees of serum free ? visible radiations were besides comparable between controls and eBL patients ( average 9.73mg/L [ scope 6.48-54.29 ] versus 11.16mg/L [ 3.95-45.06 ] ; p=0.5625 ) ( Fig.3F ) . These degrees were within the normal scopes for sensing of serum free ? visible radiation ironss ( normal scope, 3.3-19 mg/L ) and ? visible radiation ironss ( normal scope, 5.7-26.3 mg/L ) in healthy persons [ 30 ] . These findings suggest that deficiency of look of functional BCR on these B cell subsets is non due to down-regulation of the written text of heavy concatenation cistrons.
Endemic Burkitt ‘s lymphoma patients have elevated frequences of untypical ( CD19+IgD-CD27- ) memory B cells and decreased non-class switched ( CD19+IgD+CD27+ ) memory B cells
We farther determined the frequences of naif and memory B cell populations based on flow cytometric staining with IgD and CD27. As shown in Table 2, frequences of the entire B cell population ( CD19+ ) , naif ( CD19+IgD+CD27- ) and classical memory ( CD19+IgD-CD27+ ) B cell populations were comparable in eBL patients and controls. Further analysis revealed significantly higher frequences of mature naif B cells ( CD19+IgD+CD10- ) in eBL patients relative to controls ( average 60.75 % [ scope 50.59-68.48 ] versus 54.06 [ 47.55-59.38 ] ; p=0.0346 ) . However, the frequence of immature transitional B cells ( CD19+IgD+CD10+ ) was significantly higher in controls comparative to eBL patients ( average 21.42 % [ scope 15.83-27.72 ] versus 11.86 % [ scope 4.28-18.33 ] ; p=0.0023 ) .
Within the memory B cell compartment, the frequence of non-class switched memory ( CD19+IgD+CD27+ ) B cells, of import in bacterial or viral defence [ 31 ] , was significantly higher in controls as compared with eBL patients ( average 13.66 % [ 12.84-16.50 ] versus 7.90 % [ scope 6.13-12.46 ] ; p & A ; lt ; 0.0001 ) . However, the frequence of untypical ( CD19+IgD-CD27- ) memory B cells was significantly higher in eBL patients as compared to controls ( average 15.63 % [ scope 10.23-22.20 ] versus 10.77 % [ scope 6.99-14.29 ] ; p=0.0304 ) . These informations suggest that eBL patients have perturbed memory B cell homeostasis.
Following the observations that the memory B cell pool is perturbed in eBL patients, we hypothesized that there would be disturbances in the frequence of memory B cells subsets which are associated with control of blood-borne pathogens [ 31 ] . To prove this hypothesis, we carried out a flow cytometric analysis based on the look of CD19, CD27, CD10 and CD5. As shown in Table 2, there was no statistical difference in the frequence of CD19+CD27+CD5- between the controls and eBL patients, nevertheless, the frequence of CD19+CD27+CD5+ memory B cell subsets was significantly higher in the controls relative to eBL patients ( average 11.42 % [ scope 8.64-14.51 ] versus 6.91 % [ scope 3.34-10.51 ] ; p=0.0058 ) . Likewise, the frequence of CD19+CD27-CD5+ memory B cells was besides significantly higher in controls comparative to the eBL patients ( average 33.40 % [ 28.85-38.23 ] versus 21.90 % [ scope 14.97-29.06 ] ; p & A ; lt ; 0.001 ) .
Further word picture of CD5+ memory B cells based on co-expression of CD10 revealed that the frequence of CD19+CD5+CD10- B cells were significantly higher in controls comparative to the eBL patients ( average 26.70 % [ scope 22.80-30.15 ] versus 22.40 % [ scope 17.65-27.91 ] ; p=0.0486 ) . In contrast, the frequence of CD19+CD27-CD5-CD10- memory B cells were significantly higher in eBL patients as compared to the controls ( average 79.74 % [ scope 68.92-85.45 ] versus 64.67 % [ range56.01-69.74 ] ; p & A ; lt ; 0.0001 ) .
Correlation between EBV viral tonss and B cell sub-populations
Elevated EBV viral tonss have been associated with Ig ( Ig ) -null B cells in other EBV-associated conditions [ 11,25 ] . However, contrary to this old observation, we did non detect any association between BCR-negative B cells and EBV viral tonss ( r=-0.2332, p=0.2842 ) , proposing that the elevated EBV viral tonss reported in eBL patients may non be restricted to EBV continuity in BCR-negative B cells entirely.
Many surveies have shown that EBV is restricted to the CD5- B cell memory B cell subsets in peripheral blood [ 11,12,24,31 ] . Therefore, we investigated whether there is an association between EBV viral tonss and frequences of different B cell subsets. As shown, EBV viral tonss were negatively correlated with frequences of IgD+CD27+ ( r=-0.5880, p=0.0064 ) ( Fig.4A ) , CD27+CD5+ ( r=-0.5948, p=0.0045 ) ( Fig.4B ) and CD27+CD10- ( r=-0.4376, p=0.0417 ) ( Fig.4C ) and positively correlated with the frequence of CD27-CD5-CD10- ( r=0.4952, p=0.0191 ) ( Fig.4D ) . These consequences illustrate that elevated EBV viral tonss in eBL patients are associated with higher frequences of CD19+27-CD5-CD10- B cells.
Endemic Burkitt ‘s lymphoma is a extremely aggressive B cell lymphoma thought to originate from arrested ripening of originative centre B cells [ 7,16,19 ] . Previous observations raised the possibility that elevated EBV viral tonss reported in eBL patients may be due to increased circulation of BL tumour cells in peripheral circulation [ 4 ] . In add-on, recent surveies suggest that BL tumour cells may be derived from BCR-deficient B cell precursors [ 13,14,17 ] . To prove these possibilities, we analyzed B cell phenotypes and EBV viral tonss in eBL patients and in age-matched controls. Our consequences demonstrate that although there were striking differences in peripheral blood B cell subsets and EBV viral tonss in eBL patients compared to controls, BL tumours cells do non go around in peripheral blood of eBL patients. These findings besides provide a direct presentation of increased frequence of BCR-deficient B cells in peripheral blood of eBL patients and controls, farther proposing that there is profound disturbance of B cell homeostasis in eBL patients and kids from malaria-endemic parts.
While B cell receptors are a requirement for the coevals and endurance of mature B cells and in finding the size or the frequence of lymphoid compartments during immune responses [ 15,19 ] , the presence of BCR-deficient B cells in the peripheral blood of patients with EBV-associated upsets have been reported [ 11,12 ] . Our informations further confirm these old findings, and extend those observations by showing the presence of BCR-deficient B cells in the peripheral blood of eBL patients. This population represents apoptosis-prone B cell subsets that are usually eliminated through negative choice during originative centre reactions [ 9,17 ] . Although the exact mechanisms that lead to the enlargement of these B cell subsets in peripheral circulation of eBL patients are still non clearly understood, both EBV infection and/or tumor-associated immunosuppression are both factors that may be interfering with originative centre or peripheral negative choice check-points, ensuing in peripheral circulation of Ig-null B cells [ 13,14,17 ] . However, our informations show that elevated EBV viral burden was non associated with the frequence of BCR-deficient B cell subset.
We besides report observation of BCR-deficient B cells in the peripheral circulation of kids with symptomless P. falciparum malaria infection, proposing that kids from malaria-endemic parts besides experience aberrances in B cell development that may predispose them to develop eBL [ 26,32 ] . Although the exact mechanisms that lead to accretion of these subsets in peripheral blood of these kids is ill-defined, one possibility is that uninterrupted antigenic stimulation due to chronic infection with P. falciparum may interfere with peripheral or originative centre negative choice check-points, leting the release of BCR-deficient B cells into peripheral blood [ 8,11 ] . Alternatively, the long-run effects of EBV viral reactivation, common in malaria-endemic scenes [ 6 ] , may take to increased release of EBV virions that infect immature BCR-negative B cells and deliver them from programmed cell death, therefore taking to development of malignant B cell ringers. In fact, elevated frequences of immature B cells have been reported in kids from malaria-endemic scenes [ 26 ] . This subset is non merely susceptible to EBV infection as compared to memory B cells, but besides expresses elevated degrees of microRNA ( hsa-miR-127 ) , which is of import in barricading B cell distinction [ 16,33 ] . Furthermore, stimulation of this sub-population with unmethylated bacterial DNA ( CPG ) through Toll-like receptor 9 ( TLR-9 ) leads to constitutive look of activation-induced deaminase ( AID ) cistrons, critical in potentiating c-myc translocation of import in eBL lymphomagenesis [ 19,33 ] . P. falciparum besides generates a ligand for TLR-9 of import in acknowledgment of unmethylated double-stranded DNA-hemozoin composite and this may drive enlargement of latently infected B cells, therefore predisposing persons from malaria-endemic scenes to develop lymphomas [ 34 ] .
Although, we report a positive correlativity between EBV viral tonss with frequences of CD19+27-CD5-CD10- B cells in eBL patients, a recent survey in HIV+ patients demonstrated association of EBV viral burden with enlargement of immature transitional B cells [ 35 ] . Furthermore, EBV infection of natural slayer cells, monocytes and T-cells is common characteristic in patients enduring from EBV lympho-proliferative upsets [ 25 ] . Together, these informations suggest that in patients with elevated EBV viral burden, EBV infection is non restricted to memory B cells. Hence, farther surveies are needed to clarify the cellular beginning of elevated EBV viral tonss reported in eBL patients.
Previous surveies have shown that enlargement of CD19+IgD-CD27- memory B cells is associated with impaired immune responses during infective infections and in autoimmune diseases [ 36 ] . Recently, surveies in both HIV and P. falciparum infections have associated enlargement of this subset with immune hypo-responsiveness [ 32,37 ] . We besides report enlargement of this subset in eBL patients as compared to controls, with frequences in eBL patients comparable to those observed in autoimmune diseases and bacterial infections [ 36 ] . In add-on, consistent with old studies in HIV and autoimmune patients [ 31,36 ] , we report decreased frequence of non-class-switched memory B cells in eBL patients, farther proposing that chronic immune activation may take to B cell disfunctions in eBL patients. However, the effects of these disturbances on functional unsusceptibility in eBL patients were beyond the range of the current survey.
In decision, we show that there are major disturbances in peripheral B cell homeostasis in eBL patients, with our consequences back uping the impression that malignant B cell ringers in eBL tumours may be derived from BCR-deficient B cells. In add-on, elevated EBV viral tonss in eBL patients are associated with CD19+CD27-CD5-CD10- B cell sub-populations but non due to increase of BL tumour cells in peripheral circulation. Hence, future surveies on eBL lymphomagenesis should concentrate on the molecular and phenotypic markers of the BCR-deficient B cell subsets described in the current survey, in add-on to placing the niche of EBV. This will supply critical penetration into eBL pathogenesis and besides aid in polishing curative attacks.