We describe here the establishment and molecular characterization of novel human EBV-positive DLBCL cell line with plasmacytic differentiation obtained from a patient with atypical B-cell chronic lymphoproliferative disease with plasmacytic features.
Human B-lymphoid cell lines are extensively used world wide as models in studies of various aspects of B cell biology and as a tools in research on the pathogenesis of leukemia and lymphoma. Nevertheless, the efficiency establishment of new leukemia and lymphoma cell line is rather low and remains a random process 
. Sometimes, the spontaneous proliferation and immortalization of normal or malignant cells in vitro
is due to EBV infection. It is well known for several decades that infection of normal B cell with EBV leads to establishment of lymphoblastoid cell line, thus confirming its oncogenic potential 
. EBV is associated to several malignancies in vivo
and may lead to spontaneous growth and transformation of malignant cells in vitro
; nevertheless, lymphoproliferative cell lines from CLL-like chronic lymphoproliferative disorders have never been reported, unless infected by EBV or in prolymphocytoid transformation 
. The establishment of VR09 cell line was likely due to EBV infection; in fact, as EBV was already present in patient’s B cells, we can reasonably assume that the infection drove the transformation into DLBCL once cultured in vitro
and injected into mice, thus excluding a culture-dependent contamination by EBV. Indeed, the Association between EBV infection and indolent B-cell neoplasms, such as chronic lymphocytic leukemia, marginal zone lymphoma, lymphoplasmacytic lymphoma has been reported 
Thus, the positivity of EBV in VR09 cell line is not surprising. Moreover, some studies showed that cases of DLBCL developed from low-grade lymphoproliferative diseases are EBV-positive and that EBV may have a potential role, although not yet completed defined, in the progression of the indolent disease 
Unfortunately, we lack much information about the patient; however, bone marrow cell morphology and immunophenotype are in agreement with the diagnosis of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. Low-grade B-cell lymphoproliferative disorders, such as CLL/small lymphocytic lymphoma and marginal zone B-cell lymphoma may have overlapping features, thus making the differential diagnosis sometimes difficult. Furthermore, many types of small B lymphoid neoplasms can display plasmacytic differentiation and a phenotype resembling lymphoplasmacytic lymphoma; the latter consists of small lymphocytes, plasmacytoid lymphocytes and plasma cells expressing IgM and pan-B-cell antigens, such as CD19, CD20 and CD22, and usually negative for CD5, CD10 and CD23. As we lack information about serum IgM paraprotein, we concluded as a case of atypical B-chronic lymphoproliferative disease with plasmacytic differentiation, rather than a plasmacytic lymphoma.
Among lymphoproliferative disorders, VR09 cell line presents features of DLBCL with plasmacytic differentiation, with medium to large sized and plasmablastic/plasmacytic-like cells, high Ki-67 index and activated phenotype according to Hans’ and new immunostaining algorithm 
. Moreover, cells are positive for MUM1, CD38, and CD138, thus suggesting a terminal-B cell differentiation.
Although lymphoplasmacytic lymphoma and other lymphoproliferative disease with plasmacytic differentiation may develop into diffuse large B cell lymphoma, similarly to CLL, the frequency of transformation is low 
. Consequently, VR09 cell line can be useful as a model of DLBCL variant corresponding to a very late or activated stage of B cell maturation 
, i.e. the early post-germinal stage differentiation, not entirely terminally differentiated.
Despite the aggressive transformation of indolent disease is not always clonally related to original disease, some data are indirectly consistent with the hypothesis of clonal evolution: in fact, VR09 cell line maintains the immunophenotype and plasmacytoid features of the primary cells at diagnosis, displays the presence of the same clonal gene rearrangement found in patient’s DNA and has a tumorigenic capacity in vivo
; the latter feature strongly suggests its malignant behaviour. VR09 cell line grow subcutaneously without dissemination in peripheral blood and no engraftment when it is administrated i.v. This strange peculiarity could be simply due to the in vivo
model used, i.e. Rag2−/−
mice. They are immunodeficient mice with C57B16/10 background devoid of functional B and T cells because of the complete lack of function of the V(D)J recombinase enzyme system, and displaying deficient innate immunity due to the absence of γ chain of interleukin-2-receptor 
. This model has the advantage to show a stable phenotype without developing spontaneous tumors, as compared to SCID mice 
. Indeed, VR09 cell line injection led to 100% incidence of developing tumors in vivo
, always maintaining the same features.
We found trisomy of chromosome 12 in our model. This aberration is often detected in chronic lymphocytic leukemia 
and in other chronic B cell malignancies, including sometimes lymphoplasmacytic lymphoma. We also found an additional chromosomal abnormality involving chromosomes 7 and 9 after secondary culture: genetic alterations of cell lines are usually stable over the time, but it is debated whether cell line may acquire additional rearrangements in culture 
We also found a synonymous variant of CD79B involving the insertion of Alanine in the exon 2b of variant 2. This variant may be physiological since it has been detected in other isoforms; however, it has never been described before. Moreover, a recent study suggested that also synonymous variants may be involved in the pathogenesis of some diseases 
; therefore, one could speculate that this novel variant of CD79B gene could be involved in the pathogenesis of lymphomas. VR09 cell line did not display the common missense mutations of Card11, usually involved in the constitutive activation of NF-kB pathway in some cases of human activated-B-cell like DLBCL 
. However, the detection of synonymous mutations of Card11 reveals the accuracy of sequencing method. Interestingly, VR09 cell line showed unmutated Myd88 gene, differently from what described in recents reports 
, thus suggesting that lymphomas with plasmacytic features may have different gene patterns.
In conclusion, VR09 is a new cell line of activated DLBCL with plasmacytic differentiation that grow as solitary tumor once injected s.c. in immunodeficient mice. This model could be useful for further studies about the development of DLBCL in patients with low-grade B-cell lymphoproliferative disorders with plasmacytic differentiation, which is a rare but possible event in clinical practice.