Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines
Authors: Penas Eva Maria Murga , Hinke Juliane , Behrmann Petra , Janjetovic Snjezana , Schilling Georgia , Bokemeyer Carsten , Dierlamm Judith 
Published: October 2019

Abstract
Abstract 4452
The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).
FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.
As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).
Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).
Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated.

Disclosures:
No relevant conflicts of interest to declare.