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Characterization of Epstein Barr virus latency pattern in Argentine breast carcinoma.

Lorenzetti MA, De Matteo E, Gass H, Martinez Vazquez P, Lara J, Gonzalez P, Preciado MV, Chabay PA - PLoS ONE (2010)

Bottom Line: Epstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes.EBV presence was confirmed by PCR.LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Laboratory, Pathology Division, Ricardo GutiƩrrez Children Hospital, Ciudad de Buenos Aires, Argentina. marioloren@yahoo.com.ar

ABSTRACT

Introduction: Epstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes. Since in breast carcinoma this pattern is not yet fully described, our aim was to characterize EBV latency pattern in our EBV positive breast carcinoma series.

Methods: The study was conducted on 71 biopsies of breast carcinoma and in 48 non-neoplastic breast controls. EBNA1, LMP2A and LMP1 expression was assessed by immunohistochemistry with monoclonal antibodies, while viral genomic DNA and EBERs RNA transcripts expression was performed by in situ hybridization. EBV presence was confirmed by PCR.

Results: EBV genomic DNA and EBNA1 expression were detected in 31% (22/71) of patients specifically restricted to tumor epithelial cells in breast carcinoma while all breast control samples were negative for both viral DNA and EBNA1 protein. LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts.

Conclusions: These findings suggest that EBV expression pattern in the studied biopsies could be different from those previously observed in breast carcinoma cell lines and lead us to suggest a new, EBNA1, LMP2A positive and LMP1 and EBERs negative latency profile in breast carcinoma in our population.

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Immunohistochemical detection of LMP2A and double immunohistochemical staining for LMP2A and CK7.A) Ductal breast carcinoma with a high number of LMP2A positive cells. Positive signal for LMP2A is restricted to the cytoplasm and membrane of tumor epithelial cells (1000X). B) LMP2A negative ductal carcinoma tissue (1000X). C) Ductal breast carcinoma. Positive double signal for LMP2A (brown signal, dotted arrow) and CK7 (blue signal, full arrow) restricted to the cytoplasm and membrane of the same tumor epithelial cells (1000X).
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pone-0013603-g003: Immunohistochemical detection of LMP2A and double immunohistochemical staining for LMP2A and CK7.A) Ductal breast carcinoma with a high number of LMP2A positive cells. Positive signal for LMP2A is restricted to the cytoplasm and membrane of tumor epithelial cells (1000X). B) LMP2A negative ductal carcinoma tissue (1000X). C) Ductal breast carcinoma. Positive double signal for LMP2A (brown signal, dotted arrow) and CK7 (blue signal, full arrow) restricted to the cytoplasm and membrane of the same tumor epithelial cells (1000X).

Mentions: Twenty two out of 71 (31%) cases were EBV positive by EBERs specific PCR. To discard the possibility that the positive signal was given by EBV infected infiltrating B-cells within the homogenized tissue, all samples were assayed for viral DNA genome by means of BamH1W DNA ISH and the results matched the 22 EBV EBERs PCR status for all the 71 cases. Nuclear staining corresponding to EBV DNA genome positive hybridization was observed in the nucleus of about 40% of tumor cells in each biopsy, but not in infiltrating B-cells (Figures 1A and B). Breast non-neoplastic controls and the HL case used as EBV negative control were all negative for EBV genome by BamH1W DNA ISH (Figure 1C). EBNA1 expression was analyzed by IHC and all 22 (31%) of 71 EBV DNA positive cases were found to be positive. Granular nuclear staining was observed in tumor epithelial cells in a similar percent as EBV genome hybridization with both clones (1H4 and 2B4), but it was absent in infiltrating B-cells (Figures 2A and C). All non-neoplastic controls as well as the EBV negative HL were negative for both clones of EBNA1 (Figures 2B and D). Although it was reported that the 2B4 clone might not be completely specific for EBV due to cross-reactivity with tumor proteins [20] all our results with the 2B4 clone correlated exactly with the 1H4 clone and also with PCR and EBV DNA genome hybridization results. We then focused on EBV-positive cases by BamH1W DNA ISH to evaluate EBV protein expression and characterize their latency pattern. Therefore, in these cases we analyzed EBERs by EBERs ISH, and LMP1 and LMP2A expression by IHC. LMP2A was detected in 16 (73%) of the 22 EBV DNA positive cases. Positive staining was observed in the cytoplasm and membrane in a variable percentage of tumor epithelial cells but similarly to EBNA1, it was not detected in bystander B-cells (Figure 3A). All control samples were negative for LMP2A (Figure 3B).


Characterization of Epstein Barr virus latency pattern in Argentine breast carcinoma.

Lorenzetti MA, De Matteo E, Gass H, Martinez Vazquez P, Lara J, Gonzalez P, Preciado MV, Chabay PA - PLoS ONE (2010)

Immunohistochemical detection of LMP2A and double immunohistochemical staining for LMP2A and CK7.A) Ductal breast carcinoma with a high number of LMP2A positive cells. Positive signal for LMP2A is restricted to the cytoplasm and membrane of tumor epithelial cells (1000X). B) LMP2A negative ductal carcinoma tissue (1000X). C) Ductal breast carcinoma. Positive double signal for LMP2A (brown signal, dotted arrow) and CK7 (blue signal, full arrow) restricted to the cytoplasm and membrane of the same tumor epithelial cells (1000X).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2962632&req=5

pone-0013603-g003: Immunohistochemical detection of LMP2A and double immunohistochemical staining for LMP2A and CK7.A) Ductal breast carcinoma with a high number of LMP2A positive cells. Positive signal for LMP2A is restricted to the cytoplasm and membrane of tumor epithelial cells (1000X). B) LMP2A negative ductal carcinoma tissue (1000X). C) Ductal breast carcinoma. Positive double signal for LMP2A (brown signal, dotted arrow) and CK7 (blue signal, full arrow) restricted to the cytoplasm and membrane of the same tumor epithelial cells (1000X).
Mentions: Twenty two out of 71 (31%) cases were EBV positive by EBERs specific PCR. To discard the possibility that the positive signal was given by EBV infected infiltrating B-cells within the homogenized tissue, all samples were assayed for viral DNA genome by means of BamH1W DNA ISH and the results matched the 22 EBV EBERs PCR status for all the 71 cases. Nuclear staining corresponding to EBV DNA genome positive hybridization was observed in the nucleus of about 40% of tumor cells in each biopsy, but not in infiltrating B-cells (Figures 1A and B). Breast non-neoplastic controls and the HL case used as EBV negative control were all negative for EBV genome by BamH1W DNA ISH (Figure 1C). EBNA1 expression was analyzed by IHC and all 22 (31%) of 71 EBV DNA positive cases were found to be positive. Granular nuclear staining was observed in tumor epithelial cells in a similar percent as EBV genome hybridization with both clones (1H4 and 2B4), but it was absent in infiltrating B-cells (Figures 2A and C). All non-neoplastic controls as well as the EBV negative HL were negative for both clones of EBNA1 (Figures 2B and D). Although it was reported that the 2B4 clone might not be completely specific for EBV due to cross-reactivity with tumor proteins [20] all our results with the 2B4 clone correlated exactly with the 1H4 clone and also with PCR and EBV DNA genome hybridization results. We then focused on EBV-positive cases by BamH1W DNA ISH to evaluate EBV protein expression and characterize their latency pattern. Therefore, in these cases we analyzed EBERs by EBERs ISH, and LMP1 and LMP2A expression by IHC. LMP2A was detected in 16 (73%) of the 22 EBV DNA positive cases. Positive staining was observed in the cytoplasm and membrane in a variable percentage of tumor epithelial cells but similarly to EBNA1, it was not detected in bystander B-cells (Figure 3A). All control samples were negative for LMP2A (Figure 3B).

Bottom Line: Epstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes.EBV presence was confirmed by PCR.LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Laboratory, Pathology Division, Ricardo GutiƩrrez Children Hospital, Ciudad de Buenos Aires, Argentina. marioloren@yahoo.com.ar

ABSTRACT

Introduction: Epstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes. Since in breast carcinoma this pattern is not yet fully described, our aim was to characterize EBV latency pattern in our EBV positive breast carcinoma series.

Methods: The study was conducted on 71 biopsies of breast carcinoma and in 48 non-neoplastic breast controls. EBNA1, LMP2A and LMP1 expression was assessed by immunohistochemistry with monoclonal antibodies, while viral genomic DNA and EBERs RNA transcripts expression was performed by in situ hybridization. EBV presence was confirmed by PCR.

Results: EBV genomic DNA and EBNA1 expression were detected in 31% (22/71) of patients specifically restricted to tumor epithelial cells in breast carcinoma while all breast control samples were negative for both viral DNA and EBNA1 protein. LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts.

Conclusions: These findings suggest that EBV expression pattern in the studied biopsies could be different from those previously observed in breast carcinoma cell lines and lead us to suggest a new, EBNA1, LMP2A positive and LMP1 and EBERs negative latency profile in breast carcinoma in our population.

Show MeSH
Related in: MedlinePlus