Viral Associations

Epstein-Barr Virus

The presence of EBV is most frequently determined by in situ hybridization (ISH) for EBERs which are abundantly expressed, small, untranslated RNAs.20 Commercial kits are available for EBER ISH, which can be performed on routinely processed, formalin-fixed, paraffin-embedded tissue sections (Figure 34-2). An alternative or complementary approach is PCR analysis for the EBV genome. This can be qualitative or quantitative. Qualitative PCR can be performed for specific regions of the EBV genome that have strain-specific differences (type 1 or A vs type 2 or B), like EBNA2 and EBNA3C (Figure 34-2). Although Type 1 EBV is more transforming in vitro and is almost invariably associated with PTLD, AIDS lymphomas can be infected by either strain. Quantitative PCR is most frequently performed using Taqman chemistry or SYBR Green and melting-curve analysis using DNA from whole blood, serum, blood mononuclear cells, or cerebrospinal fluid to monitor EBV viral loads in patients with EBV-associated lymphomas.19,25 Another useful method is RT-PCR for EBV gene expression characterization, which can distinguish among different types of latency.26 This assay, however, is used more commonly for research than for clinical purposes. Alternatively, the different types of latency can be distinguished by immunohistochemistry using antibodies to several different EBV antigens, in particular to LMP1 and EBNA2.

Kaposi Sarcoma-Associated Herpesvirus

The most common method for detecting KSHV infection is PCR, although antibodies are now available for KSHV detection by immunohistochemistry. Although different PCR primers are used by different laboratories, the most commonly used primer set is called "KS330" and targets the ORF26 region of the viral genome.27 This primer set was described with the original identification of KSHV in 1994 and used in most early studies. Another primer set with high sensitivity and specificity targets the K9 open reading frame of the KSHV genome.28 While some studies suggest that quantitative KSHV assays are useful for monitoring Kaposi's sarcoma evolution, the utility for monitoring KSHV-associated lymphomas is still unclear.

Figure 34-2. Detection of Epstein-Barr virus. (a) In situ hybridization for EBER is positive in an AIDS-related immunoblastic lymphoma. Numerous large cells with black staining can be seen. (b) PCR analysis for the presence of EBV using primers for the EBNA3C region,which distinguishes type 1 and 2 (also called A and B) EBV in a panel of AIDS lymphomas. Type 1 isolates show a PCR band of 153 base pairs (bp; lower arrow),and type B isolates result in a band of 246 bp (upper arrow). Lanes 1 through 12 represent different AIDS-related lymphomas. Cases 1 and 7 were negative for EBV, and all other cases were positive. In AIDS lymphomas, both strains are common, while in PTLD, EBV type 1

is involved in the vast majority of cases. NC is a negative (uninfected) control, and PC is a positive control for Type 2 EBV. (c) EBV Southern blot showing genomic DNA digested with BamH I,followed by electrophoresis,transfer and hybridization with a radiolabeled probe for the terminal repeat region of EBV. C represents the EBV negative cell line, HL-60. Lanes 1 through 5 are analysis of five cases of PTLD showing monoclonal rearrangements for EBV (arrowheads). Case two has two distinct bands, indicating two different EBV-positive clonal populations.

Figure 34-2. Detection of Epstein-Barr virus. (a) In situ hybridization for EBER is positive in an AIDS-related immunoblastic lymphoma. Numerous large cells with black staining can be seen. (b) PCR analysis for the presence of EBV using primers for the EBNA3C region,which distinguishes type 1 and 2 (also called A and B) EBV in a panel of AIDS lymphomas. Type 1 isolates show a PCR band of 153 base pairs (bp; lower arrow),and type B isolates result in a band of 246 bp (upper arrow). Lanes 1 through 12 represent different AIDS-related lymphomas. Cases 1 and 7 were negative for EBV, and all other cases were positive. In AIDS lymphomas, both strains are common, while in PTLD, EBV type 1

is involved in the vast majority of cases. NC is a negative (uninfected) control, and PC is a positive control for Type 2 EBV. (c) EBV Southern blot showing genomic DNA digested with BamH I,followed by electrophoresis,transfer and hybridization with a radiolabeled probe for the terminal repeat region of EBV. C represents the EBV negative cell line, HL-60. Lanes 1 through 5 are analysis of five cases of PTLD showing monoclonal rearrangements for EBV (arrowheads). Case two has two distinct bands, indicating two different EBV-positive clonal populations.

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