The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

Latent and productive viral infections are at the extremes of the spectrum of virus-cell interactions that are thought to play a major role in the ability of such important human pathogens as human immunodeficiency virus (HIV) to elude host defenses and cause disease. The recent development of PCR-based methods to amplify target sequences in individual cells in routinely fixed tissues affords opportunities to directly examine the subtle and covert virus-ceDl relationships at the latent end ofthe spectrum that are inaccessible to analysis by conventional in situ hybridization techniques. We have now used PCR in situ with in situ hybridization to document latent and permissive HIV infection in routinely fixed and paraffinembedded tissue. In one of the first specimens we examined, a tumor biopsy from an HIV-infected individual, we found many of the lymphocytes and lymphocytes infiltrating the tumor had HIV DNA that was detectable only by PCR in situ. The fraction of positive cells varied regionally, but there were foci where most of the cells contained HIV DNA. Most of these lymphocytes and macrophages are latently infected, as we could detect HIV RNA in fewer than one in a thousand ofthese cells. We also detected HIV RNA, surprisingly, in 6% of the tumor cells, where the number of copies of viral RNA per cell was equivalent to productively infected cell lines. The alternative states of HmV-gene expression and high local concentration of latently infected lymphocytes and monocytes revealed by these studies conceptually supports models of lentiviral pathogenesis that attribute persistence to the reservoir of latently infected cells and disease to the consequences of viral-gene expression in this population. The magnitude of infection of lymphocytes documented in this report is also consistent with the emerging view that HIV infection per se could contribute substantially to depletion of immune cells in AIDS.Human immunodeficiency virus (HIV), the etiological agent of AIDS, can establish productive or latent infections in CD4+ lymphocytes and monocytes in culture, and these alternative states of viral-gene expression could readily account for the devastating consequences of infection -and the formidable difficulties in developing a protective vaccine (1-3). Latently infected cells could escape detection and destruction by host defenses and disseminate infection in and between individuals in the face of natural or vaccine-induced immunity. With activation of viral-gene expression, the immune system will eventually be destroyed, and the individual will succumb to a variety of opportunistic infections and unusual tumors. One critical prediction of this reconstruction of pathogenesis is that in vivo one can show that there is a population of infected cells that harbor the HIV provirus in a transcriptionally silent state with no detectable viral RNA and a second population in which viral transcripts are abundant. We have now used PCR-based technologies with singlecell resolution, originally developed for studies o...

We determined HER2 protein overexpression by immunohistochemical analysis and HER2 gene amplification by fluorescence in situ hybridization (FISH) in 215 formalin-fixed, paraffin-embedded breast tumors. Pathologist concordance for immunohistochemical scoring, and HER2 status concordance, as determined by immunohistochemistry and FISH, were high for immunohistochemical 3+, 1+, and 0 tumors but poor for 2+ tumors. Consensus immunohistochemical scores correlated with absolute and chromosome 17 (CEPI 7)-corrected HER2 gene copy number Among HER2-nonamplified tumors, the immunohistochemical score and mean absolute chromosome 17 (CEP17) copy number were weakly correlated. Seventeen tumors were HER2-amplified using absolute HER2 gene criteria but nonamplified when corrected for chromosome 17 polysomy (8 of these were immunohistochemical 2+). Assessment of benign epithelium within the immunohistochemical slides revealed either no staining or basolateral membrane staining, suggesting normal HER2 protein expression. Twenty tumors showing similar basolateral HER2 immunostaining were all low-moderate grade, tubule-forming, and HER2-nonamplified (17) or borderline amplified (3). Additional studies relating changes in HER2 gene content due to amplification or chromosome 17 polysomy and HER2 protein expression may be helpful to pathologists who interpret HER2 immnuohistochemical slides. Breast tumors scored at 2+ should be analyzed by FISH, preferably using a dual-probe FISH assay capable of distinguishing HER2 gene amplification from chromosome 17 polysomy.

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Titers of antibody to cytomegalovirus (CMV) of 529 persons whose blood had been supplied to 51 selected patients who underwent open-heart surgery were determined by indirect hemagglutination (IHA) and IgM-specific indirect immunofluorescence (IFA). Twenty-eight patients showed evidence of active CMV infection after transfusion (seroconversion or a fourfold rise in titer by IHA), whereas 23 showed no serological change. Patients with active CMV infections had received, on average, a greater number of blood units (12.9 vs. 7.9), of which more were seropositive (6.9 vs. 3.5), than did patients who showed no serological change. Those seropositive units of blood that had been transfused into the group that showed evidence of active infection, however, had a lower geometric mean titer than did those transfused into the group that showed no serological change (1:654 vs. 1:1,360). Seven (1.3%) of the 529 blood donors had CMV-specific IgM titers (by IFA) of greater than or equal to 1:16; each of the seven recipients of their blood subsequently showed evidence of active CMV infection. This study suggests that donor blood with high IHA titers may prevent transmission of CMV infection, whereas blood from donors with IgM antibody to CMV may transmit CMV.