Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load.

Epstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.

Accumulation of p53 in infectious mononucleosis tissues.

Epstein-Barr virus (EBV) infects lymphocytes, where it persists indefinitely for the life of the host; whether the virus interacts with p53 to maintain itself in these cells is unknown. Lymphoid biopsy samples from 10 patients with infectious mononucleosis (IM) were examined for expression of p53 by immunohistochemistry. Accumulation of p53 was detected in all 10 cases, primarily in large lymphocytes of the expanded paracortex. The presence of EBV was confirmed in all 10 cases by EBER1 (EBV-encoded RNA) in situ hybridization, whereas 11 non-IM control samples lacked significant EBER1 and did not express p53 in paracortical lymphocytes. Interestingly, EBV infection alone does not cause accumulation of intracellular p53, because many more cells expressed EBER1 than p53 in the IM tissues. To determine whether p53 was confined to the subset of infected cells in which viral replication was occurring, BZLF1 immunostains were performed. Viral BZLF1 was detected in 8 of 10 IM tissues; however, the paucity and small size of the BZLF1-expressing lymphocytes suggests that they are not the same cells overexpressing p53. To further examine the relationship between p53 and EBV gene expression, the tissues were studied for latent membrane protein 1 (LMP1) expression by immunohistochemistry. Viral LMP1 was observed in the large paracortical lymphocytes of all 10 cases of IM, indicating co-localization of p53 and LMP1 in these cells. Our findings confirm that p53 overexpression is not specific for nodal malignancy and that p53 accumulation is characteristic of IM. Because p53 was not coexpressed in the same cells as BZLF1, it appears that BZLF1 is not directly responsible for p53 accumulation. Nevertheless, co-localization of p53 and LMP1 in activated-appearing lymphocytes suggests that EBV infection is responsible for p53 accumulation. HUM PATHOL 31:1397-1403.

Absence of Epstein-Barr virus in lymphomatoid papulosis. An immunohistochemical and in situ hybridization study.

BACKGROUND AND DESIGN: Lymphomatoid papulosis (LyP) and cutaneous Hodgkin's disease share many clinical, histopathologic, and immunohistochemical features. Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several lymphoid malignancies, including Hodgkin's disease. Given the similarities between LyP and Hodgkin's disease, we asked if EBV could be detected in lesions of LyP. We examined 31 specimens of LyP that were obtained from 24 patients for evidence of EBV by in situ hybridization to EBER1 transcripts and for immunohistochemistry of viral latent membrane protein 1 (LMP1). RESULTS: In no instance there was there any evidence of EBV gene products by either in situ hybridization or immunohistochemistry. CONCLUSIONS: The absence of EBV in LyP suggests that this virus is not operative in the pathogenesis of LyP. Furthermore, it suggests that LyP and Hodgkin's disease may not share the same molecular mechanisms despite their phenotypic similarities.

Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation.

Ninety-five cases of adenocarcinoma of the stomach were evaluated for the presence of Epstein-Barr virus (EBV) using a sensitive in situ hybridization assay targeting Epstein-Barr virus-encoded RNA 1 (EBER1) transcripts. EBER1 was detected in 11 of 95 (12%) of cases. When present, the virus was localized to malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in normal-appearing gastric epithelium or intestinal metaplasia. The EBV DNA was monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant transformation. The presence of EBV was strongly associated with increased numbers of tumor-infiltrating T lymphocytes; however, EBV was not associated with prolonged survival. Neither p53 nor bcl-2 were consistently detected in the EBV-associated tumors. Specifically, 6 of 11 EBV-positive carcinomas had accumulation of p53 protein by immunohistochemical analysis, which was similar to the prevalence of p53 accumulation in EBV-negative specimens and suggests that EBV infection does not substitute for p53 mutations during tumorigenesis. The bcl-2 oncoprotein was expressed in a third of the carcinoma specimens tested, but bcl-2 expression did not correlate with the presence of EBV or with expression of EBV latent membrane protein 1. In conclusion, EBV infection appears to precede malignant transformation in a significant fraction of gastric carcinomas, but neither bcl-2 expression nor p53 accumulation appear to be consistently associated with the presence of the virus.

Epstein-Barr virus is detected in undifferentiated nasopharyngeal carcinoma but not in lymphoepithelioma-like carcinoma of the urinary bladder.

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.

Lymphomas of the oral soft tissues are not preferentially associated with latent or replicative Epstein-Barr virus.

OBJECTIVES: Epstein-Barr virus is periodically shed in the saliva of persons infected by the virus. Epstein-Barr virus has been implicated in the pathogenesis of certain subtypes of lymphoma, particularly high-grade lymphomas. Because high-grade subtypes represent the majority of lymphomas that arise in oral soft tissues, we hypothesized that Epstein-Barr virus might be preferentially associated with oral lymphomas. STUDY DESIGN: A series of 34 oral lymphomas were diagnosed according to the revised European-American classification scheme. They were examined for the presence of latent Epstein-Barr virus by EBER1 in situ hybridization and for expression of the Epstein-Barr virus replicative protein, BZLF1, by immunohistochemistry. RESULTS: Epstein-Barr virus EBER1 transcripts were detected in 11 of 31 oral lymphomas including 7 of 10 AIDS-related lymphomas and only 4 of 21 lymphomas that occurred in nonimmunocompromised persons. The Epstein-Barr virus-containing lymphomas were all high-grade histologic subtypes, that is, diffuse large cell, immunoblastic, or Burkitt's lymphomas. In contrast, Epstein-Barr virus was not detected in any of five low-grade oral lymphomas. In the single case of T-cell lymphoma in this study, EBER1 was expressed in the tumor cells. A switch from viral latency to replication, as measured by EBV BZLF1 expression, was identified in rare lymphoma cells in only four cases. This rate of viral replication was not higher than what has been reported in lymphomas arising at other anatomic sites. Although one of our lymphomas arose at a site of previous oral hairy leukoplakia, there was no other evidence that Epstein-Barr virus replication predisposed to development or persistence of oral lymphomas. CONCLUSIONS: These data suggest that even though Epstein-Barr virus is frequently found in oral secretions, neither latent nor replicative Epstein-Barr virus is present more commonly in oral lymphomas than in lymphomas arising in other anatomic sites, when controlling for immunodeficiency status.

Diagnosis of Epstein-Barr virus associated nasopharyngeal carcinoma using fine-needle aspiration biopsy and molecular diagnostics.

Molecular technology is being utilized increasingly for diagnostic purposes by practicing pathologists. Techniques such as Southern blot, in situ hybridization, and polymerase chain reaction have recently been introduced to the clinical laboratory setting. We describe a case of nasopharyngeal carcinoma that highlights the potential utility of DNA technology to secure an accurate diagnosis of a fine-needle aspiration biopsy. In this patient, cytologic examination of a cervical lymph node aspirate strongly suggested the possibility of a nasopharyngeal carcinoma. Needle aspirate material was submitted for molecular genetic detection of the Epstein-Barr virus (EBV) genome. Nine micrograms of DNA were isolated, and the presence of clonal EBV DNA was detected by the Southern blot technique. The presence of clonal EBV supported the cytologic diagnosis of nasopharyngeal carcinoma. Subsequent biopsy of a nasopharyngeal mass revealed undifferentiated carcinoma, and in situ hybridization revealed that EBV was restricted to the malignant epithelial cells. This case illustrates how molecular technology can provide new information that is useful in diagnostic cytopathology.

Absence of a reservoir of Epstein-Barr virus (EBV) in normal tongue epithelium.

We examined human tongue epithelium and serum samples at autopsy for evidence of latent Epstein-Barr virus (EBV) infection. Although clinical serology revealed anti-EBV antibodies in most sera indicating past EBV infection, we found no Epstein-Barr nuclear antigen (EBNA)-coding sequences in tongue tissue by polymerase chain reaction (PCR), or Epstein-Barr-encoded RNA (EBER1) by in situ hybridization. Tongue epithelium does not appear to be a natural reservoir for latent EBV in immunocompetent hosts.

Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Sternberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity.

One hundred twenty-five cases of Hodgkin's disease from the United States (79), Mexico City (31), and Costa Rica (15) were analyzed for the presence of Epstein-Barr virus (EBV) by in situ hybridization to EBER1 transcripts. EBV was more frequently detected in the Reed-Sternberg (RS) cells of mixed cellularity Hodgkin's disease (37 of 48 [77%]) compared with the nodular sclerosis subtype (19 of 71 [27%], P < .001). The presence of EBV was also associated with Hispanic ethnicity (P < .001). In a multivariate analysis, patient age, gender, and geographic location were less predictive of EBV positivity than were mixed cellularity histology (odds ratio = 8.3) and Hispanic ethnicity (odds ratio = 4.3). Southern blot analysis of EBV terminal repeat fragments using the Xho1a probe showed that the viral DNA was monoclonal in 17 of 17 cases having EBER1-positive RS cells. By comparison, EBV DNA was not detected by Southern analysis in 20 cases lacking EBER1 in RS cells, even when occasional background lymphocytes expressed EBER1. Because clonal viral DNA was so readily detected in EBER1-positive cases, the EBV genome is probably amplified at least 50-fold in the infected RS cells. Monoclonality of EBV DNA implies that the RS cells were infected before malignant transformation.

New clinical roles for pharmacists: a study of role expansion.

This study assessed the legitimacy of expanded roles for pharmacists with different status audiences. Pharmacy is a profession in transition and is characterized by considerable ambiguity and uncertainty concerning its status as a health care profession. Significant changes have occurred within the profession of pharmacy in the past few decades which have led to loss of function, social power and status. The response of the profession has been a movement toward a patient-oriented, clinical role for pharmacists. Hypotheses concerning level of support for expanded roles were derived from two conflict-based models of professionalization: a power model which focuses on conflict between professions and the central role of power in defining occupational territory; and a process model which focuses on conflict of interest and diversity within a profession and the development of 'segments' which struggle for control of a profession's direction. Data were collected by self-administered questionnaires sent to California pharmacists, physicians and nurses. Respondents were asked to indicate level of support for 20 role activities for pharmacists working in two practice settings (community and hospital). Pharmacy faculty were the most supportive of the clinical role activities, followed by practicing pharmacists, nurses and physicians. Physicians and nurses were more antagonistic toward clinical activities in the community than hospital practice setting, and were most antagonistic toward role activities which require independent judgement or autonomous action relevant to patient care on the part of the pharmacist. Differences were also noted in support for clinical role activities within the pharmacists' group. The effect of experience in working with a clinical pharmacist on support for clinical role activities is also discussed.