HPV Infection, but Not EBV or HHV-8 Infection, Is Associated with Salivary Gland Tumours.

Benign and malignant salivary gland tumours are clinically heterogeneous and show different histology. Little is known about the role of human herpes virus 8 (HHV-8), Epstein-Barr virus (EBV), and human papillomavirus (HPV) infection in salivary gland neoplasms. We investigated the presence of the three viruses in formalin-fixed, paraffin-embedded tissue samples in a cohort of 200 different salivary gland tumours. We performed EBV-LMP-1 and HHV-8 and p16 immunohistochemistry, a specific chip based hybridization assay for detection and typing of HPV and a chromogenic in situ hybridization for EBV analysis. Only one case, a polymorphic low-grade carcinoma, showed HHV-8 expression and one lymphoepithelial carcinoma was infected by EBV. In 17 cases (9%) moderate or strong nuclear and cytoplasmic p16 expression was detected. The HPV type was investigated in all of these cases and additionally in 8 Warthin's tumours. In 19 cases HPV type 16 was detected, mostly in Warthin's tumour, adenoid cystic carcinoma, and adenocarcinoma NOS. We concluded that HHV-8 infection and EBV infection are not associated with salivary gland cancer, but HPV infection may play a role in these tumour entities.

Human papilloma virus (HPV) is not implicated in the etiology of Warthin's tumor of the parotid gland.

CONCLUSION: The lack of human papilloma virus (HPV) sequences as well as potential HPV-activated cells such as cells that would be p16- and Ki-67 positive does not support a role of HPV in the pathogenesis of this lesion. OBJECTIVE: The exact etiopathogenesis of Warthin's tumor of the parotid gland is still unclear. The aim of the present study was to evaluate if HPV could play a role in the development of this parotid lesion. METHODS: Tissue samples from 40 Warthin's tumors of the parotid gland were investigated by PCR followed by in situ hybridization. The immunohistochemical expression of p16 and the dual immunostaining of p16 and Ki-67 were evaluated in all samples. RESULTS: Strong and diffuse p16 immunoreactivity was found in 7 of the 40 cases (17.5%). In situ hybridization showed a diffuse episomal signal in those samples. However, PCR could not reliably detect the presence of HPV genes. Furthermore, p16-expressing epithelial cells were mostly negative for the proliferation marker Ki-67.

Is human papilloma virus associated with salivary gland neoplasms? An in situ-hybridization study.

OBJECTIVE: HPV can infect cells of epithelial origin and is closely associated with carcinomas. Studies investigating its presence in salivary gland neoplasms are few and conflicting. METHODS: Detection of HPV types 16 & 18 was done on 34 formalin-fixed, paraffin-embedded archival material of different salivary gland neoplasms using Digene HPV types 16 & 18 probe using in situ hybridization technique. RESULTS: Eight of neoplastic salivary gland specimens were positively infected by HPV types 16 & 18. Seven of them were benign (4 Warthin's tumour, 2 pleomorphic adenoma and one myoepithelioma), in addition to one malignant specimen (lymphoma). Correlation was found between the incidence of HPV infection and histological differentiation of salivary gland neoplasms. CONCLUSIONS: An association exists between HPV infections and salivary gland neoplasms. However, given the sparse pattern of reactive cells, it cannot be confirmed that this virus is implicated in the aetiology of this group of tumours.

Lymphadenoma of the salivary gland: clinicopathological and immunohistochemical analysis of 33 tumors.

Lymphadenomas (LADs) are rare salivary gland tumors. Their clinicopathologic characteristics and etiopathogenesis are poorly understood. We examined 33 LADs in 31 patients (17 women and 14 men) aged 11-79 years (median 65 years). There were 22 sebaceous LADs in 21 patients (9 women and 12 men) and 11 non-sebaceous LADs in 10 patients (8 women and 2 men). Two patients had synchronous double tumors. Twenty-six tumors (79%) arose in parotid, three in the neck, and two each in submandibular gland and oral cavity. Extraparotid tumors were seen in 2 of 21 (10%) patients with sebaceous and 4 of 10 (40%) patients with non-sebaceous LADs. Seven of twenty-three (30%) patients had immunosuppressive therapy for unrelated diseases. The tumors were well circumscribed, encapsulated (n=28, 84%) painless masses, varying in size from 0.6 to 6 cm (median 2.2). The cut surfaces were gray-tan to yellow, homogeneous and multicystic (n=24, 72%). The epithelial cells were basaloid, squamous and glandular, forming solid nests, cords, tubules, and cysts. Sebaceous differentiation was restricted to sebaceous lymphadenoma. The epithelial cells expressed basal cell markers (p63, 34BE12, and/or CK5/6, 18/18, 100%) and the luminal glandular cells expressed CK7 (12/12, 100%). Myoepithelial cells were absent (n=10/16, 63%) or focal. The lymphoid stroma was reactive, with germinal centers in 28 (84%). There was no evidence of HPV (0/11), EBV (0/7), and HHV-8 (0/8). Malignant transformation to sebaceous and basal cell adenocarcinoma was seen in one patient each. None of the 11 patients with follow-up (1-8 years) recurred. In summary, sebaceous and non-sebaceous LADs are benign, encapsulated, solid and cystic tumors affecting older adults. Non-sebaceous LADs affect women and extraparotid sites more frequently than sebaceous LADs. Altered immune status may have a role in their etiopathogenesis. Multiple synchronous tumors, origin in buccal mucosa, and malignant transformation may rarely occur.

High prevalence of Human Herpes Virus 8 (HHV-8) in patients with Warthin's tumors of the salivary gland.

BACKGROUND: Warthin's tumor is a common benign neoplasm of the salivary gland. Human Herpes Virus 8 (HHV-8) is the etiologic agent for all forms of Kaposi's sarcoma (KS), and HHV-8 DNA is present in saliva, suggesting that non-sexual transmission is associated with latent infection of the salivary gland. OBJECTIVES: To provide insights into the HHV-8 cell tropism, the presence of HHV-8 was investigated in a series of Warthin's tumors of the salivary gland and corresponding adjacent normal tissue. STUDY DESIGN: Forty-three patients with Warthin's tumors (cystadenolymphoma) were tested for the presence of HHV-8 DNA, and corresponding adjacent normal tissue samples were obtained from 15 patients. DNA was extracted from the paraffin-embedded tissues. A nested polymerase chain reaction (PCR) assay was applied, and the positive samples were confirmed by direct sequencing. RESULTS: HHV-8 DNA was detected in 19 out of 43 (44%) salivary gland tumor samples. Among the 15 cases with paired samples, 9 were HHV-8-positive for both samples, 4 were HHV-8-negative for both samples while in two cases HHV-8 was detected only in the tumor specimens. CONCLUSIONS: HHV-8 is frequently detected in adenoid salivary neoplasms, suggesting a significant role of the virus in the etiopathogenesis of the disease. Larger studies are required to investigate the role of HHV-8 in the development or progression of Warthin's tumors.

Low prevalence of transfusion transmitted virus (TTV)-like DNA sequences in cystadenolymphoma and pleomorphic adenoma of the salivary glands.

Titers of transfusion transmitted virus (TTV)-like DNA in saliva samples have been reported 100-1,000 times higher than those of the corresponding sera, suggesting viral transmission by saliva droplets. The present study was conducted to determine whether TTV-like DNA sequence elements play a role in the pathogenesis of cystadenolymphoma or pleomorphic adenoma and if the parotid or the submandibular gland is a major source of TTV persistence. Sixty-two archival salivary gland samples (16 cystadenolymphomas, 13 pleomorphic adenomas, and 33 controls) and 23 corresponding saliva samples were examined using a polymerase chain reaction (PCR) for TTV DNA. All PCR products that displayed DNA bands were sequenced. Leder's stain and immunohistochemistry (anti-CD8, anti-CD20, anti-CD45R0, anti-CD68, and anti-Ki67/MiB1) were applied to detect possible changes associated with findings of TTV-like DNA sequences. Tissue displayed TTV-like DNA sequences in 8.1% (5/62; saliva: 47.8%, 11/23). Tissue that contained TTV-like DNA sequences was histologically indistinguishable from samples lacking such DNA. TTV appears to be only a bystander in cystadenolymphoma, pleomorphic adenoma, and other salivary gland affections. Neither of the glands seems to be a major source of TTV persistence.

Role of Epstein-Barr virus and cytomegalovirus in the etiology of benign parotid tumors.

BACKGROUND: Pleomorphic adenomas and Warthin's tumors are the two most common benign parotid tumors. Previous studies investigating the role of viruses in tumorigenesis of these neoplasms have been conflicting. The aim of this study was to determine whether Epstein-Barr virus (EBV) or cytomegalovirus (CMV) might play a role in the pathogenesis of pleomorphic adenomas and Warthin's tumors. METHODS: Paraffin-embedded surgical specimens of 24 pleomorphic adenomas, 10 Warthin's tumors, and 13 normal parotid tissues were obtained from the University of California-San Francisco Pathology Department. Genomic DNA was extracted from the specimens, and primers for connexin 26, a gap junction protein, were used to confirm the integrity of this DNA. The presence or absence of EBV and CMV DNA within the samples was determined with PCR-based assays, in which radiolabeled primers were used for maximal sensitivity of detection. RESULTS: PCR analysis of serially diluted control DNA revealed that using radiolabeled primers, five copies of viral DNA could be detected. By use of this method, we showed that none of the 24 pleomorphic adenomas, 10 Warthin's tumors, or 13 normal parotid samples contained EBV DNA or CMV DNA. CONCLUSIONS: These results do not support CMV or EBV as etiologic factors in pleomorphic adenomas or Warthin's tumors. In addition, normal parotid seems not to harbor either of these viruses. Future studies with larger numbers of specimens are needed to confirm these findings.

[Clinical study of bilateral parotid tumors].

We studied retrospectively 212 patients with parotid tumors who were treated in our hospital between October 1981 and March 1998. One hundred seventy-two of the tumors were benign, and 40 of them were malignant. The tumors were bilateral in 13 patients. Since 1992, we have treated at least 1 bilateral parotid tumor patient per year, and the number of patients with bilateral parotid tumors has tended to increase. Histologically, adenolymphomas occurred in 11 patients, and there was one occurrence of pleomorphic adenoma and one occurrence of basal cell adenoma. Eighty-five percent of all bilateral parotid tumors were adenolymphomas, and the bilateral parotid tumors comprised twenty percent (11 of 53 patients) of all adenolymphomas that we encountered. Among the 13 patients with bilateral parotid tumors, 1 patient experienced them heterochronously. In 7 of the 13 patients the tumor on the opposite side was found by diagnostic imaging. One patient showed recurrence in both parotid glands 4 years after initial surgery. Comparing bilareral adenolymphomas with unilateral adenolymphomas, there was no significant difference in the age or sex of the patients. Regarding bilateral adenolymphoma, 4 patients showed a solitary tumor on either side, 4 patients showed a solitary tumor on one side and multiple tumors on the other side, and 4 patients showed multiple bilateral tumors. Regarding unilateral adenolymphoma, 38 patients showed solitary tumors and 4 patients showed multiple tumors. Bilateral adenolymphomas were more multicentric than unilateral adenolymphomas. Epstein-Barr virus-encoded RNA (EBER) was detected in 11 of the 12 bilateral adenolymphomas and in 18 of 35 patients with unilateral adenolymphoma, by in situ hybridization. EBER was detected more frequently in the multiple unilateral adenolymphomas than in the solitary unilateral adenolymphomas. Based on our experience, the bilateral parotid tumor is not rare. Care should be taken to observe the other side of the parotid gland with parotid tumors that are suspected adenolymphomas. Imaging may be helpful for the detection of bilateral tumors. A relationship may exist between Epstein-Barr virus and adenolymphoma multicentricity.

Warthin's tumour is not an Epstein-Barr virus related disease.

BACKGROUND: The histogenesis of Warthin's tumour (WT) is controversial. A possible role for Epstein-Barr virus (EBV) has been suggested. MATERIALS AND METHODS: Twenty formaldehyde-fixed, paraffin-embedded blocks of WT from the parotid gland were examined for the presence of EBV. In situ hybridisation was performed using EBV encoded small nuclear RNAs (EBER1/2) probes labelled with fluorescein isothiocyanate. An EBV-positive P3HR-1 cell line processed to paraffin wax was used as a positive control and a brain section as negative control. RESULTS: EBER1/2 could not be found in the neoplastic epithelial cells in any of the tumours nor in the adjacent normal parotid tissues. Individual positive lymphocytes were present in 7 tumours. CONCLUSIONS: These results indicated that EBV is not involved in the pathogenesis of WT.

[Detection of Epstein-Barr viral genome in tumor cells of Warthin's tumor of parotid gland].

OBJECTIVE: To evaluate the relations between Warthin's tumors of salivary gland and Epstein-Barr virus (EBV). METHODS: 75 cases of salivary gland Warthin's tumor and 20 cases of normal salivary gland tissue were examined for to detect EBV genoma by using the polymerase chain reaction (PCR) of DNA, 62 cases of single Warthin's tumor and 13 cases of multiple/bilateral Warthin's tumor's. RESULTS: EBV DNA was detected in 13 cases of single Wathin's tumors, 9 cases of multiple/bilateral Warthin's tumor and 3 cases of normal salivary gland. CONCLUSION: There was a close relationship between EBV and multiple/bilateral Warthin's tumors.

EBER oligonucleotide RNA in situ hybridization in EBV associated neoplasms.

In virus associated diseases identification of viruses in cells can contribute to the understanding of the pathogenesis and may also help to establish the diagnosis. In the present communication, the effects of the microwave pretreatment (MWP) and that of the proteinase-K enzymatic predigestion (PKD) on EBER RNA oligonucleotide in situ hybridization (EBER-RNA-ISH) (EBER: Epstein-Barr-Encoded-(Early)-RNA) were studied. The efficacy of two EBV detecting methods, latent membrane protein-1 (LMP-1) immunohistochemistry and EBER-RNA-ISH were also compared. Our results show that microwave pretreatment enhances the intensity of the ISH signals and preserves significantly better the structure of the tissues compared with enzymatic predigestion. EBER-RNA-ISH, mainly in the nasopharyngeal carcinoma cases, showed a more frequent positivity than the immunohistochemical reaction for LMP-1, however in case of the Warthin's tumor only the LMP-1 protein was expressed.

Molecular characterization of Warthin tumor.

OBJECTIVE: Warthin tumor of the salivary gland is composed of oncocytic epithelium with a prominent follicular lymphoid infiltrate. The purpose of this study was to characterize the clonality of this lymphoid component by means of polymerase chain reaction technology. STUDY DESIGN: DNA was isolated from paraffin-embedded tissue from 20 cases of typical Warthin tumor of the salivary gland and amplified by polymerase chain reaction to assess B- and T-cell clonality. RESULTS: No dominant clonal populations were identified in any tumor. However, minor clonal expansions of both B and T cells were detected in up to 50% of tumors (immunoglobulin H, 50%; T-cell antigen receptor beta, 10%; T-cell antigen receptor gamma, 5%). No tumors showed evidence of bcl-2 proto-oncogene translocation, whereas 95% contained detectable Epstein-Barr virus DNA. CONCLUSION: The B- and T-cell components of Warthin tumor are polyclonal with oligoclonal expansion of both T and B cells in some lesions.

No significant role of Epstein-Barr virus in the tumorigenesis of Warthin tumor.

To determine the significance of Epstein-Barr virus (EBV) in the tumorigenesis of Warthin tumor, formaldehyde-fixed and paraffin-embedded specimens of 21 tumors from 18 patients were examined by polymerase chain reaction (PCR)-Southern hybridization, in situ hybridization (ISH), and immunohistochemistry. PCR-Southern hybridization revealed the presence of EBV DNA in 13 of the 21 tumors (61.9%). However, ISH for EBV RNA showed that nuclei of the neoplastic epithelial cells of all tumors were negative. Although ISH for EBV DNA revealed that the nuclei of the neoplastic epithelial cells were positive in 4 of the 21 tumors (19.0%), the positive cells were sparsely distributed and there was no evidence of monoclonal proliferation of EBV positive neoplastic epithelial cells. Moreover, immunohistochemical reactions with antibodies against latent membrane protein 1 (LMP1) and EBV nuclear antigen 2 (EBNA2), were negative in all cases. Judging from these findings, we conclude that EBV does not play a major role in the tumorigenesis of Warthin tumor.

No Epstein Barr and cytomegalovirus DNA found in salivary gland tumours.

A series of 219 (106 malignant and 113 benign) salivary gland tumours was investigated by in situ hybridisation (ISH) to detect Epstein-Barr-virus (EBV) and cytomegalovirus (CMV) DNA. Normal salivary gland did not show hybridisation signals for these viruses. Tumours presenting hybridisation signals in ductal and myoepithelial cells and 17 Warthin's tumours were further studied by PCR, but none of these tumours contained EBV or CMV DNA. Our series did not contain lymphoepithelial carcinomas, which are EBV-associated tumours. The results suggest that factors other than EBV or CMV are involved in carcinogenesis of primary salivary gland tumours.

Adenolymphoma and non-Hodgkin's lymphoma of the salivary glands and oral cavity in immunocompetent patients are not associated with latent Epstein-Barr virus.

The presence of Epstein-Barr virus (EBV) was studied in specimens of 50 primary non-Hodgkin's lymphomas (NHL) of the salivary gland and the oral cavity and 11 solitary adenolymphomas of the parotid gland, using EBER-1/2 in situ hybridisation and by immunohistochemistry for the detection of latent membrane-protein-1 (LMP-1). None of the patients were tested for HIV-infection, nor were there any clinical signs to suspect HIV-infection. In one adenolymphoma, few reactive EBER-1/2 positive cells were detected. In this case staining for LMP-1 was negative. In one oral B-cell NHL, EBER-1/2 positive lymphoma cells were identified; these cells also expressed LMP-1. None of the 31 oral (30 B-cell and one T-cell) and 18 salivary gland (all B-cell) NHLs and none of the 10 adenolymphomas were EBER-1/2 positive or expressed LMP-1. These results indicate that EBV is not involved in the pathogenesis of oral and salivary gland primary NHL and adenolymphoma in immunocompetent patients.