Case 1 -

Undifferentiated Nasopharyngeal Carcinoma (Lymphoepithelioma) Ricardo V. Lloyd and Arie Perry

Clinical History A 47-year-old man presented with an enlarged cervical lymph node which was excised. Gross Description The enlarged cervical lymph node was firm and tan measuring 3 cm in diameter. Case 1 - Figure 1 Case 1 - Figure 2 Case 1 - Figure 3 Microscopic Description The carcinoma consisted of poorly differentiated tumor cells with large vesicular nuclei, prominent nucleoli, and indistinct cell borders infiltrating the lymph nodes in small clusters and large syncytial nests. Discussion Epstein-Barr virus (EBV) is a herpes virus composed of a 172-Kb pair viral genome. The virus is transmitted in saliva and is distributed worldwide. In Western countries in which some segments of the population have a delayed exposure to the virus until adolescence or young adulthood, primary infection is manifested as infectious mononucleosis. During primary infection up to 10% of peripheral blood B cells may be infected. EBV may infect non-B cells possibly via CD21 expression. EBV exists in latent and lytic states. It is in the latent state that EBV is associated with clinically significant diseases. After infection, about 10% of the latently infected B lymphocytes become immortalized which requires several EBV proteins including EB nuclear antigen-1 (EBNA-1), a specific DNA binding protein; EBNA-2, a specific transcriptional transactivator of viral and cellular genes and the latency membrane protein-1 (LMP-1), a transmembrane phosphoprotein. In contrast, some proteins such as ZEBRA or BZLF-1, the EBV replication activator, is associated with a switch from latent to lytic infections. Epstein-Barr virus has been directly implicated in the pathogenesis of acute infectious mononucleosis and has been implicated in the development of many disease processes including nasopharyngeal carcinoma (Table 1). Table 1. Diseases Associated With EBV Infectious mononucleosis Post-transplant lymphoproliferative disorders B or T cell lymphoma Nasopharyngeal carcinoma Hairy leukoplakia Gastric carcinoma Hodgkin's disease AIDS-associated lymphomas Burkitt's lymphoma Richter's syndrome X-linked lymphoproliferative disease Lymphomatoid granulomatosis Primary lymphoepithelioma-like carcinoma of the lungs ISH has been extremely useful in showing the localization of viral RNA or DNA in specific neoplasms and other EBV-related diseases [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40] . The Epstein-Barr encoded RNAs (EBER) genes are expressed early during latent EBV infection of lymphocytes. These genes code for small RNAs which are expressed at levels of 106 to 107 copies/cell. They are short transcripts of approximately 170 nucleotides and have minimal homology with cellular RNA. Although their function is unknown, their abundant presence provides ideal targets for ISH detection. In situ hybridization can be performed with various types of probes, but oligonucleotide probes for EBER RNAs are commonly used. In addition to EBER-1 and EBER-2 probes, EBV cDNA probes and oligonucleotide probes such as NOT-1 have been used to detect EBV infection in tissues. EBV proteins such as LMP and EBNA can also be detected by immunohistochemistry. In our experience ISH for EBER-1 and 2 are the most sensitive methods, because of the high copy numbers of these small RNAs. Nasopharyngeal carcinoma is relatively common in Southern China and Southeastern Asia, but is rare in most other parts of the world. It is strongly associated with EBV in all areas of the world (Table 2). Table 2. Geographic Association of EBV With Nasopharyngeal and Sinonasal Carcinomas Diagnosis - Location Gulley et al. [21] Lopategui et al. [9] NPC-US 21/24 (88%) -- NPC-Hong Kong 10/11 (91%) -- SNUC-Western -- 0/11 (0%) SNUC-Asia -- 7/11 (64%) NPC - nosopharyngeal carcinoma SNUC - sinonasal undifferentiated carcinoma In addition to EBER, there is restricted expression of EBNA-1 and sometimes LMP-1, but not other members of the EBNA family [2]. In a study of 846 paraffin-embedded tissue specimens for EBERs Hamilton-Dutoit and Pallisen [8] found EBER-1 RNA in 100% of cases of nasopharyngeal carcinomas from Denmark (7/7), and Greenland (10/10), and 94% from China (65/69). All 11 cases of salivary gland lymphoepithelioma-like carcinomas from Greenlandic eskimos were also positive for EBER-1 [8]. In a study of 22 patients with sinonasal undifferentiated carcinoma, Lopategui et al. reported that 7 of 11 Asian patients but 0of 11 Western patients were positive for EBV EBER-1, suggesting that either genetic predisposition or environmental cofactors played an important role in determining the strength of the association of this carcinoma with EBV [9]. Leung et al. detected EBERs in 7/29 sinonasal carcinomas including adenocarcinomas and cylindic cell carcinomas [11]. The detection of EBV genome in sinonasal and salivary gland lesions may be related to the techniques that are used, since a majority (13/20) of inverted sinonasal papillomas were found to be positive for EBV by solution PCR in one study [15]. EBV has been reported in other carcinomas including gastric esophageal, thymic, lung and salivary gland tumors [16, 17, 18, 19, 20] . Most of the carcinomas with EBV positivity have a lymphoid stroma, and the ISH procedure usually shows positive staining in the carcinoma cells and not in the lymphoid stroma. Interestingly, not all cases of lymphoepithelioma-like carcinomas express EBV as was recently shown in a series of bladder carcinomas [21]. In the study of Shibata and Weiss, there was a higher percentage of men (21%) than women (3%) who had EBV in their gastric tumors [16]. In the recent study of 11 Chinese patients with pulmonary lymphoepithelioma-like carcinomas, Chan et al. [20] observed EBV genome in all patients. In contrast while all other eight Asian patients with pulmonary lymphoepithelial carcinomas reported in the previous literature were EBV positive, none of the four Caucasian patients were positive [20] again implicating genetic and environmental factors with EBV-related carcinomas in these patients. Other recent studies have found EBV in lung lymph epithelioma-like carcinoma in Chinese patients but not in patients from Western countries [22]. Does the presence of EBV in nasopharyngeal carcinoma (NRC) have prognostic significance? One recent study has suggested that patients with EBV-positive NPC had increased survival compared to EBV-negative ones [23]. EBV is frequently associated with lymphoproliferative disease in immunocompromised patients, including transplant recipients and HIV positive individuals. In organ transplant recipients, B cell lymphoproliferative disease is almost always associated with EBV. In AIDS patients EBV is consistently associated with primary central nervous system lymphoma and with Hodgkin's disease. Various investigators have used ISH, PCR, or Southern hybridization to detect EBV genome in post-transplant lymphoproliferative disorders (PTLPD) [24, 25, 26, 27, 28] . In situ hybridization studies have shown that EBER is expressed in almost all cases of PTLPD or in lymphomas arising in these patients (Table 3). Table 3. EBV RNA Detected in Various Lymphomas Diagnosis Chang et al. [3] Hamilton- Dutoit et al. [8] Teramoto et al. [34] Angioimmunoblastic lymphadenopathy 26/27 (96%) -- 8/12 (67%) Follicular lymphoma 0/20 (0%) -- 5/25 (20%) Hodgkin's disease 11/23 (48%) 46/121 17/28 (38%, 61%) 17/32 (53%) Large cell lymphoma 1/20 (5%) -- 27/145 (19) T-cell lymphoma 2/11 (18%) 18/67, 24/37 (27%, 65%) 22/71 (31%) Transplant lymphoma 11/11 (100%) 16/18 (89%) --- Burkitts lymphoma - Africa -- 12/12 (100%) --- Burkitts lymphoma - Denmark -- 0/29 (0%) --- Burkitts lymphoma - China -- 0/3 (0%) --- In a recent series EBER-1 was detected in most cases of PTLPD (16/18). When lymphocytic infiltrates are detected in a transplanted organ, it may be difficult to distinguish between an EBV-driven PTLPD and rejection, so the EBER ISH analysis along with immunostaining for B and T cells can allow rapid characterization of the infiltrate [28]. Recent studies done identified a subset of EBV-negative PTLPD patients [29]. In a study by Nelson et al [29], the number of EBV-negative patients had increased in recent years (Table 4). Table 4. Year of Diagnosis for EBV-Negative and Positive PTLD Year of Diagnosis EBV-Negative (number of specimens) EBV-Positive (number of specimens) 1982-1985 0 12 1986-1990 1 45 1991-1996 17 58 Nelson, et al. Am J Surg Pathol 24:375-385, 2000 Some of these patients did respond to decreased immunosuppression similar to EBV-positive cases, suggesting that EBV positivity need not be an absolute criteria for the diagnosis of PTLPD [29]. Oral hairy leukoplakia (OHL) is a lesion occurring on the lateral aspects of the tongue predominantly in immunosuppressed patients with AIDS. Histologically, there are Cowdry A inclusions which appear as basophilic intranuclear inclusions that marginate host-chromatin into a beaded pattern. Hairy leukoplakia might be the presenting lesion for HIV seropositive individuals, and in an HIV-seropositive patient, the AIDS syndrome is likely to develop within three years [30]. Patients with OHL may have HPV and HIV localized in the lesion in addition to EBV. Most of the early evidence suggested that OHL was an active infection since EBV genome was not present in the basal cell layer [31, 32, 33] , indicating direct infection of upper epithelial cells with virus from salivary or adjacent replicating infected cells. However, a recent study using in situ PCR detected EBV in the basal and parabasal layers of the squamous epithelium and suggested that AIDS might represent a reactivation of latent lingual infection associated with a marked increase in viral copy numbers in the mature superficial squamous cells [33]. EBV has been associated with various types of lymphomas including predominantly B cell lymphomas, some T cell lymphomas, lymphomatoid granulomatosis and Hodgkin's disease [34, 35, 36, 37, 38, 39, 40, 41] . Cheng and Weiss detected EBER in 26/27 cases of angioimmunoblastic lymphadenopathy, and in 11/23 cases of HD, but in only a few B and T cell lymphomas (Table 1). In a recent large series from Japan, more T cell lymphomas were commonly associated with EBV than B cell lymphomas [34]. Similarly, in Chinese peripheral T cell lymphoma, EBV is frequently associated with these neoplasms (24/39 or 62% in series of Zhou et al. [35]). EBV is associated with Hodgkin's disease in 30 to 95% of cases [39] (Table 5). Table 5. Geographic Association of EBV with Lymphoid Neoplasms Diagnosis Western Africa South America Asia Burkitt's lymphoma 5%-20% 95% 25%-70% 28% Hodgkin's disease 40%-50% -- 50%-95% 65% Nasal T Cell Lymphoma 95% -- 95% 95% Weiss and Chang. Adv Anat Pathol 3:1-15, 1996 In one series, it was most commonly associated with mixed cellularity type (68%) followed by nodular sclerosis (24%) and was uncommon in lymphocyte predominance type (0%). In contrast, patients with HIV-associated H.D. almost always express EBV in the Reed-Stenberg cells and their variants [36, 37, 38, 39] . Pulmonary lymphomatoid granulomatosis (LYG) is frequently associated with EBV infection [40]. Combined ISH for EBV and immunohistochemistry for lymphoid markers has shown that the neoplastic B cells are the ones infected with EBV in this condition [40], indicating that some cases of LYG are B cell lymphomas associated with EBV infection. A few case reports of methotrexate therapy in patients with rheumatoid arthritis or dermatomyositis have been associated with the development of EBV associated lymphoproliferative disorders [42, 43] . In all of these cases the lymphoadenopathy regressed after discontinuation of methotrexate, suggesting that the therapy was a major factor in generating these lesions. Recent studies have also found clonal proliferation of EBV genome and latent EBV infection in leiomyosarcomas from pediatric AIDS patients and from immunosuppressed organ transplant recipients [44, 45] . 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