Association of Epstein-Barr Virus with Lymphoepithelioma-Like Carcinoma of the Lung in Southern China

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Anatomic Pathology / LYMPHOEPITHELIOMA-LIKE CARCINOMA OF LUNG

Association of Epstein-Barr Virus With Lymphoepithelioma-Like Carcinoma of the Lung in Southern China

An-jia Han, MD, Min Xiong, MD, and Yong-sheng Zong, MD

Key Words: Epstein-Barr virus; Lymphoepithelioma-like carcinoma; Lung neoplasm Downloaded from https://academic.oup.com/ajcp/article/114/2/220/1757846 by guest on 01 October 2021

Abstract Epstein-Barr virus (EBV) is the cause of infectious Having reviewed the data on 3,663 consecutive mononucleosis and is associated with African Burkitt cases of primary lung carcinoma in southern China, we lymphoma and nasopharyngeal carcinoma.1-4 In recent years, a found that 32 cases could meet the criteria for close relationship has been found between EBV and lymphoepithelioma-like carcinoma (LELC) of the lung. lymphoepithelioma-like carcinoma (LELC), which has To study the relationship between pulmonary LELC and histopathologic features similar to undifferentiated nasopha- Epstein-Barr virus (EBV) infection, we used in situ ryngeal carcinoma, especially those arising from foregut- hybridization and immunohistochemistry techniques to derived organs, such as lung, stomach, salivary gland, and detect the EBV-encoded small nonpolyadenylated RNA thymus.5-8 Although there are several reports about pulmonary (EBER), latent membrane protein 1 (LMP1), and viral LELC associated with EBV, the number of cases studied is capsid antigen (VCA) in 32 cases of LELC and 19 cases very limited. Having reviewed the data on 3,663 cases of lung of non-LELC lung carcinoma. Of the 32 cases, 30 cancer in Southern China where nasopharyngeal carcinoma is (94%) showed EBER positivity. Of the 30 EBER- prevalent, we found that only 32 cases could meet the criteria positive pulmonary LELC cases, 16 and 7 expressed for LELC. It is necessary to study the relationship between LMP1 and VCA, respectively. In contrast with LELC, pulmonary LELC and EBV infection in this geographic area. none of the 19 cases of non-LELC lung carcinoma showed EBER-, LMP1-, or VCA-positive signals in carcinoma cells. The results demonstrate that there is a Materials and Methods close relationship between EBV infection and pulmonary LELC. EBV infection may have an essential While reviewing 3,663 consecutive cases of pulmonary role in the tumorigenesis of pulmonary LELC. EBV carcinoma specimens that were collected by the Department of latent infection is the main status in pulmonary LELC Pathology, Sun Yat-sen University of Medical Sciences and its except for individual EBV entering into a lytic cycle. Tumor Hospital (Guangzhou, China), Sun Yat-sen Memorial Hospital (Guangzhou), and the First Affiliated Hospital of Guangzhou Medical College, from August 31, 1985, to September 1, 1995, we found 32 cases of LELC that met the 1999 World Health Organization criteria9 and staged them according to the 1997 TNM staging system.10 In addition, 19 cases of non-LELC (7 cases of squamous cell carcinoma, 2 cases of adenocarcinoma, 8 cases of large cell carcinoma, and 2 cases of small cell carcinoma) were included in our study as a control group. None of the 51 patients with primary pulmonary carcinoma had other history of organ carcinoma, especially

220 Am J Clin Pathol 2000;114:220-226 © American Society of Clinical Pathologists Anatomic Pathology / ORIGINAL ARTICLE nasopharyngeal carcinoma. These tissues were fixed in 10% labeled streptavidin-biotin detection kit (Code No. k681, neutral formalin and processed as a regular procedure. DAKO), and incubated with primary monoclonal antibody at 4°C for 12 hours. After washing with a 0.01-mol/L concentra- In Situ Hybridization tion of phosphate-buffered saline, the sections were incubated Paraffin sections, 4 µm thick, were mounted onto with link antibody at 37°C for 30 minutes and washed with a microscopic glass slides, which had been treated with 3- 0.01-mol/L concentration of phosphate-buffered saline, and aminopropyl-triethoxysilane (DAKO, Carpinteria, CA), labeled streptavidin was added at 37°C for 30 minutes. The deparaffinized, rehydrated in serially graded ethanol (100%, sections were colorized with 0.5% diaminobenzidine and 95%, 70%, 50%), immersed in 0.05% diethyl pyrocarbonate counterstained in 0.5% methyl green, air dried, and mounted for 6 min, and digested with 0.1 mg/mL of proteinase K for with glycerol gelatin. 30 minutes at 37°C. After washing with 0.05% diethyl pyro- The working concentrations of primary antibody for

carbonate, the sections were dehydrated through serially detection of latent membrane protein 1 (LMP1; OT 17-2, Downloaded from https://academic.oup.com/ajcp/article/114/2/220/1757846 by guest on 01 October 2021 graded ethanol (75%, 95%, 100%) and air dried. Next, 20 µL Organon Teknika, Durham, NC), viral capsid antigen (VCA; of fluorescein isothiocyanate (FITC)-labeled oligonucleotide OT rabbit 27-3, Organon Teknika), B lymphocytes (CD20 or probe (Code No. Y017, DAKO) was added. An acid-cleaned L26, DAKO, M755), and T lymphocytes (CD45RO or coverglass was placed on top, and the sections were incu- UCHL1, DAKO, M742) were 1:100, 1:200, 1:100, and 1:100, bated at 37°C for 2 hours. After hybridization, the sections respectively. were washed in blocking solution (0.1% Triton X-100 The positive results for LMP1, VCA, and phenotypes of [Sigma, St Louis, MO], a 1-mol/L concentration of lymphocytes were shown by brown-yellow in the cytoplasm tris[hydroxymethyl]aminomethane [Tris] hydrochloride, and and cell membrane. a 3-mol/L concentration of sodium chloride) at room temper- The B95-8 cell line carrying EBV was used as a positive ature for 15 minutes and incubated with 1% alkaline phos- control for LMP1 and VCA. A 0.01-mol/L concentration of phatase–conjugated rabbit anti-FITC antisera (DAKO, Code phosphate-buffered saline was used instead of primary anti- No. K046; 1% alkaline phosphatase–conjugated rabbit antibody LMP1 and VCA as the negative control in each run. FITC antisera; a 1.5-mol/L concentration of Tris hydrochloride, pH 7.5; and 5% bovine serum albumin) for 60 minutes. Statistical Analysis The sections were washed with 2× Tris-buffered saline (a 1- The chi-square test and the Fisher exact test were used as mol/L concentration of Tris hydrochloride, and a 3-mol/L appropriate. concentration of sodium chloride, pH 7.6) for 6 minutes. The sections were colorized with 100 µL of 5-bromo-4-chloro-3- indolylphosphate/4-nitro blue tetrazolium chloride Results (BCIP/NBT; DAKO, Code No. K046; 2% BCIP/NBT; 0.1% levamisole, a 0.05-mol/L concentration of magnesium chloride, and a 1-mol/L concentration of Tris hydrochloride, pH Histopathologic Features of Pulmonary LELC 9.5) in a dark room for 20 minutes, and the reaction was The clinical features for 32 patients with pulmonary stopped with running tap water. The slides were counter- LELC are given in ❚Table 1❚. The patients’ ages ranged from stained in 0.5% methyl green, air dried, and mounted with 39 to 73 years (mean, 54.4 ± 10.1 years), and the glycerol gelatin (Sigma). male/female ratio was 2.2:1. The majority of pulmonary The positive result for EBV-encoded small nonpoly- LELC have histopathologic features similar to those of adenylated RNA (EBER) by in situ hybridization was shown undifferentiated nasopharyngeal carcinoma. The tumor was by a dark brown color in the nuclei of cells. characterized by confluent sheets or small clusters of tumor The EBER-positive nasopharyngeal carcinoma specimen cells showing a syncytial arrangement with an ill-defined was used as a positive control. A 1-mol/L concentration of cytoplasmic outline, interspersed by wide stromal tissue Tris-buffered saline was used instead of the EBER probe as septa bearing an intense lymphoplasmacytic cell infiltration, the negative control in each run. including a few small lymphocytes percolating between tumor cells. Tumor cell nuclei were round, oval, or elon- Immunohistochemistry gated, with mildly irregular nuclear borders, delicate chro- The sections were deparaffinized, rehydrated in serially matin, and 1 or 2 distinct eosinophilic nucleoli ❚Image 1❚. A graded ethanol, and heated in citric buffer (a 0.01-mol/L foreign body and tuberculoid granulomatous reaction occur- concentration of citric acid, pH 6.0) once for 5 minutes in a ring in the periphery of the neoplastic nest was found in microwave oven for antigen retrieval. Then they were washed some cases. It is interesting that 2 cases LELC were accom- with distilled water, blocked with blocking solution from the panied by focal squamous epithelium differentiation. Spindle

❚Table 1❚ Clinical Data for Cases of Lymphoepithelioma-Like Carcinomas of the Lung

Case No./Sex/Age (y) Tumor Location Stage (TNM) EBER LMP1 VCA

1/M/72 RLL IB (T2 N0 M0) + – – 2/F/67 LUL IB (T2 N0 M0) + + – 3/M/44 LUL IIIA (T2 N2 M0) + – – 4/M/41 LUL IIIA (T2 N2 M0) + + – 5/F/66 RLL IA (T1 N0 M0) + – – 6/F/63 LH IB (T2 N0 M0) + – – 7/M/58 LUL IB (T2 N0 M0) – ND ND 8/M/56 LUL IIIA (T2 N2 M0) + – + 9/F/66 RML IA (T1 N0 M0) + – – 10/F/44 RML IIIA (T3 N1 M0) + – – 11/M/53 LLL IIB (T2 N1 M0) + – –

12/M/53 RUL IIA (T1 N1 M0) + + – Downloaded from https://academic.oup.com/ajcp/article/114/2/220/1757846 by guest on 01 October 2021 13/M/73 LLL IB (T2 N0 M0) + + – 14/M/58 RLL IIA (T1 N1 M0) + – – 15/F/49 RML IA (T1 N0 M0) + + + 16/M/45 RML IIIA (T2 N2 M0) + – – 17/M/58 RML IIIA (T3N1M0) + + – 18/M/54 LLL IIB (T2 N1 M0) + + – 19/M/61 LLL IB (T2 N0 M0) + + + 20/M/64 LLL IB (T2 N0 M0) + – – 21/F/55 RUL IIB (T2 N1 M0) + + + 22/F/43 LUL IIIA (T1 N2 M0) + + – 23/F/53 RM and LL IV (T2 N 2M1) + + + 24/M/60 RLL IIIA (T2 N2 M0) + + – 25/M/57 LLL IIB (T2 N1 M0) + + – 26/M/56 LUL IIB (T2 N1 M0) + – – 27/M/54 LLL IIIA (T3 N2 M0) + + – 28/F/39 LLL IIB (T3 N0 M0) + + + 29/M/45 RML IIIA (T1 N2 M0) + + – 30/M/45 RML IIIA (T2 N2 M0) + – + 31/M/71 LUL IB (T2 N0 M0) + – – 32/M/57 RML IB (T2 N0 M0) – ND ND

EBER, Epstein-Barr virus–encoded small nonpolyadenylated RNA; LH, left hilum of lung; LLL, lower lobe of left lung; LMP1, latent membrane protein 1; LUL, upper lobe of left lung; ND, not done; RLL, lower lobe of right lung; RML, middle lobe of right lung; RUL, upper lobe of right lung; VCA, viral capsid antigen. tumor cells showed a palisade arrangement in the periphery staining for LMP1 was positive in 16 cases (53%). LMP1 of the carcinoma nest. In particular, tumor cells in 1 case expression was heterogeneous, with patchy areas of tumor were arranged in trabeculae including 1 or 2 layers of carci- cells showing strong membranous and cytoplasmic staining noma cells. ❚Image 3❚. Of 30 EBER-positive LELC cases, 7 (LMP1-positive, 5 cases; LMP1-negative, 2 cases) showed VCA expres- In Situ Hybridization sion. VCA signals occurred in a minority of carcinoma cells Of 32 cases of pulmonary LELC, EBER transcription and in sporadic lymphocytes in the stroma ❚Image 4❚. No was detected in 30 cases (94%). EBER expression was hetero- LMP1 or VCA expression was found in the 19 non-LELC geneous, varying from case to case, even from area to area in 1 cases. As far as LMP1 and VCA expressions were concerned, case. The 4 kinds of EBER expression patterns, which were the significant difference between LELC with EBER positivity based on variable signal distribution, were inner nuclear and non-LELC was found by using the chi-square test (P < membrane, perinucleoli, diffused, and miscellaneous ❚Image .005). The lymphocyte surface marker in the stroma of 2❚. In addition, sporadic lymphocytes within the stroma of 2 pulmonary LELC was predominantly CD45RO. Only scant cases showed EBER positivity. However, no signals occurred lymphocytes expressed CD20. in normal bronchi, bronchioles, or alveolar epithelium. In Sex and clinical stage did not correlate significantly with contrast with LELC, none of the 19 cases of non-LELC expression of EBER, LMP1, or VCA (P > .05). showed EBER signals. There was a significant difference between the LELC and non-LELC in EBER expression (chi- square, P < .005). Discussion Immunohistochemical Staining LELC is a recently recognized entity arising from Of 30 EBER-positive LELC cases, immunohistochemical foregut-derived organs, which is similar to undifferentiated

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❚Image 1❚ Lymphoepithelioma-like carcinomas of the lung: ❚Image 2❚ Almost all carcinoma cells showed Epstein-Barr histologic examination showing a large island of tumor cells virus (EBV)-encoded small nonpolyadenylated RNA (EBER) infiltrated by intense lymphoplasmacytic cell population positivity in the nucleus. (in situ hybridization for detecting (H&E, ×100). EBER transcription, ×50).

❚Image 3❚ Patchy areas of carcinoma cells showing strong ❚Image 4❚ A minority of carcinoma cells expressed viral latent membrane protein 1–positive signals in the cell capsid antigen (immunohistochemical stain, ×50). membrane and cytoplasm (immunohistochemical stain, ×50).

nasopharyngeal carcinoma. Serologic, immunohistochemical, majority of EBERs are located within the cell nucleus where nucleic acid hybridization, and in situ hybridization studies they are complexed with the cellular protein La.14,15 have shown a strong relationship between undifferentiated LELC of the lung first was reported by Begin and asso- nasopharyngeal carcinoma and EBV infection.11-13 At ciates in 1987.5 A total of 74 cases, including our 32 cases, present, the reliable, highly sensitive, and specific method for have been reported in the English literature ❚Table 2❚.16-27 detection of latent EBV in paraffin sections is EBER in situ The patients ranged in age from 8 to 78 years. Forty-seven hybridization. Because there is the most abundant EBER patients were men, 27 patients were women. Of the patients, transcription (up to 107 copies per cell) during latent EBV 66 were Asian, mainly Chinese. Only 8 patients were white. infection, EBERs have a stable secondary structure, and the Of 65 cases in Asians, 63 (97%) were positive for EBV DNA

© American Society of Clinical Pathologists Am J Clin Pathol 2000;114:220-226 223 Han et al / LYMPHOEPITHELIOMA-LIKE CARCINOMA OF LUNG or RNA by in situ hybridization. In contrast, none of the 7 (3.6%) among 137 resected lung carcinomas in Hong Kong. cases in whites showed EBV DNA or RNA positivity by in Therefore, LELC is a rare distinct entity in lung carcinoma. situ hybridization. This result shows a strong relationship Up to now, there is no report about the incidence of LELC in between LELC of the lung and EBV infection in the Asian lung carcinoma in whites. As far as other types of lung carci- population. The incidence of LELC in lung carcinoma is noma were concerned, we found less relationship with EBV 0.87% (32/3,663) in our study. Higashiyama et al23 found only infection, although a few studies showed that a small portion 2 cases of LELC in 1,358 cases (0.15%) of pulmonary carci- of pulmonary squamous cell carcinoma expressed EBER1 by nomas in Japanese patients. Pittaluga et al19 found 5 cases in situ hybridization.27,28 Pittaluga et al19 and Wong et al20

❚Table 2❚ All Lymphoepithelioma-Like Carcinomas of the Lung Reported in the English Literature (August 23, 1987 to January 31, 1999) Downloaded from https://academic.oup.com/ajcp/article/114/2/220/1757846 by guest on 01 October 2021

Methods for No. of Cases No. of Sex Studying EBV With EBV Cases Author (year) Infection Infection Reported M F Age (y) Race Follow-Up

Begin et al5 Serology (VCA- 1 1 0 1 40 Asian Recurred at 1 y (1987) IgG, IgA, and EA- after resection; D-IgG) lymph node metastasis at 2 y; died at 3 y Butler et al16 Serology 2/2 4 1 3 56-72 White, 3; 3 alive and well 18 (1989) ISH (DNA) 1 Chinese, 1 mo-9 y; 1 recurrence 18 mo after resection Gal et al17 Serology 1 1 1 0 68 Chinese Alive and well at (1991) PCR 1 1 y ISH 1 Miller et al18 ISH (DNA) 0 1 0 1 65 White Bone metastasis 13 (1991) mo after resection Pittaluga et al19 ISH (DNA) 5 5 5 0 33-73 Chinese 2 alive and well at (1993) SBA 2/2 12 and 24 mo Wong et al20 ISH (EBER) 9 9 8 1 33-71 Chinese All alive at 23-52 mo (1995) IHC LMP1 4 EBNA2 0 SBA 8/8 Wockel et al21 ISH 0 1 0 1 47 White Not reported (1995) PCR 0 Serology 1 Chan et al22 ISH (EBER) 9 9 4 5 38-72 Chinese 4 alive and (1995) well at 5-24 mo; 1 died at 4 mo; 1 metastasis at 18 mo Higashiyama PCR 2 2 2 0 55, 65 Japanese Alive at 55 and 54 mo et al23 (1995) ISH (DNA) 2 (EBER1) 1 IHC (LMP) 0 (EBNA2) 0 Ferrara and ISH 0 2 1 1 64, 78 White 1 died at 48 mo; 1 Nappi24 (1995) alive and well at 18 mo Frank et al25 IHC (LMP) 0 1 1 0 67 White No recurrence 8 mo (1997) after resection Curcio et al26 ISH (EBER1) 1 1 0 1 8 Chinese Alive and well at 20 (1997) mo Chen et al27 ISH (EBER1) 5 5 2 3 43-66 Taiwanese Alive 38 mo-6.5 y (1998) IHC (LMP1) 1

EA-D, early antigen, diffuse component; EBER, EBV-encoded small nonpolyadenylated RNA; EBNA, Epstein-Barr nuclear antigen; EBV, Epstein-Barr virus; ISH, in situ hybridization; IHC, immunohistochemistry; LMP, latent membrane protein; PCR, polymerase chain reaction; SBA, Southern blot analysis; VCA, viral capsid antigen.

224 Am J Clin Pathol 2000;114:220-226 © American Society of Clinical Pathologists Anatomic Pathology / ORIGINAL ARTICLE provided evidence by Southern blot analysis that EBV exists Address correspondence to Dr Han: Dept of Pathology, Sun as a single clone episome in neoplastic cells, suggesting that Yat-sen University of Medical Sciences, 74, Zhongshan Road II, Guangzhou, 510089, Peoples’ Republic of China. EBV infection may have an essential role in early stages of pulmonary LELC. LMP1 is an important tumor-transformation protein that can inhibit epithelial differentiation and change epithelial References phenotype.29 Of 30 cases of EBER-positive LELC in our study, 16 (53%) expressed LMP1. The detection rate for 1. Evans AS, Nuderman JC, McCollum RW. Seroepidemio- 20,23,27 logic studies of infectious mononucleosis with EB virus. N LMP1 is higher than that reported by other authors. The Engl J Med. 1968;279:1121-1127. reasons include materials from different areas and the use of 2. 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