Original Article

Modern Pathology (2004) 17, 125–130, advance online publication, 19 November 2003; doi:10.1038/modpathol.3800040

EBV-associated, extranodal NK-cell lymphoma, nasal type of the breast, after heart transplantation

Lawrence Tsao1, Hediya Y Draoua1, Mahesh Mansukhani1, Govind Bhagat1 and Bachir Alobeid1

1Department of Pathology, Columbia University, New York, NY, USA

Correspondence: B Alobeid, Department of Pathology, Hematopathology Division, 14-229 Vanderbilt Clinic, 630 West 168th Street, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. E-mail: ba2024@columbia.edu

Received 8 July 2002; Revised 20 October 2003; Accepted 21 October 2003; Published online 19 November 2003.

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Abstract

Post-transplantation lymphoproliferative disorders (PTLDs) are predominantly Epstein–Barr virus (EBV)-associated B-cell lymphoproliferations. PTLDs of T-cell lineage are rare, mostly reported after renal transplantation and show less frequent association with EBV. NK-cell lymphomas after transplantation (NK-cell PTLDs) are very rare; only five cases are reported so far in the English literature, all developed after renal transplantation. We describe a case of EBV-associated, extranodal NK-cell lymphoma of nasal type, involving the breast in a cardiac allograft recipient 5 years after transplantation. The neoplastic cells are positive for CD2, cytoplasmic CD3, CD7, CD43, CD56, TIA-1 and p53; and negative for surface CD3 and CD57. Analysis of T-cell receptor beta and gamma genes fails to show clonal rearrangement. EBV studies show clonal episomal integration of EBV and latency II pattern (EBER-1+, LMP-1+, EBNA-1+, EBNA-2-). In conclusion, NK-cell PTLDs are rare complications that arise relatively late after solid organ transplantation, show strong association with EBV, and can follow an aggressive clinical course. To the best of our knowledge, we present the first reported case of NK-cell PTLD after cardiac transplantation and the unifying clinical and diagnostic features of NK-cell PTLDs occurring after solid organ transplantation.

Keywords:

nasal-type NK/T-cell lymphoma, natural killer cell lymphoma, post-transplantation lymphoproliferative disorder, cardiac transplantation, EBV-associated lymphoma

Post-transplantation lymphoproliferative disorders (PTLDs) are a well-recognized complication of solid organ and bone marrow transplantation1, 2, 3 that arise secondary to immunosuppression, and are associated with significant morbidity and mortality. PTLDs are a clinically and morphologically heterogeneous group of diseases that account for up to 25% of post-transplantation malignancies.4 Most PTLDs are of B-cell lineage and show strong association with EBV. PTLDs of T-cell lineage are more commonly seen in Asia5 and are rarely reported in Western countries.6, 7 NK-cell PTLDs are a very rare entity with only a handful of reported cases in the literature. Under the current WHO classification,3 T- and NK-cell PTLDs are considered under the monomorphic lesions and classified similar to T- and NK-cell lymphomas arising in immunocompetent patients. T- and NK-cell lymphomas can have overlapping features, including the immunophenotype. In fact, some T-cell lymphomas can upregulate NK-cell-associated markers (CD56 and CD57) and downregulate surface CD3.8, 9 However, while T-cell lymphomas usually show clonal T-cell receptor (TCR) gene rearrangements, the TCR gene in NK-cell lymphomas is usually in germline configuration.9, 10, 11 Although clonal chromosomal abnormalities (deletions of 6q and trisomy 7) have been recently described in a few cases of NK-cell lymphoma,12, 13, 14, 15 these are not specific for NK-cell lymphomas.

Generally, NK-cell neoplasms, including those occurring in immunocompetent individuals, are relatively rare. The most common and well characterized are the nasal/nasal-type NK-cell lymphomas. These lymphomas are typically clinically aggressive and show an angiocentric/angiodestructive growth pattern with zonal necrosis8, 10 and the following immunophenotype: CD2+, cytoplasmic CD3alt epsilon+, CD56+, surface CD3-, TIA-1+ and frequently CD7+. TCR proteins are not expressed on the cell surface and p53 protein is frequently overexpressed.16 EBV infection is also present in the vast majority of cases.3, 17, 18 Clinically, two-thirds of patients with localized disease can be successfully treated, but many (50%) relapse and some (25%) progress to a highly fatal disseminated systemic disease.8 As a subtype, nasal-type NK-cell lymphomas have a worse prognosis.8

In this report, we describe a case of EBV-associated, extranodal NK-cell lymphoma of nasal-type, involving the breast after cardiac transplantation. NK-cell PTLDs are extremely rare in the literature. Our review of the literature reveals only five reported cases, all developing after renal transplantation.4, 5, 6, 7, 19 The first reported case is from the United States in 1998.7 Since then, only four other cases of NK-cell PTLDs have been reported (three in Asia and one in Austria). To the best of our knowledge, our case is the first NK-cell PTLD to be described after cardiac transplantation.

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Case report

The patient was a 20-year-old female of Korean descent with a history of orthotopic heart transplant for anthracycline-induced heart disease, secondary to chemotherapy for a Burkitt lymphoma. She presented with a right breast mass approximately 5 years post-transplantation.

The patient was diagnosed with a Burkitt lymphoma at the age of 5 years in an outside institution, where she presented with a large intestinal mass. At the time, bone marrow involvement was seen on biopsy (we could not review the pathology of Burkitt lymphoma and the patient's EBV status was not documented). She was treated with a chemotherapy regimen, which included adriamycin, and subsequently developed progressively worsening heart failure secondary to the chemotherapy, eventually requiring cardiac transplantation. At this time, serological studies for EBV showed that the patient had already been infected with EBV based on serum levels of IgG. Post-transplantation, her immunosuppressive therapy included cyclosporin A, prednisone and azathioprine. Serial EBV-enzyme immunoassays for IgG immediately after transplant showed increasing titers from 1:640 to 1:2560. Her post-transplantation course was relatively uncomplicated with minimal to no chronic rejection of the heart (ISHLT Grade 0–1A).

At 5 years after transplantation, she presented with a right breast mass, bilateral axillary lymphadenopathy, and a focal lesion in the right lobe of the liver on MRI. An excisional biopsy of the breast mass was performed, but the axillary lymph nodes and liver lesion were not biopsied. After diagnosis of PTLD was made on the breast biopsy, the patient was treated with reduction of immunosuppression and chemotherapy. However, there was no clinical response and the patient expired approximately 1 month after admission. Limited autopsy of the heart revealed cardiac involvement by lymphoma.

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Materials and methods

Morphological analysis was performed on standard H&E-stained sections from formalin-fixed and paraffin-embedded tissue. Immunohistochemical analysis (IHC) was performed on formalin-fixed and paraffin-embedded sections using the Dako Envision plus system for detection (DAKO, Carpinteria, CA, USA) and the immunostains listed in Table 1. Molecular analysis of the TCR beta gene (TCR beta gene) was performed on frozen tissue by Southern blot (SB) analysis using the Jbeta-2 primer and restriction enzymes BamHI, HindIII. Paraffin-embedded tissue was analyzed for TCR gamma (TCR italic gamma) gene rearrangement using PCR-heteroduplex analysis with polyacrylamide gel electrophoresis using the V2 V9, V10/11, J1/2, JP and JP1/P2 primers as reported by Bottaro et al.20 EBV infection status was detected by IHC (Table 1) and by in situ hybridization (ISH) for EBV-encoded RNAs (EBER 1-2, Ventana INFORM EBER, Tucson, AZ, USA) and by SB analysis of EBV episomal integration, using a probe for EBV termini. Formalin-fixed, paraffin-embedded tissue was cut to 5 mum thick sections and processed for fluorescence in situ hybridization (FISH) using a paraffin pretreatment kit supplied by Vysis (Downers Grove, IL, USA). The pretreated tissue sections were hybridized using spectrum orange-labeled alpha satellite CEP7 probe (Vysis, Downers Grove, IL, USA) by standard methods. Images were captured using Cytovision probe capturing system (Applied Imaging, Santa Clara, CA, USA).


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Results

The breast biopsy showed an angiocentric and angiodestructive infiltrate of atypical mononuclear cells in a background of zonal necrosis (Figure 1a). The cells were medium to large with vesicular nuclei and variably prominent nucleoli. Numerous apoptotic bodies and mitotic figures were present (Figure 1b). The complete results of the immunophenotyping are shown in Table 1. The neoplastic cells expressed CD2, CD3 (faint cytoplasmic), CD7, CD43, CD45, CD56 and TIA-1 (Figure 1c, d). EBER by ISH was strongly positive in the vast majority of neoplastic cells (Figure 1f) and by IHC, the neoplastic cells expressed LMP-1 and EBNA-1, but not EBNA-2. Approximately 70% of the neoplastic cells were positive for MIB-1 and over 50% showed nuclear staining for p53 (Figure 1e). The molecular analysis by SB showed germline configuration of the TCR beta gene (Figure 2a), and PCR of the TCR italic gamma gene demonstrated a polyclonal pattern (data not shown). The SB analysis for EBV clonality showed monoclonal episomal integration of EBV in the neoplastic cells (Figure 2b). The cytogenetic analysis by FISH showed normal complement of chromosome 7.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Excisional breast biopsy showing NK-cell lymphoma in a patient after cardiac transplant. (a) Low magnification (times 100) of angiocentric pattern of infiltration. (b) Higher magnification (times 400) of angiodestructive infiltration with numerous mitotic figures and apoptotic bodies. (c–e) Immunohistochemistry showing membranous staining for CD2 (c) and CD56 (d), and aberrant expression of p53 protein (e). (f) ISH with EBER 1-2 showing strong positive reaction in almost all the neoplastic cells.

Full figure and legend (824K)

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

(a) SB analysis using TCR Jbeta-2 probe showing germline configuration (lane 1, patient; lane 2, negative control; lane 3, positive control). (b) SB analysis of EBV episomal integration showing monoclonal band (arrows).

Full figure and legend (117K)

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Discussion

In this report, we describe a case of NK-cell lymphoma of nasal-type that occurred in the breast of a cardiac transplant patient with the typical features of a NK-cell lymphoma of nasal type, including clinical presentation (aggressive course and extranodal location), morphology (angiocentric growth with zonal necrosis), immunophenotype (ie CD2+, cytoplasmic CD3alt epsilon+, CD7+, CD56+, TIA-1+), a high proliferation rate (Ki-67 labelling index of 70%) and TCR gene rearrangement profiles (germline TCR beta by SB and polyclonal TCR italic gamma by PCR). Although the SB analysis of TCR beta was performed using only the J-beta2 primer without the Cbeta probe, the TCR italic gamma gene, generally believed to rearrange prior to TCR beta,21 is polyclonal by PCR,22 suggesting a true NK-cell lineage. In addition, the neoplastic cells in our current case show evidence of EBV infection and overexpression of the p53, both frequently seen in nasal/nasal-type NK-cell lymphoma.3, 16, 17, 18

Reports of NK-cell PTLDs are extremely rare in the literature, all (6/6) developed relatively late after transplantation (1–8 years) and most (5/6) presented predominantly at extranodal sites. The vast majority are positive for EBER (5/6) and LMP-1 (4/5). This is in contrast to T-cell PTLDs, as was shown in a recent review of 76 cases, the vast majority (61/76) were negative for EBV.23 Of the cases with EBNA-2 studies, the majority (3/4) of cases showed lack of EBNA-2 expression consistent with a latency II pattern. The expression of EBNA-2 is studied in three reports.4, 5, 6 Stadlmann et al6 (Table 2, Case 2) and Mukai et al4 (Table 2, Case 4) both report lack of EBNA-2 expression consistent with latency II. Hoshida et al5 report EBNA-2 expression in two cases consistent with latency III. However, one of the two cases appears to be from Mukai et al4 (Table 2, Case 4) with some immunophenotypic discrepancies. Nevertheless, the latency II pattern, also reported in Hodgkin's lymphoma and nasopharyngeal carcinoma,24, 25 has been reported in NK-cell PTLDs.4, 6 Although the exact role of EBV infection in the pathogenesis of NK-cell PTLDs is unknown, the strong association (5/6) suggests a possible pathogenic role for EBV in NK-cell PTLDs. In addition, the strong EBV association suggests that, similar to bone marrow transplant patients, early prophylactic treatment of EBV infection may help prevent the development of NK-cell PTLDs.26


As with nasal/nasal-type NK-cell lymphoma occurring in immunocompetent individuals, p53 overexpression/mutation is also commonly (3/4) seen in NK-cell PTLDs. Hoshida et al27 reports two cases (Table 2, Cases 4 and 5) of NK-cell PTLDs in which mutations of the p53 tumor suppressor gene are studied using PCR. Of the two cases, only one (Table 2, Case 4) contains mutations in the p53 gene. In addition, in two of two cases (Table 2, Cases 2 and 6) studied, p53 overexpression has been demonstrated by IHC. Unfortunately, we were unable to study the p53 gene by PCR due to limitations of available tissue. However, since p53 protein overexpression may not be associated with p53 gene mutations,28 future studies of NK-cell PTLDs should evaluate both p53 gene mutations and protein overexpression.

Nasal-type NK-cell lymphoma is more commonly seen in Asia and Latin America.5 Although the ethnicity of the patients developing NK-cell PTLDs in the reported cases is not known, three are from Asian countries (Japan and Hong Kong) and are likely patients of Asian ethnicity. Our patient is also of Asian ethnicity. Thus, NK-cell PTLDs, like NK-cell lymphoma, may show a racial predisposition (4/6) in Asian patients.

Clinically, the course of NK-cell PTLDs varies from complete remission after chemotherapy (3/6) to death due to disease or complications of disease within months of diagnosis (3/6) (Table 2). Those without systemic dissemination (Cases 2, 4 and 5, Table 2) seem to have the best outcome. Thus, early detection and intervention is necessary.

In conclusion, NK-cell neoplasms are very rare forms of monomorphic PTLDs that can arise late, not only after renal transplantation, but after cardiac transplantation as well, and present predominantly at extranodal sites. Although not entirely clear, a racial predisposition may exist in Asians. Diagnosis of a true NK-cell neoplasm is difficult and immunophenotypic and molecular genetic studies are essential. In addition, in contrast to T-cell PTLDs, the vast majority of these lesions are EBV associated with LMP-1 expression, suggesting a role for EBV infection in the pathogenesis of NK-cell PTLDs. Mutations in the p53 tumor suppressor gene can be seen, but its role in NK-cell PTLDs is unclear. Lastly, while most NK-cell PTLDs follow an aggressive clinical course with poor response to therapy, cases with limited disease may respond well to aggressive intervention. Therefore, early detection and aggressive intervention is essential.

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