Adenotonsillar histopathology after organ transplantation

Adenotonsillar histopathology after organ transplantation ROBERT A. WILLIAMSON, MD, ROBERT Y. HUANG, MD, and NINA L. SHAPIRO, MD, Los Angeles, California

OBJECTIVES: The increasing number of surviving pediatric organ transplant recipients has resulted in a new clinical controversy surrounding the significance of adenotonsillar hypertrophy. The objective of this study is to evaluate adenotonsillar specimens, understand characteristic histopathology, and to examine the frequency and significance of this finding in this population. METHODS: Twenty-one cases of pediatric transplant recipients with adenoidal and/or tonsillar hypertrophy were reviewed retrospectively in a tertiarycare setting. Particular attention was given to the histopathology of their surgical specimens, including any evidence of posttransplantation lymphoproliferative disorders (PTLD). RESULTS: Using morphologic, immunohistochemical, and molecular genetic analyses, 15 (71%) of 21 patients were noted to have Epstein-Barr virus (EBV)-related lymphoid hyperplasia, including 1 case (4.7%) of PTLD. Six (29%) of 21 had evidence of reactive follicular hyperplasia not related to EBV. Bcell and T-cell markers were nearly uniformly positive when tested for, except in the single patient with PTLD, who exhibited polymorphic, polyclonal B-cell morphology. Kappa and lambda light-chain clonality markers were positive in 11 (92%) of 12 patients. CONCLUSIONS: EBV-related lymphoid hyperplasia is frequently associated with adenotonsillar hypertrophy in pediatric organ transplant patients (71% of our cases); 92% of those cases tested exhibit polyclonal B-cell populations. PTLD, an important cause of morbidity and mortality in this population, represented approximately 5% of our cases. The

From the Division of Head and Neck Surgery, Department of Surgery, UCLA School of Medicine. Presented at the Annual Meeting of the American Academy of Otolaryngology–Head and Neck Surgery, Washington, DC, September 24-27, 2000. Reprint requests: Nina L. Shapiro, MD, Division of Head and Neck Surgery, UCLA School of Medicine, Room 62-158, Center for Health Sciences, 10833 LeConte Avenue, Los Angeles, CA 900951624; e-mail, [email protected]. Copyright © 2001 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. 0194-5998/2001/$35.00 + 0 23/1/116447 doi:10.1067/mhn.2001.116447

remainder of cases represent follicular hyperplasia unrelated to EBV or lymphoproliferative abnormalities. Characteristic histopathologic findings are presented. (Otolaryngol Head Neck Surg 2001;125: 231-40.)

As immunosuppressive agents for organ transplant recipients have become more potent and more successful, the number of surviving pediatric solid-organ transplant recipients has significantly increased. This has resulted in a new clinical controversy surrounding the management of adenotonsillar hypertrophy (ATH) in this population. In particular, organ transplant recipients have up to a 50fold increased risk of developing lymphoma and other posttransplantation lymphoproliferative disorders (PTLDs).1 A recent study from our institution recommended prompt excision of enlarged tonsil and adenoid tissue in the pediatric population as a means of facilitating early diagnosis and treatment of PTLD.2 PTLD represents a broad spectrum of disorders, ranging from localized polyclonal lymphoid proliferations to disseminated monoclonal malignant lymphomas.3,4 It has been shown to occur in between 3% and 27% of organ transplant patients.5,6 High-dose immunosuppressant agents, young age, liver versus kidney transplant, and EBV-seronegativity are associated with a higher incidence of PTLD.7 Mortality rates in those affected may be as high as 20%.8 Although the histopathology of PTLD has been well-summarized in the Society for Hematopathology Workshop in 1997,9 it has not been extensively characterized in adenotonsillar specimens. Moreover, other causes of non-PTLD related adenotonsillar histopathology in organ transplant recipients have also not been previously well-documented. This study seeks to examine characteristic histopathology of adenotonsillar specimens in pediatric organ transplant recipients with ATH, and begin to identify the frequency and significance of these findings. METHODS Subjects A retrospective review of pediatric patients who had undergone solid-organ transplantation at the University of California, Los Angeles was conducted. Patients underwent adenoidectomy and/or tonsillectomy for clinical indications specified below, following standard informed consent for the 231

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Table 1. Categories of PTLD from the 1997 Society for Hematopathology Workshop9 Early lesions Reactive plasmacytic hyperplasia Infectious mononucleosis-like PTLD Polymorphic Polyclonal (rare) Monoclonal Monomorphic B-cell lymphoma T-cell lymphoma Other T-cell rich/Hodgkin’s disease-like Plasmacytoma-like Myeloma

procedures, and surgical specimens were processed according to established pathology protocols for this patient population. Institutional Review Board approval for the retrospective, histopathologic analysis of the surgical specimens therefore was not required. Patients who presented with signs and/or symptoms of adenotonsillar hypertrophy, and who subsequently underwent tonsillectomy, adenoidectomy, or both, were included. There were 21 subjects studied during the period of March 1998 through November of 1999. Clinical Indicators Signs and symptoms of ATH were noted and recorded. Any history of snoring, obstructive sleep disorder, or obstructive sleep apnea syndrome were also noted. In some cases, polysomnography was obtained. Any clinical signs associated with PTLD were noted, including fever, malaise, poor appetite, weight loss, and irritability. Finally, tonsillar hypertrophy was classified based on the system that 4+ tonsils represented greater than 75% obstruction of the oropharynx, 3+ represented less than 75% obstruction, 2+ represented normal-sized but visible tonsils, and 1+ represented nonvisible tonsils. Specimen Processing Tissue specimens obtained at surgery were sent fresh and labelled individually in preparation for immunophenotyping (either by flow cytometry or frozen-section immunohistochemistry), routine histologic examination, and relevant molecular genetic studies. Standard immunohistochemical processing as described below was carried out individually on each specimen and recorded. Histopathology Techniques Histopathologic diagnosis was made according to the recommendations of the Society for Hematopathology Workshop

1997 classifying posttransplantation lymphoproliferative disorders (Table 1).9 Tissue was examined for gross and microscopic interpretation, including standard hematoxylin-eosin staining at low and high magnification. Nodal architecture, cytology, differentiation, hyperplasia, and polarization of follicles were examined and recorded. Any evidence of an atypical lymphoid infiltrate was noted and carefully described. Next, a standard immunohistochemical battery was performed using B-cell, T-cell, and Epstein-Barr viral markers. CD20 marker antibodies were used to label B-cells, and kappa and lambda light-chain antibody probes were used to determine B-cell clonality. The presence of both light-chain types was considered indicative of a polyclonal B-cell population. T-cell antibody probes included primarily CD3, and in some cases, CD43. In situ hybridization for EBV-encoded RNA (EBER) was performed on tissue specimens to determine the presence of the virus, and in some cases EBV latent membrane protein (LMP) was also stained for and noted. The single specimen with histologically confirmed PTLD underwent further testing including flow cytometry with specific cell markers. Markers CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD56, CD19, CD20, CD22, CD23, and CD20Kappa and CD20Lambda were used to further characterize PTLD. RESULTS

The 21 patients studied were all treated at UCLA Medical Center from March 1998 through November 1999. Clinical characteristics including transplantation data and immunosuppressant medications are listed in Table 2. There were 11 patients who had undergone liver transplantation, 9 patients who had undergone kidney transplantation, and 1 cardiac transplant recipient. Mean age at the time of transplantation was 3 years 9 months and ranged from 7 months to 14 years 6 months. Mean age at the time of adenotonsillectomy was 8 years 10 months and ranged from 4 years 6 months to 16 years 4 months. Eighteen of 21 patients presented with symptoms of obstructive sleep disorder or nasal airway obstruction. Of the remaining 3 patients, one had recurrent tonsillitis and a second had asymptomatic, asymmetric tonsillar enlargement. The final patient had a history of abdominal PTLD after liver transplantation and was noted on examination to have tonsillar hypertrophy without recurrent infections or symptoms of obstructive sleep disorder. There were 3 common histopathologic and immunohistochemical categories into which the specimens fell. EBV-related hyperplasia was found in 14 of 21 specimens, accounting for 67% of the cases. PTLD was the

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Table 2. Clinical and transplantation characteristics Patient number/ Sex/Age at adenotonsillectomy

1/Female/4y6mo 2/Male/5y

Type of transplant/ Age at transplantation

7/Male/6y4mo 8/Female/7y1mo 9/Female/9y7mo 10/Female/5y3mo

Liver/7 mo Liver (twice)/8 mo and 2y7mo Liver/11 mo Liver/8 mo Liver/9mo Liver (twice)/7mo and 13mo Liver/1y Liver/1y2mo Liver/7mo Kidney/2y4mo

11/Male/6y8mo 12/Male/12y

Kidney/2y3mo Kidney/10y8mo

13/Male/12y8mo 14/Male/14y1mo 15/Male/15y1mo

Kidney/10y5mo Kidney/2y4mo Kidney/7y

16/Male/16y4mo

Kidney/7y2mo

17/Male/10y 18/Female/4y4mo

Heart/4y1mo Liver/7mo

19/Male/11y

Kidney/9y3mo

20/Male/16y7mo

Kidney (twice)/4y and 11y11mo Liver/1y2mo

3/Female/5y7mo 4/Male/5y7mo 5/Male/5y9mo 6/Male/6y4mo

21/Male/10y9mo

EBV-serology (Pretransplant)

Immunosuppression

VCA IgG <1:10 VCA IgM >1:20 VCA IgG >1:2560 EBNA AB >1:20 VCA IgG <1:10 VCA IgG <1:10 Not available VCA IgG <1:10 VCA IgG <1:10 Not available Not available VCA IgM <1:20 VCA IgG >1:2560 EBNA AB >1:20 VCA IgM <1:20 VCA IgG <1:10 VCA IgM <1:20 VCA IgG >1:2560 EBNA AB >1:20 Not available Not available VCA IgM <1:20 VCA IgG <1:10 EBNA AB <1:5 VCA IgM <1:20 VCA IgG <1:10 EBNA AB <1:5 VCA IgG 1:160 VCA IgG <1:10 VCA IgM <1:20 EBNA AB <1:5 VCA IgM <0.9 VCA IgG >5.0 EBNA AB <1:5 VCA IgM <1:20 VCA IgG >1:2560 EBNA AB >1:20 VCA IgM <1:20 VCA IgG <1:10 EBNA AB <1:10

Histopathology

Tac, Pred Tac, Pred

EBV-related hyperplasia EBV-related hyperplasia

Cyc, Aza, Pred Cyc, Pred Cyc, Aza Cyc, Aza, Pred

EBV-related hyperplasia EBV-related hyperplasia Lymphoid hyperplasia EBV-related hyperplasia

Cyc Cyc, Aza Cyc, Pred Cyc, Pred, Myc

Lymphoid hyperplasia EBV-related hyperplasia Lymphoid hyperplasia EBV-related hyperplasia

Cyc, Pred, Myc Cyc, Pred, Myc

EBV-related hyperplasia Lymphoid hyperplasia

Cyc, Pred, Myc Cyc, Pred Cyc, Pred, Myc

EBV-related hyperplasia PTLD EBV-related hyperplasia

Tac, Pred, Myc

EBV-related hyperplasia

Tac, Pred Tac, Pred

Lymphoid hyperplasia EBV-related hyperplasia

Cyc, Pred, Myc

Lymphoid hyperplasia

Cyc, Pred, Myc

EBV-related hyperplasia

Cyc, Pred

EBV-related hyperplasia

VCA, Viral capsid antigen; EBNA AB, Epstein-Barr nuclear antigen. Reference values for antibody titers: IgM <20 = negative, :>1:20 = positive. EBNA AB <1.5 = negative, :>1:5 = positive. IgG <1.10 = negative, :>1:640 or 4-fold increase in antibody titer = positive. Reference values for patient 19: IgG <0.9 = negative, >1.0 = positive, and 0.901 to 0.999 are equivocal. Immunosuppressant medications: cyc, cyclosporine; pred, prednisone, myc, mycophenolate, tac, tacrolimus.

least common histopathologic category, occurring in 1 (4.7%) of 21 specimens. Reactive follicular hyperplasia, unrelated to EBV, was noted in 6 (29%) of 21 specimens. Characteristic histopathologic and immunohistochemical features of EBV-related hyperplasia included preservation of follicular architecture and polarization, with variable hyperplasia involving follicular and interfollicular areas (Fig 1). Atypical lymphoid infiltrates were not noted in these specimens. Eleven of 12 specimens tested for kappa and lambda light-chain markers were positive (92%) (Fig 2), indicating polyclonality. Bcell markers CD20 and T-cell markers CD3 and CD43 were nearly uniformly positive when tested for in these specimens (11 of 12, 92%), indicating both B-cell and Tcell populations were present. In situ hybridization for

EBER, specific for EBV-encoded RNA, was uniformly present when tested for in these specimens (12 of 12, 100%), though the amount of staining in the specimens was variable in different areas (Fig 3). EBV LMP was less reliable as an indicator and was negative in 5 of 6 of these specimens. These features were consistently demonstrated in both tonsil and adenoid specimens. Reported histopathologic features of those cases with nonEBV related follicular hyperplasia include similar preservation of follicular architecture and polarization without evidence of atypical lymphoid infiltrates. Hyperplasia appeared to be limited to follicular areas, but some interfollicular expansion was often noted (Fig 4). These specimens also demonstrated polyclonal B-cell and T-cell populations with positive kappa and lambda light-chain

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A

B Fig 1. EBV-related lymphoid hyperplasia. Note preservation of nodal architecture, with follicular and interfollicular hyperplasia. There is no atypical lymphoid infiltrate. (Hematoxylin and eosin stain; low [A] and high [B] magnification.)

staining, but were notably negative when tested for EBER and EBV-LMP. Histopathologic features in this category also did not significantly differ between tonsils and adenoids from the same patient. The single specimen with PTLD was notable for diffuse effacement of normal follicular architecture by a polymorphous proliferation of small lymphocytes, immunoblasts, plasma cells, and plasmacytoid

lymphocytes (Fig 5). Focal necrosis was also demonstrated in some areas. In situ hybridization for EBER showed strong nuclear staining (Fig 6). Kappa and lambda light-chain markers were both positive, suggesting polyclonality in the B-cell proliferation. Final histopathologic interpretation was consistent with PTLD, polymorphous type. Subsequent serology studies on this patient revealed

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A

B Fig 2. EBV-related lymphoid hyperplasia. A, Kappa light-chain immunohistochemistry positive. B, Lambda light-chain immunohistochemistry positive. (Hematoxylin counterstain; high magnification.)

elevated anti-EBV IgM antibodies (1.48 relative enzyme-linked immunosorbent assay units, normal <0.9 and elevated anti-EBV IgG antibodies (>5, normal <0.9). EBV titers by polymerase chain reaction were 870 copies of viral DNA (normal 0-5). These data were suggestive of an acute EBV infection. Features were again evident in tonsillar and adenoid tisssue.

DISCUSSION

The histopathology and incidence of adenotonsillar hypertrophy in pediatric organ transplant patients has not been previously emphasized in the literature. Several studies have specifically examined the clinical course of PTLD in this population.10,11 Its incidence ranges from 0.8%12 to as high as 27% in cases of organ transplantation treated with FK506 (tacrolimus).13,15

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Fig 3. EBV-related lymphoid hyperplasia. EBER immunohistochemistry strongly positive. (Hematoxylin counterstain; high magnification.)

The incidence and importance of adenotonsillar hypertrophy in these patients, whether related to PTLD or not, has also not been well-characterized. Risk factors for the development of PTLD have been studied extensively. Data from a large series of patients at the University of Pittsburgh (5732 adult and pediatric patients) showed that EBV seronegativity, type of transplant (liver, heart, heartlung, and lung were higher risk than kidney), immunosuppressant regimen, and young age at the time of transplant were associated with higher risk for developing PTLD.11 Although this study did not specifically address the indications for adenotonsillectomy in this population, other investigators have recommended early intervention in the form of adenotonsillectomy in any pediatric organ transplant recipient with adenotonsillar hypertrophy, regardless of clinical presentation, as an important maneuver in diagnosing PTLD.2,13 As the clinical presentation of PTLD can be broad, ranging from adenotonsillar hypertrophy with recurrent tonsillitis to progressive upper airway obstruction with or without multiorgan involvement and systemic symptoms, a high index of suspicion and consideration of the aforementioned risk factors is essential. The association between EBV and PTLD has been well-described in the literature.14,15 EBV is a herpesvirus that infects B lymphocytes in vivo and induces polyclonal activation and proliferation.16,17 In the normal host, after a predictable antibody response to infec-

tion, EBV-specific memory cytotoxic T-cells then control the lymphoproliferation, resulting in a self-limited process.1 In organ transplant recipients, the immunosuppressant agents limit the T-cell response, and EBVinduced polyclonal proliferation proceeds unchecked. Immunosuppression directed at the cellular T-cell response is the major risk factor for the development of EBV-related PTLD,1 and certain agents specifically directed at T-cells (eg, OKT3 and other anti–T cell antibodies) are known to potentiate the development of PTLD.5,10 Primary EBV infection in children after orthotopic liver transplantation occurs in as many as 67% of cases,18 and up to 90% of cases of PTLD in the early posttransplant period are triggered by primary EBV infection.3 Though a causative role for EBV has not been defined, there does appear to be an association between the presence of the Epstein-Barr virus and adenotonsillar hypertrophy in the pediatric organ transplant population. We noted that patients who had serologic evidence of exposure to EBV before transplantation were more likely to develop non–EBV-related lymphoid hyperplasia as the cause for their adenotonsillar hypertrophy. In addition, patients with more recent (posttransplantation) exposure to EBV were more likely to develop EBV-related lymphoid hyperplasia, and possibly PTLD. This association, however, did not achieve statistical significance in our patient population. Of 21 pediatric solid organ transplant recipients,

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A

B Fig 4. Non–EBV-related lymphoid hyperplasia. A, Nodal architecture and follicular polarization are well-preserved. Hyperplasia is predominantly follicular, with some interfollicular expansion noted. No atypical infiltrate is seen. (Hematoxylin and eosin; low magnification.) B, EBER immunohistochemistry negative. (Hematoxylin counterstain; high magnification.)

11 (52%) of 21 underwent liver transplantation, 9 (43%) of 21 underwent kidney transplantation, and 1 (4.7%) of 21 underwent cardiac transplantation. The median age was 3 years 9 months at the time of transplantation and 8 years 10 months at the time of adenotonsillectomy. Though the sample size was small, it is representative of

the pediatric population of organ transplant recipients at a tertiary care center; however, we caution against applying these observations to older patients, nontransplant immunosuppressed patients, or bone marrow transplant recipients. There was a single case of PTLD in this population, representing 4.7% of the patients

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A

B Fig 5. Posttransplantation lymphoproliferative disorder. Note diffuse effacement of normal follicular architecture with sheets of small lymphocytes, immunoblasts, plasma cells, and plasmacytoid lymphocytes. Areas of focal necrosis and marked cytologic atypia are seen. (Hematoxylin and eosin; low [A] and high [B] magnification.)

studied. This is consistent with other studies in the literature, where PTLD has been shown to occur in between 0.8% and 33%, with a mean of approximately 7% in pediatric populations and 2% in adults.3 In our series of 21 pediatric patients, adenotonsillar hypertrophy was associated with lymphoid hyperplasia related

to EBV in 71% of cases. This is consistent with other studies in the literature that demonstrate a central role for EBV in transplant recipients.3,5,7 Based on immunohistochemical markers for cell type and clonality, a number of important factors were noted. First, B-cell populations in those cases of EBV-

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Fig 6. Posttransplantation lymphoproliferative disorder. EBER immunohistochemistry positive. (Hematoxylin counterstain; high magnification.)

related hyperplasia were often polyclonal as indicated by the presence of both kappa and lambda light-chain clonality markers in 92% of our patients. This is consistent with the known polyclonal activation of B cells by EBV infection.1 We were not able to address whether the hyperplasia was associated with acute or reactivated/latent EBV infection. It is possible that some of these cases represented acute EBV infections with atypical or delayed presentations due to the potent immunosuppressive regimens, often including high-dose steroid therapy. EBV infection most commonly occurs in early childhood, and the most common presentation is asymptomatic seroconversion.1 Thus, we cannot draw conclusions on the temporal relationship of EBV infection in those cases of lymphoid hyperplasia that we observed. Second, EBER appeared to be a more reliable histologic marker for the presence of EBV in adenotonsillar specimens. It was noted in all specimens tested, whereas EBV-LMP appeared to be less reliable as a marker and was present in only 17% of EBV-related cases. Harris et al9 recommend testing for the presence of EBV using EBER in situ hybridization as the preferred method; they noted that EBV-LMP and EBNA– (used on frozen sections) detected some but not all EBV+ cases as these antigens are not uniformly expressed. In our review of 21 pediatric solid organ transplant recipients who underwent adenotonsillectomy for symptoms of airway obstruction, we found a high per-

centage (71%) of EBV-related hyperplasia in adenotonsillar specimens. One patient (4.7%) demonstrated PTLD caused by an acute EBV infection. EBV-related hyperplasia is a potential precursor of PTLD in this patient population and needs to be investigated further. Young patients receiving solid organ transplants are at high risk for developing EBV infection, with the donor organ often being the source. EBV may be harbored in adenotonsillar tissue as a primary locale for viral replication. CONCLUSION

With increasing long-term survival after solid-organ transplantation, areas of transplant-related morbidity and mortality are being addressed. PTLD, and its precursor, EBV-related hyperplasia, carry high morbidity and mortality risks in this patient population. We identified a preponderance of EBV-related hyperplasia in adenotonsillar specimens in our patient group. Surveillance of pediatric organ transplant recipients with respect to symptoms of adenotonsillar hypertrophy needs to be addressed. Further work needs to be done to elucidate clinical implications of EBV-related hyperplasia. In addition, it will be important to develop standardized protocols for evaluating these patients both clinically and in processing tissue specimens and subjecting them to immunohistochemical and molecular genetic analysis. A prospective study to identify pediatric organ transplant recipients with ATH and risk fac-

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tors for EBV-related hyperplasia and PTLD is currently being conducted at our institution.

10.

We would like to thank Thomas Calcaterra, MD, for his help and support of this project. REFERENCES 1. Basgoz N, Preiksaitis JK. Posttransplant lymphoproliferative disorder. Infect Dis Clin North Am 1995;9:901-23. 2. Huang RY, Shapiro NL. Adenotonsillar enlargement in pediatric patients following solid organ transplantation. Arch Otolaryngol Head Neck Surg 2000;126:159-64. 3. Ho M. Risk factors and pathogenesis of posttransplant lymphoproliferative disorders. Transplant Proc 1995;27(suppl. 1): 38-40. 4. Rustgi VK. Epstein-Barr viral infection and posttransplantation lymphoproliferative disorders. Liver Transplant Surg 1995;1:100-4. 5. Newell KA, Alonso EM, Whitington PF. Posttransplant lymphoproliferative disease in pediatric liver transplantation. Transplantation 1996;62:370-5. 6. Cacciarelli TV, Green M, Jaffe R. Management of post-transplant lymphoproliferative disorder (PTLD) in pediatric liver transplant recipients receiving primary tacrolimus therapy. Transplantation 1998;66:1047-52. 7. McDiarmid SV, Jordan S, Lee GS, et al. Prevention and preemptive therapy of posttransplant lymphoproliferative disease in pediatric liver recipients. Transplantation 1998;66:1604-11. 8. McDiarmid S, Goss J, Seu P, et al. One hundred children treated with tacrolimus after primary orthotopic liver transplantation. Transplant Proc 1998;30:1397-401. 9. Harris NL, Ferry JA, Swerdlow SH. Posttransplant lymphopro-

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