Objectives: Posttransplant lymphoproliferative disorders are classified as monomorphic, poly­morphic, early lesions, or Hodgkin lymphoma. Bone marrow staging examination is recommended in posttransplant lymphoproliferative disorder patients. However, information about bone marrow involvement in these disorders is scarce. We evaluated 19 transplant patients with post­transplant lymphoproliferative disorder to investi­gate incidence of bone marrow involvement, associated morphologic changes, and prognosis.

Materials and Methods: We retrospectively assessed bone marrow findings of 19 transplant patients with posttransplant lymphoproliferative disorder who underwent bone marrow staging at Baskent University from 1985 to 2013. Clinical and pathologic data were reviewed from the medical records. Follow-up information was obtained from medical records or communication with patients or families. Data collected including age, sex, Epstein-Barr virus status, immunosuppressive therapy, elapsed time from transplant to diagnosis of posttransplant lymphoproliferative disorder, B symptoms, number of extranodal sites, involvement of different organs, Ann Arbor clinical staging, hematologic parameters, and serum lactate de­hydrogenase levels.

Results: There were 5 of 19 patients (26.3%) who had bone marrow involvement with posttransplant lymphoproliferative disorder, including 2 patients diagnosed with posttransplant lymphoproliferative disorder by lymph node biopsy and 1 patient each diagnosed by native liver biopsy, nasopharyngeal biopsy, or allograft liver biopsy. In 4 patients, there was monomorphic posttransplant lympho­proliferative disorder subtype and 1 patient had early lesion posttransplant lymphoproliferative disorder subtype. In 10 of 19 patients (52.6%), Epstein-Barr virus was detected with in situ hybridization, including 3 patients with bone marrow involvement who were diagnosed with Burkitt lymphoma (n = 1), diffuse large B-cell lymphoma (n = 1), or early lesion (n = 1).

Conclusions: Patients with posttransplant lympho­proliferative disorder have high incidence of bone marrow involvement and high mortality rates. Therefore, bone marrow examination may be important in the diagnosis and staging evaluation of posttransplant lymphoproliferative disorder.

Key words : Epstein-Barr virus, Posttransplant lymphoproliferative disorder, Staging

Introduction

Posttransplant lymphoproliferative disorder (PTLD) is a heterogeneous group of abnormal lymphoid proliferations that occur after solid-organ transplant or hematopoietic transplant. Most PTLDs are B-cell proliferations that are positive for Epstein-Barr virus (EBV). However, PTLDs of T- and/or natural-killer-cell lineage have been documented. According to the present World Health Organization classification system, PTLDs are classified as early lesions, polymorphic, monomorphic, or Hodgkin lymphoma-type. The monomorphic PTLDs have been sub­classified, based on morphologic, immuno­phenotypic, genetic, and clinical features, into diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, plasma cell myeloma, plasmacytoma-like lesions, and various subtypes of T- and natural-killer-cell lymphomas.^1,2

Recommendations for staging PTLD currently are based on Ann Arbor clinical staging criteria. Bone marrow examination is an integral part of the Ann Arbor staging system for all lymphoproliferative malignancies. In non-Hodgkin lymphoma, Ann Arbor stage III to IV disease is an adverse prognostic feature in the International Prognostic Index. Bone marrow involvement by PTLD defines stage IV disease, and similar to immunocompetent patients with non-Hodgkin lymphoma, patients with advanced-stage PTLD have worse prognosis and shorter overall survival. Although bone marrow involvement by monomorphic PTLD is uncommon, there have been few studies documenting the frequency of bone marrow involvement in this patient population. Although some studies have shown that 40% of patients with PTLD have bone marrow involvement at staging, most studies have not addressed this issue in the context of the different subtypes of PTLD.³ Elimination of routine bone marrow biopsies in newly diagnosed patients would require a sensitive surrogate predictor of bone marrow infiltration because of the potential prognostic significance of bone marrow involvement.

In this study, we analyzed the clinicopathologic characteristics of patients with PTLD who were evaluated and treated at our institute, focusing on the frequency of bone marrow involvement by PTLD. In addition, we assessed the use of hematologic parameters (hemoglobin level and platelet count) and lactate dehydrogenase level as predictors of bone marrow involvement in patients with PTLD.

Materials and Methods

Case selection and clinical characteristics
We retrospectively analyzed results of 19 patients who were diagnosed with PTLD after solid-organ transplant and who underwent bone marrow examination during staging evaluation at Baskent University from 1985 to 2013. The study was approved by the Ethical Review Committee of the institute.

Clinical and pathologic data were reviewed from the medical records of patients who had PTLD. Follow-up information was obtained from medical records or by direct communication with patients or families. Data were collected including age, sex, EBV status, immunosuppressive therapy, time from transplant to diagnosis of PTLD, B symptoms, number of extranodal sites, involvement of different organs, Ann Arbor clinical stage, hematologic parameters, and serum lactate dehydrogenase level.

A clinical and radiographic staging approach was used in conjunction with bone marrow assessment, in accordance with the American Joint Committee on Cancer non-Hodgkin lymphoma staging system (AJCC Staging Manual, Sixth Edition).⁴ Morphologic features were assessed using sections of formalin-fixed, paraffin-embedded bone marrow biopsies stained with hematoxylin-eosin. All staging bone marrow biopsies considered positive for lymphomatous involvement showed morphologic evidence of disease on sections stained with hematoxylin-eosin and/or immunohistochemical staining.

Immunohistochemistry and in situ hybridization
Immunohistochemical stains performed on bone marrow biopsies included primary antibodies CD20, CD79a, PAX5, MUM1, CD138, BCL2, BCL6, CD30, CD3, CD2, CD5, CD7, CD4, and CD8 after heat-induced antigen retrieval and visualization (Envision plus, DAKO, Carpinteria, CA, USA) and diamino benzidine. The EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using the supplied protocol from the manufacturer (Bond-max system, Leica Microsystems, Buffalo Grove, IL, USA).

Statistical analyses
Data analyses was performed with statistical software (SPSS for Windows, version 16.0, SPSS Inc., Chicago, IL, USA). Statistical comparisons between patient subgroups were performed using chi-square and Fisher exact tests for proportions. The Mann-Whitney test and 1-way analysis of variance were used to analyze differences in hemoglobin level, platelet count, and lactate dehydrogenase level between patients with and without bone marrow involvement. Statistical significance was defined by P < .05.

Results

Posttransplant lymphoproliferative disorder case characteristics and staging bone marrow results
The incidence of PTLD in our transplant population was 1.2% in our previous study.⁵ The mean age of the 19 patients (14 men and 5 women) at PTLD diagnosis was 28.3 ± 18.5 years (range, 1 - 52 y), with 5 patients (26.3%) being younger than 18 years. Organs transplanted included kidney in 11 patients (57.8%) and liver in 8 patients (42.1%). All pediatric patients had liver transplant. The mean time from transplant to diagnosis of PTLD was 61.9 ± 71.8 months (range,4 - 250 mo) (Table 1).

Based on the World Health Organization classification of PTLD, the subtypes of PTLD were monomorphic in 18 patients (94.7%) and early lesion in 1 patient (5.2%). In the 18 patients with mono­morphic subtype, 14 patients had DLBCL and 1 patient each had Burkitt lymphoma, primary cutaneous CD30-positive T-cell lymphoma, peripheral T-cell lymphoma (not otherwise specified), and T-cell acute lympho­blastic leukemia. There was 1 patient who showed early lesion PTLD in 2 different lymph nodes with plasmacytic hyperplasia and infectious mono­nucleosis like (IM-like) features; in this patient, we diagnosed early lesion PTLD with IM-like features in bone marrow 3 years after detecting early lesion PTLD in lymph nodes.

Bone marrow involvement was detected in 5 of 19 PTLD cases (26.3%): 4 of 5 monomorphic PTLDs (80%) (3 DLBCLs and 1 Burkitt lymphoma) and 1 of 5 early lesion PTLDs (20%) (Table 2). None of the patients who had T-cell monomorphic PTLD (n = 3) had bone marrow involvement.

All patients (n = 19) underwent a single organ transplant before developing PTLD. In 1 patient, primary cutaneous CD30-positive T-cell lympho­proliferative disorder recurred and was restaged with bone marrow examination.

The EBV status of the PTLD analyzed by EBER-ISH was determined in 19 cases. In the EBV-negative cases, 2 of 9 patients (22.2%) exhibited bone marrow involvement, compared with 3 of 10 EBV-positive cases (30%), and this difference was not statistically significant.

In 14 of 19 patients who had PTLD (73.6%), there was extranodal disease involving the gastrointestinal tract (6 patients), lung (3 patients), skin (2 patients), native liver (1 patient), allograft liver (1 patient), mediastinum (1 patient), nasopharynx (1 patient), spleen (1 patient), breast (1 patient), omentum (1 patient) and soft tissue (1 patient). Disseminated PTLD was observed in 2 of 19 PTLD patients, and 4 patients had multiorgan involvement. Involvement of extra­nodal sites was more common in cases without bone marrow involvement (11 of 14 patients [78.5%]) compared with cases with bone marrow involvement (3 of 5 patients [60%]), but this difference was not statistically significant.

Time from transplant to PTLD diagnosis in patients with bone marrow infiltration was 14.8 ± 10.8 months (range, 4 - 32 mo) and in patients without bone marrow involvement was 78.7 ± 77.1 months (range, 5 - 250 mo). Time from transplant to PTLD diagnosis was shorter in patients with bone marrow involvement compared with patients without bone marrow involvement, and this difference was statistically significant (P < .05) (Table 1).

Bone marrow infiltration was observed in 3 patients (60%) who underwent induction therapy. This difference was not statistically significant when compared with patients who did not receive induction therapy.

Tacrolimus was the most common immuno­suppressive medicine in patients who had bone marrow involvement compared with other agents. A statistically significant difference was observed between tacrolimus and other immunosuppressive agents between patients who had or did not have bone marrow involvement (P < .05) (Table 1).

There were 9 of 19 PTLD cases (47.3 %) who presented with a high clinical stage (III or IV) before bone marrow staging. The staging of bone marrow biopsy affected the final stage in only 2 of 19 cases (10.5%). In the 4 monomorphic PTLDs (21%) with bone marrow involvement at staging, bone marrow involvement changed the Ann Arbor stage in 2 cases, from stage II to IV (1 case) and from stage I to IV (1 case). Hemoglobin and lactate dehydrogenase levels were not significantly different between PTLD cases with or without bone marrow involvement (Table 3).

Survival in patients with bone marrow involvement (74.4 ± 50.7 mo [range, 28 - 155 mo]) was longer than in patients without bone marrow involvement (60.57 ± 57.6 mo [range, 1 - 180 mo]), but this difference was not statistically significant.

All patients with bone marrow involvement were alive at mean follow-up 74.4 ± 50.7 months (range, 28 - 155 mo). In contrast, 42.8% patients without bone marrow involvement had died at mean 49.8 ± 52.4 months (range, 1 - 131 mo). This result can be due to death of the patients not from PTLD; all patients died because of complications of liver or kidney transplant.

Bone marrow involvement in PTLD was not related to the subtype of PTLD, patient age, sex, EBV status of the PTLD, type of organ transplanted, B symptoms, or donor type.

Discussion

The incidence of PTLD in our transplant population was 1.2% in our previous study.⁵ Bone marrow involvement in PTLD patients was observed in 26.3% patients in the current study, and 22% cases had monomorphic PTLD. Montanari and coworkers1 reported a 23.5% incidence of bone marrow involvement at PTLD diagnosis, and this was confirmed in our study. When we compared the patients with or without bone marrow involvement, we found that the mean age of the former group was lower, and this finding is consistent with results in the literature.⁶

In pediatric PTLD patients, Maecker and associates reported 15% incidence of bone marrow involvement at diagnosis.⁷ In the current study, 2 of 5 pediatric patients (40%) had bone marrow PTLD involvement.

Male predominance was observed in patients with and without bone marrow involvement in our study. A study performed with liver transplant patients showed that bone marrow PTLD was significantly more likely to present in male patients.⁸

The time from transplant to diagnosis of PTLD in our series varied, ranging from < 4 months to > 20 years. Time from transplant to PTLD was shorter in patients with bone marrow involvement. A study performed with renal transplant patients showed findings similar to our study,⁶ but another study in solid-organ transplant patients did not detect a statistically significant relation between time from transplant to PTLD and bone marrow involvement.¹

The PTLDs are believed to have a tendency for extranodal organ involvements. However, factors that affect the spread of the disease are not well defined. Another finding in the current study was the lower incidence of extranodal involvement in patients with than without bone marrow involvement. Similar to our study, Montanari and coworkers showed less bone marrow involvement at patients with extranodal PTLD compared with patients with nodal PTLD.¹ The underlying pathophysiologic mechanisms responsible for this inverse relation between extranodal and bone marrow involvement are unclear and require further investigation.

In 14 patients who had extranodal PTLD, only 1 patient had allograft involvement (liver) coincident with bone marrow involvement, which is consistent with a previous study.⁸ In addition, consistent with the literature, we observed bone marrow involvement coincident with involvement of the native liver and spleen in 1 patient.

Survival was significantly longer in patients with than without bone marrow involvement. This finding can be explained by the observation that patients who did not have bone marrow involvement had PTLD diagnosis later than patients with bone marrow involvement. Thus, there is more likelihood to have other risk factors causing shorter survival such as infection and rejection during the longer period.

The high incidence of bone marrow involvement in patients with PTLD and high mortality rate of PTLD patients suggest that bone marrow examination may be important in the diagnosis and staging evaluation of PTLD. Thus, more detailed and larger studies are needed to clarify prognostic factors in these patients and understand the pathophysiology of bone marrow involvement in patients with PTLD.

References:

1. Montanari F, O'Connor OA, Savage DG, et al. Bone marrow involvement in patients with posttransplant lymphoproliferative disorders: incidence and prognostic factors. Human Pathol. 2010; 41(8):1150-1158.
2. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (WHO Classification of Tumours, Volume 2). 4th ed. Lyon: The International Agency for Research on Cancer; 2008.
3. Cho MC, Park CJ, Huh J, et al. Bone marrow findings of 23 Korean post-transplant lymphoproliferative disorder patients: a single-center experience. Clin Transplant. 2012;26(4):E381-E387.
4. FL Greene, DL Page, ID Fleming. AJCC (American Joint Committee on Cancer) Cancer Staging Manuel. 6th ed. Chicago, IL: Springer Publishing Co; 2002.
5. Akar Özkan E, Özdemir BH, Deniz EE, Tunca MZ, Haberal M. Posttransplant lymphoproliferative disorder after liver and kidney transplant. Exp Clin Transplant. 2014;12(suppl 1):142-148.
6. Izadi M, Fazel M, Saadat SH, Taheri S. Bone marrow involvement by lymphoproliferative disorders after renal transplantation: PTLD. Int. Survey. J Cancer Res Ther. 2012;8(1):62-67.
7. Maecker B, Jack T, Zimmermann M, et al. CNS or bone marrow involvement as risk factors for poor survival in post-transplantation lymphoproliferative disorders in children after solid organ transplantation. J Clin Oncol. 2007;25(31):4902-4908.
8. Khedmat H, Taheri S. Bone marrow involvement by lympho­proliferative disorders post liver transplantation: PTLD Int Survey. Acta Med Indones. 2012;44(3):207-213.