In situ hybridization for Epstein-Barr virus NotI repeats in posttransplant lymphoproliferative disorder.

Epstein-Barr Virus-associated posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. In PTLD, B-cell expansions range from reactive hyperplasias to large cell lymphomas and are often associated with active Epstein-Barr virus (EBV) infection. The lymphoproliferations may infiltrate transplant allografts and therefore may need to be distinguished from acute cellular rejection (ACR). A total of 36 tissue specimens from 11 transplant patients (six kidney, two heart, three liver) with PTLD were studied for EBV content by automated in situ hybridization (ISH) on formalin-fixed or Bouin's-fixed, paraffin-embedded tissue using a synthetic 3' terminally biotin-labeled oligonucleotide DNA probe from the EBV NotI tandem repeat region. The NotI repeat is abundantly transcribed during productive EBV infection and may encode an EBV early antigen. EBV serologies from the 11 patients showed seven primary acute infections and one acute reactivation. Two serologic studies indicated infection of indeterminant onset, and serology was not performed on one patient. Histologically, seven patients presented with polymorphous infiltrates in transplant allograft biopsies, three of which progressed to disseminated monomorphous cell populations and death within 3 to 6 wk. Tissues examined by ISH from all 11 patients showed nuclear staining for EBV in the atypical lymphoid infiltrates (34/36 specimens). The nuclear signal ranged from a stippled pattern of positivity to homogeneous nuclear staining and was localized predominantly in follicular center cells and immunoblasts, although some smaller lymphocytes also contained the viral genome. ISHs performed on 31 allograft biopsies with ACR from 24 transplant patients (six kidney, five heart, 13 liver) without clinical evidence of PTLD and with serological evidence of past EBV infection were negative for the virus. Cell lines containing EBV in the productive state (EB3 and P3HR1) were positive with ISH for NotI, while a latently infected cell line (Raji) was negative. These data indicate that ISH with the NotI probe identifies amplified genome in EBV infections and is useful in discriminating the atypical infiltrate of EBV-associated PTLD from that seen in ACR.     

Primary pleural effusion posttransplant lymphoproliferative disorder: Distinction from secondary involvement and effusion lymphoma

Pleural effusion presentation of posttransplant lymphoproliferative disorder (PTLD) is relatively uncommon. Most examples of effusion-based PTLD have been secondary to widespread solid organ involvement, and are associated with an aggressive clinical course. We report on a case of primary effusion PTLD in a 70-yr-old male liver transplant recipient with a history of hepatitis B infection. Cytomorphologically, the pleural fluid specimen showed a monomorphous population of intermediate to large-sized transformed lymphoid cells, with irregular multilobated nuclear contours and readily identifiable mitotic figures. Flow cytometric immunophenotypic studies revealed a CD5-negative, CD10-negative, lambda immunoglobulin light chain-positive, monoclonal B-lymphocyte (CD19-positive/CD20-positive) population. The immunocytochemical stain for CD30 antigen was negative. In situ hybridization study for Epstein-Barr virus (EBV) early RNA (EBER) and Southern blot analysis for EBV terminal repeat sequences were both positive. Southern blot analysis for human herpes virus-8 (HHV-8) was negative. No solid-organ PTLD was identified, and the cytologic results supported the diagnosis of primary effusion PTLD. Immunosuppression was decreased, and 8 mo following the diagnosis of pleural fluid PTLD, the patient was stable and his pleural effusion had markedly diminished. Recognition of primary effusion PTLD and its distinction from PTLD secondarily involving the body fluids and from other lymphomas is important, since the behavior and prognosis appear different.     

Heart allograft involvement with Epstein-Barr virus-associated posttransplant lymphoproliferative disorder.

We describe a 60-year-old man who underwent heart transplant and died 5 months later. At autopsy, the patient was found to have posttransplant lymphoproliferative disorder (PTLD), which was not suspected ante mortem. The PTLD involved the lung, lymph nodes, spleen, and kidney and the intima of right coronary artery of the transplanted heart. Epstein-Barr virus infection was demonstrated on direct gel and dot blot after polymerase chain reaction amplification. In situ hybridization for Epstein-Barr virus DNA confirmed the presence of Epstein-Barr virus-infected lymphocytes in the intima of the right coronary artery of the cardiac allograft. To our knowledge, this case represents the first report of involvement of a heart allograft by PTLD in which Epstein-Barr virus infection of the atypical cells is documented. Although primary allograft involvement by PTLD has rarely been reported in heart transplant recipients, recognition that this may occur is important, since the lymphoid infiltrate of PTLD must be distinguished from the lymphoid infiltrate of acute rejection to avoid inappropriate therapy.     

Posttransplant lymphoproliferative disorders: A fine-needle aspiration biopsy study

Fine-needle aspiration biopsy (FNAB) has been used with high sensitivity and specificity in the diagnosis of both Hodgkin's and non-Hodgkin's lymphoma. However, studies of FNAB of posttransplant lymphoproliferative disorders (PTLDs) are rare. The clinical course of 593 allograft recipients (cardiac, 288; renal, 250; lung, 50; and heart/lung, 5) was reviewed. Twenty-six patients developed PTLD with an overall incidence of 4.4%. Of these patients, 12 underwent FNAB. Their age ranged from 33–67 yr (mean, 55 yr). The interval between transplantation and FNAB ranged between 2–14 mo (average, 8.4 mo). The lungs were the most common site aspirated (7 cases), followed by lymph nodes (3 cases) and other extranodal sites (2 cases, liver and paraspinal mass). The cytologic features of these aspirates could be classified into two categories: a polymorphous smear composed of a spectrum of mature and immature lymphocytes with scattered plasma cells and histiocytes; and a monotonous population of large lymphoid cells consistent with malignant lymphoma, large-cell type. Surgical biopsies were available in 10 (83.3%) cases and confirmed the FNAB diagnosis. In summary, FNAB appears to be a highly sensitive and specific diagnostic tool in patients with PTLD. Diagn. Cytopathol. 16:392–395, 1997. © 1997 Wiley-Liss, Inc.     

Atypical cells in a voided urine cytology specimen in a renal transplant recipient

Voided urine is routinely collected from renal transplant patients to screen for polyomavirus. In rare cases, atypical lymphoid cells can be detected in voided urine and raise the suspicion of post‐transplant lymphoproliferative disorder (PTLD). However, further immunohistochemistry of the cell block and flow cytometry is frequently limited by the low cellularity and poor preservation of voided urine. Therefore, PTLD of the renal allograft is usually diagnosed from tissue biopsy or nephrectomy specimens. Herein, we report a rare case of atypical cells in a voided urine cytology specimen from a kidney transplant recipient. Needle core biopsy of the renal allograft showed monomorphic PTLD. Diagn. Cytopathol. 2017;45:69–72. © 2016 Wiley Periodicals, Inc.     

Reexamining post-transplant lymphoproliferative disorders: Newly recognized and enigmatic types

Post-transplant lymphoproliferative disorders (PTLD) are a known risk for both solid organ transplant and stem cell transplant recipients. Overall transplant recipients have a six fold increase in risk for developing any kind of non-Hodgkin lymphoma and PTLDs occur in up to 10% of SOT recipients. Several new entities have been accepted or renamed in the 2018 update of the WHO classification of tumors of hematopoietic and lymphoid neoplasms, including florid follicular hyperplasia and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT-lymphoma) (excluding common locations such as stomach and salivary gland). Other more rare types of PTLD have been reclassified including EBV-positive mucocutaneous ulcer, which is now a recognized diagnosis in its own right and should not be considered polymorphous PTLD. In this paper newly recognized PTLD entities and more unusual PTLDs will be examined.     

Post-transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) are the second most frequent neoplasia in transplant patients after skin carcinomas. They occur following both solid organ transplants (SOT) and haematopoietic stem cell transplants (HSCT). Most PTLD in solid organ recipients are of host origin, whereas in HSCT recipients they are most often of donor origin. The EBV status of the recipient and the donor, the type of transplant, the type of immunosuppressive therapy used, and the time after transplant are all important parameters that have been associated with the incidence and the type of PTLD. Although most PTLD are B-cell lesions up to 15% may be T-cell or NK-cell type. Most PTLD are associated with EBV, but EBV-negative PTLD are also clearly recognized. In the 2008 WHO classification of lymphoid neoplasms (ref) PTLD are subclassified according to their histological and immunophenotypic characteristics into early lesions, polymorphic type, monomorphic type, and classical Hodgkin lymphoma-type. Overall PTLD mortality rates are around 50%, but new therapies that include early treatment with rituximab and novel anti-EBV therapies promise better outcomes.     

Posttransplant lymphoproliferative disorders in lung transplant patients: the Cleveland Clinic experience.

PTLD is a well-recognized complication of organ transplantation. Large series of heart, renal, and liver transplants have been examined for the incidence and behavior of PTLD. However, reports of the incidence and characteristics of PTLDs in lung transplant (LTx) patients are few. We report our experience with PTLDs in a large series of LTx recipients at a single institution and compare them to other solid organ transplant recipient PTLDs seen at our institution. Twenty-eight patients were found to have PTLD, of whom 8 were lung transplant recipients. We evaluated nine PTLD specimens from these 8 patients for their histology, immunophenotype (CD20, CD3, EBV-LMP1), EBER status by in situ hybridization, and clinical features. The incidence of PTLD was 3.3% (8/244 patients). The time to development of PTLD, after transplant, was short (median time, 7 mo). All were of B-cell lineage. Overall, EBV was demonstrated in 77.7% (7 of 9 specimens) of PTLDs. All specimens tested for clonality were found to be monoclonal. Five patients died, with a median time to death of only 4.6 months. PTLDs in LTx patients are EBV-associated B-cell, predominantly monoclonal lymphoid lesions similar to other solid organ transplant PTLDs. Compared with other solid organ transplant recipients with PTLD at our institution, PTLDs in LTx patients have a propensity to involve the transplanted organ (P =.001, Fisher's exact test), occur earlier after transplant (P =.003, Wilcoxon test), and have a shorter survival (P =.002, log rank test). Reasons for this may include the relatively higher level of immunosuppression required in these patients and limited options in decreasing it. Although the incidence is low, careful early monitoring of lung transplantation patients is warranted because of the poor prognosis of patients developing this complication.     

Bone marrow transplantation in the rat. I. Histologic correlations and quantitation of cellular infiltrates in acute graft-versus-host disease.

In an attempt to define which cytologic manifestations of inflammation are characteristic of acute graft-versus-host disease (aGVHD), the authors have analyzed hematologic reconstitution in the bone marrow, spleen, and blood of bone marrow transplant recipients and correlated these events to concomitant cytologic changes in the parenchymal target organs. After bone marrow transplantation from Lewis to BN strain, the strongest inflammatory changes were observed in the liver, a "model" parenchymal target organ for aGVHD in this strain combination. The inflammatory episode of the aGVHD in the liver was characterized by an early lymphoid blastogenesis, the presence of large granular lymphocytes (LGLs), lymphocytosis, and some monocytosis, lacking or significantly less prominent in the liver of syngeneic BN to BN recipients. Concomitantly with the infiltration of the liver with LGLs and lymphocytes, these cells were depleted from blood; and with their disappearance from the liver, they appeared in the recipient spleen. Lack of lymphoid blastogenesis in the bone marrow of allograft recipients and similar though less prominent cytologic changes in the syngeneic graft recipients, make it difficult to differentiate aGVHD-associated changes from normal reconstitution in the lymphoid tissue paper; the minimal changes in the blood make this organ the least suitable site for the monitoring of the aGVHD in the rat.     

Pleural posttransplantation lymphoproliferative disorder following liver transplantation

A case of posttransplantation lymphoproliferative disorder (PTLD) involving the pleura is reported. The patient was a 57-year-old man who underwent liver transplantation 2 years prior to the development of PTLD. The PTLD was pleural-based and was first detected by radiologic studies as a pleural effusion. Transbronchial biopsy and cytologic examination of 2 pleural fluid specimens were nondiagnostic. Subsequent open-wedge biopsy revealed a monomorphic PTLD, composed of large immunoblasts with plasmacytoid differentiation. Immunohistochemical studies demonstrated B-cell lineage with expression of monotypic cytoplasmic immunoglobulin kappa light chain and CD79a, and absence of T-cell antigens. Immunohistochemical and in situ hybridization studies demonstrated Epstein-Barr virus protein and RNA, respectively. No evidence of human herpesvirus 8 DNA was detected by polymerase chain reaction. We report this case because pleural-based PTLD is rare. The diagnosis of this entity is made more difficult by the fact that PTLD is often underrepresented in pleural fluid cytology samples.     

Expression of Epstein-Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease.

BACKGROUND. Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) develops in 1 to 10 percent of transplant recipients, in whom it can be treated by a reduction in the level of immunosuppression. We postulated that the tissue expression of the small RNA transcribed by the EBER-1 gene during latent EBV infection would identify patients at risk for PTLD. METHODS. We studied EBER-1 gene expression in liver specimens obtained from 24 patients 2 days to 22 months before the development of PTLD, using in situ hybridization with an oligonucleotide probe. Control specimens were obtained from 20 recipients of allografts with signs of injury due to organ retrieval, acute graft rejection, or viral hepatitis in whom PTLD had not developed 9 to 71 months after the biopsy. RESULTS. Of the 24 patients with PTLD, 17 (71 percent) had specimens in which 1 to 40 percent of mononuclear cells were positive for the EBER-1 gene. In addition, 10 of these 17 patients (59 percent) had specimens with histopathological changes suggestive of EBV hepatitis. In every case, EBER-1-positive cells were found within the lymphoproliferative lesions identified at autopsy. Only 2 of the 20 controls (10 percent) had specimens with EBER-1-positive cells (P < 0.001), and such cells were rare. CONCLUSIONS. EBER-1 gene expression in liver tissue precedes the occurrence of clinical and histologic PTLD. The possibility of identifying patients at risk by the method we describe here and preventing the occurrence of PTLD by a timely reduction of immunosuppression needs to be addressed by future prospective studies.     

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.     

Post-transplant lymphoproliferative disorder associated with immunosuppressive therapy for renal transplantation in rhesus macaques (Macaca mulatta)

Human post-transplant lymphoproliferative disorder (PTLD) is an abnormal lymphoid proliferation that arises in 1–12% of transplant recipients as a consequence of prolonged immunosuppression and Epstein–Barr viral infection (EBV). Nonhuman primates, primarily rhesus macaques (Macaca mulatta), have been used extensively in research models of solid organ transplantation, as the nonhuman primate immune system closely resembles that of the human. Lymphocryptovirus of rhesus monkeys has been characterized and shown to be very similar to EBV in humans in regards to its cellular tropism, host immune response, and ability to stimulate B lymphocyte proliferation and lymphomagenesis. Thus, it appears that the NHP may be an appropriate animal model for EBV-associated lymphoma development in humans.The clinical management of post-transplant nonhuman primates that are receiving multiple immunosuppressive agents can be complicated by the risk of PTLD and other opportunistic infections. We report 3 cases of PTLD in rhesus macaques that illustrate this risk potential in the setting of potent immunosuppressive therapies for solid organ transplantation.     

Posttransplant lymphoproliferative disorders in heart-lung transplant recipients: primary presentation in the allograft

Heart-lung transplant recipients are predisposed to acute rejection episodes, bronchiolitis obliterans, and opportunistic infections. In 9.4% of recipients at the University of Pittsburgh, a posttransplant lymphoproliferative disorder (PTLD) developed, and in 60% of cases, it presented in the allografted lungs and was associated with primary infection by Epstein-Barr virus (EBV). The PTLD is histologically indistinguishable from a primary pulmonary lymphoma and consists of a mixed population of large lymphoid cells, immunoblasts, and plasma cells. Two cases of PTLD were monoclonal with immunohistochemical and Southern blot analysis. Despite this, there was clinical recovery with reduced immunosuppression and acyclovir. We discuss the role of EBV in the development of PTLD and the pathogenesis of primary presentation in the allograft.     

Polymorphous lymphoproliferative disorder: a clinicopathological analysis

Lymphoproliferative disorder (LPD) with polymorphous composition of proliferation (polymorphous LPD), containing large lymphoid cells together with small lymphocytes, plasma cells, macrophages, and/or eosinophils, is found in individuals with immunodeficiency conditions. Clinicopathological findings in 19 cases of polymorphous LPD registered with the Osaka Lymphoma Study Group, Osaka, Japan, were analyzed; they represented 0.4% of the registered cases. In six cases, there was a history of rheumatoid arthritis; five of them had received immunosuppressive agents. There were no acquired immunodeficiency syndrome cases or organ transplant recipients. Southern blotting and/or polymerase chain reaction (PCR)-based clonality analysis revealed monoclonal B cell and T cell proliferation in eight and six cases (B- and T-LPD), respectively, and polyclonality in one. In B-LPD, there was polymorphous proliferation, containing large B-lymphoid cells, while medium-to-large T lymphoid cells with occasional eosinophilic infiltration were seen in T-LPD. Epstein–Barr virus (EBV) was detected in three of eight B-LPD, four of six T-LPD, and one of one polyclonal LPD. The prognosis was not favorable; the 3-year overall survival rate was 49.7?±?17.3%. Thus, polymorphous LPD is relatively rare in Japan and is a heterogeneous disease with monoclonal proliferation of B or T cells; additionally, it is occasionally EBV-associated, and behaves as an aggressive lymphoma.     

Atypical lymphoplasmacytic and immunoblastic proliferation from rheumatoid arthritis: a case report

A case of atypical lymphoplasmacytic and immunoblastic proliferation (ALPIBP) in the lymph nodes associated with well-documented rheumatoid arthritis (RA) is presented. A 68-year-old Japanese female with a 6-year history of RA presented with right neck lymphadenopathy of 3 months duration. A biopsy specimen showed paracortical hyperplasia and numerous lymphoid follicles. On high-power field, the paracortical area was diffusely infiltrated by a polymorphous population consisting of numerous mature plasma cells, plasmacytoid cells, immunoblasts, including Hodgkin-like cells, small- to medium-sized lymphocytes, and histiocytes. Immunohistochemical study demonstrated that immunoblasts usually were CD20+, and a portion of them was CD30+. The histomorphological findings of the present case are similar to those of methotrexate (MTX)-induced atypical lymphoproliferative disorders (LPDs) in some aspects. However, Epstein-Barr virus-encoded small RNA-positive cells were not identified by in situ hybridization. The polytypic nature of B lymphocytes also was demonstrated by immunohistochemistry and polymerase chain reaction. Moreover, there was no history of MTX therapy in the present case, indicating that MTX-induced, LPD-like ALPIB may occur even in the RA patients not treated with MTX therapy.     

Lymphoproliferative disease after transplantation

Herein we describe 7 cases of posttransplantation lymphoproliferative disease (PTLD), 5 in men and 2 in women (aged from 25 to 62 years), occurring from 4 months to 12 years (mean, 7 years) after transplantation. Our patients were recipients of kidney, kidney and pancreas, heart, and autologous peripheral haematopoetic stem cells. Four cases were diagnosed as monomorphic and three as polymorphic type of PTLD according to the WHO classification. Monoclonal immunoglobuline heavy chain gene rearrangement was detected in two monomorphic lesions and one polymorphic lesion by polymerase chain reaction (PCR). In the two cases of polymorphic and the one case of monomorphic PTLD, the presence of EBV was visualised by immunohistochemical staining of some transformed lymphoid cells for latent membrane protein (LMP) of EBV. The presence of type A EBV was demonstrated by PCR. The patients were treated by reduction or discontinuation of immunosuppression and by chemotherapy. In 2 cases, a part of the organ affected by lymphoma (sigmoid colon and pancreas) was surgically resected. Four patients died of causes related to PTLD (2 to 15 months after the diagnosis), mainly of infectious complications. Two other patients who achieved remission died of unrelated causes. Only the youngest man is alive and in the complete remission 10 months after the diagnosis of PTLD.     

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.     

Using Epstein-Barr Viral Load Assays To Diagnose,Monitor, and Prevent Posttransplant Lymphoproliferative Disorder

Summary: Epstein-Barr virus (EBV) DNA measurement is being incorporated into routine medical practice to help diagnose, monitor, and predict posttransplant lymphoproliferative disorder (PTLD) in immunocompromised graft recipients. PTLD is an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. Validated protocols, commercial reagents, and automated instruments facilitate implementation of EBV load assays by real-time PCR. When applied to either whole blood or plasma, EBV DNA levels reflect clinical status with respect to EBV-related neoplasia. While many healthy transplant recipients have low viral loads, high EBV loads are strongly associated with current or impending PTLD. Complementary laboratory assays as well as histopathologic examination of lesional tissue help in interpreting modest elevations in viral load. Circulating EBV levels in serial samples reflect changes in tumor burden and represent an effective, noninvasive tool for monitoring the efficacy of therapy. In high-risk patients, serial testing permits early clinical intervention to prevent progression toward frank PTLD. Restoring T cell immunity against EBV is a major strategy for overcoming PTLD, and novel EBV-directed therapies are being explored to thwart virus-driven neoplasia.