Section 2 -

Epstein-Barr Virus and Lymphoproliferative Disorders in Iatrogenic Immunodeficiency Steven H. Swerdlow

Post-Transplant Lymphoproliferative Disorders (PTLD) Introduction PTLD represent lymphoid/plasmacytic proliferations that follow solid organ/bone marrow/stem cell transplantation and that are not due to some other definable cause. [1] Their incidence and clinical spectrum vary greatly probably related to many different factors. Most cases present within one year following transplant but "late" cases occur after many years. Presentations include infectious mono-like, localized tumor masses, disseminated disease, allograft dysfunction, and subtle disease with non-specific symptoms. Therapy for PTLD varies widely based on the clinical situation, available therapeutic options, and institution. Decreasing patients' levels of immunosuppression, anti-viral agents, rituxan, novel therapies and conventional lymphoma therapy are among the varied therapeutic modalities that have been utilized. Outcomes vary widely depending on many features; however, overall a mortality of up to 50-80% is reported. Some studies report much better results. Pathology of PTLD PTLD have a wide pathologic spectrum. Some biopsies show architectural preservation of the underlying tissues, whereas most show architectural destruction. Many of the former cases are not considered PTLD by many. PTLD also vary cytologically. Some are composed of very heterogeneous cell populations including small and large lymphocytes with round to irregular nuclear contours and plasma cells whereas other cases are composed of a much more homogeneous population of B-cells, T-cells or plasma cells. A minority of PTLD are polyclonal but most have variably dominant monoclonal lymphoid/plasma cells. Clonal B-cell populations are much more frequent than clonal T-cell populations. A subset of those with monoclonal populations also demonstrate additional genotypic abnormalities such as mutations in oncogenes. Post-tranplant lympho(plasmacytic) lesions according to the WHO [1] Early lesions Reactive plasmacytic hyperplasia Infectious-mononucleosis-like PTLD Polymorphic PTLD Monomorphic PTLD (classify according to lymphoma classification) B-cell neoplasms T-cell neoplasms Hodgkin lymphoma & Hodgkin-like PTLD Oncogene/tumor suprressor gene abnormalities occur in PTLD at least of monomorphic type but their overall frequency is low. C-MYC rearrangements are associated with a minority of monomorphic B-cell PTLD. [2, 3, 4] P53 mutations are also uncommon. [3] More frequent cases have p53 expression (wild-type). N-RAS mutations have not been as widely studied but have been reported in 2/2 myeloma type PTLD and 1/3 immunoblastic lymphoma type PTLD but in 0/23 PH/P-PTLD. [3] In contrast, BCL-6 mutations are more common, being reported in 43% of P-PTLD and 90% M-PTLD. [5] Although somewhat controversial, the presence of BCL-6 mutations are reported to be an adverse prognostic indicator in terms of survival and response to decreased immunosuppression. Most PTLD are associated with latent EBV infection with some reporting all cases to be positive. The EBV is usually of type A rather than the mix of types A and B as seen in HIV-associated neoplasms. Although expression of different latency associated proteins varies and not all cells fall neatly into a standard category, most cases appear to have latency pattern type III with some of type II (HL-like) and others of type I. There is also often evidence of a lytic infection as well. However, EBV negative PTLD must be recognized and their frequency appears to be increasing. LeBlond, et al reported 29% B-PTLD are EBV-. [6] In our experience, from 1982-1990 only 1/58 biopsies with PTLD were EBV negative, whereas from 1991-1996 17/75 (23%) were EBV negative and, in unreported data from 1997- 2004, 34/109 patients (31%) had EBV negative PTLD. [7] EBV negative PTLD have distinctive features. [6, 7] They occur later following transplantation, have an increased frequency of monomorphic (lymphoma-like) PTLD (81-100% M-PTLD) and are reported by some to be associated with an adverse prognosis. T-cell PTLD also have a higher relative incidence of EBV negativity with only about 20-30% T-cell PTLD EBV positive (series vary). The etiology for the majority of the EBV negative cases is uncertain. Other viruses such as HHV-8/KSHV have been identified in only rare cases. Whether some unidentified virus is responsible or whether they have a pathogenesis like lymphomas in immunocompetent hosts remains to be established (see below). EBV- PTLD can respond to decreased immunosuppression. In our series, there were 7/17 patients treated with decreased immunosuppression without chemo- or radiation therapy. [7] Of those 7 patients, 5 had non-surgical complete remissions and were alive at 3-26 months, 1 had a surgical complete remission and was alive at 68 months and 1 had a partial remission but died at 15 months. Other patients who failed this conservative therapy would have received more aggressive therapies. Further Dissecting the Monomorphic B-cell PTLD and the Impact of EBV The monomorphic PTLD are one of the most common types of PTLD with the majority of the cases resembling a diffuse large B-cell lymphoma (DLBCL). As noted above, this is also the type of B-cell PTLD most likely to be EBV-. DLBCL are also well known to be a very heterogeneous "entity." These observations raise a number of questions. How homogeneous are the monomorphic B-cell PTLD and how do they compare to transformed B-cell lymphomas seen in the normal host? What is the impact of EBV on the histogenetic spectrum of the monomorphic B-cell PTLD? Histogenetic Spectrum of B-cell PTLD and the Impact of EBV Monomorphic B-cell PTLD are a heterogeneous entity in terms of their histogenesis, just like DLBCL in general. Johnson, et al (AJSP, in press) demonstrated that they range from having a germinal center (GC) to late GC/early post-GC to post-GC phenotype. The rather scanty literature in this area has shown conflicting results. [8, 9] The EBV+ cases were less likely to be of GC type than the EBV- cases which is consistent with many observations of the effect of EBV on B-cells. Some examples include the finding in transgenic mice that EBV LMP1 induces extrafollicular B-cell differentiation and blocks GC formation, the observation that EBV infection induces many cytokines including IL-6 which downregulates bcl-6 and leads to plasma cell differentiation, gene expression profiling of EBV infected cells clusters them with IgM-stimulated B-cells rather than with GC cells and the observation that EBV infection of B-cells leads to downregulation of CD10. Statistically significant survival differences between the phenotypic groups could not be identified; however, the groups are small and there are other confounding variables. Comparison of our non-Burkitt monomorphic PTLD with DLBCL in presumably immunocompetent hosts [10] and HIV-associated DLBCL (including immunoblastic, plasmablastic and primary effusion lymphoma cases) [11] suggests that the EBV negative cases are more like lymphomas in immunocompetent hosts than the EBV positive cases and that the HIV-associated cases are intermediate between the two groups. The Contribution of Gene Profiling Studies We also undertood a small pilot gene profiling study of monomorphic B-cell PTLD to further investigate the impact of EBV on these cases. Unsupervised clustering separated the EBV+ from EBV- cases. 54 transcripts were over-expressed in EBV+ PTLD relative to EBV- PTLD which included several that have been associated with a virally induced immune response. 269 transcripts were underexpressed in EBV+ relative to the EBV- cases including 31 signaling molecules with the largest subset representing the CD79a/CD79b B-cell receptor and its downstream signaling molecules. Comparison with literature on in vitro EBV modulated gene expression in B-cell lines showed a much greater concordance than would be expected by chance alone. [12, 13] These results, which must be interpreted with great caution, provide additional evidence that EBV positive and EBV negative PTLD are distinctive and that the former show changes known to be associated with in vitro EBV infection of B-cells. This highlights the impact of EBV on the PTLD and suggests that the EBV- cases may not be associated with any virus. Methotrexate-Associated Lymphoproliferative Disorder (MLPD) The best known of the other iatrogenic associated lymphoproliferative disorders is that related to methotrexate. [14] About 85% of the patients have received the methotrexate as therapy for rheumatoid arthritis. The disorder occurs after the patients have been on methotrexate for an average of 3 years. The impact of the rheumatoid arthritis itself on lymphoma risk also must be taken into account here. It is controversial if there is an increased incidence of non-Hodgkin lymphomas in rheumatoid arthritis but there does appear to be an increased incidence of Hodgkin lymphoma. The pathologic spectrum of MLPD is similar to the PTLD; however, the distribution of cases differs. Many MLPD are of monomorphic type with many more DLBCL-like (~35%) than of Burkitt type. Geographic necrosis is one of the pathologic features of these cases. Only rare T-cell cases are reported. A small proportion of cases resemble polymorphic PTLD or "lymphoplasmacytic" proliferations. About 25% are of Hodgkin lymphoma type (or Hodgkin-like) which is more common than what is seen in the post-transplant setting. Some small B-cell lymphomas are also reported. Up to about 50% are EBV positive. After discontinuing methotrexate, up to about 50% of patients with MLPD have a complete response with others showing either a partial response or no response at all. Responses are reported in some of the Hodgkin and Hodgkin-like cases. Some responders do relapse and some series report fewer responses. Many reported patients have received chemotherapy without a trial of methotrexate withdrawal. The patients with EBV positive disease are more likely to respond than the EBV negative cases, although, as with the PTLD, the latter may also go into complete remission. Other Iatrogenic-Asssociated EBV+ LPD In addition to occasional LPD reported in other settings, a series of five patients with an EBV-associated B-cell LPD in patients with CLL related to presumptive iatrogenic immunosuppression related to Fludarabine therapy has been reported. [15] The LPD occurred 2-12 months after completion of the Fludarabine therapy. Three of four were clonally distinct from the CLL. Two received no therapy and spontaneously regressed (one recurred), one had a surgical excision only and was alive with no evidence of disease, one received anti-viral therapy and died without disease. Pathologically, three biopsies resembled a polymorphic PTLD (one with a Hodgkin lymphoma-like area), two biopsies resembled a monomorphic PTLD, one a Hodgkin type PTLD and one relapse biopsy resembled "lymphomatoid granulomatosis." Prospective identification of these cases is problematic as oftentimes these patients have received more than one drug, plus one has to exclude the possibility of transformation of the CLL or a coexistent lymphoma (Richter's syndrome). Selected References Harris NL, Swerdlow SH, Frizzera G, Knowles DM. Post-transplant lymphoproliferative disorders. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:264-269. Dotti G, Fiocchi R, Motta T, et al. Epstein-Barr virus-negative lymphoproliferate disorders in long-term survivors after heart, kidney, and liver transplant. Transplantation. 2000;69:827-833. Knowles DM, Cesarman E, Chadburn A, et al. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood. 1995;85:552-565. Nelson BN, Nalesnik MA, Locker JD, Swerdlow SH. Posttransplant lymphoproliferation disorders (PTLD) in the adult: The Pittsburgh experience. Laboratory Investigation. 1997;76:761-761. Cesarman E, Chadburn A, Liu YF, Migliazza A, Dalla-Favera R, Knowles DM. BCL-6 gene mutations in posttransplantation lymphoproliferative disorders predict response to therapy and clinical outcome. Blood. 1998;92:2294-2302. Leblond V, Davi F, Charlotte F, et al. Posttransplant lymphoproliferative disorders not associated with Epstein-Barr virus: a distinct entity? J Clin Oncol. 1998;16:2052-2059. Nelson BP, Nalesnik MA, Bahler DW, Locker J, Fung JJ, Swerdlow SH. Epstein-Barr virus-negative post-transplant lymphoproliferative disorders - A distinct entity? Am J Surg Pathol. 2000;24:375-385. Capello D, Rossi D, Gaidano G. Post-transplant lymphoproliferative disorders: molecular basis of disease histogenesis and pathogenesis. Hematol Oncol. 2005;23:61-67. Paessler M, Kossev P, Tsai D, et al. Expression of SHP-1 Phosphatase Indicates Post-Germinal Center Cell Derivation of B-Cell Posttransplant Lymphoproliferative Disorders. Lab Invest. 2002;82:1599-1606. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103:275-282. Carbone A, Gloghini A, Larocca LM, et al. Expression profile of MUM1/IRF4, BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas. Blood. 2001;97:744-751. Baran-Marszak F, Fagard R, Girard B, et al. Gene array identification of Epstein Barr virus-regulated cellular genes in EBV-converted Burkitt lymphoma cell lines. Lab Invest. 2002;82:1463-1479. Cahir-McFarland ED, Carter K, Rosenwald A, et al. Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells. J Virol. 2004;78:4108-4119. Harris NL, Swerdlow SH. Methotrexate-associated lymphoproliferative disorders. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:270-271. Abruzzo LV, Rosales CM, Medeiros LJ, et al. Epstein-Barr virus-positive B-cell lymphoproliferative disorders arising in immunodeficient patients previously treated with fludarabine for low-grade B-cell neoplasms. Am J Surg Pathol. 2002;26:630