Virus-Associated Lymphoma

Viral Biology

EBV is a gammaherpesvirus transmitted mainly through saliva. Infected B cells are driven to proliferate until an immune response controls infection. Thereafter, the viral genome persists for life in memory B lymphocytes. These cells elude immune surveillance in part because of very restricted viral gene expression and antigen presentation. Occasionally there is activation of viral lytic gene expression, but T cell–mediated immune function keeps such proliferation in check.

In vitro, EBV immortalizes B cells such that they grow indefinitely as lymphoblastoid cell lines (LCLs) ( Fig. 87.1 ). LCLs are tumorigenic in immunodeficient mice. In LCLs, viral genomes are present as circular, double-stranded deoxyribonucleic acid (DNA) episomes within the nucleus. The viral proteins required for immortalization include Epstein-Barr virus nuclear antigen-1 (EBNA1), a sequence-specific DNA-binding protein important in the maintenance of the viral episome; EBNA2, a transcription factor that has many effects similar to those of activated Notch receptors; and latent membrane protein-1 (LMP1), a constitutively activated member of the tumor necrosis factor receptor superfamily, which most closely resembles CD40. LMP1 activates the nuclear factor-κB (NFκB) pathway, which modulates cell proliferation and apoptosis. Several other EBV proteins are also required for immortalization. Although EBV immortalization of B cells in vitro may offer some insights into tumorigenesis, some caution is required in using LCL as a tumor model. Most EBV tumors, including tumors of B-lineage cells, do not express many of the viral genes required for lymphocyte immortalization. The only tumors that express the full complement of viral proteins required for immortalization are those that arise in the most profoundly immunocompromised patients. It has been suggested that there is an inverse relationship between cellular mutations and viral gene expression in tumors.

EBV gene expression may directly drive proliferation or inhibit apoptotic pathways, as illustrated by lymphocyte immortalization. However, viral gene expression may also perturb normal lymphocyte biology. Thus LMP1 expression upregulates activation induced (cytidine) deaminase expression, which facilitates somatic hypermutation and immunoglobulin class switching. LMP1 expression may also be important in the conversion of naive B cells to postgerminal center memory B cells. LMP2A allows B cells that lack normal immunoglobulin expression to escape regulatory checkpoints and survive.

Epidemiology of Viral Infection

EBV infection is ubiquitous. The vast majority of adults are infected worldwide. Primary infection most often occurs in childhood and is asymptomatic, but it may present as infectious mononucleosis in older children and adults. Other possible determinants of symptomatic primary infection include genetic factors and possibly the size of the viral inoculum.

Strain differences in EBV are well recognized. There is general agreement that the type 1 strain EBV is most common worldwide and in tumors. However, the importance of these strain differences with regard to lymphomagenesis remains poorly understood. In addition, variations in the regulatory regions or coding regions of a variety of other genes, including EBNA1, LMP1 , and ZTA , have been recognized and suggested to play a role in lymphomagenesis. A simple classification of latent viral gene expression recognizes three patterns, as shown in Table 87.2 .

Epstein-Barr Virus Detection in Clinical Specimens

Detection of virus in tumor cells requires demonstration of viral DNA, RNA, or protein in tumor cells. Although polymerase chain reaction (PCR) makes detection of viral DNA straightforward, the ubiquity of EBV infection and its persistence in a tiny fraction of B cells that harbor EBV means that EBV DNA is readily detected in many specimens that include normal lymphocytes. In pathology laboratories, immunohistochemistry for LMP1 is commonly employed and is sensitive for the detection of EBV in Hodgkin lymphoma (HL). In a variety of other EBV-associated B- and T-cell malignancies, expression of LMP1 is more variable. Thus failure to detect LMP1 expression does not exclude the presence of EBV, except perhaps in HL. In situ hybridization to detect viral RNA in tissue specimens has emerged as the pathology laboratory standard. The viral RNAs targeted are the EBV-encoded RNA (EBER) RNAs that encode short non-coding RNA transcripts expressed at high copy number per cell. Whereas viral DNA is typically present at less than 100 copies/cell in tumor tissue, EBER RNAs are present in very high numbers (millions of copies).

EBV-positive cancers typically have detectable viral DNA in the blood, making cell-free or plasma EBV DNA an attractive tumor marker. Detection of EBV DNA in whole blood may reflect the presence of viral DNA in cells or in plasma. In cells the viral DNA may be present as linear double-stranded DNA molecules replicated by the viral DNA polymerase (to be packaged into virions) or as latent circular double stranded DNA plasmids associated with nuclear cell DNA and replicated by cellular enzymes with cell cycle. Virion DNA and viral DNA from latently infected cells that has been released following cell death is also measured in plasma. When blood has been fractionated and EBV DNA in peripheral blood mononuclear cells (PBMC) compared with EBV DNA in plasma, the correlation with EBV-associated disease is stronger with plasma. It should be noted that many patients had detectable EBV DNA in PBMC and plasma without any EBV-associated disease (i.e., no EBV tumor nor evidence of infectious mononucleosis or other virus-related syndrome). Virion DNA is never methylated and recent reports have suggested that the presence of viral DNA in the absence of CpG methylation may make it less likely that a malignancy is the source of the viral DNA. Further refinement of diagnostic tools that characterize EBV DNA methylation may be useful for improving the specificity of plasma EBV DNA for EBV-associated malignancies.

Association with Particular Types of Lymphoma

Some lymphoma types are nearly 100% EBV associated, including endemic Burkitt lymphoma (BL), extranodal natural killer (NK)/T-cell lymphoma of the nasal type, early posttransplantation lymphoproliferative disorder (PTLD), lymphomatoid granulomatosis, diffuse large B-cell lymphoma (DLBCL) associated with chronic inflammation, EBV-positive DLBCL of older adults, and AIDS primary central nervous system (CNS) lymphoma (PCNSL). Other lymphoma types are variably EBV associated. These include classic HL, PTLDs occurring many months or years after transplantation, and systemic AIDS-related lymphoma.

Some lymphoma types appear never or almost never to be EBV associated, including most indolent B-cell lymphomas, although there is growing evidence that exceptions do exist, particularly in the setting of immunocompromise. Thus, the spectrum of EBV-associated lymphomas continues to expand, and the presence of EBV might be an indication to consider an underlying immune defect in the patient.

Other lymphomas are typically not EBV associated but have the interesting feature of being associated with EBV upon transformation or upon development of de novo secondary lymphomas. For example, chronic lymphocytic leukemia (CLL) can transform into EBV-positive HL, and patients with angioimmunoblastic T-cell lymphoma (AITL) can rarely develop EBV-positive DLBCL.

Table 87.3 lists the lymphomas that have been associated with EBV, associated cofactors, viral antigen expression, and an estimate of the percentage of tumors within each lymphoma subtype that harbor viral genomes. The table serves to illustrate the range of EBV lymphomas as well as host features that increase risk, but it is not meant to be comprehensive.

Posttransplantation Lymphoproliferative Disorder

PTLD is a group of lymphoproliferative disorders ranging from polyclonal lymphoid hyperplasia to lymphomas that arise in patients after solid organ or hematopoietic stem cell transplant (HSCT). PTLD, especially in the first few months after transplant, is highly associated with EBV ( Fig. 87.2A and B ). EBV gene expression in PTLD most commonly corresponds to latencies II and III. Broad expression of viral proteins is seen only in immunosuppressed hosts, reflecting that many of these proteins are immunogenic and commonly targeted by cytotoxic T cells.

B cells that harbor EBV are able to proliferate in the setting of posttransplant immunosuppression, at least in part because of decreased T-cell surveillance. HSCT patients who receive grafts that have been T-cell depleted develop EBV-associated PTLD at very high rates. Treatment of rejection in solid organ transplant recipients with agents such as the monoclonal antibody OKT3, which targets CD3 ⁺ cells, is associated with markedly increased risk for PTLD. Treatment strategies such as reduction of immunosuppression, use of rituximab, and infusion of EBV-specific cytotoxic T lymphocytes (CTL) have been effective in treating or preventing PTLD. A recent publication describing “off-the-shelf” third-party adoptive cell therapy for EBV-PTLD found overall response rates of 68% (for HSCT patients) and 54% (for solid organ transplantation), which is comparable to rates reported for donor-derived EBV-CTL (see box on Epstein-Barr Virus–Associated Positive Posttransplant Lymphoproliferative Disorder ).

Hodgkin Lymphoma

Approximately 30% of classic HL tumors in the United States and Europe are EBV-associated (see Fig. 87.2C and D ). Epidemiologic studies suggest that individuals with a history of symptomatic infectious mononucleosis are at increased risk for EBV-associated HL, but not for EBV-negative HL or other lymphomas. The period of risk peaks at about 2 years but continues to be elevated for at least 10 years after symptomatic mononucleosis. In addition, specific HLA genes have been associated with an increased risk for EBV-associated HL.

Higher EBV associations are seen in Latin America, Africa, and parts of Asia. Factors associated with EBV tumor positivity include mixed cellularity and lymphocyte-depleted classic HL histologic subtypes, male sex, low socioeconomic background, history of symptomatic infectious mononucleosis, and Hispanic ethnicity. Organ and hematopoietic stem cell transplant recipients, patients with primary immunodeficiencies, and HIV-positive patients are more likely to develop HL than the general population, and approximately 90% of the tumors in these settings are EBV-associated.

The EBV gene expression pattern in HL is latency II, even when HL occurs in immunocompromised populations. LMP1 and LMP2A may mimic signaling of B-cell receptors and thus protect B cells lacking functional immunoglobulin expression from apoptotic signaling. Approximately 20% of HLs lack productive immunoglobulin gene rearrangements. These tumors appear to be exclusively EBV-associated.

Patients with EBV-positive HL quite reliably have EBV DNA detected in cell-free blood (plasma or serum) in the setting of active disease. Failure to clear EBV DNA from plasma or serum with therapy has been associated with inferior failure-free survival.

Currently, treatment regimens for EBV-positive HL do not differ from those used for EBV-negative HL. However, novel treatment strategies involving adoptive therapy with autologous EBV-specific T cells targeting type II latency LMP antigens appear to be associated with durable complete responses and low toxicity in some patients with EBV-positive HL, even in the setting of relapsed/refractory disease.

Burkitt Lymphoma

Endemic BL is nearly 100% associated with EBV, whereas sporadic and HIV-associated BL are much more variably EBV-associated (see Table 87.3 and Fig. 87.2E and F ). Viral expression is latency I (i.e., EBNA1 is the only viral protein consistently expressed). The defining feature of BL is a translocation between c-Myc on chromosome 8 and one of the immunoglobulin genes on chromosome 2, 14, or 22. It has been generally presumed that falciparum malaria is a cofactor in endemic BL, and the distribution of BL in Africa corresponds to the distribution of holoendemic malaria. However, little is understood of the pathogenesis or interaction between these infectious cofactors. The characteristic presentation of BL is different in the endemic versus sporadic settings, but there is no evidence to link these presentations specifically with the virus. Data has been presented suggesting that EBV DNA measurements in plasma may have utility in facilitating diagnosis, assessing prognosis, and monitoring tumor response in East Africa. The virus-tumor association does not currently guide therapy.

Virus and Tumor Epidemiology

KSHV (HHV-8) is a gammaherpesvirus that, unlike EBV, has a low prevalence worldwide. The virus is endemic in certain areas, such as in sub-Saharan Africa and the Middle East, and has an intermediate prevalence in Mediterranean countries. Transmission is believed to be predominately through saliva. Similar to EBV, KSHV latently infects B cells, and viral genes that promote cell survival are implicated in lymphomagenesis.

KSHV was discovered in Kaposi sarcoma but is also present in rare lymphoproliferative diseases, including primary effusion lymphoma (PEL) ( Fig. 87.3A and B ) and multicentric Castleman disease (MCD). Other KSHV-positive lymphomas have been characterized, all of which tend to occur in severely immunocompromised individuals and carry a poor prognosis. PEL occurs almost exclusively in HIV-positive patients, typically when CD4 counts are less than 100/mm ³ . However, PEL has been reported very rarely in solid organ transplant patients and elderly men with some degree of immunocompromise. KSHV is always present in PEL. In HIV patients with PEL, tumor cells are usually dually infected with EBV as well. By contrast, HIV-negative PEL is usually EBV-negative. KSHV-associated MCD, although much more common in HIV-infected populations, also occurs in the general population.

MCD is a nonclonal lymphoproliferative disorder typically involving the mantle zone of lymph nodes and the spleen. MCD can be a KSHV-associated or idiopathic, without detectable KSHV. In KSHV-associated MCD, the virus appears to drive λ light chain rearrangement in the lymphocytes that give rise to Castleman disease. These cells express a broad range of KSHV lytic antigens, and high KSHV copy numbers are present in blood. Evolution into or coassociation with an aggressive lymphoma, often of plasmablastic phenotype, is not uncommon. Within the HIV population, nearly all cases of MCD are KSHV-associated; this viral association is not as strong among HIV-negative MCD patients. High expression of viral interleukin-6 (IL-6) is thought to contribute to the systemic inflammation seen in this disorder. The KSHV-associated lymphomas that arise in association with MCD are not nodal equivalents of PEL; rather, these plasmablastic lymphomas (see E-Slide VM03957) are uniformly EBV-negative and express IgM λ.