Pathology of lymphoma in HIV

Diffuse large B cell lymphomas (DLBCL)

These lymphomas occur in both HIV-infected and uninfected individuals, but there are some differences between these two groups. In the context of HIV, DLBCLs have been divided on the basis of cellular morphology into centroblastic and immunoblastic categories. The immuoblastic type is mostly seen in people with AIDS, and is more frequently associated with EBV infection, with reported rates approaching 90%. EBV is most commonly detected in diagnostic pathology laboratories using in situ hybridization for EBERs (Figure 1B), which are abundantly expressed, non-coding viral RNAs. Patients with the immunoblastic variant usually have very advanced AIDS and are significantly immunosuppressed, so the frequency of this subtype has dramatically decreased with the widespread use of cART in the US and Europe. The reason why these lymphomas occur in people with more advanced immunodeficiency is that EBV within the tumors cells expresses a number of proteins, including LMP1 and EBNA2 that are oncogenic but also immunogenic, in what has been termed a Latency III pattern (reviewed in (12)). In contrast, cases with centroblastic morphology occur in individuals with or without AIDS, and these are further subdivided into germinal center and non-germinal center subtypes in both patient populations. However, in people with AIDS, the clinical significance of this sub-classification remains controversial and may be treatment dependent (13-15). Differences in DLBCLs occurring in HIV+ patients when compared to DLBCL occurring in immunocompetent individuals include more frequent extranodal presentation, a larger proportion of the germinal center subtype and a more common association with EBV in HIV (30% vs. <5%). One recent study of 70 AIDS-related DLBCL showed that EBV positivity was independently associated with a higher 2-year overall mortality, and recommended incorporating EBV status with IPI in prognostic categorization (16), although this association has not been found in other studies (13).

A diagnosis of DLBCL can be made from biopsies based on a loss of normal tissue architecture, with infiltration of large B-cells. B-cell origin should be confirmed by immunohistochemistry for a B-cell antigen, such as PAX5 or CD20. For classification into subtypes by cell of origin, molecular approaches such as gene expression profiling are more reliable, however since this is not always available as part of routine diagnosis, surrogate immunohistochemistry can be used, as illustrated in Figure 2 (17). In the context of a lymphoma occurring in the setting of HIV, immunohistochemistry with Ki67 is also useful to evaluate the proliferation index, which can have prognostic significance; individuals with tumors with higher proliferation appear to respond better to chemotherapy (13). In addition, while many DLBCLs can have very high proliferation rates, in some instances Ki67 may help differentiate a DLBCL from a Burkitt’s lymphoma.

Primary central nervous system (CNS) lymphoma is a subtype of DLBCL that is much more common in HIV-infected individuals, and is very similar to the immunoblastic variant. Tumor cells have an immunoblastic morphology, frequent EBV infection, and it has has declined in incidence with cART.

Burkitt’s lymphomas (BL)

This is another lymphoma type that can be present in people with or without HIV infection. Three epidemiologic subtypes are recognized (endemic, sporadic and AIDS related). BL accounts for around 14% of AIDS–related NHL and is more commonly seen with HIV infection than with other forms of immunodeficiency such as transplant recipients (12, 18). This disease typically occurs in less immunosuppressed patients with HIV, peaking at CD4 lymphocyte counts above the laboratory cutoff for AIDS onset (200 cells/μL) (19). In HIV-infected patients, BL may have the typical histologic features of BL, which are characterized by cohesive sheets of malignant cells which are small to intermediate-sized and contain moderately abundant basophilic cytoplasm and round, regular nuclei possessing two to five distinct nucleoli. Classical pathologic features of BL are the presence of abundant mitotic figures, and numerous, evenly distributed tingible body macrophages with abundant clear cytoplasm that lead to a starry-sky pattern (Figure 3). However, individuals with HIV can also have BL with atypical features, such as some showing plasmacytoid differentiation and others exhibiting greater nuclear polymorphism. In the past, these cases were referred to as atypical Burkitt’s or Burkitt-like lymphoma, but these categories are no longer favored, and a separate category had been described, called B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (see below) (10).

Immunohistochemically, BL is characterized by positivity for B-cell antigens, CD10, and BCL6, and negativity or only weak positivity for BCL2. Importantly, BL is one of the fastest growing tumors in humans, which can be shown with Ki67 immunohistochemistry that will stain practically all the tumor cells.

Translocation of MYC into one of the immunoglobulin (Ig) loci is the molecular hallmark of BL. The most common translocation is a t(8;14), involving the MYC and immunoglobulin heavy chain (IGH) genes, but in 10% of the cases it can involve MYC and one of the Ig light chain genes. The method most commonly used to demonstrate this translocation in the clinical setting is fluorescent in situ hybridization (FISH) using a break-apart probe, which will show a split signal regardless of the translocation partner. This translocation leads to deregulated expression of MYC. While in rare cases this translocation cannot be demonstrated by FISH, it is likely that MYC is nevertheless deregulated in true BL. Mutations in the MYC regulatory and coding regions also occur in BL (20-24), and may also lead to abnormal expression or prolonged protein stability. If no demonstrable MYC translocation is identified, the histology and phenotype must be otherwise completely typical for a diagnosis of BL.

B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL

The designation of B-cell lymphoma, unclassifiable (BCL-U), with features intermediate between DLBCL and BL, has been given to high grade lymphomas that do not fit cleanly into the DLBCL or BL categories (25). Previously, some of these cases were classified as atypical or Burkitt-like lymphoma. The presence of or absence of MYC translocations is not sufficient to place into this category otherwise typical cases of BL or DLBCL, respectively. This category is rather confined to cases with an unusual morphology or phenotype. BCL-U appears to be a heterogeneous category, but is useful for classification purposes. Some of these may belong to a separate molecular category ascribed to lymphomas with MYC translocations with a complex karyotype, with additional translocations in oncogenes such as BCL-6 and BCL-2. Some of these cases may correspond to those cases classified by histology as BL, but upon gene expression profiling do not have a Burkitt signature (26, 27). These cases include both EBV-positive and negative cases, but the proportion is not clear.

Classical Hodgkin’s lymphoma (CHL)

While not considered an AIDS-defining malignancy, CHL is increased in incidence in HIV-infected individuals (28), and the proportion of CHL appears to have increased in patients with longer survival and better immunological status (28). This disease occurs in both HIV-infected and uninfected individuals, however there are some differences in these two patient populations. One of these differences is the association with EBV; approximately one third of CHLs are positive for EBV in immunocompetent individuals, but almost all cases are positive for EBV in HIV-infected patients. In addition, while the nodular sclerosis subtype is more common in the general population, the mixed cellularity or lymphocyte depleted forms of CHL comprise the vast majority of cases in HIV+ patients (10, 29).

Primary effusion lymphoma (PEL)

This lymphoma subtype occurs primarily, albeit not exclusively, in HIV-infected individuals. The presence of KSHV within the tumor cells is a diagnostic criterion for this disease, and the vast majority also contain EBV (10, 30, 31). Case reports have described patients presenting with a primary lymphomatous effusion that lack KSHV, but this appears to be a different disease entity, which has been called KSHV- or HHV-8-negative PEL (32-35). A recent report of two cases and review of the literature describes 48 cases of KSHV-negative PEL, with 21% being positive for EBV, and 22% for the hepatitis C virus (HCV). Remarkably, where clinical information was available, all these KSHV-negative cases occurred in HIV-negative individuals and had a median age at diagnosis of 74 years (36), highlighting that this is a disease that is different than PEL. Classical PEL is characterized, in addition to KSHV infection, by the lack of expression of B cell-associated antigens and immunoglobulins (Ig), in spite of being a lymphoma of B cell origin based on the presence of monoclonal immunoglobulin gene rearrangements. This lack of B cell antigens in a neoplastic cell of B cell origin is reminiscent of the Reed-Sternberg cells of CHL. Diagnostically, the tumors cells are large, with features that can be immunoblastic or anaplastic, but immunophenotypically are difficult to classify. They do express CD138, which is an antigen present in differentiated B cells, such as plasma cells and multiple myeloma. The presence of KSHV is most easily documented by immunohistochemistry for the KSHV nuclear antigen LANA (encoded by ORF73), which is commercially available.

PEL is a rare disease, accounting for less than 5% of all HIV-related NHLs. The most common presentation is as an effusion involving one or more of the pleural, peritoneal and pericardial spaces. However, about one third of the cases can show dissemination to extracavitary sites. In addition to classical PEL, KSHV can be found in the tumor cells of some rare cases of AIDS-related NHL with no evidence of body cavity involvement. These have been called solid or extracavitary PEL. These represent approximately 5% of all AIDS-NHLs and they typically have the morphology of immunoblastic lymphoma but like PELs, they frequently lack expression of B-cell antigens and are commonly co-infected with EBV (37).

Plasmablastic lymphoma (PBL)

This aggressive malignancy was first reported in the oral cavity of HIV-infected individuals (38), but can also occur in other sites, as well as in conjunction with other immunodeficient states (39). There may be an uneven geographic distribution since this lymphoma subtype seems to be particularly common in HIV-infected patients in India (40). The vast majority of cases in the oral cavity are EBV-infected, but in other sites, EBV can be found in around 75% of cases. The immunophenotype is that of plasma cells. There is expression of plasma cell antigens like MUM1 and CD138, but usually no expression of B-cell antigens like CD20 and CD79a. In contrast to the immunophenotype seen in PEL, there is expression of monotypic cytoplasmic immunoglobulin in the majority of cases. The stringency of the criteria used for classification of PBL has varied over time, leading some studies to use a very strict definition (including presentation in the oral cavity and presence of EBV) which has resulted in this tumor being considered extremely rare (38), to a more general definition provided by the WHO that accepts EBV-negative cases and extra-oral presentation, as long as the morphology and immunophenotype is that of B immunoblasts or plasma cells (41). A recent report of five cases with a review of the literature identified 248 PBL cases, out of which 157 were in HIV-positive patients, 43% were outside the oral cavity and EBV was identified in 86% (42). This is a highly aggressive tumor with a median survival of around 14 months. Approximately half of the cases have been shown to have a MYC translocation (43). This study failed to detect rearrangements of other oncogenes altered in lymphoma (BCL2, BCL6, MALT1, PAX5), but gains in these loci were found in over a third of the cases.

Lymphoma arising in KSHV-associated multicentric Castleman’s disease (MCD)

These lymphomas mainly occur in HIV positive patients (44) and are very rare. They were initially called plasmablastic lymphomas (44). However, they are different from plasmablastic lymphoms (described above) in that they are KSHV-positive but EBV- negative. Lymphomas arising in KSHV-associated MCD are also different from PEL in that they are EBV-negative, and express IgMλ cytoplasmic immunoglobulin (while PELs do not express Ig). These lymphomas do not contain mutations in the immunoglobulin genes, are thought to arise from naïve B-cells rather than terminally differentiated B-cells as in PEL. A different entity has also been reported, called germinotropic lymphoproliferative disorder, in which germinal center B-cells are co-infected with EBV and KSHV (45).

Polymorphic B-cell lymphomas

These are rare lesions but morphologically resemble polymorphic post-transplantation lymphoproliferative disorders (PTLDs) (46).