—  SPECIALTY CONFERENCE  —

Hematopathology

Case 3 - Primary Effusion Lymphoma

Amy Chadburn
New York Presbyterian Hospital
New York, New York


Click on each slide thumbnail image for an enlarged view
Clinical History:
A 40 year homosexual man presented with shortness of breath. Chest X-ray showed bilateral pleural effusions. A diagnostic procedure was performed.

Kodachrome of a Giesma stained cytospin; H and E stained section of the cell block.


Case 3 - Figure 1
Case 3 - Figure 2
Case 3 - Figure 3 - LANA

Findings:
Cytospin: The tumor cells are pleomorphic with large, multiple nuclei and prominent nucleoli. The cells have abundant basophilic cytoplasm. Apoptotic cells are present.

Cell block: Shows sheets of large pleomorphic cells. Some cells are immunoblastic in appearance with a single large nucleus, a central prominent nucleolus and relatively abundant eosinophilic cytoplasm. Other cells are multinucleated with multiple prominent nucleoli. Apoptotic cells, cellular debris and numerous mitotic figures are present.

Immunophenotype: IHC: CD45+, CD19-, CD20-, CD22-, sIg/cIg-, CD3-, CD5-, CD10-, CD15-, HLA-DR+, CD30+, CD38+, CD138+, epithelial membrane antigen (EMA)+, BCL-6-, LANA+.
In situ hybridization: EBER+

Molecular genetics:
Southern blot hybridization:IgH R
c-myc G
EBV clonal		PCR:TCRg non-clonal
EBV +
KSHV (HHV-8)+

Additional clinical information and follow-up:
The patient was found to be HIV-positive 5 years prior to admission and was on AZT. He had been diagnosed with CMV retinitis in the past, but had no history of Kaposi's sarcoma. His CD4 count at diagnosis was 118/dl and his LDH was only slightly elevated. Additional radiographic studies showed no tumor mass, lymphadenopathy or hepatosplenomegaly. In spite of aggressive combination chemotherapy the patient died 4 months after diagnosis.

Comment:
Primary effusion lymphoma (PEL) was initially described as a distinct entity in 1995 by Cesarman, et al. Although cases with the morphologic, immunophenotypic and genotypic characteristics of PEL had been previously described in the literature, the identification of the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) within the tumor cells was instrumental in defining this neoplasm as a unique disease process. Primary effusion lymphomas occur primarily in HIV-positive individuals. They are very rare tumors, accounting for only approximately 3% of all lymphomas occurring in HIV-positive individuals and less than 0.4% of those diagnosed in HIV-negative persons.

Patients diagnosed with PEL are usually HIV-positive homosexual males in their early 40s with low CD4 counts and/or a previous history of an opportunistic infection. Approximately 40% of patients have or develop Kaposi's sarcoma during their clinical course. The neoplasm presents as an effusion usually involving one or more of the pleural, abdominal and pericardial spaces, but has also been found to involve tissue spaces around foreign material. In the majority of cases, despite extensive examination, no other site of disease is found. Although PELs occur primarily in HIV-positive individuals, they occasionally occur in HIV-negative individuals. The HIV-negative patients tend to be older and the tumor cells are usually negative for the Epstein Barr virus (see below). The disease is aggressive with a median survival of four months in spite of aggressive combination chemotherapy. New treatments, however, are now being tried with better success, including treatment with interferon-alpha and AZT.

Primary effusion lymphoma cells are characteristically striking in appearance as this case demonstrates. In general PELs are morphologically composed of large, pleomorphic cells exhibiting features which bridge those of immunoblastic lymphoma and anaplastic lymphoma. The cells have large nuclei with prominent nucleoli and a moderate to abundant amount of amphophilic to basophilic cytoplasm. A variable number of cells will have features reminiscent of Reed-Sternberg cells or are "wreath" – like similar to the cells seen in anaplastic large cell lymphoma; still others have highly irregular lobated nuclei imparting a "jelly-fish" like appearance.

Immunophenotypically PELs cells express CD45, but in general lack B and T cell lineage associated antigens such as CD19, CD20 and CD3. Although the vast majority of cases lack surface immunoglobulin expression, some cases have been reported to express surface or cytoplasmic immunoglobulin. The cells, however, express a variety of activation associated antigens including CD30, CD38, CD71 and epithelial membrane antigen (EMA); the cells also usually express the class II antigen, HLA-DR. In addition, the cells are usually CD138 (syndecan-1) and MUM1/IRF4 positive, but lack expression of BCL-6. Although the tumor cells are immunophenotypically indeterminate, molecular studies have shown that the vast majority of cases contain a monoclonal rearrangement of an immunoglobulin gene, clearly defining their B cell origin. Furthermore, most tumor cells exhibit somatic hypermutations in the immunoglobulin genes and in the BCL-6 gene. These genetic findings as well as the expression of MUM1/IFR4 and CD138, expressed by terminally differentiated B cells, and the lack of expression of BCL-6, a marker of germinal center cells, suggesting that most, if not all, PELs originate from post-germinal center, preterminally differentiated B cells.

The majority of PELs, between 70-95% of reported cases, also contain the Epstein Barr virus (EBV). The EBV infection is monoclonal and may be of either EBV type 1 (A) or type 2 (B). Similar to Burkitt's lymphoma, but in contrast to the majority of EBV-related lymphoproliferative disorders such as Hodgkin lymphoma and the majority of post-transplantation lymphoproliferative disorders, PELs exhibit EBV latency type I with the virus expressing only EBNA1, but none of the transforming genes such as LMP1 or EBNA2. Furthermore, PELs appear to only rarely contain structural alterations of the major oncogenes and tumor suppressor genes, such as c-myc , H-, K-, N-ras and p53. These findings suggest that KSHV may play a transforming role in PELs. A significant number of cases, however, contain mutations in the 5' end non-coding region of the BCL-6 gene. In addition, cytogenetic studies have identified abnormalities in chromosome 7 (most frequently trisomy 7), chromosome 8, chromosome 12 and the long arm of chromosome 1.

Primary effusion lymphoma is only one of several KSHV-related lymphoproliferative disorders. It has been well established that KSHV is associated with the developed of Multicentric Castleman's disease (MCD) in nearly all cases occurring in HIV-positive individuals and in approximately 40-50% of HIV-negative patients. In addition, another unique lymphoma known as plasmablastic lymphoma has been described which is also composed of KSHV-positive tumor cells. Although both PELs and plasmablastic lymphomas occur at an increased incidence in patients with MCD (for PEL the incidence is 15 times greater than the general HIV-positive patient population), they are clearly different disease entities. Plasmablastic lymphomas, although KSHV positive, and in contrast to PELs, are EBV negative, express high levels of cytoplasmic immunoglobulin, are only weakly CD30 positive and do not contain mutations in the immunoglobulin genes. Thus, plasmablastic lymphomas are thought to arise from naïve IgM lambda expressing B cells rather than terminally differentiated B cells as is the case with PELs. However, whether PELs can occur as only a solid tissue tumor is more controversial. Random case reports and rare reports within a large series of HIV-related non-Hodgkin's lymphomas suggest that these neoplasms can occur outside (and remain outside) body cavities. We have recently examined seven KSHV-positive non-Hodgkin's lymphomas occurring only in extra-cavity sites and have found these lesions to be similar to classic PELs.

Because PEL tumor cells contain a large KSHV viral load and because these cells can be grown in cell culture, PEL cell lines have been established which have been used to further characterize the virus and to study the pathogenesis of this disease. KSHV is composed of several open reading frames that encode for a variety of gene products, including homologues to cellular genes involved in signal transduction (i.e. GPCR; ORF74), cell cycle regulation (i.e. viral cyclin D; ORF72), inhibition of apoptosis (i.e. vFLIP; K13) and cell growth (i.e. viral interleukin 6; K2). Some of these gene products most likely are involved in the pathogenesis of PEL or other KSHV-related diseases. The KSHV ORF73 encodes for the latency associated nuclear antigen (LANA; LNA-1), the only protein consistently shown to be highly expressed by KSHV infected cells. This protein can be reliably detected by immunohistochemistry in formalin-fixed, paraffin embedded tissue sections. LANA is central to KSHV pathogenesis for not only does it mediate episome persistence and binds KSHV to mitotic chromosomes, it also modulates the expression of both viral and cellular genes.

Differential diagnosis:
It is important to consider PEL in the differential diagnosis of a pleomorphic tumor presenting as an effusion particularly if the patient is HIV-positive. Immunostaining for LANA (ORF73) or PCR analysis for KSHV should detect the virus.

Other non-Hodgkin's lymphoma occurring as an effusion:
Negative studies for KSHV: IHC for LANA, PCR for KSHV

  1. In HIV-positive individuals most often Burkitt's lymphoma
    1. Monotonous cell population with medium – sized nuclei, scant basophilic cytoplasm with prominent vacuoles
    2. Expression of B cell antigens
    3. Surface immunoglobulin expression
    4. CD10 and BCL-6 positive
    5. The presence of a tumor mass makes the diagnosis less likely
    6. c-myc rearrangement
  2. DLCL
    1. Cells less pleomorphic
    2. B cell or T cell antigen expression
    3. Usually are EBV negative (though some PELs are EBV negative and some DLCLs are EBV positive)
    4. Usually have a tumor mass
  3. Pyothorax-associated lymphoma (PAL)
    1. History of pleural inflammation (often due to tuberculosis)
    2. Present as a solid tumor mass
  4. Hodgkin lymphoma (HL) / ALCL – exceedingly rare, if ever
    1. CD15 expression and lack of EMA and CD45 by HL
    2. Expression of ALK-1 and T cell lineage antigens for ALCL

Carcinoma occurring as an effusion:
Negative studies for KSHV: IHC for LANA, PCR for KSHV

  1. Tumor cells positive for epithelial markers other than EMA
  2. Lack of CD45 expression.

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