Kaposi sarcoma herpesvirus/human herpesvirus-8–negative effusion-based lymphoma: Report of 3 cases and review of the literature

Authors

  • Jingnan Xiao MD, PhD,

    1. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
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  • Suzanne M. Selvaggi MD,

    1. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
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  • Catherine P. Leith MD,

    1. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
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  • Sean A Fitzgerald MD,

    1. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
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  • Jimmie Stewart III MD

    Corresponding author
    1. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
    • Corresponding author: Jimmie Stewart, III, MD, Cytopathology Laboratory/D4-207b, University of Wisconsin Hospital and Clinics, 600 Highland Avenue, Madison, WI 53792; Fax: (608) 263-6453; jj.stewart@hosp.wisc.edu

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Abstract

BACKGROUND

Primary effusion lymphoma (PEL) is a rare subtype of large B-cell lymphoma that arises in body cavities without detectable tumor masses. PEL is universally associated with Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus-8 (HHV8). Despite overlapping features, KSHV/HHV8-negative effusion-based lymphoma is a distinct entity from PEL. To date, 52 cases have been reported. The authors report 3 additional cases received in their laboratory from 2007 to 2012.

METHODS

Clinical data, cytomorphologic features, and immunophenotypic features of the 3 cases were described and compared with those reported in the literature.

RESULTS

The cells in HHV8-negative effusion lymphoma commonly revealed large cell, immunoblastic morphology and B-cell immunophenotype. The 3 cases demonstrated cytomorphologic and immunophenotypic variability. Cytomorphologically, 1 case contained large, highly atypical cells with a moderate amount of cytoplasm, round nucleus, coarsely granular chromatin, and a single macronucleolus. The other 2 cases had medium to large atypical cells with high nuclear-to-cytoplasmic ratios, slightly irregular to cleaved nuclei, and multiple conspicuous nucleoli. One case had a null phenotype with aberrant cytokeratin expression. B-cell phenotype was established by clonal immunoglobulin heavy-chain rearrangement using polymerase chain reaction, whereas the other 2 cases demonstrated a B-cell phenotype by flow cytometry and immunohistochemical staining. All 3 cases were negative for both HHV8 and Epstein-Barr virus.

CONCLUSIONS

HHV8-negative effusion lymphoma exhibits clinical, cytomorphologic, and immunophenotypic variability. Cases with a null-phenotype can be particularly challenging. When effusion lymphoma is suspected, ancillary tests are helpful. Moreover, HHV8 detection is critical in differentiating PEL and HHV8-negative effusion lymphoma, because they have overlapping features yet different prognoses. Cancer (Cancer Cytopathol) 2013;121:661–9. © 2013 American Cancer Society.

INTRODUCTION

Primary effusion lymphoma (PEL) is a rare subtype of diffuse large B-cell lymphoma according to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.[1] It predominantly involves body cavities and presents as pleural, peritoneal, or pericardial effusions. Whereas the vast majority of PEL cases occur in patients who are infected with human immunodeficiency virus (HIV), rare cases have been reported in patients who received solid organ transplantation and in patients with chronic hepatitis C. PEL is universally associated with Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus-8 (HHV8). Notably, many patients have concurrent Epstein-Barr virus (EBV) infection. Morphologically, the lymphoma cells exhibit features bridging immunoblastic, anaplastic, or plasmablastic large cell lymphoma. They are usually negative for B-cell markers, such as cluster of differentiation 19 (CD19), CD20, and CD79a, but they may be positive for CD45 and for activation and plasma cell-related markers, such as CD30, CD38, and CD138. The prognosis is generally poor.[1]

In the past 17 years, sporadic cases of effusion lymphoma without detectable KSHV/HHV8 have been described.[2-41] Similar to PEL, these patients present with lymphomatous effusion without a detectable mass, lymphadenopathy, or organomegaly. However, HHV8 is negative in all these reported cases, and most cases occur in patients without HIV infection. Moreover, a high percentage of cases occur in patients who have cirrhosis and other comorbidities, such as cardiovascular diseases. Unlike PEL, the lymphoma cells most commonly have large cell, immunoblastic type morphology and a B-cell immunophenotype. The prognosis of this entity appears to be more favorable than that of PEL. Most cases are referred to in the literature as “HHV8-unrelated PEL-like lymphoma.” Most recently, the term of “KSHV/HHV8-negative effusion-based lymphoma” has been proposed by Alexanian et al.[2] To date, 52 cases have been reported in the literature, the majority of which have been from Japan.

Here, we report a series of 3 cases of HHV8-negative effusion-based lymphoma among the effusion specimens received in our laboratory from 2007 to 2012. We describe the variability in clinical, cytomorphologic, and immunophenotypic features of these 3 cases and compare them with the reported 52 cases in the literature. The diagnostic challenges when evaluating lymphomatous effusion specimen also are discussed.

MATERIALS AND METHODS

Current Case Series

Three cases of effusion-based lymphomas without detectable masses or lymphadenopathy were identified among all body fluid specimens received in the University of Wisconsin Hospital and Clinics Cytopathology Laboratory from 2007 to 2012. ThinPrep (Hologic Inc., Bedford, Mass) slides and cell blocks were prepared from the pleural and pericardial fluids. The immunophenotype was determined by flow cytometry and immunohistochemical methods. Molecular studies, including karyotyping, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR), were performed at the Wisconsin State Laboratory of Hygiene and ARUP Laboratories, Utah, respectively. Clinical data were obtained by chart review.

Review of the Literature

The terms “effusion lymphoma,” “primary effusion lymphoma,” “HHV8-negative effusion lymphoma,” and “HHV8-unrelated primary effusion lymphoma-like lymphoma” were used to identify relevant cases published up to March 2013. Of the 52 cases identified in the literature, 13 cases had detailed descriptions of cytomorphologic features. The immunophenotype was available in all 52 cases. Other ancillary studies, such as cytogenetic and molecular tests, were performed in a fraction of reported cases. Cytomorphologic features, including cell size, cytoplasm, nuclear size, nuclear contour, chromatin pattern, presence or absence of prominent nucleoli, binucleation, and multinucleation, were documented and compared. Immunophenotype determined by flow cytometric analysis and/or immunohistochemical studies was also included in this review.

RESULTS

Current Case Series

Case 1

A man aged 55 years with a history of alcoholic cirrhosis presented with 5 days of fever, chills, and abdominal pain. Abdominal imaging revealed a complex, septated, and rim-enhancing collection of ascitic fluid. Paracentesis yielded purulent fluid, which grew anaerobic Gram-positive rods. No malignancy was detected. He was subsequently discharged with oral antibiotics, and an intra-abdominal drain was placed. However, he continued to have bloody ascitic fluid and worsening weakness with 40-pound weight loss over a period of 5 weeks. He was readmitted and was prepared for an abdominal washout of the abscess and evaluation for liver transplantation. Repeat imaging demonstrated some improvement of the abdominal abscess, but incidentally revealed a right-sided pleural effusion. No evidence of a mass or lymphadenopathy was identified on imaging. The pleural fluid was highly cellular and had a predominance of very large, discohesive cells with slight pleomorphism. Cytomorphology demonstrated that the cells had high nuclear/cytoplasmic ratios; centrally or eccentrically located, round nuclei containing coarse chromatin with a single macronucleolus; and a small to moderate amount of amphophilic cytoplasm. Apoptotic bodies and nuclear debris were readily identifiable (Fig. 1A,B). Cytoplasmic pigment, cytoplasmic vacuoles, multinucleation, and intranuclear inclusions were not noted. Flow cytometric analysis of the fluid revealed high background staining and a lack of expression of lineage-specific markers. On the basis of the cytomorphologic features and indeterminate immunophenotype by flow cytometry, the differential diagnosis was broad and included malignant melanoma, undifferentiated carcinoma, malignant mesothelioma, germ cell tumor, and effusion lymphoma. Immunohistochemical staining for CD3, CD20, CD30, CD68, CD79a, PAX-5, CD138, CD45, ALK, CD117, the cytokeratin 8 marker CAM 5.2, cytokeratin AE1/AE3, HHV8, vimentin, S100, HMB45/MART, calretinin, and WT-1 was performed on the cell block prepared from the fluid. The large atypical cells were diffusely and strongly positive for vimentin. CAM 5.2 was positive in a small subset of cells (Fig. 1C,D). The cells were negative for all other antibodies, including HHV8 as well as for EBV-encoded RNA (EBER) by in situ hybridization. Because of the ambiguous phenotype, the patient underwent a second thoracentesis, and the pleural fluid was sent directly for cytogenetic and gene arrangement studies. It is noteworthy that cytogenetic studies identified an abnormal clone with a complex composite karyotype, including a t(8;14) translocation. In addition, the v-myc myelocytomatosis viral oncogene homolog/immunoglobulin heavy chain (MYC/IGH) fusion, which is associated with translocation t(8;14)(q24;q32), was detected by FISH analysis. Gene arrangement studies by PCR demonstrated a clonal IGH rearrangement, thus establishing the diagnosis of B-cell lymphoma by molecular methods. His HIV status, hepatitis B virus status, and hepatitis C virus (HCV) status all were negative. Given his liver status and nonhealing abdominal abscess, he was considered to be a poor candidate for chemotherapy and received only palliative care. He died approximately 4 months later because of liver failure and sepsis.

Figure 1.

(A,B) Right pleural fluid (case 1) reveals a predominance of very large, discohesive cells with small to moderate amounts of cytoplasm and large, round nuclei with coarsely granular chromatin and a single distinct macronucleolus (Papanicolaou stain in A, H & E stain in B; original magnification ×600). (C,D) These large cells are strongly positive for vimentin and are focally positive for the cytokeratin 8 marker CAM 5.2 (original magnification ×200).

Case 2

A man aged 95 years with a history of hypertension, basal cell carcinoma, and atrial fibrillation presented with a large left pleural effusion. Thoracentesis was performed, and the fluid was sent for cytologic evaluation. The pleural fluid was cellular and consisted of numerous, medium to large, discohesive, round cells. These cells contained a small to moderate amount of basophilic cytoplasm and round to slightly irregular nuclei with coarse chromatin and multiple conspicuous nucleoli. Binucleated forms were noted. There were admixed small lymphocytes and rare neutrophils (Fig. 2A,B). Immunophenotypic analysis by flow cytometry identified a κ light chain-restricted monoclonal B-cell population. These cells were positive for CD19, CD20, CD22, and CD10 (in a subset of tumor cells), and they were negative for CD5, CD23, and CD38. Immunohistochemical staining on the cell block preparation demonstrated that these tumor cells were negative for HHV8 and CD30. In addition, EBER was negative according to in situ hybridization. The patient subsequently underwent computed tomography scans of the chest, abdomen, and pelvis, which revealed no lymphadenopathy or mass lesions. He received treatment with rituximab alone. Approximately 5 years later, he developed shortness of breath, tachypnea, and wheezing. A chest x-ray demonstrated a marked right pleural effusion. The cytomorphology and immunophenotype of the second effusion specimen were similar to those of the previous fluid, with the exception of the loss of CD10 expression in the second specimen. Notably, the tumor cells were repeatedly negative for HHV8 and EBER. Therefore, his recurrent presentation was consistent with relapsed effusion lymphoma, although the site of effusion occurred in the contralateral pleural cavity. He again received treatment with rituximab alone, and most of his symptoms have resolved. He is now 101 years old.

Figure 2.

(A,B) Left pleural fluid (case 2) reveals medium to large, discohesive, round cells with a small to moderate amount of cytoplasm and smooth to slightly irregular nucleus with multiple, conspicuous nucleoli. Binucleated forms were present (Papanicolaou stain in A, H & E stain in B; original magnification ×600).

Case 3

A woman aged 69 years presented to the Emergency Department with acute pancreatitis and pericardial tamponade. Her medical history was significant for thyroid cancer status post-thyroidectomy and radiation and gallstone pancreatitis. She was initially managed in the Trauma and Life Support Center and had drainage of the pericardial effusion. The fluid was sent for cytologic evaluation and revealed numerous, polymorphous, medium to large, atypical lymphocytes. The lymphoid cells had small amounts of cytoplasm and irregular nuclei with coarse chromatin and multiple conspicuous nucleoli. Occasional binucleated forms were noted (Fig. 3A,B). Flow cytometric analysis of the pericardial fluid identified a λ light chain-restricted monoclonal B-cell population with bright expression of CD19, CD20, and CD38. Immunohistochemical staining was performed on the cell block and demonstrated that the lymphoma cells were strongly positive for CD20 but negative for HHV8 (Fig. 3C,D). EBER also was negative by in situ hybridization. Further imaging studies demonstrated no evidence of lymphadenopathy or mass lesions. A subsequent staging bone marrow biopsy was negative for involvement by lymphoma. She received 6 cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Her pericardial effusion resolved, and no recurrence of effusion or other evidence of hematopoietic malignancy has been observed since diagnosis.

Figure 3.

(A,B) Pericardial fluid (case 3) reveals numerous, polymorphous, medium to large, atypical lymphocytes with a small amount of cytoplasm and slightly irregular nucleoli with multiple, conspicuous nucleoli that resemble immunoblasts. Occasional binucleated forms are noted (Papanicolaou stain in A, H & E stain in B; original magnification ×600). (C,D) Tumor cells are strongly positive for the B-lymphocyte antigen CD20 and negative for human herpesvirus-8 (HHV-8) (original magnification ×200).

Cytomorphology

The common cytomorphologic features shared by the majority of cases (including our case series) were large cell size, coarse chromatin, single or multiple conspicuous nucleoli, and the presence of binucleated or multinucleated tumor cells. Nevertheless, there was significant cytologic variability among cases in the literature and our current series. For example, cytoplasmic vacuoles were present in some of the cases in the literature but were not observed in any of our cases. The chromatin pattern ranged from finely distributed, vesicular to coarse. Most cases demonstrated single or multiple conspicuous nucleoli. Case 1 in our series had a distinct single macronucleus (Fig. 1).

The cytomorphologic features of our 3 cases are summarized in Table 1, and those of the previously reported cases are listed in Table 2. The denominators in Table 2 are the number of cases in which the respective cytologic features were described in the reported cases.[2-41]

Table 1. Summary of the Cytomorphologic Features of the Three Cases in the Current Series
Cytomorphologic FeatureCase 1Case 2Case 3
Cell sizeLargeMedium to largeMedium to large
Amount of cytoplasmSmallModerateModerate
Cytoplasmic vacuolesAbsentAbsentAbsent
Nuclear location   
CentralPresentPresentPresent
EccentricPresentPresentPresent
Nuclear membraneRegularSlight irregularIrregular
ChromatinCoarseCoarseCoarse
Prominent nucleoliPresent (macronucleus)PresentPresent
BinucleationAbsentPresentPresent
Apoptotic bodiesPresentPresentPresent
Mitotic figuresRareRareRare
Table 2. Comparison of the Cytomorphologic Features of Cases of Human Herpesvirus-8–Negative Effusion Lymphoma From the Literature Review and the Current Series
 No. of Positive Cases/Total No. (%)
Cytomorphologic FeatureLiterature ReviewCurrent Series
Large cell size13/13 (100)3/3 (100)
Cytoplasmic vacuoles5/5 (100)0/3 (0)
Irregular nuclear membrane11/13 (84.6)2/3 (66.7)
Presence of multilobated nuclei4/4 (100)0/3 (0)
Prominent nucleoli12/12 (100)3/3 (100)
Presence of binucleated or multinucleated cells3/3 (100)2/3 (66.7)
Coarse chromatin6/10 (60)3/3 (100)

Immunophenotype

Two of our 3 cases (66.7%) expressed CD19 and CD20, compared with 84.6% of the reported cases that were positive for B-cell markers. Case 1 in our series was negative for CD45, B-cell markers, and T-cell markers but aberrantly expressed cytokeratin in a subset of cells, as demonstrated by CAM 5.2 immunohistochemical staining (Fig. 1). B-cell lineage for case 1 was determined by PCR gene rearrangement studies. EBV and HHV8 were negative in all 3 cases.

The detailed immunophenotype of our 3 cases is listed in Table 3. Table 4 summarizes the immunophenotype from the reported cases. The denominators are the numbers of cases in which respective markers were performed in the case reports.

Table 3. Summary of Lymphoma Characteristics in Cases From the Current Series
CaseImmunophenotypeHHV8EBVCytogeneticsFISH
  1. Abbreviations: CK, cytokeratin; EBV, Epstein-Barr virus; FISH, fluorescence in situ hybridization; HHV8, human herpesvirus-8; κ, kappa light chain restricted; λ, lambda light chain restricted; MYC/IGH, v-myc myelocytomatosis viral oncogene homolog/immunoglobulin heavy chain; ND, not done.

1Vimentin, CK (focal)Complex karyotypeMYC/IGH fusion
2CD19, CD20, CD10, κNDND
3CD19, CD20, CD38, λNDND
Table 4. Comparison of the Immunophenotype of Cases of Human Herpesvirus-8–Negative Effusion Lymphoma From the Literature Review and the Current Series
No. of Positive Cases/Total No. (%)
MarkerLiterature ReviewCurrent Series
  1. Abbreviations: CD20, cluster of differentiation; EBV, Epstein-Barr virus; HHV8, human herpesvirus-8.

  2. a

    Pan-B–cell markers include CD19, CD20, CD22, CD79a, and cytoplasmic or surface immunoglobulins.

CD2037/52 (71.2)2/3 (66.7)
Pan-B–cell markersa44/52 (84.6)2/3 (66.7)
Cytokeratin1/2 (50)1/3 (33.3)
EBV12/43 (27.9)0/3 (0)
HHV80/52 (0)0/3 (0)

DISCUSSION

Lymphomatous effusion in body cavities accounts for approximately 10% to 15% of malignant effusions.[42, 43] Prompt and accurate diagnosis of such cases relies heavily on cytologic evaluation of the specimen. It is not usually a diagnostic challenge in patients who have known clinical history of lymphoma, however, it can be particularly challenging when the body cavity is the primary site of involvement. Several hematologic malignancies are known to potentially involve serous cavities, such as Burkitt lymphoma, anaplastic large-cell lymphoma, B-cell and T-lymphoblastic lymphoma, PEL, and HHV8-negative effusion-based lymphoma. Because immunophenotyping is crucial for further classification of any hematologic malignancy, an effort must be made to ensure that there is sufficient sample to perform ancillary studies, such as flow cytometric analysis and cytogenetic and molecular tests.

PEL was first introduced by Nador et al[44] as a separate entity to distinguish it from other malignant lymphomas that secondarily involve body cavities. The overwhelming majority of these relatively few cases were body cavity-based lymphomas without an identifiable tumor mass. Most of the cases occurred in immunocompromised patients, such as those with HIV infection. Notably, all cases had KSHV/HHV8 infection; lacked a c-myc gene arrangement; and exhibited overlapping morphologic features of immunoblastic, anaplastic, or plasmablastic large cell lymphomas.[45] Immunophenotypically, they usually are negative for B-cell markers (eg, CD19, CD20, CD22, and CD79a) but may be positive for CD45 and for activation and plasma cell-related markers (eg CD30, CD38, and CD138).[1] PEL was subsequently included as a distinct entity as a mature B-cell neoplasm in the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues.[1]

The incidence of KSHV/HHV8-negative effusion-based lymphoma is even lower compared with that of PEL. A review of the literature revealed a total of 55 cases (including the 3 cases in this study). Morphologically, the lymphoma cells in most reported cases were predominantly large cells with irregular nuclear contours, coarse chromatin, and single or multiple conspicuous nucleoli. Some cells contained cytoplasmic vacuoles. Lymphoma cells in cases 2 and 3 from our series exhibited medium to large cells and immunoblastic morphology, whereas the cells in case 1 demonstrated distinctive cytologic features, such as large to very large cell size, round nucleus, coarse chromatin, and a single macronucleus. Cytoplasmic vacuoles were absent in all 3 cases. Immunophenotypically, the majority of HHV8-negative effusion-based lymphomas (84.6%) express B-cell markers, such as CD19, CD20, CD22, and κ or λ light chain restriction. Hence, our cases 2 and 3 fall into this category. Case 1, however, had no expression of CD45 or any lineage-specific markers according to both flow cytometric analysis and immunohistochemical studies. B-cell clonality was only established by molecular methods. It is noteworthy that a subset of tumor cells expressed cytokeratin in a CAM 5.2-positive case (Fig. 1). To our knowledge, this is the second case to demonstrate aberrant cytokeratin expression in HHV8-negative effusion-based lymphoma.[7] Therefore, this case highlights the challenge in the workup of a lymphomatous effusion and the importance of a multimodality approach, particularly for cytogenetic and molecular studies, in arriving at a definite diagnosis.

The pathogenesis of HHV8-negative effusion-based lymphoma remains elusive. It has been suggested that HCV and EBV play a role in the lymphomagenesis, because approximately 30% and 28% (Table 4) of the reported cases had concurrent HCV and EBV infection, respectively. A significant proportion of patients have an underlying medical condition (cirrhosis, cardiovascular dysfunction, etc), leading to fluid overload.[2] Notably, a few cases (including case 1 in this report) demonstrated MYC/IGH fusion associated with t(8;14)(q24;q32) or amplification of c-MYC. Complex chromosomal abnormalities are detected in quite a few cases. In addition to translocations involving 8q24, which contains the c-myc gene, gains of 3q13-27, 10q21-23, and Yq have been reported.[30]

To date, there is no standard treatment for either PEL or HHV8-negative effusion-based lymphoma. CHOP is the mostly common chemotherapy used for both entities. Rituximab (R-CHOP) has been added for cases with CD20-positive tumor cells. Nevertheless, the prognosis of PEL is generally poor, with a median survival of 4 months and a 1-year survival rate <20%.[1] On the basis of limited reported cases, the outcome of patients with HHV8-negative effusion-based lymphoma seems to be more favorable, with a median survival of 10 months and a 1-year survival rate of 35%.[30] Most recently, there have been case reports of complete remission with effusion drainage alone or by pleurodesis.[34, 37, 38] Case 2 in received rituximab therapy alone and achieved a complete remission that lasted more than 5 years until his recent relapse. Case 3 received R-CHOP therapy with a continuing remission of more than 1 year. Case 1 died of causes unrelated to lymphoma, because he had multiple comorbidities, such as liver failure and a nonhealing abdominal abscess.

In conclusion, HHV8-negative effusion-based lymphoma is a separate entity from its counterpart, HHV8-positive PEL, despite many overlapping clinical and cytomorphologic features. We report 3 cases to demonstrate the cytomorphologic and immunophenotypic variability within this entity. Detection of HHV8 in the tumor cells is the key to distinguishing HHV8-negative effusion lymphoma from PEL, because the prognoses can be quite different.

FUNDING SUPPORT

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

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