Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-associated polyclonal body cavity effusions that mimic primary effusion lymphomas

Abstract

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is involved in the pathogenesis of Kaposi's sarcoma (KS) and 2 lymphoproliferative diseases, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease (MCD). PEL is defined as a liquid-phase lymphoma harboring monoclonal rearrangements of immunoglobulin (Ig) variable genes or, more rarely, of T-cell receptor (TCR) genes. As a rule, these elements are used for PEL diagnosis together with the demonstration of HHV-8 infection of the tumor clone. However, as pointed out by Boulanger and colleagues in the 1 July 2005 issue of the International Journal of Cancer,1 evidence of clonality in PEL is not being systematically demonstrated.2, 3 In their study, the question of clonality in PEL is addressed by performing molecular analysis of cellular (B-cell and T-cell) and viral (HHV-8 and EBV) clonalities in primary tumors and PEL cell lines and by finding a pattern of B-cell monoclonality in all PEL samples in association with a variable pattern of viral clonality for HHV-8 (monoclonal, bi-, oligoclonal pattern). We wish to extend the debate by reporting our observations on morphology, immunophenotype and cellular clonality of a series of HHV-8-positive body cavity effusions not fulfilling the definition of PEL. Indeed, these effusions showed the presence of nonneoplastic cells and/or atypical lymphoid cells, instead of a frank lymphoma, and T/B-cell polyclonality, instead of monoclonality. The hyperplastic/poly-oligoclonal vs. neoplastic/monoclonal nature of KS and MCD has been extensively discussed, but monoclonality has been clearly demonstrated in at least a fraction of individual lesions.4, 5, 6 On the other hand, while the true neoplastic nature of PEL and its monoclonal origin are well-recognized, the question whether PEL begins as a polyclonal proliferation of HHV-8-infected lymphoid cells with a subsequent evolution to a monoclonal proliferation remains to be clarified. This point is of interest for diagnostic purposes and for PEL pathogenesis implications, including the role of HHV-8 as lymphomagenic agent. Recently, we investigated a group of patients with MCD and KS (n = 2) or MCD only (n = 2) or KS only (n = 1); subjects with KS showed cutaneous and visceral involvement. All 5 patients developed recurrent and massive HHV-8-positive body cavity effusions, often in multiple sites, and died shortly after diagnosis. The main clinical features at the time of effusion development and the pathological and molecular/viral characteristics of effusion cells are shown in Table I. Three patients were HIV-positive (2 males, 1 female) and 2 HIV-negative (1 male, 1 female). Cytological examination of effusion specimens disclosed the absence of lymphoma cells in all cases. In the context of MCD or MCD and KS (Cases 1, 2, 4, 5), the effusion cell population was found to include 5–30% of atypical lymphoid cells with plasmacytic features and reminiscent of PEL. Atypical cells expressed HHV-8 latency-associated nuclear antigen (LANA). Moreover, a fraction (about 15–30%) of these atypical cells was found to express viral IL-6 (vIL-6) and clear features of apoptosis. Double immunohistochemistry showed that most HHV-8 LANA-positive cells displayed a B-cell phenotype with a variable expression of CD79a (∼5%) and CD138 (∼80%) B-cell markers, and cytoplasmic IgM and λ light chain restriction. CD20 was negative in all cases. In the case with KS only (Case 3), the pattern of LANA/v-IL6 positivity was restricted to very rare mononuclear cells (2% of the total effusion cell population), focally coexpressing CD68R. Clonality was ascertained by investigating Ig heavy and light chain variable gene and TCR variable gene rearrangements, using multiple PCR strategies.7 A polyclonal pattern of rearrangements was observed in all cases (Table I). Failure to demonstrate monoclonality of HHV-8-infected cells was improbably due to sensitivity, since the proportion of LANA-positive lymphoid cells was, in all cases, higher (≥2% of total cellular population) than the minimal required by the method (10 clonal cells in a background of 10,000 cells). Unsuccessful identification of clonal rearrangements could be due to the occurrence of somatic mutations that impair primers annealing. To rule out this possibility, all samples were tested using multiple primer sets, annealing at different positions. False-negative results could also be ascribed to the occurrence of illegitimate recombination events or other genetic alterations involving the Ig loci, as previously reported in PEL8, 9 and other B-cell malignancies. But the probability that, in all cases, these alterations involved both the heavy and the light chain loci was extremely low. To determine the presence of HHV-8 sequences in effusion cells, PCR analyses were conducted using primers specific for 2 nonoverlapping regions of the HHV-8 genome.10 All cases were found to harbor HHV-8 sequences by first-round PCR, and the viral load could be subsequently assessed in 4 of the 5 cases by real-time PCR, as previously described.11 As reported in Table I, effusion cells of 3 patients (Cases 2, 4 and 5) were found to harbor a high-viral load (≥220,000 genome equivalents (GE)/105 cells), whereas the effusion sediment of Case 1 showed a lower HHV-8 copy number (378 GE/105 cells). PCR analyses performed using EBNA-2-specific primers revealed the presence of EBV sequences in 1 effusion sample (Case 3). Our observations suggest that patients with MCD and/or KS (not exclusively in association with HIV infection) may develop recurrent HHV-8-positive body cavity effusions other than PEL, although with a similar poor outcome. Their common morphologic and molecular features appear to identify a nonlymphomatous entity that can be characterized as follows: (i) constant presence of HHV-8, with high-viral load, and very rare coinfection by EBV; (ii) low fraction of LANA/v-IL6 positive lymphomononuclear cells; (iii) polymorphous inflammatory-type background of LANA-negative leukocytes (abundant T-cells, few B-cells, monocytes/macrophages, and plasma cells); (iv) constant pattern of B-cell/T-cell policlonality. In the context of MCD only or of MCD/KS, we detected HHV-8 but no EBV, and LANA/v-IL6 expression in atypical lymphoid cells with a partial expression of B-cell lineage antigens of a mature phenotype. In the setting of KS only, we found HHV-8 DNA in association with EBV DNA, and LANA/v-IL6 in nonatypical mononuclear cells with features of monocytes. HHV-8-positive effusions described in our study might be designated pseudo-PEL since they share many clinicopathologic features with PEL (recurrent effusions sustained by a liquid-phase proliferation of HHV-8-infected cells within the body cavity, and poor prognosis). Effusions without lymphoma cells have been reported in KS and/or MCD both before12, 13, 14 and after15, 16, 17, 18 the discovery of HHV-8, but they were only rarely explored exhaustively for their clinical, morphologic, immunophenotypic, viral and molecular characteristics. Monotypic (IgM/λ) LANA-positive lymphoid cells (found in Cases 1, 4 and 5) evoke the plasmablasts of uncertain malignant capacity of MCD in lymphoid organs,6 and therefore, these pseudo-PEL cases might represent the liquid-phase counterpart of MCD. On the other hand, the effusion with rare non-B/non-T, LANA/CD68R-positive mononuclear cells and a prevalent inflammatory-type reaction (Case 3) is reminiscent of tissue-based early KS lesions,19 and therefore, it might represent the effusional form of KS.20 The pseudo-PEL with LANA/CD138-positive cells (Case 2) with almost null immunoglobulin expression is more similar to the standard pattern of PEL. Another observation is that polyclonal pseudo-PEL is often EBV-negative by striking contrast with monoclonal PEL, which is frequently dually infected by EBV and HHV-8. Negativity for EBV has been previously reported in HHV-8-positive effusions without evidence of monoclonality2, 15, 16 and in extranodal plasmablastic lymphoma in the context of MCD associated with body cavity effusions.21 The identification of HHV-8 in polyclonal effusions that mimic PEL is notable in light of the association between HHV-8 and PEL, and suggests a putative model of PEL lymphomagenesis. By analogy with MCD and KS that begin as a polyclonal disease with subsequent evolution to a clonal entity in some cases, individual PEL lesions may arise by neoplastic transformation of intracavitary HHV-8-infected polyclonal precursors. Although we did not observe a progression to full-blown/clonal PEL, the molecular evidence of B-cell clonality in HHV-8-positive effusions with low proportions (<20%) of atypical lymphoid cells1, 15 further supports this hypothesis. It remains to be verified whether pseudo-PEL represent an early/polyclonal stage of PEL, effusional MCD or effusional KS or, otherwise, the expression of a transient manifestation of an HHV-8-driven infectious process localized into a compartment of the body (HHV-8-associated polyserositis). Some of these hypotheses are not mutually exclusive. From the available evidence, body cavity effusions containing HHV-8-infected cells represent a heterogeneous group of lesions including PEL (monoclonal and with HHV-8-infected lymphoma cells) and pseudo-PEL (polyclonal and with HHV-8-infected atypical lymphomononuclear cells). Yours sincerely, Valeria Ascoli, Maria Luisa Calabrò, Kostas Giannakakis, Massimo Barbierato, Luigi Chieco-Bianchi, Roberta Gastaldi, Pasquale Narciso, Gianluca Gaidano, Daniela Capello