British Journal of Haernatology, 1995, 91, 918-920

SHORT REPORT

Distribution of Kaposi’s sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain

CRISTINA PASTORE,’ ANNUNZIATA GLOGHINI,’ GISELLA VOLPE,

UMBERTO M A Z Z A ,

JOSEP NOFV~DEDEU,~ EUGENIO LEONARD0,4

GIUSEPPE SAGLIO, ’” ANTONINO cARBONE2 AND GIANLUCA GAIDANO’

‘Laboratorio di Medicina e Oncologia Molecolare, Dipartimento di Scienze Biomediche e Oncologia Umana, Italy; ’Dipartimento di Scienze Cliniche e Biologiche; and ‘CNR-CZOS, Universith di Torino, Torino, Italy; Divisione di Anatomia Patologica, I.R.C.C.S. - C.R.O., Aviano, Italy; 3Departament d’Hematologia,

Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 4Divisione di Anatomia Patologica, Azienda Ospedaliera Sun Luigi, Orbassano-Torino, Italy

2

Received 2 June 1995; accepted for publication 27 July 1995

Summary. In this study we have tested the distribution of Kaposi’s sarcoma herpesvirus (KSHV) DNA sequences throughout the spectrum of lymphoid neoplasia in Italy and Spain. 180 cases of lymphoid malignancies representa- tive of the major histologic and immunophenotypic cate- gories of B- and T-cell tumours were analysed by means of a polymerase chain reaction-based assay. KSHV sequences were consistently absent in all categories of lymphoid malignancies studied, with the exception of a subset of B- cell non-Hodgkin’s lymphomas localizing in the pleural,

pericardial or peritoneal cavities, and fulfilling the diagnostic criteria of body-cavity-based lymphoma. The selective and consistent association of KSHV sequences with cases of body- cavity-based lymphoma throughout the spectrum of lym- phoid neoplasms suggests that KSHV may be involved in the pathogenesis of this peculiar type of lymphoid malignancy.

Keywords: acute lymphoblastic leukaemia, non-Hodgkin’s lymphoma, body-cavity-based lymphoma, Kaposi’s sarcoma herpesvirus, AIDS.

Lymphoid malignancies represent a markedly heterogenous group of neoplasms which are classified based on their histologic and immunophenotypic features (Harris et al, 1994). The pathogenesis of these malignancies is also heterogenous, since specific categories of lymphoid tumours associate with distinct molecular lesions (Gaidano & Dalla- Favera, 1995). Most commonly, molecular lesions of lymphoid malignancies involve activation of dominantly acting oncogenes or disruption of tumour suppressor loci. Viral infection of the turnour clone represents an additional type of genetic lesion contributing to the molecular pathogenesis of lymphoid tumours (Gaidano & Dalla- Favera. 1995). Among €?-cell neoplasms, a large body of evidence has pointed to the role played by Epstein-Barr virus (FBV) in the development of a subset of non-Hodgkin’s lymphomas (NHL), including Burkitt’s lymphoma and a

Correspondence: Dr Gianluca Gaidano. Laboratorio di Medici~ e Oncologia Molecolare-Medicina Interna 5A. Dipartimento di Scienze Biomediche e Oncologia Umana, Universita di Torino, Ospedale San Luigi, Regione Gonzole 10. Orbassanc-Torino 10043, Italy.

918

fraction of human immunodeficiency virus (HIV) related NHL. Among T-cell tumours the consistent clonal integra- tion of HTLV-I genomes in cases of adult T-cell lymphoma/ leukaemia suggests that HTLV-I is an absolute requirement for the pathogenesis of this tumour type.

Recently, DNA sequences belonging to a putative novel virus have been isolated from cases of Kaposi’s sarcoma in the United States (Chang et al, 1994). Because of sequence homology to other herpesviruses, namely EBV and herpes- virus saimiri, the term Kaposi’s sarcoma herpesvirus (KSHV) has been provisionally suggested. In the United States, analysis of several types of human neoplasia. including certain categories of lymphoid malignancies, has shown that KSHV sequences selectively associate with Kaposi’s sarcoma and with a peculiar subset of non-Hodgkin’s lymphomas (Chang et al, 1994; Cesarman et al, 1995: Huang et al, 1995). In Europe, published reports are restricted to the detection of KSHV sequences in Kaposi’s sarcoma ( h p i n et d, 1995). On this basis, the purpose of this study was to define the precise distribution of KSHV sequences throughout the spectrum of lymphoid neoplasia in Europe.

0 1995 Blackwell Science Ltd

Short Report 9 19 Table I. Distribution of KSHV DNA sequences among lymphoid malignancies in Europe.

Tumour category*

B-cell lineage Precursor B-cell ALL ECLL/small lymphocytic lymphoma Lymphoplasmacytoid lymphoma Mantle cell lymphoma Follicular lymphoma Monocytoid lymphoma MALT lymphoma Splenic marginal zone lymphoma Hairy cell leukaemia Multiple myeloma Diffuse large cell lymphomat

Centroblastic Large cell immunoblastic Body cavity based

Burkitt's lymphoma$ T-cell lineage

T-cell W T-CLL/prolymphocytic leukaemia Peripheral T-cell lymphoma5 Cutaneous T-cell lymphoma

Total cases

KSHV positive/tested

0132 0110

017 0113 011 0116 012

0112

013

015

Of22 012 1 313 0114

*ALL, acute lymphoblastic leukaemia; CLL, chronic lymphocytic leukaemia: MALT, mucosa associated lymphoid tissue.

t Including eight cases associated with HIV infection. *Including five cases associated with HIV infection. 5 Including four cases of CD30' anaplastic large cell lymphoma.

Toward this aim, we tested the presence of KSHV sequences by a polymerase chain reaction (PCR) based assay in a relatively large panel of Bcell and T-cell tumours representative of the major categories defined by the Revised European-American Classification of Lymphoid Neoplasms (Harris et al, 1994; Table I). Peripheral blood, bone marrow aspirates, body cavity effusions, and samples of lymph nodes or other involved organs from 180 patients were collected during standard diagnostic procedures. Patients were referred to institutions within Italy and Spain (i.e. Divisione di Patologia Medica, Ospedale San Luigi, Universita di Torino, for lymphoid malignancies arising in the general population: and Divisione di Anatomia Patologica, C.R.O., Aviano, or Hospital de la Santa Creu i Sant Pau, Barcelona, for lymphoid malignancies in HIV-infected individuals). For most cases the fraction of malignant cells in the pathologic specimen was at least 60%, as determined by cytofluoro- metric analysis of cell surface markers, antigen-receptor gene rearrangement analysis, or both. Genomic DNA was extracted by the 'salting out' method and precipitated with ethanol (Miller et al, 1988). All samples included in the study were subjected to PCR analysis using primers KS330233-F (S'-AGCCGAAAGGATTCCACCAT-3') and KS330233-R (5'- TCCGTGTTGTCTACGTCCAG-3') which amplify a 23 3 bp fragment from the KS330 Barn region of KSHV (Chang et al, 1994). For all cases, control amplifications were performed with primers derived from the K-RAS and/or the p53 exon 6 genomic sequences (Ballerini et al, 1993). PCR was performed with lOOng of genomic DNA, 20 pmol of each primer, 20OpM dNTPs, 10m Tris-HC1 (pH 8.8). 5OmM KCl, 1 m~ MgC12, 0.01% gelatin, 1 U Taq polymerase, in a h a 1 volume of 2 5 ~ 1 . 30 cycles of denaturation (94"C), annealing (58°C for KS330233; 48°C for K-RAS; and 62°C for p53 exon 6), and extension (72°C) were performed in a

Fig 1. PCR analysis of KSHV DNA sequences in representative lymphoid neoplasms. Genomic DNA was subjected to PCR amplification using the KS330233 primers derived from the KSHV sequence and yielding a 233 bp amplimer. The PCR reactions were run onto a 2% agarose gel for visualization by ethidium bromide staining. A positive signal was detected in samples of body-cavity-based lymphoma (cases 816,817 and 8 18). Cases of B-cell lineage acute lymphoblastic leukaemia (cases 195, 281 and 325). EceU chronic lymphmytic leukaemia (case 460). MALT lymphoma (cases 167 and 418), and T-cell acute lymphoblastic leukaemia (cases 324 and 800) scored negative. A positive control (POS), represented by the KS330Bam clone (Chang et al, 1994). and a negative control (NEG), represented by the NC9 B lymphoblastoid cell line, were also included in the analysis. Sizes of molecular markers (MM) are 1230,1033,653, 517,453, 394,298,234 and 154 bp (from top to bottom). Cases 816, 817 and 818 have also been included in Gaidano et al(1995, unpublished).

0 1995 Blackwell Science LM, British Journal of Haematdogy 91: 918-920

920 Short Report temperature controller. The reaction was visualized by ethidium bromide staining on a 2% agarose gel. The identity of the KS330233 amplimer was verified by restriction digest.

Preliminary experiments showed that all 180 cases of lymphoid malignancies included in the study could be amplified with K-RAS and/or p53 exon 6 primers, thus ruling out DNA degradation or the presence of a PCR inhibitor in the samples analysed. The results of the analysis of KSHV sequences are summarized in Table I and representative cases are shown in Fig 1. All cases of precursor B cell neoplasia (i.e. Ecell lineage acute lympho- blastic leukaemia), plasma cell tumours (i.e. multiple myeloma and plasma cell leukaemia), and T-cell malignan- cies scored negative for KSHV sequences. Among neoplasms of mature B cells, detection of KSHV sequences clustered with cases of NHL (3/3) presenting as body cavity effusions and cytologically resembling diffuse large cell lymphoma.

Upon thorough revision of clinical, morphologic and immunophenotypic features, the KSHV positive NHL were found to fulfill the clinico-pathologic definition of body- cavity-based lymphoma (Knowles et al, 1989; Walts et al, 1990; Green et al, 1995). Indeed. as peculiar of body-cavity- based lymphoma, a lymphomatous effusion of the pleura, pericardium or peritoneum in the absence of solid lympho- matous masses represented the exclusive localization of these NHL cases both at diagnosis and throughout their clinical course. The B-cell derivation of these NHL was based upon the expression of mRNA coding for monotypic light chain immunoglobulins in the cell cytoplasm (data not shown). All the KSHV-positive body-cavity-based lympho- mas included in the study had developed in HIV-infected individuals, among whom body-cavity-based lymphomas appear to develop more frequently than in the general population (Green et al, 1995). Since the three body-cavity- based lymphomas analysed constituted of a virtually pure tumour population, it is likely that the detected KSHV sequences were indeed harboured by the tumour clone. Future studies by in situ technologies are needed to clarify this issue.

Overall, this study defines that the distribution of KSHV sequences throughout the spectrum of lymphoid neoplasia is highly selective, being restricted to body-cavity-based lymphomas. Other categories of lymphoid malignancies, arising either in the general population or in HIV-infected individuals, are consistently devoid of KSHV DNA. Together with other biological peculiarities of body-cavity-based lymphomas, namely expression of lymphoid activation antigens, positivity for KSHV DNA sequences may contribute to the clinico-pathologic definition of lymphomas growing in fluid phase (Knowles et d, 1989; Walts et d, 1990; Green et d. 1995). The consistent and selective association of KSHV sequences with body-cavity-based lymphoma suggests a novel genetic pathway in lymphomagenesis, thus further adding to the molecular heterogeneity of lymphoid malig- nancies. Since the development of body-cavity-based lym- phomas is virtually restricted to HIV-infected individuals and lymphoid tumours in the immunocompetent host are consistently devoid of KSHV DNA, it may be postulated that the genetic pathway involving KSHV requires a

profoundly disrupted immune function in order to lead to transformation of lymphoid cells.

ACKNOWLEDGMENTS

This work has been supported by grants from VIII Progetto AIDS, I.S.S.. Rome, Italy (grants 9304-32 and 9306-17). C.P. is being supported by a fellowship from A.I.R.C., Milan, Italy. G.V. is being supported by a fellowship from the Fondazione Piera. Pietro, e Giovanni Ferrero, Alba, Italy. J.N. is supported by the Institut de Recerca de I'Hospital de Sant Pau, Barcelona, Spain.

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0 1995 Blackwell Science Ltd, British Journal ofhlaernatologu 91: 918-920