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Keywords:

  • primary effusion lymphoma cell lines;
  • HHV-8-positive primary effusion lymphoma cell lines;
  • AIDS-related non-Hodgkin's lymphoma;
  • HHV-8 infection;
  • EBV infection

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

In this study we report on the establishment and characterization of two novel lymphoma cell lines (CRO-AP/3 and CRO-AP/5) which carry infection by human herpesvirus type-8 (HHV-8) and have derived from AIDS-related primary effusion lymphoma (PEL). These two cell lines are representative of different virologic subtypes of PEL, i.e. HHV-8+/EBV PEL in the case of CRO-AP/3 and HHV-8+/EBV+ PEL in the case of CRO-AP/5. Consistent with the diagnosis of PEL, both CRO-AP/3 and CRO-AP/5 expressed indeterminate (i.e. non-B, non-T) phenotypes although immunogenotypic studies documented their B-cell origin. Both cell lines are devoid of genetic lesions of c-MYC, BCL-2 and p53 as well as gross rearrangements of BCL-6. Detailed histogenetic characterization of these novel PEL cell lines suggests that PEL may derive from a post-germinal centre B cell which has undergone pre-terminal differentiation. The CRO-AP/3 and CRO-AP/5 cell lines may provide a valuable model for clarifying the pathogenesis of PEL. In particular, these cell lines may help understand the relative contribution of HHV-8 and EBV to PEL growth and development and may facilitate the identification of recurrent cytogenetic abnormalities highlighting putative novel cancer related loci relevant to PEL.

Primary effusion lymphoma (PEL) represents a peculiar lymphoma entity which consistently harbours infection by human herpesvirus type-8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus [KSHV]) ( Cesarman et al, 1995 ; Carbone & Gaidano, 1997). At the clinico-pathological level, PEL is characterized by liquid growth in the serous body cavities associated with spreading along the serous membranes without infiltrative or destructive growth patterns ( Komanduri et al, 1996 ; Carbone et al, 1997a ; Morassut et al, 1997 ). Typically, identifiable tumour masses are absent throughout the clinical course of PEL ( Carbone et al, 1996 ; Nador et al, 1996 ). Morphologically, PEL bridges immunoblastic and anaplastic features and frequently displays a certain degree of plasmacell differentiation (Carbone et al, 1996; Nador et al, 1996 ; Gaidano et al, 1997b ). Although PEL usually displays an indeterminate (non-B, non-T) phenotype, immunogenotypic studies have unequivocally defined the B-cell origin of this lymphoma ( Cesarman et al, 1995 ; Carbone & Gaidano, 1997).

Despite the progressive accumulation of a certain body of evidence regarding the biology of PEL, several issues remain to be answered. For example, the precise histogenetic derivation of PEL remains unclear, as well as the detailed reasons for its peculiar growth pattern. Also, studies aimed at defining the relative pathogenetic contribution of HHV-8, which consistently infects PEL, and of Epstein-Barr virus (EBV), which frequently infects PEL, have been hampered by the rarity of EBV negative PEL models for in vitro studies ( Arvanitakis et al, 1996 ; Komanduri et al, 1996 ; Boshoff et al, 1998 ). As derived from the example of other non-Hodgkin's lymphoma (NHL) categories, novel insights into the biology of PEL may be gained from the establishment and subsequent characterization of PEL cell lines, providing an inexhaustible source of biological material amenable to multiple investigations.

Here we report the establishment and characterization of two novel PEL cell lines. The two cell lines are derived from HIV-infected individuals and are representative of different virologic subtypes of PEL, namely HHV-8+/EBV and HHV-8+/EBV+. Detailed immunophenotypic and molecular characterization of these novel PEL cell lines indicate that PEL may be histogenetically related to a post-germinal centre B cell which has undergone pre-terminal differentiation. These PEL cell lines may provide a valuable model for investigating PEL pathogenesis and behaviour in vitro and in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patients

The CRO-AP/3 and CRO-AP/5 PEL cell lines were derived from two distinct HIV-infected patients, defined as case 1 and case 2, respectively. Selected clinical characteristics of both patients have been included in a previous report ( Spina et al, 1998 ).

Case 1 was a 42-year-old HIV-positive homosexual male without a previous history of Kaposi's sarcoma. At the clinical level, the lymphoma presented exclusively as a malignant peritoneal effusion.

Case 2 was a 35-year-old HIV-positive homosexual male with a previous history of Kaposi's sarcoma. At the clinical level, the lymphoma presented exclusively as a malignant pleural effusion.

The assignment of both cases to PEL was based on multiple clinico-pathological and biological criteria as reported previously (reviewed by Jaffe, 1996; Carbone & Gaidano, 1997).

Pathological samples

Samples of lymphomatous effusion were collected under sterile conditions during standard diagnostic procedures. After centrifugation, the effusion sediments were used to prepare smears and cell blocks. For morphological studies, cytospin preparations were routinely fixed and stained by the Papanicolaou method. For cell block preparation, cell pellets were Bouin fixed and treated according to a standard regimen used at the Division of Pathology of the Centro di Riferimento Oncologico. The sectioned material from cell blocks was stained with haematoxylin and eosin.

Establishment of cell lines and cell culture procedures

Lymphoma cells obtained from ascitic fluid (case 1) and pleural effusion (case 2) were separated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and washed twice in sterile phosphate-buffered saline. Subsequently, cells were cultured in RPMI 1640 (GIBCO, Paisley, Scotland) supplemented with 20% heat-inactivated fetal bovine serum (FBS, ICN Biomedicals Inc., Costa Mesa, Calif.), 2 m ML-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Irvine Scientific, Santa Ana, Calif.) at 37°C in the presence of 5% CO2. After 3 weeks (case 1; CRO-AP/3) or 6 weeks (case 2; CRO-AP/5) the cells started to grow spontaneously. Cultures were fed twice weekly with the above-mentioned medium. Once established (defined as in vitro growth for more than 40 passages), the cell lines were continuously cultured in RPMI 1640–10% FBS at 37°C in the presence of 5% CO2.

Immunophenotypic analysis

The immunophenotype of PEL primary samples and derived cell lines was determined either by immunocyto-histochemistry (on cytospins and cell blocks) or by direct and indirect immunofluorescent flow cytometry using the FACScan fluorescent activated cell sorter (Becton Dickinson, Mountain View, Calif.) as previously described ( Carbone et al, 1996, 1997b). The monoclonal antibodies (MoAb) used in this study (together with their sources) are listed in Table I. A phenotypic analysis of EBV latent gene expression (latent membrane protein 1 [LMP 1] and EBV-encoded nuclear antigen 2 [EBNA 2]) was also performed on cytospins and cell blocks, as previously reported ( Carbone et al, 1997b ).

Table 1. Table I. Immunophenotype of PEL cell lines and corresponding primary tumour samples. −, negative; +, positive < 25%; ++, positive 25–50%; +++, positive 50–75%; ++++, positive > 75%. The following B-cell and T-cell associated markers were consistently negative: k (A8B5/D), λ (TB28-2/BD), IgM (R1/69/D), IgD (IgD26/D), IgG (A57H/Ser), CD10 (W8E7/BD), CD19 (Leu12/BD), CD22 (HD39/BOE), CD40 (89/JB), CD45RA (MB1/CB), CD79a (JCB117/D), CD3 (Leu4/BD), CD5 (Leu1/BD).* D, Dakopatts A/S (Glostrup, Denmark); BD, Becton Dickinson (Mountain View, Calif.); Ser, Serotec (Serotec, Oxford, U.K.); CB, Clonab Biotest (Dreiech, Germany); BS, The Binding Site (Birmingham, U.K.); V WS, V Workshop on Leukocyte Typing; BOE, Boehringer (Mannheim, Germany); JB, Dr J. Banchereau (Centre de Reserche, Schering-Plough, Dardilly, France); IT, Immunotech (Marseille, France).Thumbnail image of

In order to better define the stage of B-cell maturation of PEL and to gain better insights into the biological basis of PEL growth pattern, the PEL-derived cell lines and primary samples were investigated by immunocytochemistry for the expression of molecules selectively associated with late stages of B-cell differentiation (see Table II) as well as molecules implicated in cell-to-cell and cell-to-extracellular matrix (ECM) interactions (see Table III).

Table 2. Table II. Plasma-cell associated antigens in PEL cell lines and corresponding primary tumour samples. −, negative; +, positive < 25%; ++, positive 25–50%; +++, positive 50–75%; ++++, positive > 75%.* Ser, Serotec (Oxford, U.K.); D, Dakopatts A/S (Glostrup, Denmark); VI WS, VI Workshop on Leukocyte Typing.Thumbnail image of
Table 3. Table III. Adhesion molecules in PEL cell lines and the corresponding primary tumour samples. −, negative; +, positive < 25%; ++, positive 25–50%; +++, positive 50–75%; ++++, positive > 75%.* TCS, T-Cell Sciences Inc. (Cambridge, Mass.); D, Dakopatts A/S (Glostrup, Denmark); AS, A. Sonnenberg (NKI, Amsterdam, The Netherlands); BD, Becton Dickinson (Mountain View, Calif.); Ser, Serotec (Oxford, U.K.); IT, Immunotech (Marseille, France); Mon, Monosan (Uden, The Netherlands); JTA, J. Thomas August (The Johns Hopkins University School of Medicine, Baltimore, Md.).Thumbnail image of

Cytogenetic analysis

Chromosomal studies were performed on both cell lines (CRO-AP/3 and CRO-AP/5) by the trypsin-Giemsa banding method. Well-spread metaphases were photographed and arranged according to the recommendation of an International System for Human Cytogenetic Nomenclature ( ISCN, 1995) for cancer cytogenetics. Seven to 15 karyotypes per cell line were prepared.

As a first step, conventional cytogenetic analysis was performed on both cell lines. Subsequently, to assess doubtful findings, FISH analysis of metaphase spreads was performed using biotinylated DNA-painting probes according to the method of Pinkel et al (1986 ).

Cytogenetic analysis of PEL parental samples could not be performed due to the lack of sufficient numbers of metaphase spreads.

DNA extraction and immunogenotypic analysis

Genomic DNA was prepared by cell lysis and proteinase K digestion, followed by ‘salting out’ extraction and ethanol precipitation ( Miller et al, 1988 ). In order to establish the clonality of the CRO-AP/3 and CRO-AP/5 PEL cell lines, we performed immunoglobulin (Ig) heavy and light chain gene rearrangement analysis by Southern hybridization as previously described ( Ballerini et al, 1993 ; Volpe et al, 1996 ). The configuration of the JH locus was investigated by Southern hybridization of a human JH-specific probe to genomic DNA digested with EcoRI, HindIII and BamHI ( Ballerini et al, 1993 ). The configuration of the Jκ locus was investigated by hybridization of BamHI digested DNA to a Jκ-specific DNA probe ( Ballerini et al, 1993 ).

Analysis of viral infection

The presence of HHV-8 infection was assessed by multiple approaches, including PCR and Southern blot analysis of genomic DNA. PCR was performed with primers KS330233-F (5′-AGCCGAAAGGATTCCACCAT-3′) and KS330233-R (5′-TCCGTGTTGTCTACGTCCAG-3′) as previously reported ( Gaidano et al, 1996 ). Briefly, PCR was performed with 100 ng genomic DNA, 25 pmol of each primer, 200 μmol/l dNTPs, 10 mmol/l Tris-HCl (pH 8.8), 50 mmol/l KCl, 1 mmol/l MgCl2, 0.01% gelatin, 1 U Taq polymerase (Perkin-Elmer, Norwalk, Ct., U.S.A.), in a final volume of 25 μl. 35 cycles of denaturation (94°C), annealing (58°C) and extension (72°C) were performed in a Hybaid Omn-E thermocycler. For confirmatory purposes, samples scored positive by PCR were further tested by Southern blot hybridization of BamHI digested genomic DNA to the radiolabelled probe KS631Bam ( Chang et al, 1994 ). The HHV-8 viral load was assessed by semiquantitative PCR analysis performed according to a previously reported method ( Carbone et al, 1996 ).

Infection by EBV was investigated by multiple approaches, including PCR and Southern blot analysis of genomic DNA ( Gaidano et al, 1996 ). Analysis of EBV by PCR was performed with primers SL-1 (5′-GGACCTCAAAGAAGAGGGGG-3′) and SL-3 (5′-GCTCCTGGTCTTCCGCCTCC-3′), representative of the EBV nuclear antigen (EBNA)-1 gene. 35 cycles of PCR were performed as described above (annealing temperature 55°C). Southern blot analysis was performed with a probe representative of the EBV genomic termini (5.2 kb BamHI-EcoRI fragment isolated from the fused BamHI terminal fragment NJ-het) which, upon BamHI digestion, enables the definition of the clonality status of EBV infection ( Weiss et al, 1987 ).

The presence of HIV infection in the PEL cell lines and in the parental tumour samples was assessed by PCR analysis using primers derived from a highly conserved DNA sequence of the HIV clone HXB2, as previously reported ( Simmonds et al, 1990 ). Primers amplified a portion of the gp120 gene and their sequence was as follows: V3(a), 5′-TACAATGTACACATGGAATT-3′, and V3(d), 5′-ATTACAGTAGAAAAATTCCCC-3′. 30 cycles of PCR were performed as described above (annealing temperature 50°C).

Analysis of genetic lesions

Gross rearrangements of BCL-2, BCL-6 and c-MYC, as well as mutations of p53, c-MYC first exon–first intron border, and BCL-6 5′ noncoding regions were investigated by a combination of molecular approaches as previously reported ( Ballerini et al, 1993 ; Volpe et al, 1996 ; Gaidano et al, 1997a ).

Southern blot analysis of gross rearrangements of BCL-2, BCL-6 and c-MYC was performed with the following molecular probes: pFL-1, pFL-2 and pB16 (for BCL-2) ( Cleary et al, 1986 ; Tsujimoto & Croce, 1986); Sac4.0 and Sac0.8 (for BCL-6) ( Ye et al, 1993 ; Gaidano et al, 1994 ); MC413RC (for c-MYC) ( Dalla-Favera et al, 1982 ).

Mutations of p53, c-MYC first exon–first intron border, and BCL-6 5′ non-coding regions were investigated by a two step approach, including PCR–Single Strand Conformation Polymorphism (PCR-SSCP) followed by DNA direct sequencing analysis of positive samples ( Gaidano et al, 1991 , 1997a; Ballerini et al, 1993 ). Briefly, PCR-SSCP was performed with 100 ng genomic DNA, 10 pmol of each primer, 2.5 μmol/l dNTPs, 37 kBq of [α-32P]dCTP (Amersham, U.K.; specific activity, 111 TBq/mmol), 10 mmol/l Tris-HCl (pH 8.8), 50 mmol/l KCl, 1 mmol/l MgCl2, 0.01% gelatin, 0.5 U Taq polymerase (Perkin-Elmer, Norwalk, Ct., U.S.A.), in a final volume of 10 μl. 30 cycles of denaturation (94°C), annealing (annealing temperatures were optimized for each pair of primers), and extension (72°C) were performed in a Hybaid Omn-E thermocycler. Samples were heated at 95°C for 5 min, chilled on ice, and immediately loaded (3 μl) onto a 6% acrylamide-TBE gel containing 10% glycerol. The sequence of oligonucleotides corresponding to p53 exons 5–9 was as follows ( Gaidano et al, 1991 ): p5-5, 5′-TTCCTCTTCCTGCAGTACTC-3′, and P5-3, 5′-ACCCTGGGCAACCAGCCCTGT-3′ (for p53 exon 5); P6-5, 5′-ACAGGGCTGGTTGCCCAGGGT-3′, and P6-3, 5′-AGTTGCAAACCAGACCTCAG-3′ (for p53 exon 6); P7-5, 5′-GTGTTGTCTCCTAGGTTGGC-3′, and P7-3, 5′-GTCAGAGGCAAGCAGAGGCT-3′ (for p53 exon 7); P8-5, 5′-TATCCTGAGTAGTGGTAATC-3′, and P8-3, 5′-AAGTGAATCTGAGGCATAAC-3′ (for p53 exon 8); P9-5, 5′-GCAGTTATGCCTCAGATTCAC-3′, and P9-3, 5′-AAGACTTAGTACC-

TGAAGGGT-3′ (for p53 exon 9). The sequence of oligonucleotides corresponding to c-MYC first exon-first intron border (fragment F and fragment G) was as follows ( Ballerini et al, 1993 ): F5′, 5′-GCACTGGAACTTACAACACC-3′, and F3′, 5′-GGTGCTTACCTGGTTTTCCA-3′ (for fragment F); G5′, 5′-CTGCCAGGACCCGCTTCTCT-3′, and G3′, 5′-TTTACCCCGATCCAGTTCTG-3′ (for fragment G). The sequence of oligonucleotides corresponding to BCL-6 5′ non-coding regions (fragments E1.10, E1.11 and E1.12) was as follows ( Migliazza et al, 1995 ): E1.21B, 5′-CTCTTGCCAAATGCTTTG-3′, and E1.24, 5′-TAATTCCCCTCCTTCCTC-3′ (for fragment E1.10); E1.23, 5′-AGGAAGGAGGGGAATTAG-3′, and IP1.6, 5′-AAGCAGTTTGCAAGCGAG-3′ (for fragment E1.11); IP1.7, 5′-TTCTCGCTTGCAAACTGC-3′, and E1.26, 5′-CACGATACTTCATCTCATC-3′ (for fragment E1.12).

For DNA sequencing of BCL-6 5′ non-coding regions, a unique PCR product encompassing fragments E1.10, E1.11 and E1.12 (nucleotides +404 to +1142) was amplified by primers E1.21B and E1.26. DNA direct sequencing of the amplified PCR fragment was performed with appropriate primers using a commercially available kit (ThermoSequenase, Amersham Life Sciences, Amersham, U.K.). α-33P-labelled terminator dideoxynucleotides were included in the sequencing mixture. Both strands were sequenced for each DNA fragment analysed.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Establishment of cell lines

The PEL cell lines CRO-AP/3 and CRO-AP/5 have been established from two patients with NHL presenting as primary lymphomatous effusions. Based on morphological, immunophenotypic and genotypic data (see below), the two NHLs were classified as AIDS-PEL. Both cell lines have been grown for > 40 passages.

Morphological and immunophenotypic features

Conventional morphological studies on cytospin preparations and paraffin-embedded sections from cell blocks obtained from the CRO-AP/3 and CRO-AP/5 cell lines revealed cell pleomorphism and heterogeneity in nuclear size and shape. The spectrum of the morphological features of CRO-AP/3 and CRO-AP/5 extended from immunoblast-like cells with plasmacytoid features, to anaplastic, multilobated or multinucleated large cells resembling Reed-Sternberg cells of Hodgkin's disease ( Figs 1 and 2).

image

Figure 1. Fig 1. CRO-AP/3 cell line derived from an AIDS-related HHV-8+/EBV primary effusion lymphoma. Tumour cells display pleomorphism and heterogeneity in nuclear shape. A giant cell, resembling the Reed-Sternberg cell of Hodgkin's disease, is also present. Cytospin preparation, haematoxylin–eosin stain. (Original magnification × 250.)

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Table I shows the results of the comparative immunophenotypic analysis performed on CRO-AP/3 and CRO-AP/5 and the parental primary samples from which they were derived.

CRO-AP/3 and CRO-AP/5 and the corresponding parental samples displayed superimposable phenotypic features. Tumour cells exhibited an indeterminate immunophenotype, since they lacked surface Ig and B- and T-cell-associated antigens. Conversely, both cell lines and the parental tumour samples expressed leucocyte common antigen (LCA) and various markers associated with activation, including CD30, CD38 and EMA.

Expression of plasma cell associated markers

CRO-AP/3 and CRO-AP/5, as well as the corresponding parental primary samples, were analysed with a panel of plasma cell associated markers ( Table II). As a first step, the CD138/syndecan-1 (CD138/syn-1) antigen was tested with several antibodies (B-B4, B-B2, MI15, 1D4) recognizing different epitopes of the CD138/syn-1 molecule. As expected ( Gaidano et al, 1997b ), both CRO-AP/3 and CRO-AP/5, as well as the corresponding parental samples, scored positive for CD138/syn-1 when stained with the B-B4 MoAb. Conversely, only CRO-AP/3 and case 1, but not CRO-AP/5 and case 2, stained positive with anti-CD138/syn-1 MoAb recognizing different epitopes (B-B2, MI15, 1D4). These data suggest a certain degree of heterogeneity in the expression pattern of CD138/syn-1 in different PEL patients.

Subsequently, CRO-AP/3 and CRO-AP/5, as well as the corresponding parental tumour samples, were investigated with several MoAb recognizing plasma cell associated antigens other than CD138/syn-1 (VS38c, MC186, MI37). CRO-AP/3 and case 1 scored positive for all markers tested, whereas CRO-AP/5 and case 2 scored positive only for the MI37 marker.

Expression of adhesion molecules

CRO-AP/3 and CRO-AP/5, as well as the corresponding parental primary samples, were tested for selected adhesion molecules known to mediate cell-to-cell and cell-to-ECM interactions. The results are summarized in Table III. With one exception, the PEL cell lines CRO-AP/3 and CRO-AP/5, as well as the corresponding parental samples, displayed a superimposable profile of adhesion molecules characterized by positive expression of α4β1, α6β1, LFA-1, LFA-3, ICAM-1 and H-CAM complexes. The αvβ5 integrin scored positive in case 1 and CRO-AP/3 but negative in case 2 and CRO-AP/5.

Cytogenetic characteristics

Fifteen metaphase spreads of CRO-AP/3 and CRO-AP/5 were evaluated for chromosome number and structure and 10 karyotypes were prepared (data not shown). According to the ISCN (1995), the complete karyotype of CRO-AP/3 was: 47, XY, inv(1)(p31q21), ?add(3)(q13), add(4)(p16), del(5)(p13p15), del(5)(q15q31), ?der(7) t(7;12;?) (7q ter[RIGHTWARDS ARROW] 7p22::12q11[RIGHTWARDS ARROW]12q21::?), ?del(9)(q21q31), del(11)(q13q22), t(14;19)(q13;?q13), del(18)(q21), add(22)(q13), +mar.

The complete karyotype of CRO-AP/5 was 49, X, −Y, del(4)(q24), der(12) dup (12)(q13q?15) add(12)(q24), del(14)(q24), + 4 mar.

These findings were confirmed by FISH analysis (data not shown).

Clonal analyis

Immunogenotypic studies of the JH and Jκ loci demonstrated that CRO-AP/3 and CRO-AP/5 display a monoclonal rearrangement of the Ig genes ( Table IV and Fig 3 ). For each cell line an identical pattern of Ig gene rearrangement was observed in the corresponding parental tumour sample. These data demonstrated the monoclonal B-cell origin of these cell lines despite the fact that CRO-AP/3 and CRO-AP/5 failed to express the most common B-cell-associated surface antigens.

Table 4. Table IV. Genetic lesions and viral infection in PEL cell lines and the corresponding primary tumour samples. Abbreviations: Ig, immuoglobulin; HHV-8, human herpesvirus type-8; EBV, Epstein-Barr virus; HIV, human immunodeficiency virus. +, positive; −, negative. Copy number of HHV-8 sequences are given in parentheses.* As assessed by multiple approaches, including PCR and Southern blot analysis of genomic DNA.Thumbnail image of
image

Figure 3. and case 1 were both negative for EBV infection, whereas an identically sized band of EBV infection, consistent with monoclonal infection, is detectable in CRO-AP/5 and case 2.

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Viral infection

Both CRO-AP/3 and CRO-AP/5 and their parental tumour samples scored positive for HHV-8 infection ( Table IV and Fig 4). The approximate copy number of HHV-8 sequences was 15–30 copies of HHV-8 DNA per cell in the case of CRO-AP/3, and 30–60 copies of HHV-8 DNA per cell in the case of CRO-AP/5 (data not shown). The HHV-8 DNA copy number detected in CRO-AP/3 and CRO-AP/5 was superimposable to that detected in the corresponding parental tumour samples ( Table IV).

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Figure 4. Fig 4. Polymerase chain reaction (PCR) analysis of infection by HHV-8 in PEL cell lines CRO-AP/3 and CRO-AP/5 and in parental tumour samples (case 1 and case 2, respectively). PCR reactions were run onto a 2% agarose gel for visualization by ethidium bromide. Positive (POS) and negative (NEG) controls were included in all experiments. All cell lines and parental tumour samples were positive, yielding a 233 base-pair amplimer.

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Infection by EBV scored positive in CRO-AP/5 and in the corresponding parental sample, whereas it was negative in CRO-AP/3 and case 1 ( Table IV; Fig 3). By Southern blot analysis, CRO-AP/5 and its parental tumour sample displayed a unique band of EBV infection, consistent with a monoclonal pattern of infection (Fig 3 ). Analysis of EBV latent gene expression revealed that case 2 harboured small numbers of tumour cells which expressed LMP 1, but not EBNA 2 (latency II), whereas tumour cells from CRO-AP/5 lacked both proteins ( Table I).

Infection of the tumour clone by HIV was negative in both CRO-AP/3 and CRO-AP/5, as well as in the corresponding parental tumour samples ( Table IV).

Genetic lesions of proto-oncogenes and tumour suppressor genes

Molecular studies (summarized in Table IV) showed that the CRO-AP/3 and CRO-AP/5 cell lines, as well as the parental tumours from which they were derived, were devoid of gross rearrangements of c-MYC, BCL-2 and BCL-6. No p53 mutations were detected in the cell lines or their respective parental tumours. Conversely, both CRO-AP/3 and CRO-AP/5, as well as their parental tumours, harboured mutations of the 5′ noncoding region of BCL-6. The detailed characterization of BCL -6 5′ mutations of CRO-AP/3 and CRO-AP/5 is reported in Table V.

Table 5. Table V. Characteristics of mutations of BCL-6 5′ non-coding regions in PEL cell lines CRO-AP/3 and CRO-AP/5. * The position of the mutated nucleotide is given in parentheses.Thumbnail image of

Classification of cell lines

Based on morphological, immunophenotypic, virological and genetic features, CRO-AP/3 and CRO-AP/5 are representative of AIDS-related PEL ( Cesarman et al, 1995 ; Nador et al, 1996 ; Carbone & Gaidano, 1997). CRO-AP/3 and CRO-AP/5 accurately represent the parental tumour clones, based on the results of phenotypic analysis as well as the identity of the pattern of Ig gene rearrangements detected in cell lines and parental samples.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

In this report we have established two novel HHV-8+ lymphoma cell lines, termed CRO-AP/3 and CRO-AP/5. The definition of these cell lines as AIDS-related PEL, as well as the accurate representation of the original tumour clone, was based on the results of our multiparameter investigation which included analysis of morphology, immunophenotype, karyotype and molecular profile of genetic lesions and viral infection. The two cell lines are representative of two distinct virologic subtypes of PEL, i.e. HHV-8+/EBV PEL (CRO-AP/3) and HHV-8+/EBV+ PEL (CRO-AP/5).

The PEL cell lines established in this study may provide a relevant tool to address several issues which concern the nature of the disease. As an example, results derived from our characterization of the CRO-AP/3 and CRO-AP/5 cell lines may help to define the precise stage of B-cell differentiation reflected by PEL and may clarify the histogenesis of this lymphoma. In this respect, it is notable that both CRO-AP/3 and CRO-AP/5 harbour mutations of BCL-6 5′ noncoding regions, which occur frequently among B-cell malignancies that are histogenetically related to the germinal centre (GC) ( Gaidano et al, 1997a ; Migliazza et al, 1995 ). The association of PEL with BCL-6 mutations indicate that this lymphoma may derive from a B-cell subset which has transited through the GC.

Another issue regarding PEL histogenesis is based on the consistent expression of CD138/syn-1 by PEL cells, as documented by this and other studies ( Carbone & Gaidano, 1997; Gaidano et al, 1997b ). Since CD138/syn-1 selectively stains terminal B-cell differentiation ( Wijdenes et al, 1996 ), it is conceivable that PEL reflects a post-GC B cell which has undergone pre-terminal differentiation. Intriguingly, however, our data suggest a certain degree of heterogeneity in the differentiation stage of different PEL cases. In fact, plasmacell-associated markers other than CD138/syn-1, such as the VS38c antigen, stained positive in the HHV8+/EBV cell line CRO-AP/3 whereas they stained negative in the HHV8+/EBV+ CRO-AP/5 cell line. Preliminary data on a larger number of cases confirm such heterogeneity among PEL and indicate that it may be related to the status of EBV infection of the tumour (unpublished observations). Furthermore, the results of our analysis of the CD138/syn-1 molecule with numerous antibodies recognizing different epitopes of the antigen are consistent with a certain degree of heterogeneity in the biochemical structure of CD138/syn-1 in different PEL cases.

Independent of histogenetic studies, other applications may also be suitable for the CRO-AP/3 and CRO-AP/5 cell lines. In particular, since CRO-AP/3 and CRO-AP/5 are representative of distinct virologic subtypes of PEL, i.e. HHV8+/EBV+ and HHV8+/EBV, these cell lines may be helpful in detailing the relative role of HHV-8 and of EBV in PEL pathogenesis and progression both in vitro and in vivo. Results derived from this study suggest that the role of EBV, if any, does not markedly influence the adhesion properties of PEL cells. In fact, CRO-AP/3 and CRO-AP/5 share a similar pattern of integrin expression when considering the adhesion molecules tested in this study. The only exception is represented by the αvβ5 complex, which is expressed by the EBV CRO-AP/3 cell line, but not by the EBV+ CRO-AP/5 cell line. Future studies on larger panels of cases will define whether αvβ5 expression in PEL is modulated by EBV infection.

Another potential application of the CRO-AP/3 and CRO-AP/5 cell lines is the putative identification of subtle cytogenetic abnormalities, which may be specifically and recurrently associated with PEL and which may be visualized only by advanced cytogenetic techniques requiring large numbers of actively dividing cells. The identification of such cytogenetic abnormalities is expected to play a pivotal role in the definition of the precise molecular pathway leading to PEL pathogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This work was supported by ISS, Programma nazionale di ricerca sull'AIDS 1997–Progetto Patologia clinica e terapia dell'AIDS (no. 30A.0.10 and no. 30A.0.62), Rome, Italy, and by Fondazione CRT, Torino, Italy. D.C. and S.Q. are supported by a fellowship from Fondazione Piera, Pietro e Giovanni Ferrero, Alba, Italy.

The MoAb CD40/89 was kindly provided by Dr J. Banchereau, Centre de Recherche Schering-Plough, Dardilly, France. The MoAb CD44/H4C4 was kindly provided by Dr J. Thomas August, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

The authors thank Fulvio Coletto for photographic assistance.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Arvanitakis, L., Mesri, E.A., Nador, R.G., Said, J.W., Asch, A.S., Knowles, D.M., Cesarman, E. (1996) Establishment and characterization of primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus. Blood, 88, 2648 2654.
  • 2
    Ballerini, P., Gaidano, G., Gong, J.Z., Tassi, V., Saglio, G., Knowles, D.M., Dalla-Favera, R. (1993) Multiple genetic lesions in acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma. Blood, 81, 166 176.
  • 3
    Boshoff, C., Gao, S.-J., Healy, L.E., Matthews, S., Thomas, A.S., Coignet, L., Warnke, R.A., Strauchen, J.A., Matutes, E., Kamel, O.W., Moore, P.S., Weiss, R.A., Chang, Y. (1998) Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice. Blood, 91, 1671 1679.
  • 4
    Carbone, A., Cilia, A.M., Gloghini, A., Canzonieri, V., Pastore, C., Todesco, M., Cozzi, M., Perin, T., Volpe, R., Pinto, A., Gaidano, G. (1997a) Establishment of HHV-8 positive and HHV-8 negative lymphoma cell lines from primary lymphomatous effusions. International Journal of Cancer, 73, 562 569.
  • 5
    Carbone, A. & Gaidano, G. (1997) HHV-8 positive body cavity-based lymphoma: a novel lymphoma entity. British Journal of Haematology, 97, 515 522.
  • 6
    Carbone, A., Gaidano, G., Gloghini, A., Pastore, C., Saglio, G., Tirelli, U., Dalla-Favera, R., Falini, B. (1997b) BCL-6 protein expression in AIDS-related non-Hodgkin's lymphomas: inverse relationship with Epstein-Barr virus-encoded latent membrane protein-1 expression. American Journal of Pathology, 150, 155 165.
  • 7
    Carbone, A., Gloghini, A., Vaccher, E., Zagonel, V., Pastore, C., Dalla Palma, C., Branz, F., Saglio, G., Volpe, R., Tirelli, U., Gaidano, G. (1996) Kaposi's sarcoma-associated herpesvirus DNA sequences in AIDS-related and AIDS-unrelated lymphomatous effusions. British Journal of Haematology, 94, 533 543.
  • 8
    Cesarman, E., Chang, Y., Moore, P.S., Said, J.W., Knowles, D.M. (1995) Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body cavity-based lymphomas. New England Journal of Medicine, 332, 1186 1191.
  • 9
    Chang, Y., Cesarman, E., Pessin, M.S., Lee, F., Culpepper, J., Knowles, D.M., Moore, P.S. (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science, 266, 1865 1869.
  • 10
    Cleary, M.L., Galili, N., Sklar, J. (1986) Detection of a second t(14;18) breakpoint cluster region in human follicular lymphomas. Journal of Experimental Medicine, 164, 315 320.
  • 11
    Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D., Gallo, R.C., Croce, C.M. (1982) Human c-myc oncogene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proceedings of the National Academy of Sciences of the United States of America, 79, 7824 7827.
  • 12
    Gaidano, G., Ballerini, P., Gong, J.Z., Inghirami, G., Neri, A., Newcomb, E.W., Magrath, I.T., Knowles, D.M., Dalla-Favera, R. (1991) p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America, 88, 5413 5417.
  • 13
    Gaidano, G., Carbone, A., Pastore, C., Capello, D., Migliazza, A., Gloghini, A., Roncella, S., Ferrarini, M., Saglio, G., Dalla-Favera, R. (1997a) Frequent mutation of the 5′ noncoding region of the BCL-6 gene in acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas . Blood, 89, 3755 3762.
  • 14
    Gaidano, G., Gloghini, A., Gattei, V., Rossi, M.F., Cilia, A.M., Godeas, C., Degan, M., Perin, T., Canzonieri, V., Aldinucci, D., Saglio, G., Carbone, A., Pinto, A. (1997b) Association of KSHV positive primary effusion lymphoma with expression of the CD138/syndecan-1 antigen. Blood, 90, 4894 4900.
  • 15
    Gaidano, G., Lo Coco, F., Ye, B.H., Shibata, D., Levine, A.M., Knowles, D.M., Dalla-Favera, R. (1994) Rearrangements of the BCL-6 gene in acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas: association with diffuse large cell subtype . Blood, 84, 397 402.
  • 16
    Gaidano, G., Pastore, C., Gloghini, A., Cusini, M., Nomdedéu, J., Volpe, G., Capello, D., Vaccher, E., Bordes, R., Tirelli, U., Saglio, G., Carbone, A. (1996) Distribution of human herpesvirus-8 sequences throughout the spectrum of AIDS-related neoplasia. AIDS, 10, 941 949.
  • 17
    ISCN (1995) An International System for Human Cytogenetic Nomenclature (ed. by F. Mitelman). Karger, Basel.
  • 18
    Jaffe, E.S. (1996) Primary body-cavity-based AIDS-related lymphomas: evolution of a new disease entity. American Journal of Clinical Pathology, 105, 141 143.
  • 19
    Komanduri, K.V., Luce, J.A., McGrath, M.S., Herndier, B.G., Ng, V.L. (1996) The natural history and molecular heterogeneity of HIV-associated primary malignant lymphomatous effusions. Journal of Acquired Immune Deficiency Syndrome, 13, 215 226.
  • 20
    Migliazza, A., Martinotti, S., Chen, W., Fusco, C., Ye, B.Y., Knowles, D.M., Offit, K., Chaganti, R.S.K., Dalla-Favera, R. (1995) Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma . Proceedings of the National Academy of Sciences of the United States of America, 92, 12520 12524.
  • 21
    Miller, S.A., Dykes, D.D., Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16, 1215.
  • 22
    Morassut, S., Vaccher, E., Balestreri, L., Gloghini, A., Gaidano, G., Volpe, R., Tirelli, U., Carbone, A. (1997) HIV-associated HHV-8 positive primary lymphomatous effusions: radiological findings in six patients. Radiology, 205, 459 463.
  • 23
    Nador, R.G., Cesarman, E., Chadburn, A., Dawson, D.B., Ansari, M.Q., Said, J.W., Knowles, D.M. (1996) Primary effusion lymphoma: a distinct clinicopathologic entity associated with the Kaposi's sarcoma-associated herpes virus. Blood, 88, 645 656.
  • 24
    Pinkel, D., Straume, T., Gray, J.W. (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proceedings of the National Academy of Sciences of the United States of America, 83, 2934 2938.
  • 25
    Simmonds, P., Balfe, P., Ludlam, C.A., Bishop, J.O., Leigh Brown, A.J. (1990) Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1. Journal of Virology, 64, 5840 5850.
  • 26
    Spina, M., Gaidano, G., Carbone, A., Capello, D., Tirelli, U. (1998) Highly active antiretroviral therapy in HHV-8 related body-cavity-based lymphoma. AIDS, 12, 955 956.
  • 27
    Tsujimoto, Y. & Croce, C.M. (1986) Analysis of the structure, transcripts and protein products of BCL-2, the gene involved in human follicular lymphoma. Proceedings of the National Academy of Sciences of the United States of America, 83, 5214 5218.
  • 28
    Volpe, G., Vitolo, U., Carbone, A., Pastore, C., Bertini, M., Botto, B., Audisio, E., Freilone, R., Novero, D., Cappia, S., De Giuli, P., Mazza, U., Resegotti, L., Palestro, G., Saglio, G., Gaidano, G. (1996) Molecular heterogeneity of B-lineage diffuse large cell lymphoma. Genes, Chromosomes and Cancer, 16, 21 30.
  • 29
    Weiss, L.M., Strickler, J., Warnke, R., Purtilo, D.T., Sklar, J. (1987) Epstein-Barr virus DNA in tissues of Hodgkin's disease. American Journal of Pathology, 129, 86 91.
  • 30
    Wijdenes, J., Voojis, W.C., Clément, C., Post, J., Morard, P., Vita, N., Laurent, P., Sun, R.-X., Klein, B., Dore, J.-M. (1996) A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1. British Journal of Haematology, 94, 318 323.
  • 31
    Ye, B.H., Rao, P.H., Chaganti, R.S.K., Dalla-Favera, R. (1993) Cloning of bcl-6, the locus involved in chromosome translocations affecting band 3q27 in B-cell lymphoma . Cancer Research, 53, 2732 2735.