Lymphomas with testicular localisation show a consistent BCL-2 expression without a translocation (14;18): a molecular and immunohistochemical study.

The presence of the BCL-2 protein was studied in nine non-Hodgkin's lymphomas with testicular localisation. A consistent presence of the BCL-2 protein was found. The chromosomal translocation (14;18) was seen neither by cytogenetic analysis (n = 4) nor by polymerase chain reaction amplification and Southern blotting (n = 9). Therefore, this translocation is not responsible for the presence of the BCL-2 protein in non-Hodgkin's lymphomas with testicular localisation. We suggest that the presence of the BCL-2 protein in these lymphomas is related to the differentiation stage of the B-lymphocytes or may play a role in the pathogenesis of these lymphomas. The consistent finding of the BCL-2 protein in lymphomas with testicular localisation may support the clinical observation that these lymphomas are a separate entity.

extremely rare. They may occur as a primary manifestation or in the context of dissemination of nodal NHL (secondary testicular NHL) (Doll and Weis, 1986;Hamilton and Horwich, 1988;Martenson et al., 1988;Lukes and Collins, 1992). Thus far, there are no criteria to discriminate between these two. These NHLs of the testis are usually diffuse large-cell NHLs according to the Working Formulation for lymphomas (Doll and Weis, 1986;Hamilton and Horwich. 1988;Martenson et al., 1988;Lukes and Collins, 1992). The prognosis of NHL with testicular localisation is poor; most patients die of disseminated NHL within 2 years. In addition, they show a specific pattern of metastases, e.g. extranodal sites such as the upper airways, central nervous system and bones are especially involved (Doll and Weis, 1986;Hamilton and Horwich. 1988).
In follicular NHL, diffuse large-cell NHL and NHL of the gastrointestinal tract, the BCL-2 protein is found using immunohistochemistry (Ngan et al., 1988;Pezzella et al., 1990b;Zutter et al., 1991;Gaulard et al., 1992;Kondo et al., 1992;LeBurn et al., 1992). Presence of the BCL-2 protein is also reported in normal lymphoid cells and is demonstrated in precursor cells of all haematopoietic lineages, memory B cells and plasma and mantle zone B cells Nunez et al. 1991;Pettersson et al. 1992). In nearly all follicular NHLs, independent of the presence of the t(14;18), the BCL-2 protein is present (Ngan et al., 1988 , 1990b;Gaulard et al., 1992). In 22-80% of diffuse large-cell NHLs the BCL-2 protein is found and is not restricted to those with the t(14;18) (Ngan et al., 1988;Pezzella et al., 1990b;Zutter et al., 1991;Kondo et al., 1992). NHLs of the gastrointestinal tract, neoplasms in which t(14;18) occurs infrequently, exhibit the BCL-2 protein in about 50% of cases (Kondo et al., 1992;LeBurn et al., 1992). Thus, the BCL-2 protein is found in normal lymphoid cells and in many different histological subtypes of NHL independent of the presence of the t(14;18).
In the context that NHLs with testicular localisation show a separate clinical identity among NHL, we investigated the presence of the t(14;18) by combining cytogenetic analysis (n = 4), polymerase chain reaction (PCR) and Southern blotting (n = 9). The presence of the BCL-2 protein was studied using immunohistochemistry on frozen tissue sections (n = 9).

Samples
Tumour samples were collected in The Netherlands from nine patients with NHL localised in the testis. The tumours were classified according to the Working Formulation for lymphomas by a panel of pathologists (National Cancer Institute sponsored study of classification of non-Hodgcin's lymphomas, 1982). Staging was according to the Ann Arbor classification (Carbone et al., 1971). Representative frozen tissue sections were used for immunohistochemical and immunofluorescence analysis (Lambrechts et al., 1992).

Immunohistochemistrv
Frozen tissue sections were fixed in acetone (100%) for 10 min. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide in phosphate-buffered saline (PBS), containing 0.2% bovine serum albumin (BSA). Expression of the BCL-2 protein was studied using a mouse monoclonal antibody, BCL-2 lOOa (kindly provided by Drs F Pezzella and DY Mason; Pezzella et al., 1990b). Visualisation was performed using an indirect peroxidase assay with a streptavdin-biotin-conjugated goat anti-mouse immunoglobulin antibody as a second step. Hyperplastic lymph nodes were used as positive controls. DB-2 and I ada r_nooddn's lymphoma AC Lambrechts et a Immunofluorescence Surface marker analysis was performed by direct immunofluorescence with fluorescein isothiocyanate (FITC)conjugated goat anti-human IgM, IgD, IgG, IgA and lambda serum and tetramethylrhodamineisothiocyanate (TRITC)conjugated goat anti-human kappa and lambda serum (Nordic Immunological Laboratories Tilburg, The Netherlands). Double staining was performed with TRITC-conjugated goat anti-human kappa plus FITC-conjugated goat anti-human lambda or with the FITC-conjugated antibody against the heavy chain expressed by the malignant B-cell plus TRITCconjugated anti-kappa or anti-lambda.

Cytogenetic analysis
Cytogenetic analysis was performed on metaphase spread chromosome of four cases according to standard procedures (Gibas et al., 1984). The chromosomes were identified with G banding (GTG banding) and described according to ISCN 1991(Mittelman, 1991. DNA extraction and polymerase chain reaction (PCR) analjysis High molecular weight DNA was isolated from frozen tissue sections of all NHLs with testicular localisation. DNA was digested with HindIII and analysed for t(14;18) and immunoglobulin heavy-chain rearrangement (IgH) using Southern blotting and probing with BCL-2 (Bakhshi et al., 1985) and the joining region of IgH allele (JH) (Takahasi et al., 1980). In addition, PCR was used to detect small amounts of t(14;18)-positive cells. PCR primers were designed for the major breakpoint region (mbr: 5'-GGTGGTTTGACCTTTAGA-3') of the BCL-2 gene and the consensus region of the JH genes (5'-TGAGGAGACG-GTGACC-3') (Lambrechts et al., 1992). As a control to DNA quality the interleukin-3 gene of the DNA was amplified in 25 cycles, as described previously (Lambrechts et al., 1992). For the detection of t(14;18) a total of 0.5-1.Oig of DNA in a reaction volume of 25-501lI was subjected to 30 cycles of PCR amplification using an automated Perkin-Elmer/Cetus DNA thermal cycler (Gouda, The Netherlands).
In each experiment positive and negative controls were included. As positive controls different dilutions of DNA from a mbr t(14;18)-positive cell line (SU-DHL-6) or DNA from a mcr t(14;18)-positive lymph node biopsy was used. Amplified samples were analysed as described using the BCL-2 and JH probes (Lambrechts et al., 1992).

Diession
NHLs localised in the testis are high-grade malignant NHLs and usually of B-lymphocyte origin. They constitute approximately 1% of all lymphomas and have a specific clinical course of disease. They metastasise to uncommon sites for other lymphomas, for instance the central nervous system (Doll and Weis, 1986;Hamilton and Horwich, 1988). Cytogenetic analysis of four of the NHLs with testicular localisation revealed many different and complex chromosomal abnormalities but no chromosomal translocation (14;18) as found in other histological subtypes of NHL Figue 2 A representative example of the imnunohistochemical detection of the BCL-2 protein on frozen tissue sections of a non-Hodgkin's lymphoma with testicular localisation (case 3, Table   I).
BCL-2 and besdw non4iod9bn's yphomas AC Lanbrechts et al 76 (Aisenberg et al., 1988: Rowley, 1988Gnresser and Lennert, 1990;Pezzella el al.. 1990a;Lambrechts et al.. 1992;Limpens et al., 1992). In addition, we used PCR analysis, a technique able to detect one t(14;18)-positive cell out of 100 000 normal cells, to evaluate nine cases of NHL with testicular localisation for the presence of t(14;18)-positive cells. No t(14;18)positive cells were detected in these NHLs. The presence of the BCL-2 protein in different histological subtypes of NHL without t(14;18) was the reason for evaluating the involvement of the presence of the BCL-2 protein in NHL with testicular localisation as well (Kondo et al., 1992;LeBurn et al., 1992). While the BCL-2 protein was present in normal lymphoid cells. no BCL-2 protein was reported in normal testis : Nunez et al., 1991Pettersson et al.. 1992). Our data show the presence of the BCL-2 protein in all of the NHLs with testicular localisation studied. Thus. in all cases of NHL with testicular localisation we studied, the BCL-2 protein is consistently present without a t(14;18). In spite of the relatively small number of cases, this finding supports the clinical observation that NHL with testicular localisation may represent a separate subgroup of large-cell lymphomas which is distinct from progressed follicular lymphoma. The presence of the BCL-2 protein may represent a differentiation stage of the B lymphocyte or a functional subpopulation of B lymphocytes.