Lack of BCL10 mutations in germ cell tumors and B cell lymphomas.

The BCL10 gene has recently been cloned from the chromosomal translocation t(1;14)(p22;q32) in a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and was implicated in the pathogenesis of this and several other tumor types (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999). BCL10 is a cellular homolog of the equine herpesvirus-2 E10 gene, which contains an amino-terminal caspase recruitment domain (CARD) and plays a role in apoptosis. Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999 also showed that BCL10 exhibits hypermutations in MALT lymphomas with t(1;14) as well as frequent mutations in 45% of B and T cell lineage lymphomas without the 1p22 chromosomal rearrangements. In addition, they reported BCL10 mutations in cell lines derived from several solid tumor types including three each of male germ cell tumors (GCTs) and mesotheliomas, suggesting that it may be commonly involved in the pathogenesis of many human malignancies. The 1p22 region is affected by frequent deletions in male GCTs (Mathew et al. 1994xMathew, S., Murty, V.V.S., Bosl, G.J., and Chaganti, R.S.K. Cancer Res. 1994; 54: 6265–6299PubMedSee all ReferencesMathew et al. 1994) and mesotheliomas (Lee et al. 1996xLee, W.C., Balsara, B., Liu, Z., Jhanwar, S.C., and Testa, J.R. Cancer Res. 1996; 56: 4297–4301PubMedSee all ReferencesLee et al. 1996), suggesting that inactivation of a critical gene in this region may play a role in the genesis of these tumors.In order to validate a possible role for BCL10 in male GCT and lymphoma pathogenesis, we performed mutation analysis of its coding region in a panel of 59 GCTs (41 primary tumors and 18 cell lines) (Murty et al. 1994xMurty, V.V.V.S., Bosl, G.J., Houldsworth, J., Meyers, M., Mukherjee, A.B., Reuter, V., and Chaganti, R.S.K. Oncogene. 1994; 9: 2245–2251PubMedSee all ReferencesMurty et al. 1994), 15 MALT lymphomas, and 15 follicular lymphomas (Offit et al. 1993xOffit, K., Parsa, N.Z., Gaidano, G., Filippa, D.A., Louie, D., Pan, D., Jhanwar, S.C., Dalla-Favera, R., and Chaganti, R.S.K. Blood. 1993; 82: 2157–2162PubMedSee all ReferencesOffit et al. 1993). The coding region of BCL10 was amplified as described (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999) and SSCP analysis was performed by standard methods (Orita et al. 1989xOrita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. Genomics. 1989; 5: 874–879Crossref | PubMed | Scopus (2705)See all ReferencesOrita et al. 1989). We reasoned that if BCL10 is the target gene deleted, it should exhibit mutations in cases with loss of heterozygosity (LOH), and we included 11 such tumors in the panel of 41 primary GCTs (Mathew et al. 1994xMathew, S., Murty, V.V.S., Bosl, G.J., and Chaganti, R.S.K. Cancer Res. 1994; 54: 6265–6299PubMedSee all ReferencesMathew et al. 1994). Among the cell lines studied, we also included the Tera-1, Tera-2, and GCT-44 cell lines studied by Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999. Conformational changes were noted with high frequency in both GCTs and lymphomas in exon 1 and in 11 cases in exon 3 (5 GCTs and 6 lymphomas) (Table 1Table 1). No variants were found in either tumor in exon 2. All SSCP variants in exon 3 in both tumors were identical while exon 1 variants exhibited a complex pattern (Figure 1AFigure 1A). In order to determine if the SSCP variants represent somatic mutations or genetic polymorphisms, we analyzed paired normal-tumor DNAs by SSCP in 14 of 15 exon 1 variants and all 5 exon 3 variants in GCTs. We found identical SSCP variants in all cases, suggesting that these represent genetic polymorphisms.Figure 1Genetic Analysis of BCL10 in Germ Cell Tumors (GCTs) and B Cell Lymphomas(A) PCR-SSCP analysis. (a) and (c), GCT; (b) and (d), lymphoma. (a) and (b), exon 1; (c) and (d), exon 3.2. Arrows indicate conformational changes. N, normal; T, tumor; CL, cell line; tumor numbers are indicated on top.(B) PCR sequence analysis. (a) and (b), Tera-1; (c), GCT-44; (d) and (e), Tera-2; (f), T-243A; (g)–(i), 240A. Panels (a)–(e) show absence of mutations in GCT cell lines in relation to Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999 report, where boxes indicate base change and upward arrow indicates insertion positions. Positions in coding region of cDNA are: (a), 172 bp; (b), 653 bp; (c), 172 bp; (d), 58 bp; (e), insertion at 499 bp. Panel (f), A/G heterozygosity at position 2 of codon 213. Panels (g)–(i) indicate loss of heterozygosity at codon 5 (underlined) in tumor and cell line 240A. Panel (g), normal DNA; panel (h), tumor DNA; panel (i) cell line DNA. The residual peak of G in panel (h) indicates the presence of contaminating normal cells.(C) Multiplex RT-PCR analysis of BCL10 expression in GCT. Primer from the 5′ coding region (5′-GGACCCGGAAGAAGCGCCATCTCC-3′ and 5′-AAGTAGTCTAACAATTTTCCAGCCC-3′) spanning two different exons were used to amplify a 249 bp PCR product. PCR was performed utilizing AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). The 450 bp PCR product represents β-actin as control. Marker, PhiX HaeIII; tumor and cell line numbers are shown on top.View Large Image | View Hi-Res Image | Download PowerPoint SlideTable 1Analysis of BCL10 Sequence Alterations in GCT and NHLTumor TypeNo. StudiedSSCP VariantsPosition of Sequence Variation in cDNA (amino acid)Germ cell tumor59Exon 1–Intron 115 (25.5%)703 G/T (Ala/Ser) 714 G/C (Leu/Leu) Intron 1, 58 G/CExon 35 (8.5%)1328 G/A (Gly/Glu)Non-Hodgkin's Lymphoma30Exon 1–Intron 110 (33.3%)703 G/T (Ala/Ser) 714 G/C (Leu/Leu) Intron 1, 58 G/CExon 36 (20.0%)1328 G/A (Gly/Glu)To identify the nature of alterations associated with conformation variations, we determined the sequences of 9 exon 3 (paired normal-tumor DNAs in 5 GCTs and 4 lymphomas) variants and 12 exon 1 variants (paired normal-tumor DNAs in 7 GCTs and 4 lymphomas) (Table 1Table 1). We found identical sequences in both normal and tumor DNAs in each case. Although we did not find SCCP variants in the 3 GCT cell lines (Tera-1, Tera-2, and GCT-44), we sequenced the exons and found no sequence alterations (Figure 1BFigure 1B). This analysis also identified the sequence variation reflected in SSCP variants. The region amplified for exon 1 exhibited 3 different polymorphisms, 2 in the coding region and one in 5′ intron 1. These included G/T in the first base of codon 5, G/C at the third base of codon 8, and a C/G polymorphism in intron 1. The polymorphism in exon 3 was G/A at second base of codon 213. Consistent with the reported high frequency of LOH at the 1p22 region in GCT, the tumor and the cell line from GCT-240A were reduced to homozygosity at all polymorphic loci in exon 1–intron 1 compared to germline heterozygosity (Figure 1BFigure 1B). Thus, these data are at variance with the results reported by Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M.-Q., Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999. The changes noted by them, mostly truncations or missense mutations supported a tumor suppressor gene (TSG) role for BCL10. Our data contradict this presumption in GCTs and B cell lymphomas, and provide evidence for polymorphisms in BCL10 coding region.Lack of mutation in the BCL10 coding region may not exclude it as a deleted TSG. Other genetic or epigenetic mechanisms such as alterations in promoter or methylation patterns can silence gene expression (1xBaylin, S.B., Herman, J.G., Graff, J.R., Vertino, P.M., and Issa, J.P. Adv. Cancer Res. 1998; 72: 141–196Crossref | PubMedSee all References, 2xForget, B.G. Ann. N.Y. Acad. Sci. 1998; 30;850: 38–44Crossref | Scopus (91)See all References). In order to examine this possibility, we employed a multiplex RT-PCR strategy to analyze BCL10 expression. This analysis revealed detectable levels of expression in 14 of 16 GCTs (10 cell lines and 6 primary tumors) examined. BCL10 message could not be detected in the remaining 2 cells lines, 577M-F and 2102E-R, after 45 cycles of PCR amplification (Figure 1CFigure 1C). Thus, the present analysis rules out a role for BCL10 as a TSG in the majority of GCTs. On the basis of the data presented, we conclude that BCL10 is not a target TSG at 1p22 in male GCTs or B cell lymphomas.‡To whom correspondence should be addressed (e-mail: vvm2@columbia.edu).