Chromosomal translocation detected by bcl- I and bc2-2 rearrangement in low-grade B-cell lymphomas in a European population M.M.OTT, H.K.MULLER-HERMELINK, B.SCHMITT & A.C.FELLER Uepartrnent uf Pathology, University of Wiirzburg, Germany Uete of submission 27 December 1990 Accepted for publication 9 April 1991
OTT M . M . ,
MULLEK-HERMELINK
H . K . , SCHMITT B . & FELLER
A.C.
(1991) Histopathology 19, 163-167
Chromosomal translocation detected by bcl-1 and bcl-2 rearrangement in low-grade B-cell lymphomas in a European population Twenty-nine cases of non-Hodgkin's lymphoma of low-grade malignancy in a European population were investigated for the presence of bcl-2 and bcl-1 gene rearrangement. The cases were classified according to the Kiel classification. It was shown that bcl-2 gene rearrangements were exclusively confined to centroblastic-centrocytic lymphomas. bcl- 1 rearrangements were found in two cases of chronic lymphocytic leukaemia. As the chromosomal translocation t( 14;1 8) is reported to occur in up to 8 5% of folIicular lymphomas, our results provide additional evidence that the differentiation of low-grade B-cell lymphomas according to the Kiel classification defines biologically distinct entities. Keywords: malignant lymphoma, bcl rearrangement, Kiel classification
Introduction In different histological subtypes of non-Hodgkin's lymphoma, three non-random chromosomal abnormalities have been described, all involving the chromosomal band 14q32. The translocation t(14;18) in particular is closely associated with follicular lymphoma. Cytogenetic studies in the USA have reported a frequency of this translocation in follicular lymphomas of 63-82%'.'. Molecular analyses oft( 14;18)have shown a rearrangement of the bcl-2 gene on chromosome 18 to the JHlocus on chromosome 14. as detected by Southern blotting and hybridization with probes coding for different regions of the bcl-2 gene. The frequency of bcl-2 rearrangement in follicular lymphomas is equivalent to the t( 14;18) translocation described in cytogenetic studies34. On the other hand, further studies have also revealed detectable bcI-2 rearrangements in a large number of diffuse large cell lymphomas probably of follicular centre cell origin'. A significantly lower frequency of bcl-2 rearrangement was found in follicular
lymphomas in Japan, where this subtype is relatively rareh. While t(14;18) is one of the most constant chromosomal abnormalities in non-Hodgkin's lymphoma, a second less frequent translocation t( 11;14)is also non-randomly found in non-Hodgkin's lymphomas of low-grade malignancy, especially in lymphocytic lymphomas. In this translocation a rearrangement of the bcl-1 gene on chromosome 11 to the JH-locus on chromosome 14 occursx. Using DNA-probes for bcl-l and bcl-2, the present study aimed to determine the frequency and distribution in a European population of the chromosomal translocations t(14;18) and t ( l 1 ; 1 4 ) in malignant non-Hodgkin's lymphomas of low-grade malignancy, classified according to the Kiel classification. It was attempted to substantiate further the morphological differentiation of the lymphoma subtypes known to be of different cellular origin, as recently reported by Wotherspoon and coworkers'.
Materials and methods Address for correspondence. Dr Alfred CFeller. Department of Pathology, University of Wurxburg. losef-Schneider-Str. 2, 8700 Wiirzburg, Germany.
Lymph node biopsies of 29 patients with non-Hodgkin's lymphoma of low-grade malignancy were classified by 163
164
M.M.Ott et al.
routine histology. All cases were immunophenotyped with monoclonal antibodies (CD2, CD3. CD5, CD10, CD22, CD23). Histological diagnoses followed the criteria of the Kiel classification. Seventeen cases were diagnosed as centroblastic-centrocytic (cb-cc) lymphoma with a follicular or follicular and diffuse growth pattern. Seven cases were classified as centrocytic lymphoma; and six cases, comprising four cases of chronic lymphocytic leukaemia (B-CLL) and two of lymphoplasmacytic/cytoid (LP) immunocytoma, were classified as lymphocytic lymphoma. High molecular weight DNA was isolated from frozen tissue samples according to standard methods: 10 pg of DNA was digested with suitable restriction enzymes (Bam HI, Hind 111, Eco RI, SstI, Pstl) and size-fractionated in 0.7% agarose gels. After Southern blotting to nitrocellulose filters"', hybridization was carried out in a solution of SO% formamide, 5 x Denhardt's, 4 x SSC and 100 pg salmon sperm DNA at 42°C with [32P]-labelled DNA probes. The filters were washed to a stringency of 68°C in 0.1 x SSC and 0.1% SDS prior to autoradiography. JH-rearrangement was studied using a probe for the immunoglobulin heavy chain joining region (2.5 kb Sau 3A fragment''), kindly provided by P.Leder, Boston, MA, USA. The bcl-1 and bcl-2 probes were a gift of C.M.Croce, Philadelphia, PA, USA12.'3, the pFL-2 probe for the minor breakpoint region of the bcl-2 gene was kindly provided by M.Cleary, Stanford, CA, USAI4. TcR genes were studied using a TcR-P-probe (770 bp constant region fragment from Jurkat, generously provided by T.W.Mak, Toronto, Canada'').
Table 1. Rearrangement profile in centroblastic-centrocytic lymphomas
Probe Case no.
The results of the DNA analyses are shown in detail in Tables 1 & 2 . All cases had a detectable JH-rearrangement, indicating that sufficient and representative DNA was extracted. TcR-cp rearrangement was not found in a single case. bcl-2 rearrangement could be detected by hybridization with the bcl-2 probe for the major breakpoint region in nine out of 1 7 cb-cc cases (Figure 1).In three other cases the detectable breakpoints occurred at the second breakpoint location on chromosome 18 (minor breakpoint region), as demonstrated by hybridization with probe pFL-2 (Figure 2). Five cases of cb-cc exhibited no detectable bcZ-2 rearrangement with either probe bcl-2 or with probe pFL-2. None of the centrocytic and lymphocytic lymphomas showed bcl-2 rearrangement. The translocation t( 11:14) was found in two cases of B-CLL with probe bcl-1 (Figure 3).
JH
bcl-2
pFL2
bcl- 1
+ + + + + + + + + + +
8 9 10 11 12 13 14 15 16 17
+ =rearranged: - =germ line Table 2. Rearrangement profile in immunocytoma. chronic lymphocytic leukaemia and centrocytic lymphoma
Probe Case no.
Results
CB
cfr
Immunocytoma
18
-
19
-
JH
bcl-2
+ +
-
Chronic lymphocytic leukaemia 20 21 22 23
+ + + +
Centrocytic lymphoma 24 25 26
27 28 29 30
-
-
+ + + + + +
+
+ =rearranged: - =germ line
-
-
-
pFL2
bcl- 1
bcl rearrangement in B-cell lymphomas
Figure 1. Southern blot analysis of bcl-2 major breakpoint rearrangements in low-grade B-cell lymphomas: DNA from cases 2, 3 , 1 0 and 27 (Tables 1 & 2) and from placenta (P) was digested with Sstl and hybridized with probe bcl-2. Rearranged bands are arrowed.
165
Figure 3. Southern blot analysis of bcl-I rearrangements in lowgrade B-cell lymphomas: DNA from cases 22, 23, 24 and 25 (Table 2) and from placenta (P) was digested with Bam HI and hybridized with probe bcl-1. Rearranged bands are arrowed.
Discussion
Figure 2. Southern blot analysis of bcl-2 minor breakpoint rearrangements in centroblastic-centrocytic lymphomas: DNA from cases 4 and 6 (Table I ) and from placenta (P) was digested with Bam HI and hybridized with probe pFL-2. Rearranged bands are arrowed.
Malignant non-Hodgkin’s lymphoma can be classified according to a variety of criteria, resulting in a variety of classification systems. The major tool of the Kiel classification is the differentiation of biologically distinct entities based on detailed cytological evaluation and, thus, it is of interest to study the relation between established chromosomal translocations and these lymphoma classes. The most important finding of this study was that the chromosomal translocation t( 14;18),detected by bcl-2 rearrangement, is confined to a distinct lymphoma entity among the low-grade B-cell lymphomas: 70% of cb-cc showed this translocation, whereas none of the other cases had detectable bcl-2 rearrangement. There were no significant differences in morphology or immunophenotype between the cb-cc lymphomas that were positive and those that were negative for bcl-2 rearrangement. The overall frequency found in our study was similar to that described in the USA for follicular lymphoma^.^ Thus, it is clear that the majority of these follicular lymphomas are also of germinal centre cell origin. However, individual follicular lymphoma cases may fall into the centrocytic lymphoma group, exhibiting a follicular growth pattern but otherwise possessing a distinct cytological picture and immunophenotypic
I66
M.M,Ott et al.
properties. The overall frequency found for bcl-2 rearrangement in Japan, however, is much lower. Amakawa rt al.’ describe bcl-2 rearrangement in one-third of patients with follicular lymphoma, classified according to the Working Formulation. Because the Working Formulation is based on criteria different from those of the Kiel classification, a comparison of individual cases is difficult, particularly for follicular lymphomas. The major differencebetween cases in Japan and cases in the USA and Europe remains the overall percentage of detectable bcl-2 rearrangements. The total incidence of cb-cc lymphoma is much higher in Europe than Japan, while in the USA i t is even higher. Our results and those of Wotherspoon et al.’ indicate that there may be distinct but different transforming events in the development of germinal centre cell lymphomas. It has been shown that bcl-2 rearrangement occurs during VDJ-rearrangement in early B-cell differentiation.I6 Since the expression of the bcl-2 gene may provide cel!s with a growth advantage” it seems justified to designate t( 14;18j as the first transforming event. This translocation is exceptionally found in nonneoplastic germinal centre cellslx. Thus, a second yet unknown transforming event may induce tumour growth. Whether the bcl-2 negative cb-cc lymphomas have undergone different transforming events is also a n unsolved question. It seems possible that a minor subgroup is not detected with the available probes, which do not completely cover the entire breakpoint cluster region. In individual cases chromosomal analyses demonstrated t( 14:18)that had not been detected by molecular hybridization techniques‘. Moreover, a number of cases may be missed using Southern blot analysis due to limited sensitivity. A more sensitive method for the detection of bcl-2 rearrangements might be the polymerase chain reaction (PCR).As this method is able to detect the event also in reactive follicles”, it seemed doubtful to us that PCR would be of use in the present study. The results clearly demonstrate that t(14;18) is confined to a well-defined entity among low-grade B-cell lymphomas. With respect to the bcl- 1 rearrangement, our results are in line with a number of reports in which this translocation had been detected in cases of B-cell chronic lymphocytic leukaemia. In our sample of centrocytic lymphomas not a single case of bcl-l rearrangement could be shown, although Williams et d.” recently detected the translocation in some cases. Earlier reports have also obviously included cases of centrocytic lymphoma with t ( l l ; l 4 ) , which were in part classified as nodular poorly differentiated lymphocytic lymphomas2”. It becomes clear, therefore, that a varying percentage of
centrocytic lymphomas have the t( 1 1;14)chromosomal aberration, and further investigation may show that subgroups exist within this morphologically homogeneous entity. Our results, especially on bcl-2 rearrangement and centroblastic-centrocytic lymphomas, further stress the value of the Kiel classification and clearly document that we are dealing with biologically distinct lymphoma entities. Further investigations will have to reveal whether the absence of bcl-2 rearrangement in these lymphomas is of clinical relevance, since-in the highgrade lymphomas at least-a different clinical course was found in patients with bcl-2 rearrangement compared with those without”.
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft, grant DFG Fe 221/1-4.
References 1. Yunis JJ, Martin MO. Kaplan ME. Ensrud KM, Howe RR. Theologides OA. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphoma. N. Engl. 1. Med. 1982: 307; 1231-1236. 2. Levine EG. Arthur DC. Frizzera G. Peterson BA. Hurd DD. Bloomfield CD. There are Differences in cytogenetic abnormalities among Histologic subtypes of the non-Hodgkin’s lymphomas. Blood 1985: 6; 1414-1422. 3 . Tsujimoto Y. Cossman J. Jaffe E. Croce CM. Involvement of the brl2 gene in human follicular lymphoma. Science 1985: 228; 14401443. 4. Weiss LM. Warnke RA. Sklar J , Cleary ML. Molecular analysis of the chromosomal translocation in malignant lymphomas. N. Engl. /. Men. 1987: 317; 1185-1189. 5. Aisenberg AC. Wilkes BM, Jacobson JO. The bcl-2 gene is rearranged in many diffuse B-cell lymphomas. Blood 1988: 71; 969-9 72. 6. Amakawa R. Fukuhara S. Ohno H et al. Involvement of bcl-2 gene in Japanese follicular lymphoma. Blood 1989: 3; 787-791, 7. Yunis JJ. Oken MM. Theologides A. Howe RR. Kaplan ME. Recurrent Chromosomal defects are found in most patients with non-Hodgkin’slymphoma. Cancer Genet. Cytogertet. 1984; 13; 1728. 8. Tsujimoto Y. Junis J. Onorato-Showe L. Erikson 1. Nowell PC. Croce CM. Molecular cloning of the chromosomal breakpoint of Rcell lymphomas with the t(l1:14) chromosome translocation. Science 1984: 224; 1403-1406. 9. Wotherspoon AC. Langxing P. Diss TC. lsaacson PG. A genotypic study of low grade B-cell lymphomas including lymphomas of mucosa associated tissue (MALT)./. I’athol. 1990: 162; 1 3 5-1 40. 10. Southern EM. Detection of specific sequences among the DNA fragments separated by gel electrophoresis. \. Mol. Biol. 1975: 98; 503-517. 11. Ravetch JV. Siehenlist U. Korsmeyer S. Waldmann T. Leder P. Structure of the human immunoglobulin p locus: characterization of embryonic and rearranged J and D genes. C d l 198 1 : 27; 583-591.
brl rearrungenlent in H-cell lymphomas
12. Tsujimoto Y. Jaffe 1;. Cossman J. Gorham J , Nowell PC. Croce CM. Clustering of breakpoints on chromosome 1 1 in human B-cell neoplasms with the t( 11;14) chromosome translocation. Nuture 1985: 31 5; 340-343. 13. Tsujimoto Y . Yunis J . Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic H-cells with the t( 14;18) chromosome translocation. Sciericc, 1984; 226; 1097-1 099. 14. Cleary DA. Galili N , Sklar J. Detection of a second t(14;18) breakpoint cluster region in human Iollicular lymphomas. I. Exp. Med. 1986; 164; 3 1 5-320. 1 5. Yoshikai Y, Anatoniou D. Clark SP et al. Sequence and expression of transcripts of the human T-cell receptor [{-chain genes. Nutun, 1984: 312; 521-524. 16. Tsujimoto Y . Gorham J , Cossman J , Jaffe E, Croce CM. The t( 14; 18) chromosome translocations involved in H-cell neoplasms result from mistakes in VDJ-joining. Scicriw 1985; 229; 1390-1 393.
167
17. McDonell TJ, Deane N . Platt FM et d.bcl-l-immunoglobulin transgenic mice demonstrate extended B-cell survival and follicular lymphoproliferation. Cell 1989; 57: 79-88. 18. De long I). Limpens CEJM. Van Krieken JHJM.Van Ommen CJB. Kluin PM. The origin of follicle centre cell lymphomas. 3rd Meeting of the European Association for Haematopathology, Wurzburg. October. 1990. 19. Williams ME. Westerman CD, Swerdlow SH. Genotypic characterization of centrocytic lymphoma: frequent rearrangement of the chromosome 11 bcl-l locus. Hlood 1990; 76; 1387-1 39 I . 20. Koduru PRK, Offit K. Filippa DA. Molecular analysis of breaks in bcl-1 proto-oncogene in B-cell lymphomas with abnormalities of l l q 1 3 . Oncogene 1989: 4; 929-934. 21. Yunis J J , Mayer MG, Arnesen MA. Aeppli DP. Oken MM, I+izzera (2. bcl-2 and other genomic alterations in the prognosis of largecell lymphoma. N.Engl. ]. Med. 1989: 320; 1047- 1054.
OTT M . M . ,
MULLEK-HERMELINK
H . K . , SCHMITT B . & FELLER
A.C.
(1991) Histopathology 19, 163-167
Chromosomal translocation detected by bcl-1 and bcl-2 rearrangement in low-grade B-cell lymphomas in a European population Twenty-nine cases of non-Hodgkin's lymphoma of low-grade malignancy in a European population were investigated for the presence of bcl-2 and bcl-1 gene rearrangement. The cases were classified according to the Kiel classification. It was shown that bcl-2 gene rearrangements were exclusively confined to centroblastic-centrocytic lymphomas. bcl- 1 rearrangements were found in two cases of chronic lymphocytic leukaemia. As the chromosomal translocation t( 14;1 8) is reported to occur in up to 8 5% of folIicular lymphomas, our results provide additional evidence that the differentiation of low-grade B-cell lymphomas according to the Kiel classification defines biologically distinct entities. Keywords: malignant lymphoma, bcl rearrangement, Kiel classification
Introduction In different histological subtypes of non-Hodgkin's lymphoma, three non-random chromosomal abnormalities have been described, all involving the chromosomal band 14q32. The translocation t(14;18) in particular is closely associated with follicular lymphoma. Cytogenetic studies in the USA have reported a frequency of this translocation in follicular lymphomas of 63-82%'.'. Molecular analyses oft( 14;18)have shown a rearrangement of the bcl-2 gene on chromosome 18 to the JHlocus on chromosome 14. as detected by Southern blotting and hybridization with probes coding for different regions of the bcl-2 gene. The frequency of bcl-2 rearrangement in follicular lymphomas is equivalent to the t( 14;18) translocation described in cytogenetic studies34. On the other hand, further studies have also revealed detectable bcI-2 rearrangements in a large number of diffuse large cell lymphomas probably of follicular centre cell origin'. A significantly lower frequency of bcl-2 rearrangement was found in follicular
lymphomas in Japan, where this subtype is relatively rareh. While t(14;18) is one of the most constant chromosomal abnormalities in non-Hodgkin's lymphoma, a second less frequent translocation t( 11;14)is also non-randomly found in non-Hodgkin's lymphomas of low-grade malignancy, especially in lymphocytic lymphomas. In this translocation a rearrangement of the bcl-1 gene on chromosome 11 to the JH-locus on chromosome 14 occursx. Using DNA-probes for bcl-l and bcl-2, the present study aimed to determine the frequency and distribution in a European population of the chromosomal translocations t(14;18) and t ( l 1 ; 1 4 ) in malignant non-Hodgkin's lymphomas of low-grade malignancy, classified according to the Kiel classification. It was attempted to substantiate further the morphological differentiation of the lymphoma subtypes known to be of different cellular origin, as recently reported by Wotherspoon and coworkers'.
Materials and methods Address for correspondence. Dr Alfred CFeller. Department of Pathology, University of Wurxburg. losef-Schneider-Str. 2, 8700 Wiirzburg, Germany.
Lymph node biopsies of 29 patients with non-Hodgkin's lymphoma of low-grade malignancy were classified by 163
164
M.M.Ott et al.
routine histology. All cases were immunophenotyped with monoclonal antibodies (CD2, CD3. CD5, CD10, CD22, CD23). Histological diagnoses followed the criteria of the Kiel classification. Seventeen cases were diagnosed as centroblastic-centrocytic (cb-cc) lymphoma with a follicular or follicular and diffuse growth pattern. Seven cases were classified as centrocytic lymphoma; and six cases, comprising four cases of chronic lymphocytic leukaemia (B-CLL) and two of lymphoplasmacytic/cytoid (LP) immunocytoma, were classified as lymphocytic lymphoma. High molecular weight DNA was isolated from frozen tissue samples according to standard methods: 10 pg of DNA was digested with suitable restriction enzymes (Bam HI, Hind 111, Eco RI, SstI, Pstl) and size-fractionated in 0.7% agarose gels. After Southern blotting to nitrocellulose filters"', hybridization was carried out in a solution of SO% formamide, 5 x Denhardt's, 4 x SSC and 100 pg salmon sperm DNA at 42°C with [32P]-labelled DNA probes. The filters were washed to a stringency of 68°C in 0.1 x SSC and 0.1% SDS prior to autoradiography. JH-rearrangement was studied using a probe for the immunoglobulin heavy chain joining region (2.5 kb Sau 3A fragment''), kindly provided by P.Leder, Boston, MA, USA. The bcl-1 and bcl-2 probes were a gift of C.M.Croce, Philadelphia, PA, USA12.'3, the pFL-2 probe for the minor breakpoint region of the bcl-2 gene was kindly provided by M.Cleary, Stanford, CA, USAI4. TcR genes were studied using a TcR-P-probe (770 bp constant region fragment from Jurkat, generously provided by T.W.Mak, Toronto, Canada'').
Table 1. Rearrangement profile in centroblastic-centrocytic lymphomas
Probe Case no.
The results of the DNA analyses are shown in detail in Tables 1 & 2 . All cases had a detectable JH-rearrangement, indicating that sufficient and representative DNA was extracted. TcR-cp rearrangement was not found in a single case. bcl-2 rearrangement could be detected by hybridization with the bcl-2 probe for the major breakpoint region in nine out of 1 7 cb-cc cases (Figure 1).In three other cases the detectable breakpoints occurred at the second breakpoint location on chromosome 18 (minor breakpoint region), as demonstrated by hybridization with probe pFL-2 (Figure 2). Five cases of cb-cc exhibited no detectable bcZ-2 rearrangement with either probe bcl-2 or with probe pFL-2. None of the centrocytic and lymphocytic lymphomas showed bcl-2 rearrangement. The translocation t( 11:14) was found in two cases of B-CLL with probe bcl-1 (Figure 3).
JH
bcl-2
pFL2
bcl- 1
+ + + + + + + + + + +
8 9 10 11 12 13 14 15 16 17
+ =rearranged: - =germ line Table 2. Rearrangement profile in immunocytoma. chronic lymphocytic leukaemia and centrocytic lymphoma
Probe Case no.
Results
CB
cfr
Immunocytoma
18
-
19
-
JH
bcl-2
+ +
-
Chronic lymphocytic leukaemia 20 21 22 23
+ + + +
Centrocytic lymphoma 24 25 26
27 28 29 30
-
-
+ + + + + +
+
+ =rearranged: - =germ line
-
-
-
pFL2
bcl- 1
bcl rearrangement in B-cell lymphomas
Figure 1. Southern blot analysis of bcl-2 major breakpoint rearrangements in low-grade B-cell lymphomas: DNA from cases 2, 3 , 1 0 and 27 (Tables 1 & 2) and from placenta (P) was digested with Sstl and hybridized with probe bcl-2. Rearranged bands are arrowed.
165
Figure 3. Southern blot analysis of bcl-I rearrangements in lowgrade B-cell lymphomas: DNA from cases 22, 23, 24 and 25 (Table 2) and from placenta (P) was digested with Bam HI and hybridized with probe bcl-1. Rearranged bands are arrowed.
Discussion
Figure 2. Southern blot analysis of bcl-2 minor breakpoint rearrangements in centroblastic-centrocytic lymphomas: DNA from cases 4 and 6 (Table I ) and from placenta (P) was digested with Bam HI and hybridized with probe pFL-2. Rearranged bands are arrowed.
Malignant non-Hodgkin’s lymphoma can be classified according to a variety of criteria, resulting in a variety of classification systems. The major tool of the Kiel classification is the differentiation of biologically distinct entities based on detailed cytological evaluation and, thus, it is of interest to study the relation between established chromosomal translocations and these lymphoma classes. The most important finding of this study was that the chromosomal translocation t( 14;18),detected by bcl-2 rearrangement, is confined to a distinct lymphoma entity among the low-grade B-cell lymphomas: 70% of cb-cc showed this translocation, whereas none of the other cases had detectable bcl-2 rearrangement. There were no significant differences in morphology or immunophenotype between the cb-cc lymphomas that were positive and those that were negative for bcl-2 rearrangement. The overall frequency found in our study was similar to that described in the USA for follicular lymphoma^.^ Thus, it is clear that the majority of these follicular lymphomas are also of germinal centre cell origin. However, individual follicular lymphoma cases may fall into the centrocytic lymphoma group, exhibiting a follicular growth pattern but otherwise possessing a distinct cytological picture and immunophenotypic
I66
M.M,Ott et al.
properties. The overall frequency found for bcl-2 rearrangement in Japan, however, is much lower. Amakawa rt al.’ describe bcl-2 rearrangement in one-third of patients with follicular lymphoma, classified according to the Working Formulation. Because the Working Formulation is based on criteria different from those of the Kiel classification, a comparison of individual cases is difficult, particularly for follicular lymphomas. The major differencebetween cases in Japan and cases in the USA and Europe remains the overall percentage of detectable bcl-2 rearrangements. The total incidence of cb-cc lymphoma is much higher in Europe than Japan, while in the USA i t is even higher. Our results and those of Wotherspoon et al.’ indicate that there may be distinct but different transforming events in the development of germinal centre cell lymphomas. It has been shown that bcl-2 rearrangement occurs during VDJ-rearrangement in early B-cell differentiation.I6 Since the expression of the bcl-2 gene may provide cel!s with a growth advantage” it seems justified to designate t( 14;18j as the first transforming event. This translocation is exceptionally found in nonneoplastic germinal centre cellslx. Thus, a second yet unknown transforming event may induce tumour growth. Whether the bcl-2 negative cb-cc lymphomas have undergone different transforming events is also a n unsolved question. It seems possible that a minor subgroup is not detected with the available probes, which do not completely cover the entire breakpoint cluster region. In individual cases chromosomal analyses demonstrated t( 14:18)that had not been detected by molecular hybridization techniques‘. Moreover, a number of cases may be missed using Southern blot analysis due to limited sensitivity. A more sensitive method for the detection of bcl-2 rearrangements might be the polymerase chain reaction (PCR).As this method is able to detect the event also in reactive follicles”, it seemed doubtful to us that PCR would be of use in the present study. The results clearly demonstrate that t(14;18) is confined to a well-defined entity among low-grade B-cell lymphomas. With respect to the bcl- 1 rearrangement, our results are in line with a number of reports in which this translocation had been detected in cases of B-cell chronic lymphocytic leukaemia. In our sample of centrocytic lymphomas not a single case of bcl-l rearrangement could be shown, although Williams et d.” recently detected the translocation in some cases. Earlier reports have also obviously included cases of centrocytic lymphoma with t ( l l ; l 4 ) , which were in part classified as nodular poorly differentiated lymphocytic lymphomas2”. It becomes clear, therefore, that a varying percentage of
centrocytic lymphomas have the t( 1 1;14)chromosomal aberration, and further investigation may show that subgroups exist within this morphologically homogeneous entity. Our results, especially on bcl-2 rearrangement and centroblastic-centrocytic lymphomas, further stress the value of the Kiel classification and clearly document that we are dealing with biologically distinct lymphoma entities. Further investigations will have to reveal whether the absence of bcl-2 rearrangement in these lymphomas is of clinical relevance, since-in the highgrade lymphomas at least-a different clinical course was found in patients with bcl-2 rearrangement compared with those without”.
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft, grant DFG Fe 221/1-4.
References 1. Yunis JJ, Martin MO. Kaplan ME. Ensrud KM, Howe RR. Theologides OA. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphoma. N. Engl. 1. Med. 1982: 307; 1231-1236. 2. Levine EG. Arthur DC. Frizzera G. Peterson BA. Hurd DD. Bloomfield CD. There are Differences in cytogenetic abnormalities among Histologic subtypes of the non-Hodgkin’s lymphomas. Blood 1985: 6; 1414-1422. 3 . Tsujimoto Y. Cossman J. Jaffe E. Croce CM. Involvement of the brl2 gene in human follicular lymphoma. Science 1985: 228; 14401443. 4. Weiss LM. Warnke RA. Sklar J , Cleary ML. Molecular analysis of the chromosomal translocation in malignant lymphomas. N. Engl. /. Men. 1987: 317; 1185-1189. 5. Aisenberg AC. Wilkes BM, Jacobson JO. The bcl-2 gene is rearranged in many diffuse B-cell lymphomas. Blood 1988: 71; 969-9 72. 6. Amakawa R. Fukuhara S. Ohno H et al. Involvement of bcl-2 gene in Japanese follicular lymphoma. Blood 1989: 3; 787-791, 7. Yunis JJ. Oken MM. Theologides A. Howe RR. Kaplan ME. Recurrent Chromosomal defects are found in most patients with non-Hodgkin’slymphoma. Cancer Genet. Cytogertet. 1984; 13; 1728. 8. Tsujimoto Y. Junis J. Onorato-Showe L. Erikson 1. Nowell PC. Croce CM. Molecular cloning of the chromosomal breakpoint of Rcell lymphomas with the t(l1:14) chromosome translocation. Science 1984: 224; 1403-1406. 9. Wotherspoon AC. Langxing P. Diss TC. lsaacson PG. A genotypic study of low grade B-cell lymphomas including lymphomas of mucosa associated tissue (MALT)./. I’athol. 1990: 162; 1 3 5-1 40. 10. Southern EM. Detection of specific sequences among the DNA fragments separated by gel electrophoresis. \. Mol. Biol. 1975: 98; 503-517. 11. Ravetch JV. Siehenlist U. Korsmeyer S. Waldmann T. Leder P. Structure of the human immunoglobulin p locus: characterization of embryonic and rearranged J and D genes. C d l 198 1 : 27; 583-591.
brl rearrungenlent in H-cell lymphomas
12. Tsujimoto Y. Jaffe 1;. Cossman J. Gorham J , Nowell PC. Croce CM. Clustering of breakpoints on chromosome 1 1 in human B-cell neoplasms with the t( 11;14) chromosome translocation. Nuture 1985: 31 5; 340-343. 13. Tsujimoto Y . Yunis J . Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic H-cells with the t( 14;18) chromosome translocation. Sciericc, 1984; 226; 1097-1 099. 14. Cleary DA. Galili N , Sklar J. Detection of a second t(14;18) breakpoint cluster region in human Iollicular lymphomas. I. Exp. Med. 1986; 164; 3 1 5-320. 1 5. Yoshikai Y, Anatoniou D. Clark SP et al. Sequence and expression of transcripts of the human T-cell receptor [{-chain genes. Nutun, 1984: 312; 521-524. 16. Tsujimoto Y . Gorham J , Cossman J , Jaffe E, Croce CM. The t( 14; 18) chromosome translocations involved in H-cell neoplasms result from mistakes in VDJ-joining. Scicriw 1985; 229; 1390-1 393.
167
17. McDonell TJ, Deane N . Platt FM et d.bcl-l-immunoglobulin transgenic mice demonstrate extended B-cell survival and follicular lymphoproliferation. Cell 1989; 57: 79-88. 18. De long I). Limpens CEJM. Van Krieken JHJM.Van Ommen CJB. Kluin PM. The origin of follicle centre cell lymphomas. 3rd Meeting of the European Association for Haematopathology, Wurzburg. October. 1990. 19. Williams ME. Westerman CD, Swerdlow SH. Genotypic characterization of centrocytic lymphoma: frequent rearrangement of the chromosome 11 bcl-l locus. Hlood 1990; 76; 1387-1 39 I . 20. Koduru PRK, Offit K. Filippa DA. Molecular analysis of breaks in bcl-1 proto-oncogene in B-cell lymphomas with abnormalities of l l q 1 3 . Oncogene 1989: 4; 929-934. 21. Yunis J J , Mayer MG, Arnesen MA. Aeppli DP. Oken MM, I+izzera (2. bcl-2 and other genomic alterations in the prognosis of largecell lymphoma. N.Engl. ]. Med. 1989: 320; 1047- 1054.
