Proceedings of The National Academy of Sciences, 1992
We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocati... more We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocation in human follicular lymphomas. Although lymphoid tissues from all transgenic mice contained high levels of human Bcl-2 protein, transgene expression was differentially regulated within the B- and T-cell compartments of lines derived from various founder mice. We have characterized the phenotypes of two lines of bcl-2 transgenic mice (line 2 and line 6) in which bcl-2 transgene expression was restricted primarily to the T- or B-cell lineages, respectively. Analysis of line 6 lymphocytes revealed a polyclonal expansion of B cells, and these B cells exhibited prolonged survival in vitro. In line 2 mice, numbers of T cells in the peripheral lymphoid tissues were more moderately elevated despite enhanced T-cell survival in vitro. Line 2 transgenic mice also showed significantly increased proportions of thymocytes with a mature phenotype. Taken together, these findings suggest different roles for bcl-2 in the in vivo regulation of B- and T-cell development and homeostasis.
Proceedings of The National Academy of Sciences, 1992
The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to ex... more The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend cellular lifespan through inhibition of apoptosis, or programmed cell death. However, the physiological role of the Bcl-2 protein in lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcl-2 protein in both cortical and medullary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resistant to apoptosis mediated by corticosteroids and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.
The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnor... more The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.
Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements... more Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements in approximately 8% of B-cell non-Hodgkin lymphomas (NHL) belonging to low-grade as well as diffuse aggressive histologies. The BCL6 gene, which encodes a zinc-finger transcription repressor protein and which maps to chromosomal band 3q27, is deregulated in t(3;14)(q27;q32) and other translocations by substitution of its transcription regulatory sequences by those of genes on the partner chromosomes. To delineate the cytogenetics and investigate the nature and consequence of BCL6 involvement in the spectrum of 3q27 aberrations seen in NHL, we analyzed a panel of 53 NHL tumors with 3q27 aberrations for BCL6 gene rearrangements and a subset of 32 of these for mutations. We identified four new recurring translocations involving 3q27, in addition to the previously recognized t(3;14)(q27;q32) and its variant, t(3;22)(q27;q11). Histologically, the 3q27 breaks were represented by 4% mantle cell lymphomas, 38% follicular center cell lymphomas, and 58% diffuse large B-cell lymphomas. Approximately 50% of the tumors exhibited BCL6 rearrangements, whereas 87.5% showed mutations in the 5' noncoding region which contains the transcription regulatory sequences. These results demonstrate that a substantial proportion of cytogenetically detected 3q27 breaks in NHLs do not represent BCL6-associated translocations. They also suggest alternate breakpoints which may lead to BCL6 deregulation, or involvement of other genes in 3q27 translocations. The frequent BCL6 mutation in these tumors is consistent with our previous observation of hypermutation of the 5' noncoding region of the gene in lymphomas arising in the germinal-center B-cells.
We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series ... more We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series of non-Hodgkin's lymphomas (NHLs), which suggested loss of candidate tumor suppressor genes (TSGs). In order to define these deletions at the molecular level, we performed an LOH analysis of a panel of paired normal and tumor DNAs comprising 13 cases of diffuse lymphoma with a large cell component (DLLC) and 18 cases of Burkitt's lymphoma (BL). The loci tested comprised eight polymorphic probes mapped to 9p (D9S33, D9S25, IFNB, IFNA, IFNW, D9S126, D9S3, and D9S19) and seven polymorphic probes mapped to 9q (D9S29, ASS, AKI, ABL, D9S10, D9S7, and D9S14). In this analysis, among cases informative for all loci in each subset, 5/13 (38%) DLLC and 4/18 (22%) BL showed LOH at 9p loci, whereas 5/13 (38%) DLLC and 3/18 (16%) BL showed LOH at 9q loci. Among the 9p loci partial homozygous or heterozygous losses were observed in 20–50% of informative cases of DLLC at D9S25, IFNB, IFNA, IFNW, D9S126, and D9S3, whereas in BL, losses at these loci ranged from 0% to 11%. Among the 9q loci, heterozygous losses were observed in >20% of informative cases of DLLC at D9S7 (23%) and D9S29 (27%), whereas no losses were seen at these two loci in BL. These data demonstrate a high level of molecular deletion in DLLC, but not in BL, suggesting that loss of one or more TSGs on chromosome 9 plays an important role in DLLC development.
Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphom... more Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphomas with a large cell component (DLLC), and was found to be of prognostic significance at the time of diagnosis. To incorporate these observations into current cytogenetic and clinical prognostic models, 76 cases of DLLC with known BCL6 status were analyzed. Cytogenetic indicators of progression, including trisomy 7, trisomy 12, del(6)(q21q25), and structural alterations of 17p were less frequent in BCL6 rearranged DLLC compared to BCL6 germline tumors. Despite a 93% overall survival at median follow-up of 30 months, a trend for continued relapse resulted in a projected freedom from progression for the BCL6 rearranged cohort of 66% at 4 years, compared to 39% for the BCL6 germline cohort. Six cases among the BCL6 rearranged group lacked additional cytogenetic indicators of progression and remained free of disease at follow-up in excess of 7 years, whereas BCL6 rearranged cases with increasing numbers of cytogenetic aberrations showed decreased intervals free from progression of disease. These results suggest that BCL6 rearrangement should be combined with other known clinical and cytogenetic indicators in prognostic analyses of patients with DLLC.
About 40 percent of non-Hodgkin's lymphomas are diffuse lymphomas with a large-cell compo... more About 40 percent of non-Hodgkin's lymphomas are diffuse lymphomas with a large-cell component (DLLC). Current therapy can induce a long-term remission in half the patients with DLLC, but more intensive treatment has the potential to improve outcome, particularly in patients at high risk for treatment failure. Clinical and cytogenetic markers can identify subgroups at high or low risk. Rearrangement of a novel candidate proto-oncogene, bcl-6, is a possible prognostic indicator in DLLC. We performed Southern blot hybridization to detect bcl-6 and bcl-2 gene rearrangement in samples of lymphoma from 102 patients with B-cell DLLC. The results were correlated with the patients' histologic features, age, disease stage, tumor sites and bulk of disease, serum lactate dehydrogenase level, and treatment outcome. Rearranged bcl-6 was found in 23 cases, and rearranged bcl-2 in 21 cases. Nineteen of the patients with rearranged bcl-6 had extranodal DLLC, two had primary splenic lymphomas, and only one had bone marrow involvement. Thirty-six months after diagnosis, the proportion with freedom from progression of disease was projected to be 82 percent (95 percent confidence interval, 66 to 98 percent) among the patients with rearranged bcl-6, as compared with 56 percent (95 percent confidence interval, 43 to 70 percent) for the patients with germ-line bcl-6 and bcl-2 and 31 percent (95 percent confidence interval, 8 to 53 percent) for the patients with rearranged bcl-2. The status of the bcl-6 gene was an independent prognostic marker of survival and freedom from disease progression in a multivariate model and added predictive value to established prognostic signs. Rearrangement of the bcl-6 gene correlated with a favorable clinical outcome in DLLC and may thus serve as a prognostic marker in patients with this form of malignant lymphoma.
Cytogenetic analysis was performed on 363 biopsy specimens with histologically confirmed diffuse ... more Cytogenetic analysis was performed on 363 biopsy specimens with histologically confirmed diffuse large B-cell lymphoma (DLBCL), consecutively ascertained at the Memorial Sloan-Kettering Cancer Center, New York, between 1984 and 1994. Among 248 samples successfully ...
The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnor... more The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.
Proceedings of The National Academy of Sciences, 1992
We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocati... more We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocation in human follicular lymphomas. Although lymphoid tissues from all transgenic mice contained high levels of human Bcl-2 protein, transgene expression was differentially regulated within the B- and T-cell compartments of lines derived from various founder mice. We have characterized the phenotypes of two lines of bcl-2 transgenic mice (line 2 and line 6) in which bcl-2 transgene expression was restricted primarily to the T- or B-cell lineages, respectively. Analysis of line 6 lymphocytes revealed a polyclonal expansion of B cells, and these B cells exhibited prolonged survival in vitro. In line 2 mice, numbers of T cells in the peripheral lymphoid tissues were more moderately elevated despite enhanced T-cell survival in vitro. Line 2 transgenic mice also showed significantly increased proportions of thymocytes with a mature phenotype. Taken together, these findings suggest different roles for bcl-2 in the in vivo regulation of B- and T-cell development and homeostasis.
Proceedings of The National Academy of Sciences, 1992
The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to ex... more The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend cellular lifespan through inhibition of apoptosis, or programmed cell death. However, the physiological role of the Bcl-2 protein in lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcl-2 protein in both cortical and medullary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resistant to apoptosis mediated by corticosteroids and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.
The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnor... more The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.
Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements... more Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements in approximately 8% of B-cell non-Hodgkin lymphomas (NHL) belonging to low-grade as well as diffuse aggressive histologies. The BCL6 gene, which encodes a zinc-finger transcription repressor protein and which maps to chromosomal band 3q27, is deregulated in t(3;14)(q27;q32) and other translocations by substitution of its transcription regulatory sequences by those of genes on the partner chromosomes. To delineate the cytogenetics and investigate the nature and consequence of BCL6 involvement in the spectrum of 3q27 aberrations seen in NHL, we analyzed a panel of 53 NHL tumors with 3q27 aberrations for BCL6 gene rearrangements and a subset of 32 of these for mutations. We identified four new recurring translocations involving 3q27, in addition to the previously recognized t(3;14)(q27;q32) and its variant, t(3;22)(q27;q11). Histologically, the 3q27 breaks were represented by 4% mantle cell lymphomas, 38% follicular center cell lymphomas, and 58% diffuse large B-cell lymphomas. Approximately 50% of the tumors exhibited BCL6 rearrangements, whereas 87.5% showed mutations in the 5' noncoding region which contains the transcription regulatory sequences. These results demonstrate that a substantial proportion of cytogenetically detected 3q27 breaks in NHLs do not represent BCL6-associated translocations. They also suggest alternate breakpoints which may lead to BCL6 deregulation, or involvement of other genes in 3q27 translocations. The frequent BCL6 mutation in these tumors is consistent with our previous observation of hypermutation of the 5' noncoding region of the gene in lymphomas arising in the germinal-center B-cells.
We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series ... more We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series of non-Hodgkin's lymphomas (NHLs), which suggested loss of candidate tumor suppressor genes (TSGs). In order to define these deletions at the molecular level, we performed an LOH analysis of a panel of paired normal and tumor DNAs comprising 13 cases of diffuse lymphoma with a large cell component (DLLC) and 18 cases of Burkitt's lymphoma (BL). The loci tested comprised eight polymorphic probes mapped to 9p (D9S33, D9S25, IFNB, IFNA, IFNW, D9S126, D9S3, and D9S19) and seven polymorphic probes mapped to 9q (D9S29, ASS, AKI, ABL, D9S10, D9S7, and D9S14). In this analysis, among cases informative for all loci in each subset, 5/13 (38%) DLLC and 4/18 (22%) BL showed LOH at 9p loci, whereas 5/13 (38%) DLLC and 3/18 (16%) BL showed LOH at 9q loci. Among the 9p loci partial homozygous or heterozygous losses were observed in 20–50% of informative cases of DLLC at D9S25, IFNB, IFNA, IFNW, D9S126, and D9S3, whereas in BL, losses at these loci ranged from 0% to 11%. Among the 9q loci, heterozygous losses were observed in >20% of informative cases of DLLC at D9S7 (23%) and D9S29 (27%), whereas no losses were seen at these two loci in BL. These data demonstrate a high level of molecular deletion in DLLC, but not in BL, suggesting that loss of one or more TSGs on chromosome 9 plays an important role in DLLC development.
Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphom... more Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphomas with a large cell component (DLLC), and was found to be of prognostic significance at the time of diagnosis. To incorporate these observations into current cytogenetic and clinical prognostic models, 76 cases of DLLC with known BCL6 status were analyzed. Cytogenetic indicators of progression, including trisomy 7, trisomy 12, del(6)(q21q25), and structural alterations of 17p were less frequent in BCL6 rearranged DLLC compared to BCL6 germline tumors. Despite a 93% overall survival at median follow-up of 30 months, a trend for continued relapse resulted in a projected freedom from progression for the BCL6 rearranged cohort of 66% at 4 years, compared to 39% for the BCL6 germline cohort. Six cases among the BCL6 rearranged group lacked additional cytogenetic indicators of progression and remained free of disease at follow-up in excess of 7 years, whereas BCL6 rearranged cases with increasing numbers of cytogenetic aberrations showed decreased intervals free from progression of disease. These results suggest that BCL6 rearrangement should be combined with other known clinical and cytogenetic indicators in prognostic analyses of patients with DLLC.
About 40 percent of non-Hodgkin's lymphomas are diffuse lymphomas with a large-cell compo... more About 40 percent of non-Hodgkin's lymphomas are diffuse lymphomas with a large-cell component (DLLC). Current therapy can induce a long-term remission in half the patients with DLLC, but more intensive treatment has the potential to improve outcome, particularly in patients at high risk for treatment failure. Clinical and cytogenetic markers can identify subgroups at high or low risk. Rearrangement of a novel candidate proto-oncogene, bcl-6, is a possible prognostic indicator in DLLC. We performed Southern blot hybridization to detect bcl-6 and bcl-2 gene rearrangement in samples of lymphoma from 102 patients with B-cell DLLC. The results were correlated with the patients' histologic features, age, disease stage, tumor sites and bulk of disease, serum lactate dehydrogenase level, and treatment outcome. Rearranged bcl-6 was found in 23 cases, and rearranged bcl-2 in 21 cases. Nineteen of the patients with rearranged bcl-6 had extranodal DLLC, two had primary splenic lymphomas, and only one had bone marrow involvement. Thirty-six months after diagnosis, the proportion with freedom from progression of disease was projected to be 82 percent (95 percent confidence interval, 66 to 98 percent) among the patients with rearranged bcl-6, as compared with 56 percent (95 percent confidence interval, 43 to 70 percent) for the patients with germ-line bcl-6 and bcl-2 and 31 percent (95 percent confidence interval, 8 to 53 percent) for the patients with rearranged bcl-2. The status of the bcl-6 gene was an independent prognostic marker of survival and freedom from disease progression in a multivariate model and added predictive value to established prognostic signs. Rearrangement of the bcl-6 gene correlated with a favorable clinical outcome in DLLC and may thus serve as a prognostic marker in patients with this form of malignant lymphoma.
Cytogenetic analysis was performed on 363 biopsy specimens with histologically confirmed diffuse ... more Cytogenetic analysis was performed on 363 biopsy specimens with histologically confirmed diffuse large B-cell lymphoma (DLBCL), consecutively ascertained at the Memorial Sloan-Kettering Cancer Center, New York, between 1984 and 1994. Among 248 samples successfully ...
The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnor... more The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.
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