Insertion of c-Myc into Igh induces B-cell and plasma-cell neoplasms in mice

We used gene targeting in mice to insert a His(6)-tagged mouse c-Myc cDNA, Myc(His), head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Emu. The insertion of Myc(His) mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, Myc(His) transgenic mice developed B-cell and plasma neoplasms, such as IgM(+) lymphoblastic B-cell lymphomas, Bcl-6(+) diffuse large B-cell lymphomas, and CD138(+) plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed Myc(His), and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of Myc(His), suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19(Arf)-Mdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that Myc(His) insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Myc-activating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.     

Inactivation of Myc in murine two-hit B lymphomas causes dormancy with elevated levels of interleukin 10 receptor and CD20: implications for adjuvant therapies

Overexpression of c-Myc and inactivation of p53 are hallmarks of human Burkitt's lymphomas. We had previously showed that transduction of murine p53-null bone marrow cells with a Myc-encoding retrovirus is sufficient for B lymphomagenesis. To address the role of Myc in tumor sustenance, we generated lymphomas induced by the Myc-estrogen receptor fusion protein (MycER). Engrafted hosts were continuously treated with the ER ligand 4-hydroxytamoxifen (4-OHT) to allow tumor formation. Subsequent inactivation of MycER via 4-OHT deprivation resulted in tumor stasis but only partial regression. At the cellular level, dormant neoplastic lymphocytes withdrew from mitosis and underwent further B-cell differentiation. Concomitantly, they up-regulated genes involved in lymphocyte proliferation and survival, most notably interleukin 10 receptor alpha (IL10Ralpha) and CD20, the target for antibody therapy with Rituxan. We found that overexpression of IL10Ralpha affords significant proliferative advantages and in 4-OHT-deprived animals correlates with eventual tumor relapse. Both dormant and relapsing tumors maintain IL10Ralpha expression suggesting that they might be sensitive to emerging drugs targeting the IL-10 pathway. Up-regulation of CD20 following Myc inactivation was also observed in immortalized human lymphocytes. Importantly, in this system, Myc(OFF)CD20(HIGH) cells were more prone to Rituxan-induced apoptosis than Myc(ON)CD20(MED). Thus, targeting Myc, while moderately effective on its own, shapes the phenotype of dormant neoplastic cells and sensitizes them to adjuvant molecular therapies.     

Elevated Mdm2 expression induces chromosomal instability and confers a survival and growth advantage to B cells

Mdm2, a regulator of the p53 tumor suppressor, is frequently overexpressed in lymphomas, including lymphomas that have inactivated p53. However, the biological consequences of Mdm2 overexpression in lymphocytes are not fully resolved. Here, we report that increased expression of Mdm2 in B cells augmented proliferation and reduced susceptibility to p53-dependent apoptosis, which was due to inhibition of p53 and suppression of p21 expression. Notably, developing and mature B cells from Mdm2 transgenic mice had an increased frequency of chromosomal/chromatid breaks and/or aneuploidy. This Mdm2-mediated genome instability occurred at a similar frequency as that in B cells overexpressing the oncogene c-Myc, but the chromosomal instability was not further enhanced when Mdm2 and c-Myc were overexpressed together. Elevated Mdm2 expression alone increased the occurrence of B-cell transformation in vivo and cooperated with c-Myc overexpression, resulting in an acceleration of B-cell lymphomagenesis. In addition, the frequency of p53 mutations was reduced, but not eliminated, in lymphomas arising in Mdm2/Emu-myc double transgenic mice. Therefore, increased Mdm2 expression facilitated B-cell lymphomagenesis, in part, through regulation of p53 by altering B-cell proliferation and susceptibility to apoptosis, and by inducing chromosomal instability.     

Lymphocyte markers in non-Hodgkin's lymphomas.

The lymphocyte marker pattern of non-Hodgkin's lymphoma cells was related to current concepts of lymphoma classification. In a series of 28 lymphomas lymphocyte markers indicated that 2 were of histiocytic origin, 2 were unclassifiable, none were derived from T cells and the remainder were B-cell neoplasms. The immunoglobulin heavy chain associated with the B-cell tumours was gamma in one case, alpha in one case but was mu in the majority of cases, reflecting the predominance of this heavy chain, together with delta chains, on normal lymph node lymphocytes in man. delta chains accompanied mu chains on the tumour cells in 6/17 lymphomas in which anti-delta staining was performed. delta chains were not found on any lymphomas other than well differentiated diffuse lymphocytic types. There was evidence of a reduction in surface immunoglobulin, Fcgamma and C3 receptors on undifferentiated lymphoma cells. T lymphocytes of normal morphology were present in all lymphomas except one, and were more numerous in follicular lymphomas than in diffuse tumours.     

Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations

Inactivating Tp53 mutations are frequent genetic lesions in human tumors that harbor genomic instability, including B lineage lymphomas with IG translocations. Antigen receptor genes are assembled and modified in developing lymphocytes by RAG/AID-initiated genomic rearrangements that involve the induction of DNA double strand breaks (DSBs). Although TP53 inhibits the persistence of DSBs and induces apoptosis to protect cells from genomic instability and transformation, the development of spontaneous tumors harboring clonal translocations has not been reported in mice that only lack wild-type Tp53 protein or express Tp53 mutants. Tp53-deficient (Tp53(-/-)) mice succumb to T lineage lymphomas lacking clonal translocations but develop B lymphoid tumors containing immunoglobulin (Ig) translocations upon combined inactivation of DSB repair factors, RAG mutation or AID overexpression; mice expressing apoptosis-defective Tp53 mutants develop B cell lymphomas that have not been characterized for potential genomic instability. As somatic rather than germline inactivating mutations of TP53 are typically associated with human cancers and Tp53 deletion has cellular context dependent effects upon lymphocyte transformation, we generated mice with conditional Tp53 deletion in lineage-committed B lymphocytes to avoid complications associated with defective Tp53 responses during embryogenesis and/or in multi-lineage potential cells and, thereby, directly evaluate the potential physiological role of Tp53 in suppressing translocations in differentiated cells. These mb1-cre:Tp53(flox/flox) mice succumbed to lymphoid tumors containing Ig gene rearrangements and immunophenotypes characteristic of B cells from various developmental stages. Most mb1-cre:Tp53(flox/flox) tumors harbored clonal translocations, including Igh/c-myc or other oncogenic translocations generated by the aberrant repair of RAG/AID-generated DSBs. Our data indicate that Tp53 serves critical functions in B lineage lymphocytes to prevent transformation caused by translocations in cell populations experiencing physiological levels of RAG/AID-initiated DSB intermediates, and provide evidence that the somatic TP53 mutations found in diffuse large B-cell lymphoma and Burkitt's lymphoma may contribute to the development of these human malignancies.     

Molecular analysis of mucosa-associated lymphoid tissue (MALT) lymphoma of ocular adnexa.

Lymphomas of mucosa-associated lymphoid tissue (MALT) are a distinct subgroup of extranodal B-cell non-Hodgkin's lymphomas. Most studies have failed to demonstrate the clonal rearrangement of BCL-1, BCL-2 or c-MYC genes for MALT lymphomas. Further, alteration of the p53 gene is rarely demonstrated in low-grade MALT lymphomas, but can be detected in high-grade disease. Lymphomas of the ocular adnexa represent approximately eight percent of all extranodal lymphomas, most of which are MALT lymphomas, but few studies had explored the alterations of BCL-1, BCL-2, c-MYC and p53 genes specifically for ocular MALT lymphomas. We investigated the changes to BCL-1, BCL-2, c-MYC and p53 genes in these lymphomas for Taiwanese patients. Clonal rearrangement for immunoglobulin heavy-chain (IgH), BCL-1, BCL-2, c-MYC and p53 genes was examined for 16 cases of ocular MALT lymphoma. Restriction-length polymorphism and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of the DNA, corresponding to exons 5 through 9, followed by DNA sequencing, were utilized to analyze the possible mutations of the p53 gene for these tumors. Thirteen of the cases revealed rearranged IgH genes using Southern blotting or PCR. No rearrangement of BCL-1, BCL-2, c-MYC or p53 genes was discovered, with point mutation of the p53 gene in one case. As for other types of MALT lymphomas, BCL-1, BCL-2 and c-MYC genes are not implicated in the pathogenesis of the ocular sub-group. Although alteration of the p53 gene is rare for low-grade ocular MALT lymphoma, its role in disease progression merits further research.     

Gene mutation analysis of sinonasal lymphomas in Indonesia

Sinonasal lymphomas comprise NK/T-cell (NKTCL) type and B-cell type with unique geographical development. In this study, mutations of p53, K-ras, c-kit, beta-catenin, and bak gene were analyzed using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) followed by direct sequencing in 41 sinonasal lymphomas (27 NKTCL and 14 B-cell type) from Indonesia. In situ hybridization study with EBER-1 probe revealed that 85% of NKTCL cases were EBV positive, but none of B-cell type was EBV positive. Frequency of mutations in p53, K-ras, c-kit, beta-catenin, and bak gene was 62.9%, 0%, 11.1%, 18.5%, and 25.9%, respectively, in NKTCL, and 71.4%, 0%, 23.1%, 21.4%, and 57.1%, respectively, in B-cell cases, showing that mutation frequency in all genes was higher in B-cell than in NKTCL cases. These findings suggest that gene mutations might be the driving-force for B-cell lymphoma, whereas combined EBV infection and gene mutations contribute to NKTCL development in Indonesia.     

p53 protein accumulation and p53 gene alterations (RFLP, VNTR and p53 gene mutations) in non-invasive versus invasive human transitional bladder cancer

Eleven non-invasive and 24 invasive transitional cell bladder cancers were analysed for molecular alterations to the p53 gene and nuclear accumulation of the p53 protein. 9% (1/11) of non-invasive rumours and 21% (5/24) of invasive tumours revealed nuclear accumulation in more than 50% of the tumour cells. PCR analysis of D17S30 showed loss of heterozygosity (LOH) in invasive tumours (3/24; 12%). Two invasive tumours harboured point mutations in exon 6 and exon 7, respectively (8%). Our results indicate that p53 protein overexpression correlates with tumour progression, p53 gene mutations and LOH detected by PCR.     

p53 Mutation in B-Cell Lymphoid Neoplasms with Reference to Oncogene Rearrangements Associated with Chromosomal Translocations

We investigated mutations of the p53 tumor suppressor gene in B-cell lymphoid neoplasms with reference to oncogene rearrangements associated with specific chromosomal translocations. These included 15 patients with a BCL1/PRAD1 gene rearrangement and/or PRAD1 overexpression, 45 with a BCL2 rearrangement, 2 with a BCL3 rearrangement, 24 with a BCL6 rearrangement, and 6 with both BCL2 and BCL6 rearrangements. Thirty-six patients lacked detectable oncogene rearrangements. Genomic DNA was isolated from involved tissues or leukemic cells obtained at diagnosis and/or at relapse, and established cell lines. Polymerase chain reaction-mediated single-strand conformation polymorphism analysis and direct sequencing were performed to analyze abnormalities of the p53 gene. We detected p53 gene alterations in 18 of 128 patients, representing 21 of the total 151 materials analyzed. In the total of 66 patients with an oncogene rearrangement studied at diagnosis, only one had a mutation; however, 6 of 37 patients studied at relapse showed p53 mutations. Sequential analysis revealed that the p53 mutation was closely associated with transformation from follicular lymphoma to large cell lymphoma, exclusively in BCL2 -positive lymphoma cases. Two of 13 mutations observed in oncogene rearrangement-positive cases and cell lines were transitions at CpG dinucleotides. In contrast, the relationship between p53 mutations and clinical behavior in oncogene rearrangement-negative cases was variable; 5 patients including one with indolent follicular lymphoma were positive for p53 mutation at initial presentation, and 2 of the 5 showed prolonged disease-free survival. Our findings suggest that p53 alteration exhibits diverse functions in the development and progression of B-cell tumors related to the presence or absence of oncogene rearrangement, and that chemotherapy-related influences may be involved in the occurrence of progression-associated p53 mutations.     

SV40 and p53 as team players in childhood lymphoproliferative disorders

Simian virus 40 (SV40) is known to be potently oncogenic and can induce several types of tumours, such as lymphoma. p53 was discovered as a cellular partner of the SV40 large T-antigen, the oncoprotein of this virus. There have not been many studies on SV40 and p53 in lymphomas and the ones that exist, are controversial. A comparison of these two components in lymphoma has not been reported previously. We examined 91 lymphomas [60 B-cell non-Hodgkin's lymphomas (B-NHLs), 19 B-cell acute lymphoblastic leukemias (B-ALLs), 7 B-cell precursor acute lymphoblastic leukemias and 5 T-cell acute lymphoblastic leukemias] for the presence of SV40. Overall, 40 samples from 12 B-NHL/19 B-ALL patients were additionally investigated for p53 mutation in the hot-spot exons 5 to 8. Overall, we found 62/91 lymphomas to be SV40-positive, among them 16/19 B-ALLs and 38/60 B-NHLs. SV40 was absent in 147 of the 149 blood control samples. We found 11 p53 mutations in 19 B-ALL patients: 5 in exon 5 (codons 132, 141, 143, 155 and 181), 4 in exon 7 (codons 236, 238 and 248), 2 in exon 8 (codon 273). In B-NHL patients we found p53-mutations in 9/12 samples: 6 of these in 3 lymph nodes (LNs). One LN harboured 3 different p53 mutations: Exon 5 (codon 132), exon 6 (codon 213) and exon 8 (codon 288). Another LN showed 2 different p53 mutations: Exon 6 (codon 213) and exon 8 (codon 285). Except for 1 nonsense mutation in an LN of a B-NHL patient, all 20 mutations were missense mutations, 2 were homozygous, both found in B-NHL-samples, and one of these (codon 175) is known to cause the global denaturation of p53. All occur in the DNA-binding domain of p53. All specimens showing a p53 mutation, were SV40-positive. p53 mutaions found in LNs of B-NHL patients harbour high SV40 copy numbers. Our data strongly support an important role for SV40, as well as a strong association of SV40 and p53 in childhood lympho-proliferative disorders.     

Sensitivity to myc-induced apoptosis is retained in spontaneous and transplanted lymphomas of CD2-mycER mice

To study the effects of the Myc oncoprotein in a regulatable in vivo system, we generated lines of transgenic mice in which a tamoxifen inducible Myc fusion protein (c-mycER) is expressed under the control of the CD2 locus control region. Activation of the Myc oncoprotein resulted in both proliferation and apoptosis in vivo. Lines with a high transgene copy number developed spontaneous lymphomas at low frequency, but the tumour incidence was significantly increased with tamoxifen treatment. Surprisingly, we found that cellular sensitivity to Myc-induced apoptosis was retained in tumours from these mice and in most lymphoma cell lines, even when null for p53. Resistance to Myc-induced apoptosis could be conferred on these cells by co-expression of Bcl-2. However, acquired resistance is clearly not an obligatory progression event as sensitivity to apoptosis was retained in transplanted tumours in athymic mice. In conclusion, lymphomas arising in CD2-mycER mice retain the capacity to undergo apoptosis in response to Myc activation and show no phenotypic evidence of the presence of an active dominant inhibitor.     

Intraclonal molecular heterogeneity suggests a hierarchy of pathogenetic events in Burkitt's lymphoma

Background: Burkitt's lymphoma is a B-cell neoplasm characterized by a chromosomal translocation involving the c-myc gene. BL may carry, besides the c-myc translocation, several other lesions including a) mutations in c-myc, b) mutations in bcl-6, c) mutations in p53 and d) EBV genomes. In this report we describe a unique study of the timing of these genetic lesions during the evolution and progression of Burkitt's lymphoma.Materials and methods: From each of two patients with Burkitt's lymphoma, we established three different cell lines – from different sites or at different times in the clinical course of the disease (diagnosis and relapse). Chromosomal aberrations were analyzed by karyotyping and the presence of molecular lesions determined by Southern blot, PCR, SSCP and sequence analyses.Results: In each patient all the clones carry identical c-myc translocations, identical bcl-6 status (wild type or mutant) and the same productive VDJ rearrangement. However, within each individual patient, we could demonstrate the presence of intraclonal variation with respect to EBV, p53 mutations and c-myc mutations.Conclusions: c-myc translocation and bcl-6 mutations appear to be early events, mutations in the coding region of c-myc occur early but are an ongoing event, while mutations in the p53 gene seem to occur later. Discrete clonal bands reflecting independent EBV infection were observed in the cell lines from one HIV-associated Burkitt's lymphoma, suggesting the possibility that EBV infection may occur as a late event, at least in some HIV associated lymphomas.     

Mutations of p53, c-kit, K-ras, and beta-catenin gene in non-Hodgkin's lymphoma of adrenal gland.

Malignant lymphoma of the adrenal gland is a rare disease, usually with diffuse large cell morphology and B-cell immunophenotype, and often associated with Epstein-Barr virus infection. In this study, mutations of p53, c-kit, K-ras, and beta-catenin gene were analyzed in 17 cases (13 males and four females with ages ranging from 25 to 84 years) of such lymphomas by polymerase chain reaction-single strand conformation polymorphism followed by direct sequencing. Selected exons in each gene, representing hot spots, were analyzed. All 44 mutations detected were single-nucleotide substitutions and 33 were missense mutations. Nineteen mutations were detected in exon 5 and / or 7 of the p53 gene in nine of 17 cases (52.9%) and 21 in exon 11 and / or 17 of the c-kit gene in 10 of 14 cases (71.4%). Bilateral adrenal lesions in one case who had not received any adjuvant therapy showed different mutational patterns of the p53 and c-kit genes, suggesting different clonal evolution of lymphoma between the left and right sides. Mutation at codon 13 of the K-ras gene was detected in one of 14 cases (7.1%), and in exon 3 of the beta-catenin gene in three of 12 cases (25%). All but one mutation were transition mutations, indicating that some endogenous mutagens act in lymphomagenesis in the adrenal gland. Our results suggest that p53 and c-kit gene mutations might play a role in adrenal lymphomagenesis.     

Mutations of p53, c-kit, K-ras, and β-Catenin Gene in Non-Hodgkin's Lymphoma of Adrenal Gland

Malignant lymphoma of the adrenal gland is a rare disease, usually with diffuse large cell morphology and B-cell immunophenotype, and often associated with Epstein-Barr virus infection. In this study, mutations of p53 , c- kit , K- ras , and β-catenin gene were analyzed in 17 cases (13 males and four females with ages ranging from 25 to 84 years) of such lymphomas by polymerase chain reaction-single strand conformation polymorphism followed by direct sequencing. Selected exons in each gene, representing hot spots, were analyzed. All 44 mutations detected were single-nucleotide substitutions and 33 were missense mutations. Nineteen mutations were detected in exon 5 and/or 7 of the p53 gene in nine of 17 cases (52.9%) and 21 in exon 11 and/or 17 of the c-kit gene in 10 of 14 cases (71.4%). Bilateral adrenal lesions in one case who had not received any adjuvant therapy showed different mutational patterns of the p53 and c- kit genes, suggesting different clonal evolution of lymphoma between the left and right sides. Mutation at codon 13 of the K- ras gene was detected in one of 14 cases (7.1%), and in exon 3 of the β-catenin gene in three of 12 cases (25%). All but one mutation were transition mutations, indicating that some endogenous mutagens act in lymphomagenesis in the adrenal gland. Our results suggest that p53 and c- kit gene mutations might play a role in adrenal lymphomagenesis.     

Association of p53 mutations,microvessel density and neoangiogenesis in pairs of colorectal cancers and corresponding liver metastases

p53 suppressor gene mutations are a well known step which occurs in the late stages of the complex tumourigenesis of colorectal cancer. A deregulation of p53 protein function may be associated with increased neovascularization and aggressive tumour growth. In vitro studies have shown that these genetic alterations cause a loss of wild-type p53-induced anti-angiogenetic control and could possibly induce expression of the neoangiogenic vascular endothelial growth factor (VEGF). Therefore, this in vivo study was performed to assess p53 mutations, i.e. hot spots in exons 4-9, in primary colorectal cancers and in corresponding liver metastases in order to test whether there is an association between p53 mutated tumours with increased microvessel density (MVD) and VEGF overexpression. Twenty-two tissue samples taken from primary colorectal cancers and the corresponding liver metastases were immediately snap-frozen in liquid nitrogen and fixed in formaldehyde. After DNA extraction exons 4-9 were amplified and directly sequenced. Cryostat sections were stained immunohistochemically using antibodies against VEGF, CD34, and p53 protein. A modified semiquantitative Weidner score and interactive computerized image analysis was used to assess MVD. Overexpression of immunohistochemically detected p53 protein was found in 7 of the 11 primary tumours and liver metastases (64%). Sequencing showed 3 out of 11 primary tumours (27%) and 5 out of 11 liver metastases (46%) to have p53 point or frameshift mutations; these samples tested immunohistochemically positive for p53 protein. Two p53 mutations in samples of liver metastases were not detectable in the corresponding primaries. We detected one frameshift mutation in exon 4 that has not yet been described in the literature. Tumour samples with p53 mutations and increased VEGF immunoreactivity were associated with higher MVD (p<0.01 and p<0.05, respectively). However, there was no association detected immunohistochemically between p53 and MVD as well as p53 mutations and VEGF overexpression. Our data demonstrate specific genetic alterations in the coding regions of p53 suppressor gene in both primary colorectal cancers and corresponding liver metastases, these alterations are associated with an increase in MVD, but not in VEGF overexpression. In addition, a novel frameshift mutation in both colorectal cancer and metastasis is described.