Anna Teresa Maiolo

B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-κB p50

Abstract A B cell lymphoma-associated chromosomal translocation, t(10;14)(q24;q32), juxtaposes the immunoglobulin Cα 1 locus to a novel gene, lyt -10. The normal lyt -10 cDNA codes for a 98 kd protein which displays amino-terminal homology with the rel (DNA-binding) domain of the NF-κB-rel family of transcription factors and carboxy-terminal homology with the NF-κB p50 precursor protein, including the putative proteolytic cleavage domain (poly-G) and the ankyrin-like repeat domains. The lyt-10 protein can bind to κB sequences in vitro, although with different specificity from NF-κB p50, and in vitro DNA-binding is activated by removal of the ankyrin domain. Chromosomal translocation generates an lyt -10- Cα 1 fusion gene coding for a protein that retains the rel effector domain, lacks the ankyrin regulatory domain, and binds κB sequences in vitro, suggesting its constitutive activation in vivo. Analogous rearrangements of the lyt -10 gene have been found in an additional three cases of lymphoid neoplasia. The lyt -10 gene defines a new subfamily (rel/poly-G/ankyrin) of NF-κB-rel transcription factors with potential for oncogenic activation in human cancer.

Thrombotic and hemorrhagic complications in essential thrombocythemia. A retrospective study of 103 patients

A retrospective study of 103 patients with essential thrombocythemia was carried out to evaluate the incidence of thrombohemorrhagic complications and establish whether there were any correlations between these events and clinical or laboratory data. At onset or during the course of the disease, 26 patients (25.2%) presented thrombotic and 12 (11.6%) hemorrhagic complications: among the latter, six patients had gastrointestinal bleeding during antiaggregant therapy. No significant correlations were observed between thrombohemorrhagic complications and platelet count, age, sex, platelet function, bleeding time, or therapeutic regimen. However, there was a statistically significant correlation between a positive patient history for thrombotic events and an increase in thromboses. In agreement with other authors, it is believed that the best approach in asymptomatic patients is strict surveillance without treatment. Chemotherapy and/or treatment with antiaggregant agents should be reserved for symptomatic patients or patients with a positive history for thrombotic events.

Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma.

Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the α-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

CEREBRAL BLOOD FLOW AND METABOLISM IN THERAPEUTIC INSULIN COMA.

Abstract The cerebral blood flow, cerebral metabolic rate of oxygen and glucose and R.Q. were studied in 14 subjects suffering from schizophrenia in the active stage, during a cycle of Sakel insulin therapy. The basal conditions, the state of marked hypoglycemia, the deep coma and the return of consciousness after administration of glucose were studied. Starting from basal data within normal limits, a significant increase of cerebral blood flow in coma was particularly observed. Furthermore, while reduction of the cerebral glucose utilization was noted, increasing progressively from the state of marked hypoglycemia at 90′ to coma, the oxygen utilization did not show any considerable variations on the average, although falling in some cases. These results are discussed in relation to the data reported in the literature.

Immunohistochemical analysis of cyclin D1 shows deregulated expression in multiple myeloma with the t(11;14)

The t(11;14)(q13;q32) chromosomal translocation, the hallmark of mantle cell lymphoma (MCL), is recurrently found in multiple myelomas (MM) by means of conventional cytogenetics. Unlike MCL, recent molecular studies of MM-derived cell lines with t(11;14) have indicated that the breakpoints are highly dispersed over the 11q13 region; however, the fact that cyclin D1 is generally overexpressed in these cell lines suggests that this gene is the target of the translocation. To evaluate further the involvement of cyclin D1 in MM, we used immunohistochemistry and fluorescence in situ hybridization to investigate cyclin D1 expression and the presence of chromosome 11 abnormalities in a representative panel of 48 MM patients (40 at diagnosis and 8 at relapse). Cyclin D1 overexpression occurred in 12/48 (25%) of cases; combined immunohistochemistry and fluorescence in situ hybridization analyses in 39 patients showed cyclin D1 positivity in all of the cases (7/7) bearing the t(11;14), in two of the 13 cases with trisomy 11, and in one of the 19 cases with no apparent abnormalities of chromosome 11. Our data indicate that the t(11;14) translocation in MM leads to cyclin D1 overexpression and that immunohistochemical analysis may represent a reliable means of identifying this lesion in MM.

Analysis of FGFR3 gene mutations in multiple myeloma patients with t(4;14)

The t(4;14)(p16.3;q32) in multiple myeloma (MM) leads to an apparent deregulation of the FGFR3 and WHSC1/MMSET genes. FGFR3 mutations, known to be associated with genetic skeletal disorders, have also been identified in a few cases of MM (mainly cell lines) with t(4;14). We investigated FGFR3 mutations in a series of 53 MM cases; 11 cases with t(4;14) and FGFR3 overexpression were analysed using reverse transcription polymerase chain reaction, while the remaining cases were studied at DNA level. The Arg248Cys mutation, which is associated with some lethal forms of skeletal disorders, was found in one case with t(4;14). Our results indicate that FGFR3 mutations occur in only a small fraction of MM cases with t(4;14).

Constitutive expression of lymphoma-associated NFKB-2/Lyt-10 proteins is tumorigenic in murine fibroblasts

The NFKB-2 (Lyt-10) gene codes for an NF-κB-related transcription factor containing rel-polyG-ankyrin domains. Rearrangements of the NFKB-2 locus leading to the production of 3′ truncated NFKB-2 proteins are recurrently found in lymphoid neoplasms, particularly cutaneous lymphomas. Such mutant NFKB-2 proteins have lost the ability to repress transcription that is typical of NFKB-2 subunit p52, and function as constitutive transcriptional activators. To verify whether the expression of abnormal NFKB-2 proteins can lead to malignant transformations in mammalian cells, we transfected human lymphoblastoid cell lines and murine fibroblasts (Balb/3T3) with expression vectors carrying the cDNAs coding for normal NFKB-2p52, Lyt-10Cα or LB40 proteins, which are representative of the abnormal types found in lymphoma cases. The expression of both normal and mutant NFKB-2 proteins has a lethal effect on lymphoblastoid cells and a cytotoxic effect was also observed in murine fibroblasts. The fibroblast cell lines expressing Lyt-10Cα or LB40, but not those expressing normal NFKB-2p52, were capable of forming colonies in soft agar. The analysis of individual clones revealed that cloning efficiency correlated with the expression levels of the abnormal proteins. Injection of the Lyt-10Cα-transfected Balb cells in SCID mice led to tumor formation in all of the animals, whereas no tumors were observed in the mice injected with control or NFKB-2p52-transfected cells, thus indicating that abnormal NFKB-2 protein expression is tumorigenic in vivo. Our results show that mutant NFKB-2 proteins can lead to the transformed phenotype, and support the hypothesis that alterations in NFKB-2 genes may play a role in lymphomagenesis.

Frequency of RAS and p53 mutations in acute promyelocytic leukemias

The frequency of RAS and p53 mutations was investigated in 30 acute promyelocytic leukemias by single strand conformation polymorphism analysis and direct sequencing of genomic DNA. Only two cases bore N-RAS codon 12 mutations and none had p53 mutations responsible for aminoacid substitutions. It would, therefore, seem that neither RAS nor p53 are involved in acute promyelocytic leukemogenesis.

Identification of a tumor-associated mutant form of the NF-κB RelA gene with reduced DNA-binding and transactivating activities

Alterations of NF-κB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for κB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-κB members, such as p50, cREL and IκBα. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma

Chromosomal rearrangements of the MYC locus, which often involve the IG loci, are recurrent events in multiple myeloma (MM) and plasma cell leukemia (PCL). We used dual‐color fluorescence in situ hybridization (FISH) to characterize the breakpoint locations of chromosomal translocations/rearrangements involving the MYC locus at 8q24 found in a panel of 14 MM cell lines and 70 primary tumors (66 MM and 4 PCL). MYC locus alterations were observed in 21 cases: MYC/IG (mainly IGH@) fusions in 11 cell lines and three patients (2 MM and 1 PCL), and extra signals and/or abnormal MYC localizations in seven patients (5 MM and 2 PCL). Fourteen of these cases were investigated by FISH analyses by use of a panel of BAC clones covering about 6 Mb encompassing the MYC locus. The breakpoints were localized in a region 100–250 kb centromeric to MYC in four cases, a region 500–800 kb telomeric to the gene in four cases, and regions ≥ 2 Mb centromeric or telomeric to MYC in five cases. Two different breakpoints were detected in the KMS‐18 cell line, whereas the insertion of a MYC allele was found in a complex t(16;22) chromosomal translocation in the RPMI8226 cell line. Our data document a relatively high dispersion of 8q24 breakpoints in MM.