bcl-2 gene expression in hematopoietic cell differentiation.

Nonrandom translocations with breakpoint at band q21 on chromosome 18 might cause bcl-2 gene deregulation and might contribute to neoplastic transformation in human lymphomas. As the pattern of expression of bcl-2 in hematopoietic cells is still unclear, we have measured the level of the corresponding messenger RNA (mRNA) in a variety of myeloid and lymphoid cell malignancies not usually associated with the t(14;18) translocation. Molecular genetic analysis showed that bcl-2 was rearranged in only 2 of 77 patients: one was affected by hairy cell leukemia and one by diffuse small cleaved cell lymphoma with peripheral blood invasion. Although in rare cases of myeloid leukemia fairly high levels can be found, the expression of bcl-2 appears to be typical of certain lymphoid malignancies. High levels of bcl-2 mRNA had been found, previously, in established pre-B-cell lines. However, in fresh specimens, the peak level of bcl-2 expression shifts to a more differentiated cell type, represented by the long-living B lymphocytes that are found in most cases of chronic lymphocytic leukemia. bcl-2 gene product might have a role in prolonging cell survival and, even in the absence of translocations, might contribute to some of the biologic features that are typical of this disorder.

Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.

In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV-6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.

Kappa light chain gene rearrangement in a T-cell lymphoma.

Forty-three patients were studied to determine whether light chain gene rearrangements may occur in hematopoietic cells not pertaining to the B-lineage. In only one patient, affected by T-cell lymphoblastic lymphoma, one kappa light chain allele was rearranged. Neither at the protein level nor at the RNA level the rearranged gene was expressed. These data confirm that, although rarely, kappa light chain gene rearrangements may occur in neoplastic T-cells. Furthermore, as in our patient Ig heavy chain genes retained a germline configuration, the present data demonstrate that kappa light chain gene rearrangements may occur regardless of Ig heavy chain gene arrangement.

Atypical pattern of light chain gene rearrangement in hairy cell leukemia.

Molecular genetic analysis was exploited to determine the lineage of the neoplastic cells in nine patients affected by hairy cell leukemia (HCL). In all cases the B-lineage of the cells was confirmed at the molecular level. In four cases a relatively advanced maturation stage was suggested by the expression of lambda light chain genes. Surprisingly, in two patients lambda light chain gene rearrangement was observed in spite of a germ-line kappa light chain gene. In at least one case the rearrangement was productive, as a full length messenger RNA (mRNA) was shown by Northern blot analysis and lambda light chain-restricted surface immunoglobulins (sIg) were found. These data suggest that exceptions to the hierarchy that regulates light chain gene rearrangements are not uncommon in this type of leukemia and that molecular genetic analysis should include lambda gene locus to determine more precisely the lineage origin of some leukemic cell populations.

Down-regulated c-myb expression inhibits DNA synthesis of T-leukemia cells in most patients.

We have investigated the functional relevance of c-myb expression for DNA synthesis in patients' T-leukemia cells. [3H]Thymidine incorporation assays of 32 patients' leukemia cells exposed in vitro to c-myb sense or antisense oligodeoxynucleotides served to define two groups of patients: a responder group whose leukemia cells showed 2- to 16-fold lower levels of [3H]thymidine incorporation in c-myb antisense-treated cultures than in c-myb sense-treated cultures (20 patients) and a nonresponder group whose cells showed comparable [3H]thymidine incorporation levels in either c-myb sense- or antisense-treated cultures (12 patients). Down-regulation of c-myb mRNA levels in cells exposed to c-myb antisense oligodeoxynucleotides was comparable in both groups of patients, indicating that differential sensitivity to c-myb antisense oligodeoxynucleotides was not due to differential uptake of these oligodeoxynucleotides. DNA polymerase alpha mRNA levels were down-regulated in cells from the responders but were unaffected in the nonresponder group. These results suggest that c-myb is required for DNA synthesis in cells of many but not all T-leukemia patients and that leukemia cells in which DNA synthesis is not inhibited despite down-regulation of c-myb expression may have undergone some genetic change(s) that obviate(s) the requirement for myb protein.

[Identification of human herpesvirus HHV-6 sequence in a case of Hodgkin's disease by polymerase chain reaction]. / Identificazione di sequenze del virus erpetico umano HHV-6 in un caso di malattia di Hodgkin mediante reazione polimerasica a catena.

In the last decades new approaches to the diagnosis and treatment of Hodgkin's Disease (HD) have contributed to improved rates of survival and cure but the pathogenesis of the disease still remains unknown. Data have been collected suggesting a relationship between viral infections and HD. HD patients with evidence of Epstein-Barr virus genomes in their affected tissues have been recently reported. Human Herpes Virus six (HHV-6) is a newly isolated virus, derived from patients with lymphoproliferative disorders. In order to investigate the possible role of this virus in the pathogenesis of HD we looked for a specific segment of HHV-6 genome by means of the polymerase chain reaction (PCR) in tissue samples obtained from peripheral blood and lymphnodes in HD patients: one clearly positive case has been identified. This result is the first indication of HHV-6 sequences associated with a case of HD and raises the possibility that this virus might be involved in the pathogenesis of this lymphoproliferative disorder. The relationship between HHV-6 and HD therefore warrants further investigation.

T-cell receptor genes expression in B-cell leukaemias.

Using Northern-blot analysis we have studied the expression of T-cell receptor (TCR) alpha, beta and gamma chain genes in primary cells obtained from 36 leukaemic patients. Fifteen patients had myeloid leukaemias, two had T-cell leukaemias, and 19 leukaemias corresponding to various stages of B-lymphocyte differentiation. We observed that truncated TCR beta mRNAs were produced in B-cells at relatively high levels even in the absence of detectable gene rearrangements. Ti alpha mRNAs of abnormal size were also frequently found. Such transcripts were not detectable in total RNA extracted from leukaemic myeloid cells. Factors allowing Ti alpha and beta gene transcription must be active in leukaemic pre-B and B cells but not in myeloid cells. Neither B-lineage nor myeloid leukaemias expressed TCR gamma gene at detectable levels.

[Expression of immunoglobulin genes and T lymphocyte antigen receptors in a case of acute lymphatic leukemia]. / Espressione dei geni delle immunoglobuline e del recettore antigenico T-linfocitario in un caso di leucemia linfatica acuta.

Molecular genetic analysis was performed on DNA extracted from blast cells of a patient with acute lymphoblastic leukemia. While k light chain genes were in a germ line configuration rearranged bands were found using both JH and TCR beta probes, thus making a lineage diagnosis uncertain. Northern blot analysis showed that both mu heavy chain and TCR beta chain genes were transcribed in blast cells; TCR beta gene, however, gave a truncated transcript whereas Ig heavy chain gene cluster produced a full length mu mRNA. These data show that the accuracy of lineage diagnosis can be improved if expression studies are performed in association with molecular genetic analysis.

Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia.

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

Prognostic significance of "short-term" effects of chemotherapy on MYC and histone H3 mRNA levels in acute leukemia patients.

We have found that administration of chemotherapy alters expression of growth-regulated genes in leukemia blast cells. To determine if such changes might be correlated with therapeutic outcome, we studied steady-state mRNA levels of MYC and histone H3 in the leukemic blasts of patients just prior to and 24 hr after the administration of the first doses of antileukemic drug therapy. Among nine patients with acute myelogenous leukemia, mRNA levels of MYC and histone H3 were reduced in five patients, and hematologic remission was achieved in three of these individuals. No remission was obtained in the four patients without reduction in MYC and histone H3 mRNA. Among acute lymphocytic leukemia patients, the mRNA levels of MYC and/or histone H3 were reduced by the therapy in seven of nine patients. A complete hematologic remission was obtained in five of them, and a partial remission was obtained in the other two. No remission was obtained in the patients in which MYC and H3 mRNA levels were unaffected by the therapy. These studies are of interest because they suggest that a decrease in the mRNA levels of MYC and histone H3 24 hr after a single dose of antineoplastic drugs may predict which patients will achieve complete remission; lack of reduction in these mRNAs correlates with failure to achieve remission. In addition, these studies also provide further proof of the heterogeneity of altered growth regulation among human leukemias.