Activated fos oncogene in rat embryo fibroblasts transformed by ras and myc oncogenes

Rat embryo fibroblasts (REF) were transformed by simultaneous gene transfer of the complementary oncogenes ras and myc using the calcium phosphate coprecipitation method. Cell lines derived from transformation foci expressed in addition to ras and myc cellular oncogene fos while normal REF did not express ras, myc and fos according to the hybridization methods used. The transformed cell lines produced colonies in soft agar and tumors in newborn syngeneic rats. From one tumor a cell line was established which was characterized by a high level of fos gene expression.     

Non-radioactive detection of immunoglobulin and T cell receptor gene rearrangement in acute lymphoblastic leukaemia

Rearrangements of the heavy chain immunoglobulin gene and T cell receptor beta gene were investigated in 25 patients suffering from precursor B cell acute leukaemia and six patients suffering from T cell acute leukaemia using biotinylated DNA probes. All precursor B acute leukaemia patients had IgH gene rearrangements and 63% of those studied also had TCR beta gene rearrangements. All T cell acute leukaemia patients had TCR beta gene rearrangements and germline IgH configuration. Dilution experiments indicated that DNA from leukaemic cells representing 1-2% of a tested sample could be detected using this technique which compares favourably to radioactive DNA probes.     

Transformation of rodent immortalized and embryo cells by oncogenes. II. Rat embryo fibroblasts (REF)

Rat embryo fibroblasts were transformed by co-transfer of two plasmids carrying the oncogenes ras and myc, respectively. In contrast to immortalized cells gene transfer of ras alone was not sufficient for transformation of rat embryo cells. Embryo cells transformed by both oncogenes showed an altered morphology and produced colonies in soft agar. Cell lines were established from transformation foci. These cells are characterized by a high proliferation capacity, they expressed the oncogenes ras and myc and produced regressing tumors in syngeneic newborn rats. Oncogene expression was demonstrated by in situ and blot hybridization. A cell line was established from a rat tumor induced by oncogene transformed cells. This cell line, designated FTD5, showed in addition to an increased expression of the transferred oncogenes ras and myc an elevated expression of the cellular oncogene fos.     

The use of DNA probes to monitor minimal residual disease in childhood acute lymphoblastic leukaemia

DNA probes to both the joining region (JH) of the immunoglobulin heavy chain gene (IgH) and to the beta chain of the T-cell antigen receptor complex (TCR) have been used as tumour-specific markers to monitor the rearrangements of the IgH chain gene and the TCR beta gene in the blast cells of children presenting with acute lymphoblastic leukaemia (ALL) of B or T cell origin. Blast cells from 68 children with early B cell ALL and eight with T-ALL were examined at presentation, at day 28 after commencement of therapy and at varying times thereafter. An additional 43 patients (42 with B cell ALL, one with T-ALL) were studied both at presentation, at completion of their 2-year treatment course and 3 months later. Twelve patients, drawn from both these groups, were studied at relapse as were a further eight patients in whom an extramedullary relapse had occurred. Persistence of clonally-derived cells as a predictor of early relapse was seen in the day 28 bone marrows of 11/76 newly-diagnosed children (nine early B and two T-ALL) followed by rapid, overt relapse in four of the early B ALL cases. No minimal residual disease (MRD) was detected in bone marrows from any of the 43 patients completing their 2-year treatment course, but six of these subsequently relapsed at varying time periods thereafter. Identical patterns of rearrangement at both presentation and relapse were seen in most cases. Oligoclonality, or multiple IgH chain gene rearrangements was seen in the blast cells of 15% of patients with early B cell ALL. No correlation between oligoclonality, high white count, unfavourable phenotype or abnormal karyotype could, however, be ascertained.     

α, β and γ T-cell receptor genes: rearrangements correlate with haematological phenotype in T cell leukaemias

We have studied the arrangement of the alpha, beta and gamma T cell receptor (TCR) genes in 27 patients with T cell lymphoproliferative disorders. Nine patients had acute lymphoblastic leukaemia (T-ALL), nine patients had prolymphocytic leukaemia (PLL), six patients presented with a T-CLL/T-lymphocytosis syndrome, two patients had Sezary syndrome (SS) and one patient had HTLV-I positive T-cell leukaemia/lymphoma (ATLL). alpha TCR gene rearrangement could be demonstrated by the use of three available probes in only one case. By contrast, both beta and gamma TCR gene rearrangement could be demonstrated by Southern blot analysis of DNA samples digested with appropriate restriction enzymes in the majority of cases. In general, when rearrangements were present they involved both alleles. The proportion of rearranged chromosomes was lower in T-ALL than in other forms of T-cell leukaemia and it was lower in cases with the CD4-/CD8+ phenotype than in those with a CD4+/CD8- phenotype. In three out of 34 cases of B-cell leukaemia the TCR beta-gene but not the TCR gamma-gene was rearranged, just as in two out of 26 cases of T-cell leukaemia the immunoglobulin (Ig) heavy chain but not the light chain genes were rearranged. These data suggest that development of the machinery required for gene rearrangement may precede commitment to B or T cell lineage. The use of this technique is especially useful for the classification of cases of ALL in which the cells are negative with respect to most current phenotypic markers and in cases of T cell lymphocytosis in which the finding of a gene rearrangement identifies a monoclonal cell population.     

Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization

An mRNA in situ hybridization (ISH) method which used non-radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (18–22mer) was prepared from non-germline sequences of the rearranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal individuals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used were from the same patient. Using simultaneous fluorescent immunocytochemistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm. ISH positive cells were found in 1–4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma.     

Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes

The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.     

Immunoglobulin and T-cell receptor gene rearrangement and expression in human lymphoid leukemia cells at different stages of maturation.

The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.     

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus- host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.     

Analysis of the T-cell receptor beta-chain variable-region (V beta) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific V beta segments in CD4+ T cells of the virus-positive twins.

We analyzed the T-cell receptor (TCR) V betarepertoire in human immunodeficiency virus type 1 (HIV-1)-infected individualsat different stages of disease. To circumvent the effect of HLA and other locion the expressed TCR repertoire, we compared the TCR repertoire in nine pairs ofmonozygotic twins who were discordant for HIV infection. A semiquantitativepolymerase chain reaction (PCR) assay and flow cytometry enabled us to showdistinct differences in the V beta repertoire in the HIV-positive twin comparedwith the HIV-negative twin. By combining PCR and cytofluorometry, thesedifferences were restricted to a specific set of TCR V beta segments, withmembers of the V beta 13 family perturbed in six out of seven cases and those ofthe V beta 21 family perturbed in four out of seven cases studied. Most of theother V beta families remained unchanged. Our results provide direct evidencefor a skewed TCR repertoire in HIV infection.     

Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia.

In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and -delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.     

Unique genotypic features of infant acute lymphoblastic leukaemia at presentation and at relapse.

Acute lymphoblastic leukaemia (ALL) of infants aged less than 1 year represents a group of patients with peculiar biological features, poor response to therapy and unfavourable prognosis. In order better to characterize this type of leukaemia, we have investigated the immunoglobulin (Ig) and T-cell receptor (TCR) genes configuration of 21 infants with ALL, and compared the genotypic features with the phenotypic and karyotypic data, as well as with the clinical outcome. All cases had a pre-B phenotype; 12 (57%) of them were pre-pre-B ALL (CD10-, CD19+). Six of the 16 cases evaluated (38%) displayed chromosomal abnormalities; five had the typical translocation t(4;11)(q21;23). Eleven cases presented with a white blood cell count greater than 100 x 10(9)/l. The clinical course was unfavourable in 14 patients. The genotype of this group of ALL revealed several peculiarities. (1) Of the 21 cases, six (29%) displayed a multiple rearrangement pattern at the IgH locus. (2) In three cases (15%), the light chain genes were rearranged. (3) The TCR beta and gamma genes were rearranged in only one case (one case at the TCR beta and one at the TCR gamma locus). (4) The TCR delta chain was rearranged in eight cases (40%) and rarely deleted; the rearrangements observed were those most frequently observed in B cell-precursor ALL. Two cases were evaluated both at presentation and at relapse. While the immunophenotype had remained unmodified, comparison of Ig heavy chain gene rearrangements revealed clonal variations in both cases. Taken together, these findings further underline the biological peculiarities of infant ALL compared to ALL which occurs in older children and in adults, and stress the need of differentiated and aggressive therapeutic approach for these patients.     

金葡菌L型感染C57小鼠诱发肿瘤中c-fos、c-myc、P53基因的表达及意义

目的　探讨原癌基因c-fos、c-myc和抑癌基因P53 蛋白及mRNA在诱发小鼠肿瘤及增生组织中的表达及意义。方法　应用免疫组化和核酸原位杂交等方法检测感染细菌L型后发生肿瘤和过度增生病变的小鼠组织中癌基因c -fos、c -myc和抑癌基因P53 蛋白及mRNA表达。结果　小鼠肿瘤中c -fos、c -myc和P53 蛋白表达分别为 5 0 %、5 0 %、6 0 % ,癌前病变组织中 15 38%、4 6 15 %、38 4 6 %。mRNA阳性表达分别为 5 0 %、4 0 %、5 0 %和 15 38%、30 76 %、38 4 6 % ;结果无显著性差异。结论　细菌L型感染 ,c -fos、c-myc和P53 基因的异常表达与小鼠肿瘤的发生发展有密切关系     

NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas

The chromosomal aberration t(2;5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1⁺ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin’s disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRδ-chain gene rearranged (Dδ2–Dδ3 and Vδ1–Jδ1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRδ-chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH.     

Rearrangement of the T-cell receptor delta genes in human T-cell leukemias

Two distinct types of T-cell receptors (TCR), designated alpha beta and gamma delta, have been identified on the surface of T cells. In the adult, T cells bearing the gamma delta TCR are a minority and they have the phenotype CD3+, CD4-, CD8-/+. By using appropriate probes, rearrangements of the TCR alpha, beta, and gamma genes have been extensively investigated in a variety of lymphoproliferative disorders. Because the TCR delta gene has been cloned only recently, no comparable information exists with respect to this in human leukemias. We report the analysis of the TCR delta gene configuration in 21 T-cell acute and chronic leukemias, 40 B-cell leukemias, 4 acute myeloid leukemias of difficult classification, and 12 normal controls. The TCR delta genes were structurally modified in all T-cell disorders and in germ-line configuration in all controls and all but one case of non-T-cell leukemias tested. In one case of T-chronic lymphocytic leukemia (CD3+, CD4-, CD8+) we found rearrangement and expression of TCR gamma and delta (but not alpha and beta), suggesting that leukemic transformation took place in a cell bearing a TCR gamma delta rather than a TCR alpha beta. In two cases of pre-T-acute lymphoblastic leukemia, only delta was rearranged out of the three TCR genes tested. This finding is in keeping with the suggestion that the TCR delta gene might be the first to rearrange in T cell ontogeny, and that its mode of rearrangement may play a role in the subsequent choice of the cell between production of a TCR alpha beta or gamma delta. Thus, TCR delta chain gene analysis can provide novel information of the clonal nature of T-cell disorders, particularly if the analysis of the beta and gamma genes has not been helpful.