Direct sequence analysis of TCR Vδ2–Dδ3 rearrangements in common acute lymphoblastic leukaemia and application to detection of minimal residual disease

Summary. T cell receptor δ chain (TCRδ) gene rearrangements were studied by Southern blot analysis in 36 patients with common acute lymphoblastic leukaemia, including 14 adults and 22 children. The majority of patients (68%) had either a rearrangement or deletion of one or more TCRδ genes. The most frequent rearrangement involved a partial recombination of Vδ2 to Dδ3 (55%). Dδ2–Dδ3 rearrangements were present in five patients (14%). To investigate the TCRδ rearrangement as a tumour marker in minimal residual disease studies, presentation samples from 18 patients were amplified by PCR and directly sequenced. Although the size of the Vδ2–Dδ3 junction varied by only 40 bp, sequence analysis showed extensive diversity. This was derived from four factors: deletion of the 5'end of Dδ3 gene (15/18) and 3'end of Vδ2 gene (16/18); the presence of Dδ2 sequences (6/18); insertion of N nucleotides (15/18); association of P nucleotides with intact Vδ2 and Dδ3 genes (5/18). N nucleotides were the major feature, contributing to 75% of the junction. Dδ1 sequences were not involved. Twenty base oligonucleotide probes, constructed from the junctional sequences, were capable of detecting residual tumour cells at the 10⁻⁴ sensitivity level. Cross hybridization studies confirmed the probes to be clone specific. Longitudinal studies on patients undergoing treatment were capable of detecting tumour in remission samples.     

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.     

Multiplex PCR for TCR delta rearrangements: a rapid and specific approach for the detection and identification of immature and mature rearrangements in ALL

The preferential occurrence of immature T-cell receptor (TCR) δ rearrangements (i.e. incomplete Dδ2-Dδ3 and Vδ2-Dδ3) in B-cell precursor acute lymphoblastic leukaemia (BCP ALL) and of predominantly mature rearrangements (incomplete Dδ2-Jδ1, complete Vδ1, Vδ2, Vδ3 to Jδ1) in T-lineage ALL prompted us to establish two separate multiplex PCR systems for the identification of clonal TCRδ rearrangements. PCR products of the expected size for the specific rearrangements were detectable from a dilution of 100–1000 clonal cells in 150 000 polyclonal cells. Both multiplex PCR systems were used to analyse samples from 86 childhood BCP ALLs and 30 T-lineage ALLs. The results of the multiplex PCRs were controlled by standard PCR analyses for the individual rearrangements and Southern blots, which were identical. Only immature TCRδ rearrangements were detected in BCP ALL (59%), whereas no rearrangement was found in the remaining BCP leukaemias, thus confirming the exclusive presence of immature TCRδ rearrangements in B-lineage cells. 50% of the T-lineage ALLs contained mature rearrangements, but no immature rearrangements were found. These two multiplex PCR techniques appear to be reliable and fast aids in the analysis of clonal TCRδ rearrangements in ALL.     

Molecular characterization of illegitimate TCRδ gene rearrangements in acute myeloid leukaemia

SUMMARY. Recently, we and others have shown the occurrence of TCRδ gene rearrangements in acute myeloid leukaemia (AML). In this study we describe the molecular characteristics of these rearrangements by the polymerase chain reaction (PCR) and the direct sequencing of PCR products. 11 rearrangements were characterized in blast cell samples from six patients. We found a heterogenous pattern of TCRδ gene rearrangements with involvement of Vδ1-5 regions. These findings differ from observations in T-ALL and B-cell precursor ALL, where predominantly usage of Vδ1 and Vδ2 regions has been described. Furthermore, extensive diversity of junctional sites was observed, including addition of up to 37 N nucleotides, nucleotide deletions at junction sites of Vδ and Jδ segments and usage of up to three Dδ segments. The Dδ3 fragment was the most frequently used diversity element and was found in 10 rearrangements. Nine of the 11 rearrangements were non-functional, either incomplete or out of the reading frame. Therefore a functional TCRδ cannot be expressed in these myeloid blast cells.     

Minimal residual disease analysis for the prediction of relapse in children with standard-risk acute lymphoblastic leukaemia

We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high-risk features at diagnosis, i.e. the presenting white cell count was <50 × 10⁹/l, age 1–16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRδ and TCRγ gene rearrangements and allele-specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10⁻⁴ in 78/82 (93%) probes examined.
A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow-up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed ( P <0.001, P <0.005 and P <0.005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.     

Crosslineage TCR delta rearrangements occur shortly after the DJ joinings of the IgH genes in childhood precursor B ALL and display age-specific characteristics

In order to test the hypothesis that the most immature T-cell receptor (TCR) rearrangements occur after the DJ joining of the immunoglobulin heavy chain genes (IgH), we analysed the TCR Vδ2–Dδ3 rearrangements in precursor B-cell leukaemias (PBC ALL) from 25 children younger than 3 years at disease onset and found that most of the junctional regions had N nucleotides inserted. We then selected 14 of these PCB ALLs for DJH (DJ joining of the IgH) characterization. These joining regions showed homology-directed recombination and lack of N regions, indicating absence of terminal deoxynucleotidyl transferase (TdT) activity during their rearrangement. Most leukaemias with a DJH rearrangement without N region have no, or only one, nucleotide in the joining regions of their Vδ2–Dδ3 rearrangements. The N regions of the TCR delta rearrangements displayed 'age-specific' differences: in children younger than 3 years of age the N regions were shorter than in those older than 3 years, and the rearrangements frequently contained complete segments. We conclude that the Vδ2–Dδ3 rearrangement in childhood PCB ALLs is an early event following DJH rearrangement and that it occurs shortly before or after the first hit, leading to malignant transformation.     

Distinct ongoing Ig heavy chain rearrangement processes in childhood B- precursor acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is thought to arise from the clonal expansion of a single transformed precursor cell. However, an oligoclonal Ig heavy chain (IgH) rearrangement pattern has been observed in 30% of ALL patients and was shown to be the result of ongoing rearrangement events. The extent and nature of these ongoing rearrangement processes in individual patients has so far remained obscure. We performed a detailed analysis of leukemic VHDJH rearrangements in three children with B-precursor ALL at diagnosis and one B-lymphoid blast crisis of a child with Ph+ chronic myeloid leukemia at diagnosis and relapse. The children were selected because they presented with multiple IgH rearrangements on Southern blot analysis. Polymerase chain reaction analysis of leukemic cells from two B-precursor ALL patients showed exclusively two groups of related sequences resulting from VH gene replacement events. Most VH gene replacements involved 3' located acceptor VH genes. Analysis of cells from the other B-precursor ALL patient showed exclusively related sequences as a result of VH gene joinings to a pre-existing DJH rearrangement. In the B-lymphoid blast crisis, a single germline precursor cell had generated multiple unrelated rearrangements and additional groups of related rearrangements resulting from VH to DJH joinings. Direct proof for the VH to DJH joining mechanism was obtained by amplification of the expected preexisting DJH rearrangements. Our findings suggest that the pattern of ongoing rearrangements in an individual patient reflects the IgH rearrangement status of the precursor cell at the time of malignant transformation. Sequence analysis of VHDJH rearrangements at diagnosis may therefore allow a prediction of the reliability of complementarity determining region 3 probes for the detection of minimal residual disease.     

APLASTIC ANAEMIA PATIENTS WITH CLONAL X-CHROMOSOME INACTIVATION PATTERN IN HAEMOPOIETIC CELLS EXHIBIT POLYCLONAL TCRγ AND IgH GENE REARRANGEMENTS

Previously, we reported that 13/18 (72%) female patients with aplastic anaemia (AA) exhibited a clonal X-chromosome inactivation (XCI) pattern in all haemopoietic lineages. To study the consequences of a clonal haemopoiesis for the randomness of immunoreceptor rearrangements in lymphocytes we determined clonality of T-cell receptor gamma (TCRγ) and immunoglobulin heavy chain (IgH) gene rearrangements in purified cell fractions. Peripheral blood granulocytes, monocytes, and B and T lymphocytes from 18 female patients in remission from AA were studied by PCR for randomness of XCI and rearrangement at the IgH and TCRγ locus. 13 patients were informative at the phosphoglycerate kinase-1 (PGK₁) and monoamine oxidase A (MAOA) loci. Five of them displayed an clonal XCI pattern in all lineages studied and one patient had a clonal XCI in all lineages, except the T cells. In three cases skin biopsies were also available, exhibiting a polyclonal pattern in two of them, and a reversed skewed pattern in the third. Analysis of the rearrangement patterns at the immunoreceptor loci revealed a polyclonal ladder of bands, irrespective of XCI status in the lymphocyte populations. These results demonstrate that in AA a clonal XCI pattern of the lymphoid compartment is compatible with a polyclonal immunoreceptor rearrangement pattern.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Rearrangement patterns of immunoglobulin heavy chain (IgH) and light chain genes in acute lymphoblastic leukemia and chronic myelocytic leukemia lymphoid crisis cells showing oligoclonal IgH gene rearrangements.

We investigated leukemic cells with multiple immunoglobulin heavy chain (IgH) gene rearrangements from nine B-precursor cell acute lymphoblastic leukemia (ALL) patients and three chronic myelocytic leukemia lymphoid crisis (CML.Ly-BC) patients in order to determine detailed recombination patterns of the variable (V), diversity (D), and joining (J) region genes. Southern blot study, using DNA probes for DQ52 and 5'D region genes, was useful to distinguish VDJ recombination from DJ recombination at the level of each allele. Leukemic cells from seven out of eight CD10-positive ALL patients showed biallelic VDJ recombinations. Rearrangements of Ig kappa genes were found in only one case. Leukemic cells from all of the CML.Ly-BC patients had a DJ/(V)DJ IgH genotype. These findings suggest that the multiple IgH gene rearrangements in B-precursor cell ALL occurred as a consequence of continuing V-(V)DJ rearrangements after neoplastic transformation, and were closely related to the stage of bone marrow B-precursor cell differentiation. Multiple IgH gene rearrangements in CML.Ly-BC might take place earlier in the process of IgH gene rearrangements than is the case in B-precursor cell ALL. In this sense, the genotypic oligoclonality observed in ALL and CML.Ly-BC should be regarded not as 'true', but as 'pseudo' oligoclonal leukemia.     

Beta 3-adrenergic receptor gene polymorphism and type 2 diabetes in a Caucasian population

Summary
Aim   The β₃-adrenergic receptor (β₃-AR) is suspected to play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. A mutation in the β₃-AR gene (Trp64Arg) has been associated with the capacity of weight gain and with early onset of noninsulin dependent diabetes mellitus (type 2 diabetes). In this study we investigated the prevalence of the two β₃-AR alleles in a Caucasian population and studied the association between the β₃-AR genotype and metabolic disorders (obesity and type 2 diabetes).
Methods   Genomic DNA extracted from peripheral blood leucocytes of 200 Caucasian subjects (137 subjects with and 63 subjects without type 2 diabetes). The Mva I polymorphism of β₃-AR, which detects the Trp64Arg mutation, was determined by polymerase chain reaction (PCR). We studied the correlation between the Trp64Arg mutation and the body mass index (b.m.i. kg/m²).
Results   There was no significant difference between the patients with type 2 diabetes and control subjects in the frequency of the Arg64 allele (5.5% and 4.8%, respectively). Within the group of type 2 diabetes patients were 14 subjects with the Trp64Arg mutation (b.m.i., mean ± s.d.: 31 ± 8.5 kg/m²) and 123 without the mutation (b.m.i. 29 ± 4.8). There was no association between the β₃-AR gene polymorphism and sex, obesity, blood pressure, glycohaemoglobin concentration, proteinuria.
Conclusion   Our results suggest that the Trp64Arg mutation is not a major determinant of metabolic disorders (type 2 diabetes, obesity) and chronic complications of type 2 diabetes in a Dutch population.     

Immunoglobulin VH4 gene usage in B lymphoid leukaemias

Summary. V_H4 gene rearrangements occur in a similar proportion of cases of B lineage acute lymphoblastic leukaemia (ALL) and B chronic lymphocytic leukaemia (B CLL). However, there may be differences in the pattern of V_H4 gene usage between these disorders as is the case for V_H1 gene rearrangements. To examine this, we analysed the sequences of 24 PCR-amplified clonal V_H4 gene rearrangements from a series of 15 cases of ALL and nine cases of CLL. Five distinct groups of genes were rearranged, three of which (represented by V2-1, V71-2/V71-4, V4.21) have been described in rearranged form in normal B lymphoid tissues. The most frequently rearranged gene was V4.21 which is strongly associated with autoimmune reactivity. V71-2, V71-4 and V2-1 were more frequently rearranged in CLL than ALL. The remaining two groups (represented by V4.33, V4.35) have not previously been described in rearranged form. One of these, V4.35, was seen only in ALL rearrangements. Both V4.35 and a V_H1 gene, 20P3, which is also preferentially rearranged in ALL, are located at the 3'end of the V_H locus. The location of these genes suggests that their rearrangement may be developmentally regulated in ALL. The findings in this study confirm restricted repertoires of IgH gene rearrangement in ALL and CLL. Characterization of IgH repertoires provides a means of correlating these transformed B cell populations with normal B cell developmental compartments. Moreover, the distinctive repertoires in ALL and CLL may reflect important differences in the ontogenic timing and microenvironmental milieu of tumourigenesis in these disorders.     

Extended triple intrathecal therapy in children with T-cell acute lymphoblastic leukaemia: a report from the Israeli National ALL-Studies

Owing to the increased central nervous system (CNS) relapse risk in T-cell acute lymphoblastic leukaemia (ALL), it is unclear whether preventive cranial radiation (pCRT) can be safely omitted. In this study, pCRT was replaced by extended triple intrathecal therapy (TIT) in prednisone good early responders – medium-risk (MR) group, accounting for 76% of T-ALL patients. From 1989 to 2003, 143 T-ALL patients aged 1–18 years were enrolled in the Israel National Studies (INS) 89 ( n  = 84) and INS 98 ( n  = 59) trials, based on ALL-Berlin–Frankfurt–Munster (BFM) 86/90 and ALL-BFM 95 protocols, respectively. Five-year event-free survival (EFS) of the MR group in the INS 89 ( n  = 60) was 70 ± 5·9% and the INS 98 ( n  = 43), 83·7 ± 5·6% ( P  = 0·12); the cumulative incidence (CI) of any CNS relapse was 5·0 ± 2·8% and 2·3 ± 2·3% ( P  = 0·50), respectively. There was no difference in outcome between MR patients with a white blood cell count (WBC) ≥100 × 10⁹/l treated with extended TIT ( n  = 17) or pCRT ( n  = 10). For all T-ALL patients, 5-year EFS was 61·9 ± 5·3% in INS 89 and 72·9 ± 5·8% in INS 98, ( P  = 0·21); the CI of any CNS relapse was 7·1 ± 2·8% and 1·7 ± 1·7% ( P  = 0·142), respectively. Outcome of T-ALL MR patients given extended TIT in the context of BFM-based protocols with long-term follow-up appeared to be comparable to studies in which a larger proportion of patients was irradiated, and was associated with low risk of CNS relapse, regardless of the WBC.     

Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis

The rearrangement patterns of Ig and T-cell receptor (TcR) genes were studied by Southern blot analysis in 30 precursor B-cell acute lymphoblastic leukemias (B-ALLs) and 10 T-ALLs at diagnosis and subsequent relapse. Eight precursor B-ALLs appeared to contain biclonal/oligoclonal Ig heavy-chain (IgH) gene rearrangements at diagnosis. Differences in rearrangement patterns between diagnosis and relapse were found in 67% (20 cases) of precursor B-ALLs (including all eight biclonal/oligoclonal cases) and 50% (five cases) of T-ALLs. In precursor B-ALLs, especially changes in IgH and/or TcR-delta gene rearrangements were found (17 cases), but also changes in TcR-beta, TcR- gamma, Ig kappa, and/or Ig lambda genes (11 cases) occurred. The changes in T-ALLs concerned the TcR-beta, TcR-gamma, TcR-delta, and/or IgH genes. Two precursor B-ALLs showed completely different Ig and TcR gene rearrangement patterns at relapse, suggesting the absence of a clonal relation between the leukemic cells at diagnosis and relapse and the development of a secondary leukemia. The clonal evolution in the other 23 ALL patients was based on continuing rearrangement processes and selection of subclones. The development of changes in Ig and TcR gene rearrangement patterns was related to remission duration, suggesting an increasing chance of continuing rearrangement processes with time. These immunogenotypic changes at relapse occurred in a hierarchical order, with changes in IgH and TcR-delta genes occurring after only 6 months of remission duration, whereas changes in other Ig and TcR genes were generally detectable after 1 to 2 years of remission duration. The heterogeneity reported here in Ig and/or TcR gene rearrangement patterns at diagnosis and relapse might hamper polymerase chain reaction (PCR)-mediated detection of minimal residual disease (MRD) using junctional regions of rearranged Ig or TcR genes as PCR targets. However, our data also indicate that in 75% to 90% of ALLs, at least one major rearranged IgH, TcR-gamma, or TcR-delta band (allele) remained stable at relapse. We conclude that two or more junctional regions of different genes (IgH, TcR-gamma, and/or TcR-delta) should be monitored during follow-up of ALL patients for MRD detection by use of PCR techniques. Especially in biclonal/oligoclonal precursor B-ALL cases, the monitoring should not be restricted to rearranged IgH genes, but TcR-gamma and/or TcR-delta genes should be monitored as well, because of the extensive changes in IgH gene rearrangement patterns in this ALL subgroup.     

Total body irradiation–high-dose cytosine arabinoside and melphalan followed by allogeneic bone marrow transplantation from HLA-identical siblings in the treatment of children with acute lymphoblastic leukaemia after relapse while receiving chemotherapy: a Société Française de Greffe de Moelle study

We investigated the use of a new conditioning regimen followed by allogeneic bone marrow transplantation (BMT) for treating children with acute lymphoblastic leukaemia (ALL) after relapse within 6 months of the completion of therapy. One hundred and sixteen children with acute lymphoblastic leukaemia in second or subsequent complete remission (CR) underwent allogeneic bone marrow transplantation from HLA-identical siblings after a preparative regimen comprising total body irradiation (TBI), high-dose cytosine arabinoside and melphalan (TAM regimen). The Kaplan-Meier product-limit estimate (mean ± SE) of disease-free survival (DFS) at 7 years was 59.5 ± 9% (95% confidence interval). The estimated chance of relapse was 22.5 ± 15% with a median follow-up of 88.5 months (range 51–132). 26 patients (22.4%) died with no evidence of recurrent leukaemia, mainly from interstitial pneumonitis, veno-occlusive disease or acute graft-versus-host disease (GVHD). Three factors significantly affected DFS: acute GVHD, site of relapse and, for children in second remission after a marrow relapse, the disease status at the time of transplantation. The DFS were 59.02 ± 12.6%, 37.5 ± 19.8% and 77.4 ± 15% among patients in CR2 after a marrow relapse, in CR3 or in untreated partial marrow relapse, and in CR2 after an isolated CNS relapse, respectively. The lowest DFS was seen in children with acute GVHD grades 3–4. Two significant factors were associated with relapse: the marrow status at the time of transplantation and chronic GVHD. The relapse rate was lower among children in CR2 or with chronic GVHD. We conclude that transplantation after the TAM regimen is an effective therapy for this population with acceptable toxicity, particularly for children in second remission after a very early marrow relapse, or those with early isolated CNS involvement.     

Multidrug resistance analysis in lymphoproliferative disease of large granular lymphocytes

Multi-drug resistance (MDR) phenotype contributes to the ineffectiveness of chemotherapy. P-glycoprotein (PgP) and lung resistance protein (LRP) are proteins implicated in chemoresistance. We analysed the expression of PgP and LRP respectively in 17 and 15 cases of lymphoproliferative disease of granular lymphocytes (LDGL) including 10 cases of clonal large granular lymphocytic (LGL) leukaemia, six cases of oligoclonal ( n  = 5) and polyclonal ( n  = 1) CD3⁺ lymphoproliferation and one case of CD3⁻ NK lymphocytosis. Functional PgP activity, as determined by Rh123 dye efflux assay, was found in all the patients. The mean percentage of effluxing cells was 47 ± 22%, compared to 35 ± 8% on normal lymphocytes ( P  < 0.04). The efflux was blocked in the presence of verapamil, a PgP revertant agent. A high proportion of CD57⁺ cells (66 ± 10%) from these patients expelled Rh123. Functional PgP activity was associated with expression of MDR1 mRNA. By using immunocytochemistry, LRP expression was detected in 11/15 patients (73%). 7/10 LGL leukaemia patients presented a LRP⁺/Efflux⁺ phenotype and 5/7 had LRP⁺/Efflux⁺/MDR1 mRNA⁺ phenotype. These findings suggest that the PgP⁺/LRP⁺ phenotype is frequently observed in LDGL. Its clinical relevance in aggressive cases remains to be determined.     

Clonal expansion of Vγ3/Vδ3-expressing γδ T cells in an HIV-1/2-negative patient with CD4 T-cell deficiency

Immunological investigations were carried out in an HIV-1/2//HTLV-1-negative patient with CD4 T-cell deficiency (0.357–0.6 × 10⁹/l) and expansion of γδ T cells which accounted for 26–42% of peripheral blood lymphocytes during an observation period of 3 years. Flow cytometry analyses with a panel of available Vγ/Vδ-specific monoclonal antibodies indicated that the pathologically expanded γδ population expressed Vγ2 or Vγ3 paired with Vδ3 on the surface but lacked the expression of activation antigens such as CD38 or CD71. Cloning and sequencing of RT-PCR products obtained after amplification of cDNA with Vγ-Cγ and Vδ-Cδ specific primers confirmed the presence of a clonally expanded Vγ3/Vδ3 population in the peripheral blood of this patient. Cytotoxicity assays performed with purified γδ T cells as effectors and resting or preactivated autologous CD4 T cells as targets failed to reveal evidence for autoreactive cytotoxicity of Vγ3/Vδ3 cells as a possible mechanism of CD4 T-cell deficiency in this patient.     

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells

The vitamin D₃ derived hormone 1,25 (OH)₂ vitamin D₃ (1,25 D₃) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1,25 D₃ reduced proliferation as measured by ³H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D₃ 65% inhibition was achieved at 48 n M . The MC 1288 stereoisomer of 1,25 D₃ was more potent and had the same activity at 48 n M .
The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4.
The antiproliferative effect of liposomal 1,25 D₃ was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33.
When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)₂ vitamin D₃ may offer a novel approach to treatment of myelomonocytic leukaemia.