Different maturation capabilities of chronic lymphocytic leukaemia lymphocytes in vitro

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.     

Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1.     

The coexistence of chronic lymphocytic leukaemia and polycythaemia vera terminating in acute myeloid leukaemia

A 67-year-old man with the coexistence of CLL and PV converted after 4 years to AML is described. This rare simultaneous occurrence of both chronic lymphoid and myeloid proliferations as well as nonlymphoblastic leukaemia developing in a patient with CLL is discussed in the light of literature data.     

Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia

Tissue stromal cells interact with leukaemia cells and profoundly affect their viability and drug sensitivity. Here we show a biochemical mechanism by which bone marrow stromal cells modulate the redox status of chronic lymphocytic leukaemia (CLL) cells and promote cellular survival and drug resistance. Primary CLL cells from patients exhibit a limited ability to transport cystine for glutathione (GSH) synthesis owing to a low expression level of Xc-transporter. In contrast, bone marrow stromal cells effectively import cystine and convert it to cysteine, which is then released into the microenvironment for uptake by CLL cells to promote GSH synthesis. The elevated level of GSH enhances leukaemia cell survival and protects them from drug-induced cytotoxicity. Furthermore, disabling this protective mechanism significantly sensitizes CLL cells to drug treatment in the stromal environment. This stromal-leukaemia interaction is critical for CLL cell survival and represents a key biochemical pathway for effectively targeting leukaemia cells to overcome drug resistance in vivo.     

Karyotypes of 33 patients with clonal aberrations in chronic lymphocytic leukaemia. Review of 216 abnormal karyotypes in chronic lymphocytic leukaemia

We report on 33 unpublished patients with clonal anomalies in chronic lymphocytic leukaemia. The literature was thoroughly reviewed in order 1) to quantify the frequency of anomalies found in chronic lymphocytic leukaemia and to give new status to the rarest, 2) to determine whether a given anomaly was an additional anomaly and/or a primary anomaly, and 3) to find out whether strong associations between different anomalies exist in this disease.     

The rate of evolutionary divergence of initiation factors IF2 and IF3 in various bacterial species determined quantitatively by immunoblotting

Antibodies to Escherichia coli translational initiation factors IF2 and IF3 were used for an immunological comparison of unpurified proteins from the following genera: Salmonella, Serratia, Proteus, Aeromonas, Pseudomonas, Streptococcus, Sarcina and Bacillus. Immunological relatedness was compared by Ouchterlony double diffusion experiments and immunoblotting analysis. Immunoblotting is a quantitative technique for measuring levels of specific proteins in crude cell lysates. We have used this technique to measure immunological distance with the assumption that the levels of the various translational components are essentially the same in the different bacterial cells examined. Both immunodiffusion and immunoblotting analysis showed a similar evolutionary relationship between the various species for the two initiation factors examined: (Escherichia=Salmonella>Serratia>Proteus>Aeromonas>Pseudomonas). Little or no crossreactivity was found using either analysis with genera: Streptococcus, Sarcina and Bacillus. Using the immunoblot distance, the two initiation factors were shown to diverge at similar rates. One advantage the immunoblotting analysis has over other immunological techniques is that the antigens can be analyzed structurally. We found, for example, that the two forms of IF2 were present in all bacterial species which cross-reacted with anti-IF2, suggesting that both forms are functionally important. Because of its sensitivity, the immunoblot analysis may be more useful than other immunological techniques in studying species that are more distantly related.Abbreviations used IF initiation factor - MOPS morpholinopropane sulfonate - PAGE/SDS polyacrylamide gel electrophoresis/sodium dodecyl sulfate     

CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia.

Thrombospondin forms a 'molecular bridge' between phagocytic and apoptotic cells through interaction with alphavbeta3/CD36. We report here that engagement of CD47, a newly described thrombospondin receptor, by immobilized monoclonal antibody against CD47 or by thrombospondin induced in all B-cell chronic lymphocytic leukemia clones the cytoplasmic features of apoptosis (cell shrinkage, decrease in mitochondrial transmembrane potential and phosphatidylserine externalization) without the nuclear features (chromatin condensation, appearance of single-stranded DNA, DNA fragmentation and cleavage of poly ADP-ribose polymerase). These cytoplasmic events of apoptosis were not prevented by the addition of caspase inhibitor z-VAD-fmk, or by the presence of survival factors (such as interleukin-4 and gamma interferon) or cell activation. Morphological studies confirmed the integrity of the nucleus and showed swelling of the mitochondria. This caspase-independent death pathway may be relevant to the development of alternate therapeutic strategies in chronic lymphocytic leukemia, which remains an incurable disease.     

Expression and distribution of aphidicolin-induced fragile sites in chronic myeloid leukaemia, acute lymphocytic leukaemia and acute myeloid leukaemia

New information is revealed concerning the frequency of expression and distribution of aphidicolin-induced fragile sites in eight leukaemic patients, namely, four chronic myeloid leukaemic patients (CML), three acute lymphocytic leukaemic (ALL) patients, and one acute myeloid leukaemic (AML) patient. The cytogenetic data demonstrate a statistically significant (p less than 10(-6] increase in the frequency of aphidicolin-induced fragile sites in seven of the eight leukaemic patients compared with healthy age-matched and sex-matched controls. The chromosomal band locations of the aphidicolin-induced fragile sites from 400 metaphase spreads of these leukaemic patients reveal a nonrandom distribution in the karyotype. Some aphidicolin-induced fragile sites in these leukaemic patients were located at chromosome bands known to be induced specifically by folic acid, distamycin A, bromodeoxyuridine or azacytidine. The cross-induction of fragile sites in the leukaemic patients may be indicative of shared molecular homology in the sequence composition of nonrandom chromosomal DNA.     

Use of various mitogens and cell stimulation index in 21 patients with chronic lymphocytic leukaemia

A number of mitogens was used in 21 chronic B-lymphocytic leukaemia patients. Thymidine uptake assays were performed to evaluate cell stimulation. Phytohemagglutinin and phorbol-myristate 13 acetate were found to be the most efficient on cell proliferation. Abnormal clones were found 7 times with PMA, 4 times with PHA, twice with pokeweed, but in no case with lipopolysaccharide or proteine-A. The efficiency of PHA as a B-cell activator is likely to be due to T cell mediation.     

Fludarabine modulates composition and function of the T cell pool in patients with chronic lymphocytic leukaemia

The combination of cytotoxic treatment with strategies for immune activation represents an attractive strategy for tumour therapy. Following reduction of high tumour burden by effective cytotoxic agents, two major immune-stimulating approaches are being pursued. First, innate immunity can be activated by monoclonal antibodies triggering antibody-dependent cellular cytotoxicity. Second, tumour-specific T cell responses can be generated by immunization of patients with peptides derived from tumour antigens and infused in soluble form or loaded onto dendritic cells. The choice of cytotoxic agents for such combinatory regimens is crucial since most substances such as fludarabine are considered immunosuppressive while others such as cyclophosphamide can have immunostimulatory activity. We tested in this study whether fludarabine and/or cyclophosphamide, which represent a very effective treatment regimen for chronic lymphocytic leukaemia, would interfere with a therapeutic strategy of T cell activation. Analysis of peripheral blood samples from patients prior and during fludarabine/cyclophosphamide therapy revealed rapid and sustained reduction of tumour cells but also of CD4⁺ and CD8⁺ T cells. This correlated with a significant cytotoxic activity of fludarabine/cyclophosphamide on T cells in vitro. Unexpectedly, T cells surviving fludarabine/cyclophosphamide treatment in vitro had a more mature phenotype, while fludarabine-treated T cells were significantly more responsive to mitogenic stimulation than their untreated counterparts and showed a shift towards T_H1 cytokine secretion. In conclusion, fludarabine/cyclophosphamide therapy though inducing significant and relevant T cell depletion seems to generate a micromilieu suitable for subsequent T cell activation.     

Study of cell death in Friend leukaemia.

Cell death of splenic Friend leukaemic cells has been studied in vivo, using 125I-UdR and 3H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of 3H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8% of the splenic cellular DNA being renewed per day. Those results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

The influence of cell cycle kinetics on the radiosensitivity of Down's syndrome lymphocytes

In agreement with previous work, [60Co]gamma-irradiation shortly after phytohemagglutinin (PHA) stimulation, induces higher frequencies of chromosomal aberrations in trisomy 21 lymphocytes compared to normal controls. However, equal frequencies of chromatid aberrations are induced in fully-stimulated trisomy 21 and normal lymphocytes by irradiation during G2. We have observed that trisomic lymphocytes respond more rapidly to PHA stimulation than normal lymphocytes. Furthermore, we have observed that chromosomal radiosensitivity increases as a function of time after PHA stimulation in normal lymphocytes. When normal lymphocytes are irradiated 8 h after PHA stimulation, the frequencies of chromosomal aberrations induced are comparable to those induced in trisomy 21 lymphocytes irradiated 30 min after PHA stimulation.     

The requirement for cell surface galactosyl residues during oxidative activation of normal and chronic lymphocytic leukemia lymphocytes.

Galactose oxidase stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. Although the response of normal lymphocytes to galactose oxidase was enhanced with neuraminidase pretreatment, substantial activation of leukemic lymphocytes required pretreatment with neuraminidase. Leukemic lymphocytes exhibited maximal response to neuraminidase-galactose oxidase later than that observed in normal lymphocytes. Treatment of lymphocytes with trypsin diminished their response to galactose oxidase. When lymphocytes were pretreated with β-galactosidase to specifically remove cell surface galactosyl residues, the response to galactose oxidase was prevented. The response of normal and leukemic lymphocytes to sodium periodate was also reduced after treatment with galactose oxidase. These data support the concept that oxidation of cell surface galactosyl residues is critical during lymphocyte activation.     

Comprehensive characterization of immunoglobulin gene rearrangements in patients with chronic lymphocytic leukaemia

Previous studies have suggested a geographical pattern of immunoglobulin rearrangement in chronic lymphocytic leukaemia (CLL), which could be as a result of a genetic background or an environmental antigen. However, the characteristics of Ig rearrangements in the population from the South of France have not yet been established. Here, we studied CLL B‐cell repertoire and mutational pattern in a Southern French cohort of patients using an in‐house protocol for whole sequencing of the rearranged immunoglobulin heavy‐chain genes. Described biased usage of variable, diversity and joining genes between the mutated and unmutated groups was found in our population. However, variable gene frequencies are more in accordance with those observed in the Mediterranean patients. We found that the third complementary‐determining region (CDR) length was higher in unmutated sequences, because of bias in the diversity and joining genes usage and not due to the N diversity. Mutations found in CLL followed the features of canonical somatic hypermutation mechanism: preference of targeting for activation‐induced cytidine deaminase and polymerase motifs, base change bias for transitions and more replacement mutations occurring in CDRs than in framework regions. Surprisingly, localization of activation‐induced cytidine deaminase motifs onto the variable gene showed a preference for framework regions. The study of the characteristics at the age of diagnosis showed no difference in clinical outcome, but suggested a tendency of increased replacement and transition‐over‐transversion mutations and a longer third CDR length in older patients.     

Cutaneous squamous cell carcinoma metastatic to chronic lymphocytic leukaemia: diagnostic potential of fine needle aspiration cytology

OBJECTIVE: The phenomenon of cancer-to-cancer metastasis of cutaneous squamous cell carcinoma (SCC) and chronic lymphocytic leukaemia (CLL)/small lymphocytic lymphoma (SLL) is a rare event and only occasionally documented in the medical literature. METHODS: Two patients with SCC of the skin that were previously treated for CLL are presented. Both had palpable lymph nodes in the neck and fine needle aspiration cytology (FNAC) was performed to evaluate the pathological process. In addition, the literature on cutaneous SCC metastatic to CLL/SLL with special emphasis on the role of FNAC in this specific clinical situation was reviewed. RESULTS: On examination of the FNAC smear, cancer-to-cancer metastasis of cutaneous SCC to lymph node replaced by CLL was found. In one of the patients, light microscopy examination of the smear was complemented by immunostaining of atypical cells with cytokeratin antibodies and immunophenotyping of lymphoid cells by flow cytometry. In addition to our two patients, nine cases of cutaneous SCC metastatic to CLL/SLL have been reported in the literature, and in only one was the diagnosis made by FNAC. CONCLUSION: FNAC supported by ancillary immunocytological techniques could also be used in diagnosis of specific clinical situations such as cancer-to-cancer metastasis of the tandem of SCC-CLL/SLL.