A limited role for TP53 mutation in the transformation of follicular lymphoma to diffuse large B-cell lymphoma.

The role of TP53 mutation in transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (t-FL) was examined in a panel of 91 lymph node biopsies derived from 29 patients pre- and post-transformation. The entire TP53 coding sequence was screened and immunocytochemistry performed to determine expression of p53 and its key regulator MDM2. A total of 10 mutations were detected in eight patients (28%), although none were present at FL diagnosis. Mutations were not detected solely at the time of transformation; in three patients, mutated TP53 arose in at least one antecedent FL sample (6 months, 2.5 years and 4 years prior to transformation). Loss of heterozygosity at the TP53 locus occurred in 2/20 informative patients (only in t-FL samples). p53 staining was positive in 82% (9/11) of available biopsies with a missense mutation, and negative in 71% (45/63) with wtTP53. MDM2 expression was significantly higher in t-FL samples (mean 72% positive; 95% confidence interval (95% CI) 68-76%) than FL (mean 58% positive; 95% CI 54-62%) (P<0.001) but did not correlate with TP53 status. TP53 mutation has only a limited role in the transformation of FL, exerting a heterogeneous influence upon phenotypic change. In contrast, dysregulation of MDM2 is frequent and may provide a more rational therapeutic target..

Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma.

PURPOSE: To determine the incidence and frequency of the Bcl-2/IgH rearrangement in the peripheral blood of normal individuals to define the potential complication this may pose for the molecular monitoring of disease in patients with follicular lymphoma (FL). MATERIALS AND METHODS: The incidence and frequency of the major breakpoint cluster region rearrangement in DNA extracted from peripheral blood or lymphoblastoid cell lines from 481 normal individuals was determined using a TaqMan real-time polymerase chain reaction assay (PE Applied Biosystems, Foster City, CA). RESULTS: Twenty three percent of samples were positive for the Bcl-2/IgH rearrangement, with approximately 3% of these at levels of more than 1 in 10(4) cells. CONCLUSION: The presence of circulating Bcl-2/IgH+ cells, other than those derived from the malignant clone, could confound the detection and quantitation of minimal residual disease in patients with FL, particularly at low levels of tumor burden.

Normal values for the different classes of venous blood mononuclear cells defined by monoclonal antibodies.

We present the data obtained from routine quantitation of normal venous blood mononuclear cells using 19 monoclonal antibodies against defined mononuclear cell surface antigens. The results indicate different values for percentage and absolute numbers of T lymphocytes and of T lymphocyte subsets depending on the monoclonal antibodies used to quantify these populations. In most instances the total number of identified cells was significantly less than the total number of recovered blood mononuclear cells, which suggests the existence in blood of a null cell population. Evidence is advanced to support previous observations that at least a proportion of this population is of B cell lineage, expressing cytoplasmic immunoglobulin but lacking other class or lineage specific markers. The value of a diverse monoclonal panel in routine quantitation of peripheral blood mononuclear cells is discussed.

Lymphoid cells, small cell lung cancer cells and epithelial cells share a membrane determinant which effects cell proliferation.

Two monoclonal antibodies (mAb) 1D1 and 2A6 were obtained from a fusion following hyperimmunization with prolymphocytic leukaemia (PLL) B cells. These mAb stain a minority of B- and T-cell leukaemias and approximately 20% of peripheral blood and tonsil T and B cells, activated with a variety of mitogens. Interestingly, all small cell lung cancer (SCLC) and bladder carcinoma lines examined were also stained by both mAb. On sections of normal and malignant tissue 1D1 and 2A6 show strong but distinct reactivity with epithelium, and in the case of ID1 staining is also present on endothelial tissue. The addition of purified 1D1 and 2A6 to Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCL) and SCLC lines caused a significant increase in the rate of proliferation of these cells. Capping experiments have suggested that these two mAb, despite showing significantly different staining profiles, probably recognize distinct epitopes of the same surface molecule. These studies confirm that a lymphoid-cell associated antigen(s) detected by mAbs 1D1 and 2A6 is expressed on a wide range of normal and malignant cells and related cell lines.

Functional interaction between B cell subpopulations defined by CD23 expression.

Using the CD23 monoclonal antibody (mAb) MHM6 and sheep anti-mouse Ig bound to magnetic beads we have obtained highly purified populations of MHM6+ and MHM6- tonsil B cells. We have found that the increased expression of MHM6 reactivity seen on B cells after activation results from up-regulation of antigen on cells already weakly positive and not from expression of new antigen on the previously negative population. The strong proliferative responses of MHM6+ cells seen in the presence of anti-IgM (alpha mu) and interleukin 4 (IL4) or the CDw40 mAb G28-5, and with Staphylococcus aureus Cowan I (SAC), and to a lesser extent with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), resemble that seen among unfractionated B cells. In contrast, the MHM6- population cultured alone responds only weakly to alpha mu + G28-5 or SAC and exhibits virtually no response to alpha mu + IL4 or TPA. With all these mitogenic stimuli, tritiated thymidine uptake by the MHM6- population is augmented three- to sixfold by the addition of mitomycin C (MC)-treated MHM6+ cells. Pretreatment of cells with anti-leukocyte functional antigen 1 mAb has little effect on the subsequent proliferation of the MHM6- population but shows cell contact to be critical for the proliferation of MHM6+ cells. Such pretreatment has revealed that the functional interaction observed between MHM6+ and MHM6- cells is dependent on both cell contact and the presence of an MHM6+ cell-derived soluble component. We have found that addition of soluble CD23, purified from Epstein-Barr virus-transformed lymphoblastoid cell line supernatant, increases the proliferative response of MHM6- tonsil B cells to mitogenic stimuli in the presence of inactivated MHM6+ cells but has no effect on proliferation when MHM6+ cells are absent. By way of contrast to normal B lymphocytes, we have examined functional responses of prolymphocytic leukemia (PLL) B cells. Although these cells, when freshly isolated, show comparable levels of CD23 expression to normal B cells, this expression is not increased upon activation. In addition, in contrast to normal B cells, the PLL MHM6- population cultured alone shows a strong proliferative response to various mitogenic stimuli, comparable to that of MHM6+ or unfractionated cells, and this response is not augmented by the addition of MC-treated MHM6+ cells. Thus, a novel functional interaction is described between normal, but not leukemic, B cell populations defined by their expression of CD23.(ABSTRACT TRUNCATED AT 400 WORDS)

The variable occurrence of CD45RA on CD8+ thymocytes correlates with the presence of Mtv sequences; its expression on other thymocytes is rare.

While all thymocytes express CD45, only a small fraction (less than 3% in mice) bear the high molecular weight isoform, CD45RA. It has been suggested that these cells alone constitute the generative thymocyte lineage and should, therefore, occur within every ontogenic subset. To test this, we determined CD45RA expression among thymocyte subsets defined by CD4 and CD8. In some strains, exemplified by C57BL/Icrf, very few (less than 0.2%) T thymocytes expressed CD45RA and were mostly CD4-CD8- or CD4+CD8+, inconsistent with them constituting the generative lineage. In others, exemplified by BALB/c, CD45RA was expressed on up to 3% of T thymocytes, which were mostly CD4-CD8+. The limited occurrence of CD4-CD8+CD45RA+ thymocytes suggests that they are a nonconstitutive subset playing a role in the thymus of only some strains. Their occurrence correlates with that of Mtv proviruses within the mouse genome; however, their T cell receptor V beta repertoire is diverse, suggesting they are not uniquely selected by Mtv superantigens. We propose that they may be mature CD8 T cells, possibly responsible for introducing viral superantigens into the thymus.

Characterization of constitutive and strain-dependent subsets of CD45RA+ cells in the thymus.

We have previously shown that the occurrence of CD45RA+ adult mouse thymocytes is strain-dependent, e.g. constituting approximately 0.6% in C57BL/Icrf and approximately 2.5% in BALB/c (Huby, R. and Goff, L., 1992. Eur. J. Immunol. 22:1659). Here we show that irrespective of strain, the thymus contains approximately 0.6% CD45RA+ cells which are composed of slg+ B cells (approximately 0.4%), slg- CD4-CD8- cells (< 0.2%), and CD4+ CD8+ cells (< 0.2%). In some strains an additional CD45RA+ population, representing up to approximately 2% of all thymocytes, is present and has a CD4-CD8+ phenotype. It is this CD4-CD8+CD45RA+ subset which is responsible for the observed strain difference. In BALB/c mice, this additional population comprises approximately 90% of the CD45RA+ thymic cells. They are larger than the majority of thymocytes, with a size typical of mature, single positive cells (CD4+CD8- or CD4-CD8+). Further phenotyping for co-expression of other maturation markers showed them to be distinctive; they are CD3int-hi, i.e. as bright as other CD8 single positives, which are dimmer than CD4 single positives. In addition they are CD44hi, MEL-14dim and hi, Thy-1lo, HSAlo/-, and PNAlo, suggesting them to be amongst the most mature cells in the thymus. This was corroborated by their phenotypic similarity to CD45RA+ lymph node T cells. Furthermore, in BALB/c adult thymus sections, CD45RA+ cells are localized mainly in the medulla, consistent with a mature phenotype. Comparable with most mature thymocytes, cell cycle analysis revealed this subset to be composed of resting (G0/G1) cells. The CD4-CD8+CD45RA+ cells are amongst the most mature thymocytes and yet are indistinguishable from peripheral T cell counterparts; the possibilities that they are mature thymocytes due to exit the thymus, or that they may represent recirculating peripheral T cells, are discussed.

Kinetics of thymocyte subset development and selection revealed by cyclosporin A treatment.

Cyclosporin A (CsA) inhibits the development of mature thymocytes from their CD4+ CD8+ precursors, but may allow autoreactive cells to mature. Using 3-color flow cytometry, we have followed the progressive development of thymocytes, including potentially autoreactive cells, during CsA treatment. Numbers of CD4+ CD8+ CD3high thymocytes dropped immediately, suggesting that the generation of these mature thymocyte precursors, normally dependent upon positive selection, was inhibited by CsA. Numbers of CD4+ CD8- thymocytes also declined rapidly, but CD4 - CD8+ thymocytes were unaffected for 2 days, suggesting that the mature single-positive subsets are not symmetrically derived from a common GsA-sensitive precursor. An exceptional subset of CD8 SP thymocytes, expressing CD45RA, did not respond to CsA for about 10 days, indicating that they are distantly derived from a CsA-sensitive precursor. Apoptosis of TCR-V beta 3 + thymocytes caused by Mtv-6, quantified according to the down-regulation of CD4 and CD8 on immature thymocytes, was partially inhibited by CsA, to maximal effect within 24 hours. This did not, however, facilitate their development into mature thymocytes.

Characterization of two CD23 monoclonal antibodies with reactivity distinct from other antibodies within this cluster of differentiation.

We have produced two CD23 monoclonal antibodies (mAb), LA1 and LA2, which differ significantly in their patterns of reactivity compared to other mAb within this cluster. Unlike other CD23 mAb, LA1 and LA2 show virtually no reactivity with freshly isolated tonsil B lymphocytes or mantle zone lymphocytes in tissue section. That LA1 and LA2 are CD23 mAb is confirmed by their precipitation of a 45,000 MW surface protein from B cells and strong reactivity with a CD23 transfectant. Cross-blocking studies with four well-characterized CD23 mAb show that LA1 and LA2 recognize the same, distinct epitope of the CD23 molecule. However, similar to other CD23 mAb, expression of LA1 and LA2 increases after activation. Following removal of cells staining with the well-characterized CD23 mAb MHM6, using a highly efficient magnetic bead technique, LA1 and LA2, but not other CD23 mAb, react with a subpopulation of the remaining cells when activated with interleukin-4 (IL-4) or phorbol ester. Soluble LA1 and MHM6 both provide a co-stimulatory signal for phorbol ester-induced B-cell proliferation. This response is increased if these mAb are used to cross-link the CD23 molecule. Interestingly, despite the fact that LA2 and LA1 cross-block, LA2 has no effect on functional responses in its soluble form but can elicit a comparable increase in proliferation when cross-linked. Results presented here suggest that the novel CD23 mAb, LA1 and LA2, recognize a distinct form of the CD23 molecule, expressed only on activation. These mAb define a subpopulation of activated B cells which do not stain with other CD23 mAb.

Expression and functional role of CD23 on T cells.

We have found that approximately 10%-15% of tonsil, but not peripheral blood, T cells express the CD23 antigen following activation with 12-O-tetradecanoylphorbol 13-acetate (TPA), phytohemagglutinin (PHA) or recombinant interleukin 4. The proliferative response of tonsil T cells is significantly increased when CD23 monoclonal antibodies (mAb) are present in the cultures. In contrast, no such proliferative augmentation is seen when peripheral blood T cells are cultured in this way. Supernatant (SN) of Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBVLCL), is found to have a similar co-stimulatory effect on the proliferation of tonsil T cells to that seen with CD23 mAb. This effect is greatly diminished by preclearing SN with CD23 mAb. Similarly, SN from a CD23+ L cell transfectant augments the proliferative response of tonsil T cells to both TPA and PHA. The CD23 molecule expressed by TPA-driven T cell blasts appears identical in size to the 45-kDa glycoprotein present on EBVLCL and activated B cells. In contrast, a 42-kDa molecule is observed when CD23 is precipitated from T cells activated with PHA. The results presented here demonstrate that CD23 is expressed on activated tonsil, but not peripheral blood T cells and plays a role, via the binding of CD23 mAb and CD23+ material, present in EBVLCL and CD23+ transfectant SN, in the regulation of T cell proliferation in response to mitogens such as PHA and TPA.

Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells.

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double-negative (CD4-CD8-) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double-negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3-2C2 and RA3-3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3-2C2 and RA3-3A1 reactive) fraction of double-negative thymocytes; CD45RA- enriched populations had increased efficiency of recolonization and CD45RA- depleted populations had decreased ability to recolonize as compared with the whole CD4-CD8- fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+ CD8+ cells, we analyzed the progeny of lobes seeded with CD4-CD8-CD45RA+ fractions. After 7-9 days in organ culture the proportion of CD4+, CD8+ or CD4+ CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA-expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.

Increase in the ratio of mitochondrial Bax/Bcl-XL induces Bax activation in human leukemic K562 cell line.

The p53- and Bcl-2-negative leukemic K562 cell line showed resistant to DNA damage-induced Bax activation and apoptosis. The constitutive balanced ratio of Bax/Bcl-XL in K562 mitochondria allowed the formation of active Bax and cytochrome c release from mitochondria in the presence of a BH3-only protein, tBid, in a cell-free system. Bax transfection led to Bax undergoing a conformational change, translocation to mitochondria and homo-oligomerization but not apoptosis in the K562 cell line. After treatment with UV light, while Bcl-XL but not Bax translocated to mitochondria in K562, both Bax and Bcl-XL translocated to mitochondria in the Bax stable transfectant K/Bax cells. The increased ratio of Bax/Bcl-XL in K/Bax mitochondria led to an increased conformationally changed Bax, formation of the homo-multimer of Bax-Bax, and a reduced hetero-dimerization of Bax-Bcl-XL. Increased proportion of active Bax was accompanied with increased percentage of apoptosis. We therefore demonstrate that direct increase in the ratio of mitochondrial Bax/Bcl-XL can induce Bax activation in the p53- and Bcl-2-negative leukemic cells. Increased Bcl-XL translocation and failure in Bax translocation from cytosol to mitochondria play important roles in preventing Bax activation.

High level amplification of N-MYC is not associated with adverse histology or outcome in primary retinoblastoma tumours.

Twenty-five primary retinoblastoma tumours were analysed by real-time quantitative polymerase chain reaction to determine the genomic copy number of the N-MYC gene (2p24) relative to the copy number for REL, B2M, ALB, AF10 and MLL. Twenty-one of these tumours were shown by Comparative Genomic Hybridization to contain variable copy number increases of chromosomal material mapping to 2p. High level amplification (>30-fold) of N-MYC was found in three tumours, none of which showed adverse histological features and all patients are surviving at between 54 and 108 months post enucleation. Furthermore, the three tumours associated with metastasis and adverse patient outcome showed normal N-MYC copy number. Although high level amplification of N-MYC is an unfavourable prognostic indicator in neuroblastoma, these data show no evidence of a correlation between amplification of N-MYC and adverse outcome in retinoblastoma.

A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.

The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation.

Human thymocyte development in mouse organ cultures.

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.

Problems in the physiology of class I and class II MHC molecules, and of CD45.

1. Co-processing of alloantigens suggests that epitope-loaded MHC class I molecules may pass from tissue cells to dendritic cells. 2. Antigen-presenting cells in the thymus need some special trick in order to load their MHC class II molecules with epitopes from "intermediate concentration" self-proteins in order to induce self-tolerance in developing cells. 3. Cell-cell interactions may transmit signals simply by rearranging surface glycoproteins and thus locally perturbing a phosphorylation equilibrium. 4. The CD45 and STB1 phenotype of most cells in the thymus may be characteristic of a doomed cell.

The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma.

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B-cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13-16. Using real-time quantitative polymerase chain reaction (RQ-PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, beta2-microglobulin and CD8alpha, that lie close to REL on 2p. There was no correlation apparent between 2p13-16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real-time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.