ZAP-70 by flow cytometry: a comparison of different antibodies, anticoagulants, and methods of analysis.

BACKGROUND: The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. METHODS: We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. RESULTS: Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. CONCLUSION: Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.

Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease.

The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.

Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Frequent homozygous deletions of the D13S25 locus in chromosome region 13q14 defines the location of a gene critical in leukaemogenesis in chronic B-cell lymphocytic leukaemia.

Cytogenetic studies of B-cell chronic lymphocytic leukaemia show structural abnormalities involving the 13q14 chromosome region as the only karyotypic change in a significant proportion of tumours. This observation suggests the location of a gene important in leukaemogenesis. A series of 68 BCLL tumours have been analysed for allele loss using a series of probes from 13q14. Using intragenic polymorphic markers from the retinoblastoma predisposition gene LOH was observed in 25% of tumours including 3/6 showing cytogenetically obvious deletions of the 13q14 region and 3/6 showing translocations involving 13q14. However, three deletions with proximal breakpoints in 13q14 did not show allele loss, demonstrating that the breakpoint lay distal to RB1. Using the D13S25 locus, which lies 1.6 cM distal to RB1, allele loss was seen in 90% of tumours with structural rearrangements of 13q14 and 75% of tumours with an apparently normal karyotype. 50% of these tumours showed homozygous loss of D13S25, suggesting that a 'tumour suppressor gene' lies in this region. The more distal D13S31 locus, 1 cM distal to D13S25, was infrequently involved in allele loss demonstrating that the minimum region of overlap for homozygous deletions is approximately 1 Mbp around the D13S25 locus.

Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.

The increased or inappropriate expression of genes with oncogenic properties through specific chromosome translocations is an important event in the pathogenesis of B-cell lymphoproliferative diseases. Recent studies have found deletions or translocations of chromosome 7q to be the most common cytogenetic abnormality observed in SLVL, a leukemic variant of SMZL, with the q21-q22 region being most frequently affected. In three patients with translocations between chromosomes 2 and 7, the cloning of the breakpoints at 7q21 revealed that each was located within a small region of DNA 3.6 kb upstream of the transcription start site of cyclin dependent kinase 6 (CDK6). In each case the translocation event was consistent with aberrant VJ recombination between the immunoglobulin light chain region (Ig kappa) on chromosome 2p12 and DNA sequences at 7q21, resembling the heptamer recombination site. The t(7;21) breakpoint in an additional patient with splenic marginal zone lymphoma (SMZL), resided 66 kb telomeric to the t(2;7) breakpoints juxtaposing CDK6 to an uncharacterized transcript. In two of the SLVL patient samples, the CDK6 protein was found to be markedly over expressed. These results suggest that dysregulation of CDK6 gene expression contributes to the pathogenesis of SLVL and SMZL.

The configuration of the immunoglobulin genes in B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) lacks a consistent genetic abnormality. However, immunoglobulin V(H) gene segment mutation analysis has provided insights into the pathogenesis of these diseases and allowed the development of powerful prognostic markers. Immunoglobulin gene chromosomal translocations are rare in CLL and involve a distinct subset of genes including BCL3, BCL11A and CCND2. BCL2 translocations in CLL appear to arise via a different mechanism from comparable translocations seen in B cell non-Hodgkin lymphoma.

Assessment of the role of clonogenic B lymphocytes in the pathogenesis of multiple myeloma.

The identifiable neoplastic cell in multiple myeloma is the plasma cell, which usually synthesizes and secretes a monoclonal immunoglobulin. However, there exists the possibility that the neoplastic event has occurred in a less mature clonally-related cell, such as a B lymphocyte, prior to Ig class switching. Since the presence of such a clonogenic cell could influence design of therapy, particularly with monoclonal antibodies, we have used the analysis of tumour-related VH genes to approach this question. Cloning and sequencing of PCR products from VH genes of tumour cells obtained from 4/4 patients with myeloma revealed significant mutation of the genes as compared to germ line sequences. In all cases the mutations were scattered throughout the variable region, with a pattern which did not indicate a role for antigen in selection. Importantly for therapy, multiple VH sequences from all patients were completely homogeneous, with no intraclonal variation. These findings indicate that, although IgM-positive clonogenic cells may exist, it is unlikely that they are involved in continuous maintenance of the malignant isotype-switched cell population. One possibility is that the B-cell progenitor population has to undergo further chromosomal changes to generate the malignant cell, and that this occurs at a more mature stage; in this case, antibody therapy should be aimed primarily at the more differentiated cells.

Use of a retinoblastoma gene probe to investigate clonality in Richter's syndrome.

A 69-year-old woman presented with Rai stage 0 chronic lymphocytic leukemia. Ten years later she developed a diffuse centroblastic lymphoma involving the stomach. The surface membrane phenotype of the CLL cells was MD lambda while that of the large cell lymphoma (LCL) cells was MD kappa. The two populations had different heavy and kappa light chain rearrangements. Cytogenetic analysis of the CLL cells showed a deletion involving chromosome 13, band q14, but was unsuccessful in the LCL cells. However, use of a probe (p68 RS2.0) which recognizes a variable number tandem repeat sequence in the retinoblastoma gene, localized to chromosome 13q14, showed two alleles in the LCL cells but only one in the CLL cells. These data suggest that in this case of Richter's syndrome the CLL cells and the LCL cells are clonally distinct.

PCR analysis of polymorphisms at the D13S25 locus.

Evidence that the D13S25 locus lies close to a potential tumour suppressor gene implicated in the pathogenesis of B-CLL has been based on detection of LOH and bi-allelic loss using the pH2-42 probe. The SspI polymorphism detected by this probe has been identified by sequencing adjacent clones and a polymorphic (TA)n repeat has been found. Amplification of the region encompassing both polymorphic markers by PCR increases the informativity to 80%.

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR.

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.

Lack of somatic hypermutation of IG V(H) genes in lymphoid malignancies with t(2;14)(p13;q32) translocation involving the BCL11A gene.

The t(2;14)(p13;q32.3) involving the BCL11A and IGH genes is a rare but recurrent chromosomal aberration in B-cell malignancies. Hitherto, juxtaposition of BCL11A and IGH has only been described in B-cell chronic lymphocytic leukemia (B-CLL) and immunocytoma. As subgroups of B-CLL can be distinguished by the pattern of somatic mutation of immunoglobulin variable (V) genes we investigated four lymphomas with IGH/BCL11A involvement for IGH hypermutation. Clonal V(H) gene rearrangements were amplified; in all four cases, sequencing of the amplificates revealed the rearranged V(H) genes to lack somatic mutations. These results suggest that t(2;14)(p13;q32.3) is associated with a subset of B-CLL/immunocytoma characterized by non-mutated IG genes deriving from pre-germinal center B cells. As the translocations in both informative cases are targeted to the switch regions of the IGG2 gene, which is mainly used in T cell-independent immune responses, these translocations presumably occurred in activated B cells in the course of T cell-independent immune responses outside the germinal center.

P2X7 polymorphism and chronic lymphocytic leukaemia: lack of correlation with incidence, survival and abnormalities of chromosome 12.

The P2X7 receptor, a plasma membrane ATP-gated ion channel that plays a role in lymphocyte apoptosis, has been suggested as an important contributory factor to the pathogenesis of chronic lymphocytic leukaemia (CLL). The P2X7 gene resides on chromosome 12 and is polymorphic in the population at large (1513A/C) with the A and C alleles encoding fully active and nonfunctional proteins, respectively. We have evaluated the significance of this polymorphism by genotyping 144 patients with CLL and 348 healthy controls using a tetraprimer ARMS assay. We found no significant difference in allele frequency between patients and controls. Although patients with the C allele (A/C heterozygotes or C/C homozygotes) had a marginally shorter survival than those who were homozygous for the A allele, this difference was not significant for either the patient group considered as a whole or for IgVH-mutated/unmutated subsets. Finally, no association was found between trisomy 12 and P2X7 genotype. We conclude that the influence, if any, of P2X7 genotype on susceptibility to CLL or clinical outcome is small.

Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B.

B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.

Cytogenetic and molecular abnormalities in chronic lymphocytic leukaemia.

Genetic abnormalities are found in 50% of cases of chronic lymphocytic leukaemia (CLL) by cytogenetic analysis and in a higher percentage of patients using molecular techniques. The commonest cytogenetic abnormalities are trisomy 12 and deletions or translocations of the long arm of chromosome 13 usually involving band q14. The genetic consequences of trisomy 12 are unknown but structural abnormalities of chromosome 13q14 frequently involve hetero or homozygous loss of a region distal to the retinoblastoma gene which may be the site of a tumour suppressor gene. Trisomy 12 or loss of one copy of the retinoblastoma gene have been detected by fluorescent in situ hybridisation (FISH) in interphase cells of patients with a normal karyotype. By combining FISH with immunophenotyping, it has been found that trisomy 12 occurs in only 30 to 40% of the malignant clone, suggesting that it is a secondary event in leukaemogenesis. Trisomy 12 is strongly associated with atypical lymphocyte morphology in patients with otherwise typical CLL. Complex karyotypic abnormalities, a high percentage of abnormal metaphases and trisomy 12 but not structural abnormalities of chromosome 13 are associated with a poor prognosis at all stages of the disease. Mutations or deletions of the P53 gene are found in 10 to 15% of patients with advanced CLL and correlate with resistance to treatment and poor survival.

Chronic lymphocytic leukaemia: the nature of the leukaemic cell.

Far from being the boring, inactive, inert lymphocyte that haematologists of old perceived it to be, the chronic lymphocytic leukaemia (CLL) cell has set us many complex problems. The cell is apparently stuck in G0 in cell cycle, yet expresses many activation markers. The cells apparently manufacture many cytokines and respond in vitro to even more, yet cells entering even G1 are few. The cell surface marker profile is unique. There is apparently no normal equivalent of the CLL cell. In part, this may be because the cell is malignant; malignant cells often express aberrant markers. Consistent chromosomal abnormalities are emerging but we have no idea how these abnormalities translate into molecular mistakes that dictate the peculiar nature of the cell. CLL cells carry a characteristics set of adhesion molecules, but we cannot read their homing and recycling instructions. The outstanding irregularities of the CLL cell are its CD5 positivity and its sparse surface immunoglobulin. This ought to translate as an anergic B1 cell, perhaps programmed for autoimmunity. If the tumour cell were responsible for the patient's production of immunoglobulin or secretion of autoantibodies, then a pattern might have emerged. Alas, these are the product of the normal B cells. How the CLL cell induces these complications is unknown. Thus, despite the information contained in this review, the CLL cell remains a puzzle.

Waldenström's macroglobulinaemia.

Waldenström's macroglobulinaemia (WM) is most usefully defined as a distinct chronic lymphoproliferative disorder with characteristic marrow morphology and phenotype; although nodal morphology if available will reveal a lymphoplasmacytoid lymphoma, the presence of a significant IgM paraprotein defines the clinical features of the disease. The clonal cell is a B-cell expressing IgM, CD19, and CD20 but not IgD, CD5, CD10 or CD23 and has somatic hypermutation of immunoglobulin heavy chain variable regions consistent with a post-germinal centre origin. Treatment of WM has been dependent on alkylating agents with or without coriticosteroids for many years, supplemented by the use of therapeutic plasmapheresis in the initial stages for patients at risk from the clinical consequences of hyperviscosity. This approach to treatment results in response rates of approximately 60% with a median survival of about 60 months. There is increasing evidence to show that the purine analogues fludarabine and cladribine which are active in the treatment of patients who are resistant to alkylating agents such as chlorambucil may be able to achieve higher response rates when used as initial therapy. A prospective trial is being undertaken to compare fludarabine and chlorambucil as initial treatment; because of the effect of subsequent active treatment on patients who do not respond to the first treatment choice, the long-term outcome may be similar for both groups. Recent advances in therapy include the use of therapeutic monoclonal antibodies such as rituximab and the use of autologous or allogeneic transplant procedures for selected patients.

Lymphoid neoplasms.

The majority of B cell lymphomas, but only a minority of T cell lymphomas, are characterized by recurring chromosome translocations. Many involve the immunoglobulin or T cell receptor loci with various partner chromosomes and lead to abnormal proto-oncogene expression. Other recurring translocations result in the production of a novel fusion protein. The detection of translocations is of particular value in diagnosis and in the detection of minimal residual disease. Aneuploidy and deletion of specific chromosome regions are common secondary chromosomal events which are rarely specific to a particular type of lymphoma but provide valuable prognostic information. Analysis by G banding, 24-colour FISH and CGH provides global genomic information; however, more specifically directed investigations utilizing locus-specific FISH probes, PCR techniques or monoclonal antibodies may be more appropriate to answer particular questions regarding diagnosis and prognosis. The known molecular consequences of abnormalities and the appropriate methods of detection are discussed for each subtype of lymphoma.

An 8;14 translocation in a case of plasma cell leukemia.

We report a patient with plasma cell leukemia. Chromosomal analysis of bone marrow showed hypodiploidy with a modal number of 32, and a t(8;14). This is only the second reported case of t(8;14) in plasma cell leukemia.

A patient with monosomy 7 and polyuria.

Diabetes insipidus (DI) is a rare complication of leukaemia. An association between monosomy 7 and DI in leukaemias has been proposed. We present a case of Ph1-positive CML who developed polyuria at the time of lymphoid blast transformation associated with loss of chromosome 7. Biochemical results were not diagnostic of DI and a therapeutic trial of DDAVP was unsuccessful. Post-mortem showed a peripituitary and renal leukaemic infiltrate and although DI is a possibility, the cause of his polyuria remains unresolved.

Correlation of bone marrow colony growth in the myelodysplastic syndromes with the FAB classification and the Bournemouth score.

Erythroid and myeloid colonies were grown from the bone marrow of 81 patients with myelodysplasia and the median number of colonies correlated with the FAB classification and Bournemouth score. CFU-GM were increased in CMML compared to RAEB and RAEBt. BFU-E were higher in RA than in the other FAB subgroups. Patients with a high Bournemouth score had poorer CFU-GM and BFU-E growth than those with a low score.