EBV-associated post-transplant lymphoproliferative disorder following in vivo T-cell-depleted allogeneic transplantation: clinical features, viral load correlates and prognostic factors in the rituximab era.

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49 300 copies/mL (50-65 200 000 copies/mL) and, notably, 23% and 45% of cases, respectively, had 10 000 and 40 000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12-0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12-2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL.

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak.

Glucocorticoids (GCs) represent an important component of modern treatment regimens for fludarabine-refractory or TP53-defective chronic lymphocytic leukemia (CLL). However, GC therapy is not effective in all patients. The molecular mechanisms responsible for GC-induced apoptosis and resistance were therefore investigated in primary malignant cells obtained from a cohort of 46 patients with CLL. Dexamethasone-induced apoptosis was unaffected by p53 dysfunction and more pronounced in cases with unmutated IGHV genes. Cross-resistance was observed between dexamethasone and other GCs but not fludarabine, indicating non-identical resistance mechanisms. GC treatment resulted in the upregulation of Bim mRNA and protein, but to comparable levels in both GC-resistant and sensitive cells. Pre-incubation with Bim siRNAs reduced GC-induced upregulation of Bim protein and conferred resistance to GC-induced apoptosis in previously GC-sensitive cells. GC-induced upregulation of Bim was associated with the activation of Bax and Bak in GC-sensitive but not -resistant CLL samples. Co-immunoprecipitation experiments showed that Bim does not interact directly with Bax or Bak, but is almost exclusively bound to Bcl-2 regardless of GC treatment. Taken together, these findings suggest that the GC-induced killing of CLL cells results from the indirect activation of Bax and Bak by upregulated Bim/Bcl-2 complexes, and that GC resistance results from the failure of such activation to occur.

Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia.

Genome-wide array approaches and sequencing analyses are powerful tools for identifying genetic aberrations in cancers, including leukemias and lymphomas. However, the clinical and biological significance of such aberrations and their subclonal distribution are poorly understood. Here, we present the first genome-wide array based study of pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) that uses the computational statistical tool OncoSNP. We show that quantification of the proportion of copy number alterations (CNAs) and copy neutral loss of heterozygosity regions (cnLOHs) in each sample is feasible. Furthermore, we (i) reveal complex changes in the subclonal architecture of paired samples at relapse compared with pre-treatment, (ii) provide evidence supporting an association between increased genomic complexity and poor clinical outcome (iii) report previously undefined, recurrent CNA/cnLOH regions that expand or newly occur at relapse and therefore might harbor candidate driver genes of relapse and/or chemotherapy resistance. Our findings are likely to impact on future therapeutic strategies aimed towards selecting effective and individually tailored targeted therapies.

Incurable, invisible and inconclusive: watchful waiting for chronic lymphocytic leukaemia and implications for doctor-patient communication.

Patients with chronic lymphocytic leukaemia (CLL) find it hard to accept a diagnosis of an incurable cancer for which no treatment is recommended and which may not cause symptoms for many years. We used qualitative interviews with 12 people with CLL managed by watchful waiting, drawn from a maximum variation sample of 39 adults with leukaemia, to explore accounts of watchful waiting and implications for clinical management. Patients with CLL recalled being given little information about the condition and wanted to know more about how it might affect them in the future. The invisibility of CLL meant that some chose not to disclose the diagnosis to others. Check-ups sometimes felt cursory, causing dissatisfaction. As symptoms increased, lifestyle adaptations became essential, well before treatment was warranted. Patients with CLL on watchful waiting experience levels of depression, anxiety and quality of life similar to those in active treatment; our qualitative approach has illuminated some of the reasons for the negative psychological impacts. We relate our findings to perceptions of the illness state, doctor-patient communication, and work pressure. We recommend that specialists could better support patients by acknowledging psychological impacts of CLL, actively listening to patients' concerns, and meeting their needs for information.

Hsp90 inhibition has opposing effects on wild-type and mutant p53 and induces p21 expression and cytotoxicity irrespective of p53/ATM status in chronic lymphocytic leukaemia cells.

In chronic lymphocytic leukaemia (CLL), mutation/deletion of TP53 is strongly associated with early disease progression, resistance to chemotherapy and short patient survival. Consequently, there is a pressing need to develop novel treatment protocols for this high-risk patient group. The present study was performed to evaluate Hsp90 inhibition as a possible therapeutic approach for such patients. Primary CLL cells of defined ataxia telangiectasia mutated (ATM)/p53 status were incubated with the Hsp90 inhibitor geldanamycin (GA) and analysed by western blotting for the expression of p53, p21, MDM2 and Akt. GA downregulated overexpressed mutant p53 protein (an oncogene) and upregulated wild-type (wt) p53 (a tumour suppressor). The upregulation of wt p53 by GA was independent of ATM and was accompanied by downregulation of Akt and the active form of MDM2, indicating a possible mechanism. GA also produced a p53/ATM-independent increase in the levels of p21-a potent inducer of cell-cycle arrest. In-vitro cytotoxicity studies showed that GA killed cultured CLL cells in a dose- and time-dependent fashion irrespective of their p53/ATM status and more effectively than normal blood mononuclear cells. In summary, our findings reveal important consequences of inhibiting Hsp90 in CLL cells and strongly support the therapeutic evaluation of Hsp90 inhibitors in poor-prognosis patients with p53 defects.

Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial.

BACKGROUND: Previous studies of patients with chronic lymphocytic leukaemia reported high response rates to fludarabine combined with cyclophosphamide. We aimed to establish whether this treatment combination provided greater survival benefit than did chlorambucil or fludarabine. METHODS: 777 patients with chronic lymphocytic leukaemia requiring treatment were randomly assigned to fludarabine (n=194) or fludarabine plus cyclophosphamide (196) for six courses, or chlorambucil (387) for 12 courses. The primary endpoint was overall survival, with secondary endpoints of response rates, progression-free survival, toxic effects, and quality of life. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number NCT 58585610. FINDINGS: There was no significant difference in overall survival between patients given fludarabine plus cyclophosphamide, fludarabine, or chlorambucil. Complete and overall response rates were better with fludarabine plus cyclophosphamide than with fludarabine (complete response rate 38%vs 15%, respectively; overall response rate 94%vs 80%, respectively; p<0.0001 for both comparisons), which were in turn better than with chlorambucil (complete response rate 7%, overall response rate 72%; p=0.006 and 0.04, respectively). Progression-free survival at 5 years was significantly better with fludarabine plus cyclophosphamide (36%) than with fludarabine (10%) or chlorambucil (10%; p<0.00005). Fludarabine plus cyclophosphamide was the best combination for all ages, including patients older than 70 years, and in prognostic groups defined by immunoglobulin heavy chain gene (V(H)) mutation status and cytogenetics, which were tested in 533 and 579 cases, respectively. Patients had more neutropenia and days in hospital with fludarabine plus cyclophosphamide, or fludarabine, than with chlorambucil. There was less haemolytic anaemia with fludarabine plus cyclophosphamide (5%) than with fludarabine (11%) or chlorambucil (12%). Quality of life was better for responders, but preliminary analyses showed no significant difference between treatments. A meta-analysis of these data and those of two published phase III trials showed a consistent benefit for the fludarabine plus cyclophosphamide regimen in terms of progression-free survival. INTERPRETATION: Fludarabine plus cyclophosphamide should now become the standard treatment for chronic lymphocytic leukaemia and the basis for new protocols that incorporate monoclonal antibodies.

The role of prognostic factors in assessing 'high-risk' subgroups of patients with chronic lymphocytic leukemia.

The management of chronic lymphocytic leukemia (CLL) has historically relied on 'watchful waiting' and palliative approaches to therapy. However, the course of disease is highly variable and a substantial proportion of patients with early-stage CLL develop rapidly progressive disease requiring therapy. In recent decades, numerous clinical and biological prognostic markers that are predictive of decreased survival outcomes, disease progression and/or resistance to therapy, and that may play a role in defining the subgroups of patients with 'high-risk' CLL have been identified. At the same time, highly effective treatment modalities have become available with the advent of chemoimmunotherapy combinations and allogeneic stem cell transplantation. Thus, we are approaching an era when patients with CLL may potentially benefit from individualized risk assessments based on prognostic markers and when specific therapies may be offered to the subgroup of patients with high-risk disease. This review provides a brief overview of newer biological prognostic markers, discusses the challenges associated with identifying the subgroup of patients with high-risk CLL and further aims to provide recommendations on how prognostic markers may be used to assess high-risk subgroups in different clinical situations in CLL.

Analysis of VH gene sequences using two web-based immunogenetics resources gives different results, but the affinity maturation status of chronic lymphocytic leukaemia clones as assessed from either of the resulting data sets has no prognostic significance.

Some cellular and molecular features of chronic lymphocytic leukaemia (CLL) cells that are associated with prognosis may reflect the context within which their progenitors encountered antigen. It follows that the nature of antigen drive in CLL could influence the clinical course and we were prompted to assess the impact, if any, of affinity maturation (an antigen-driven process) on prognosis. Statistical models for assessing affinity maturation status are typically applied to V(H) gene sequence data analysed using a web-based resource like IMGT or VBASE. Since these resources differ with respect to some key relevant features, we evaluated a cohort of CLL cases by applying statistical models to V(H) data derived from both IMGT and VBASE. Important differences between the resulting data sets became apparent. These resulted from database variance and because IMGT and VBASE define complementarity-determining and framework regions (CDRs, FRs) in different ways. Thus, the numbers of mutations identified and their distribution between CDRs/FRs varied between the data sets for the majority of clones. Consequently, two different but overlapping sets of cases with evidence of affinity maturation were defined. Notwithstanding their differences, no significant associations of affinity maturation status with CD38 expression, p53 functional status or survival were identifiable in either data set.

Detection of p53 dysfunction by flow cytometry in chronic lymphocytic leukaemia.

In chronic lymphocytic leukaemia (CLL) cells, functional impairment of the p53 pathway is detectable by Western blotting as impaired up-regulation of p21 (a transcriptional target of p53) in response to ionizing radiation (IR). The type A defect is characterized by baseline p53 overexpression and is associated with TP53 mutation. The type B defect is characterized by impaired IR-induced p53 up-regulation and is associated with inactivation of the ataxia telangiectasia-mutated gene (ATM). Both abnormalities are strongly associated with adverse clinical outcome. In the present study, flow cytometry was found to be an effective alternative to Western blotting in the detection of p53 dysfunction in CLL.

Homotypic interactions protect chronic lymphocytic leukaemia cells from spontaneous death in vitro.

Chronic lymphocytic leukaemia (CLL) cells are long-lived in vivo but undergo spontaneous apoptosis when cultured in vitro. Since CLL cells associate intimately with one another at sites of tissue involvement, we examined the hitherto unproven possibility that homotypic interactions between the malignant cells might reduce their propensity to undergo spontaneous cell death. In a series of experiments in which highly pure CLL-cell populations were cultured on a non-adherent surface, cell viability was found to increase markedly with the level of crowding at the bottom of the culture vessel. The effect was observed among unevenly distributed cells within a single culture vessel and did not require direct cell-cell contact. This indicates that cell survival was being regulated in an autocrine fashion by locally acting soluble products. Conditioned medium from crowded CLL cells enhanced the survival of autologous non-crowded cells, indicating that at least some of the autocrine survival factors produced by CLL cells could accumulate in the extracellular environment. In addition, the survival of non-crowded CLL cells was markedly enhanced by co-culturing them with an excess of autologous fixed cells. This protective effect of direct cell-cell contact was mediated by specific surface structures since it was abrogated by pre-treating the fixed cells with neuraminidase. Our results provide the first direct demonstration that the survival of cultured CLL cells is enhanced by homotypic interactions. We speculate that these protective effects may contribute to the accumulation of CLL cells in vivo, and that further elucidation of the underlying mechanisms may lead to novel therapeutic strategies.

p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation.

The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21(CIP1/WAF1) after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation. (Blood. 2001;98:814-822)

Caspases influence the mode but not the extent of cell death induced by purine analogues in chronic lymphocytic leukaemia.

Caspases are known to be involved in the apoptotic killing of chronic lymphocytic leukaemia (CLL) cells by fludarabine. However, it is unclear whether these enzymes are required for the induction of such killing, or whether they simply determine the mode of cell death. To address this question, we examined the effect of the broad-spectrum caspase inhibitor Z-VAD.fmk on six different manifestations of nucleoside cytotoxicity. Our results indicate that while caspase activity is required for nucleoside-induced poly(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation, other manifestations of cell death (mitochondrial depolarization, exposure of phosphatidyl serine, cell membrane disruption and cell shrinkage) are caspase independent. By showing that caspases influence the mode, but not the extent, of nucleoside cytotoxicity, our results exclude defects in these enzymes as a mechanism of nucleoside resistance in CLL.

Role of poly(ADP-ribosyl)ation in the killing of chronic lymphocytic leukemia cells by purine analogues.

Although the nucleoside analogues fludarabine and chlorodeoxyadenosine have become important therapeutic agents in chronic lymphocytic leukemia (CLL), their effectiveness is limited by drug resistance. Because such resistance is likely to result from impaired drug-induced apoptosis, it is clearly important to understand the mechanisms involved in this process. Whereas p53 can contribute to the nucleoside-induced killing of CLL cells, recent work from this laboratory and elsewhere has shown that such killing can also occur by p53-independent mechanisms. Because poly(ADP-ribose) polymerase (PARP)-mediated NAD+/ATP depletion has been implicated in the nucleoside-induced killing of normal resting lymphocytes, we postulated that this mechanism might account for the p53-independent component of nucleoside cytotoxicity in CLL. To address this question, we used 3-aminobenzamide (3AB) at a concentration (200 microM) known to produce selective inhibition of poly(ADP-ribosyl)ation in intact cells and examined nucleoside-induced killing using a number of different end points (cell membrane disruption, cell shrinkage, mitochondrial depolarization, exposure of phosphatidyl serine, morphological changes, DNA fragmentation, and PARP-1 cleavage). In 27 of the 30 cases of CLL examined, 3AB delayed nucleoside-induced cell membrane disruption without inhibiting other manifestations of cytotoxicity. This indicates that PARP activity, rather than contributing to the induction of cell killing, was accelerating cell membrane disruption during the late stages of apoptosis. This novel observation has important implications for previous studies of PARP-mediated cytotoxicity. However, in cells from one CLL patient, 3AB inhibited all manifestations of nucleoside cytotoxicity; this was the only case in the study known to have a p53 gene defect affecting both alleles. This indicates that PARP activity can occasionally be central to nucleoside-induced killing and that such PARP-mediated killing is p53 independent.

Granulocyte-macrophage colony-stimulating factor as an autocrine survival factor for mature normal and malignant B lymphocytes.

The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.