Incidence and clinicobiologic characteristics of leukemic B-cell chronic lymphoproliferative disorders with more than one B-cell clone.

Leukemic B-chronic lymphoproliferative disorders (B-CLPDs) are generally believed to derive from a monoclonal B cell; biclonality has only occasionally been reported. In this study, we have explored the incidence of B-CLPD cases with 2 or more B-cell clones and established both the phenotypic differences between the coexisting clones and the clinicobiologic features of these patients. In total, 53 B-CLPD cases with 2 or more B-cell clones were studied. Presence of 2 or more B-cell clones was suspected by immunophenotype and confirmed by molecular/genetic techniques in leukemic samples (n = 42) and purified B-cell subpopulations (n = 10). Overall, 4.8% of 477 consecutive B-CLPDs had 2 or more B-cell clones, their incidence being especially higher among hairy cell leukemia (3 of 13), large cell lymphoma (2 of 10), and atypical chronic lymphocytic leukemia (CLL) (4 of 29). In most cases the 2 B-cell subsets displayed either different surface immunoglobulin (sIg) light chain (n = 37 of 53) or different levels of the same sIg (n = 9 of 53), usually associated with other phenotypic differences. Compared with monoclonal cases, B-CLL patients with 2 or more clones had lower white blood cell (WBC) and lymphocyte counts, more frequently displayed splenomegaly, and required early treatment. Among these, the cases in which a CLL clone coexisted with a non-CLL clone were older and more often displayed B symptoms, a monoclonal component, and diffuse infiltration of bone marrow and required early treatment more frequently than cases with monoclonal CLL or 2 CLL clones.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

CLLU1 expression has prognostic value in chronic lymphocytic leukemia after first-line therapy in younger patients and in those with mutated IGHV genes.

CLLU1, located at chromosome 12q22, encodes a transcript specific to chronic lymphocytic leukemia and has potential prognostic value. We assessed the value of CLLU1 expression in the LRF CLL4 randomized trial. Samples from 515 patients with chronic lymphocytic leukemia were collected immediately before the start of treatment. After RNA extraction and cDNA synthesis, CLLU1 expression was assessed by quantitative polymerase chain reaction. In total, 247 and 268 samples were identified as having low and high CLLU1 expression, respectively. The median follow-up was 88 months. High CLLU1 expression was significantly correlated with unmutated IGHV genes, ZAP-70 and CD38 positivity, and absence of 13q deletion (all r>0.2, P

ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia.

Recent evidence suggests that - in addition to 17p deletion - TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4-9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.

Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 with >or=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases with >or=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases with >or=5%TP53-deleted cells (por=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with >20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

Fludarabine, Cyclophosphamide and Rituximab:an effective chemoimmunotherapy combination with high remission rates for chronic lymphocytic leukaemia

BACKGROUND: Combined Fludarabine and Cyclophosphamide is now standard first-line therapy in chronic lymphocytic leukaemia (CLL) and the addition of Rituximab improves outcome.

METHODS: We adopted a modified Fludarabine, Cyclophosphamide and Rituximab (FCR) protocol in treating 39 patients (median age 57 years) with progressive or advanced CLL. Depending on CR, treatment was given for four or six cycles.

RESULT: Twenty-six patients were treatment naïve and 13 were pre-treated. Twelve patients had progressive Binet stage A, 16 stage B and 11 stage C disease. The overall response rate (ORR) was 100%, with 75% achieving CR. Neutropenia was the major toxicity in 71/187 (38%) of the cycles. There were five deaths, two from infection and three from progressive disease. Twenty-six of 31 patients have maintained their post-treatment disease status for a median of 17 months (2-41).

CONCLUSION: We conclude that FCR is a feasible, well-tolerated and effective treatment for patients with CLL.

MicroRNA antagonism of the picornaviral life cycle: alternative mechanisms of interference.

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3' untranslated region (3'UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.

Acute skin GVHD following syngeneic BMT for CLL

A 41-year-old woman received a syngeneic BMT for CLL and subsequently developed acute skin GVHD. Transfusion-related allogeneic GVHD was excluded on the basis of an unchanged HLA type in circulating lymphocytes. Short tandem repeat PCR was used to confirm syngeneicity between donor and recipient. The patient had a personal and family history of autoimmune disease which may have made her particularly susceptible to development of syngeneic GVHD. The distinction between allogeneic and syngeneic or autologous GVHD is important because of therapeutic implications.

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

Combinations of ZAP-70, CD38 and IGHV mutational status as predictors of time to first treatment in CLL.

ZAP-70, CD38 and IGHV mutations have all been reported to have prognostic impact in chronic lymphocytic leukemia (CLL), both individually and in paired combinations. We aimed to determine whether the combination of all three factors provided more refined prognostic information concerning the treatment-free interval (TFI) from diagnosis. ZAP-70, CD38 and IGHV mutations were evaluated in 142 patients. Combining all three factors, the ZAP-70-/CD38-/Mutated group showed the longest median TFI (62 months, n = 37), ZAP-70+/CD38+/Unmutated cases the shortest (11 months, n = 37) and cases discordant for > or = 1 factor, an intermediate TFI (27 months, n = 68) (p = 0.006). Analysis of discordant cases revealed values that were otherwise masked when measuring single prognostic factors. The presence or absence of cytogenetic abnormalities did not explain the variability among discordant cases. Simultaneous analysis of ZAP-70, CD38 and IGHV mutations in CLL provides more discriminatory prediction of TFI than any factor alone.

CLLU1 expression distinguishes chronic lymphocytic leukemia from other mature B-cell neoplasms

The distinction of CLL from other mature B-cell neoplasms, especially from leukemic forms of mantle cell lymphoma or splenic marginal zone lymphoma, can be difficult but has important prognostic and therapeutic implications. We measured CLLU1 (CLL upregulated gene1) mRNA by qPCR and found a highly significant difference between CLL and other lymphoid neoplasms (AUC 0.96, 95%CI 0.93-0.99). Based on our cut-off values we can predict CLL and other mature B-cell neoplasms with high probability (PPV 99% and 94%). Analysis of CLLU1 expression is a rapid and reliable tool that may facilitate the diagnosis of mature B-cell neoplasms especially in inconclusive cases.