Natural History Of Chronic Lymphocytic Leukemia Research Articles

B cell receptor immunoglobulin stereotypy in chronic lymphocytic leukemia: Key to understanding disease biology and stratifying patients

Sequence convergence, otherwise stereotypy, of B cell receptor immunoglobulin (BcR IG) from unrelated patients is a distinctive feature of the IG gene repertoire in chronic lymphocytic leukemia (CLL) whereby patients expressing a particular BcR IG archetype are classified into groups termed stereotyped subsets. From a biological perspective, the fact that a considerable fraction (∼41%) of patients with CLL express (quasi)identical or stereotyped BcR IG underscores the key role of antigen selection in the natural history of CLL. From a clinical perspective, at odds with the pronounced heterogeneity of CLL at large, patients belonging to the same stereotyped subset display consistent clinical presentation and outcome, including response to treatment, likely as a reflection of consistent biological background. Many major stereotyped subsets were recently shown to have satellites, that is, smaller subsets that are immunogenetically similar. Preliminary evidence supports that this similarity extends to shared biological and even clinical features, with important implications for patient stratification. Consequently, BcR IG stereotypy emerges as a powerful tool for dissecting the heterogeneity of CLL toward refined risk stratification and, eventually, more precise therapeutic interventions.

RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia

AbstractRecent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

Impact of Serum Immunoglobulin Subsets and Levels on Chronic Lymphocytic Leukemia Natural History: A Retrospective Multicentric Italian Experience

Introduction: Chronic Lymphocytic Leukemia (CLL) is a clinically and biologically heterogeneous disorder that can be associated with immunological dysregulation. Several prognostic factors, such as IGHV status and chromosomal aberrations as trisomy 12, del11q, del13q or del17p have been detected so far. More recent genetic mutations, such as BIRC3, SF3B1, NOTCH1 and TP53 stratify even further the prognosis and the outcome in CLL patients (pts). However all data above reported, because of expensive techniques and experience typical of big laboratories, are not available to all medical centers. Therefore we considered necessary to identify more accessible and easy to perform parameters in all laboratories, as data concerning the presence of hypogammaglobulinemia, IgM or IgG monoclonal gammopathy, whose prevalence and impact on natural history of CLL in the medical literature are controversial, contradictory and not very significant because of few numbers in cohorts. Therefore the first aim of the study was to evaluate the prevalence of monoclonal IgM/CLL, IgG/CLL and hypogammaglobulinemia compared with CLL pts with normal immunoglobulins (Ig) levels and to establish the impact on clinical outcomes based on Ig levels. The second aim was to find a correlation between each group of pts and biological data. Methods: From four different Italian centers, we collected data from 1505 pts diagnosed with typical CLL from 1987 to 2017 and we divided them in four groups based on the presence of qualitative and quantitative alterations of serum immunoglobulins. Baseline assessment and levels of serum Ig, immunofixation, chromosomal aberrations and clinical features were collected. [Table 1]. We evaluated the impact of each immunoglobulin subset on progression free survival (PFS) and overall survival (OS). Results: Once assessment was made, we included in our study 1505 pts. Median age of 65.5 was similar in all groups and data gathered from the four centers showed an overlapping rate about prevalence and time-dependent parameters in CLL pts and their related subclasses. The overall prevalence of monoclonal gammopathy was 15%, of whom 149 pts (10%) with IgG/CLL and 73 pts (5%) with IgM/CLL. Hypogammaglobulinemia was detected in 200 pts (13%), while 1083 pts (72%) had no evidence of paraprotein or hypogammaglobulinemia. The worst outcome group was identified among CLL with IgM paraprotein; moreover, this group of pts showed more advanced stages at diagnosis according to Binet staging (p<0.002) and a higher frequency of del17p with a percentage of 15% (p<0.022). [Table 1]. The worse median PFS observed was in IgM/CLL group, compared to Hypogamma/CLL, IgG/CLL, and normal/CLL: 33, 54, 62, 96 months respectively (p<0.0001). At the same time, a projection at 150-months showed an estimated OS of 62% in IgM/CLL group, versus 68% in hypogamma/CLL, 81% in IgG/CLL and 86% in normal/CLL (p<0.0001). To evaluate if the specific deficit of IgG or IgM could have any unfavourable impact on each group's prognosis, we measured the serum levels of IgM and IgG in all pts. No statistically significant differences were observed among time-dependent parameters in IgG/CLL, IgM/CLL and hypogamma groups, based on IgM and IgG levels, if deficient or normal.Finally, in the group of pts who had normal gamma-level, median PFS and TTT was reached at 79 months (p=0.006 and 0.002, respectively) in the subgroup with IgM deficiency, while no statistical difference was observed regarding OS (p=0.785). Conclusions: Our study provides data about prevalence and impact of monoclonal gammopathy and hypogammaglobulinemia on the outcomes of CLL patients. In this study, we observed that the 28% pts had any qualitative or quantitative immunoglobulins alteration, of whom 15% had monoclonal gammopathy and 13% had hypogammaglobulinemia. The worst outcomes were observed in the IgM/CLL pts in terms of PFS and OS. In conclusion, the laboratory data of serum Ig could be used as predictive markers of clinical outcomes because, for their easy availability, they could direct the prognosis of CLL pts. As a future perspective, we would like to characterize pts with monoclonal IgM component from a biological view, to correlate the subset of somatic mutations of the heavy chains of surface immunoglobulins with the monoclonal component and its prognostic significance. Disclosures Varettoni: ABBVIE: Other: travel expenses; Roche: Consultancy; Gilead: Other: travel expenses; Janssen: Consultancy. Trentin:ABBVIE: Honoraria, Other: board; Janssen: Consultancy, Honoraria, Other: Board; gilead: Consultancy; Roche: Honoraria, Other: Board.

Relationship between the expression of CD25 and CD69 on the surface of lymphocytes T and B from peripheral blood and bone marrow of patients with chronic lymphocytic leukemia and established prognostic factors of this disease.

Chronic lymphocytic leukemia (CLL) is a condition characterized by the accumulation of morphologically mature monoclonal lymphocytes B with the CD19+/CD5+/CD23+ phenotype in lymphoid tissue, peripheral blood and bone marrow. The clinical course of patients with CLL is heterogeneous, ranging from indolent to aggressive. The role of lymphocyte activation in the natural history of CLL is still a matter of discussion. The aim of this study was to determine the percentages and absolute numbers of lymphocytes B and T in peripheral blood and bone marrow of CLL patients. Moreover, we analyzed the relationship between the number of CD25-positive and CD69-positive lymphocytes and the established prognostic factors in CLL. The study included 80 untreated patients with CLL and 20 healthy subjects. The immunophenotype of peripheral blood mononuclear cells (in both groups) and bone marrow cells (solely in the CLL group) was determined by means of flow cytometry. Patients with CLL showed a higher absolute number of activated lymphocytes B with phenotypes CD19+CD25+ and CD19+CD69+, as well as a higher absolute number of CD3+CD25+ lymphocytes T than the controls. The enhanced activation of peripheral blood and bone marrow lymphocytes was associated with higher Rai stages, an increased concentration of lactate dehydrogenase and beta-2 microglobulin and the progression of the disease. The number of lymphocytes B CD19+ZAP-70+ correlated positively with the number of CD19+CD25+ B cells and CD3+CD69+ T cells. The study confirmed the association between an unfavorable prognosis and a high expression of activation markers in CLL patients. The determination of CD25+ and CD69+ lymphocytes T and B constitutes a valuable diagnostic tool, completing the cytometric evaluation of CLL.

Comparing overall survival (OS) outcomes in patients with newly diagnosed chronic lymphocytic leukemia (CLL) with normal life expectancy.

7 Background: CLL is an orphan hematologic malignancy but also accounts for the most common adult leukemia in the Western Hemisphere, with a median age at diagnosis of 72 years. Median OS for symptomatic, treatment-naïve, elderly patients with CLL is approximately 5.3 years (CLL5; Eichhorst, et al. Blood 2009), whereas the life expectancy of a 72 year old (yo) is around 12 years (US Life Tables, 2006). Ibrutinib, an oral, first-in-class, covalent Bruton’s tyrosine kinase inhibitor, significantly improves OS. Early results from a phase 1/2 study in treatment-naïve elderly patients with CLL (PCYC-1102; median age, 71 years) suggest that single-agent ibrutinib extends OS, with survival at 3 years of approximately 97% (Byrd, et al. Blood 2015). Here we compare the OS in newly diagnosed patients with CLL treated with single-agent ibrutinib or chlorambucil with the normal life expectancy of a 72 yo adult. Methods: OS outcomes for patients treated with chlorambucil from the CLL5 study conducted in Germany (Eichhorst, et al. Blood 2009) were compared with long-term OS results from PCYC-1102 (Byrd, et al. Blood 2015). Life expectancy data were obtained from US Life Tables. Results: Results from CLL5 suggest that single-agent chlorambucil has a median OS of 5.3 years (median age, 71 years), with approximately 75% of patients alive at 3 years. The median OS for ibrutinib has not been reached (median follow-up, 3 years), with an estimated progression-free survival of 96% and OS of 97% at 3 years. Life expectancy for a 72 yo healthy person at 3 years is approximately 95%. Naïve comparisons suggest that single-agent ibrutinib may be altering the natural history of CLL, providing a longer OS versus chlorambucil and a similar OS as expected in a 72 yo in the general population. Conclusions: Treatment outcomes in CLL are fundamentally changing with the advent of new targeted therapies. This indirect naïve comparison between ibrutinib-treated patients and a general population suggests we may be moving from palliative care for elderly patients with CLL to chronic care management. Implications for chronic care management of patients with CLL will be discussed.

ATM Mutations in Major Stereotyped CLL Subsets: Enrichment in Subset #2 is Associated with Unfavourable Outcome

Chronic lymphocytic leukemia (CLL) is a paradigmatic malignancy where the interplay of cell-extrinsic and cell-intrinsic factors has a major impact on disease evolution. Indeed, extrinsic triggering, e.g. antigenic stimulation through the B-cell receptor (BcR), together with intrinsic aberrations, e.g. accumulation of genetic defects, play a major role throughout the natural history of CLL. The importance of antigen involvement is underscored by the existence of 'stereotyped' BcR in up to 30% of CLL patients. Notably, CLL patients with stereotyped BcR can be grouped into different subsets, each with a subset-biased biological and clinical profile. For instance, while the clinically aggressive subset #2 (IGHV3-21/IGLV3-21, comprising both mutated (M-CLL) and unmutated (U-CLL) IGHV genes) displays a remarkably high frequency of SF3B1 mutations, subset #8, a subset with the highest risk of Richter transformation, shows a strong association with trisomy 12 and NOTCH1 mutations. ATM defects are implicated in the evolution of CLL and are associated with a dismal prognosis, however the extent to which they contribute to the genetic landscape in stereotyped subsets remains unexplored.To gain insight into this issue, we assessed the frequency of ATM mutations in 249 well-characterized CLL patients assigned to major subsets #1-8. The entire coding region of ATM (62 exons) was investigated with two different targeted deep-sequencing approaches, i.e. Haloplex technology (HiSeq, coverage ~1500X) or the Nextera XT kit (MiSeq, coverage ~4000X). A conservative variant allele frequency cut-off of 10% was selected, and mutations were validated by Sanger sequencing.Altogether, we identified 61 ATM mutations in 47/249 (19%) patients across all major subsets (Fig. 1). As expected, the majority of identified ATM mutations (n=43, 70%) have not yet been reported while the remaining 30% were listed in the HGMD or COSMIC mutation databases. The spectrum of ATM mutations included missense (n=31), nonsense (n=9), splicing (n=6), and frame-shift (n=14) mutations, and one in-frame deletion. Missense substitutions were distributed along the entire gene without any 'hotspot' region or preferred domain. The highest mutation frequency was detected in subset #2 (26%), with a significant enrichment in U-CLL vs. M-CLL cases, (13/33 vs. 8/48 subset #2 cases, respectively; p=0.021). Within poor-prognostic U-CLL subsets, ATM mutations were also frequent in subsets #6 (25%) and #7 (23%), while subsets #3, #5, #1, and #8 showed lower frequencies (17%, 17%, 13%, and 7%, respectively). The favorable prognostic M-CLL subset #4 exhibited a low frequency (7%) of ATM mutations. Notably, when comparing the two most populated subsets, i.e. #1 and #2, ATM mutations were overrepresented in the latter with a borderline significance value (p=0.086); when restricting the analysis to U-CLL #2 cases a significantly higher frequency was observed compared to #1 (13/33 vs 9/68; p=0.0045).Regarding the clinical impact of ATM defects in subset #2, we divided patients into subgroups with biallelic inactivation (def-ATM), sole 11q-, sole ATM -mutation, TP53 -aberrations and WT. While both groups with mono-allelic ATM disruption showed a significantly reduced overall survival compared to WT (median survival sole ATM -mutation, 71 months, sole 11q-, 40 months, vs. 123 months in the WT group; p=0.002 and 0.02, respectively), a non-significant reduction of overall survival was observed for patients with bi-allelic ATM aberrations (70 months, p=0.29) (Fig. 2). The few subset #2 patients with TP53 defects showed a similar survival as WT group, underscoring previous observations that TP53 dysfunction per se plays a minor role in this subset.In summary, we demonstrate that ATM mutations can be added to the list of genetic defects with a biased distribution in stereotyped subsets. The enrichment of ATM defects in subset #2 was associated with a negative impact on overall survival, suggesting a role for ATM inactivation in shaping the aggressive phenotype of this subset. This study further supports the recent suggestion that CLL development is driven by antigenic selection, coupled with preferential acquisition of specific genetic defects. The work was supported by the projects MSMT CR CZ.1.05/1.1.00/02.0068, IGA NT13493-4/2012 and TACR TE02000058. [Display omitted] [Display omitted] DisclosuresLangerak:Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. . Strefford:Roche: Research Funding. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding.

Next Generation Sequence Immunoprofiling of the T-Cell Repertoire in Chronic Lymphocytic Leukemia Supports Selection By Shared Antigenic Elements

Chronic lymphocytic leukemia (CLL) is characterized by remarkable skewing of the B-cell receptor immunoglobulin (BcR IG) repertoire, culminating in the existence of subsets of patients with stereotyped BcR IGs. This implies antigen selection in the natural history of CLL, ultimately affecting clonal behavior. Currently, limited information is available regarding the role of antigens in the selection and activation of cognate T cells, although this is relevant in light of B and T cell interactions inducing T cell tolerance. Our preliminary next-generation sequencing (NGS) studies in 11 patients assigned to stereotyped subset #4, a clinically indolent disease subgroup, indicated T-cell receptor beta chain (TRB) gene repertoire restriction and oligoclonality. Prompted by these observations, here we sought to obtain a comprehensive view of the T-cell repertoire in CLL by extending our analysis to 36 untreated CLL patients, either assigned to major stereotyped subsets [subset #4 (n=11), subset #1 (n=10), subset #2 (n=4), subset #16 (n=1)] or non-subset cases [with mutated-M (n=5) or unmutated-UM clonotypic BcR IGs (n=5)]. Starting material was PB mononuclear cells (n=27), purified CD4+ and CD8+ T-cell subpopulations (n=10), bone marrow (n=2) or lymph node (LN) tissue (n=1). Three patients were studied overtime. Multiple sample and PCR replicates, as well as 3 age-matched healthy controls were also included. TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA (BIOMED2 protocol) and subjected to paired-end NGS, designed to cover the complementarity determining region 3 (CDR3) twice/sequence. In order to further increase the accuracy of results, raw NGS reads were subjected to a purpose-built, bioinformatics algorithm, performing: (i) length and quality filtering of raw reads; (ii) merging of filtered-in paired reads via local alignment; (iii) length and quality filtering of stitched sequences. No base calls of Q-score<30 were allowed in the 75 nucleotide stretch ahead of the FGXG motif, thus further increasing the CDR3 sequencing reliability. Filtered-in sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline designed for clonotype computation and repertoire analysis. Overall, 66 samples were analyzed, producing 23,238,779 filtered-in sequences. Only productive, in-frame TRBV-TRBD-TRBJ rearrangements were included in the analysis (85.8% of filtered-in sequences, median 354,972/sample). For repertoire analysis, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) were considered (median 37,550 distinct clonotypes/sample; 13,134 expanded versus 22,550 singletons). The distribution of TRBV genes was asymmetric with only 5 genes [TRBV12-3/12-4 (8.4%), TRBV19 (6.8%), TRBV29-1 (6.6%), TRBV5-1 (6.1%), TRBV6-5 (5.2%)] collectively accounting for one-third of the repertoire, indicating functional constraints. No significant TRBV gene repertoire difference was identified across the different immunogenetic CLL subgroups analyzed. In contrast, significant (p<0.001) differences were seen between CD8+ versus CD4+ cells regarding certain genes (e.g. over-representation of TRBV7-2 in CD4+ and under-representation of TRBV5-1 in CD8+ cells), alluding to distinct immune selective processes. Notably, the TRB repertoire was significantly more oligoclonal in CLL compared to the healthy controls (median frequency of the predominant clonotype: 5.7% versus 1.27%, respectively, p<0.05), albeit with differences between cases with distinct BcR IG, perhaps reflecting their different immunopathogenesis. Overtime analysis showed persistence of most major clonotypes in 2/3 cases studied, likely in a context of persistent antigenic stimulation. Cluster analysis of all CLL cases identified 17,592 different clonotypes (excluding singletons) shared by different patients and not present in the healthy controls, arguing in favor of a disease-biased immunoprofile; formal proof of this claim will require analysis of a larger number of healthy controls, currently underway by our group. In conclusion, massive parallel sequencing documents the restricted nature of the TRB repertoire in CLL, supporting selection by shared antigenic elements. Whether these are the same antigens interacting with the malignant clone remains to be elucidated. DisclosuresStamatopoulos:Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

Recurrent Mutations within the Nfkbie gene: A Novel Mechanism for NF-κB Deregulation in Aggressive Chronic Lymphocytic Leukemia

Dysregulated NF-κB signaling appears to be particularly important in B-cell malignancies, with recurrent mutations identified within both the canonical and non-canonical NF-κB pathways, as well as in components of the B-cell receptor (BcR) and Toll-like receptor (TLR) signaling pathways. In chronic lymphocytic leukemia (CLL), although recurrent mutations have been identified in MYD88 (TLR signaling) and BIRC3 (non-canonical NF-κB pathway), their frequency is low (<3%) and hence the extent to which genetic aberrations may contribute to constitutional NF-κB activation remains largely unknown. To gain further insight into this issue, we designed a HaloPlex gene panel (Agilent Technologies) and performed targeted next-generation sequencing (NGS) (HiSeq 2000/Illumina) of 18 NF-κB genes in a discovery cohort of 124 CLL patients, intentionally biased towards poor-prognostic patients with either unmutated IGHV genes or high-risk genomic aberrations. Using a conservative cutoff of >10% for the mutant allele, we identified mutations (n=35) within 30/124 (24%) patients in 14/18 NF-κB genes analyzed. IκB genes, which encode for cytoplasmic inhibitor proteins, accounted for 20/35 (57%) mutations, with IκBε (encoded by NFKBIE) mutated in 8 patients; notably, 3/8 cases carried an identical 4bp deletion within exon 1 of NFKBIE. Prompted by these findings, we proceeded to validate our findings in an independent CLL cohort (n=168) using the same methodology as above and primarily focusing on cases with poor-prognostic features. We identified 30 mutations within 28 CLL patients in 11/18 NF-κB genes analyzed. Strikingly, 13/30 mutations were found within IκBε, with 10/13 patients carrying the same 4bp NFKBIE deletion. Notably, investigations into whether additional cases (within both the discovery and validation cohort) may harbor mutations of low clonal abundance (<10% mutant allele), led to the detection of the NFKBIE deletion in another 18 cases. Owing to the prevalence of this 4bp deletion within the NFKBIE gene, we developed a GeneScan assay and screened an additional 312 CLL cases. Collectively, 40/604 (6.6%) CLL patients were found to carry this frame-shift deletion within the NFKBIE gene, which is in line with a recent publication reporting that 10% of Binet stage B/C patients carried this mutation (Damm et al. Cancer Discovery 2014). Remarkably, the majority of these NFKBIE mutations (16/40) were found in a subgroup of patients that expressed highly similar or stereotyped BcRs and are known to have a particularly poor outcome, denoted as subset #1. This finding thus alludes to a subset-biased acquisition and/or selection of genomic aberrations, similar to what has been reported for subset #2 and SF3B1, perhaps as a result of particular modes of BcR/antigen interaction. We utilized proximity-ligation assays to test the functional impact of the NFKBIE deletion by investigating protein-protein interactions. This analysis revealed reduced interaction between the inhibitor IκBε and the transcription factor p65 in NFKBIE-deleted CLL cells; IκBε-knock-down shRNA experiments confirmed dysregulated apoptosis/NF-κB signaling. Finally, to assess whether the NFKBIE deletion could also be present in other B-cell malignancies, we screened 372 mature B-cell lymphoma cases using NGS or the GeneScan assay and found the deletion in 7/136 (5.1%) mantle cell lymphomas, 3/66 (4.5%) diffuse large B-cell lymphomas and 3/170 (1.8%) splenic marginal zone lymphomas. Taken together, our analysis revealed that inactivating mutations within the NFKBIE gene lead to NF-κB activation in CLL and potentially several other B-cell-derived malignancies. Considering the central role of BcR stimulation in the natural history of CLL, the functional loss of IκBε may significantly contribute to sustained CLL cell survival and shape the disease evolution. This novel data strongly indicates that components of the NF-κB signaling pathway may be prime targets for future targeted therapies not only in CLL but also other mature B-cell lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Clonal evolution including 14q32/IGH translocations in chronic lymphocytic leukemia: analysis of clinicobiologic correlations in 105 patients

To better define the significance of clonal evolution (CE) including 14q32 translocations involving the immunoglobulin heavy chain gene (IGH) in chronic lymphocytic leukemia (CLL), 105 patients were analyzed sequentially by fluorescence in situ hybridization (FISH) with the following panel of probes: 13q14/D13S25, 11q22/ATM, 17p13/TP53, #12-centromere and 14q32/IGH break-apart probe. CE was observed in 15/105 patients after 24–170 months (median 64). Recurring aberrations at CE were 14q32/IGH translocation in seven patients; other aberrations were 17p −, 11q −, biallelic 13q − and 14q32 deletion. CE was detected in 15/58 pre-treated patients; in contrast, none of 47 untreated patients developed CE (p < 0.0001). In two cases the appearance of 14q32/IGH translocation was first detected in the bone marrow (BM) or in the lymph node (LN) and 13–58 months later in the peripheral blood (PB). ZAP70 + and high-risk cytogenetics predicted for the occurrence of CE with borderline statistical significance (p = 0.055 and 0.07, respectively). Shorter time to first treatment (TTT) and time to chemorefractoriness (TTCR) were noted in 15 patients with CE when compared to patients without CE (TTT: 35 vs. 71 months, p = 0.0033 and TTCR: 34 vs. 86 months, p = 0.0046, respectively). Survival after the development of CE was 32 months (standard error 8.5). We arrived at the following conclusions: (i) 14q32/IGH translocation may represent one of the most frequent aberrations acquired during the natural history of CLL and (ii) it may be detected earlier in BM or LN samples; (iii) CE including 14q32/IGH translocation occurs in pre-treated patients with short TTT and TTCR; (iii) survival after CE is relatively short.

Treatment options for high-risk chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world. The natural history of CLL is extremely variable with a survival time from initial diagnosis that ranges from 2 to more than 20 years. Understanding the clinical diversity and allowing the subclassification of CLL into various prognostic groups not only assists in predicting future outcome for patients, but also helps to direct treatment decisions. Chlorambucil and fludarabine were the standard therapy for CLL for decades. Randomized studies have reported superior overall response and progression-free survival (PFS) for fludarabine compared with alkylator-based therapy and for the fludarabine-cyclophospamide (FC) combination over fludarabine alone. More recently the addition of rituximab to the FC regimen (R-FC) has shown significant improvement in overall response, PFS and overall survival compared with FC alone. However, there are patients for whom this regimen still provides less satisfactory results. Within the above studies CLL patients who have some of the poorer prognostic markers, such as unmutated IgVH genes and/or high beta-2 microglobulin (B2M), and those who fail to achieve a minimal residual disease (MRD) negative remission are likely to have a shorter PFS compared with those without these features. Various strategies have been explored to improve the outcome for such patients. These include the addition of agents to a frontline R-FC regimen, use of consolidation and consideration of maintenance. The only group that can be clearly identified pretreatment for whom conventional fludarabine-based therapies produce significantly inferior response rates, PFS and overall survival are the patients who harbour a genetic fault; deletion or mutation or a combination of deletion and mutation of tumour protein p53 (TP53). TP53 inactivation is a less common finding at first treatment but becomes much more common in fludarabine-refractory patients. Alemtuzumab and high-dose corticosteroids have been shown to be effective in this group of CLL patients. Trials combining these two agents have shown improved responses, particularly for those patients with bulky nodal disease for whom alemtuzumab alone may be insufficient. Since the duration of responses remains relatively short, suitable patients should be considered for allogeneic stem cell transplantation according to the European Group for Blood and Marrow Transplantation (EBMT) guidelines. Furthermore, there are a number of other new treatments on the horizon, including humanized antibodies directed against novel targets and small-molecule inhibitors.

Richter syndrome: molecular insights and clinical perspectives

Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). The clinical definition of RS is heterogeneous, and encompasses at least two biologically different conditions: (i) CLL transformation to a clonally related DLBCL, that accounts for the majority of cases; (ii) development of a DLBCL unrelated to the CLL clone. In clonally related RS, the pathogenetic link between the CLL and the DLBCL phases is substantiated by the acquisition of novel molecular lesions at the time of clinico-pathologic transformation. RS is not a rare event in the natural history of CLL, since the cumulative incidence of RS at 10 years exceeds 10%. Prompt recognition of RS is known to be clinically useful, and may be favoured by close monitoring of CLL patients harbouring clinical and/or biological risk factors of RS development. Conventional risk factors that are independent predictors of RS development at the time of CLL diagnosis include: (i) expression of CD38; (ii) absence of del13q14 and (iii) lymph node size > or =3 cm. Other risk factors of RS development include CD38 genotype and usage of specific immunoglobulin variable genes. The molecular pathogenesis of RS has been elucidated to a certain extent. Acquisition of TP53 mutations and/or 17p13 deletion is a frequent molecular event in RS, as it is in other types of transformation from indolent to aggressive B-cell malignancies. Additional molecular alterations are being revealed by genome wide studies. Once that transformation has occurred, RS prognosis may be predicted by the RS score, based on performance status, LDH, platelet count, tumour size and number of prior therapies. Depending on patient's age and RS score, the therapeutic options for RS may range from conventional immunochemotherapy to allogeneic bone marrow transplantation.

Systematic genomic screen for tyrosine kinase mutations in CLL

Systematic genomic screen for tyrosine kinase mutations in CLL