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In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials., (© 2024. The Author(s).)

The somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is a critical biomarker for assessing the prognosis of patients with chronic lymphocytic leukemia (CLL). Importantly, independent studies have documented that IGHV SHM status is also a predictor of responses to therapy, including both chemoimmunotherapy (CIT) and novel, targeted agents. Moreover, immunogenetic analysis in CLL has revealed that different patients may express (quasi)identical, stereotyped B cell receptor immunoglobulin (BcR IG) and are classified into subsets based on this common feature. Patients in certain stereotyped subsets display consistent biology, clinical presentation, and outcome that are distinct from other patients, even with concordant IGHV gene SHM status. All of the above highlights the relevance of immunogenetic analysis in CLL, which is considered a cornerstone for accurate risk stratification and clinical decision making. Recommendations for robust immunogenetic analysis exist thanks to dedicated efforts by ERIC, the European Research Initiative on CLL, covering all test phases, from the pre-analytical and analytical to the post-analytical, pertaining to the analysis, interpretation, and reporting of the findings. That said, these recommendations apply to Sanger sequencing, which is increasingly being superseded by next generation sequencing (NGS), further underscoring the need for an update. Here, we present an overview of the clinical utility of immunogenetics in CLL and update our analytical recommendations with the aim to assist in the refined management of patients with CLL., (© 2022. The Author(s).)

Fludarabine, cyclophosphamide, and rituximab (FCR) is highly effective initial therapy for younger patients with chronic lymphocytic leukemia (CLL); however, most eventually relapse. Duvelisib is a delta/gamma PI3K inhibitor approved for relapsed/refractory CLL. We conducted an investigator-initiated, phase 1b/2 study of duvelisib + FCR (DFCR) as initial treatment for CLL patients aged ≤65. A standard 3 + 3 design included two dose levels of duvelisib (25 mg qd and 25 mg bid). Duvelisib was given for 1 week, then with standard FCR added for up to six 28-day cycles, then up to 2 years of duvelisib maintenance. Thirty-two patients were enrolled. The phase 2 dose of duvelisib was identified as 25 mg bid. Hematologic toxicity was common, and all-grade non-hematologic toxicities included transaminitis (28%), febrile neutropenia (22%), pneumonia (19%), and colitis (6%). The best overall response rate by ITT was 88% (56% CR/CRi and 32% PR). The best rate of bone marrow undetectable minimal residual disease (BM-uMRD) by ITT was 66%. The rate of CR with BM-uMRD at end of combination treatment (primary endpoint) was 25%. Three-year PFS and OS are 73 and 93%, respectively. DFCR is active as initial therapy of younger CLL patients. Immune-mediated and infectious toxicities occurred and required active management.

RNA editing-primarily conversion of adenosine to inosine (A > I)-is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy.

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMF low signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by "omics" profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations.

Obinutuzumab (GA101) and ibrutinib show excellent efficacy for treatment of chronic lymphocytic leukemia (CLL). Preclinical investigations and a complementary safety profile were in support of testing their combined use. The exploratory CLL2-BIG-trial evaluated a sequential combination therapy following a recently proposed strategy. Two courses of bendamustine were used for debulking in patients with a high tumor load, followed by six courses of induction therapy with ibrutinib and GA101, followed by an MRD-triggered maintenance phase. The results of a pre-planned analysis at the end of the induction phase are presented. 61 patients were included, 30 previously untreated and 31 with relapsed/refractory CLL. 44 patients received bendamustine. During induction, neutropenia (14.8%) and thrombocytopenia (13.1%) were the most common CTC grade 3 and 4 events. One fatality (duodenitis) occurred. The overall response rate was 100%. 54.1% of patients achieved a partial remission, 41% a clinical complete remission (cCR) without confirmation by CT scan or bone marrow (BM) biopsy according to protocol and 4.9% a cCR with incomplete recovery of the BM. 29 patients (47.5%) had no detectable (<10 -4 ) minimal residual disease assessed by flow cytometry in peripheral blood. In conclusion, the BIG regimen is a safe and highly effective therapy for CLL.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

The discovery of almost identical or 'stereotyped' B-cell receptor immunoglobulins (BcR IG) among unrelated patients with chronic lymphocytic leukemia (CLL) cemented the idea of antigen selection in disease ontogeny and evolution. The systematic analysis of the stereotypy phenomenon in CLL revealed that around one-third of CLL patients may be grouped into subsets based on shared sequence motifs within the variable heavy complementarity determining region 3. Stereotyped subsets display a strikingly similar biology of the leukemic clones, referring to many different levels, from the immunogenetic and genetic and extending to the epigenetic and functional levels. Even more importantly, the homogeneity of stereotyped subsets has clinical consequences as patients assigned to the same stereotyped subset generally exhibit an overall similar disease course and outcome. In other words, stereotypy-based patient classification of CLL has already provided a more compartmentalized view of this otherwise heterogeneous disease and can assist in refining prognostication models. While this is relevant only for the one-third of cases expressing stereotyped BcR IG; in principle, however, the findings from further analysis of the stereotyped subsets may also contribute towards improved understanding of the remaining non-stereotyped fraction of CLL patients.

The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in chronic lymphocytic leukemia (CLL). The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR-stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; P<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15; P<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose-responsive inhibition of pAKT(Ser473) is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR-induced chemokines CCL3 and CCL4 secretion to 17% and 37%, respectively. Pre-treatment with 1 μM IPI-145 inhibits the chemotaxis toward CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR-activated signaling proteins AKT(Ser473), BAD(Ser112), ERK(Thr202/Tyr204) and S6(Ser235/236) are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B cells, sparing normal B- and T-lymphocytes.

In order to evaluate the predictive value of positron emission tomography-computed tomography (PET/CT) in discriminating the presence of a Richter's syndrome (RS) or a second malignancy (SM), as well as to evaluate its prognostic value in patients with chronic lymphocytic leukemia (CLL), we retrospectively analyzed the data of 90 patients who, in the suspicion of a RS or a SM, underwent PET/CT followed by the biopsy of the involved tissue. The median maximum Standardized Uptake Value (SUV max) in the presence of a CLL/small lymphocytic lymphoma, a diffuse large B-cell lymphoma (DLBCL), a Hodgkin lymphoma (HL), a SM were 3.5, 14.6, 7.0 and 6.3, respectively (P ⩽ 0.0001). A SUV max cutoff value ⩾ 5 showed a sensitivity, specificity, positive and negative predictive values of 88.2, 71.2, 51.3 and 94%, respectively, for the presence of a more aggressive disease (DLBCL, HL and SM). A SUV max ⩾ 5 identified also a subset of treatment naive patients with an inferior progression-free survival (P = 0.011) and overall survival (P = 0.067). These findings suggest that PET/CT may helpfully integrate the biologically-based prognostic stratification of CLL. Prospective clinical trials including larger cohorts of patients are needed to conclusively define the role and prognostic impact of PET/CT in the routine management of CLL patients.

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH-HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10(-4) and 20% in those with MRD ≥10(-4) (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5-32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ≥10(-5) was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH-HTS method may facilitate routine MRD quantification in clinical trials.

Although the risk of progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) has been well characterized, it is unknown whether other common complications associated with CLL, such as increased risk of infection, occurs in individuals with MBL. We used the Mayo CLL database to identify cohorts of individuals with newly diagnosed MBL (n=154) or newly diagnosed CLL (n=174) who resided within 50 miles of Mayo Clinic. A cohort of 689 adult patients seen for a general medical examination who resided within 50 miles of Mayo clinic and who enrolled in a case-control study of non-Hodgkin lymphoma (NHL) was used as a comparison cohort. Hospitalization with infection was more common among individuals with MBL (25/154; 16.2%), and CLL (32/174; 18.4%) than controls (18/689; 2.6%). On pooled multivariable Cox proportional hazards analysis of all 1017 patients (controls, MBL and CLL), male sex (hazards ratio (HR)=2.3; P=0.002), major co-morbid health problems (HR=1.7, P=0.04), the presence of CLL (HR=3.2, P<0.001), treatment for progressive CLL (HR=2.4, P=0.001) and the presence of MBL (HR=3.0, P=0.001) were independently associated with risk of hospitalization for infection. These results suggest the risk of serious infection in clinical MBL is substantially greater than the risk of progression requiring treatment.

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.

Immunoglobulin gene sequence analysis is widely utilized for prognostication in chronic lymphocytic leukemia (CLL) and the definition of standardized procedures has allowed reliable and reproducible results. Occasionally, a straightforward interpretation of the sequences is not possible because of the so-called 'problematic sequences' that do not fit the 'classic' interpretation and pose scientific questions at the cross-road between hematology and immunology. Thanks to a dedicated effort within the European Research Initiative on CLL (ERIC), we have now the possibility to present such cases, offer a scientific explanation and propose recommendations in terms of prognostication.

Progress in our understanding of chronic lymphocytic leukemia and its treatment has resulted in a more tailored approach to patient management, with different therapeutic regimens for different patient populations. The current standard of care has evolved from single-agent therapy with chlorambucil or cyclophosphamide, through the introduction of purine analogs to the more recent introduction of chemoimmunotherapy. Selection of appropriate initial therapy should be based primarily on patient characteristics such as age, performance status and the expected clinical course of the leukemia based on established risk factors. Achieving a complete and durable response is the major goal for fit patients; chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab would be advantageous. Alternatively, in unfit patients, controlling symptoms is the essential treatment goal and a regimen with a more favorable toxicity profile should be applied. This manuscript reviews the data that has lead to current treatment choices, advises on tailored therapies and discusses emerging trends. Data for this review was identified by a search of electronic information including Medline and PubMed databases, conference proceedings and trial registers. Critical analysis of extracted data was undertaken with attention to trial phase, treatment schedules and end points, including response rates, follow-up times, progression-free survival and overall survival.

Rituximab-containing regimens are becoming a therapeutic standard in chronic lymphocytic leukemia (CLL), so that a validation of flow cytometric minimal residual disease (MRD) quantification (MRD flow) in the presence of this antibody is necessary. We therefore compared results obtained by real-time quantitative (RQ)-PCR to MRD flow in 530 samples from 69 patients randomized to receive chemotherapy or chemotherapy plus rituximab. Quantitative MRD levels assessed by both techniques were closely correlated irrespective of therapy (r=0.95). The sensitivity and specificity of MRD flow was not influenced by the presence of rituximab. With 58.9% positive and 26.4% negative samples by both techniques, 85.3% of assessments (452/530) were qualitatively concordant between MRD flow and RQ-PCR. Discordant samples were typically negative by MRD flow and simultaneously positive close to the detection limit of the PCR assays, indicating a higher sensitivity of PCR for very low MRD levels. However, 93.8% of all samples were concordantly classified by both methods using a threshold of 10(-4) to determine MRD positivity. MRD flow and PCR are equally effective for MRD quantification in rituximab-treated CLL patients within a sensitivity range of up to 10(-4), whereas PCR is more sensitive for detecting MRD below that level.

Telomere length (TL) has been associated with outcome in chronic lymphocytic leukemia (CLL). The aim of this extensive analysis carried out on 401 CLL patients was to assess TL conclusively as a prognostic biomarker. Our study included two cohorts used as learning (191 patients) and blinded validation series (210 patients). A TL cutoff of 5000 bp was chosen by receiver operating characteristic (ROC) analysis and Youden's index in the learning series. In this series, TL< or =5000 bp was independently associated to a worse outcome for both overall survival (OS; 105.5 vs 281 months, P<0.001) and treatment-free survival (TFS; 24.6 vs 73 months, P<0.001). In the blinded validation series, TL< or =5000 bp was confirmed as an independent outcome predictor for OS (79.8 vs not reached, P<0.001) and TFS (15.2 vs 130.8 months, P<0.001). Moreover, TL< or =5000 bp independently predicted the risk of Richter's syndrome (5-year risk: 18.9 vs 6.4%, P=0.016). Within CLL subsets defined by biological predictors, TL consistently identified patient subgroups harboring unfavorable prognosis. These results demonstrate that TL is a powerful independent predictor of multiple outcomes in CLL, and contributes to refine the prognostic assessment of this disease when utilized in combination with other prognostic markers. We thus believe that this prognostic biomarker has the potential for a more widespread use in CLL.

The (-938C>A) polymorphism in the promoter region of the BCL-2 gene was recently associated with inferior time to treatment and overall survival in B-cell chronic lymphocytic leukemia (CLL) patients displaying the -938A/A genotype and may thus serve as an unfavorable genetic marker in CLL. Furthermore, the -938A/A genotype was associated with increased expression of Bcl-2. To investigate this further, we analyzed the -938 genotypes of the BCL-2 gene in 268 CLL patients and correlated data with treatment status, overall survival and known prognostic factors, for example, Binet stage, immunoglobulin heavy-chain variable (IGHV) mutational status and CD38 expression. In contrast to the recent report, the current cohort of CLL patients showed no differences either in time to treatment or overall survival in relation to usage of a particular genotype. In addition, no correlation was evident between the (-938C>A) genotypes and IGHV mutational status, Binet stage or CD38. Furthermore, the polymorphism did not appear to affect the Bcl-2 expression at the RNA level. Taken together, our data do not support the use of the (-938C>A) BCL-2 polymorphism as a prognostic marker in CLL and argue against its postulated role in modulating Bcl-2 levels.

Four chromosomal defects associated with outcome are commonly evaluated by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia (CLL), namely deletions of the 13q13-q14, 11q22 and 17p13 regions and trisomy 12. In this study, we compared a quantitative PCR method--quantitative multiplex PCR of short fluorescent fragment (QMPSF)--with FISH for the detection of these acquired aneuploidies in a series of 110 patients with Binet stage A CLL. Genes located in the deleted or gained regions were selected as target genes and amplified using a method based on the simultaneous amplification of short fluorescent genomic fragments under quantitative conditions. A chromosomal imbalance involving one or several of the four loci was detected by either method in 72 patients (65%). A chromosome 13 deletion was present in 61 patients (54%), a 11q22 deletion in nine (8%), a trisomy 12 in nine and a 17p deletion in one. FISH and QMPSF results were identical for 103 out of 110 patients and discrepancies could be explained in most cases. This study demonstrates that a quantitative multiplex PCR represents a cost-effective method that could replace FISH in CLL patients. However, although QMPSF is perfectly adapted to the detection of primary defects, care should be taken when searching for clonal evolutions present in a small proportion of tumor cells.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin V(H) gene (IgV(H))mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high ('LPL+', n=10) or low ('LPL-', n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL- B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P

B-cell chronic lymphocytic leukaemia (B-CLL) is a slowly progressing malignancy of CD5(+) B cells, for which at present no curative treatment is available. In our current study, we apply a novel bridging reagent to redirect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) to target B-CLL. A streptavidin-fused anti-CD20 single-chain variable fragment (scFv) is used in combination with biotinylated MHC class I molecules containing CMV pp65 peptide (HLA/CMV). We demonstrate that B-CLL cells coated with this CD20-HLA/CMV complex can be lysed by autologous CMV-specific CTL with similar efficiency as B-CLL cells directly loaded with CMV peptide. Killing is HLA restricted and occurs at scFv CD20 concentrations of >/=100 ng ml(-1) and HLA/CMV concentrations of >/=20 ng ml(-1). Furthermore, complex-coated B-CLL cells induce both proliferation and cytokine production (interferon gamma, tumour necrosis factor alpha and macrophage inflammatory protein-1 beta) in CMV-specific CD8(+) T cells. Hereby, a necessary step towards possible application of CD20-HLA/CMV complexes for immunotherapy of B-cell malignancies is constituted.

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. Strong and constitutive nuclear factor kappa B (NF-kappaB) activation is a characteristic of CLL cells. We examined the effects of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on CLL cells. Dehydroxymethylepoxyquinomicin completely abrogated constitutive NF-kappaB activity and induced apoptosis of CLL cells. Apoptosis induced by DHMEQ was accompanied by downregulation of NF-kappaB-dependent antiapoptotic genes: c-IAP, Bfl-1, Bcl-X(L) and c-FLIP. Dehydroxymethylepoxyquinomicin also inhibited NF-kappaB induced by CD40 and enhanced fludarabine-mediated apoptosis of CLL cells. Results of this study suggest that inhibition of constitutive and inducible NF-kappaB by DHMEQ in combination with fludarabine is a promising strategy for the treatment of CLL.

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.

The purpose of this study was to document the frequency and distribution of karyotypic changes present at diagnosis in 103 non-MALT marginal zone cell lymphoma (MZL) patients. This cytogenetic analysis of a large cohort extends previous observations and allows the identification of new cytogenetic features. Abnormalities identified in more than 15% of patients included +3/+3q (37%), 7q deletions (31%), +18/+18q (28%), 6q deletions (19%), +12/+12q (15%) and 8p deletions (15%). Trisomy 3/3q, 7q deletions, +18 and +12 were seen in different combinations in more than 30% of patients in comparison to 2% in lymphocytic lymphomas/chronic lymphocytic leukemias, 1% in mantle cell lymphomas and 7% in follicular lymphomas. The marked propensity of these abnormalities to be recurrently associated with the same tumoral clone of individual karyotypes allowed the delineation of a cytogenetic profile that may help to distinguish non-MALT MZL among other mature B-cell neoplasms. If +3/3q, +12/+12q, and 6q, 7q and 8p deletions were significantly associated with clinical prognostic factors previously reported to influence survival and time to progression, patients displaying these abnormalities did not experience a significantly shorter time to progression.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavy-chain variable region genes (IGV(H)) compared to those with mutated IGV(H) genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N=51) or unmutated (N=53) IGV(H) (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV(H) mutational status (R=0.614; P<0.0001). High LPL expression predicted unmutated IGV(H) status with an odds ratio of 25.90 (P<0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P=0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.