Integrative NGS testing reveals clonal dynamics of adverse genomic defects contributing to a natural progression in treatment-naïve CLL patients.

Large-scale next-generation sequencing (NGS) studies revealed extensive genetic heterogeneity, driving a highly variable clinical course of chronic lymphocytic leukaemia (CLL). The evolution of subclonal populations contributes to diverse therapy responses and disease refractoriness. Besides, the dynamics and impact of subpopulations before therapy initiation are not well understood. We examined changes in genomic defects in serial samples of 100 untreated CLL patients, spanning from indolent to aggressive disease. A comprehensive NGS panel LYNX, which provides targeted mutational analysis and genome-wide chromosomal defect assessment, was employed. We observed dynamic changes in the composition and/or proportion of genomic aberrations in most patients (62%). Clonal evolution of gene variants prevailed over the chromosomal alterations. Unsupervised clustering based on aberration dynamics revealed four groups of patients with different clinical behaviour. An adverse cluster was associated with fast progression and early therapy need, characterized by the expansion of TP53 defects, ATM mutations, and 18p- alongside dynamic SF3B1 mutations. Our results show that clonal evolution is active even without therapy pressure and that repeated genetic testing can be clinically relevant during long-term patient monitoring. Moreover, integrative NGS testing contributes to the consolidated evaluation of results and accurate assessment of individual patient prognosis.

Low-burden TP53 mutations in CLL: clinical impact and clonal evolution within the context of different treatment options.

Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter for debate. Herein, we describe clonal evolution scenarios of low-burden TP53 mutations, the clinical impact of which we analyzed in a "real-world" CLL cohort. TP53 status was assessed by targeted next-generation sequencing (NGS) in 511 patients entering first-line treatment with chemo- and/or immunotherapy and 159 patients in relapse before treatment with targeted agents. Within the pretherapy cohort, 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). Although their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). In a subgroup with TP53 mutations of 1% to 10% VAF, the impact on OS was observed only in patients with unmutated IGHV who had not received targeted therapy, as patients benefited from switching to targeted agents, regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with fludarabine, cyclophosphamide, and rituximab regimen in both first- and second-line treatments (median VAF increase, 14.8× and 11.8×, respectively) in contrast to treatment with less intense treatment regimens (1.6×) and no treatment (0.8×). In the relapse cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change, 1×). Sporadic cases of TP53 mutations' clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision making.

Evolution of TP53 abnormalities during CLL disease course is associated with telomere length changes.

BACKGROUND: Telomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved. METHODS: We investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity. RESULTS: At baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively). CONCLUSIONS: Our study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia.

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

Casein kinase 1 is a therapeutic target in chronic lymphocytic leukemia.

Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.

ToTem: a tool for variant calling pipeline optimization.

BACKGROUND: High-throughput bioinformatics analyses of next generation sequencing (NGS) data often require challenging pipeline optimization. The key problem is choosing appropriate tools and selecting the best parameters for optimal precision and recall. RESULTS: Here we introduce ToTem, a tool for automated pipeline optimization. ToTem is a stand-alone web application with a comprehensive graphical user interface (GUI). ToTem is written in Java and PHP with an underlying connection to a MySQL database. Its primary role is to automatically generate, execute and benchmark different variant calling pipeline settings. Our tool allows an analysis to be started from any level of the process and with the possibility of plugging almost any tool or code. To prevent an over-fitting of pipeline parameters, ToTem ensures the reproducibility of these by using cross validation techniques that penalize the final precision, recall and F-measure. The results are interpreted as interactive graphs and tables allowing an optimal pipeline to be selected, based on the user's priorities. Using ToTem, we were able to optimize somatic variant calling from ultra-deep targeted gene sequencing (TGS) data and germline variant detection in whole genome sequencing (WGS) data. CONCLUSIONS: ToTem is a tool for automated pipeline optimization which is freely available as a web application at https://totem.software .

Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.

Decreased WNT3 expression in chronic lymphocytic leukaemia is a hallmark of disease progression and identifies patients with worse prognosis in the subgroup with mutated IGHV.

The canonical Wnt pathway, dependent on ß-catenin-controlled transcription, is the most explored Wnt pathway, known to drive the malignant transformation of multiple cell types. Several reports have suggested that this pathway also participates in chronic lymphocytic leukaemia (CLL) pathogenesis. To get a better insight into the role of the Wnt/ß-catenin pathway in CLL we analysed in detail the expression of the most overexpressed Wnt ligand, encoded by the WNT3 gene, in a well-defined cohort of 137 CLL patients. Our analysis demonstrated that (i) untreated patients with more aggressive disease (with a notable exception of patients with 11q deletion) express less WNT3, (ii) WNT3 declines with disease progression in a significant proportion of patients and (iii) low WNT3 was identified as a strong independent marker indicating shorter treatment-free survival in CLL patients with IGHV mutation. Interestingly, CLL-related lymphoid cell lines, but not stromal cells, failed to respond to the ligand-induced activation of the Wnt/ß-catenin pathway. This opens the possibility that CLL cells use Wnt-3 to communicate with the cells in the microenvironment. We thus propose that the Wnt/ß-catenin pathway plays a more complex role in CLL pathogenesis than previously anticipated.

TP53 mutation analysis in chronic lymphocytic leukemia: comparison of different detection methods.

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

TP53 mutation analysis in clinical practice: lessons from chronic lymphocytic leukemia.

In leukemia, TP53 mutations are not frequent but clearly associate with impaired survival and therapy response. Here, we describe the biological and clinical consequences of TP53 dysfunction as well as the methodical aspects of TP53 analysis in chronic lymphocytic leukemia (CLL). In CLL, TP53 defects are routinely analyzed as part of disease prognostication. Deletions of TP53 locus (17p) have been uniformly detected using I-FISH for several years. Since monoallelic mutations have also been shown to have negative prognostic impact, it is recommended to examine both TP53 mutations and deletions. Several methods are used to detect TP53 mutations, and next-generation sequencing (NGS) is becoming a convenient option for routine analysis. Besides this, ultradeep NGS permits the detection of minor clones carrying TP53 mutations, even below 1%. The prognostic impact of minor TP53-defective subclones is currently unknown, nevertheless they unequivocally bear the risk of being selected by therapy. Prospective studies assessing the consequences of carrying such clones are in progress.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia.

MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.

Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones.

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.

MDM2 promotor polymorphism and disease characteristics in chronic lymphocytic leukemia: results of an individual patient data-based meta-analysis.

A number of single nucleotide polymorphisms have been associated with disease predisposition in chronic lymphocytic leukemia. A single nucleotide polymorphism in the MDM2 promotor region, MDM2SNP309, was shown to soothe the p53 pathway. In the current study, we aimed to clarify the effect of the MDM2SNP309 on chronic lymphocytic leukemia characteristics and outcome. We performed a meta-analysis of data from 2598 individual patients from 10 different cohorts. Patients' data and genetic analysis for MDM2SNP309 genotype, immunoglobulin heavy chain variable region mutation status and fluorescence in situ hybridization results were collected. There were no differences in overall survival based on the polymorphism (log rank test, stratified by study cohort; P=0.76; GG genotype: cohort-adjusted median overall survival of 151 months; TG: 153 months; TT: 149 months). In a multivariable Cox proportional hazards regression analysis, advanced age, male sex and unmutated immunoglobulin heavy chain variable region genes were associated with inferior survival, but not the MDM2 genotype. The MDM2SNP309 is unlikely to influence disease characteristics and prognosis in chronic lymphocytic leukemia. Studies investigating the impact of individual single nucleotide polymorphisms on prognosis are often controversial. This may be due to selection bias and small sample size. A meta-analysis based on individual patient data provides a reasonable strategy for prognostic factor analyses in the case of small individual studies. Individual patient data-based meta-analysis can, therefore, be a powerful tool to assess genetic risk factors in the absence of large studies.

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-2024 update.

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.

Unveiling the dynamics and molecular landscape of a rare chronic lymphocytic leukemia subpopulation driving refractoriness: insights from single-cell RNA sequencing.

Early identification of resistant cancer cells is currently a major challenge, as their expansion leads to refractoriness. To capture the dynamics of these cells, we made a comprehensive analysis of disease progression and treatment response in a chronic lymphocytic leukemia (CLL) patient using a combination of single-cell and bulk genomic methods. At diagnosis, the patient presented with unfavorable genetic markers, including notch receptor 1 (NOTCH1) mutation and loss(11q). The initial and subsequent treatment lines did not lead to a durable response and the patient developed refractory disease. Refractory CLL cells featured substantial dysregulation in B-cell phenotypic markers such as human leukocyte antigen (HLA) genes, immunoglobulin (IG) genes, CD19 molecule (CD19), membrane spanning 4-domains A1 (MS4A1; previously known as CD20), CD79a molecule (CD79A) and paired box 5 (PAX5), indicating B-cell de-differentiation and disease transformation. We described the clonal evolution and characterized in detail two cell populations that emerged during the refractory disease phase, differing in the presence of high genomic complexity. In addition, we successfully tracked the cells with high genomic complexity back to the time before treatment, where they formed a rare subpopulation. We have confirmed that single-cell RNA sequencing enables the characterization of refractory cells and the monitoring of their development over time.

ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin.

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

Distinct p53 phosphorylation patterns in chronic lymphocytic leukemia patients are reflected in the activation of circumjacent pathways upon DNA damage.

TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild-type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho-profiles induced by DNA-damaging agents (fludarabine, doxorubicin) in 71 TP53-intact primary CLL samples. Doxorubicin induced two distinct phospho-profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53-mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53-mutant samples, followed by profile II and profile I. Our study suggests that wild-type TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.

Lyn Phosphorylates and Controls ROR1 Surface Dynamics During Chemotaxis of CLL Cells.

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.

Memory B-cell like chronic lymphocytic leukaemia is associated with specific methylation profile of WNT5A promoter and undetectable expression of WNT5A gene.

Genome methylation profiles define naïve-like (n-CLL), memory-like (m-CLL), and intermediate (i-CLL) subsets of chronic lymphocytic leukaemia (CLL). The profiles can be easily determined by the analysis of the five-CpG signature. m-CLL, i-CLL, and n-CLL with the good, intermediate, and poor prognoses, respectively, differ by the somatic hypermutation status of the immunoglobulin heavy chain variable gene (IGHV), a widely used prognostic predictor in CLL. We have previously shown that the expression of WNT5A, encoding a ROR1 ligand, distinguishes patients with the worse outcome within the prognostically favourable IGHV-mutated subgroup. To analyse the mechanisms controlling WNT5A expression, we investigated the methylation status of 54 CpG sites within the WNT5A promoter and its relation to the WNT5A gene expression. In a cohort of 59 CLL patients balanced for combinations of IGHV and WNT5A statuses, we identified three promoter CpG sites whose methylation level correlated with the WNT5A expression within the IGHV-mutated subgroup. Further, we complemented our data with the methylation status of the five-CpG signature. IGHV-mutated/WNT5A-negative and IGHV-mutated/WNT5A-positive cases overlapped with m­CLL and i­CLL methylation subgroups, respectively, while most IGHV­unmutated samples were assigned to n-CLL. Median methylation levels of all the three CpG sites in the WNT5A promoter were lowest in i-CLL. Finally, a detailed analysis of m-CLL and i-CLL showed that undetectable WNT5A expression predicts longer treatment-free survival with higher statistical significance than the classification according to the five-CpG signature. To conclude, a favourable m-CLL subgroup is associated with mutated IGHV and undetectable WNT5A expression due to its promoter methylation.