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.

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.

Cerebral organoids derived from patients with Alzheimer's disease with PSEN1/2 mutations have defective tissue patterning and altered development.

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study human neural development and disease. Especially in the field of Alzheimer's disease (AD), remarkable effort has been put into investigating molecular mechanisms behind this disease. Then, with the advent of 3D neuronal cultures and cerebral organoids (COs), several studies have demonstrated that this model can adequately mimic familial and sporadic AD. Therefore, we created an AD-CO model using iPSCs derived from patients with familial AD forms and explored early events and the progression of AD pathogenesis. Our study demonstrated that COs derived from three AD-iPSC lines with PSEN1(A246E) or PSEN2(N141I) mutations developed the AD-specific markers in vitro, yet they also uncover tissue patterning defects and altered development. These findings are complemented by single-cell sequencing data confirming this observation and uncovering that neurons in AD-COs likely differentiate prematurely.

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.

Duplication of 8q24 in Chronic Lymphocytic Leukemia: Cytogenetic and Molecular Biologic Analysis of MYC Aberrations.

Chronic lymphocytic leukemia (CLL) with cytogenetics findings, such as complex karyotype and deletions of TP53 or ATM, is associated with adverse clinical outcomes. Additional chromosomal abnormalities further stratify patients into groups with diverse prognoses. Gain of 8q24 is one of the abnormalities considered as prognostically unfavorable. In our study, we performed a FISH analysis in an initial cohort of 303 consecutive CLL patients and determined the frequency of +8q to be 6.3 %. Our analysis confirmed the association with TP53/ATM aberrations and CK, as the frequency of +8q reached 26.7 % in an extended delTP53/ATM+CK cohort. M-FISH analysis enabled the identification of partner chromosomes where the segment of the duplicated 8q arm was localized. More detailed mapping of the gained 8q region using the M-BAND method determined the smallest amplified region 8q23-8qter. We observed significantly shorter overall survival (OS; 9.0 years in +8q-positive vs. 10.6 years in +8q-negative; p=0.02) and detected slightly higher MYC mRNA/protein levels in +8q-positive vs. +8q-negative patients.

Single-cell RNA sequencing analysis of T helper cell differentiation and heterogeneity.

Single-cell transcriptomics has emerged as a powerful tool to investigate cells' biological landscape and focus on the expression profile of individual cells. Major advantage of this approach is an analysis of highly complex and heterogeneous cell populations, such as a specific subpopulation of T helper cells that are known to differentiate into distinct subpopulations. The need for distinguishing the specific expression profile is even more important considering the T cell plasticity. However, importantly, the universal pipelines for single-cell analysis are usually not sufficient for every cell type. Here, the aims are to analyze the diversity of T cell phenotypes employing classical in vitro cytokine-mediated differentiation of human T cells isolated from human peripheral blood by single-cell transcriptomic approach with support of labelled antibodies and a comprehensive bioinformatics analysis using combination of Seurat, Nebulosa, GGplot and others. The results showed high expression similarities between Th1 and Th17 phenotype and very distinct Th2 expression profile. In a case of Th2 highly specific marker genes SPINT2, TRIB3 and CST7 were expressed. Overall, our results demonstrate how donor difference, Th plasticity and cell cycle influence the expression profiles of distinct T cell populations. The results could help to better understand the importance of each step of the analysis when working with T cell single-cell data and observe the results in a more practical way by using our analyzed datasets.

Clonal haematopoiesis as a risk factor for therapy-related myeloid neoplasms in patients with chronic lymphocytic leukaemia treated with chemo-(immuno)therapy.

Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p = 0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.

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.

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.

Chromothripsis in Chronic Lymphocytic Leukemia: A Driving Force of Genome Instability.

Chromothripsis represents a mechanism of massive chromosome shattering and reassembly leading to the formation of derivative chromosomes with abnormal functions and expression. It has been observed in many cancer types, importantly, including chronic lymphocytic leukemia (CLL). Due to the associated chromosomal rearrangements, it has a significant impact on the pathophysiology of the disease. Recent studies have suggested that chromothripsis may be more common than initially inferred, especially in CLL cases with adverse clinical outcome. Here, we review the main features of chromothripsis, the challenges of its assessment, and the potential benefit of its detection. We summarize recent findings of chromothripsis occurrence across hematological malignancies and address its causes and consequences in the context of CLL clinical features, as well as chromothripsis-related molecular abnormalities described in published CLL studies. Furthermore, we discuss the use of the current knowledge about genome functions associated with chromothripsis in the optimization of treatment strategies in CLL.

Identification of Clinically Relevant Subgroups of Chronic Lymphocytic Leukemia Through Discovery of Abnormal Molecular Pathways.

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world with a highly variable clinical course. Its striking genetic heterogeneity is not yet fully understood. Although the CLL genetic landscape has been well-described, patient stratification based on mutation profiles remains elusive mainly due to the heterogeneity of data. Here we attempted to decrease the heterogeneity of somatic mutation data by mapping mutated genes in the respective biological processes. From the sequencing data gathered by the International Cancer Genome Consortium for 506 CLL patients, we generated pathway mutation scores, applied ensemble clustering on them, and extracted abnormal molecular pathways with a machine learning approach. We identified four clusters differing in pathway mutational profiles and time to first treatment. Interestingly, common CLL drivers such as ATM or TP53 were associated with particular subtypes, while others like NOTCH1 or SF3B1 were not. This study provides an important step in understanding mutational patterns in CLL.

RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia.

Recent 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.

LYmphoid NeXt-Generation Sequencing (LYNX) Panel: A Comprehensive Capture-Based Sequencing Tool for the Analysis of Prognostic and Predictive Markers in Lymphoid Malignancies.

B-cell neoplasms represent a clinically heterogeneous group of hematologic malignancies with considerably diverse genomic architecture recently endorsed by next-generation sequencing (NGS) studies. Because multiple genetic defects have a potential or confirmed clinical impact, a tendency toward more comprehensive testing of diagnostic, prognostic, and predictive markers is desired. This study introduces the design, validation, and implementation of an integrative, custom-designed, capture-based NGS panel titled LYmphoid NeXt-generation sequencing (LYNX) for the analysis of standard and novel molecular markers in the most common lymphoid neoplasms (chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma). A single LYNX test provides the following: i) accurate detection of mutations in all coding exons and splice sites of 70 lymphoma-related genes with a sensitivity of 5% variant allele frequency, ii) reliable identification of large genome-wide (≥6 Mb) and recurrent chromosomal aberrations (≥300 kb) in at least 20% of the clonal cell fraction, iii) the assessment of immunoglobulin and T-cell receptor gene rearrangements, and iv) lymphoma-specific translocation detection. Dedicated bioinformatic pipelines were designed to detect all markers mentioned above. The LYNX panel represents a comprehensive, up-to-date tool suitable for routine testing of lymphoid neoplasms with research and clinical applicability. It allows a wide adoption of capture-based targeted NGS in clinical practice and personalized management of patients with lymphoproliferative diseases.

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.

Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

Higher-order immunoglobulin repertoire restrictions in CLL: the illustrative case of stereotyped subsets 2 and 169.

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Real-world data on efficacy and safety of obinutuzumab plus chlorambucil, rituximab plus chlorambucil, and rituximab plus bendamustine in the frontline treatment of chronic lymphocytic leukemia: The GO-CLLEAR Study by the Czech CLL Study Group.

Until recently, a combination of anti-CD20 antibody plus less intensive chemotherapy was a standard of care in elderly population with previously untreated chronic lymphocytic leukemia (CLL). The aim of this observational study was to retrospectively assess efficacy and safety of obinutuzumab + chlorambucil (G-Clb), rituximab + chlorambucil (R-Clb), and bendamustine + rituximab (BR) given as the frontline therapy within routine practice. The final analyzed dataset included 398 consecutive CLL patients from 10 hematology centers cooperating within the Czech CLL Study Group: 63 treated with G-Clb, 78 with R-Clb, and 257 with BR. There were no significant differences in prognostic and predictive markers among the groups. On the contrary, median age at the start of therapy and cumulative illness rating scale (CIRS) score was significantly higher in R-Clb group. Obinutuzumab plus chlorambucil regimen was preferably offered to elderly patients (compared to BR) with less severe comorbidities and lower CIRS score (compared to R-Clb). A time period when a treatment was indicated had also a strong impact on the choice of the regimen. The overall response rate reached 76% (30% complete remissions, CRs) in G-Clb, 75% (22% CRs) in R-Clb, and 85% (47% CRs) in BR group. Median event-free survival was 49.0 months for G-Clb, 20.3 months for R-Clb, and 37.0 months for BR group. Neutropenia grade ≥ 3 developed in 43% of G-Clb, 31% of R-Clb and in 49% of BR patients, grade ≥ 3 infections were recorded in 17% of G-Clb, 6.4% of R-Clb, and 17% of BR patients. In conclusion, real-world therapeutic activity of G-Clb appears to be at least comparable to prospective clinical trial data. R-Clb yields relatively good results in very old and severely comorbid patients.