Immunogenetic Architecture of Chronic Lymphocytic Leukemia at Early Stage: Insights from the O-CLL1 Cohort Davide Bagnara, Andrea Nicola Mazzarello, Monica Colombo, Ennio Nano, Niccolò Cardente, et al. Antibodies, 2026 Background/Objectives: The immunoglobulin heavy-chain variable (IGHV) gene repertoire represents a characteristic feature of chronic lymphocytic leukemia (CLL), although its configuration is not well defined at the early disease stages. The IGHV repertoire of a cohort of early CLL patients was analyzed and compared to that of a “real-world” reference cohort. Methods: Patients from the O-CLL1 observational protocol, which enrolled only Binet stage A cases within twelve months from diagnosis, were studied. IGHV/IGHJ rearrangements were sequenced and annotated following ERIC recommendations, and stereotyped subsets were assigned using ARResT/AssignSubsets. The repertoire features were compared with the dataset of a real-world cohort of patients with heterogeneous staging (CTR cohort) and with published early-diagnosis series. Results: IGHV and IGHJ gene distributions and HCDR3-length profiles in O-CLL1 closely mirrored those of CTR, indicating that the BcR IG repertoire at diagnosis is already defined rather than being selected during disease progression. Mutated IGHV (M-CLL) predominated, with a frequency of stereotyped BcR IG comparable to that of other early-diagnosis cohorts. However, within this conserved framework, subset #4 was over-represented among M-CLL from O-CLL without an increased overall IGHV4-34 gene usage, suggestive of a selective expansion rather than a recombinational bias. Subset #4 cases retained canonical HCDR3 motifs and showed time-to-first-treatment like other M-CLL, likely reflecting the younger age structure of O-CLL1. Conclusions: Early-diagnosis CLL displays a biased IGHV repertoire with stereotyped configurations characteristic of CLL, including subsets that are rare in the normal B-cell repertoire. These findings support a central role for antigen-driven selection in shaping CLL evolution.
Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia Martina Cardillo, Fabiana Ferrero, Nadia Bertola, Ennio Nano, Rosanna Massara, et al. International Journal of Molecular Sciences, 2026 Chronic lymphocytic leukemia (CLL) is a dynamic malignancy in which intraclonal subfractions differ in activation history and responsiveness to microenvironmental signals. Here, we investigated the expression and inducibility of IL-12 family receptor subunits (IL-23R, IL-12Rβ1, IL-12Rβ2) and the related receptor complexes in recirculating CLL cells, with a focus on CXCR4/CD5-defined fractions: the proliferative fraction (PF; CXCR4dim/CD5bright; most recently divided, tissue-emigrated cells) and the resting fraction (RF; CXCR4bright/CD5dim; older, quiescent cells). At baseline, IL-12Rβ1 was enriched in the PF and was associated with a higher proportion of cells expressing IL-23R and IL-12R receptor complexes. Concomitantly, RT-qPCR disclosed higher IL-12Rβ1 mRNA levels. Following antigen-independent activation with CpG or CpG + IL-15, there was a marked increase in IL-23R and IL-12Rβ1 but not in IL-12Rβ2 surface expression, resulting in preferential upregulation of the IL-23R complex over the IL-12R complex. Fraction-specific analyses showed stronger induction of IL-23R and IL-23R complex expression in PF compared with RF. These findings identify an intraclonal bias toward IL-23 responsiveness in the CLL cells with a phenotype of recently divided, tissue-emigrated cells and suggest the IL-23/IL-23R axis as a potential therapeutic target.
To Treat or Not to Treat: Navigating Early-Stage CLL in the Era of Targeted Therapy Enrica Antonia Martino, Santino Caserta, Ernesto Vigna, Giovanna Cutrona, Antonella Bruzzese, et al. European Journal of Haematology, 2026 Chronic lymphocytic leukemia (CLL) is most frequently diagnosed at early, asymptomatic stages (Rai 0/Binet A), in which a watch‐and‐wait strategy remains the standard of care, based on historical trials demonstrating no overall survival benefit from early treatment. Over the past two decades, however, substantial advances in genomic profiling—including immunoglobulin heavy‐chain variable region (IGHV) mutational status, TP53 disruption, recurrent gene mutations, and complex karyotype—have uncovered marked biological heterogeneity among early‐stage patients and substantially improved prediction of disease progression. In parallel, targeted therapies such as Bruton tyrosine kinase (BTK) inhibitors and venetoclax‐based combinations have transformed the management of symptomatic CLL, raising renewed interest in whether early intervention might favorably alter the natural history of biologically high‐risk disease. In this review, we critically examine the evolution of prognostication in early‐stage CLL, integrate contemporary molecular and clinical risk models, and summarize evidence from both historical chemotherapy‐era studies and modern early‐intervention trials. We discuss key unresolved controversies, including reliance on surrogate endpoints, the risks of overtreatment, and the persistent absence of an overall survival benefit across all early‐treatment strategies. Finally, we outline future research priorities, including refined genomic stratification, minimal residual disease‐driven (MRD)‐driven approaches, and combination targeted therapies currently under investigation. Despite renewed interest in preemptive treatment, available evidence supports continued observation for asymptomatic patients outside clinical trials.
Targeting the p53/xCT/GSH Axis with PRIMA-1Met Combined with Sulfasalazine Shows Therapeutic Potential in Chronic Lymphocytic Leukemia Martina Pasino, Andrea Speciale, Silvia Ravera, Giovanna Cutrona, Rosanna Massara, et al. International Journal of Molecular Sciences, 2025 In Chronic Lymphocytic Leukemia (CLL), mutations at the TP53 tumor suppressor gene are an important hallmark since they may strongly influence the therapeutic decision. PRIMA-1Met (also known as APR-246/Eprenetapopt) is a small molecule able to restore the wild-type (wt) p53 conformation to mutant p53 proteins and to stimulate apoptosis in tumor cells; in addition, it can deplete the glutathione reservoir, increasing reactive oxygen species (ROS) production. In this study, we investigated whether combining PRIMA-1Met with Sulfasalazine (SAS), a SLC7A11/xCT inhibitor, reduces CLL cell viability by targeting mutant p53 and the glutathione pathway. The results demonstrated that, in CLL cells, PRIMA-1Met did not restore the wt functions in the mutant p53 proteins, but it strongly reduced the antioxidant defense and induced cell death. PRIMA-1Met and SAS combination synergistically reduced cell survival regardless of p53 status and further impaired antioxidant capacity, especially in mutant p53 cells, linking their cytotoxic effect to redox imbalance. Thus, the association of PRIMA-1Met with drugs targeting the antioxidant response could represent a valid strategy to kill CLL cells carrying either wt or mutant p53.
MicroRNA Profiling as a Predictive Indicator for Time to First Treatment in Chronic Lymphocytic Leukemia: Insights from the O-CLL1 Prospective Study Ennio Nano, Francesco Reggiani, Adriana Agnese Amaro, Paola Monti, Monica Colombo, et al. Non Coding RNA, 2024 A “watch and wait” strategy, delaying treatment until active disease manifests, is adopted for most CLL cases; however, prognostic models incorporating biomarkers have shown to be useful to predict treatment requirement. In our prospective O-CLL1 study including 224 patients, we investigated the predictive role of 513 microRNAs (miRNAs) on time to first treatment (TTFT). In the context of this study, six well-established variables (i.e., Rai stage, beta-2-microglobulin levels, IGVH mutational status, del11q, del17p, and NOTCH1 mutations) maintained significant associations with TTFT in a basic multivariable model, collectively yielding a Harrell’s C-index of 75% and explaining 45.4% of the variance in the prediction of TTFT. Concerning miRNAs, 73 out of 513 were significantly associated with TTFT in a univariable model; of these, 16 retained an independent relationship with the outcome in a multivariable analysis. For 8 of these (i.e., miR-582-3p, miR-33a-3p, miR-516a-5p, miR-99a-5p, and miR-296-3p, miR-502-5p, miR-625-5p, and miR-29c-3p), a lower expression correlated with a shorter TTFT, whereas in the remaining eight (i.e., miR-150-5p, miR-148a-3p, miR-28-5p, miR-144-5p, miR-671-5p, miR-1-3p, miR-193a-3p, and miR-124-3p), the higher expression was associated with shorter TTFT. Integrating these miRNAs into the basic model significantly enhanced predictive accuracy, raising the Harrell’s C-index to 81.1% and the explained variation in TTFT to 63.3%. Moreover, the inclusion of the miRNA scores enhanced the integrated discrimination improvement (IDI) and the net reclassification index (NRI), underscoring the potential of miRNAs to refine CLL prognostic models and providing insights for clinical decision-making. In silico analyses on the differently expressed miRNAs revealed their potential regulatory functions of several pathways, including those involved in the therapeutic responses. To add a biological context to the clinical evidence, an miRNA–mRNA correlation analysis revealed at least one significant negative correlation between 15 of the identified miRNAs and a set of 50 artificial intelligence (AI)-selected genes, previously identified by us as relevant for TTFT prediction in the same cohort of CLL patients. In conclusion, the identification of specific miRNAs as predictors of TTFT holds promise for enhancing risk stratification in CLL to predict therapeutic needs. However, further validation studies and in-depth functional analyses are required to confirm the robustness of these observations and to facilitate their translation into meaningful clinical utility.
Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates Andrea N. Mazzarello, Elena Gugiatti, Vanessa Cossu, Nadia Bertola, Davide Bagnara, et al. Cancer Immunology Immunotherapy, 2024 Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.
Unraveling the Bone Tissue Microenvironment in Chronic Lymphocytic Leukemia Paolo Giannoni, Cecilia Marini, Giovanna Cutrona, Gian Mario Sambuceti, Franco Fais, et al. Cancers, 2023 Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western countries. Although characterized by the progressive expansion and accumulation of leukemic B cells in peripheral blood, CLL cells develop in protective niches mainly located within lymph nodes and bone marrow. Multiple interactions between CLL and microenvironmental cells may favor the expansion of a B cell clone, further driving immune cells toward an immunosuppressive phenotype. Here, we summarize the current understanding of bone tissue alterations in CLL patients, further addressing and suggesting how the multiple interactions between CLL cells and osteoblasts/osteoclasts can be involved in these processes. Recent findings proposing the disruption of the endosteal niche by the expansion of a leukemic B cell clone appear to be a novel field of research to be deeply investigated and potentially relevant to provide new therapeutic approaches.
Genes selection using deep learning and explainable artificial intelligence for chronic lymphocytic leukemia predicting the need and time to therapy Fortunato Morabito, Carlo Adornetto, Paola Monti, Adriana Amaro, Francesco Reggiani, et al. Frontiers in Oncology, 2023 Analyzing gene expression profiles (GEP) through artificial intelligence provides meaningful insight into cancer disease. This study introduces DeepSHAP Autoencoder Filter for Genes Selection (DSAF-GS), a novel deep learning and explainable artificial intelligence-based approach for feature selection in genomics-scale data. DSAF-GS exploits the autoencoder’s reconstruction capabilities without changing the original feature space, enhancing the interpretation of the results. Explainable artificial intelligence is then used to select the informative genes for chronic lymphocytic leukemia prognosis of 217 cases from a GEP database comprising roughly 20,000 genes. The model for prognosis prediction achieved an accuracy of 86.4%, a sensitivity of 85.0%, and a specificity of 87.5%. According to the proposed approach, predictions were strongly influenced by CEACAM19 and PIGP, moderately influenced by MKL1 and GNE, and poorly influenced by other genes. The 10 most influential genes were selected for further analysis. Among them, FADD, FIBP, FIBP, GNE, IGF1R, MKL1, PIGP, and SLC39A6 were identified in the Reactome pathway database as involved in signal transduction, transcription, protein metabolism, immune system, cell cycle, and apoptosis. Moreover, according to the network model of the 3D protein-protein interaction (PPI) explored using the NetworkAnalyst tool, FADD, FIBP, IGF1R, QTRT1, GNE, SLC39A6, and MKL1 appear coupled into a complex network. Finally, all 10 selected genes showed a predictive power on time to first treatment (TTFT) in univariate analyses on a basic prognostic model including IGHV mutational status, del(11q) and del(17p), NOTCH1 mutations, β2-microglobulin, Rai stage, and B-lymphocytosis known to predict TTFT in CLL. However, only IGF1R [hazard ratio (HR) 1.41, 95% CI 1.08-1.84, P=0.013), COL28A1 (HR 0.32, 95% CI 0.10-0.97, P=0.045), and QTRT1 (HR 7.73, 95% CI 2.48-24.04, P<0.001) genes were significantly associated with TTFT in multivariable analyses when combined with the prognostic factors of the basic model, ultimately increasing the Harrell’s c-index and the explained variation to 78.6% (versus 76.5% of the basic prognostic model) and 52.6% (versus 42.2% of the basic prognostic model), respectively. Also, the goodness of model fit was enhanced (χ2 = 20.1, P=0.002), indicating its improved performance above the basic prognostic model. In conclusion, DSAF-GS identified a group of significant genes for CLL prognosis, suggesting future directions for bio-molecular research.
A High Percentage of CD16+ Monocytes Correlates with the Extent of Bone Erosion in Chronic Lymphocytic Leukemia Patients: The Impact of Leukemic B Cells in Monocyte Differentiation and Osteoclast Maturation Paolo Giannoni, Cecilia Marini, Giovanna Cutrona, Katia Todoerti, Antonino Neri, et al. Cancers, 2022 Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.
Immunogenetic Architecture of Chronic Lymphocytic Leukemia at Early Stage: Insights from the O-CLL1 Cohort D Bagnara, AN Mazzarello, M Colombo, E Nano, N Cardente, F Ferrero, ... Antibodies 15 (2), 25 , 2026 2026
Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia M Cardillo, F Ferrero, N Bertola, E Nano, R Massara, MC Capra, ... International Journal of Molecular Sciences 27 (3), 1202 , 2026 2026
To Treat or Not to Treat: Navigating Early‐Stage CLL in the Era of Targeted Therapy EA Martino, S Caserta, E Vigna, G Cutrona, A Bruzzese, F Mendicino, ... European Journal of Haematology , 2026 2026
Asymmetric induction of IL-23R by CpG and IL-15 in proliferative CLL fractions highlights intraclonal heterogeneity in chronic lymphocytic leukemia M Cardillo, N Bertola, F Ferrero, R Massara, MC Capra, D Reverberi, ... bioRxiv, 2025.08. 27.672649 , 2025 2025
Targeting the p53/xCT/GSH Axis with PRIMA-1 Met Combined with Sulfasalazine Shows Therapeutic Potential in Chronic Lymphocytic Leukemia M Pasino, A Speciale, S Ravera, G Cutrona, R Massara, N Bertola, ... International Journal of Molecular Sciences 26 (12), 5559 , 2025 2025 Citations: 2
Regulation of mitochondrial metabolism by IL12-family cytokines in chronic lymphocytic leukemia: A novel perspective on therapeutic resistance N Bertola, F Ferrero, V Cossu, E Nano, M Colombo, R Massara, ... EUROPEAN JOURNAL OF CLINICAL INVESTIGATION 55 , 2025 2025
MicroRNA Profiling as a Predictive Indicator for Time to First Treatment in Chronic Lymphocytic Leukemia: Insights from the O-CLL1 Prospective Study E Nano, F Reggiani, AA Amaro, P Monti, M Colombo, N Bertola, F Ferrero, ... Non-coding RNA 10 (5), 46 , 2024 2024 Citations: 2
Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates AN Mazzarello, E Gugiatti, V Cossu, N Bertola, D Bagnara, S Carta, ... Cancer Immunology, Immunotherapy 73 (2), 27 , 2024 2024
Unraveling the bone tissue microenvironment in chronic lymphocytic leukemia P Giannoni, C Marini, G Cutrona, GM Sambuceti, F Fais, D de Totero Cancers 15 (20), 5058 , 2023 2023 Citations: 12
Genes selection using deep learning and explainable artificial intelligence for chronic lymphocytic leukemia predicting the need and time to therapy F Morabito, C Adornetto, P Monti, A Amaro, F Reggiani, M Colombo, ... Frontiers in oncology 13, 1198992 , 2023 2023 Citations: 20
The time to first treatment is an independent predictor of overall survival in chronic lymphocytic leukemia F Morabito, G Tripepi, FR Mauro, L Laurenti, G Reda, R Moia, ... American Journal of Hematology 98 (7), E157-E160 , 2023 2023 Citations: 1
Genes selection using deep learning and explainable artificial intelligence for chronic lymphocytic leukemia predicting the need and time to therapy S Galimberti, A Neri, C Adornetto, F Morabito, C Adornetto, P Monti, ... 2023
A high percentage of CD16+ monocytes correlates with the extent of bone erosion in chronic lymphocytic leukemia patients: The impact of leukemic B cells in monocyte … P Giannoni, C Marini, G Cutrona, K Todoerti, A Neri, A Ibatici, ... Cancers 14 (23), 5979 , 2022 2022 Citations: 8
The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study G De Luca, G Cerruti, S Lastraioli, R Conte, A Ibatici, N Di Felice, ... Hematological Oncology 40 (5), 962-975 , 2022 2022 Citations: 6
Old and new facts and speculations on the role of the B cell receptor in the origin of chronic lymphocytic leukemia D Bagnara, AN Mazzarello, F Ghiotto, M Colombo, G Cutrona, F Fais, ... International Journal of Molecular Sciences 23 (22), 14249 , 2022 2022 Citations: 24
MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells S Matis, A Grazia Recchia, M Colombo, M Cardillo, M Fabbi, K Todoerti, ... Blood Advances 6 (20), 5593-5612 , 2022 2022 Citations: 17
Characterizing features of human circulating B cells carrying CLL-like stereotyped immunoglobulin rearrangements D Bagnara, M Colombo, D Reverberi, S Matis, R Massara, N Cardente, ... Frontiers in oncology 12, 894419 , 2022 2022 Citations: 13
LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells S Matis, M Rossi, L Brondolo, M Cardillo, D Reverberi, R Massara, ... Hematological Oncology 40 (1), 41-48 , 2022 2022 Citations: 10
Lymphocyte doubling time as a key prognostic factor to predict time to first treatment in early-stage chronic lymphocytic leukemia F Morabito, G Tripepi, R Moia, AG Recchia, P Boggione, FR Mauro, ... Frontiers in oncology 11, 684621 , 2021 2021 Citations: 17
TP53 disruption as a risk factor in the era of targeted therapies: A multicenter retrospective study of 525 chronic lymphocytic leukemia cases F Morabito, G Del Poeta, FR Mauro, G Reda, P Sportoletti, L Laurenti, ... American Journal of Hematology 96 (8), E306-E310 , 2021 2021 Citations: 20
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