Immune-based therapeutic strategies have drastically changed the landscape of hematological disorders, as they have introduced the concept of boosting immune responses against tumor cells. Anti-CD20 monoclonal antibodies have been the first form of immunotherapy successfully applied in the treatment of CLL, in the context of chemoimmunotherapy regimens. Since then, several immunotherapeutic approaches have been studied in CLL settings, with the aim of exploiting or eliciting anti-tumor immune responses against leukemia cells. Unfortunately, despite initial promising data, results from pilot clinical studies have not shown optimal results in terms of disease control - especially when immunotherapy was used individually - largely due to CLL-related immune dysfunctions hampering the achievement of effective anti-tumor responses. The growing understanding of the complex interactions between immune cells and the tumor cells has paved the way for the development of new combined approaches that rely on the synergism between novel agents and immunotherapy. In this review, we provide an overview of the most successful and promising immunotherapeutic modalities in CLL, including both antibody-based therapy (i.e. monoclonal antibodies, bispecific antibodies, bi- or tri- specific killer engagers) and adoptive cellular therapy (i.e. CAR T cells and NK cells). We also provide examples of successful new combination strategies and some insights on future perspectives.

Chronic lymphocytic leukemia (CLL) is characterized by a wide spectrum of immune alterations, affecting both the innate and adaptive immunity. These immune dysfunctions strongly impact the immune surveillance, facilitate tumor progression and eventually affect the disease course. Quantitative and functional alterations involving conventional T cells, γδ T cells, regulatory T cells, NK and NKT cells, and myeloid cells, together with hypogammaglobulinemia, aberrations in the complement pathways and altered cytokine signature have been reported in patients with CLL. Some of these immune parameters have been shown to associate with other CLL-related characteristics with a known prognostic relevance or to correlate with disease prognosis. Also, in CLL, the complex immune response dysfunctions eventually translate in clinical manifestations, including autoimmune phenomena, increased risk of infections and second malignancies. These clinical issues are overall the most common complications that affect the course and management of CLL, and they also may impact overall disease prognosis.

The hypoxia-inducible factor 1 (HIF-1) and the CXCL12/CXCR4 axis regulate the interaction of chronic lymphocytic leukemia cells and the tumor microenvironment. However, the interconnections occurring between HIF-1 and the CXCL12/CXCR4 axis are not fully elucidated. Here, we demonstrate that the CXCL12/CXCR4 axis plays a pivotal role in the positive regulation of the α subunit of HIF-1 (HIF-1α) that occurs in CLL cells co-cultured with stromal cells (SC). Inhibitors acting at different levels on CXCR4 downstream signalling counteract the SC-induced HIF-1α upregulation in CLL cells, also hindering the SC-mediated pro-survival effect. HIF-1α inhibition also exerts off-tumor effects on the SC component, inducing the downregulation of target genes, including CXCL12. Consistently, our data show that pretreatment of leukemic cells and/or SC with idelalisib effectively abrogates the SC-mediated survival support. A combined on-tumor and off-tumor inhibition of HIF-1α was also observed in idelalisib-treated patients, who showed, along with a downregulation of HIF-1α target genes in leukemic cells, a significant decrease in CXCL12 serum concentration and changes in the bone marrow microenvironment. Our data demonstrate that the targeting of HIF-1α or its regulatory pathways acts at the tumor- and SC-level, and may be an appealing strategy to overcome the microenvironment-mediated protection of CLL cells.

The clinical and biological significance of clonal hematopoiesis (CH) has not been investigated in the myeloid compartment of chronic lymphocytic leukemia (CLL). By studying 488 newly diagnosed CLL through CAPP‐seq using a 28‐gene panel on granulocyte genomic DNA (gDNA), CH occurred in 231 (47.3%) patients. Cell sorting of cases that never developed Richter transformation (RT) confirmed that CH mutations, including CH‐related TP53 mutations, were restricted to the myelomonocytic compartment and absent in CLL cells, as also documented by single‐cell DNA sequencing. CH associated with shorter overall survival (OS) (hazard ratio [HR] 1.36, 95% CI 1.04–1.77, P = 0.023); specifically, TET2 mutations independently predicted inferior OS (HR 1.62, 95% CI 1.15–2.28, P = 0.01) after adjusting for age and for CLL‐related prognostic biomarkers, namely IGHV and TP53 status. Regarding therapy‐related toxicities, CH correlated with a higher incidence of Grade ≥ 3 neutropenia (P = 0.004) after venetoclax‐based regimens. Sequential samples ( n = 57) analysis showed that Bruton tyrosine kinase (BTK) and BCL2 inhibitors do not induce CH expansion, which was instead driven by chemotherapy. CH is significantly associated with a higher risk of second hematological malignancies only in chemo‐exposed patients. Single‐cell RNA sequencing of seven CH+ and six CH− CLL revealed that the T‐cell compartment of CH+ patients exhibits a less exhausted phenotype, documented by lower expression of TOX, the master regulator of T‐cell exhaustion, and a higher pro‐inflammatory profile. CH also influenced RT, since CH ASXL1 mutations independently associated with higher RT risk (HR 11.19, 95% CI 4.09–30.62, P < 0.001). Overall, CH in CLL impacts survival, therapeutic toxicity, and transformation risk while also influencing the T‐cell immune compartment.

Membrane-bound CD200 is overexpressed in chronic lymphocytic leukemia (CLL), and there is some evidence that its soluble ectodomain (sCD200) could also be involved in the pathophysiology and the disease. However, very little is known about sCD200’s prognostic significance. sCD200 was tested at diagnosis in 272 patients with CLL and in 78 age- and sex-matched healthy subjects using a specific human CD200 (OX-2 membrane glycoprotein) ELISA kit. A significantly higher concentration of sCD200 was found in CLL patients compared to controls. In our cohort, sCD200 was significantly higher in patients who were older than 66 years, with Binet stage C, unmutated IgVH and unfavorable (del11q or del17p) FISH. Time-to-first treatment and overall survival were significantly shorter in patients with higher sCD200 concentration, using as a cut-off 1281 pg/mL, the median value for sCD200 concentration in the whole CLL cohort. However, the prognostic impact of sCD200 was not confirmed in multivariate analysis. Baseline sCD200 values appeared to have an impact on the response to chemotherapy or chemo-immunotherapy, but not to targeted agents. Collectively, our data show that sCD200 serum levels correlate with more aggressive clinical and biological features and are able to predict a worse prognosis. This work supports the relevant role of CD200 not only as a diagnostic tool but also as a prognostic indicator and a potential therapeutic target in CLL.

Background. Bendamustine is a cytotoxic alkylating drug with a broad range of indications as a single agent or in combination therapy in lymphoid neoplasia patients. However, its tolerability in elderly patients is still debated. Methods: An observational, retrospective study was carried out; patients with chronic lymphocytic leukemia (CLL) or lymphoma, aged ≥ 65 years old, treated with bendamustine-based regimens in first or subsequent lines between 2010 and 2020 were considered eligible. Results: Overall, 179 patients aged ≥ 65 years were enrolled, 53% between 71 and 79 years old. Cumulative Illness Rating Scale (CIRS) comorbidity score was ≥6 in 54% patients. Overall survival (OS) at 12 months was 95% (95% confidence interval [CI]: 90–97%); after a median follow up of 50 months, median OS was 84 months. The overall response rate was 87%, with 56% complete responses; the median time to progression (TTP) was 61 months. The baseline factors affecting OS by multivariable analysis were sex, histological diagnosis, renal function, and planned bendamustine dose, while only type of lymphoma and bendamustine dose impacted on TTP. Main adverse events were neutropenia (grade ≥ 3: 43%) and infections (any grade: 36%), with 17% of patients requiring hospital admission. Conclusions: The responses to bendamustine, as well as survival, are relevant even in advanced age patients, with a manageable incidence of acute toxicity.