Abstract

Introduction

CLL patients are at increased risk for infections caused by Streptococcus pneumoniae (Spn). Both antigen induced- and natural-anti-Spn antibodies represent a key component of the anti-pneumococcal immune response. These antibodies play important roles during the early immune response to Spn by effector mechanism such as opsonophagocytosis and reduction of bacterial adherence. While hipogammaglobulinemia is commonly observed in patients, the amount and relevance of anti-Spn antibodies in CLL have not been addressed in depth. 

Objectives

To determine the level of natural and vaccine-induced anti-Spn antibodies and their role in protection during Spn-pulmonary infection in the Eµ-TCL1 mouse model of CLL.

Methods

For murine adoptive transfer (AT)-model of CLL, C57BL/6 mice (8-10 weeks old) were intraperitoneally (ip) injected with 20 x 106 leukemic cells obtained from spleens of Eµ-TCL1 mice (C57BL/6 background). To evaluate the response to vaccination, control (age-matched C57BL/6) and leukemic mice (> 60% of CD5+ CD19+ cells in peripheral blood) were ip injected with Prevnar 13 vaccine or adyuvant, and blood samples were collected 1 and 3 weeks thereafter. Plasma levels of total and anti-Spn specific IgG and IgM were determinate by ELISA. Neutrophil phagocytosis of FITC-Spn was evaluated by flow cytometry (FC). For Spn-infection experiments, groups of control and leukemic mice were intranasally infected with a total of 2 x 106 CFU of Spn serotype 3. For survival experiments, mice were daily controlled for 11 days. In other set of experiments mice were euthanized 24 h after infection and the bronchoalveolar lavage fluid (BALF) was obtained to determine: bacterial load by serial dilution in Columbia blood agar plates, cell number and phenotype by FC and cytokines by ELISA. Statistical analysis was performed with Prism v8 (GraphPad). 

Results

AT-TCL1 mice had lower levels of total IgG and similar levels of total IgM compared to control animals (n=10, p<0.05). Interestingly, AT-TCL1 mice had lower levels of natural anti-Spn IgM, that decreased as leukemic burden increased (n=10, p<0.05). The lower levels of anti-Spn IgM in AT-TCL1 serum was accompanied with a lower capacity to induce Spn-phagocytosis by control neutrophils (n=8, p<0.05). Regarding the susceptibility of the AT-TCL1 mice to infection, we found an increased mortality rate compared to Spn-infected control (n=8, p<0.05). At 24 h after infection, the BALF of AT-TCL1 mice showed a higher bacterial burden than control mice (n=12, p<0.05) and similar levels of neutrophils and classical inflammatory parameters as total protein concentration, TNF-α, IL-1β and CXCL1. Finally, we observed a poor response of anti-Spn IgM and IgG to vaccination in AT-TCL1 mice compared to adjuvant-injected mice (n=8, p<0.05).

Conclusion

Leukemic burden in AT-TCL1 mice was associated with a decrease of natural IgM anti-Spn levels, resulting in diminished opsonophagocytosis of the bacteria in vitro and a higher mortality rate and bacterial burden in vivo. As occurs with CLL patients, leukemic mice showed a very low response to conjugated vaccine suggesting a weak protection against Spn infection. This work adds new insight into the immune defects that predispose to Spn infection in CLL. 

Abstract

Although an inflammatory tumour microenvironment (TME) is known to be involved in the initiation and progression of CLL, the role of the inflammasome, the multiprotein complex that activates caspase-1 and drives maturation of proinflammatory cytokine IL-1b is not fully characterized in this disease. Transmembrane protein 176A/B (TMEM176 A/B) channels are important regulators of the NLRP3 inflammasome (Hill M, 2020). Our previous work demonstrate that TMEM176A expression is increased in primary cells isolated from active versus indolent disease and healthy donors. 

Aims: To characterize the role of the inflammasome during CLL progression and to investigate whether pharmacological inhibition of the inflammasome regulator TMEM176A enhances antitumoral responses in-vitro and in-vivo. Primary CLL cells were obtained after informed consent and stored in the Uruguayan Group of CLL Biobank. Cell culture, flow cytometry, PCR and Western blot analysis were performed. TCL-1 mice housed at Institut Pasteur de Montevideo were used for in-vivo experiments. Ethics Committee approval was granted for human and animal studies. 

Results: Increased TMEM176A expression correlates with impaired inflammasome activation in active disease as lower percentages of mature caspase-1 were observed by flow cytometry and lower Gasdermin (another surrogate for inflammasome activation) by Western blot. Furthermore, inhibition of TMEM176A with Boritinib (a pharmacologic inhibitor of TMEM176A/B proteins -Segovia and Russo, 2019-) in-vitro triggered caspase-1-dependent cell death suggesting pharmacologic modulation of the inflammasome is feasible as a therapeutic option in active CLL. 

Since TMEM176A is overexpressed in CLL cells with active disease, we stimulated leukemic cells in-vitro with CD40L+IL-4, a classical activator of B cells that has been described as a key signaling pathway in active disease (Granziero et al., 2001). Our results demonstrate that activation with CD40L+IL-4 in-vitro increases TMEM176A expression. The mainstay treatment of CLL includes BCL-2 and BTK inhibitors. To assess the effect of Boritinib combined with Ibrutinib while maintaining an "activated microenvironment" in-vitro, primary CLL cells were stimulated with CD40L+IL-4 and treated with these drugs, either alone or in combination. Our findings show that the combination of Ibrutinib and Boritinib induces inflammasome-dependent cell death in over 90% of treated patients. 

To determine whether the combination of Ibrutinib plus Boritinib, would also lead to increased anti-leukemic responses in-vivo, we performed adoptive transfer of splenocytes from Eμ-TCL1 mice. Monotherapy with Ibrutinib (78 d) and Boritinib (163 d) increased overall survival compared to mice assigned to vehicles (69 d), p = 0.0088 and 0.0044, respectively. Median survival was not reached in the combination group, p < 0.0001. Mice receiving ibrutinib plus boritinib reached day 170 of the experiment looking healthy with no ruffled fur, lethargy or hair loss. 

In this work we propose a new axis of progression in CLL associated with inflammasome activation. Our results suggest that TME-derived signals might be responsible for the upregulation of TMEM176A in active CLL, contributing to disease progression by impairing inflammasome activation and halting cell death. The combination of Boritinib plus ibrutinib enhances cell death, improves survival of treated mice and sets the ground for the clinical evaluation of the inflammasome as a potential target in CLL. 

Abstract

Introduction: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in Western countries, with an incidence of 4.8–5 per 100,000 in the US and Europe, and significantly lower in Asia (0.48 per 100,000). Latin America (LATAM) presents a complex and variable landscape for CLL incidence, with some countries like Uruguay and Argentina exhibiting rates similar to Europe, while others (e.g. Mexico, Peru, Chile) reporting lower incidences. The region’s ethnic, cultural, and economic heterogeneity leads to disparities in access to diagnostic and prognostic tools, as well as therapeutic options, especially with the increasing use of targeted agents. In 2022, the GELL-CLL cohort presented initial data from 459 patients across six countries. Here, we provide an updated analysis, expanding the cohort and further exploring treatment patterns and biomarker usage.

Objective: To describe the epidemiology, access to biomarkers, and treatment patterns in CLL patients across Latin America.  

Methodology: We conducted a retrospective cohort study of CLL patients aged ≥18 years, diagnosed and treated between 2010-2024, or diagnosed since 2000 and treated from 2010 onwards, from centers participating in the GELL-CLL registry. 

Results: A total of 958 patients from nine countries (Argentina, Chile, Colombia, Guatemala, Mexico, Paraguay, Peru, Uruguay, Venezuela) were included, with 860 eligible for analysis. Of these, 66% were treated in private institutions. The median age was 68 years (30–94), with 40.3% female. Racial distribution included 91.2% White, 0.9% African-ancestry, 0.4% Indigenous, 7.4% mixed-race, and 0.1% Asian. At diagnosis, 91.3% had an ECOG performance status of 0–1, with 73.6% Rai 0 (I: 12.4%, II: 7.5%, III: 3.7%, IV: 2.8%). Key prognostic factors revealed that 13.5% were CD38 positive, and elevated B2-microglobulin was found in 51.1%. IgVH mutational status was studied in 30.6% of patients, with 53% mutated and 47% unmutated.

At diagnosis, 77.2% of patients were under observation, with 51% requiring treatment after a median of 9 months. Of those treated, only 34% underwent cytogenetic or FISH analysis prior to therapy. Del17p was identified in 3.7% of patients, while P53 mutations were found in 7.2%.

First-line treatments included chemo-immunotherapy (56.2%), chemotherapy (28%), BTK inhibitors (11.7%), and Venetoclax-based regimens (4.2%). Remarkably, 85.8% of patients receiving chemotherapy had no prior cytogenetic or FISH testing. Differences in biomarker testing between countries were stark, ranging from 0% in Venezuela to 50% in Colombia.

With a median follow-up of 57 months (0–362), overall survival (OS) differed significantly by treatment era: OS was 116 months for 2010-2014, 127 months for 2015-2019, and was not reached for 2020-2024 (p=0.006). Four-year OS rates were 65% for chemotherapy, 81% for chemo-immunotherapy, 85% for BTK inhibitors, and 90% for Venetoclax-based regimens. 

Conclusions: This real-world analysis highlights significant disparities in CLL management across LATAM. Despite recent shifts toward targeted therapies, many patients still lack access to essential prognostic testing, potentially leading to suboptimal treatment choices. Efforts to improve biomarker availability and targeted therapy access are crucial for enhancing outcomes across the region. Expanding this cohort will further elucidate regional variations and support initiatives to address treatment inequities.

Abstract

Introduction: Patients with chronic lymphocytic leukemia (CLL) have an increased risk of developing second malignancies compared to the general population. Objectives: 1) to evaluate the frequency of second malignancies in CLL patients from a Uruguayan cohort; 2) to study the  relationship between these cancers and the clinical and biological characteristics of CLL; and 3) to determine if there is a relationship between the type of treatment (target therapy, FCR or FC)  and the frequency of the second malignancies. Method: This is a retrospective national multicenter study conducted by GURU-LLC (Uruguayan Group of CLL, “Grupo Uruguayo de Leucemia Linfoide Crónica”) with 521 patients diagnosed with CLL between 1998 – 2024. Results: The median age of this population is 70 years (range 36-87 years), 69% male and 31% female. Distribution by Binet A, B and C stages was 66%, 18% and 12% respectively (4% without data), and Rai stages 0 to IV were 45%, 24%, 13%, 8% and 5% respectively (5% without data). Eighteen percent of individuals (n=94) were diagnosed with a second malignancy.  Second neoplasms were in 85% solid tumors (n=80) and 15% hematologic malignancies (n=14). Among solid tumors, frequencies were as follows: skin (34%), prostate (16%), colon (14%), breast (11%), kidney (8%), lung (8%) and other tumors 10%. Hematologic malignancies were Richter transformation (RT), essential thrombocythemia and acute myeloblastic leukemia (AML) (79%, 14% and 7% respectively). Six percent of patients with second malignancies associated more than one neoplasm. The appearance of second malignancies occurred during the course of CLL in 56%, in 5% were present at diagnosis and in 18% they preceded the diagnosis of CLL (20% without data). Forty-five percent of the study population received treatment for their CLL, 71% with alkylating agents, and 3.7% of them developed AML. Median overall survival (OS) of CLL patients with or without second neoplasms were 88 and 138 months respectively (p=0.03). OS at 60 months was 55% for CLL with second neoplasms and 71% for individuals without neoplasm. Population with and without a second neoplasm are comparable with regard to the risk of their CLL, whether or not they received treatment, and the type of treatment received.  Second malignancy was the cause of death in 36% of individuals. Conclusions: Our results highlight the presence of second malignancies in patients with CLL. Similar to previously published data in Caucasian cohorts (Shen et al., 2021; van der Straten et al., 2023), these findings demonstrate an increased frequency of second malignancies, which negatively impacts the overall survival (OS) of CLL patients. These results emphasize the importance of regular surveillance and early diagnosis of secondary neoplasms in order to improve long-term outcomes in CLL patients .   

Figure 1: Median overall survival (OS) of CLL patients with or without second neoplasms were 88 and 138 months respectively (p=0.03).

Figure 2: Number of cases of solid tumors and hematological malignance in CLL patients.