Does Peripheral Blood Mirror Immune Tumor Microenvironment? Flow Cytometry Quantification of Peripheral Blood Lymphocyte Subpopulations and Outcomes in Patients with Diffuse Large B-Cell Lymphoma
Authors: Lage Luís Alberto de Pádua Covas , Almeida Igor Cerejo , Reichert Cadiele Oliana , de Lima Giselle Groetares , Culler Hebert Fabrício , de Freitas Fábio Alessandro , França Lilian de Souza , da Costa Raphael Rodrigues , da Silva Paola Carolina Rabesquini Marcelino , Rocha Vanderson , Costa Renata de Oliveira , da Siqueira Sheila Aparecida Coelho , Pereira Juliana 
Published: December 2024

Introduction: Diffuse large B-cell lymphoma (DLBCL) presents clinical, biological, and prognostic heterogeneity. The composition of the tumor microenvironment (TME), including cytotoxic CD8+T-lymphocytes, NK cells, and macrophages, strongly contributes for its biological diversity, being able to predict treatment response and determine prognosis. DLBCL with TME enriched in CD8+T-lymphocytes and NK cells demonstrate increased OS compared to those with TME enriched in histiocytes. Data from patients with solid tumors have established strong associations between absolute quantification of lymphocyte subpopulations (LS) in peripheral blood (PB) and prognosis. Therefore, it is hypothesized that the cellular constituents of PB may mirror the TME, and their quantification may constitute an easy, fast and cost-effective tool to access biological biomarkers predictive of therapeutic response and prognosis in malignancies. This study aims to determine the absolute quantification of LS in the PB of newly-diagnosed DLBCL patients, seeking associations with outcomes and specific clinical phenotypes.

Methods: This prospective study involved 54 newly-diagnosed DLBCL, NOS patients, treated at the University of São Paulo, Brazil, from February 2021 to December 2022. Immunocompromised patients, and those previously exposed to corticosteroids or radiotherapy were excluded. PB collection was performed before starting any therapy, for blood count on a Beckman-Coulter counter and lymphocyte immunophenotyping on a BD Facs Calibur flow cytometer. End points included OS and EFS. Survival curves were constructed using the Kaplan-Meier method. The Mann-Whitney test and the Kruskal-Wallis test were used to establish the relationship between the lymphocyte profile, clinical-laboratory variables, therapeutic response and outcomes. The Contal O'Quingley test was used to determine cut-offs for each LS capable of discriminating outcomes. Univariate analysis was performed using the Cox method and a p-value ≤ 0.05 was considered statistically significant.

Results: The median age at diagnosis was 54 years (21-90) and 55.6% (30/54) were female. Clinical stage III/IV was observed in 77.8% (42/54), 57.4% (31/54) had bulky ≥ 7 cm, 63.0% (34/54) had B-symptoms, and 53.7% (29/54) presented IPI ≥ 3. More than 85% (46/54) of cases were treated with R-CHOP. The ORR was 80.8% (95% CI: 68.6-89.7%). With a median follow-up of 21.3 months, the estimated 2-year OS and EFS were 73.3% (95% CI: 61.3-85.3%) and 64.7% (95% CI: 51.3-78.1%). The median absolute values obtained for the PB lymphocyte subpopulations were: 1026 cells/mm3 for CD3+ T-lymphocytes, 591 cells/mm3 for CD4+ T-helper lymphocytes, 412 cells/mm3 for CD8+ T-cytotoxic lymphocytes, 1.6 for CD4/CD8 ratio, 240 cells/mm3 for T-reg lymphocytes, 112 cells/mm3 for large-granular T-lymphocytes, 173 cells/mm3 for NK cells, and 70 cells/mm3 for B-lymphocytes. In univariate analysis, using a cut-off value = 367 CD8+T-cells/mm3 and 141 NK-cells/mm3, cytotoxic T-lymphodepletion was associated with decreased OS (p=0.005) and EFS (p&lt;0.001), as well as NK-lymphodepletion was associated with decreased EFS (p=0.033), but not with shortened OS (p=0.240). DLBCL patients presenting higher NK-cell counts had lower rates of chemoresistance to the R-CHOP (p=0.024), and those with higher NK-cell counts (p=0.033) and higher CD8+T-lymphocyte counts (p=0.039) had lower probability to develop relapse, progression or death, confirming the central role of these PB cellular constituents in anti-tumor immunity. Moreover, we observed that B-cell lymphodepletion was associated with an adverse clinical phenotype, including higher frequency of bulky disease (p=0.009), B-symptoms (p&lt;0.001) and advanced clinical stage (p=0.033).

Conclusion: In newly-diagnosed DLBCL patients, the lymphodepletion of cytotoxic CD8+T-cells and NK cells quantified in PB samples were associated with poor clinical outcomes, including decreased OS and EFS. Similarly, peripheral T-cytotoxic and NK lymphodepletion were associated with absence of therapeutic response to the R-CHOP regimen, as well as higher probability of relapse, progression or death. Therefore, the absolute quantification of these LS in PB may be considered potential biomarkers capable of predicting response and prognosis in DLBCL. Also, we believe it may mirror the cellular composition of the tissular immune TME.