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 (DLBCL)

Abstract

Introduction: DLBCL presents clinical, biological, and prognostic heterogeneity. The composition of the tumor TME 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 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 and fast 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 DLBCL patients, seeking associations with outcomes and specific clinical phenotypes. Methods: This study involved 54 newly-diagnosed DLBCL patients. PB collection was performed before starting any therapy, for blood count and immunophenotyping on a BD Facs Calibur flow cytometer. End points included OS and EFS. Survival curves were constructed using the KM method. The Mann-Whitney test and the Kruskal-Wallis test were used to establish the relationship between the lymphocyte profile, clinical 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 and 55.6% were female. Clinical stage III/IV was observed in 77.8%, 57.4% had bulky ≥ 7 cm, 63.0% had B-symptoms, and 53.7% presented IPI ≥ 3. The ORR was 80.8%. With a median follow-up of 21.3 months, the estimated 2-year OS and EFS were 73.3% and 64.7%. The median absolute values obtained for the PB lymphocyte subpopulations were: 1026 cells for CD3+ T-lymphocytes, 591 cells for CD4+ T-helper lymphocytes, 412 cells for CD8+ T-cytotoxic lymphocytes, 1.6 for CD4/CD8 ratio, 240 cells for T-reg lymphocytes, 112 cells for LG T-lymphocytes, 173 cells for NK cells, and 70 cells 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 < 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. 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 < 0.001) and advanced clinical stage (p = 0.033). Conclusion: In newly-diagnosed DLBCL, the lymphodepletion of CD8+T-cells and NK cells quantified in PB were associated with decreased OS and EFS. Similarly, peripheral T-cytotoxic and NK lymphodepletion were associated with absence of 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 immune TME.