Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.

Fluorescent-Activated Cell Sorting (Flow Cytometry).

Flow cytometry is a widely used diagnostic tool in many laboratories, which generates information that is essential for the diagnosis and classification of different hematolymphoid neoplasms (Reichard KK, KS, Flow cytometry in the assessment of hematologic disorders. In: Orazi A, Foucar K, Knowles DM et al (eds) Neoplastic hematopathology. Lippincott, Williams and Wilkins, Baltimore, MD, pp 119-145, 2013). Flow cytometry allows us to identify individual cells within heterogeneous populations. It is also useful for the quantification of cells, such as CD4 counts in HIV patients and CD34 stem cells on bone marrow and peripheral blood specimens. Lastly, it can also be used to describe the pattern of antigen expression on cells known as immunophenotyped (Craig FE, Foon KA, Blood 111(8):3941-3967, 2008).