P042 | FLOW CYTOMETRY IN THE ERA OF TARGETED THERAPY: EVALUATING ANTIGEN EXPRESSION IN LARGE B CELL LYMPHOMA AT DIAGNOSIS AND RELAPSE

In previous work our group described a diagnostic approach using flow cytometry (FC) to detect aberrant B-cell populations and raise suspicion of B-cell non-Hodgkin lymphoma (B-NHL). Our standard B-cell panel includes eight surface markers: Kappa, CD45, CD20, CD79b, CD5, CD19, CD10 and Lambda. With the recent introduction of targeted therapies against CD19, CD20 and CD79b, FC has gained importance as a tool to quantify antigen expression on pathological B cells. This study aims to characterize the expression of these therapeutic targets in 109 histologically confirmed cases of Large B cell lymphoma (LBCL) analyzed by FC between May 2020 and March 2025—69 at diagnosis and 40 at relapse. All samples underwent standard surface staining with additional intracellular staining performed when surface CD19 or CD79b were undetectable. Data were acquired on a DXFlex cytometer and analyzed with CyExpert software (Beckman Coulter). CD20 was highly expressed in both diagnostic and relapsed samples (median 99%, IQR 95–99%). However 5/40 relapse samples (12.5%) showed low or absent CD20 expression (≤20%), including cases following bispecific antibody therapy (n=2), rituximab maintenance for follicular lymphoma (n=2) and a primary refractory case (n=1). CD19 expression remained high except in relapses post-CAR T-cell therapy. Of six such cases evaluated at 4.5, 7, and 17 months post-CAR T, three (50%) had surface CD19 <1%. Intracellular staining in two of these cases revealed CD19 positivity in one (80%) and absence in the other, suggesting diverse mechanisms of antigen loss. CD79b expression was <1% in 13/40 relapse samples—nine not pre-treated with polatuzumab vedotin (PV), reflecting levels similar to those at diagnosis, and four of five relapsed post-PV. An additional six relapse samples had CD79b expression between 1–20%, with strong intracellular expression (98%) in all three cases that were evaluated. One illustrative case, monitored across multiple relapses (post-rituximab, PV, CAR-T, and CD20xCD3 bispecific antibody), ultimately lacked surface expression of CD19, CD20, CD79b, and light chains. A pathological population was detectable only through CD22 staining and intracellular markers (CD79a and clonally restricted light chains), underscoring the need for alternative gating strategies. While limited by sample size, our findings support the role of FC in therapeutic target quantification, guiding treatment decisions, and revealing potential mechanisms of resistance.