CD20 Antigen Density at Time of Presentation Is An Independent Predictor of Outcome in B-Cell Lymphoproliferative Disorders
Authors: Khubchandani Sapna , Mashtare Terry , Chisti Mohammad Muhsin , Wilding Gregory , Olejniczak Scott , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J. 
Published: April 2020

Abstract
The integration of monoclonal antibodies (mAbs) targeting CD20 antigen has changed the treatment paradigm of patients with B-cell non Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Current scientific efforts are aimed to: identify factors that can accurately predict clinical responsiveness to rituximab, develop ways, to augment rituximab activity, and use current biotechnology to create novel and more potent anti-CD20 antibodies. Pre-clinical studies demonstrated a direct correlation between CD20 levels and rituximab-associated complement-mediated cytotoxicity. We previously demonstrated that CD20 levels varied between different subtypes of B-cell malignancies (Olejniczak SH et al. Immunol Invest.2006; 35:93–114) raising the possibility that CD20 surface levels may correlate with the clinical behavior of B-cell lymphomas. In continuation of our work, we evaluated the correlation between quantitative CD20 expression on pathological material at diagnosis and clinical parameters/endpoints including overall survival (OS) in patients with a diagnosis of B-cell NHL and CLL. Patient’s specimens (lymph nodes, blood, or bone marrow) were identified by our tissue procurement facility and collected from 1997–2003. Surface antigen expression of CD20 was quantitated by quantitative indirect immunofluorescence assay (QIFI). Samples were analyzed using either three or four color flow cytometry with standard antibody panels. Demographic characteristics and clinical endpoint parameters (i.e. performance status, histology, stage, LDH at diagnosis, IPI/FLIPI score, treatment history, response to therapy, time to progression, OS) were collected. CD20 expression was divided into two categories using the median level as a cut off. Kaplan Meier curves were used to predict the overall survival in patients with high and low expression of CD20. A Cox proportional hazard model was used to study the effect of CD20 in multivariate analysis adjusting for age at diagnosis, response, source of quantifying CD20 expression, stage, race (Caucasian/non Caucasian), first course of treatment (CHOP/non CHOP) and sex. A total of 324 samples were analyzed, clinical parameters were available for 135 patients (24 patients with diffuse large B-cell lymphoma, 16 patients with follicular lymphoma, 48 patients with CLL and 47 patients with small lymphocytic lymphoma). Median age of the patients was 60 years. The vast majority of the patients received rituximab immunotherapy at varying time-points during their treatment history. Overall, Kaplan-Meier analysis demonstrated a trend, not statistically significant, towards better OS among B-cell lymphoma/CLL patients with high CD20 expression. However, when adjusted for histological subtype and other known predictors of survival, higher CD20 levels were associated with better OS. Using a multivariate analysis we found that patients with high CD20 expression at presentation had a statistically significant lower hazard of death compared to patients with low CD20 expression (Hazard ratio 0.27, CI 0.11 – 0.65, p value 0.0035). In addition, and as would be expected, those who achieved complete remission, partial remission or stable disease had a lower hazard of death (HR 0.05, 0.08 and 0.12 for patients in CR, PR and SD, respectively) when compared to patients with progressive disease. In summary, our data suggests that CD20 expression by QIFI at time of presentation is an independent prognostic factor of outcome in patients with B-cell malignancies. Similarly to what has been observed with other prognostic factors, validation in a larger sample of patients treated with rituximab-based therapy is necessary and ongoing to further establish the clinical value of monitoring CD20 levels by QIFI.
Table. Multivariate Analysis (OS)
Hazard 95 % CI Variable p-value Ratio Lower Upper Multivariate analyses adjusting for age at diagnosis, response, source, stage, race first course of treatment (CHOP/not-CHOP), and PS. CD20 (reference, Low) 0.0035 0.27 0.11 0.65 Response to First Treatment (reference, PD) CR &lt;.0001 0.05 0.02 0.18 PR 0.0003 0.08 0.02 0.32 SD 0.0014 0.12 0.03 0.44 Stage 0.0199 0.57 0.35 0.91 Race (reference, non-white) 0.0045 0.19 0.06 0.59 First Treatment (reference, non-CHOP) 0.0378 3.05 1.07 8.71 ECOG PS &lt;.0001 7.79 3.41 17.77
Kaplan Meier curves for survival distribution Kaplan Meier curves for survival distribution