B-Cell Lymphopenia and Circulating Neoplastic B-Cell Populations Are Associated with High Risk Disease in DLBCL: Results from the UK Remodl-B Trial
Authors: de Tute Ruth Mary , Barrans Sharon , Rawstron Andy C. , Manbridge Christopher T , Caddy Josh , Pugh Keith , Johnson Peter W.M. , Davies Andrew J , Jack Andrew 
Published: October 2019

Abstract
Background: Preliminary results from the UK REMoDL-B trial have shown that circulating neoplastic B-cells and B-lymphopenia are frequently detected by flow cytometry in presenting DLBCL patients. Uncertainty remains about the significance of these findings and how they relate to the course of clinical disease.
Aim: The aim of this study was to assess whether numerical and phenotypic peripheral B-cell abnormalities are related to advanced disease and adverse prognosis.
Methods: The study was carried out using peripheral blood samples collected from patients prior to first treatment. Peripheral blood was analysed using a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. Analysis allowed B-cell enumeration, identification of monoclonal populations and phenotypic classification as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. In addition, gene expression profiling (GEP) was performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay to classify tumours as GCB-type, ABC-type or unclassified.
Results: A total of 845 patients with an average age of 62.7 years (range 20.9-87.8) were included. A monoclonal population was detected in 174/845 (20.5%) of cases; CLL 3%, GC 7.8%, non-GC 7.7% and NOS 2.1%. Numerical abnormalities were also detected; 33% of patients had B-lymphopenia (<0.06 x 109/l) and 2.8% had B-lymphocytosis (>1 x 109/l). There was minimal overlap between numerical abnormalities and the presence of neoplastic B-cells, with 86% of cases with an abnormal population having a normal B-cell count. Circulating neoplastic cells were present in a small proportion of cases with B-lymphopenia (17/264) and a higher percentage of cases with B-lymphocytosis (8/19), as would be expected. Neoplastic B-cells were detected in 33% of cases an International Prognostic Index (IPI) score of 4-5, compared to 14% of cases with an IPI of 0-2. B-lymphopenia was present in 39% of individuals with an IPI of 4-5 compared to 24% of individuals with an IPI of 0-2. 51% (58/113) of cases with BM involvement had a circulating monoclonal population, compared to 15% (116/732) of cases with no overt marrow disease. Conversely, 66% of cases with circulating neoplastic cells did not have marrow involvement but this may, in part, be a sensitivity issue as staging was based on morphological assessment only.
Tumour GEP results were available for 690 individuals; 362 (52%) GCB, 184 (26%) ABC and 144 (21%) unclassified. 86% of populations identified in the ABC subgroup were phenotyped non-GC or NOS. Presence of a GC-population by flow was highly predictive of GCB GEP (88% GC-type populations detected were GCB cases). The number of discordant cases and CLL clones detected approximated to the numbers that would have been expected in a normal age-matched population.
Conclusion: Both the presence of circulating monoclonal B-cells and B-lymphopenia showed an association with higher risk disease. In addition, there was strong concordance between clonal phenotypes and the GEP of the underlying tumours. It is likely that in most cases the peripheral clonal populations are circulating tumour cells or a closely related precursor clone. Absolute B-cell counts are known to decline with age but there was no correlation with age in this patient group. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and the observed B-lymphopenia may be as a result of underlying immune dysfunction. Alternatively it may reflect suppression of normal B-cells by the neoplastic clone. Either way this has implications for the efficacy of immunomodulatory drugs, especially if the T-cell subsets are also affected. Multiparameter flow is highly applicable for diagnostic screening of both numerical and phenotypic B-cell abnormalities and may have a role in the prognostic assessment of DLBCL. Outcome data will be available shortly allowing the impact on progression free overall survival to be assessed.

Disclosures
Rawstron: Celgene: Honoraria; Abbvie: Honoraria; Pharmacyclics: Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria; BD Biosciences: Patents & Royalties. Johnson:Takeda: Honoraria; Pfizer: Honoraria; Janssen: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Jack:Jannsen: Research Funding.