Gene Expression and Cytokine Analyses Identify Markers of Progression from CLL-like Monoclonal B-Cell Lymphocytosis to Chronic Lymphocytic Leukemia
Authors: Blanco Gonzalo , Puiggros Anna , Sherry Barbara , Nonell Lara , Puigdecanet Eulalia , Chiu Pui Yan , Kieso Yasmine , Ferrer Gerardo , Arnal Magdalena , Rodríguez-Rivera María , Gimeno Eva , Abella Eugenia , Rai Kanti R , Abrisqueta Pau , Calvo Xavier , Bosch Francesc , Ferrer Ana , Chiorazzi Nicholas , Espinet Blanca 
Published: November 2019

INTRODUCTION: Chronic lymphocytic leukemia (CLL)-like monoclonal B cell lymphocytosis (MBL) is considered a precursor of CLL. It is found in 5-10% of elderly healthy individuals and shows a progression rate to CLL requiring therapy of 1.1% per year. A balance between microenvironmental factors and intrinsic properties of the emerging B cell clone may be decisive for the transition from MBL to CLL, although biomarkers of progression remain unknown. The objective is to describe biological markers (B cell gene expression profiles and serum cytokine levels) that predict progression from MBL to CLL.
METHODS: Gene expression profiles of clonal B cells from 14 MBL subjects (median age: 76 years, clonal B cells: 0.5-4.3 x109/L) were evaluated. With a median follow-up from analysis of 59 months (range: 10-77), 3 cases (21.4%) had progressed to CLL Binet stage A at last follow-up (clonal lymphocytosis >5x109/L, range: 6.2-7.9). Clonal B cells (CD19+CD5+) were isolated from peripheral blood by immunomagnetic methods (Miltenyi Biotec). Extracted RNA (RIN>7) was hybridized to GeneChip Human Gene 2.0 ST arrays (Affymetrix). Gene expression profiles were compared between MBL cases that progressed to CLL (P-MBL, n=3) and non-progressive MBL cases (NP-MBL, n=11). Differential gene expression was evaluated employing linear models for microarrays in R, and genes with P<0.05 and Fold Change >1.5 or <-1.5 were considered differentially expressed. To obtain insight into the functional significance of the differential genetic signatures, the Ingenuity Pathway Analysis tool (IPA, QIAGEN) was employed. On the other hand, serum levels of IL1β, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL17, IFNα, IFNγ, TNFα, GM-CSF, CCL3, CCL4, CCL19, CXCL9, CXCL10 and CXCL11 were quantified using the U-PLEX Platform (Meso Scale Discovery) and Human CXCL9/MIG Quantikine ELISA Kit (R&D Systems) in 41 MBL subjects (median age: 67 years, clonal B cells: 0.5-4.8 x109/L). With a median follow-up from analysis of 47 months (range: 0-117), 5 of them (12.2%) had progressed to CLL Binet stage A at last follow-up (clonal lymphocytosis >5x109/L, range: 6.4-17.3). Clonal B cells and cytokine levels were compared between P-MBL (n=5) and NP-MBL (N=36). For cytokine levels, the optimal cut-off values to stratify MBL cases according to their progression risk were assessed using the maxstat R package, whereas for clonal B cells a cut-off value of 3.9 x109/L was considered according to the results obtained by Kostopoulos et al (Blood Cancer J, 2017). The effect of different covariates on progression-free survival was evaluated using log-rank test. Cox proportional hazards regression models were performed to assess their independent prognostic value. P<0.05 was considered significant.
RESULTS: A total of 455 genes were differentially expressed (250 upregulated and 205 downregulated in P-MBL). IPA predicted an inhibition of apoptosis as well as proteins with tumor suppressor activity (SMARCA4) in P-MBL, besides enhanced bioenergetic processes (transmembrane potential of mitochondria) and anti-inflammatory features (activation of IL13 pathway and decreased chemotaxis of phagocytes and granulocytes) (Table 1). P-MBL displayed increased clonal B cells (4.2 vs. 1.7 x109/L, P=0.003) and levels of IL10 (1.15 vs. 0.9 pg/mL, P=0.087) as well as diminished levels of IL6 (2.04 vs. 3.75 pg/mL, P=0.041). MBL cases with ≥3.9 x109/L clonal B cells, ≥1.08 pg/mL of IL10 and ≤2.04 pg/mL of IL6 had an increased risk of progression to CLL (P<0.001, P=0.006 and P=0.034, respectively) (Figure 1, Table 2). Multivariate analysis for clonal B cells and levels of IL10 maintained significance for both factors (HR=12.8, P=0.013 and HR=10.2, P=0.047, respectively) (Table 2).
CONCLUSIONS: 1. P-MBL cases showed an inhibition of the apoptotic pathway and an activation of bioenergetic processes, which may account for the increased clonal B cells observed in this group. 2. P-MBL exhibited enhanced anti-inflammatory features, including augmented levels of the anti-inflammatory cytokine IL10. 3. Increased clonal B cells and IL10 levels predicted a higher risk of progression to CLL, suggesting that an augmented proliferative rate of clonal B cells together with a supporting tumor microenvironment are required for progression from MBL to CLL.
ACKNOWLEDGEMENTS. PI11/01621, PI15/00437, 2017/SGR437, Fundació La Caixa, Fundación Española de Hematología y Hemoterapia (FEHH).

Disclosures
Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Pharmacyctics: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding.