Serum CCL3 and CCL4 Levels Function As Novel Prognostic Markers in Diffuse Large B Cell Lymphoma.
Authors: Takahashi Koichi , Sivina Mariela , Oki Yasuhiro , Fayad Luis E , Neelapu Sattva S. , Kwak Larry W. , Xiao Lianchun , Huang Xuelin , Fu Kai , Chan Wing Chung , Vose Julie M , Kantarjian Hagop M. , Keating Michael , Burger Jan A. 
Published: October 2019

Abstract
Abstract 2709

Background:
B cell receptor (BCR) signaling is a critical growth and survival pathway in various B cell malignancies, including diffuse large B cell lymphoma (DLBCL). Upon BCR stimulation, normal and malignant B cells secrete the chemokine CCL3 and CCL4 (MIP-1 α, β) to foster B cell interactions with accessory cells. Serum and plasma level of CCL3/CCL4 is readily quantified using standard ELISAs, and was validated as a novel, robust and independent prognostic marker in chronic lymphocytic leukemia (CLL). Gene expression profiling in DLBCL revealed distinct signatures, such as the activated B cell (ABC) signature and germinal center B cell (GCB) signature. SCYA3, the gene coding for CCL3, is part of the genetic signature overexpressed in ABC phenotype. We therefore hypothesized that CCL3/CCL4 serum levels may have prognostic impact in DLBCL.

Aims:
1) To determine the association between serum CCL3/CCL4 levels and established prognostic markers including GCB/ABC signature in DLBCL patients. 2) To evaluate independent prognostic significance of serum CCL3/CCL4 levels in DLBCL patients.

Methods:
102 patients from MD Anderson (MDA cohort) and 19 patients from University of Nebraska (Nebraska cohort) with untreated DLBCL were retrospectively analyzed. Nebraska cohort was analyzed solely to investigate association between CCL3/4 levels and GCB/ABC signature. Serum samples from patients were analyzed for levels of CCL3 and CCL4 by ELISA. GCB/ABC signature was determined immune-phenotypically based on Hans' criteria.

Results:
Median age of the MDA cohort was 58 years (range: 22 to 86) and 41% was female. Thirty nine (39%) patients had an Ann Arbor Stage I/ II, and 63 (63%) had III/IV disease. Sixty five patients (64%) had low International Prognostic Index (IPI) scores (0–2) and 37 (36%) had high IPI (3–5). Mean (± SE) serum CCL3 and CCL4 levels of MDA cohort were 49.9 (± 3.63) and 224 (± 17.4) pg/ml, respectively. High serum CCL3 levels (≥40 pg/ml) correlated with higher IPI (P = 0.04), high LDH (P = 0.002), and high β2 microglobulin (P < 0.001). High serum CCL4 levels (≥225 pg/ml) correlated with higher Ann Arbor stages (P < 0.001), higher IPI (P = 0.03) and trend with high LDH (P = 0.09), and high β2 microglobulin (P = 0.067). During the median follow up duration of 14 months (range: 1 to 33), 10 patients were dead and 12 patients had progression of disease. One year overall survival (OS) of the whole cohort was 91% (95% CI: 85–97), and 1 year progression free survival (PFS) was 81% (95% CI: 74–90). Log-rank test demonstrated that high CCL3 was associated with shorter PFS (P = 0.029), and high CCL4 was associated with shorter OS (P < 0.01) and PFS (P < 0.01). When both levels were combined, CCL3high/CCL4high group (N = 27) had the worst PFS and OS (P = 0.003 and P = 0.007, both vs. CCL3low/CCL4low group; Figure 1.) Multivariate Cox proportional hazard regression revealed that high CCL4 level (HR = 3.2, P = 0.03) and high IPI score (HR: 2.5, P = 0.04) were significant and independent prognostic factors for PFS.
Total of 93 patients (74 from MDA and 19 from Nebraska cohort) had data regarding GCB/ABC signature. Mean serum levels of CCL3/CCL4 were not statistically different between GCB and ABC signature (GCB vs. ABC: CCL3 = 33.7 ± 2.5 vs. 35.5 ± 7.0, CCL4 = 207.1 ± 21.5 vs. 166.4 ± 21.4). Statistical association was not identified between serum CCL3/4 levels and GCB/ABC signature (CCL3: P = 0.46, CCL4: P = 1.0).
Post-treatment serum CCL3/4 levels were measured in 19 patients, of which 18 achieved CR and 1 with partial response. Eight patients had high CCL3 (≥ 40) before treatment, of which 7 became low post-treatment, while 7 patients had high CCL4 (≥225) before treatment and all patients became low post-treatment. Patients who started from low levels of CCL3 or CCL4 remained low post-treatment.

Conclusion:
High serum CCL3 and CCL4 levels correlate with established prognostic markers and inferior outcome in DLBCL. Serum levels of CCL3/4 did not correlate with GCB/ABC signature and prognostic impact appears to be independent from it. Serum CCL3/4 levels also correlated with treatment response in some patients especially who started with high levels pre-treatment. Therefore, serum CCL3 and CCL4 levels should be considered for prognostication and may also function as a sensitive marker to assess treatment response in DLBCL, particularly in the new era of BCR-targeting therapies.

Disclosures:
No relevant conflicts of interest to declare.