The Aggressive Molecular Signature of Peripheral Blood Chronic Lymphocytic Leukemia (CLL) Cells From Patients with Bulky Lymphadenopathy Closely Resembles That of CLL Cells From Lymph Nodes,
Authors: Mittal Amit K. , Iqbal Javeed , Gilling Christine E , Moragues Margaret , Bociek R. Gregory , Aoun Patricia , Weisenburger Dennis D , Joshi Shantaram S 
Published: October 2019

Abstract
Abstract 3868
Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy which is characterized by accumulation of CLL cells in the peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We have previously reported the gene expression profiles (GEPs) of CLL cells from these tissue sites (Blood, ASH Annual Meeting Abstracts, 2008; 112: Abstract 546) and shown that genes associated with various biologically-relevant signaling pathways (BCR, BAFF/APRIL, NFB, PI3K/AKT) were overexpressed in LN-CLL cells compared to PB-CLL and BM-CLL cells. To elucidate the association of the differentially- expressed gene signatures among PB, BM and LN-CLL with disease progression, gene expression was further studied based on clinical parameters obtained at the time of diagnosis such as chromosomal abnormalities (del 11q, del 17p and trisomy 12, and del 13q and normal karyotype as poor and good prognosis markers respectively), bulky lymphadenopathy (BLA) as a poor prognosis indicator and time to treatment <12 months as a poor prognosis indicator. GEPs showed that PB-CLL (n=7) with poor prognosis chromosomal abnormalities had higher expression levels of genes associated with BCR signaling (BTK, AKT2) and cell activation (CD83, LAG3) compared to PB-CLL (n=12) with markers of good prognosis. Furthermore, BM-CLL cells (n=6) with poor prognosis chromosomal abnormalities overexpressed genes associated with BCR signaling molecules (NFATC4, VAV1), cell activation (CD97), and the MAPK pathway (FGF20, MAP2K4) when compared to BM-CLL (n=11) with good prognosis genetics. This suggests that PB-CLL and BM-CLL cells in with aggressive disease have overexpression of survival/proliferation and activation signaling molecules. PB and LN samples were also grouped based on time to treatment. Genes associated with the MAPK pathway (MAP3K10) and cell activation (CD69) were overexpressed in PB-CLL (n=9) with a short time to treatment compared to the PB-CLL (n= 10) with a longer time to treatment. Genes related to apoptosis (CASP6, BID, and CASP4) were underexpressed in the LN-CLL with short time to treatment (n=11) versus those with a longer time (n=4), suggesting anti-apoptotic functions dominate in this poor prognosis group. GEPs were also performed on the basis of the presence or absence of BLA, one of the symptoms of aggressive CLL associated with a poor prognosis. We were specifically interested in the differential gene expression pattern between the PB-CLL with and without BLA because if the microenvironment is important in regulating prognosis, determining the expression of key genes associated with PB-CLL from BLA may reveal how the LN microenvironment supports PB-CLL. Therefore, we divided the PB-CLL samples based on the presence (n=9) or absence of BLA (n=11). Interestingly, PB-CLL cells from patients with BLA had a significantly high number (N>1300) of overexpressed genes. Most of these genes were associated with BCR signaling (CD79b1, CD79b2, CD72, SYK, BTK, BLNK, VAV1), the MAPK pathway (ELK4, MAP4K1, MAP3K10, MAP3K3), hedgehog signaling (GLI, SUFU, PTCH), chemokines (CCL21, CXCL13), and the NFκB pathway (TANK, TNFSF8, TNFSF7), indicating that PB-CLL cells from patients with BLA are highly activated compared to PB-CLL cells from those without BLA. Since this expression profile closely resembles that of LN-CLL, we infer that PB-CLL cells with high levels of signaling molecules had migrated out of LN in patients with BLA. We measured expression of one of the key players of BCR signaling, phosphorylated SYK (p-SYK), to validate that this pathway is active in the PB-CLL from BLA patients. Expression of p-SYK was significantly (p<0.05) higher in the PB-CLL from patients with BLA (n=4) compared to those without BLA (n=5) using flow cytometry indicating that BCR signaling molecule is functionally active in PB-CLL with BLA. Overall, our results provide a comprehensive overview of the gene signature associated with aggressive CLL. (This work was supported by the CLL Foundation, Houston, TX).

Disclosures:
No relevant conflicts of interest to declare.