microRNA-21 Is Differentially Expressed in the Lymphoid Tissue and Modulated By Stromal Signaling in Chronic Lymphocytic Leukemia

MicroRNAs contribute to the initiation and dissemination of malignant clone and serve as important prognostic indicators. miR-21 overexpression was linked to fludarabine resistance and unfavorable prognosis in CLL. We and others have shown that B-cell receptor (BCR) signaling upregulates miR-21 in CLL. While recent literature addresses the impact of miR-21 in the circulating malignant B cells, few studies have focused on its expression in CLL lymph nodes, where cells receive stromal pro-survival signals including via BCR. In those tissues, some microRNAs demonstrate distinct expression patterns, e.g. miR-155 is highly expressed in the proliferation centers. Here we aimed to investigate miR-21 expression in the lymphoid tissue and assessed the effect of the modeled lymphoid niche on this MIR in CLL cells in vitro.

Expression of miR-21, RNU6B and CD20 was assessed in 30 formalin fixed paraffin-embedded lymph nodes from patients with CLL and 4 control (tonsil) tissues using a modified combined FISH/IHC assay. miR-21 was differentially expressed in the lymphoid tissues of patients with CLL: miR-21 was detected in 13/30 lymph nodes (43%), with three demonstrating strong and ten - weak staining. The remaining 17 tissue specimens (56%) stained negative for miR-21. Where present, strong miR-21 expression followed focal rather than diffuse staining pattern. Foci of miR-21 did not localize to the proliferation centers as per morphologic assessment. Since microRNAs are also expressed in other cell types including stromal cells, we used CD20 to confirm that miR-21 expression was detected in the neoplastic CLL cells. miR-21 positivity in the lymphoid tissue did not predict time to first treatment or correlate with either CD38 or ZAP-70 expression. Interestingly, 2/4 control tissues demonstrated strong staining for MIR21, indicating that its expression in the lymphoid organs is not restricted to clonal neoplastic B-cells.

We further studied whether miR-21 expression was modulated by stromal signaling. Peripheral blood CLL cells (n=33) and normal B-cells (n=7) were isolated using standard Ficoll-Hypaque techniques, B-cell isolation kit and CD19 MACS microbeads, followed by cDNA synthesis and RT-PCR with specific TaqMan probes. We modeled lymph node microenvironment in vitro using CD40L-expressing or control mouse L cells, thus promoting drug resistance and survival of the peripheral blood CLL; CD40L-expressing cells induced NFκB. Consistent with previous reports, we found increased levels of miR-21 in peripheral blood CLL cells compared with normal B-cells. Co-culture of CLL cells with stroma resulted in induction of miR-21. IGHV mutational status is closely associated with BCR signaling capacity in CLL and predicts cellular reliance on microenvironmental support. We did not find a correlation between baseline miR-21 expression and IGHV mutational status in circulating CLL cells. However, CLL cells with unmutated IGHV were stronger inducers of miR-21 in response to stromal signaling. Meanwhile, CD40L-expressing stroma led to further induction of miR-155, a recognized NFκB target, but not miR-21, indicating that alternative mechanisms are responsible for stromal modulation of this microRNA.

In summary, here for the first time we successfully visualized miR-21 expression within the neoplastic B-cells in the lymphoid tissues from patients with CLL. miR-21 was induced in stromal CLL cell co-cultures, primarily in cells with unmutated IGHV. Our observations are particularly relevant in the current era where BCR signaling pathways have become key pharmacologic targets. These therapies disrupt CLL–stroma interactions leading to an egress of CLL cells to the periphery, where they are unable to proliferate. Analysis of expression of miR-21 in lymph nodes and peripheral blood of patients treated with the BCR-targeting agents may clarify its predictive role in this setting as well as shed additional light on the mechanisms involved in its regulation.