miRNA Expression Profile of B-SLL Consistent with Normal Memory B Cells:BIC/miR–155 Specific Location in Proliferation Center.
Authors: Wang Miao , Robertus Jan-Lukas , Tan Lu-ping , Harms Geert , Blokzijl Tjasso , Schakel Rikst Niynke , Kooistra Klaas , Rosati Stefano , Visser Lydia , Reijmers Rogier M. , Pals Steven T. , Kluin P.M. , van den Berg Anke 
Published: April 2020

Abstract
Introduction: B cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen. B-CLL behaves like leukemia and is found mostly in the blood. The expression of ZAP-70 and mutation status of the immunoglobulin heavy-chain gene (IgH) can serve as prognostic markers in B-CLL. ZAP-70 positive cases usually present with unmutated IgH genes and have a bad prognosis, whereas ZAP-70 negative cases mostly present with mutated IgH genes and have good prognosis. Gene expression studies of B-CLL indicated that the profile of IgH mutated and unmutated cases are similar to normal memory B cells. Several studies showed that miRNAs play important roles in pathogenesis of B-CLL and some miRNAs correlate with the prognosis in B-CLL. B-SLL is considered to be the same disease entity as CLL, but this variant is found mostly in bone marrow and the lymphatic system. The most aggressive type of B-SLL is characterized by neoplastic cells that are more responsive to B-cell receptor signaling and are characterized by proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. Until now, only a few reports have been published about the ZAP-70 expression and IgH mutation status and no data are available about the microRNA expression profile.
Methods: 33 B-SLL cases were retrieved from the pathology files. ZAP-70 expression was analyzed by using immunohistochemistry. IgH mutation status was determined using PCR followed by direct sequencing. Cases with homology of ≥98% with germline sequences were considered as unmutated and cases with homologies &lt;98% as mutated. Levels of 15 miRNAs were determined by qRT-PCR using U6 as a housekeeping gene in 28 B-SLL cases with good quality RNA. As a control we also analyzed miRNA levels in normal naïve, GC and memory B cell subsets. RNA in-situ hybridization (ISH) was used to localize the most abundantly expressed miRNAs in B-SLL tissues.
Results: 16 B-CLL cases were ZAP70+ with the vast majority of tumor cells staining positive and 17 were negative. Of the ZAP70+ cases 10 carried unmutated and 3 mutated IgH genes and 3 cases failed due to bad quality DNA. 14/17 ZAP-70- cases carried mutated IgH genes and 3 cases failed. miR-150, miR-21, miR-16, miR-92 and miR-155 were expressed at high levels in all B-SLL cases independent of the ZAP70 and IgH mutation status. The miRNA expression pattern in B-SLL was very similar to normal memory B cells. RNA-ISH for BIC, the primary transcript of miR-155, demonstrated the most abundant expression in the proliferation centers of B-SLL cases.
Conclusion: In B-SLL there is significant correlation between ZAP-70 expression and IgH mutation status similar to B-CLL cases. miRNA expression levels in B-SLL did not correlate with ZAP-70 or IgH status. The overall expression profile is very similar to normal memory B cells. BIC/miR-155 expression is observed specifically in the proliferation center of B-SLL tissues.