Assessment of Tissue and Subcellular Distribution of miRNA Expressed Differentially between ALK + and ALK- Anaplastic Large Cell Lymphoma
Meenakshi Mehrotra, Rajyalakshmi Luthra, Rajesh Singh, Rachel Sargent, Jeffery Medeiros, Keyur P Patel. MD Anderson Cancer Center, Houston, TX
Background: Anaplastic lymphoma kinase (ALK)-positive (ALK+) anaplastic large cell lymphoma (ALCL) is a distinctive type of T-cell lymphoma characterized by CD30 expression, translocations involving the ALK gene, and a favorable clinical outcome. In contrast, ALK-negative (ALK-) ALCL, although morphologically and immunophenotypically similar, lacks ALK translocations and has a worse prognosis. Recent microRNA (miRNA) profiling using whole tissue extracts revealed differential expression of miRNAs that included miR-155 with a potential role in the ALCL pathogenesis. The miRNA profiling however, is inadequate for identifying subcellular distribution of individual miRNAs, which could provide important biologic and diagnostic information. In this study, we examined tissue and cellular distribution of miR-155 in ALK+ and ALK- ALCL using in situ hybridization (ISH) analysis.
Design: Formalin-fixed and paraffin-embedded (FFPE) tissue samples of ALK+ ALCL (N=11), ALK-ALCL (N=11) and reactive lymph nodes (N=2) were processed and sectioned on super frost slides in RNAse free condition. ISH using double dioxigenin (DIG)-labeled mercury LNATm miRNA detection probes (Exiqon, Vedbaek, Denmark) was optimized for miR-155 on cell lines and FFPE tumor samples using positive control (miR-126) and scrambled control. The results were interpreted and scored by two independent observers using a scoring system ranging from 0 (negative) to 3 (strong and diffuse) based on cellular intensity and tissue distribution. qPCR was performed to correlate ISH results.
Results: ISH protocol for miR-155 was established using FFPE cell-pellets from ALK+ (Karpas, SUDHL-1) and ALK- (Mac2A) ALCL cell lines and control cell line (Jurkat). Significantly higher expression of miR-155 was noted in ALK- ALCL compared to ALK+ ALCL (median score: 3 vs 1, average score 2.3 vs 1.3, p<0.05). The tissue distribution correlated with morphologic involvement by the lymphoma. Reactive lymph node showed no expression when hybridized with DIG labeled LNA probe of miR-155. The ISH findings results were consistent with the qPCR findings for miR-155 in ALK+ and ALK- ALCL.
Conclusions: ISH studies performed on FFPE-tissue specimens show higher miR-155 expression in ALK- ALCL compared with ALK+ ALCL, consistent with the qPCR findings. The ability to visualize miRNA expression patterns and perform relative quantitation in tissue and cells provides a useful tool for the analysis of miRNA expression and for convenient diagnostic testing on FFPE-tissue sections for differentially expressed miRNAs.
Tuesday, March 5, 2013 2:00 PM
Proffered Papers: Section G, Tuesday Afternoon