DLBCL Molecular Subtyping using NanoString

To determine the molecular subtype according to the cell-of-origin (COO) classification, extracted RNA was analyzed by the NanoString nCounter FLEX gene expression profiling (GEP) system (NanoString, Seattle, Washington, USA) as previously described [18 ]. The NanoString Lymphoma Subtyping Test (LST) algorithm allows the assignment of each analyzed sample to the germinal center B-cell like (GCB) subtype, the activated B-cell like (ABC) subtype, or to be unclassified (Supplementary Table 1) [19 , 20 (link)]. In brief, the LST CodeSet consists of capture and reporter probes for 20 genes: 7 genes overexpressed in GCB DLBCL (ASB13, ITPKB, MAML3, MME, MYBL1, S1PR2, SERPINA9), 8 genes overexpressed in ABC DLBCL (CCDC50, CREB3L2, CYB5R2, IRF4, LIMD1, PIM2, RAB7L1, TNFRSF13B), and 5 housekeeping genes (ISY1, R3HDM1, TRIM56, UBXN4, WDR55). Quality and quantity of used RNA was determined by spectrophotometer (Nanodrop, Thermo Scientific). The LST CodeSet was hybridized to 500 ng of total RNA for 18 h at 65 °C. Hybridized RNA samples were loaded into the nCounter Prep Station and expression of target mRNA was finally assessed by the nCounter Digital Analyzer.