Molecular Subtype Characterization of Formalin-Fixed, Paraffin-Embedded Diffuse Large B-Cell Lymphoma Samples on the Iceplex® System.

Molecular characterization of diffuse large B-cell lymphoma (DLBCL) provides important prognostic, and potentially therapeutic, information. Although the original classification of DLBCL into germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes was based on microarray gene expression profiling, this technique is not currently feasible in a clinical setting. Surrogate studies to assign classification, such as immunohistochemistry, have shown variability between laboratories and may not yield reliable results. Therefore, we have developed a novel multiplex real-time assay on the automated ICEPlex® System which allows differentiation between germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes of DLBCL from formalin-fixed, paraffin-embedded (FFPE) specimens.

A 17-gene panel was constructed from genes utilized previously in DLBCL subtype classification (Wright classifier and Lossos outcome predictor). Primers were designed for this panel, and two reference genes, using extension tag amplification (ETA). This method allows amplification from FFPE tissue with a template size less than 100 nucleotides. Thirty de novo DLBCL clinical samples, with paired frozen tissue and FFPE tissue, from patients at our institution were selected. Gene expression profiling using microarrays was performed on frozen tissue, and molecular subtype was assigned based on Wright classification. The ICEPlex® assay was performed on isolated RNA from two cells lines, one GCB subtype and one ABC subtype. The ICEPlex® assay was also completed on RNA extracted from three DLBCL clinical samples, and molecular subtype was determined for each sample. Analysis of FFPE clinical samples and cell lines is underway using the multiplex assay on the ICEPlex® instrument for determination of molecular subtype.

The real-time 19-plex expression profiling assay for DLBCL molecular subtyping was developed and optimized on the ICEPlex® System. Samples from two cell lines and from three clinical samples with frozen tissue were analyzed. The assay identified the molecular subtype of both cell lines. All three clinical samples were classified into the identical molecular subtype by the multiplex assay and the microarray gene expression profile. Results from the profiling of additional FFPE clinical samples and cell lines using the multiplex assay will be presented.

A novel 19-plex quantitative real-time expression profiling assay designed for DLBCL molecular classification into GCB or ABC subtypes was developed. This assay correctly assigned DLBCL cell lines and clinical samples into the correct subtype based on microarray gene expression profiling as the gold standard. Further work involves assessment of additional clinical DLBCL FFPE samples. This potentially robust multiplex assay may provide rapid molecular classification that is feasible for routine laboratory use in a clinical setting. * ICEPlex® System is for Research Use Only. Not for clinical diagnostic use.

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