Measurement of gene expression biomarkers by qNPA from archived NSCLC FFPE: Prognosis of 5-year survival.
11098 Background: There are vast archives of formalin fixed paraffin-embedded (FFPE) tissue samples that are clinically annotated offering great research potential. However, the technology currently available to assess gene expression is limited to fresh, frozen, tissue. Recently, a method for measuring gene expression from FFPE using the quantitative Nuclease Protection Assay (qNPA) has been published in a model of diffuse large B-cell lymphoma. It was demonstrated that identical results were obtained from small amounts of FFPE as from matched frozen tissue, or from freshly fixed versus 18 year archived FFPE. METHODS: This study used the qNPA assay to measure gene expression in archived FFPE primary tumor samples of patients with stage 1 NSCLC for whom the survival outcomes are known (n=86). HTG lysis buffer is added to the sample; nuclease protection probes that are complementary to the mRNA of interest are then added to the solution. The probes hybridize to all RNA, soluble and cross-linked. After hybridization, S1 nuclease was added and destroys all nonspecific, single strand nucleic acid, producing a stoichiometric amount of target-mRNA to probe duplexes. Base hydrolysis releases the probe from these duplexes. Probes were transferred to a programmed ArrayPlate, detection linker added, and both probes and detection linkers were captured onto the array. The ArrayPlate was washed, HRP-labeled detection probe added, incubated, washed, and chemiluminescent substrate was added. Finally, the ArrayPlate was imaged, to measure the expression of each gene in all the wells. RESULTS: Mantel-Cox analysis indicates that the detection of increased expression of G-CSF (p = 0.07; H.R. =1.904; 95% CI=0.9003-4.028) and Leptin (p=0.09; H.R. =1.910; 95% CI=0.9299-3.924) individually suggest an improved survival. Age, gender and T size were found to not be significant in this data set. CONCLUSIONS: These results suggest an improved survival advantage in patients with an elevated native GCSF level in stage 1 NSCLC that is consistent with the survival benefits associated with the prophylactic treatment of GCSF for chemosensitivity in stage III or IV NSCLC patients. These results are currently being assessed using a larger cohort. [Table: see text].
Joshi, MM; Seligmann, B; Sabalos, C; Harpole, DH
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