StaRT-PCR expression measurement of the c-myc x E2F1/p21WAF/CIP1 malignancy index and lung cancer associated genes to augment morphologic diagnosis of lung cancer in pleural effusion cell samples

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Pleural effusions often present a diagnostic dilemma to physicians. Frequently, pleural effusions represent a benign process such as congestive heart failure or inflammation from infection. On the other hand, malignancy is known to cause up to 40 % of pleural effusions (Monte, S.A. et al., Acta Cytologica. 31:448,1987). Unfortunately, only 50-60 % of malignant pleural effusions are accurately diagnosed by thoracentesis alone (Johnston, W.W. et al., Cancer. 56:905, 1985; Fenton, K.N. and Richardson, J.D., American Journal of Surgery. 170: 69, 1995). Previously, an interactive gene expression, the c-myc x E2F1/p21^WAF1/CIP1 (malignancy) index, measured by Standardized Reverse Transcription Polymerase Chain Reaction (StaRT-PCR™) was identified that augments cytomorphological diagnosis of malignancy in fine needle aspiration (FNA) or bronchial brush samples of solid tumors (Warner, K.A. et al., J. Mol. Diag., 5:176, 2003). In this study, we tested the hypothesis that the malignancy index would augment diagnosis in cytologic pleural effusion samples obtained at thoracentesis. Cytomorphologic confirmation of malignant cells in the pleural fluid was used as a gold standard for comparison. Of nineteen samples from fifteen patients, total RNA was quantified in fifteen. Adequate total RNA (range 1.2-200μg) for reverse transcription (RT) was obtained from eleven of the fifteen samples. In one of the eleven samples, no cDNA was obtained despite an estimated 413ng of total RNA per RT reaction. The remaining four samples were not analyzed for total RNA prior to RT. These results confirm that adequate RNA to conduct gene expression studies may be extracted from the majority of pleural effusions. Currently, the malignancy index is being analyzed by StaRT-PCR™ in the fourteen samples from which cDNA was obtained. To date, four of the samples have been analyzed for the malignancy index using the mean of triplicate values of each gene (i.e. c-myc, E2F1, and p21). Using the malignancy index, malignant samples have a 1,000-fold higher value than non-malignant samples. We propose that use of this gene expression index will aid diagnosis and improve sensitivity and specificity of current methods, particularly in circumstances where clinicians have a high index of suspicion for malignancy. JCW, ELC, and KAW have equity interest in Gene Express, Inc. which produces and markets StaRT-PCR reagents.