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Specimen quality constraints for a molecular diagnostic test using gene expression ratios
Author(s) -
Rienstra Rachel G.,
Gordon Gavin,
Van Oss Branden,
Colquitt Richard,
Glickman Jonathan,
Bueno Raphael,
Richards William
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a216
Subject(s) - rna , adenocarcinoma , lung cancer , gene , gene expression , biology , microbiology and biotechnology , pathology , cancer , medicine , genetics
Differential diagnosis of adenocarcinoma (ADCA) of the lung from malignant pleural mesothelioma (MPM) is sometimes difficult by routine pathology. We developed a method based on the ratios of selected gene expression levels to create simple diagnostic tests. Three expression ratios comprising six genes (selected by transcriptional profiling of tumor‐rich samples) can be combined to accurately distinguish tumor‐rich samples of these two cancers (Cancer Res 62:4963,2002). We investigated the minimum tumor content required in a test sample for an accurate gene ratio test result. Total RNA obtained from 6 ADCA and 7 MPM samples was diluted with RNA obtained from uninvolved lung and pleura, respectively, from the same resection specimens to create sets of test samples estimated to contain between 0% and 70% tumor RNA in 10% increments (2 μg RNA per sample). All samples were reverse‐transcribed (Taq‐Man) and expression levels of the six diagnostic genes ( calretinin [GenBank #X56667], claudin‐7 [AJ011497], VAC‐ β [X16662], TACSTD1 [M93036], MRC OX‐2 antigen [X05323], and TITF‐1 [U43203]) were measured using quantitative real‐time RT PCR (SYBR‐Green; 40 cycles). The ratio test accurately diagnosed ADCA samples containing at least 10% tumor RNA and MPM samples containing at least 20% tumor RNA. Misdiagnosis of both tumor types occurred below these levels, however, highlighting the importance of specimen quality assessment as the clinical utility of this test is further evaluated. Supported by NCI‐CA105249