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[1613] Validation of a 10 Gene Multiplex Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) Assay To Detect the Primary Site of Metastatic Carcinoma of Unknown Origin (CUP)

CK Ibrahim, J Baden, C Major, A Mazumder, K Gray, CE Sheehan, B Varadarajalu, MS Ross, DM Jones, T Nazeer, TA Jennings, JS Ross. Albany Medical College, Albany, NY; Veridex, LLC, Warren, NJ

Background: Despite advances in biomedical imaging and minimally invasive surgical techniques, the determination of the primary site in CUPs remains a challenge in clinical practice. There has been significant interest in the use of molecular techniques to complement traditional morphology in the assessment of these metastatic lesions.
Design: Using transcriptional profiling, literature searches and a quantitative RT-PCR technique (Veridex, LLC), a 10 gene classifier was developed on 205 formalin fixed paraffin embedded (ffpe) CUPs to predict the primary site of origin. The RT-PCR assay detects primary tumor sites as: breast, colon, small and non-small cell lung, ovary, prostate, pancreas and other. The 10 gene classifier was subsequently tested on 48 CUPs not included in the discovery set. In the current study, the 10 gene classifier was validated on an additional 10 CUPs whose site or origin was assessed by immunohistochemistry (IHC) and confirmed by follow-up clinical studies.
Results: The qRT-PCR technique featured an initial one-out cross validation accuracy of 78% and a 76% accuracy with the initial test set. The primary sites for the 10 case new validation set were lung (3), colon (3), gallbladder (1), endometrium (1), head and neck (1) and ovary (1). The qRT-PCR assay correctly identified the primary tumor site in 8 (80%) of the cases. The endometrial and head and neck primary tumors were classified as other by the qRT-PCR assay. The decision algorithm prediction averaged 83% (range 37% to 100%). In one case of poorly differentiated metastatic gallbladder cancer, the qRT-PCR assay predicted a small cell lung cancer origin while the IHC was not conclusive. In another case of metastatic adenocarcinoma with enteric features originating in the lung, the qRT-PCR assay predicted colorectal rather than pulmonary origin. IHC classification was not conclusive in this case with negative stains for both cytokeratin 20 and TTF-1.
Conclusions: The 10 gene multiplex qRT-PCR assay designed to identify the primary tumor site in cases of CUP had a high accuracy of 80% in this validation study using ffpe tissues. Further evaluation of this qRT-PCR technique on larger cohorts of patients is warranted to confirm the high accuracy and the potential clinical utility of the technique.
Category: Techniques

Monday, March 26, 2007

Poster # 224, Monday Morning