A quantitative reverse transcriptase polymerase chain reaction assay to identify metastatic carcinoma tissue of origin

Abstract

20024 Background: Carcinoma of unknown primary (CUP) wherein metastatic disease presents without an identifiable primary tumor site represents approximately 3–5% of all cancers. Identifying the origin of the primary tumor in patients with CUP can enable rational choice of therapeutic regimens. We developed an optimized set of ten gene markers for a quantitative reverse transcriptase polymerase chain reaction (qRTPCR) assay, and demonstrated high accuracy in predicting the tissue of origin when used on with formalin-fixed, paraffin-embedded (FFPE) metastatic carcinoma samples. Methods: Twenty-three putative tissue-specific markers for lung, colon, pancreas, breast, prostate and ovarian carcinomas were nominated by querying a gene expression profile database and by performing a literature search. Ten of these marker candidates were then selected based on validation by qRTPCR on 205 FFPE metastatic carcinomas of known tissue origin. Next, we optimized the RNA isolation and qRTPCR methods for these ten markers, and tested the qRTPCR assay on two sets of FFPE metastatic tumors. Results: We applied the 10-gene qRTPCR assay to a set of 260 metastatic tumors of known origin, generating an overall accuracy of 78%. Furthermore we tested an independent set of 48 metastatic samples, including thirty-seven samples where either the tissue of origin was known or which initially presented as CUP but were subsequently resolved. In these 48 samples, our assay demonstrated an accuracy of 76%. Conclusions: Our results suggested that optimized ten-gene markers qRTPCR assay reliably predicts tissue of origin of metastatic carcinomas in FFPE tissues. Such assay can significantly improve the rate of tissue of origin identification for carcinoma of unknown primary. [Table: see text]