Abstract
BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.
Highlights
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) tests that measure transcript abundance of selected genes in clinical specimens have been demonstrated to increase cancer diagnostic accuracy and enable ‘‘personalized medicine’’ through selection of the most effective treatment for each cancer [1,2,3]
A key challenge is that current clinical pathology sample collection and processing procedures focus on formalin fixation and paraffin embedding (FFPE) and fresh/fresh-frozen tissues rarely are available for molecular analysis
To address the challenge of analyzing clinical samples that yield a low amount of RNA, we previously described a competitive multiplex PCR method, in which all reference and target genes are first co-amplified with internal standards mixture (ISM) in a first round of PCR, followed by amplification of individual gene in the second round [20]
Summary
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) tests that measure transcript abundance of selected genes in clinical specimens have been demonstrated to increase cancer diagnostic accuracy and enable ‘‘personalized medicine’’ through selection of the most effective treatment for each cancer [1,2,3]. One way to address the challenge of interfering substances is to incorporate quality control in qPCR through measurement of each analyte relative to a known number of competitive template internal standard (IS) copies. This quality control method is recommended by regulatory agencies, including the EPA [9], ISO [10], and FDA [11], and is implemented in many FDA-approved single analyte RT-qPCR tests [12,13,14]. Samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed
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