A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples

Abstract

Background: Reverse 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.

Methods: We 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.

Results: Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R(2)>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.

Conclusions: This 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.

Figure 1. Schematic illustration of the probe design (A) and pre-amplification PCR (B).

Native template (NT) binding hydrolysis probes were labeled with FAM. Internal standard (IS) binding hydrolysis probes were labeled with Quasar 670. (A) For each gene, NT and IS had the same primer binding sites but there was a 4–6 bp difference in probe binding sites. (B) Varying concentrations of internal standards mixture (ISM) relative to cDNA were used to ensure that NT: IS ratio was >1∶10 and <10∶1.

Figure 2. Observed compared to expected positive PCR with limiting dilution.

Frequency of observed relative to expected positive PCR signal was measured. Poisson analysis was used to calculate expected positive frequency. Results from the average of nine replicates at each of 10 internal standard mixture dilution points (40, 20, 10, 7, 4, 2, 1, 0.7, 0.4, 0.1 molecules/µl) averaged across the four genes (ACTB, MYC, E2F1, CDKN1A) were compiled and plotted. Each gene plot is presented in Figure S2 in File S1.

Figure 3. Observed compared to expected E2F1 NT molecule values measured by two-color fluorometric assay in dilution series samples.

Linearity graphs (A, C, E) and amplification plots of E2F1 (B, D, F). (A, B) Serial dilution of external standards mixture (ESM, 1/1 mixture of NT/IS) from 10⁻¹¹ M through 10⁻¹⁷ M (triplicate measurements, with error bars). (C, D) NT dilution relative to constant IS from 1/1 NT/IS (10⁻¹² M) down to 1/80 (NT/IS) (triplicate measurements with error bars). (E, F) IS dilution relative to constant NT from 1/1 NT/IS (10⁻¹³ M) down to 1/80-fold (one replicate). NT: native template. IS: internal standard.

Figure 4. Internal standards control for PCR inhibition by EDTA.

MYC and ACTB were measured in the presence of varying EDTA concentration. (A) Quantification cycle (Cq) values of MYC IS, MYC NT, ACTB IS, ACTB NT. (B) Molecules of each gene and normalized value of MYC/10⁶ ACTB molecules (triplicate measurements) analyzed in benign, non-FFPE lung cDNA reverse transcribed with gene specific primers. NT: native template. IS: internal standard. FFPE: formalin-fixed, paraffin-embedded. The asterisk (*) indicates that Cq values were undetermined by software.

Figure 5. External standards mixture controls for inter-experimental variation in fluor signal or quantification cycle (Cq) selection.

(A–D) Effect of diluting labeled probe with unlabeled probe on measurement of MYC in benign, non-FFPE lung cDNA reverse transcribed with gene specific primers (triplicate measurements, with error bars). (A, B) NT labeled probe diluted with NT unlabeled probe. (C, D) IS labeled probe diluted with IS unlabeled probe. (E) Effect of inter-day variation in threshold selection on measurement of MYC and ACTB in surgically removed, FFPE sample 8 (SM8). NT: native template. IS: internal standard. FFPE: formalin-fixed, paraffin-embedded. The asterisk (*) indicates that Cq values were undetermined by software.

Figure 6. Validation of two-color fluorometric assay in 20 surgically removed, FFPE lung samples.

(A) lung cancer diagnostic test (LCDT) index values by diagnostic class. (B) receiver operator characteristic curve (ROC) of LCDT index.