Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies

Abstract

Background: The Oncotype DX Prostate Cancer Assay is a multi-gene RT-PCR expression assay that was developed for use with fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies containing as little as 1 mm of prostate tumor in the greatest dimension. The assay measures expression of 12 cancer-related genes representing four biological pathways and 5 reference genes which are algorithmically combined to calculate the Genomic Prostate Score (GPS). This biopsy-based assay has been analytically and subsequently clinically validated as a predictor of aggressive prostate cancer. The aim of this study was to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria.

Results: The lowest quartile of RNA yields from prostate needle biopsies (six 5 μm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as 0.005 ng to 320 ng). Analytical accuracy was excellent with average biases at qPCR inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively.

Conclusions: The Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. The assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer.

Figure 1

Calculation of Genomic Prostate Score (GPS). The aggregate expression of 5 reference genes was used to reference normalize the expression of the 12 cancer-related genes. Normalized gene expression was used to calculate the individual group scores: stromal group score, cellular organization score, androgen groups score and proliferation score. Each of those group scores is algorithmically combined to calculate the unscaled Genomic Prostate Score (GPSu); the GPSu is then scaled to a 100-unit range GPS. A negative coefficient in the calculation of the GPS is associated with better outcome whereas a positive coefficient is associated with poorer outcome.

Figure 2

Estimates of RNA yields in the smallest diagnostic biopsies. (a) RNA from 46 formalin-fixed prostate needle core biopsy specimens collected between 2003 and 2008 by the Cleveland Clinic was extracted and RNA yields were measured using the RiboGreen method. Tumor volumes were measured and all results were adjusted to represent RNA yields in the target tumor volume of 0.0225 mm³ (1 mm tumor length x 0.75 mm width of diagnostic biopsies x 0.030 mm tumor depth). (b) AUA low and intermediate risk patients treated with radical prostatectomy at Cleveland Clinic between 1999 and 2007 with available biopsy tissues were included in the study. In total, RNA from 167 diagnostic biopsies was extracted and RNA yields were measured using the RiboGreen method. Tumor volumes were measured and all results were adjusted to represent RNA yields in the target tumor volume of 0.0225 mm³ (1 mm tumor length x 0.75 mm width of diagnostic biopsies x 0.030 mm tumor depth).

Figure 3

Boxplots for the RT-PCR positive control during analytical and clinical validation studies. Boxplots summarizing performance of each gene over analytical and clinical validation for RT-PCR positive control (prostate cancer FPE pool) representing 18 RT-PCR control plates stratified by gene (ARF1, ATP5E, CLTC, GPS1 and PGK1 are reference genes). Each RT-PCR control plate contains one positive control and each gene is measured in triplicate. Standard deviations ranged from 0.19 Cp to 0.33 Cp. The box represents the inter-quartile range, the line in the box represents the median and the diamond is centered at the mean. The whiskers represent minimum and maximum values observed.

Figure 4

Boxplots of genomic DNA detection and qPCR positive controls. Boxplots summarizing the performance of genomic DNA detection and qPCR positive controls over Analytical and Clinical Validation. The box represents the inter-quartile range, the line in the box represents the median and the diamond is centered at the mean. The whiskers represent minimum and maximum values observed.