Biomarker discovery for colon cancer using a 761 gene RT-PCR assay
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Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco type DX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application.
RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery.
We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.
KeywordsLaboratory Information Management System Formalin Fixed Paraffin Recurrence Free Interval Stromal Response Automate Liquid Handler
Over the last decade, many studies have applied gene expression analysis to identify biomarkers for prognostic and/or predictive information in relation to human disease [1, 2, 3, 4]. RNA for these studies has come from either frozen or formalin fixed paraffin embedded (FPE) tissue. RNA from frozen tissues is generally regarded as the most desirable for molecular assays, since if collected correctly it is generally intact and can be analyzed by a wide variety of standard molecular biology techniques. However, FPE tissue is the most widely available source of tumor tissue as it is the product of standard tissue processing procedures followed by surgical pathology laboratories. It is clear that RNA obtained from FPE tissue is not full length and the extent of degradation increases with storage time . Therefore, it is generally considered to be challenging to extract RNA from archival FPE tissue for analysis by standard molecular biology techniques.
With the development of automated liquid handling and DNA microarrays, high throughput screening using hundreds of samples and hundreds or thousands of genes has become routine in many laboratories. DNA microarrays offer the advantage of simultaneously assessing the relative expression level of thousands of genes with a relatively small amount of starting RNA. However, DNA microarray measurements are limited in dynamic range, specificity and reproducibility, leading to high false positive and false negative biomarker discovery rates. As currently configured, DNA microarray technology also requires high quality RNA. Alternatively, reverse-transcription polymerase chain reaction (RT-PCR) technology offers the advantages of high accuracy and reproducibility, and precise quantitation over a wide dynamic range. To overcome the issue of fragmented RNA in FPE tissue specimens, assays can be optimized for short amplicons so the RNA from FPE tissue can be successfully analyzed .
It has been suggested that there is a bottleneck in scaling up TaqMan® RT-PCR using archival FPE samples to analyze beyond 30 genes . On the contrary, we demonstrated that TaqMan® RT-PCR biomarker screening using FPE samples is not necessarily limited to small candidate gene sets and have performed studies with up to 192 genes [5, 7, 8]. Using this technology we developed and commercialized a 21 gene panel that predicts the likelihood of cancer recurrence in early stage breast cancer patients . We have now scaled our TaqMan® RT-PCR screening process to assay 768 wells of data per patient sample. Here we describe the methodology that was used to identify prognostic biomarkers in stage II/III colon cancer .
In this paper we report the use of this high throughput, highly parallel TaqMan® RT-PCR process to screen RNA extracted from colon cancer FPE clinical trial specimens (NSABP C01 and C02 studies). We focused on selecting candidate genes known to be involved in pathways related to colon cancer and genes from published expression profiling data sets relating to colon cancer prognosis and response to therapy. Our results indicate that this approach yields high quality expression data that can be used for simultaneous evaluation of hundreds of candidate genes in defined cohorts of patients to identify prognostic and predictive colon cancer biomarkers.
Large scale multiplexed, gene-specific reverse transcription
Once we confirmed the high complexity priming reaction was working consistently in FPET RNA, we prepared a gene specific primer pool containing 761 unique reverse primers for the biomarker discovery study using NSABP C01 and C02 clinical trial specimens.
To be included in the final study analysis, samples had to pass pathology, clinical and laboratory data QC requirements. To meet pathology acceptance criteria, a minimum of 5% of the tissue present in each sample was required to be invasive cancer cells. All samples were dissected to enrich tumor tissue and minimize non-tumor elements.
Sample exclusion criteria
Incomplete sample data
Total in final dataset
Using a semi-automated extraction process, two lab technicians extracted RNA from 48 samples per day. Paraffin was removed from the tissue manually with xylene, followed by ethanol washes. After a proteinase K digestion and phenol-chloroform purification, the remaining RNA extraction steps were performed on an automated liquid handler using a plate-based protocol. The purified RNA was then quantified using an automated RiboGreen fluorescence assay (Invitrogen, Carlsbad, CA.). The resulting files containing the RNA quantification data were automatically collected by the laboratory information management system (LIMS). The LIMS used those data to generate an RNA concentration normalization work-list, which was then executed by an automated liquid handler during the reverse transcription reaction assembly procedure. The reverse transcription reaction was completed using an MJ Research thermocycler (Bio-Rad, Hercules, CA). Finally, quantitative PCR reactions were assembled at a rate of 64 plates (32 patient samples) per day. All automated liquid handlers were obtained from Tecan (TECAN Schweiz AG, Männedorf, Switzerland). This system yielded a total of 24,576 real time quantitative PCR reactions to be performed per day using four ABI PRISM 7900 HT instruments (Applied Biosystems, Foster City, CA.).
Expression data normalization
Although the two assay plates for each patient RNA were run on the same ABI Prism 7900 instrument to minimize process variability, data from each sample had to be internally normalized to allow all study specimens to be compared without being confounded by relative variability in RNA quality, quantity or process variability. This was accomplished by subtracting the averaged expression values from 6 reference genes (CLTC, NEDD8, RPLPO, RPS13, UBB, UBC) from the expression values for each gene in each sample. This method has previously been shown to effectively compensate for variability associated with RNA degradation in FPE material of different ages and qualities . Normalization genes were chosen on the basis of low expression variation among patient samples, robust CT signals (CT < 35) and lack of association with clinical outcome. As part of our quality control and acceptance criteria, the average expression CT value for the 6 reference genes for each patient sample had to be less than 35. Within the final evaluable dataset of 270 patient samples, the average reference signal was 29.5 CT (SD = 1.39 CT) with the lowest observed reference signal at 33.7 CT, indicating good overall signal response and relative invariance in reference genes among patients.
Gene groups and genes correlated to recurrence free interval (RFI) in colon cancer
After data quality control and reference normalization, approximately 19% of the cancer related genes tested in this study were found to have a significant (p < 0.05) correlation with Recurrence Free Interval (RFI) by univariate regression analysis . Approximately one quarter of these genes would be expected to be false positives . While a large number of candidate genes showed significant correlations with clinical outcome (on both the raw and normalized CT measurements), the average reference signal was shown not to be correlated with disease recurrence.
The development of a clinically validated test that could determine the risk of recurrence or death from stage II/III colon cancer and the likelihood of benefit from standard chemotherapy regimens is highly desirable but complex. The process begins with biomarker discovery and ends with a clinical validation study with prospectively defined endpoints. Since the method by which an mRNA species is measured will have a profound effect on the success of such a validation study, it is important to characterize and maintain the assay's performance, particularly its reproducibility and quantitative precision. Consequently, we have adopted RT-PCR, the most robust gene expression method available for gene discovery studies. Although nearly 150 genes were found to be significantly related to RFI in this study, some markers will prove to be false positives and true markers will vary in how robustly they correlate with outcome. It is therefore important to evaluate the candidate genes identified here by conducting further independent studies to identify truly useful disease biomarkers. Only after consistent association with clinical outcomes in multiple independent studies should genes be considered for inclusion in an assay used to make clinical decisions. Employing a single technology consistently throughout biomarker discovery and into clinical testing has the advantage of reducing the time required to fully validate and commercialize a multi-gene clinical decision-making tool.
RT-PCR is often carried out using oligo-dT priming to generically reverse transcribe mRNA from the polyA tail. However, this technique is unsuccessful with degraded RNA, such as that extracted from FPE tissue. We have previously shown that RT-PCR using gene-specific priming can be successfully applied to FPE tissue as old as 30 years . As described here, we have now increased the scale of screening using this technique, to 761 genes. We further demonstrate that gene specific priming for 761 assays can be successfully combined into a single RT reaction that results in precise, sensitive and reproducible quantitative PCR for biomarker discovery.
DNA microarrays are a popular technology for biomarker discovery because one can quickly examine the expression of hundreds or thousands of genes. RT-PCR has often been subsequently used to verify the results of microarray data, since it offers much higher sensitivity, specificity, reproducibility and a greater quantitative dynamic range. The present results demonstrate that RT-PCR can also be applied to highly parallel gene expression analysis if robotic processes and assay miniaturization are used. We were able to extract and quantify RNA and generate expression data for 761 unique assays for more than 300 patients in less than 5 weeks.
While screening these patient specimens, it was important to monitor potential sources of variability such as primer and probe stability and gene specific primer pool stability. We used an FPE colon RNA pool as a reference sample and generated a baseline CT value for each of the 761 assays. This FPE colon RNA pool reference sample was then included during reverse transcription with every patient sample batch and used to monitor process stability throughout the study. Over the 5 week period, variability within the reference sample remained low, indicating that all patient samples were being analyzed with a stable assay process. Analysis of one of the reference genes, RPLPO, which was assayed on both plates for every patient sample, also highlighted the internal consistency of the process throughout the study.
The robustness of this technology is evidenced by results from hierarchical clustering of all 761 genes which identified known pathways and gene group clusters that one would expect to be co-expressed. One of the largest was a "stromal response" gene group containing genes that are associated with wound healing and are thought to be representative of fibroblast activation, or the 'stromal response' within tumor stroma. Stromal response is becoming increasingly recognized as a marker of invasion and poor clinical outcome in several different classes of solid tumors [16, 17, 18, 19, 20]. A number of genes within this group encode proteins that compose or regulate extracellular matrix including BGN, SPARC, CTGF, THBS, VIM, and COL1A1. Several focal adhesion and actin-binding protein genes grouped together to form another distinct cluster, including CALD1, TAGLN, TLN, MYH11, MYLK and CNN. Because MYH11, MYLK and CNN are genes specific to muscle, they could represent a myofibroblast (MF) signature. The myofibroblast cell type has been implicated as a driver of tumor progression . Another cluster might be called an "epithelial/secreted" gene group; within this group are genes known to be markers of epithelial cells or code products secreted by them, such as SERPINB5 (maspin), KRP19, KLK10 and LAMB2. A cluster containing GBP1, GBP2, G1P2, IFIT, CD8A, CD8A, HLA-DRPB1 and CXCR4 represents an immune/interferon-inducible group. Another group contains genes that represent acute response to stimuli, or "early response" genes such as EGR1, EGR3, RhoB, FOS and NR4A. An inflammatory response gene group was also identified, containing genes such as ICAM1, IL1B, IL-8, IL6, OSM and S100A8. A small group of intestinal specific genes such as MUC2, MUC5B, pS2 (TFF1) and TFF3 are also highly correlated in expression. Lastly, it was gratifying to see that the expression of CDH1 and CAPN1 were correlated with one another (Pearson's correlation = 0.48) since CAPN1 cleaves CDH1 . Given the biological connection between these genes, one may have expected this correlation to be higher – possibly indicating specific post-translational control mechanisms may have a role in defining steady state protein levels.
The aim of this study was to identify gene biomarkers that predict recurrence-free interval in patients with Stage II and Stage III colon cancer. Approximately 19% of the 761 genes showed a significant (p < 0.05) association with RFI by univariate Cox proportional hazards regression analysis. It is highly unlikely that any one gene will be able to predict clinical outcome or response to therapy to the extent that it will be useful to oncologists. A successful diagnostic tool is much more likely to consist of a panel of genes and an algorithm weighting and combining each gene contribution into one value that defines the unique risk of recurrence and potential for therapeutic response in each patient. This concept is supported by the observation that several different biological pathways were shown to be associated with RFI in this study.
We have demonstrated that RT-PCR can be scaled to enable studies testing hundreds of candidate genes for biomarker discovery in hundreds of archival FPE cancer biopsy specimens. Analysis of the data from this study has shown it to be biologically plausible and consistent with known pathways and gene groups identified to be important in cancer. We are applying this technology to colon cancer with the aim of developing a predictive and prognostic clinical test for patients with this disease.
Archival colon tumor FPE tissue blocks were provided by the NSABP from both the C01 "A Clinical Trial To Evaluate Postoperative Immunotherapy And Postoperative Systemic Chemotherapy In The Management Of Resectable Colon Cancer" and C-02 "A Protocol To Evaluate The Postoperative Portal Vein Infusion Of 5-Fluorouracil And Heparin In Adenocarcinoma Of The Colon" clinical trials. Patients were enrolled in these trials between 1977–1983. Samples used in this study are representative of the general study populations for both trials.
Tissue sectioning and macrodissection
Histotechnologists wore gloves at all times when handling tissue blocks. Before and after each block was sectioned, any debris was removed from the microtome with a disposable cotton swab or brush. All appliances (brush, forceps, knife, knife holder base) were wiped with an RNase Zap wipe followed by a soft cloth wetted with de-ionized water. A tissue floatation bath was used to help eliminate wrinkles and distortions in sections being mounted to glass microscope slides. When dissection was required to remove significant non-tumor elements, a representative H&E stained slide was used as a guide to mark tumor and non-tumor portions of three 10 micron unstained slides. Tumor tissue was then scraped away from the non tumor material and placed into an extraction tube. The tumor tissue from all 3 sections was placed into the same tube.
RNA was extracted from the tumor-enriched portion of three 10 micron sections per patient block. In order to scale sample throughput to 48 samples/batch, the RNA extraction procedure used a semi-automated method performed on a TECAN robotic liquid handler (TECAN Schweiz AG, Männedorf, Switzerland). The samples originated in individual 1.5 ml Eppendorf tubes and paraffin was removed by incubating with lab grade xylene for 5 minutes. Tissue was then pelleted by centrifugation at room temperature (+18°C to +25°C) for 5 minutes at approximately 14,000 RPM. The xylene was removed and the procedure repeated. The tissue pellet was washed by inverting several times with 200 proof ethyl alcohol and again pelleting by centrifugation at room temperature. The ethyl alcohol wash step was repeated. Immediately prior to adding Proteinase K, samples were inspected for residual alcohol; if any alcohol was visible, it was aspirated without disturbing the tissue pellet. Proteinase K digestion was performed using reagents from the MasterPure® Purification kit (Epicentre, Madison, WI). Samples were incubated at +65°C for 2 hr with Proteinase K. Protein and genomic DNA were removed by manual addition of an equal volume of acid-phenol: chloroform and the removal of the upper aqueous phase after centrifugation for 5 minutes at approximately 10,000 RPM. Samples tubes were transferred to a TECAN liquid handler where purification was completed using the mirVana™ RNA purification kit (Ambion, Austin, Texas) on a 96 well glass fiber filter plate. Purification was followed by DNase I treatment on the same 96 well filter plate.
RNA was quantified using the RiboGreen fluorescence method as described by the kit manufacturer (Invitrogen, Carlsbad, CA.).
Candidate gene selection
Candidate genes were obtained from published gene expression profiling data relating to colon cancer prognosis and response to therapy, biological pathways known to be important in cancer [23, 24, 25, 26, 27, 28] and suggestions by our collaborators at the NSABP. The full gene list, accession numbers and associated primer and probe oligo sequences are provided [see Additional file 3].
TaqMan® primer and probe design
The reference sequence for each gene included in the study was obtained from the NCBI Entrez website. TaqMan® RT-PCR primers and probes were designed using an automated in-house primer design module. The complete list of assay primers and probes is shown in Additional file 3. Oligonucleotides were purchased from Biosearch Technologies Inc. (Novato, CA), Integrated DNA Technologies (Coralville, IA) and Eurogentec (San Diego, CA). Dual labeled TaqMan® probes had 5'FAM as a reporter and either 3'BHQ-1 or 3'BHQ-2 as a quencher. Amplicon size was limited to a maximum of 90 bp.
Reverse transcription was performed using the Omniscript kit (Valencia, CA) for RT-PCR. For initial investigations, reverse primers (each at 100 μM) were collected into sub-pools of 94–96 primers. An aliquot from each sub-pool was added together to create the final GSP pool (each primer at 100 nmol/L). For the clinical study, reverse primers (each at 1 mM) were first collected in sub-pools of 89–96 primers and tested for priming performance before being combined into a master 761 gene-specific primer pool (each primer at 1 μmol/L). For both the initial investigations and the clinical study, each primer in the RT reaction was at a concentration of 50 nmol. Our standard procedure was to add RNA to the RT reaction at 12.5 ng/ul which equates to 1 ng/well (cDNA) for quantitative PCR. In each case, the RT reaction was performed in a single tube with the GSP pool (ie up to 761 reverse primers). The resulting cDNA was distributed equally among the wells of a 384 well plate, and the appropriate forward and reverse primer and probe were added to each assay well.
TaqMan® gene expression analysis
For each patient sample, two 384-well plates were used. Assays for 7 potential reference genes were included on both plates, with all other gene assays randomly distributed in single assay wells. RT-PCR assays for three K-ras gene mutations and one BRAF gene mutation were included in the assay panel along with each corresponding wild type allele assay. Therefore, 757 normal gene alleles were assayed and 4 mutant genes were assayed, bringing the total number of unique assays to 761. TaqMan® RT-PCR was performed according to instructions of the manufacturer, using Applied Biosystems Prism (ABI) 7900 HT instruments. Reactions were performed in a 5 μl volume with cDNA equivalent to 1 ng total RNA. Final primer and probe concentrations were 0.9 μmol/L (primers) and 0.2 μmol/L (probe). For the K-ras mutation assays a blocker oligomer was added to the primer and probe pool at a final concentration of 3.6 μmol/L (mutant 1) 3.6 μmol/L (mutant 2) and 12.95 μmol/L (mutant 3). These blockers are added to inhibit amplification of the non-mutant allele, permitting specific amplification of the mutant allele. PCR cycling conditions were 95°C for 10 minutes for one cycle, 95°C for 20 seconds, and 60°C for 45 seconds for 40 cycles. A reference sample (pooled colon FPET RNA) was assayed throughout the study to ensure reagent and process stability. As a negative control, wells without any template were also assayed every two weeks to ensure that no exogenous nucleic acid contaminations occurred.
Unsupervised hierarchical clustering
The unsupervised hierarchical clustering of genes was performed using 1-Pearson R as the distance measure for gene expression and the un-weighted pair-group average as the amalgamation method .
We are grateful to the NSABP for providing the FPE tissue samples. We kindly thank Andrew Dei Rossi, Debjani Dutta, Mylan Pho and John Morlan for their assistance with preparation of reagents and samples; Mei-Lan Liu for her helpful advice and suggestions; Robyn Loverro and Kenneth Hoyt for the development of robotics systems, Joel Robertson for IT support; Xitong Li for the primer design module; Jeanne Yue for her assistance in generating the figures and Melanie Finnigan, William Hiller, and Teresa Oeller from Division of Pathology, NSABP for tissue sectioning.
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