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(American Journal of Pathology. 2004;164:35-42.)
© 2004 American Society for Investigative Pathology

Technical Advance

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

Development and Performance of a 92-Gene Reverse Transcriptase-Polymerase Chain Reaction Assay

Maureen Cronin^*, Mylan Pho^*, Debjani Dutta^*, James C. Stephans^*, Steven Shak^*, Michael C. Kiefer^*, Jose M. Esteban and Joffre B. Baker^*

From Genomic Health Incorporated,^* Redwood City; and Providence–St. Joseph Medical Center, Burbank, California

	Abstract

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Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-µm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

We therefore have sought to develop RT-PCR assays that measure FPE RNA and are optimized with respect to sensitivity, accuracy, reproducibility, and precision. The capability of gene expression analysis to provide diagnostic information of greatest utility hinges on measuring the contributions of multiple genes, often dozens or more.^11-17 Consequently, we have also sought to develop RT-PCR assays of FPE RNA that measure the expression of many genes at once from small amounts of archival tumor blocks. The present study describes results with 48- and 92-gene assays.

	Materials and Methods

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Tissue Specimens

Archival breast tumor FPE blocks and matching frozen tumor sections were provided by Providence–St. Joseph Medical Center, Burbank CA. Fixed tissues were incubated for 5 to 10 hours in 10% neutral-buffered formalin before being alcohol-dehydrated and embedded in paraffin.

RNA Extraction Procedure

RNA was extracted from three 10-µm FPE sections per each patient case. Paraffin was removed by xylene extraction followed by ethanol wash. RNA was isolated from sectioned tissue blocks using the MasterPure Purification kit (Epicenter, Madison, WI); a DNase I treatment step was included. RNA was extracted from frozen samples using Trizol reagent according to the supplier’s instructions (Invitrogen Life Technologies, Carlsbad, CA). Residual genomic DNA contamination was assayed by a TaqMan (Applied Biosystems, Foster City, CA) quantitative PCR assay (no RT control) for ß-actin DNA. Samples with measurable residual genomic DNA were resubjected to DNase I treatment, and assayed again for DNA contamination.

FPE Tissue RNA Analysis

RNA was quantitated using the RiboGreen fluorescence method (Molecular Probes, Eugene, OR), and RNA size was analyzed by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

TaqMan Primer and Probe Design

For each gene, we identified the appropriate mRNA reference sequence (REFSEQ) accession number and accessed the consensus sequence through the NCBI Entrez nucleotide database. RT-PCR primers and probes were designed using Primer Express (Applied Biosystems, Foster City, CA) and Primer3 programs.¹⁸ Oligonucleotides were supplied by Biosearch Technologies Inc. (Novato, CA) and Integrated DNA Technologies (Coralville, IA). Amplicon sizes were preferably limited to less than 100 bases in length (see Results). Fluorogenic probes were dual-labeled with 5'-FAM as a reporter and 3'-BHQ-1 as a quencher.

Reverse Transcription

Reverse transcription (RT) was performed using a SuperScript First-Strand Synthesis kit for RT-PCR (Invitrogen Corp., Carlsbad, CA). Total FPE RNA and pooled gene-specific primers were present at 10 to 50 ng/µl and 100 nmol/L (each), respectively.

TaqMan Gene Expression Profiling

TaqMan reactions were performed in 384-well plates according to instructions of the manufacturer, using Applied Biosystems Prism 7900HT TaqMan instruments. Expression of each gene was measured either in duplicate 5-µl reactions using cDNA synthesized from 1 ng of total RNA per reaction well, or in single reactions using cDNA synthesized from 2 ng of total RNA, as indicated. Final primer and probe concentrations were 0.9 µmol/L (each primer) and 0.2 µmol/L, respectively. PCR cycling was performed as follows: 95°C for 10 minutes for one cycle, 95°C for 20 seconds, and 60°C for 45 seconds for 40 cycles. To verify that the RT-PCR signals derived from RNA rather than genomic DNA, for each gene tested a control identical to the test assay but omitting the RT reaction (no RT control) was included. The threshold cycle for a given amplification curve during RT-PCR occurs at the point the fluorescent signal from probe cleavage grows beyond a specified fluorescence threshold setting. Test samples with greater initial template exceed the threshold value at earlier amplification cycle numbers than those with lower initial template quantities.

Normalization and Data Analysis

To compare expression profiles between specimens, normalization based on six reference genes was used to correct for differences arising from variability in RNA quality and total quantity of RNA in each assay. A reference C_T (threshold cycle) for each tested specimen was defined as the average measured C_T of the six reference genes. In an approach similar to what has been described by others, six reference genes were selected for use from among 10 candidate reference genes tested in this assay.¹⁹ Reference gene candidates were selected from among those well-known in the literature as commonly constitutively expressed genes across a wide range of tissues and biological conditions. The six genes selected for the final analysis showed the lowest levels of expression variability among the patient specimens tested. An average of six reference genes was used to minimize the risk of normalization bias that can result from variation in expression of any single reference gene.²⁰ Relative mRNA level of a test gene within a tissue specimen was defined as 2^CT+10.0, where C_T = C_T (test gene) - C_T (mean of six reference genes). In the figures, unless indicated otherwise, normalized expression is represented on a scale in which the average expression of the six reference genes is 10, corresponding to a mean C_T of 30.7.

Statistical Analysis

Correlation of gene expression analyses was done using Pearson linear correlation. Cluster analysis was done using 1-Pearson R as the distance metric and single linkage hierarchical clustering.

	Results

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FPE Tissue RNA Fragmentation Increases with Archive Storage Time

Capillary electrophoresis analysis of RNA extracted from archival FPE breast cancer specimens shows that the RNA exists primarily as fragments of less than 300 bases in length. This is consistent with findings of others.^6,21 Figure 1 presents RNA-sizing results from specimens archived for substantially different durations. As shown, breast cancer tissue RNA archived for 1 year had larger average molecular weight than RNA archived for 6 or 17 years (note detectable 18S RNA at 2000 bases in the 1-year-old specimens). All of these specimens came from one source (Providence Hospital, Burbank, CA) and throughout this 17-year period all specimens were fixed using the same formalin fixation protocol (see Materials and Methods for details). This therefore suggests that fragmentation of FPE tissue continues to occur after specimens are dehydrated and embedded in wax.

View larger version (79K): [in this window] [in a new window]   Figure 1. Size distribution of FPE tissue RNA from 12 tumor specimens. Total RNA was extracted from breast cancer specimens as described in Materials and Methods. One µl from each RNA extract (1/30 of the sample) was analyzed using an Agilent 2100 Bioanalyzer, RNA 6000 Nanochip. Lanes 1–4, 5–8, and 9–12 contain RNA from samples archived 1, 6, and 17 years, respectively. Lanes M1 and M2 contain two different sets of molecular weight marker RNA (sizes denoted in bases).

Expression of 92 different genes was profiled (single well per gene) across 62 different FPE breast cancer specimens that had been archived from 1 to 17 years. All specimens yielded an adequate quantity of RNA for analysis. The mean and median raw C_T for all patients and genes was 33.2 and 32.5, respectively. Raw C_T values ranged from 24 to 40 (the latter being the default upper limit PCR cycle number that defines failure to detect a signal as set by the manufacturer).

To be able to compare RT-PCR data from different tissue specimens, it is necessary to correct for relative differences in input RNA quantity and quality. These differences arise primarily from the variability inherent in processing surgical tissue specimens, including relative mass of tissue and the time between surgery and formalin fixation. A secondary consideration is the cumulative variability accrued while processing each sample from RNA extraction through quantitation, reverse transcription to cDNA, and PCR. This correction is routinely accomplished by normalizing raw expression values relative to a set of genes that vary little in their median expression among different tissue specimens (reference genes). Our observation that RNA continues to degrade with increased archive storage (Figure 1 , above) raised the question whether RT-PCR signals tend to decay with increased archive storage, and if so, whether normalization to reference genes could compensate for this trend. Figure 2 shows the mean expression (±SD) relative to the six reference genes for all 92 genes.

View larger version (32K): [in this window] [in a new window]   Figure 2. Expression ranges for 92 genes in 62 breast cancer specimens. TaqMan RT-PCR was used to measure mRNA levels as described in Materials and Methods, and expression relative to six reference genes. The mean and mean SD of the expression values across all tested patients is shown for each gene. Each box represents the mean mRNA level for all tested tumor specimens and the error bars indicate the SD of all measurements for that gene. Expression values (y axis) are normalized relative to the reference genes expressed as log base 2 values.

View larger version (19K): [in this window] [in a new window]   Figure 3. A and B: Mean CT values for 92 genes in 62 patient samples as a function of paraffin block archive storage time. The x axis shows the year each specimen was archived. The y axis shows mean expression values for all tested genes. Each symbol represents a separate patient. A: Raw mean CT expression values for all specimens. B: Expression values after normalization relative to six reference genes, as described in Materials and Methods. Reference genes were ß-ACTIN, CYP1, GUS, RPLPO, TBP, and TFRC. Lines: Linear regression best fit.

The amplicon size was greater than 90 bases for 10 of the 12 genes. Therefore, when possible, we redesigned probe and primer sets to fit within the relatively narrow range of 70 to 85 bases. We found that with the redesigned probe and primer sets normalization corrected for the archive storage-related bias. Thus, optimally, amplicon sizes not only must be limited in length but also the lengths of test gene and reference gene amplicons must be effectively homogeneous.

Results of a 92-Gene Assay: Clusters of Co-Expressed Genes

Subsets of the 92 tested genes would be expected to co-express based on results published by others. The presence or absence of these expected clusters of co-expressed genes can serve as one check of the expression data validity. The DNA array-based gene expression profiles published by Sorlie and colleagues¹¹ identified four clusters of co-expressed genes in human breast cancer, likely representing different cell type subclasses. We included 11 genes from among these four cluster groups in our 92-gene panel. Table 1 shows that our assay clearly identifies the same gene clusters as Sorlie and colleagues.¹¹ For example, the correlation coefficient for HER2 and GRB7 expression using our assay was 0.71. The HER2 gene is often amplified in breast cancer and the GRB7 gene lies nearby at this locus.²² For none of the other genes represented in Table 1 was there a strong indication of co-expression with these two genes. Similarly, the basal epithelial gene cluster represented by GRO1, KRT5, and KRT17 all had pair-wise correlation coefficients for their expression values between 0.6 and 0.8.

To validate the performance of our quantitative RT-PCR assay at measuring gene expression in FPE tissues, we compared the measured mRNA levels of the diagnostic marker genes estrogen receptor (ER), progesterone receptor (PR), and HER2, to their respective protein levels as determined by standard diagnostic assays based on IHC, for all 62 primary breast cancer FPE specimens. In addition, 17 specimens that were analyzed by RT-PCR and IHC for HER2 were also analyzed for HER2 gene amplification by fluorescent in situ hybridization (FISH). IHC and FISH were performed by an independent clinical diagnostics laboratory.

Figure 4, A to C , shows that a significant correlation exists between mRNA levels and (IHC-based) protein levels for each of these genes. For ER (Figure 4A) and PR (Figure 4B) , the concordance between RT-PCR and IHC was further compared by defining a cut point to differentiate RT-PCR-positive and -negative receptor status. The derived interassay concordance values for ER and PR were 94% and 84%, respectively (Table 2) .

View larger version (15K): [in this window] [in a new window]   Figure 4. A–C: RT-PCR compared with IHC and FISH assays for ER, PR, and HER2 in 62 FPE breast tumors. The RT-PCR expression data from the 92-gene assay of 62 patient specimens are normalized relative to the reference genes and presented on a log base 2 scale. IHC and FISH assays were performed by an independent diagnostics reference laboratory. The RT-PCR and IHC/FISHlaboratories were each blinded with respect to the data generated by the other laboratory at the time the analyses were performed. In each panel, normalized expression values are on the y axis and the IHC or FISH scores are on the x axis. A: Normalized ER expression compared with percentage of cells scored as ER+ by IHC. Open circles are ER- by IHC and filled circles are IHC ER+. Expression scores above 28.0 are ER+ and below are ER-. B: Normalized PR expression compared with percentage of cells scored as PR+ by IHC. Open circles are PR- by IHC and filled circles are IHC PR+. Expression scores above 27.2 are PR+ and below are PR-. C: Normalized HER2 expression compared with IHC scoring (0 to +3 scale). Open circles are HER2-negative and filled circles are HER2-positive. Expression scores above 211.5 are HER2+ and below are HER2-. FISH scores are noted for each case tested. NS, FISH was not scored.

Gene Expression Profiles Compared between FPE and Frozen Tissue RNAs

RT-PCR is often used as a standard against which to test other gene expression measurement methods, for example DNA array methods.^25,26 Similarly, we sought to compare RT-PCR-based gene expression profiles from FPE tissue RNA with those from unfixed tissue RNA. For this purpose we identified FPE and frozen samples prepared from the same breast tumor in 1995. As shown in Figure 5 , the RNA from the frozen tissue remained relatively intact, as indicated by detectable 28S and 18S ribosomal RNA bands. In contrast, much of the RNA from the FPE tissue was smaller than 200 bases in length. Nevertheless, we note that in our experience frozen tissues often yield RNA that is similarly fragmented. The specimens chosen here were deliberately selected for their well-preserved RNA. We have explored in some detail the effect of tissue processing on gene expression profiles using fresh placenta tissue as a model. RNA isolated from placenta tissue transferred shortly after delivery into RNAlater (Ambion, Inc., Austin, TX) preservative was compared with frozen tissue and with tissue fixed using a variety of fixing times and conditions. Expression profiles were found to be highly concordant among tissues processed by all of these methods (manuscript in preparation).

View larger version (36K): [in this window] [in a new window]   Figure 5. RNA size analysis of paired frozen and FPE tissue RNAs. Total RNA was extracted from frozen and FPE tissue specimens as described in Materials and Methods then analyzed for size profile using an Agilent 2100 Bioanalyzer, RNA 6000 Nanochip. One µl from each RNA preparation (1/30 of the sample) was analyzed. Lanes 2 and 3 contain frozen and FPE RNA, respectively, from the same breast tumor (from a 1995 surgery). Lanes M1 and M2 contain different sets of molecular weight RNAs (band sizes denoted in bases).

View larger version (23K): [in this window] [in a new window]   Figure 6. A–B: Comparison of RT-PCR expression profiles of 48 genes from paired frozen and FPE tissue RNAs. A: RNA was extracted from the frozen and FPET specimens and mRNA levels were determined by TaqMan quantitative RT-PCR as described in Materials and Methods. Results are plotted in a bar graph. Each bar represents normalized expression relative to the reference genes, and the mean of three measurements. B: Pearson correlation for all 48 genes between the two tissue preparation methods is 0.91.

	Discussion

Top Abstract Materials and Methods Results Discussion References

In this study, two multianalyte assays one with 48 genes and the other with 92 genes, were designed and tested in different experiments with preserved human breast cancer tissue. In the first case, all of the genes profiled in FPE RNA yielded measurable values, and the overall profile was validated by its similarity to that generated with well-preserved RNA from matched fresh-frozen tissue. In the second experiment using a 92-gene assay only one of the tested genes failed to yield a signal.

Measured levels of ER, PR, and HER2 mRNAs were concordant with the levels of the respective proteins as measured by IHC at an independent clinical reference laboratory. Approximately 90% concordance was obtained when RT-PCR expression results for ER and PR were dichotomized into positive and negative values and compared to ER- and PR-positive and -negative assignments based on IHC. It is noteworthy, that similar levels of concordance were found in other comparative studies of IHC versus RT-PCR.^33,34 Interestingly, those studies differed from the present study in that the RT-PCR analyses of ER and PR were performed with RNA from unfixed breast tissue.

Although the number of different mRNA species measured per RT-PCR assay panel as described in the present study could be extended to hundreds of different mRNAs, it will likely remain impractical in the near future to develop RT-PCR assays for the tens of thousands of genes it is possible to measure with DNA array technology. On the other hand, little evidence exists to date that DNA arrays can effectively be applied to FPE tissue RNA analysis.³⁵ RT-PCR and DNA array technologies complement each other in this respect for clinical research. Data from array experiments on limited numbers of frozen tissue specimens generate candidate gene sets of tens to hundreds of genes^10-13 that can then be screened at high resolution in multiplexed RT-PCR experiments on relatively large numbers of available FPE tissue specimens.

IHC remains the standard gene expression assay that is widely used in diagnostic clinical applications despite its numerous weaknesses that include variation in sensitivity from field to field, dependence on fixation conditions, and lack of calibrated quantitation.³⁶ The advantages of RT-PCR with respect to reproducibility, quantitation, sensitivity, dynamic range, and multianalyte capability, make this a promising diagnostic technology for immediate future application.

	Footnotes

 
Address reprint requests to Joffre Baker, Genomic Health, Inc., 301Penobscot Dr., Redwood City, CA 94063. E-mail: joff{at}genomichealth.com

Accepted for publication October 2, 2003.

	References

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				L. Harris, H. Fritsche, R. Mennel, L. Norton, P. Ravdin, S. Taube, M. R. Somerfield, D. F. Hayes, and R. C. Bast Jr American Society of Clinical Oncology 2007 Update of Recommendations for the Use of Tumor Markers in Breast Cancer J. Clin. Oncol., November 20, 2007; 25(33): 5287 - 5312. [Abstract] [Full Text] [PDF]

				O. Loudig, E. Milova, M. Brandwein-Gensler, A. Massimi, T. J. Belbin, G. Childs, R. H. Singer, T. Rohan, and M. B. Prystowsky Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) Nucleic Acids Res., August 1, 2007; 35(15): e94 - e94. [Abstract] [Full Text] [PDF]

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				S. Paik Development and Clinical Utility of a 21-Gene Recurrence Score Prognostic Assay in Patients with Early Breast Cancer Treated with Tamoxifen Oncologist, June 1, 2007; 12(6): 631 - 635. [Abstract] [Full Text] [PDF]

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				S. Paik, G. Tang, S. Shak, C. Kim, J. Baker, W. Kim, M. Cronin, F. L. Baehner, D. Watson, J. Bryant, et al. Gene Expression and Benefit of Chemotherapy in Women With Node-Negative, Estrogen Receptor-Positive Breast Cancer J. Clin. Oncol., August 10, 2006; 24(23): 3726 - 3734. [Abstract] [Full Text] [PDF]

				S. Uccella, R. Cerutti, D. Vigetti, D. Furlan, R. Oldrini, I. Carnevali, G. Pelosi, S. La Rosa, A. Passi, and C. Capella Histidine Decarboxylase, DOPA Decarboxylase, and Vesicular Monoamine Transporter 2 Expression in Neuroendocrine Tumors: Immunohistochemical Study and Gene Expression Analysis J. Histochem. Cytochem., August 1, 2006; 54(8): 863 - 875. [Abstract] [Full Text] [PDF]

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				K. Hamatani, H. Eguchi, K. Takahashi, K. Koyama, M. Mukai, R. Ito, M. Taga, W. Yasui, and K. Nakachi Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens J. Histochem. Cytochem., July 1, 2006; 54(7): 773 - 780. [Abstract] [Full Text] [PDF]

				C. Delfour, P. Roger, C. Bret, M.-L. Berthe, P. Rochaix, N. Kalfa, P. Raynaud, F. Bibeau, T. Maudelonde, and N. Boulle RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells J. Mol. Diagn., May 1, 2006; 8(2): 157 - 169. [Abstract] [Full Text] [PDF]

				S. Paik Methods for Gene Expression Profiling in Clinical Trials of Adjuvant Breast Cancer Therapy Clin. Cancer Res., February 1, 2006; 12(3): 1019s - 1023s. [Abstract] [Full Text] [PDF]

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