Expression profiling with RNA from formalin-fixed, paraffin-embedded material
- Andrea Oberli†1,
- Vlad Popovici†2,
- Mauro Delorenzi2, 3,
- Anna Baltzer1,
- Janine Antonov1,
- Sybille Matthey1,
- Stefan Aebi1,
- Hans Jörg Altermatt4 and
- Rolf Jaggi1Email author
© Oberli et al; licensee BioMed Central Ltd. 2008
Received: 17 October 2007
Accepted: 19 April 2008
Published: 19 April 2008
Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.
We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.
We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.
Breast cancer has been widely studied in the past and molecular characterization has increased the understanding of biological pathways that are altered during neoplastic transformation of cells [1–4]. However, the findings based on molecular profiling have not yet altered diagnosis, and decisions about treatment still rely mostly on histopathological and immunohistochemical techniques which are at best semi-quantitative [5, 6]. Currently, many patients with primary, non-metastatic breast cancer with positive estrogen receptor (ER) status undergo several cycles of chemotherapy, although a substantial proportion of them does not benefit from it. Presently, no conventional parameters exist for many patients which allow to identify individuals who will benefit from chemotherapy. Personalized diagnosis on the basis of highly specific molecular analyses has the potential to improve the situation of many patients by optimizing medication, and at the same time, sparing others from unnecessary treatment regimens.
DNA chip studies are based on measuring gene expression for many genes in parallel [1, 4, 7, 8]. Most protocols for gene expression analysis on the basis of DNA chips are sensitive to RNA degradation and RNA must be isolated from freshly prepared or FF tumor material. As a consequence, material is fairly limited and often originates from convenience samples of heterogeneous patients. Many of these studies including meta-analyses have revealed genes and biological functions of their products which are relevant for classification and prognosis [9, 10]. However, many samples were derived from patients who did not participate in clinical studies and their treatment regimens were not standardized. Therefore, follow up data must still be interpreted with caution.
Obviously, procedures based on formalin-fixed, paraffin-embedded (FFPE) material would greatly facilitate and speed up research in this area as large amounts of highly valuable material and clinical data have already been collected. In many cases, FFPE blocks are still available and they could be used for a molecular analysis. Especially material from clinical trials would allow investigating distinct clinical questions with existing material rather than material from newly designed studies.
Many efforts are currently made to individualize diagnosis of breast cancer by including molecular parameters into diagnosis. Fresh frozen material would obviously be ideal for a molecular analysis by gene expression measurements but it may be difficult to implement novel procedures which complicate current workflows of daily routine. Procedures based on FFPE material would be more feasible as they do not interfere with current protocols and they do not affect routine diagnosis as material for molecular analysis could be collected after standard diagnosis has been terminated. Only relatively few molecular approaches have been described which are based on FFPE material. For example, Paik and co-workers have established a recurrence score (RS, Oncotype DX), it allows to quantify the likelihood of distant recurrence and to predict the magnitude of chemotherapy benefit [11, 12].
It is generally accepted that molecular profiles which reflect primarily biological characteristics of tumor cells, may complement clinical and histopathological diagnosis, resulting in a more detailed characterization of individual tumors, a pre-requisite for better treatment decisions. In this study we present the development of a novel procedure for RNA isolation from FFPE material and an optimized workflow for expression measurements by QPCR.
Human breast cancer samples
Human breast cancer specimens were divided into two aliquots, one of which was processed for histological diagnosis by fixation with formalin and embedding in paraffin. FFPE material was obtained from the Institute of Pathology (University of Bern) and the Pathology Länggasse, Bern. Tissue (3–5 mm thick slices of tumor) was fixed over night in buffered formalin and processed for paraffin embedding in a Tissue Processing Center TPC 15 (Medite Medizintechnik, Germany). The second aliquot was frozen on dry ice and stored at -80°C. Fresh frozen material was obtained from the Tumorbank Bern. Both, FF and FFPE samples were checked by hematoxylin and eosin staining and only samples with more than 50% tumor cells were used for this study. An informed consent to use the material for research was obtained from all the patients.
Intact RNA was isolated from four 25 μm thick kryo-sections of approximately 0.5 cm2. The tissue was homogenized in 420 μl lysis buffer (4 M guanidinium thiocyanate, 30 mM Tris pH 8.0, 1% Triton-X-100), 8.0,1 using a TissueLyser (Mixer Mill, Retsch GmbH, Haan, Germany) at 15 Hz for 3 min. Total RNA was bound to silica-based columns (Epoch Biolabs, Huston Texas), treated with DNase I (30 Kunitz units for 20 min. at room temperature; Qiagen, Hilden, Germany), washed once with lysis buffer (containing 30% ethanol) and once with 20 mM NaCl (containing 20% ethanol) and eluted in 50 μl 10 mM Tris pH 7.4, 0.1 mM EDTA and stored at -20°C. RNA quantity was measured on an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and quality assessed by capillary electrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) using Agilent RNA 6000 Series Nano kits.
cDNA synthesis and QPCR
QPCR assays. QPCR assays (Assays on Demand) were from Applied Biosystems (Palo Alto, CA). Reverse primers from each assay were used for the synthesis of gene-specific cDNAs. They were provided separately by Applied Biosystems. Three assays (IGBP5_short, IGBP5_medium, IGBP5_long) were designed manually.
Data processing and determination of breast cancer classification scores
where R represents the reference value and was taken as the mean of Ct' values of 5 selected reference genes (GAPDH, GUSB, RPLP0, TFRC, UBB, see Results section for details). The approach guarantees that all ΔCt values are positive and upper bounded by max_val (set to 33 for all the results reported here).
where the gene symbols stand for the corresponding ΔCt values.
The Total score, together with the group scores as computed above, are used in all subsequent discussions.
Isolation of RNA from FFPE material
Parameters affecting the RNA quality and QPCR
RNAs isolated from FFPE material according to our own protocol were also compared to RNA derived of kryo-preserved material of the same tumors in a different way. The arithmetic mean of the five reference genes (GAPDH, GUSB, RPLP0, TFRC and UBB) was used for normalizing expression values of all the genes in each RNA. Normalized expression values were compared between intact and FFPE-derived RNA for each gene and each tumor [see Additional File 1]. Good conservation of inter-tumor differences were observed between kryo-preserved and FFPE samples for most genes.
Clinical and molecular parameters of breast cancers. Clinical and molecular parameters are given for each breast cancer used in this study. Module scores for each tumor were calculated from the results based on intact RNA (FF material) and based on RNA isolated from FFPE material according to our own method. N.A., data not available.
Module Score (FF/FFPE)
The similarity between the results generated from intact and partially fragmented RNA was also assessed by calculating Pearson correlation coefficients between the scores of both RNAs. Correlation coefficients (and corresponding p-values and 95% confidence intervals) were 0.966 (p = 2.071*10-8, CI = 0.893; 0.989), 0.856 (p = 9.32*10-5, CI = 0.597; 0.954) and 0.833 (p = 2.177*10-4, CI = 0.541; 0.946) for ER, HER2 and Proliferation scores, respectively. The corresponding Spearman correlations were 0.938 (p < 2.2*10-16), 0.851 (p = 1.167*10-4) and 0.867 (p = 2.048*10-5), respectively.
RNA was quantified by dot blot hybridization , semi-quantitative PCR  and more recently, by QPCR [24, 18, 26, 13, 17, 33, 32] and other methods [28–30]. RNA derived of FFPE material is not only partially hydrolyzed but also chemically modified: formalin reacts with nucleotides leading to the formation of methylol groups in nucleobases. These groups tend to further react and form intra- and inter-molecular methylene bridges in RNA, DNA [34, 35, 31] and protein . As a result, reverse transcription is impaired and threshold cycle values (Ct values) increase during subsequent QPCR.
The protocol for RNA isolation described here was complemented by adding a separate demodification step which involves incubation at elevated temperature in a buffer containing ammonium chloride which favors the reversion of methylol groups to amino groups in nucleobases. It does not only improve the efficiency of downstream applications (mainly reverse transcription), it also improves the recovery of RNA from FFPE sections. RNA yield and quality can be further improved by extensive digestion of FFPE material with protease in a buffer containing guanidinium thiocyanate. Reverse transcription in the presence of gene-specific primers prevents the initiation of cDNA synthesis inside amplicons and therefore, cDNA made in the presence of gene-specific primers is a better template for QPCR than cDNA made from random primers (Fig. 2). Several papers have demonstrated that QPCR with primers coding for short amplicons are more efficient than primers coding for long amplicons [17, 20, 24, 13, 32].
Finally, normalization of raw data is used to eliminate or at least reduce the effect of poorer quality of starting RNA. Various approaches of normalization were proposed in the literature [37, 14, 38, 32]. They are based on calculating relative expression values: expression levels of genes of interest are expressed relative to the expression of one or a panel of several suitable reference genes. An ideal reference gene has a stable expression level in all the samples under investigation. As such "ideal" reference gene normally does not exist, the mean or median expression level of several suitably chosen reference genes is used as a relatively stable reference Ct value. We used a formalized approach to characterize all candidate reference genes. Candidate reference genes were ranked according to their standard deviations of raw Ct values in RNA from FF and FFPE material. The final rank of each candidate reference gene was taken as the mean of the two ranks obtained with RNA from intact and FFPE material. Genes with higher ranks were excluded as reference genes.
We also applied GeNorm  to characterize candidate reference genes: ACTB and RPS11 had poorest stability measure M  for FFPE-derived RNA and RPL7A had a poor stability measure when RNA from FF material was tested (data not shown). For these reasons GAPDH, GUSB, RPLP0, TFRC and UBB were used as reference genes in this study.
Our own RNA isolation protocol was compared to RNA that was isolated from the same material but according to commercial protocols and products (Qiagen RNeasy FFPE and ncLysis system of Applied Biosystems). Additional products for FFPE material from commercial providers (e.g. Stratagene, Ambion) were tested and the results obtained with our own protocol were superior to all tested commercial products (data not shown).
We determined module scores for each of the 14 tumors in this study. The limited number of samples does not allow statements about the clinical significance of module scores but they can be used to compare scores computed from intact RNA from FF material and RNA isolated from FFPE according to our own protocol. Pearson correlations between these RNAs in the 14 tumors were 0.966, 0.856 and 0.833 for ER, HER2 and Proliferation scores, respectively. As kryo-preserved RNA and RNA from FFPE material always originated from different portions of the same tumor, a certain variation of gene expression cannot be excluded and, as a consequence, part of the observed variability between kryo and FFPE material may be attributed to biological heterogeneity in the tumors. The three module scores were combined to a Total score. The Total score is similar to the recurrence score described by Paik , with high expression of genes related to proliferation and HER2 and low expression of ER-related genes indicating higher risk.
The data generated from FF and FFPE material were also compared to ER and HER2 levels assessed by IHC results from the same tumors. Three tumors (#15, #18, #20) were ER-negative and one was strongly HER2-positive (#6) (Tab. 2). The same tumors had low ER scores when assessed by QPCR (Fig. 8). Tumor #6 had a high HER2 score and an intermediate ER score. These results are in good agreement with the expected distribution of the three scores [15, 39]. By comparing QPCR based data with well known tumor subtypes allowed to validate the protocols developed here, even if no new biological findings are provided. The primary issue of this work was to document that stable and robust expression values can be determined from FFPE-derived RNA which are close to the values computed from intact RNA of the same tumors. The optimization and validation of the scoring procedure remains an important issue but obviously, the available number of samples is not sufficient to deal with this aspect and it will be addressed separately and on a larger collection of samples.
While IHC results are at most semi-quantitative, QPCR-based results reflect more accurately the expression level of genes in question. The module scores proposed here integrate quantitative gene expression data from several genes, this makes the resulting scores more robust than measurements based on single genes. QPCR is not only quantitative, it is also very sensitive over a large dynamic range. The number of genes which can be measured by QPCR is not limited and additional genes and module scores can be included in the analysis if this will be required.
Importantly, certain predictive parameters still cannot be determined with current technologies. For example, breast cancers are classified into histological grade 1, 2 or 3. This grading most likely reflects the proliferative state of tumor cells . Grading may be especially important as high grade tumors seem to respond more favorably to chemotherapy than low grade tumors. Unfortunately, many tumors are histological grade 2 and for those tumors the benefit is not clear. Paik and co-workers documented that their recurrence score (RS) was also predictive for a response to chemotherapy . The RS defined by Paik and coworkers is composed of 16 test genes mainly representing ER response genes, proliferation-associated genes, HER2-related genes and invasion genes and 5 genes for normalization [11, 41].
The results presented in this study reveal that RNA isolated from FFPE material according to the protocol developed in our laboratory can be used for expression measurements by QPCR although the RNA is partially degraded. The optimized isolation and de-modification procedures combined with a normalization procedure results in stable and robust gene expression data. Robustness of results was further increased by computing scores from several genes representing the hormonal and the proliferation status of the tumor. Molecular profiling from FFPE material may be of interest for routine diagnostics in the near future as FFPE material is always available . Similarly, molecular profiling from FFPE material may be of great interest in the context of existing and newly planed clinical trials for which only formalin-fixed samples exist.
fresh frozen tissue
formalin-fixed, paraffin-embedded tissue
quantitative polymerase chain reaction.
The authors thank Dr. R. Haener, University of Bern for helpful suggestions on formalin-induced modification of RNA, Drs. M. Müller, C. Meier and A. Günthert for providing tumors for the tumorbank, Drs. A. Fleischmann and H. Burger for providing FFPE tumor material, and Dr. M. Schobesberger for critical reading of the manuscript. This work was supported by the Swiss Cancer League (to RJ and MD), the NCCR "Molecular Oncology" (to MD), the Bernese Cancer League and Applied Biosystems, Rotkreuz Switzerland (to RJ). Kryo-preserved material was provided by the Tumorbank Bern. The Tumorbank Bern is sponsored by the Department of Clinical Research, the Institute of Pathology, University of Bern, and the Bernese Cancer League. Informed consent was provided from the patients for all the samples used in this study.
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