Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues

Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.     

Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)–embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT–embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis. (J Histochem Cytochem 57:849–860, 2009)     

Genome-Wide Massively Parallel Sequencing of Formaldehyde Fixed-Paraffin Embedded (FFPE) Tumor Tissues for Copy-Number- and Mutation-Analysis

Background

Cancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies.

Methodology/Principal Findings

Here we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples.

Conclusions/Significance

The application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.     

Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.     

Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.     

Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches

Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. Phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortunately, the development of molecular tests for clinical use is constrained by the limited availability of fresh frozen, clinically annotated samples. Here we report phosphopeptide analysis in human archival formalin-fixed, paraffin-embedded (FFPE) cancer samples based on immobilized metal affinity chromatography followed by liquid chromatography coupled with tandem mass spectrometry and selected reaction monitoring techniques. Our results indicate the equivalence of detectable phosphorylation rates in archival FFPE and fresh frozen tissues. Moreover, we demonstrate the applicability of targeted assays for phosphopeptide analysis in clinical archival FFPE samples, using an experimental workflow suitable for processing and analyzing large sample series. This work paves the way for the application of shotgun and targeted phosphoproteomics approaches in clinically relevant studies using archival clinical samples.     

A Method to Evaluate Genome-Wide Methylation in Archival Formalin-Fixed,Paraffin-Embedded Ovarian Epithelial Cells

Background

The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.

Methodology/Principal Findings

Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.

Conclusions/Significance

We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.     

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.     

Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10(-15)). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.     

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

RNA expression analysis from formalin fixed paraffin embedded tissues

Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.     

Transcriptional profiling of formalin fixed paraffin embedded tissue: pitfalls and recommendations for identifying biologically relevant changes

Expression profiling techniques have been used to study the biology of many types of cancer but have been limited to some extent by the requirement for collection of fresh tissue. In contrast, formalin fixed paraffin embedded (FFPE) samples are widely available and represent a vast resource of potential material. The techniques used to handle the degraded and modified RNA from these samples are relatively new and all the pitfalls and limitations of this material for whole genome expression profiling are not yet clarified. Here, we analyzed 70 FFPE tongue carcinoma samples and 17 controls using the whole genome DASL array covering nearly 21000 genes. We identified that sample age is related to quality of extracted RNA and that sample quality influences apparent expression levels in a non-random manner related to gene probe sequence, leading to spurious results. However, by removing sub-standard samples and analysing only those 28 cancers and 15 controls that had similar quality we were able to generate a list of 934 genes significantly altered in tongue cancer compared to control samples of tongue. This list contained previously identified changes and was enriched for genes involved in many cancer-related processes such as tissue remodelling, inflammation, differentiation and apoptosis. Four novel genes of potential importance in tongue cancer development and maintenance, SH3BGL2, SLC2A6, SLC16A3 and CXCL10, were independently confirmed, validating our data. Hence, gene expression profiling can be performed usefully on archival material if appropriate quality assurance steps are taken to ensure sample consistency and we present some recommendations for the use of FFPE material based on our findings.     

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.