Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays

We recently developed a sensitive and flexible gene expression profiling system that is not dependent on an intact poly-A tail and showed that it could be used to analyze degraded RNA samples. We hypothesized that the DASL (cDNA-mediated annealing, selection, extension and ligation) assay might be suitable for the analysis of formalin-fixed, paraffin-embedded tissues, an important source of archival tissue material. We now show that, using the DASL assay system, highly reproducible tissue- and cancer-specific gene expression profiles can be obtained with as little as 50 ng of total RNA isolated from formalin-fixed tissues that had been stored from 1 to over 10 years. Further, tissue- and cancer-specific markers derived from previous genome-wide expression profiling studies of fresh-frozen samples were validated in the formalin-fixed samples. The DASL assay system should prove useful for high-throughput expression profiling of archived clinical samples.     

Flow cytometric analysis of ploidy in solid neoplasms: comparison of fresh tissues with formalin-fixed paraffin-embedded specimens

The DNA content of both fresh and formalin-fixed, paraffin-embedded tissues from 30 solid neoplasms was analyzed by flow cytometry. Cells from a single paraffin block of each tumor were disaggregated according to the method of Hedley et al. Cells were sampled from multiple sites of each fresh specimen. Fresh and deparaffinized cells were stored frozen and prepared for cytometric analysis by the technique of Vindelov et al. Thirteen fresh specimens showed DNA diploidy/tetraploidy and 17 showed DNA aneuploidy. Twenty-six of the 30 paraffin-embedded samples were concordant; aneuploid peaks present in histograms from fresh tissue specimens were absent in the 4 paraffin-embedded specimens that were discordant. For 2 of the discordant cases, a hyperdiploid peak that was absent in the initial paraffin-embedded samples was evident on analysis of cells from additional paraffin blocks, indicating ploidy heterogeneity. Peridiploid aneuploid peaks present in the other 2 fresh samples were not observed in initial or additional deparaffinized specimens. Although fresh tissues are preferred for ploidy examination by flow cytometry, formalin-fixed paraffin-embedded specimens, despite a few inherent pitfalls, are generally useful for retrospective studies that compare the ploidy status of solid neoplasms to therapeutic response and prognosis.     

Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman)

Real time quantitative RT-PCR (Taqman) identified specifically BVD virus in freshly processed formalin-fixed paraffin-embedded tissue sections and archival samples up to 7 years old. Samples included tissue from both acutely and persistently infected animals. To assess RNA degradation due to tissue handling and processing, freshly collected tissues from a calf persistently infected with BVD virus were stored at 4 degrees C or room temperature for up to 1 week prior to 24 h formalin fixation and routine histologic processing. Samples stored at 4 degrees C for up to a week prior to fixation were positive while samples stored at room temperature were positive at 74 h but became negative after 96 h. Fixation of fresh tissue in formalin for 1 week prior to processing resulted in a mild decrease in signal strength compared with tissue fixed for 24-48 h. Real time RT-PCR improves diagnosis of BVD infection by allowing prospective and retrospective identification of BVD virus in tissues processed routinely and stored for histologic evaluation.     

Validation of tissue microarray technology in breast carcinoma

The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.     

A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples

Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer-ase chain reaction (PCR) amplification from formalin-fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln-flxed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non-isotopic method, temperature gradient gel elec-trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.     

Detection of human papillomavirus type 16 DNA sequences in archival cervical tissues by the polymerase chain reaction.

We have evaluated the polymerase chain reaction for the detection of viral DNA sequences in paraffin-embedded archival tissues. In 63 frozen cervical biopsy specimens that were taken from premalignant and invasive lesions, Southern blotting detected human papillomavirus (HPV) type 16 DNA in 28 (44%) of the samples. In the polymerase chain reaction analysis of the formalin-fixed, paraffin-embedded mirror biopsy specimens, 46 (73%) of the tissues were found to be positive for HPV type 16. In three Southern blotting-positive cases, the DNA of the paraffin-embedded sections was too scant or too degraded to allow the detection of HPV DNA by the polymerase chain reaction. In 21 Southern blotting-negative cases, HPV type 16 DNA could be demonstrated in the archival sections by the polymerase chain reaction technique--a sensitivity improvement of more than 80% over the standard method of HPV detection in tissues.     

Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study

There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin-fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin-fixed and paraffin-embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin-fixed and paraffin-embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at -80 degrees C for several years. Formalin-fixed, paraffin-embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin-fixed tissue. Genomiphi and TaKaRa Ex Taq methods were also assayed in paraffin blocks and in formalin-fixed samples with limited success.     

Efficient Genetic Analysis of Microdissected Samples by Agarose-Bead Method: Alterations of β-Catenin Gene in Fundic Gland Polyp and Heterotopic Gastric Mucosa of Duodenum

Molecular genetic analyses of archival formalin-fixed, paraffin-embedded (FFPE) pathological specimens taken at biopsy or autopsy, are occasionally compromised because the DNA molecules therein are inevitably degraded. Furthermore, since these tissue samples comprise various cell types, the analyses based on mixtures of such heterogeneous populations often fail to reflect the nature of the affected cells. In the present study, to elucidate the contribution of β-catenin gene mutation to the fundic gland polyp and the heterotopic gastric mucosa in the duodenum, we successfully introduced an agarose-bead mediated technique as an effectual tool for retrospective morphology-oriented genetic analyses. Microdissected samples were embedded in low-melting agarose, and directly treated with proteinase K. A fragment of the agarose-bead was used as a template for polymerase chain reaction to analyze β-catenin mutation. Of the six cases of heterotopic gastric mucosa in the duodenum associated with fundic gland polyps, one showed a common 1-bp missense mutation at codon 37 shared by both the fundic gland polyp and the heterotopic gastric mucosa. Alternatively, a 1-bp silent mutation at codon 33 and missense mutation at codon 32 were identified only in the heterotopic gastric mucosa. Agarose-bead mediated technique shows superior sensitivity to the previously described techniques and is an effectual tool for retrospective morphology-oriented genetic analyses using a large number of archival pathological samples stored for long periods in the pathology laboratory.     

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.     

Genotyping of Helicobacter pylori strains in formalin-fixed or Formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens.

Different genotypes of Helicobacter pylori can play a role in the development of atrophic gastritis, peptic ulcer disease, and gastric carcinomas. In this study the authors developed polymerase chain reaction assays for the detection and identification of vacA (s- and m-regions), cagA, and iceA genotypes of H. pylori in formalin-fixed or formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens. Polymerase chain reaction products were analyzed by reverse hybridization on a line-probe assay. Complete genotyping was achieved in 26 of 28 samples (93%), and multiple genotypes, indicating the presence of multiple strains, were detected in nine samples (32%). This genotyping method offers the possibility for long-term retrospective studies on H. pylori genotypes and gastric pathology in the same archival gastric tissue specimens.     

SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples

Global genome amplification from formalin-fixed tissues is still problematic when performed with low cell numbers. Here, we tested a recently developed method for whole genome amplification termed "SCOMP" (single cell comparative genomic hybridization) on archival tissues of different ages. We show that the method is very well suited for formalin-fixed paraffin-embedded samples obtained by nuclei extraction or laser microdissection. The polymerase chain reaction (PCR) products can be used for subsequent comparative genomic hybridization, loss of heterozygosity studies, and DNA sequencing. To control for PCR-induced artifacts we amplified genomic DNA isolated from 20 nuclei of archival formalin-fixed, paraffin-embedded nonpathological lymph nodes. Subsequent comparative genomic hybridization revealed the expected balanced profiles. For loss of heterozygosity analysis by microsatellite PCR 60 to 160 cells were sufficient. In comparative experiments the approach turned out to be superior to published degenerated oligonucleotide-primed-PCR protocols. The method provides a robust and valuable tool to study very small cell samples, such as the genomes of dysplastic cells or the clonal evolution within heterogeneous tumors.     

Endosialin (CD248) is a marker of tumor-associated pericytes in high-grade glioma.

Gliomas are the most frequent primary tumors of the central nervous system in adults. The most prevalent and aggressive subclass of these is glioblastoma multiforme, which is characterized by massive neovascularization. Endosialin (CD248) has generated interest as a target for antiangiogenic therapy following reports that its expression is upregulated on angiogenic endothelial cells. We demonstrate here that endosialin is not expressed in normal human adult brain but is strongly upregulated in the angiogenic vasculature of all high-grade glioma specimens examined. However, by taking advantage of a technique which allows for multiple fluorescent labeling of formalin-fixed paraffin-embedded archival sections, we demonstrate unambiguously that endosialin is not expressed by the glioma endothelial cells but on closely associated perivascular cells. With increasing awareness that targeting pericytes is an attractive adjunct in antiangiogenic therapy, this finding has important implications for understanding the molecular mechanisms regulating angiogenesis in these highly vascularized tumors.