From Formalin-fixed Paraffin-embedded Tissue Research Articles

A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

BackgroundRNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA.ResultsWe have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon.ConclusionsThe multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

Genotyping of DNA Samples Isolated from Formalin-Fixed Paraffin-Embedded Tissues Using Preamplification

DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is often fragmented and cross-linked and is therefore difficult to genotype. To enable this source of DNA for genotyping analysis using Taqman probes, we tested whether enrichment of the target genes would increase the amount of available DNA. For enrichment of the target genes, we used preamplification by means of diluted Taqman assays. To establish the appropriateness of preamplification, we used DNA extracted from paraffin-embedded tissue and compared the genotyping results of a series of single nucleotide polymorphisms assessed in DNA samples with and without preamplification. In a subset of patients, DNA was isolated from both blood and FFPE tissue to test the reliability of genotyping results derived after preamplification. We found an increase in call rate after preamplification and a convincing concordance in genotype. Based on our findings, we can safely conclude that preamplification of DNA isolated from paraffin-embedded tissue is a valuable and reliable method to optimize genotyping results.

A Molecular Profile of Focal Segmental Glomerulosclerosis from Formalin-Fixed, Paraffin-Embedded Tissue

Focal segmental glomerulosclerosis (FSGS) is a common form of idiopathic nephrotic syndrome defined by the characteristic lesions of focal glomerular sclerosis and foot process effacement; however, its etiology and pathogenesis are unknown. We used mRNA isolated from laser-captured glomeruli from archived formalin-fixed, paraffin-embedded renal biopsies, until recently considered an unsuitable source of mRNA for microarray analysis, to investigate the glomerular gene expression profiles of patients with primary classic FSGS, collapsing FSGS (COLL), minimal change disease (MCD), and normal controls (Normal). Amplified mRNA was hybridized to an Affymetrix Human X3P array. Unsupervised (unbiased) hierarchical clustering revealed two distinct clusters delineating FSGS and COLL from Normal and MCD. Class comparison analysis of FSGS + COLL combined versus Normal + MCD revealed 316 significantly differentially regulated genes (134 up-regulated, 182 down-regulated). Among the differentially regulated genes were those known to be part of the slit diaphragm junctional complex and those previously described in the dysregulated podocyte phenotype. Analysis based on Gene Ontology categories revealed overrepresented biological processes of development, differentiation and morphogenesis, cell motility and migration, cytoskeleton organization, and signal transduction. Transcription factors associated with developmental processes were heavily overrepresented, indicating the importance of reactivation of developmental programs in the pathogenesis of FSGS. Our findings reveal novel insights into the molecular pathogenesis of glomerular injury and structural degeneration in FSGS.

Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays

The current transition in cancer therapy from general treatment approaches, based mainly on chemotherapy and radiotherapy, to more directed approaches that aim to inhibit specific molecular targets has brought about new challenges for pathology. In the past, classical assignment of pathology consisted of tumor diagnosis and staging for further therapy decisions; nowadays, pathologists are asked to predict possible therapeutic results by detecting and quantifying therapeutic targets in tumors such as the human epidermal growth factor receptor 2 (HER2). The best approach to analyze such molecular targets is to provide a tumor-specific protein expression profile prior to therapy. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow fast, precise, cheap, and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling quantitative measurement of proteins. Recently, methods for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues have become available. In this article, we demonstrate how the use of RPPA to analyze signaling pathways from FFPE tissues may improve quantification of therapeutic targets and diagnostic markers in the near future.

Let-7 family miRNAs regulate estrogen receptor alpha signaling in estrogen receptor positive breast cancer

In order to understand how microRNAs (miRNAs) regulate breast cancer tumorigenesis, a miRNA expression microarray screening was performed using RNA from formalin-fixed paraffin-embedded (FFPE) breast tissues, which included benign (n = 13), ductal carcinoma in situ (DCIS) (n = 16), and invasive ductal carcinoma (IDC) (n = 15). Twenty-five differentially expressed miRNAs (P < 0.01) were identified, of which let-7 family miRNAs were down-regulated in human breast cancer tissues at stages of DCIS and IDC compared to benign stage. We further found that there was an inverse correlation between ER-α expression and several members of let-7 family in the FFPE tissues. Next, we performed bioinformatics analysis and found that let-7 miRNA sequences match sequence in the 3'-UTR of estrogen receptor alpha (ER-α), suggesting ER-α may be a target of let-7, which was further confirmed by a number of experimental assays, including luciferase assay, protein expression, and mRNA expression. Overexpression of let-7 miRNAs in ER-positive breast cancer MCF7 cell line negatively affected ER-α activity. As expected, dampening of the ER-α signaling by let-7 miRNAs inhibited cell proliferation, and subsequently triggered the cell apoptotic process in MCF7 cells. In conclusion, our findings indicate a new regulatory mechanism of let-7 miRNAs in ER-α mediated cellular malignant growth of breast cancer.

Abstract 3026: miR-203 and miR-205 expression profiles are predictive of high grade non-invasive urothelial carcinoma progression

Abstract Introduction: MicroRNA (miRNA) has been shown to play a role in regulating the expression of genes involved in tumor suppression and oncogenesis. High-grade, non-muscle-invasive urothelial carcinoma of the bladder (UCB) harbors a significant risk of progression to muscle invasion. In this study, we sought to identify miRNAs differentially expressed in non-progressive high-grade non-muscle-invasive UCB as compared to pathologically identical lesions that progress to muscle invasion. Methods: Total RNA was extracted from formalin fixed paraffin-embedded tissue (FFPE) of high-grade non-muscle-invasive (TaG3/T1G3) UCB lesions. Patients were categorized into two groups: non-progressors with at least 3 years follow-up or progressors advancing to ≥T2. Exclusion criteria were previous tumors of the upper tract, prior systemic chemotherapy or previous pelvic irradiation. Total RNA was pooled for microarray analysis from a subset of the non-progressor and progressor groups. Data was analyzed using the LOWESS normalization method. Expression profiles of miRNAs were verified with qRT-PCR on 89 high-grade non-muscle-invasive UCB (68 non-progressors and 21 progressors). Logistic regression analysis was performed to identify miRNAs predictive of tumor progression. Results: MicroRNA microarray data identified a panel of miRNAs (25) with potential involvement in the progression to invasion of high-grade non-muscle-invasive UCB. 36% of these miRNAs were upregulated and 64% were downregulated in the progressor samples. To validate differential expression of these miRNAs, qRT-PCR was performed on the 89 FFPE tissue samples. Logistic regression analysis of qRT-PCR data revealed decreased expression of miR-203 and miR-205 as predictive of progression (p = 0.031 and p = 0.045, respectively). Subsequent qRT-PCR with miR-203 and miR-205 on 31 muscle-invasive tumor samples (≥T2) showed expression profiles that were similar to those of the non-invasive progressors. Conclusions: In this study, we have shown that miR-203 and miR-205 can identify patients with non-muscle- invasive UCB at high risk to progress to muscle-invasive disease. Our findings also suggest that such tumors have a biomolecular profile similar to muscle-invasive lesions. Further validation studies are underway to investigate additional miRNAs and determine the mechanism by which these miRNAs promote tumor invasion and progression. These results provide an important tool that can be used to prognosticate the clinical behavior of a very challenging subset of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3026.

Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

Extraction of Proteins from Formalin‐Fixed, Paraffin‐Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis

This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue.

Detection of BRCA1 and BRCA2 Ashkenazi Jewish Founder Mutations in Formalin-Fixed Paraffin-Embedded Tissues Using Conventional PCR and Heteroduplex/Amplicon Size Differences

In many families with histories suggestive of BRCA1- or BRCA2-related disease, the proband is deceased. Reliable assessment of archived tissue blocks not amenable to full gene sequencing would be helpful. In this study, a polymerase chain reaction (PCR) assay using primers that bracket the BRCA mutation site and microfluidics-based detection of heteroduplex/amplicon size differences was developed to circumvent artifacts associated with low quality DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Genomic DNA was extracted from 100 FFPE specimens from patients that had previously undergone BRCA gene sequence analysis on blood specimens. Conventional PCR amplification products were differentiated using the Agilent 2100 Bioanalyzer. One FFPE specimen failed to amplify the wild-type alleles for all three sites and was therefore called indeterminate. All 62 FFPE specimens with known Ashkenazi Jewish founder mutations had both the wild-type and the correct mutated allele amplified, including one specimen that failed to amplify the mutant allele in other real-time PCR assays. Appropriately, 21 FFPE specimens known to have other BRCA1/2 mutations and 16 without any mutation had only the wild-type allele correctly amplified for each target. Therefore, by changing the primer location and detecting amplicons via heteroduplexes formed by size differences, we identified mutations from FFPE tissues missed using real-time methods.

Identification of the first case of germline duplication of BRCA1 exon 13 in an Italian family

In this work we report for the first time a family in Italy with the BRCA1 ins6kbEx13 mutation, a recurrent founder mutation originating from northern Britain. After the initial identification of exon 13 duplication by multiplex ligation-dependent probe amplification (MLPA assay), we confirmed the identity of the alteration with the previously published BRCA1 ins6kbEx13 mutation, by mutation specific PCR and RT-PCR assays and by haplotype analysis. As rarely reported previously, the MLPA assay was also used to examine DNA from formalin fixed paraffin embedded (FFPE) normal tissues of other affected subjects in the family and it was the only effective method to perform a complete segregation analysis of the BRCA1 ins6kbEx13 mutation in the family. A combination of different approaches including MLPA analysis, haplotype analysis and LOH study on tumor samples of all the affected members allowed to reassess the maternal transmission of the mutation expected by the pedigree analysis. Moreover, detailed morphological analysis of breast cancers of BRCA1 ins6kbEx13 mutation carriers demonstrated a rare histological variant of breast carcinomas that has never been described in patients carrying BRCA1 mutations. Our study confirms the MLPA technique as a reliable and effective method for a primary screening for BRCA1 rearrangements also by using FFPE tissues and strongly suggests that histo-pathological, immunonohistochemical and molecular information from FFPE tumor tissues should be more often considered and integrated into routine diagnostic practice of hereditary tumors.

Validation of an Array CGH Test for HER2 Status Using Tumor-Targeted DNA Extraction from Formalin Fixed Tissue: Comparison with Fresh Frozen Tissue Results.

Abstract Background: Recent clinical validation of a commercially available array CGH test (HerScan™) for determination of HER2 status in 97 cases of newly diagnosed breast cancer showed high accuracy and 97% concordance with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) results meeting the CAP/ASCO validation requirements for HER2 testing. However, the validation study was performed using fresh frozen tissue, a requirement which precludes the clinical use of array CGH in the majority of breast cancer workups. In this study, 55 additional cases with known HER2 status by IHC and FISH were analyzed by array CGH using a tumor-targeted DNA extraction method designed to achieve optimal results from formalin fixed paraffin embedded (FFPE) tissue.Methods: Tumor tissue from 55 cases of invasive ductal, invasive lobular, and one mucinous carcinoma was visualized and marked on H&amp;E stained sections cut from the FFPE block, with targeted areas captured by slide scraping or 4mm punch. Following proteinase K digestion, DNA was extracted from known HER2 positive (n=25), HER2 negative (n=25), and HER2 equivocal (n=5) samples. Verification of high molecular weight DNA was accomplished by agarose gel electropheresis followed by analysis of the tumor genomes using the HerScan test, a 1524 clone whole genome BAC microarray including 64 probes targeting the HER2/TOP2A amplicons on chromosome 17q.Results: Concordance equivalent to fresh frozen tissue results (&amp;gt;95%) was found between HER2 status obtained by IHC/FISH and array CGH on FFPE tissue. Five cases with equivocal results by IHC/FISH were resolved by array CGH and found to have unamplified partial chromosome 17 polysomy (n=4) and normal HER2 status (n=1). In addition, chromosome 17 status, overall genomic stability, and the presence of co-amplified genes including EGFR, MYC, MDM2, CCND1, and PDGFRalpha were clearly revealed by microarray evaluation in all cases.Conclusions: Array CGH analysis of DNA extracted from FFPE tissue provides a robust and accurate alternative for the clinical determination of HER2 status in newly diagnosed breast cancer. In addition, the complete genomic evaluation of core biopsy specimens afforded by array CGH allows clinicians to deliver personalized cancer care through incorporation of prognostic markers and potential therapeutic targets into treatment planning and prediction of disease outcome. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6038.

Amplification of mRNA from Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues without 3' Bias and Multiplex Expression Analysis on Flow-Through Microarrays.

Abstract Background: FFPE samples are an abundant sample resource for validation of expression-based assays and are will suited for routine clinical testing. Analysis on microarrays enables the analysis of many transcripts from small samples. However, mRNA in FFPE sections is degraded and cross-linked, with RNA fragment sizes of a few hundred bases or less, and consequently standard methods of microarray sample preparation are not reliable unless probes are very strongly biased to the 3' end. It is not always possible to design high quality probes within this restricted region of mRNAs. Amplification of target sequences any place within transcripts would permit greater flexibility in probe design and optimization of probe sets for reliable multiplex analysis.Material and Methods: Total RNA extracted from FFPE samples was subjected to two rounds of amplification with the ExpressArt TRinucleotide kit (AmpTec), which preferentially primes cDNA synthesis near the 3'-ends of mRNAs (rather than at poly-A sites) and selects for mRNAs against ribosomal RNAs. Biotin-labeled anti-sense RNA (aRNA) was prepared from the second round cDNA with the Illumina® TotalPrep™ RNA kit (Applied Biosystems). The labeled aRNA was hybridized to probes immobilized in singlet on flow-through microarrays on the Ziplex Automated Workstation (Xceed Molecular). Performance was assessed by the analysis of titration mixtures prepared from mRNA from breast cancer and colon cancer FFPE tissue blocks.Results: Median CVs of technical replicates were less than 25%. Linear responses were observed in the analysis of the titration mixtures, and all correlations between predicted and observed expression values of the titration mixtures had R2 values greater than 0.8. The data indicated that the minimum detectable expression difference was less than two-fold. Similar signal intensities were observed for 3'-biased probes and probes several hundred bases from the 3'end, confirming the lack of 3' bias in the amplification.Discussion: The results demonstrate the feasibility of amplifying and quantifying sequences at any position within transcripts in degraded mRNA from FFPE samples. Expression differences of less than two-fold may be analyzed with tens or hundreds of probes for translational research and clinical assay development on the Ziplex Workstation. Many probes may be tested in parallel to optimize probe sets for specific tests. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3052.

A High-Sensitivity, Medium-Density, and Target Amplification–Free Planar Waveguide Microarray System for Gene Expression Analysis of Formalin-Fixed and Paraffin-Embedded Tissue

Many microarray platforms and their associated assay chemistries do not work properly with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples, a feature that severely hampers the use of microarrays in oncology applications, for which FFPE tissue is the routine specimen. Furthermore, the limited sensitivity of most microarray platforms requires time-consuming and costly amplification reactions of the target RNA, which negatively affects clinical laboratory work flow. We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR). The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was <10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates. The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.

Expression profiling of formalin-fixed paraffin embedded primary human uveal melanomas using DASL matrices

Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue analyzed by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two uveal melanoma (UM) clinical-histopathological parameters (metastasis, cell type). A total of 43 FFPE UM were used. The expression of RPL13a, a ribosomal protein gene, for each sample was used to evaluate the quality of RNA extracted from FFPE tissue. Gene expression values generated from the array were analyzed using the GeneSpring GX software (Agilent). Immunohistochemistry was used in order to validate transcriptional findings at the protein level. A total of 106 genes were identified with (P < 0.05, Welch ANOVA test) a difference in transcript abundance for the metastasis clinical parameter. Furthermore, we identified 64 genes with a statistically significant (P < 0.05) difference in transcript abundance between the spindle and epithelioid cell types. Each individual sample for both groups (metastasis, cell type) exhibited distinct transcriptional profiles that were separated on a PCA. Positive nuclear immunostaining for LIG4-metastasis, ErbB3-cell type was found to be associated with better patient prognosis and outcome. To the best of our knowledge, this is the first time that a successful retrospective analysis has been done with UM FFPE RNA. This data may lead to future customized therapeutic targets, which may improve the now unchanged mortality rate of this particular malignancy.

MiRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue

Objective Micro RNA (miRNA) is a class of small noncoding RNA that plays a major role in the regulation of gene expression, which has been related to cancer behavior. The possibility of analyzing miRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. Our aim is to compare miRNA profiles from formalin-fixed paraffin embedded (FFPE) tissues with fresh-frozen prostate cancer tissues. Methods The expression of 14 miRNAs was determined by quantitative real time polymerase chain reaction (qRT-PCR) in 5 paired fresh-frozen and FFPE tissues, which were representative of prostate carcinoma. Results There was a very good correlation of the miRNA expression of miR-let7c and miR-32 between the fresh-frozen and FFPE tissues, with Pearson's correlation coefficients of 0.927 ( P = 0.023) and 0.960 ( P = 0.010), respectively. For the remaining miRNAs, the correlation was good with Spearman correlation coefficient of 0.638 ( P < 0.001). Conclusion Analysis of miRNAs from routinely processed and stored FFPE prostate tissue is feasible for some miRNAs using qRT-PCR. Further studies should be conducted to confirm the reliability of using stock tissues for miRNA expression determination.

2‐D PAGE and MS analysis of proteins from formalin‐fixed, paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues. However, 2-D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2-D PAGE separation and MS identification of full-length proteins extracted from FFPE skeletal muscle tissue. The 2-D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2-D maps of proteins from FFPE tissue following standard mass-compatible silver staining. Protein spots from both FFPE and frozen tissue 2-D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2-D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh-frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full-length proteins from FFPE tissues might be suitable to 2-D PAGE-MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological-fixed tissues.

Reliability and Reproducibility of a RNA Preamplification Method for Low-density Array Analysis From Formalin-fixed Paraffin-embedded Breast Cancer Samples

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.

Risk assessment of relapse in Swiss participants of BIG 1-98 with grade 2 early breast cancer by gene expression profiling using RNA from formalin-fixed, paraffin embedded (FFPE) tissue.

Abstract Abstract #1073 BIG 1-98 is a randomized controlled clinical trial for postmenopausal patients with early, estrogen and/or progesterone receptor (ER/PgR) positive breast cancer. It compares adjuvant therapy with 5 years of tamoxifen (T) or letrozole (L), two years of T followed by 3 years of L (T→L) or vice versa (L→T). L improved disease free survival (DFS) over T by 18% (hazard ratio 0.82, JCO 2007;25:486ff). Switzerland contributed 598/8010 patients. The patient and tumor characteristics of Swiss patients was similar to the entire BIG 1-98 population. The purpose of this study was to test a prognostic score in RNA derived from FFPE tissues.&amp;#x2028; Scores representing ER and progesterone receptor (PgR) function, proliferation (PRO14, AURKA/STK15, BIRC5/SURV, CCNB1, CCNB2, CCNE2, CDC2, ENPF, KIF20A, MKI67, MYBL2, ORC6L, PRC1, SPAG5, TOP2A), and HER-2 were built from multiple genes for each score. Genes were selected according to an analysis of publicly available cDNA array databases. In a prior set of 82 pairs of breast cancer samples the Pearson correlation coefficients of the scores between fresh frozen and FFPE tissues were at least 0.94. Cut-offs for ER and HER-2 scores were defined as the antimodes of fitted bimodal Gaussian distributions; the ER and HER-2 scores closely agreed with immunohistochemical (IHC) data (kappa(ER)=0.868, kappa(HER-2)=0.836).&amp;#x2028; FFPE tissues were available from 437 Swiss BIG 1-98 patients. RNA of sufficient quantity and quality was extracted from 388 blocks (89%). Forty-seven genes were quantified by QRTPCR on TaqMan Low Density Arrays (TLDAs) as reported previously (Proc AACR 2008, Abstract 974).&amp;#x2028; The PRO14 score was positively correlated with the histological grade. PRO14 scores greater than the median and PGR scores lower than the median were associated with shorter DFS. In patients with grade 2 tumors, PRO14 scores were significant predictors of DFS in univariate Cox regression analyses. Multivariate analyses will be presented. Our findings are in agreement with an analysis of fresh frozen tissues of Belgian participants in BIG 1-98 (European Breast Cancer Conference 2008, Abstract 481).We show that RNA can be extracted from FFPE in sufficient quantity and quality to be used in QRTPCR using TLDAs. This opens the possibility of using FFPE tumor banks from controlled clinical trials for the investigation of gene expression patterns. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1073.

Differential Wnt pathway activation in triple negative breast cancers comparative to HER2 and hormone positive breast cancers identified from formalin-fixed, paraffin-embedded tissues.

Abstract Abstract #43 Background: Patients afflicted with triple negative (TN) breast cancer (BC) are typically younger and have the worst prognosis of any BC subtype as a result of its aggressive and invasive phenotype. TN BC has been well studied with respect to the associative and prognostic values of its mRNA expression profiles. We have used the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) platform to characterize 502 genes from 96 BC patients sourced from formalin-fixed, paraffin-embedded (FFPE) tissues. RNA extracted from FFPE tissues is highly fragmented and typically unsuited for expression platforms, but recent advances have allowed for accurate expression profiling of RNA from FFPE tissues. This 96 patient cohort included 21 TN BC patients identified by immunohistochemistry (IHC); differential analysis of TN expression was integrated with known pathway mechanisms and identified activation of the Wnt pathway in TN tumors.&amp;#x2028; Material and Methods: The Illumina DASL assay was used to measure mRNA expression levels from BC patients obtained from St. Mary's Hospital, Montreal, Quebec. RNA was extracted from three 5µm tissue sections from FFPE blocks using the RNA High Pure Kit (Roche). Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR, q-value &amp;lt; 0.01) or a Bonferroni corrected two-tailed student's t-test (p-value &amp;lt; 0.05). IHC concordance was measured by mRNA expression fold-change for positive versus negative IHC categories.&amp;#x2028; Results: 165 samples from 96 patients were characterized for mRNA expression on 1,488 probes across 502 genes in the DASL assay. Quality control limited the analysis to 150 samples from 91 patients over 500 genes. Comparison of the TN subtype relative to HER2 positive and hormone positive breast carcinomas identified up-regulation [fold-change] of MMP7 [2.9], MMP9 [2.0], MMP1 [1.8], NOTCH1 [1.5], FZD7 [1.5], PPARD [1.3], CCND2 [1.2], MET [1.3], MYC [1.5], and MYCN [2.0] in the TN subtype. Most of these genes are either a component or downstream target of the Wnt signaling pathway. Down-regulated relevant genes included ESR1 [-8.4], PGR [-3.9], ERBB2 [-3.4] as expected, as well as ERRB3, ERBB4, GLI3, TFF1, LAF4/AFF3, and AR.&amp;#x2028; Discussion: Down-regulation of TFF1 suggests inhibition of the ERK/MAPK pathways which have complex effects on cell-cell adhesion. In combination with over-expression of MMPs 1, 7, and 9, these changes may contribute to the invasive nature of TN BCs. GLI3 down-regulation may attenuate Hedgehog signaling potentially contributing to Wnt activation, further leading to cell migration, invasion, and decrease in cell adhesion to cognate integrin-matrix interaction Up-regulation of both the FZD7 receptor as well as the Wnt targets MMP7, MYC, MYCN, PPARD, and CCND2 strongly suggest Wnt pathway activation in the TN subtype. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 43.

Molecular differences in triple negative breast cancer between race/ethnicities.

Abstract Abstract #2089 Background: A disparity in prognosis of triple negative (TN) breast cancer (BC) has been observed between African American (AA) and Caucasian (CAU) race/ethnicities afflicted with this aggressive and invasive BC subtype. Etiological understanding of these differences involves accounting for several factors associated with phenotype and genotype. Here, we address the latter using the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) assay to quantify mRNA expression of 512 breast cancer related genes in a cohort of 24 CAU and 56 AA TN BC tissues sourced from formalin-fixed, paraffin-embedded (FFPE) blocks.&amp;#x2028; Material and Methods: The DASL assay was used to measure mRNA expression levels from FFPE sourced tissues in both cohorts of self-identified patients. CAU BC patients were obtained from St. Mary's Hospital, Montreal, Quebec and AA BC patients were obtained from Grady Hospital, Atlanta, Georgia. RNA extraction used the RNA High Pure Kit (Roche) and was taken from archival FFPE tissues either 5µm tissue sections or cores. Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR) less than 1% and a two fold-change criteria to determine differential regulation.&amp;#x2028; Results: In all, 33 genes were found differentially expressed between AA and CAU TN BC tumor samples, 32 of which were upregulated in the AA cohort, only 1 of which was upregulated in the CAU group. The upregulated gene in CAU TN BC was TFF1. Upregulated genes in the AA cohort (order of statistical significance according to SAM software) were KIF20A, EP300, AURKB, FGF4, C14orf155, USP22, EPOR, ZNF668, SCNN1G, MAPT, FLNB, EP400, LTA, ACOT11, RBP3, CSF3, E2F2, TGFB1, CCNE1, L1CAM, NDP, VWF, RHOB, FEN1, BIN1, KRT17, CDC42EP4, SERPINF1, CHI3L2, NES, BCL2, and RERG.&amp;#x2028; Discussion: TFF1 upregulated in the CAU population, has been indicated as biomarker of favorable prognosis in endocrine therapy in clinical studies which is consistent with race/ethnicity disparities. The remaining genes upregulated in the AA cohort include transcription factors E2F2 and RBP3/E2F1 both with cyclin binding domains which may interact with CCNE1, extracellular and adhesion related genes KRT17, L1CAM and FGF4, genes associated with cell cycle AURKB, EP400, and EP300 (activator of HIF-1A). Several RAS related genes were also found differentially expressed in the AA cohort including RHOB, RERG, BIN1, and EPOR. Moreover, it is worth mentioning that BCL2 which is expressed in the aggressive mammary cancer cell line MCF-7 was also found upregulated in the AA cohort. These initial findings suggest that several differentially regulated genes between AA and CAU race/ethnicities may account for the disparity in outcomes resultant in these populations. These initial data warrant further investigation which is currently ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2089.