From Formalin-fixed Paraffin-embedded Blocks Research Articles

Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.

Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1-240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6-72 h) for cancer patients.

DNA isolation from achieved formalin-fixed paraffin-embedded tissues in a series of 212 thyroid carcinoma cases: the influence of preanalytical factors on DNA quantity and purity

ObjectiveThe present study aimed to investigate the influence of several important preanalytical factors (storage period of the tumor block, maximal diameter of the tumor circled area, tumor volume and tumor...

Abstract P2-03-03: A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues

Abstract The Xpert® Breast Cancer STRAT4 (STRAT4) is a CE-IVD marked, semi-quantitative, cartridge-based RT-qPCR assay for the detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs from formalin fixed, paraffin embedded (FFPE) breast tumors. The assay is fast (< 2 hrs), reproducible, robust, and easy to perform. The aim of this multicenter clinical study was to assess the performance characteristics of the STRAT4 assay relative to central lab immunohistochemistry (IHC) for ER, PgR, HER2, and Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification. Methods: A total of 200 archived primary invasive breast cancer FFPE blocks were sourced from Indivumed for this study. From each block, twelve (12) adjacent tissue sections (4-µm thickness) on slides were prepared for pathological H&E confirmation to define tumor area, and for testing by STRAT4, IHC (ER, PgR, HER2,Ki67), and HER2 FISH. Standard STRAT4 lysate preparation using a single unstained slide per specimen and testing on N=84, N=68, and N=48 samples was performed at 3 independent sites, respectively (2 US and 1 EU). A single slide from each specimen was also processed using the recommended concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH testing was performed by a central academic reference laboratory in the US. For a given sample, STRAT4 data generated using the standard lysate procedure was included for concordance analysis when all target gene test results were valid. In cases where the standard lysate preparation yielded indeterminate test results for any target, data from the concentrated lysate preparation was used for the data analysis. Receiver Operating Characteristic (ROC) analysis, overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC/FISH for HER2) were determined for ESR1,PGR, ERBB2, and MKi67. Results: Of the 200 samples tested by STRAT4, all samples generated valid results for ESR1 and ERBB2, 199 of 200 samples were valid for PGR, and 198 of 200 samples were valid for MKi67 using the standard or concentrated lysate preparation protocol. One sample failed to generate results for both ER and PgR IHC. Twelve samples failed to yield HER2 FISH results. The STRAT4 success rate and results concordance with IHC were comparable across study sites. OPA between STRAT4 and IHC was 97% for ESR1, 88.9% for PGR, 93.3% for HER2 (92.4% for IHC and FISH), and 90.7% for MKi67 (excluding IHC 10-20% staining). Areas under the ROC curves were 0.9922 for ESR1, 0.9509 for PGR, 0.9958 for ERBB2, and 0.9395 for MKi67. Conclusion: STRAT4 measurements for ESR1, PGR, ERBB2 and MKi67 mRNA expression are robust and highly concordant with IHC (IHC/FISH for HER2). The technical portion of the assay is easily performed in < 2 hrs including hands-on time using standard FFPE tissue sections. Xpert STRAT4 offers local pathology labs an alternative to centralized, subjective IHC/FISH tests that require a higher level of expertise. Further investigations correlating STRAT4 markers directly with clinical outcomes in independent cohorts are in progress. Citation Format: Wu NC, Wong E, Acca B, Birkmeier J, Tran L, Zhao S, Wong W, Chu VC, Ho K, Malek M, Lu C, Ge G, David K, Quigley NB, Beqaj SS, Davenport S, Weidler J, Bates M, Press M. A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-03.

Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue.

The demand for nucleic acid and protein derivatives from formalin-fixed paraffin-embedded (FFPE) tissue has greatly increased due to advances in extraction and purification methods, making these derivatives available for numerous genomic and proteomic platforms. Previously, DNA, RNA, microRNA (miRNA), or protein derived from FFPE tissue blocks were considered “unfit” for such platforms, as the process of tissue immobilization by FFPE resulted in cross-linked, fragmented, and chemically modified macromolecules. We conducted a systematic examination of nucleic acids and proteins co-extracted from 118 FFPE blocks sampled from the AIDS and Cancer Specimen Resource (ACSR) at The George Washington University after stratification by storage duration and the three most common tumor tissue types at the ACSR (adenocarcinoma, squamous cell carcinoma, and papillary carcinoma). DNA, RNA, miRNA, and protein could be co-extracted from 98% of the FFPE blocks sampled, with DNA and miRNA “fit” for diverse genomic purposes including sequencing. While RNA was the most labile of the FFPE derivatives, especially when assessed by RNA integrity number (RIN), it was still “fit” for genomic methods that use smaller sequence lengths, e.g., quantitative PCR. While more than half of the protein derivatives were fit for proteomic purposes, our analyses indicated a significant interaction effect on the absorbance values for proteins derived from FFPE, implying that storage duration may affect protein derivatives differently by tumor tissue type. The mean absorbance value for proteins derived from more recently stored FFPE was greater than protein derived from older FFPE, with the exception of adenocarcinoma tissue. Finally, the fitness of one type of derivative was weakly associated with the fitness of derivatives co-extracted from the same FFPE block. The current study used several novel quality assurance approaches and metrics to show that archival FFPE tissue blocks are a valuable resource for contemporary genomic and proteomic platforms.

The Effect of PT Link and Waterbath Antigenic Retrieval Procedures On The Expression of Common Receptors In Breast Cancer Cases

<p><strong>Background:</strong> Antigen retrieval (AR) techniques play a major role in determining the quality and functional state of tissue proteins recovered from formalin fixed paraffin embedded blocks (FFPEB). Different techniques are used each with its merits and drawbacks.<br /><strong>Materials and Method:</strong> FFPEB from cases diagnosed as breast cancer were examined for the expression of estrogen- receptor (ER), progesterone receptor (PR) and HER2 by immunohistochemical techniques using two different AR methods.<br /><strong>Results:</strong> Fifty cases, 49 were from females, the age ranged from 28–85 years were examined. The expression of ER using water bath and PT link (pretreatment system) as antigen retrieval methods was equal, both methods showed 20(40%) positive cases and 30(60%) negative cases, whereas, the expression of PR was found positive in 18 cases (36%) and negative in 32 (64%) when water bath was used, in comparison to10 positive (20%) and 40 negative (80%) when PT link was used (P value 0.312).The expression of HER2 was as follows; water bath HER2 weak positive 20 cases (40%), moderate expression 10 cases (20%), overexpression 2 cases (4%). PT link weak expression 13 cases (26%), moderate expression 15 cases (30%), overexpression 4cases (8%); yet, the total number of negative cases and positive cases was equal for both methods (p-value 0.035).<br />Conclusion: Antigen retrieval methods were found to affect the expression of common receptors in breast cancer. The magnitude of this effect was found to be significant in PR and HER2 receptors, however, the different methods of antigen retrieval did not affect the expression of ER. The appropriate and suitable retrieval methods have to be chosen individually for each receptor.</p><p><br /><strong>Keywords:</strong> Antigen retrieval, ER, PR, HER2 expression, PT link, waterbath.</p>

Patterns of PD-1, PD-L1, and PD-L2 expression in pediatric solid tumors.

Significant antitumor effects have been observed in a variety of malignancies via blockade of immune checkpoints. Interaction of programmed death 1 (PD-1) with its ligands PD-L1 and PD-L2 suppresses T-cell function and restricts immune-mediated tumor killing. We examined expression of these proteins in children with solid tumors, as expression may serve as biomarkers of response to this class of drugs. Sections cut from formalin-fixed paraffin-embedded (FFPE) tissue blocks were processed and evaluated for PD-1, PD-L1, and PD-L2 by immunohistochemistry (IHC) as well as by mRNA expression. A semiquantitative 0-5 IHC scoring system (0 = negative to 5 = very high) was applied, with scores incorporating combined prevalence of tumor cell and nontumor cell labeling. Expression profiling was performed using the NanoString nCounter™ system. Data analysis was performed using quantile normalization. All quantile-normalized data underwent subsequent log10 transformation. One hundred twenty-four FFPE blocks were included in the analysis. PD-1, PD-L1, and PD-L2 IHC were not evaluable in 8, 0, and 12 blocks, respectively. PD-1, PDL-1, and PDL-2 expression was negative to moderate by both IHC (range 0-3) and mRNA expression (range 0-2.62). Correlation between IHC score and mRNA expression was poor for all three tested proteins (PD-1, r2 = 0.06; PDL-1, r2 = 0.007; and PDL-2, r2 = 0.15). Expression of PD-1, PD-L1, and PD-L2 is low in pediatric solid tumors. At low levels of expression, IHC score and mRNA expression correlate poorly. Current and planned clinical trials will determine whether this low level of expression predicts limited response to immune checkpoint inhibitors.

Extraction of miRNAs from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues.

miRNAs are small non-coding RNAs that regulate gene expression and are involved in numerous diseases ranging from osteoporosis, cardiovascular disease, and numerous cancer indications. Most of the clinical material that is accessible for study is archival FFPE blocks. Due to the harsh nature of fixation and embedding procedures involved in preserving clinical material, these samples are heavily fragmented and chemically cross-linked by formalin. miRNAs, due to their small size and increased stability, are easier to retrieve and study in these precious tissues. Therefore, miRNAs have become useful tools in the diagnosis, prognosis, and prediction of diseases. Due to their increased importance, isolation of miRNAs from FFPE material is extremely clinically relevant. Here, we describe the best method for miRNA extraction from FFPE tissue blocks and determine the Qiagen miRNeasy FFPE kit to be the best kit for this task.

Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas.

BRAF and NRAS mutation detection is crucial for advanced melanoma treatment. Our aim was to evaluate how different characteristics from formalin-fixed paraffin-embedded (FFPE) samples, age of the block or DNA concentration could influence the success of BRAF and NRAS mutational screening. DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis). BRAF and NRAS were sequenced by Sanger sequencing. Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them. BRAF was mutated in 55% (29/53) and NRAS in 11% (5/45) of the primary melanomas sequenced. DNA concentration correlated with the tumor area used for DNA extraction (mm2) (adj p-value<0.01, r=0.73). The age of the block did not affect sequencing success. In 60% of samples kept for more than 10 years, both BRAF and NRAS were successfully sequenced. Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

Validation of a Next-Generation Sequencing Cancer Panel for Clinical Mutation Profiling in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: A previously developed NGS-based assay (ENGAUGE™-Cancer-DLBCL), which consists of the genes BRAF, BTK, CARD11, CD79A, CD79B, EZH2, KRAS, MYD88, NOTCH1, PIM1, SYK, TNFAIP3 and TP53 was analytically validated for clinical use in patients with Diffuse large B-cell lymphoma. We analyzed the performance of this assay with respect to the reproducibility, accuracy, precision, linearity, as well as limits of detection and quantification in a clinical reference laboratory environment in tumor sections taken from formalin-fixed paraffin-embedded (FFPE) tissue blocks.Methods: Illumina TrueSeq® Custom Amplicon on MiSeq was used for this study. The basic design of the experiments comprised the evaluation of 26 samples in replicate starting from genomic DNA isolated from a pooled sample in which each replicate underwent independent library preparation and was analyzed in a multiplexed run in the same flow cell. We repeated the analysis of the same 26 samples three times on different days (run-to-run reproducibility). All reactions were performed in triplicate with both positive and negative controls, and detection thresholds for each gene were chosen by receiver-operator curve analysis. The sequence data analysis was performed with NextGene, Variant Studio and Diagnovus’s proprietary bioinformatics analysis software. Unsupervised clustering and component analysis were performed to examine the variance and correlation of gene profiles amongst patients and sections within FFPE blocks. Sensitivity and Specificity were also calculated.Results: In all the sequencing runs performed in the validation studies, the average library coverage from clinical FFPE samples was 1847. The allele frequency for 22 out of 23 (96%) previously identified mutations was concordant with those observed by Sanger sequencing. With respect to limits of detection, the NGS assay can detect mutations present at 5-6% allele frequency with at least 95% confidence. Extraction of total DNA yielded high-quality DNA ranging from 250 to 500 ng per sample. Unsupervised clustering and principal component analysis revealed a strong correlation between blocks from the same patient, as well as between tumor samples from the same block. Using only 250ng total dsDNA input, the ENGAUGE™-Cancer-DLBCL NGS assay is capable of detecting all mutations including single base substitutions and small indels, with 5% LOD, ≥95% intra‐run reproducibility (precision), 100% inter‐run reproducibility, and with ≥95% analytical sensitivity and specificity. 17 critical variants with frequencies ranging from 5% to 100% (10 SBS, 4 deletions, and 3 deletions with insertions) were detected in 26 tumor FFPE samples. The most frequently observed mutations were NOTCH1 (27%), CARD11 (19%) and TP53 (19%).Conclusions: Extensive reagent and analytical performance studies were conducted to evaluate the reliability and reproducibility of a gene test panel (ENGAUGE™-Cancer-DLBCL). Our validation study demonstrates that this Illumina TrueSeq® Amplicon panel combined with Diagnovus’s proprietary bioinformatics analysis software provides a highly sensitive and sensitive test with a reportable analytical range of 5% to 100% mutation allele frequency.This assay is suitable for screening diffuse large B-cell patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations. DisclosuresNo relevant conflicts of interest to declare.

Long-term stability of RNA from FFPE brian tissue post-sectioning on slides.

e22142 Background: Analysis of nucleic acids (NA) from formalin fixed paraffin embedded (FFPE) tissue can provide detailed information about gene sequence mutational status, which may be important for oncology treatment decisions. FFPE specimens also have utility for retrospective analyses. Potential degradation of NA during formalin fixation, paraffin embedding processes and possible continued deterioration during subsequent storage may diminish utility of FFPE tissue for these purposes. Using real-time PCR, this study investigated the functional stability of RNA from brain FFPE tissue sections on slides over an extended time period after sectioning. Methods: Brain biopsy specimens obtained from glioblastoma patients with informed consent were used to prepare blocks with standard formalin fixation and paraffin embedding techniques. Slides were made from the FFPE blocks and stored at room temperature until testing. RNA was extracted from sequential slides within one week of sectioning for a zero time and then at 4, 8 and 12 months. Reverse transcription PCR was performed, and real-time PCR was analyzed on the ABI7900 to detect EGFRvIII mutation and cABL gene. RNA Integrity Analysis was performed with an Agilent Bioanalyzer. Results: Consistent qualitative results were obtained with EGFRvIII mutant positive specimens (n =10) and wild type (wt) specimens (n =10) from slides stored up to twelve months at room temperature compared to the initial testing (95% agreement). One wt specimen showed negative results for the first three time points but a low positive result at 12 months, possibly due to tumor content change in the different sections of the FFPE block. Ct values for EGFR (wt and mutant) and cABL genes did not increase during the storage period. RNA integrity number (RIN) indicated the degradation of RNA during FFPE processing, although no further significant degradation occurred during the course of the experiment. Conclusions: The results of this study indicated that although the RNA was impacted by the tissue preparation, fixation, and processing steps, for the brain FFPE slide specimens, target genes with amplicon size up to 124bp could be detected with minimum degradation for up to 12 months when slides were stored at room temperature.

Abstract 5455: Toxic heavy metal content in lung tissue samples from non-small cell lung cancer patients

Abstract Background: Cigarettes, food, water, and air pollution can contain toxic heavy metals, such as cadmium (Cd), arsenic (As), mercury (Hg), and lead (Pb). A cigarette contains approximately 1-2 ug of Cd, 40-120 ng of As, 2.95-10.2 ng of Hg, and 1-2 mg/kg of Pb because the tobacco plant concentrates metals from soil and water. The accumulation of toxic heavy metals inhaled from smoking may be associated with lung cancer. We surveyed tissue from current/former smokers with non-small cell lung cancer (NSCLC) for heavy metal content. Methods: Tissue collected from treatment-naïve stage I and II NSCLC patient samples that underwent surgical resection was analyzed by inductively coupled plasma mass spectroscopy (ICP-MS) for concentration of Cd, As, Hg, and Pb. 16 cases were matched for stage, histology, gender, age, and race. Concentrations were measured from two 20 micron thick slides each from formalin-fixed, paraffin-embedded (FFPE) tissues: primary lung cancer, benign adjacent lung, and where applicable to malignant lymph node, benign adjacent lymph node, benign lymph node, and distant non-malignant lung tissue. Negative controls were paraffin without tissue from FFPE blocks. Paired samples t-tests were used to make intra-case comparisons. The nonparametric Mann-Whitney U and the Wilcoxon signed-rank test were used to examine inter-case differences. Results: The median patient age was 64.7 (range 36.4-75.4). There were 15 primary lung cancer cases with sufficient tissue for analysis: 8 were men, 7 were stage I, 7 had adenocarcinoma, 8 had squamous cell carcinoma, 8 were former smokers, 7 were current smokers, 9 were Caucasian, and 6 were African-American. A total of 75 tissue samples were analyzed with an average sample weight of 2.9 mg. With the lower limit of detection at 0.001 ug/g, the median concentrations in ug/g for Cd, As, Hg, and Pb in primary lung tumor were 0.058 (range undetectable [Not]-0.316), 0.019 (range Not-0.078), 0 (range Not-0.015), and 0 (range Not-0.026); respectively. In distant non-malignant lung samples, the median concentrations in ug/g for Cd, As, Hg, and Pb were 0.204 (range Not-2.500), 0.025 (range Not-0.070), 0 (range Not-0.012), and Not; respectively. There were no significant inter-case differences for the heavy metal concentrations in primary lung tumor and stage, histology, gender, and race. However, distant non-malignant lung samples had significantly higher Cd levels compared to primary lung tumor (p=0.013). Conclusion: Using ICP-MS, concentrations of Cd, As, Hg, and Pb could be measured in low quantities of FFPE tissue. Significantly higher Cd content was observed in distant non-malignant lung tissue compared to primary lung tumor. There appears to be higher levels of Cd and As than Hg and Pb in the lung tissues of current/former smokers with NSCLC. Analysis of lung tissues from clinically-matched non-cancer patients is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5455. doi:1538-7445.AM2012-5455

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.

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.

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.