Formalin-fixed And Paraffin-embedded Tissue Samples Research Articles

A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens

Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology.

Differential HLA class I subunit (A, B, C heavy chain and β2-microglobulin) expression levels in normal tissues

Human leukocyte antigen (HLA) class I subunit expression level in primary and metastatic lesions has been characterized in many cancer types utilizing formalin-fixed and paraffin-embedded (FFPE) tissue sections as substrates in immunohistochemical reactions. The evaluation of the results of these studies has been hampered by the scant information about HLA class I subunit expression level in normal tissues. To address this unmet need, we have analyzed the HLA class I subunit expression level in FFPE sections of normal tissues.Two tissue microarray (TMA) blocks were constructed from archived FFPE tissue samples of a wide number of human normal tissues. The expression level of HLA-A, HLA-B, HLA-C heavy chains and β2-microglobulin (β2-M) was evaluated by IHC staining, with mAb HC-A2, mAb HC-10, and mAb NAMB1, respectively. The staining was scored according to its intensity.According to their staining patterns with the three mAbs tested, normal tissues can be divided into four groups: (i) tissues displaying moderate/strong staining patterns, (ii) tissues displaying barely detectable staining patterns, (iii) tissues displaying differential staining patterns, and (iv) tissues with no detectable staining. The ubiquitous expression pattern for HLA-A, B, C heavy chain and β2-M was found only at the endothelial level; the stroma was negative except for fibroblasts in all the tissues analyzed. Our data suggest that, contrary to the general postulate, HLA class I subunit expression is not detectable in all nucleated cells. This information provides a useful background to evaluate changes in HLA class I subunit expression associated with the malignant transformation of cells.

Immunohistochemical Characterization of Immune System Cells in Lymphoid Organs from Roe and Fallow Deer.

Roe and Fallow deer are common wild ruminants widely distributed in Italy. Infectious diseases of these species can potentially pose health risks to domestic animals and humans. However, few studies have been conducted in which immune system cells in these species were phenotyped. The aims of this study were to determine the cross-reactivity of a wide anti-human panel of commercial antibodies on formalin-fixed and paraffin-embedded (FFPE) samples and to describe the distribution of roe and fallow deer main immune cell subsets in the lymph nodes and spleen. Twenty retromandibular lymph nodes (RLNs) and spleen samples were collected from 10 roe deer and 10 fallow deer and were tested by a panel of 12 commercial anti-human antibodies. The CD79a, CD20, CD3, Iba-1, MAC387, and AM-3K antibodies were successfully labeled cells in cervine tissue, while the Foxp3 and the CD68 did not show suitable immunostaining. This study supplies the first immunohistochemical description of immune cell subpopulations in non-pathological spleen and RLNs from roe and fallow deer and provides an easily repeatable manual IHC protocol to immunolocalize cervine B-, T-cells, and macrophages subsets in FFPE tissue samples.

Effects of processing, storage time, and tissue volume in the retrieval and quality of RNA and the expression level of RNU6

Background: Molecular analyses of tumor RNA expression have become widely used both for research and clinical purposes. Tumoral tissue preservation is a critical step to ensure accuracy of molecular-based diagnostics, for which, formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source of clinical samples. MicroRNAs are ideal biomarkers in FFPE-tissues, in whose expression evaluation RNU6 is one of the genes used as a normalizer. Our aim was to determine, in FFPE tissue samples, the effects of length of storage and corresponding volume of each studied sample, on the RNA retrieval, quality and concentration, as well as their correlation to the expression level of RNU6. Methods: Fifty tissue blocks with a mean length of tissue storage of 30 months (SD=±12.07, 95% CI= 27.4-34.3). were included. Total RNA was isolated, absorbance and concentrations were determined and correlated with length of storage and volume of tissue. RT-qPCR for RNU6 was performed and their Ct results were correlated to the same parameters. Results: There was a direct correlation between the concentration and quality of the obtained RNA, and an inverse correlation between the tissue storage time and the RNA quality. The volume of tissue studied was not correlated with the RNA quality or concentration. The RNA quality and the length of tissue storage directly correlated to the RNU6 expression level, while RNA concentration and the volume of tissue studied did not affect it. Conclusions: There is an association between longer FFPE tissue storage time with lower RNA quality and lower RNU6 expression level.

Optimized protocol for metabolomic and lipidomic profiling in formalin-fixed paraffin-embedded kidney tissue by LC-MS

Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines [LPE]) remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.

MiR-221-5p acts as an oncogene and predicts worse survival in patients of renal cell cancer

BackgroundRenal cell carcinoma(RCC) is one of the most common malignancies in kidney, and usually leads to poor prognosis. Therefore, identifying novel biomarkers for predicting the progression and prognosis of RCC is essential. The purpose of this study is aimed to evaluate the function of miR-221-5p in RCC and the clinical value of miR-221-5p in RCC prognosis after surgery. Materials and methodsIn our study, RT-qPCR, wound scratch assay, cell proliferation assay, transwell assay, and flow cytometry assay were performed to explore miR-221-5p expression level and its proliferation, migration and apoptosis in clear cell RCC(ccRCC). Besides, we collected 196 formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients who received partial or radical nephrectomy from May 2006 to October 2016 at Shenzhen Traditional Chinese Medicine Hospital and People's Liberation Army 303 Hospital. The relative levels of miR-221-5p from the FFPE tissue samples was detected by RT-qPCR. The Kaplan-Meier method, Cox regression analyses, and ROC curve analysis were performed to approve the effect of the miR-221-5p expression on patient survival. ResultsIn our study, we found that miR-221-5p is significantly upregulated in ccRCC tissues and ccRCC cell lines. Moreover, miR-221-5p promotes cell proliferation, mobility, and inhibits cell apoptosis in 786-O and ACHN cell lines. The Kaplan-Meier analysis indicated that patients with high expression of miR-221-5p had a significantly poor prognosis (P = 0.013). The Cox regression analyses showed that patients with high expression of miR-221-5p remained to have a shorter overall survival (P = 0.025). The ROC curve of miR-221-5p expression combined with tumor stage showed an area under the curve of 0.658 (P < 0.001). ConclusionOur results indicated that miR-221-5p might not only be an oncogene in ccRCC cells but also might be an independent prognosis factor of ccRCC.

Immunohistochemistry on old archival paraffin blocks: is there an expiry date?

Few studies have focused on antigen preservation in formalin-fixed and paraffin-embedded (FFPE) tissue in old archival material and additional studies are required, especially considering that these samples are an irreplaceable...

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM)

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

Abstract 4919: Evaluating the gene expression of frozen tissue-derived prognostic signatures in FFPE colorectal cancer samples

Abstract OBJECTIVE: Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II and stage III patients remains a challenging problem in cancer research. Most available clinical samples are Formalin Fixed and Paraffin Embedded (FFPE). Nanostring nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high throughput measurement of mRNA expression from FFPE tissue samples. In this study, to identify an optimal platform for the gene expression profiling of FFPE CRC samples, we compared the expression of genes that make up published frozen tissue-derived prognostic signatures measured by these two platforms. METHODS: FFPE primary tumor tissue blocks were identified from 500 patients with stage II or III CRC and complete pathological information. We removed samples with neoadjuvant therapy, inadequate clinical follow-up or tumor specimens, samples with failure of RNA extraction and highly degraded samples. We identified 194 eligible FFPE-derived CRC primary tumors with a 7.4-year mean follow up. To measure the gene expression of the 194 samples, we designed a custom nCounter codeset using 536 gene elements from multiple published frozen tissue-derived prognostic signatures for CRC. We also performed gene expression profiling using the HTA platform on a subset of 84 of the 194 samples. For the nCounter data, samples with low total average count or with more than 20% genes having lower average count than the synthetic negative control genes were omitted to ensure adequate data quality. RESULTS: In total, for CRC samples from 42 patients, the gene expression data of 516 common genes measured by both platforms were of sufficient quality for comparative analysis. Based on HTA platform-derived data, we found that (1) 36 and 97 of the 516 common genes are significantly associated with overall survival (OS) and disease-free survival (DFS) at the single gene level (FDR &amp;lt; 0.05), respectively; and (2) two of the nine reported multi-gene signatures, for which sufficient information from the original papers enabled such evaluation, can divide patients into high-risk and low-risk groups with significantly different DFS (p ≤ 0.05). Based on nCounter platform-derived data, no individual gene was found to be significantly associated with survival at the single gene level (FDR &amp;lt; 0.05), but one of the nine published multi-gene signatures can divide patients into two groups with significantly different OS (p ≤ 0.05). Our results also showed moderate correlations between paired FFPE samples measured by nCounter and HTA platforms (the median correlation coefficient is 0.52). CONCLUSION: Our data demonstrate that while both platforms may identify gene or gene set expression differences associated with survival outcomes, the HTA appears to provide a more robust gene expression analysis dataset when using genes selected from published gene signatures. Citation Format: Jing Zhu, Natasha G. Deane, Keeli B. Lewis, Chandrasekhar Padmanabhan, Mary K. Washington, Kristen K. Ciombor, Cynthia Timmers, Richard M. Goldberg, R. Daniel Beauchamp, Xi Chen. Evaluating the gene expression of frozen tissue-derived prognostic signatures in FFPE colorectal cancer samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4919.

High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing.

The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases.

Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

High‐throughput RNA sequencing of a formalin‐fixed, paraffin‐embedded autopsy lung tissue sample from the 1918 influenza pandemic

Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Detection of genetic variation in KCNQ1 gene by high-resolution melting analysis in a prospective-based series of postmortem negative sudden death: comparison of results obtained in fresh frozen and formalin-fixed paraffin-embedded tissues

High-resolution melting (HRM) analysis is a recently developed molecular technique proved to be applicable for detection of genetic variation, notably in sudden cardiac death. In certain circumstances, especially in postmortem genetic investigations, the formalin-fixed and paraffin-embedded (FFPE) tissues are the only DNA source available. The present study aimed to develop HRM assays, optimized for the analysis of FFPE tissues, to detect sequence variations in KCNQ1 exons in a prospective population-based series of postmortem negative sudden death and to compare the results between the paired freshly frozen and FFPE tissue samples simultaneously obtained from the same case. The analyses were conducted in each case of sudden death involving cases younger than 35years with no significant morphological anomalies particularly with no cardiac structural disease and with negatives toxicological investigations. HRM analysis was successfully optimized for 13 of the 16 exons of the KCNQ1 gene. All mutated samples were correctly identified by HRM whatever the type of tissue tested. However, for FFPE samples, HRM indicated more positive samples than classical sequencing, used in parallel, due to the degradation of DNA by formalin fixation. This is the first postmortem study of KCNQ1 mutation detection with HRM on DNA extracted from FFPE samples with adapted protocol. Despite the false-positive detection, we concluded that the use of HRM as a screening method with FFPE samples to analyze KCNQ1 mutations can reduce the number of sequencing reactions and, thus, results in substantial time and cost savings.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

10575 Background: Cancer of unknown primary (CUP) constitutes 3%-5% of all newly diagnosed cancer cases. It presents a major diagnostic challenge as knowing the tumor tissue of origin (ToO) of the cancer is crucial for choosing the optimal treatment. MicroRNAs are a family of non-coding, regulatory RNA genes involved in development and differentiation that were shown to be involved in carcinogenesis. MicroRNAs, highly stable in clinical samples and tissue-specific, serve as ideal biomarkers for cancer diagnosis. Our first generation assay identifies the ToO using a set of 48 microRNAs measured on a qRT-PCR platform and differentiates 25 different tumor types. We present here the development and validation of a second generation clinical assay that can identify 42 different tumor types using a custom array platform. Methods: Over 1300 primary and metastatic tumor formalin-fixed and paraffin-embedded (FFPE) tissue samples were used for the training and development of the assay. High-purity RNA was extracted using proprietary protocols. Expression levels of all known and additional Rosetta’s proprietary microRNAs were profiled using a custom array platform. A combination of a decision tree with a KNN classifier was developed to identify the ToO based on the expression of 64 microRNAs. A validation set of more than 500 independent FFPE tissue samples of primary tumors and metastases was analyzed blindly and classified using the developed assay. Results: The assay is able to identify 42 tumor types that include carcinomas, soft tissue tumors, lymphoma and other malignancies with very high accuracy. Based on the validation set, the sensitivity (positive agreement) for identifying the tumor origin is 85%. In more than 80% of the cases, a single answer is reported, and for these cases, the sensitivity is 90%. Conclusions: Previous studies have highlighted the tissue-specificity of microRNA expression, and have demonstrated their potential use for classification of human malignancies. An enlarged panel of 42 ToO that can be identified with high accuracy promises improved utility for the diagnosis of cancers of unknown/uncertain primary.

Multiplex Quantum Dot ISH Measurement of Gene Expression in Tissue Microarrays Identifies HOXA9 and DNMT3A as Markers of Poor Response to Chemotherapy in Patients with Acute Myeloid Leukaemia

Microarray-based expression profiling has identified prognostic gene signatures for many cancers and validation is required in clinical samples. However, most clinical material is in the form of formalin fixed and paraffin embedded tissue (FFPET) in which gene expression analysis is problematic. We have developed a generic quantum dot (QD) based multiplexed in-situ hybridization (ISH) method enabling quantitative localization of multiple mRNA targets in FFPET. We expand on our previous work introducing a method for standardization of ISH signal, enabling comparative measurement of gene expression across multiple samples. This was applied to tissue microarrays (TMAs) using archived trephine biopsies from patients with acute myeloid leukaemia (AML) to identify prognostic genes. A total of 15 TMAs were prepared using FFPET samples from 240 patients with AML diagnosed and treated between 1994 and 2005 at Manchester Royal Infirmary (Manchester, UK). For the analysis, 192 patients were included as the remainder either died before, during or immediately after one course of chemotherapy or there was incomplete data collection. The median age was 52 years (range 17–77) and all patients received intensive chemotherapy according to standard UK MRC AML protocols. Three cores were taken from each sample for TMA preparation. A standard was prepared using a cell pellet obtained from whole blood white cells which was embedded, in triplicate, in each TMA. QD-ISH was performed for nine genes recognized to be of prognostic value in AML. Triplex QD-ISH using QD labeled anti-sense cDNA oligonucleotides was performed for the following targets: Bcl2, survivin and XIAP; DNMT1, DNMT3A and DNMT3B; HOXA4, HOXA9 and Meis1. Signal intensity for each gene was measured using spectral imaging. Scrambled sense cDNA oligonucleotides were used to measure the level of background staining for each gene in each core. Background noise was corrected for by dividing expression levels of anti-sense probes by that of the scrambled probe, for both samples and standards. This enabled direct comparison between TMAs as gene expression values of samples were normalized against the standard. The mean expression of each gene was calculated for each patient, divided into quartiles and correlated with clinical outcome data. Statistical analysis was performed using contingency tables, the chi-square test and Mann Whitney-U. Overall survival (OS) and disease free survival (DFS) were displayed using the Kaplan-Meier method and Cox regression was performed for univariate and multivariate analysis. The OS in this cohort of patients was 43% at 5 years with 80% achieving complete remission (CR) after induction chemotherapy. Patient age (<60 years), WCC (<100×109/l) at diagnosis, cytogenetics (good and intermediate risk) and low HOXA4 expression (median 577 [95%CI 325–828]) were all associated with improved OS (p<0.0001; p=0.02; p<0.0001; p=0.013) and DFS (p<0.0001; p=0.013; p<0.0001; p=0.025) on univariate and multivariate analysis. High expression of HOXA9 (median 0.843 [0.145–7.479]; p<0.0001) and DNMT3A (median 1.305 [0.073–5.477]; p=0.04) were associated with failure to achieve CR. High Meis1 expression was found to be of borderline significance for poor response to chemotherapy (median 0.716 [0.051–7.840]; p=0.05). Expression levels of the remaining 5 genes did not show any correlation with CR, DFS or OS. These findings are consistent with recently published data regarding the prognostic significance of various new markers. In line with others we have demonstrated low expression of HOXA4 is an independent good prognostic marker in adult AML. Although high expression of HOXA9, DNMT3A and Meis1 was associated with inferior CR rates in our study, OS and DFS were not adversely affected. This may be related to improvements and more aggressive clinical practice (eg stem cell transplantation) over recent years which can overcome potential deleterious gene effects. These results demonstrate that the application of a standardized, quantitative multiplex QD-ISH can be used for identification of prognostic markers in FFPET samples. The advantages of this method include its application to TMAs which allows high sample throughput, use of archived materials and its transferability across a spectrum of malignancies.

Extraction of Phosphorylated Proteins from Formalin-Fixed Cancer Cells and Tissues

Deregulated signal pathways are promising targets for cancer therapy as they can drive tumour growth, sur- vival, invasion, and metastasis. In most hospitals around the world, histopathological cancer diagnosis is based upon for- malin-fixed and paraffin-embedded (FFPE) tissues. In this study, we evaluated whether phosphorylated signaling proteins can be extracted from cells and tissues after formalin fixation. Three epidermal growth factor (EGF) receptor-positive cell lines were stimulated with EGF for various time points and then either fixed with formalin or left unfixed before protein extraction. Using Western blot, we were able to detect all phosphorylated proteins analyzed, including activated (phos- phorylated) extracellular signal-regulated kinase (ERK) 1 and 2. There were no differences between formalin-fixed and unfixed cells. Because ERK1/2 kinases are involved in HER2-mediated signal transduction, we applied our methodology to study ERK1/2 and phospho-ERK1/2 in 20 primary breast cancers using protein lysate microarray technology. We found a statistically significant correlation between HER2 status and ERK1/2 expression; however, activation (phosphory- lation) of ERK1/2 was independent of HER2 expression. Our study demonstrates the successful extraction of phosphory- lated proteins from FFPE tissue samples. Monitoring phosphorylated signaling proteins in FFPE tissues, as applied here, may allow us in the near future to infer the activity levels of proteins in particular pathways as starting point for the design of individual combination therapy regimens without changing the routine clinical workflow for tissue analysis.