Formaldehyde-fixed Paraffin-embedded Samples Research Articles

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 μg/μL) protein preparation for mass spectrometry (MS) based analysis, in which 2 μg/μL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 μg/μL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

Abstract 2679: Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues

Abstract Introduction: Imaging mass cytometry (IMC) enables highly multiplexed assessment of more than 30 proteins at a time for the quantification of cell phenotypes within the tumor microenvironment of formaldehyde fixed-paraffin embedded (FFPE) tumor tissue. This methodology provides a deep understanding of the immune-contexture while preserving their spatial information in regards to malignant cells. We report a detailed workflow for optimization of a 27-plex immune-oncology (IO) panel, immune-phenotyping, and spatial analysis of tumor tissue. Methods: IO-panel includes 27 antibodies targeting lymphoid (CD3e, CD4, CD8a, CD19, CD45RO, LAG3, ICOS, Granzyme-B), myeloid (CD11b, CD14, CD33, CD68, CSF1R, IDO-1), immune-regulatory (HLA-DR, OX-40, VISTA, TIM-3, CD73, B7-H3), epithelial (Cytokeratin), proliferative (Ki-67), and constitutive (GAPDH, NaKATPase, Vimentin, aSMA, Histone H3) markers that were selected by singlet automated chromogenic immunohistochemistry (IHC) staining. BSA-free formulation from the same clones of antibodies were isotope-metal conjugated and manual indirect immunofluorescence (IF) staining was used to confirm the stability of metal-tagged antibodies. The optimization by singlet-IF and multiplexed-IMC staining was performed with normal and malignant tissues (4mm core TMA with tonsil, placenta, prostate, breast carcinoma, ovarian carcinoma, and endometrioid carcinoma tissues). All images from IHC (HALO software), indirect IF (InForm software), and IMC (MCD viewer software) slides were evaluated by two pathologists. Cell densities and spatial analysis of a breast carcinoma core was performed using HALO software. Results: All antibodies included in the panel were individually optimized by IHC under the same protocol conditions. A significant correlation of optimal antibody concentrations where observed across the different staining techniques (IHC vs IF, r=0.51; IHC vs IMC, r= 0.54; IF vs IMC, r=0.992; all p values &amp;lt;0.01). The breast carcinoma core (total area=0.308mm2) was segmented into epithelial (area=0.216 mm2) and stroma (area=0.092mm2) compartments by digital image analysis. Individual cell segmentation was obtained with based on iridium nuclei signal (total cells=2420). Automated immune-phenotyping showed 20 different cell phenotypes with a predominance of malignant cells expressing B7-H3 (120.1 cells/mm2) and T-lymphocytes expressing LAG3 (3.2 cells/mm2). The average distance between proliferative cytotoxic T-lymphocytes to malignant cells (MCs) was higher when MCs were proliferating (22.94 μm, and 55.4 μm, respectively). Conclusions: IMC is a powerful platform for highly multiplexed detection of protein and cell phenotypes quantification in FFPE samples. Our developed IMC panel and digital image analysis of tissues allow the quantification of immune cells and spatial analysis of the tumor tissue microenvironment. Citation Format: Pedro Rocha, Luisa Solis, Edwin Parra, Ou Shi, Barbara Mino, Vidya C. Sinha, Amanda Rinkenbaugh, Auriole Tamegnon, Mak Duncan, Wei Lu, Mei Jiang, Lakshimi Kakarala, Jared K. Burks, Helen Piwnica-Worms, Cara Haymacker, Michael Teztlaff, Ignacio Wistuba, Alejandro Francisco-Cruz. Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2679.

Abstract 179: Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus)

Abstract Comprehensive genomic profiling (CGP) of tumor samples by next-generation sequencing is used to support clinical and translational research into the genetic variants that serve as biomarkers for diagnosis, prognosis and potential therapeutic response. However, as these assays grow in size to meet the expanding demands of users, it is challenging to maintain performance in the face of limited sample input necessitated by small sample volumes and to provide a simple and fast sample to report workflow with limited hands-on time. We, therefore, developed OCA Plus to meet user needs for a large CGP assay with excellent performance. Gene content was prioritized based on potential clinical relevance and variant prevalence in solid tumors. Over 500 genes were selected including genes in the indication statements of approved drug labels, clinical guidelines, and in the enrollment criteria of clinical trials. In addition, driver genes were selected in key pathways including DNA repair and immune checkpoint response. Amplicon design strategies were optimized accordingly for key hotspots, full coding sequences or copy number variation (CNV). The assay used Ion AmpliSeq™ technology with manual library preparation or automated templating on the Ion Chef™ System and sequencing on the Ion GeneStudio™ S5 platform. Twenty ng of purified DNA was routinely used as input. An automated tumor-only workflow for variant calling and sample quality reporting was provided within Ion Reporter™ Software. Streamlined access to reporting of variant relevance was enabled by Oncomine™ Reporter. In development studies of cancer cell line and formaldehyde-fixed, paraffin-embedded (FFPE) tumor samples, the assay displayed excellent uniformity (98% and 94%, respectively). Detection of single nucleotide variants and indels in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Detection of CNV gain and loss in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Assessment of tumor mutational burden (TMB) using publicly available whole-exome cancer sequencing data as well as test cell lines and FFPE samples showed high concordance with whole exome sequencing (R2 &amp;gt; 0.90). MSI sensitivity and specificity was &amp;gt;95% as tested using a diverse set of tumor samples. Targeted fusions were reported with 100% sensitivity and specificity when tested with commercially available controls. Total time from purified DNA to end of sequencing was &amp;lt; 2 days with &amp;lt; 3 hours of hands-on time and the time from post-sequencing to report generation was &amp;lt; 2 hours. Oncomine Comprehensive Assay (OCA Plus) was developed to support CGP and routine clinical research in oncology. The assay design and informatics workflow were optimized to support low input and rapid sample-to-report turn-around time, which will accelerate clinical and translational research. Citation Format: Vinay Mittal, Jennifer Kilzer, Dinesh Cyanam, Janice Au-Young, Santhoshi Bandla, Gary Bee, Sameh El-Difrawy, Aren Ewing, Rajesh Gottimukkala, Mohit Gupta, Nickolay Khazanov, Anelia Kraltcheva, Amir Marcovitz, Scott Myrand, Rose Putler, Yu-Ting Tseng, Warren Tom, Cristina Van Loy, James Veitch, Paul Williams, Elaine Wong-Ho, Huimin Xie, Chenchen Yang, Zheng Zang, Seth Sadis. Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 179.

Pathophysiology of Intracranial Aneurysms: COX-2 Expression, Iron Deposition in Aneurysm Wall, and Correlation With Magnetic Resonance Imaging.

The pathophysiology of development, growth, and rupture of intracranial aneurysms (IAs) is only partly understood. Cyclooxygenase 2 (COX-2) converts arachidonic acid to prostaglandin H2, which, in turn, is isomerized to prostaglandin E2. In the human body, COX-2 plays an essential role in inflammatory pathways. This explorative study aimed to investigate COX-2 expression in the wall of IAs and its correlation to image features in clinical (1.0T, 1.5T, and 3.0T) magnetic resonance imaging (MRI) and ultra-high-field 7T MRI. The study group comprised 40 patients with partly thrombosed saccular IAs. The cohort included 17 ruptured- and 24 unruptured IAs, which had all been treated microsurgically. Formaldehyde-fixed paraffin-embedded samples were immunohistochemically stained with a monoclonal antibody against COX-2 (Dako, Santa Clara, CA; Clone: CX-294). We correlated Perls Prussian blue staining, MRI, and clinical data with immunohistochemistry, analyzed using the Trainable Weka Segmentation algorithm. Aneurysm dome size ranged between 2 and 67 mm. The proportion of COX-2 positive cells ranged between 3.54% to 85.09%. An upregulated COX-2 expression correlated with increasing IA dome size (P=0.047). Furthermore, there was a tendency of higher COX-2 expression in most ruptured IAs (P=0.064). At all field strengths, MRI shows wall hypointensities due to iron deposition correlating with COX-2 expression (P=0.022). Iron deposition and COX-2 expression in IAs walls correlate with signal hypointensity in MRI, which might, therefore, serve as a biomarker for IA instability. Furthermore, as COX-2 was also expressed in small unruptured IAs, it could be a potential target for specific medical treatment.

Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.

Introduction and ObjectivesOncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples.Materials and MethodsThe RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.ResultsFGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.ConclusionsWe developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors.

Abstract 4902: Detecting low abundant mutations in circulating cell free DNA and FFPE samples

Abstract Detection of low abundant alterations in oncogenes from circulating cell free DNA (cfDNA) samples or formaldehyde fixed-paraffin embedded samples (FFPE) has been a challenge for the community. Additionally, cfDNA and FFPE samples are typically limited in the amount of material that can be obtained, and often the FFPE samples are highly degraded, compounding the ability to sequence them and to perform accurate variant calling. To overcome these challenges, we have developed a single tube, multiplexed amplicon sequencing technology that employ hundreds of primer pairs for amplification of targeted loci, producing ready-to-run libraries for Illumina sequencing platforms. The resulting amplicons are less than 150 bp in length, enabling amplification and sensitive detection of mutations from cfDNA-sized DNA fragments or highly damaged DNA. A 200+ amplicon panel across 55 genes was developed to target known clinically relevant oncological mutations. The panel design encompasses single exons (e.g. BRAF) as well as comprehensive exon coverage of entire genes (e.g. TP53). DNA was extracted from FFPE, and cfDNA was extracted from fresh plasma. DNA was quantified by qPCR using 2 primer pairs targeting ALU repeat regions. 10ng of DNA input was used to perform the multiplexed PCR. Libraries were quantified by qPCR and sequenced with MiSEQ V2 reagents, paired end reads. Alignment was performed using Burrows-wheeler Aligner (BWA) and variant calling was performed with at least two validated publicly available tools and confirmed manually by IGV. A variety of tools were used to validate allele technology was used to validate the sequencing data. We interrogated 80 FFPE samples using our amplicon technology.The percent of on-target bases was over 95% and the coverage uniformity was over 98%, where uniformity is the percent based covered over 20% of the mean coverage. Out of the 40 colorectal cancer samples tested, 17 showed mutations in KRAS. Out of the 20 melanoma samples (Cure line) also analyzed, 9 were positive for BRAF V600E. The presence of those mutations was confirmed using myTprimerTM KRAS and BRAF qPCR assay. The limit of detection of the assay was set at 3% due to the inherent noise caused by the damage from the FFPE samples. For cell lines with known mutations, the sensitivity was 0.5%. No false positive or false negative were reported. 20 FFPE samples from different cancer types were also assessed. Mutations were identified in ERBB2 (cervical cancer), ALK (lung cancer) and TP53 (colon cancer) genes, as well as MET amplification (lung cancer). Cell free DNA provides a non-invasive tool to monitor cancer progression and treatment efficacy. Tumor DNA, matching blood DNA and cfDNA from 10 cancer patients will be subjected to our multiplex PCR oncology panel. Data of this study will be presented. Our results show that this unique multiplex PCR panel is an excellent tool to assess multiple oncogenes in limiting clinical samples, enabling high throughput, cost effective NGS analysis. Citation Format: Julie Laliberte, Catherine Couture, Vladimir Makarov, Cassie Schumacher, Sukhinder Sandhu, Laurie Kurihara, Timothy Harkins. Detecting low abundant mutations in circulating cell free DNA and FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4902. doi:10.1158/1538-7445.AM2015-4902

Abstract 1115: Targeted RNA sequencing improves transcript analysis in cancer samples

Abstract RNA sequencing (RNA-seq) is a transcriptome profiling technology that provides multiple levels of insight into the genome. In addition to expression levels (transcript abundance), it generates endpoints such as alternative splicing, somatic mutations and rearrangements, which may have functional consequences in cancer. Although somatic mutations are generally identified by DNA sequencing, RNA-seq has the advantage of detecting allele-specific expression affecting a variant allele, as well as functional chimeric transcripts that result from structural rearrangements. Compared to microarray technologies, RNA-seq can provide additional information about novel transcripts. Due to the complexity of the human transcriptome and the variability of gene abundance, the cost of whole transcriptome sequencing to achieve sufficient coverage to detect these types of alterations remains high. To explore the feasibility of a more cost-effective method, we compared the performance of three different RNA-seq methods: whole-transcriptome-, exome-, and targeted RNA-seq, using RNA derived from cancer cell lines and Formaldehyde Fixed-Paraffin Embedded (FFPE) samples. For whole-transcriptome preparation, we used the Illumina TruSeq Stranded mRNA and total RNA kits for cell line and FFPE samples, respectively. Exome-RNAseq was performed using the Illumina Access kit. The libraries from whole-transcriptome RNAseq were subjected to hybridization capture using OncoPanel-an Agilent SureSelect baitset of 500 cancer-related genes. Compared to whole-transcriptome, exome- and targeted-RNA-seq demonstrated (1) higher coding exon coverage and multiplexing capability; (2) reduced rRNA composition to 1%; (3) comparable gene abundance information and (4) over 90% of reads aligned to coding exon regions in FFPE samples, compare to ∼30% when using whole transcriptome method. In conclusion, we demonstrated that exome- and targeted RNA-seq provide a cost-effective way to analyze a subset of the transcriptome. Furthermore, targeted RNA-seq can be highly multiplexed and is therefore amenable for large-scale tumor profiling in clinical or research settings. Citation Format: Ling Lin, Ryan Abo, Deniz Dolcen, Rachel Paquette, Angelica Laing, Luc de Waal, Aaron Thorner, Matthew Ducar, Liuda Ziaugra, Bruce Wollison, Marc Breneiser, William Hahn, Matthew Meyerson, Paul Van Hummelen, Laura MacConaill. Targeted RNA sequencing improves transcript analysis in cancer samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1115. doi:10.1158/1538-7445.AM2015-1115

Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity

BackgroundMassively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings.MethodsUsing identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.ResultsWe show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations.ConclusionsThe application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

GAPDH, YWHAZ, and RRN18S as control reference genes for gene expression studies on renal cell carcinoma (RCC) formaldehyde-fixed paraffin-embedded (FFPE) tissue samples.

389 Background: It is mandatory to have a control reference gen (RG) to correctly measure gene expression by real-time quantitative PCR (qPCR). The purpose of this study was to test a panel of 12 RGs (Human Endogenous Control Gene Panel, tataabiocenter) in order to select and validate the most appropriate control genes for expression studies on FFPE RCC tissues. Methods: qPCR followed by Normfinder and geNorm-based analysis was employed (GenEx Standard). The study was performed on 9 selected RCC tumor samples with different local stage (T1, T2 and T3). The most representative RCC histologies were also collected; clear-cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC) and cromophobe renal cell carcinoma (cRCC). A commercial pool (Biochain) of 5 cases of normal kidney was analyzed too. All samples were measured in triplicate. Expression levels of RGs: GAPDH, TUBB, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP, GUSB and HPRT1 were measured by qPCR on a Light Cycler 480 (Roche) utilizing Light Cycler 480 SYBR Green I Master (Roche). Results: The analysis of experimental data showed that RRN18S is the most stable gene found in our FFEP RCC samples followed by GUSB and TBP. In contrast, ACTB was found to be the least stable gene between our samples. GAPDH together with YWHAZ was showed as the pair of genes introducing the least systematic error into data normalization (M-value &lt; 1) needed to conduct expression gene studies further. Conclusions: These data suggest that GAPDH, YWHAZ and RRN18S are the most suitable RGs for gene expression profile studies in FFEP RCC. Standardization of RGs will help us to compare results from translational studies on RCC FFPE samples genes profiling. No significant financial relationships to disclose.

Abstract 2196: Genome-wide analysis of DNA methylation patterns in formaldehyde-fixed paraffin-embedded (FFPE) human tumor specimens

Abstract Formalin-fixed paraffin-embedded samples (FFPE) are in many case, the only available source of biological materiel available for translational studies. However the quantity and quality of nucleic acid extracted from FFPE samples is very often challenging (small amount and high level of degradation), which makes genomic analysis in general, and epigenomics analysis in particular, difficult to impossible. Here we are showing that we developed a robust, unbiased and cost effective method for genome-wide analysis of DNA methylation of FFPE DNA. Our method, named STAMP for Sequence Tag Analysis of Methylation Patterns, uses a recombinant protein containing a methyl-CpG binding domain (MBD) to precipitate methylated DNA fragments that are then identified by massively parallel sequencing using the SOLiD sequencer (ABI). We generate ∼15-million sequence tags per specimen and utilize an analytical platform comprised of custom-written R-language algorithms to study genomic DNA methylation patterns. We used this technology to compare the methylation patterns of matched fresh frozen (FF) and FFPE tumor specimens. Tissues had been stored for as long as 6 years. Because the STAMP method utilizes fragmented DNA as starting material we hypothesized that this method would be well-suited to the study of FFPE DNA methylation. Using this method, we found the methylation patterns of the FFPE and matched FF specimens were highly correlated. We successfully detected previously known methylated patterns and found hundreds of novel methylated regions. DNA methylation patterns were observed at all scales across the genome: from whole chromosomes to individual genes. We found that densely methylated elements are often highly conserved or closely associated with gene coding regions and promoters. We identified distinct patterns of DNA methylation surrounding the transcription start and termination sites of all genes. Genome-wide analyses of DNA methylation patterns of FFPE samples using the STAMP assay opens up the possibility to analyze large repositories of tumor specimens as well as samples that have been collected during routine clinical work and clinical trials. Large scales studies of DNA methylation using existing FFPE specimens linked to treatment outcomes may be rapidly translated to new epigenetic prognostic measures for cancer patients. 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 2196.

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

BackgroundCancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies.Methodology/Principal FindingsHere we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples.Conclusions/SignificanceThe application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.