Formalin-fixed Paraffin-embedded RNA Research Articles

Abstract 5781: Development of RNA-Seq library preparation method for highly degraded FFPE RNA and ultra-low RNA input

Abstract RNA sequencing (RNA-Seq) has been widely used for investigating genetic variations, splicing, and gene expression in cancer research. However, RNA-Seq library preparation from archived formalin fixed paraffin embedded (FFPE) samples tends to fail due to both limited quantity and high degradation. We aimed to overcome the sample quality and quantity limitations by leveraging both in-house cDNA synthesis modules and the unique, single-stranded ligation strategy of the IDT xGen cfDNA & FFPE DNA Library Prep chemistry and workflow. This approach delivers high library conversion of input molecules and maximizes high quality data from sequencing. To represent the types of challenging cancer samples that may be lost during traditional RNA-Seq analyses, we collected thirty-seven low quality RNA FFPE samples (RIN <3) from lung, breast, colorectal, thyroid tumors and more. In addition, twenty-one frozen tissue RNA samples and Nine RNA reference materials were used as controls. Regardless of sample origin, 25 ng of total RNA were taken for RNA-Seq library preparation. In-house cDNA synthesis modules were used for the 1st and 2nd strand cDNA synthesis followed by Illumina library generation using IDT’s xGen™ cfDNA & FFPE DNA Library Prep Kit. All sample types generated sufficient high-quality libraries (>1 ug) for hybridization capture enrichment. The RNA-seq libraries were captured using IDT’s custom designed panel with 56 known fusion targets. We detected all expected fusion targets from the tested samples. No correlation was observed between sample quality and the number of genes detected between the different types of samples including the low quality FFPE. To test the lower limits of the workflow, a degraded FFPE sample and a Universal Human Reference RNA (UHR) were used to construct libraries with inputs from 1ng to 10pg. High quality libraries were successfully constructed with as low as 10pg of UHR and 50pg of the FFPE RNA sample. We demonstrated the flexibility of our current cfDNA & FFPE DNA Library Prep whose high conversion rate could be used for constructing libraries from low quality and degraded FFPE RNA in conjunction with our in-house cDNA synthesis modules. Libraries were successfully constructed from ultralow RNA input which could be leveraged for cfRNA applications. The high RT efficiency enables processing FFPE samples with very low-quality scores and provides users with an effective solution for their degraded RNA samples challenges. Citation Format: Ekaterina Star, Noreen Karim, Berlin DeGroen, Michael Salgado, David Wang, Jing Li, Katica Ilic, Sarah Beaudoin, Christopher Vakulskas, Bosun Min, Peilin Chen. Development of RNA-Seq library preparation method for highly degraded FFPE RNA and ultra-low RNA input [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5781.

Characterizing immune-genomic signatures associated with response to immune checkpoint inhibitors (ICI) in patients with metastatic urothelial carcinoma (mUC).

e16582 Background: Response rates with ICI are modest at around 20% in mUC and there is a lack of validated biomarkers to select pts who are likely to derive benefit from ICI. There is an unmet need to develop biomarkers of response and resistance to avoid unnecessary treatment (Tx) and spare physical and financial toxicity. Here we report tumor immune-genomic characteristics associated with response and resistance to ICI in mUC. Methods: We selected 58 mUC pts who received at least 2 cycles of ICI (atezolizumab or pembrolizumab) between 2015-2020 and had pre-Tx archival formalin fixed paraffin-embedded (FFPE) tissues available. Pts were categorized by their response to ICI as responders (R) (Complete Response (CR), Partial Response (PR), and Stable Disease (SD)) (N = 30) or non-responders (NR) (Progressive Disease (PD)) (N = 28). Tumor was macrodissected from FFPE slides and RNA extraction was done using Qiagen Rneasy FFPE kit. 100ng of RNA was used for transcriptomic analysis using the Nanostring PanCancer IO 360 panel. Data normalization and analysis was carried out using nSolver 4.0. Results: Differential expression analysis comparing (R) vs (NR) displayed upregulation of gene signatures associated with Immunoproteasome, Antigen presenting machinery (APM), IFN Gamma, PD-L1, and IFN downstream. Additionally, the tumor inflammation signature (TIS), which is trained to predict response to ICI, was upregulated in (R) vs (NR) (Table 1). Single gene analysis showed trend towards upregulation of ISG15 (logFC = 0.7821, p = 0.00337), TAP1(logFC = 0.7649, p = 0.00422), and STAT1 (logFC = 0.6053, p = 0.00510) in (R), P-values, were above significance threshold after adjustment for multiple comparisons. Receiver operator characteristic (ROC) analysis with area-under the curve (AUC) scores assess capacity of gene signatures to predict response to ICI (Table 1). In our cohort, the median overall survival for (R) was 44.1 months compared to 24.5 months in NR (p = 0.0100). Conclusions: In our cohort of mUC pts treated with ICI, we found an association between signatures of tumor immunogenicity (APM and Immunoproteosome) and anti-tumor immune activity (IFN Gamma, IFN Downstream, TIS) in pre-Tx tissue with favorable response to therapy (including SD). These findings are hypothesis generating and need to be further validated in prospective trials. Ongoing genomic and immunologic correlative studies in our cohort will further help understand comprehensive biomarkers of response and resistance to ICI in pts with mUC. [Table: see text]

Application of the 3\u2032 mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue

BackgroundArchival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation.ResultsThe total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67).ConclusionIn this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

Chemical capping improves template switching and enhances sequencing of small RNAs.

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.

Abstract 4276: Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer

Abstract In proof of concept clinical studies, selecting for patients that are the most likely to respond to a given therapy will increase the chance of demonstrating clinical benefit for a new investigational agent. Archived Formalin-Fixed, Paraffin-Embedded (FFPE) tissue taken at the time of first biopsy and cancer diagnosis are a ready source of material for such patient selection assays; however if the selection assay is RNA-based sequencing based assay, FFPE tumor tissues are typically refractory to standard methods. Here we describe Target Enrichment, an RNA-sequencing technique that enabled analysis of FFPE tumor tissue. The Target Enrichment technique provides the necessary, high quality gene expression data required for identifying the mesenchymal subtype of CRC. In this experiment, RNA was extracted from a set of 192 histologically characterized, archived colorectal cancer (CRC) FFPE tumor tissues with sufficient representation of all Consensus Molecular Subtypes of CRC. A major issue with FFPE tissue is that nucleic acids are not well preserved in the fixation process. The RNA extraction method was carefully optimized in advance toward improving yield and integrity of the total RNA. In order to accommodate the low FFPE RNA integrity, we used Target Enrichment, a technique derived from whole-exome sequencing that isolates and amplifies library fragments corresponding to only the transcribed regions of interest. After testing several protocols, we established a Target Enrichment technique to remove low-quality library fragments that would otherwise interfere and impair sequencing quality. This method allowed us to generate high-quality sequencing data for samples that proved challenging using traditional RNA-seq methods. Fresh-frozen tissue samples were used as a benchmark and our Target Enrichment technique showed comparable performance across multiple sequencing quality metrics including mapping rate, expression profiling efficiency, number of genes detected, also the pass rate. Furthermore, data generated using our Target Enrichment technique aligned with the known frequencies of the Consensus Molecular Subtypes of CRC. Overall, our Target Enrichment technique provides a protocol for generating high quality RNA-seq libraries from highly degraded FFPE RNA. Citation Format: Maximilian Rogers-Grazado, Joachim Theilhaber, Jack Pollard, Alexei Protopopov. Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer [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 4276.

Abstract B46: Development of a fully integrated sample-to-report system for a pan-cancer application

Abstract Introduction: We have developed a new sample-to-report solution for testing solid tissue cancers using the Ion Torrent System and Software. The assay is designed for research applications from either formalin-fixed, paraffin-embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy. The new sequencer is a fully automated system requiring minimal hands-on time and touch points for a user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types. Methods: The assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biologic variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. The assay is optimized for use with the Ion Torrent System. With minimal hands-on time (~15 minutes), the user prepares the system with prefilled reagents strips to start a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: The assay is designed to detect somatic variants in 50 unique genes, where 45 of these genes include substitutions, deletions, and insertions. 14 genes are targeted for copy number variation, and 19 genes are drivers of gene fusions and alternate splice forms. The content has been selected based on published accounts of target actionability and prevalence across multiple cancer types. An example of run setup is shared along with timing (~30min) to input sample information using the Torrent Suite Software. Data are shown from a multiplexed sequencing run using FFPE DNA (10 ng) and RNA (10ng) as input. The automated FFPE run on the Ion Torrent System completed in about 30 hours. Data are also presented from a sequencing run that included multiple liquid biopsy libraries utilizing cell-free total nucleic acid (cfTNA) extracted from plasma. In this run mode, variant calling is optimized for highly sensitive and specific variant calling below 1% allelic frequency for all targeted variant types assay including CNVs, fusions, and alternative splice forms and was accomplished in less than 24 hours. Conclusion: The assay and system enable detection of key oncology variants in 50 genes using both solid tissue and liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in a single 24-hour period with minimal hands-on time and touch-points from the user. This system is not yet released but is in development. Citation Format: Kelli Bramlett, Ru Cao, Jeff Schageman, Yanchun Li, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Oana Lungu, Varun Bagai, Desiree Martin, Khalid Hanif, Jian Gu. Development of a fully integrated sample-to-report system for a pan-cancer application [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B46.

SQUAMOUS CELL CARCINOMA

Background: Head and Neck Squamous cell carcinoma is the sixth most common cancer globally with increasing frequency in developing countries. Despite huge advancement in surgery, radiotherapy and chemotherapy there is a little changed in the overall survival rate for patient with HNSCC over the past few decades. Due to its late diagnosis and lack of availability of reliable biomarker for this disease, its incidence is still on rise. Aims &Objectives: This study was aimed to study the expression of miR-21 in the tumor genesis of HNSCC. The objective of the study is to analyze the expression profile of miR-21 in HNSCC, to study the miRNA expression profile of miR-21 between control and tumor samples, to study the expression profile of miR-21 benign tumors and different categories of HNSCC Tumors on the basis of Histological Differentiation, gender-based Comparison of Benign and Malignant HNSCC Tumors, age-based Comparison of Benign and Malignant HNSCC Tumors, tumor Sitebased Comparison of Benign and Malignant HNSCC Tumors. Study Design: Case-control study. Study Setting: The University of Lahore. Period: June -2014 June -2105. Materials & Methods: In this research, 43 Formalin-fixed paraffin embedded (FFPE) tissue samples (31 malignant HNSCC samples and 12 benign tumors from the same region) of both genders and aged 15-80 years were included in this study. 31 cases were malignant tumors were further consisted of 14 well-, 11 moderately- and 6 poorly differentiated tumors. Total RNA was extracted using PureLink FFPE RNA Isolation Kit and Two-Step RT-PCR was performed. TaqMan primer/ probe sets were used for the target miRNA- 221, while RNUB6 was the normalization control.By calculating __Ct and fold change difference according to Livak method. late onset disease the Relative quantification was done to determine the level of expression of miRNA-221. Tumor site did not show any effect on miR-21 expression levels. Results: Our results showed that the malignant samples have higher expression level of miR-21 then benign control samples. Significantly higher expression was observed in moderately and poorly categories of HNSCC. Gender-based expression showed that females had higher level of expression, while it was found that its expression is high in late onset disease. Tumor site did not show any effect on miR-21 expression levels. Conclusion: Our miRNA expression profile provides a potential strategy for finding new head and neck squamous cell carcinoma (HNSCC) molecular targets. miR-21 could be regarded as potential diagnostic marker in HNSCC. 

Feasibility of developing reliable gene expression modules from FFPE derived RNA profiled on Affymetrix arrays.

The reliability of differential gene expression analysis on formalin-fixed, paraffin-embedded (FFPE) expression profiles generated using Affymetrix arrays is questionable, due to the high range of percent-present values reported in studies which profiled FFPE samples using this technology. Moreover, the validity of gene-modules derived from external datasets in FFPE microarray expression profiles is unknown. By generating matched gene expression profiles using RNAs derived from fresh-frozen (FF) and FFPE preserved breast tumors with Affymetrix arrays and FF/FFPE RNA specific amplification-and-labeling kits, the reliability of differential expression analysis and the validity of gene modules derived from external datasets were investigated. Specifically, the reliability of differential expression analysis was investigated by developing de-novo ER/HER2 pathway gene-modules from the matched datasets and validating them on external FF/FFPE gene expression datasets using ROC analysis. Spearman's rank correlation coefficient of module scores between matched FFPE/frozen datasets was used to measure the reliability of gene-modules derived from external datasets in FFPE expression profiles. Independent of the array/amplification-kit/sample preservation method used, de-novo ER/HER2 gene-modules derived from all matched datasets showed similar prediction performance in the independent validation (AUC range in FFPE dataset; ER: 0.93–0.95, HER2: 0.85–0.91), except for the de-novo ER/HER2 gene-module derived from the FFPE dataset using the 3'IVT kit (AUC range in FFPE dataset; ER: 0.79–0.81, HER2: 0.78). Among the external gene modules considered, roughly ~50% gene modules showed high concordance between expression profiles derived from matching FF and FFPE RNA. The remaining discordant gene modules between FF and FFPE expression profiles showed high concordance within matching FF datasets and within matching FFPE datasets independently, implying that microarrays still require improved amplification-and-sample-preparation protocols for deriving 100% concordant expression profiles from matching FF and FFPE RNA.

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens.

-Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18-24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

Abstract LB-263: Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths. There are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have less than a 7% 5-year survival rate and the median survival from the time of diagnosis is less than 12 months. Next-Generation Sequencing (NGS) together with in vitro/in vivo functional studies is likely to identify and validate new biomarkers of PDAC onset, progression and therapy resistance. Importantly, the substantial archives of formalin-fixed, paraffin-embedded (FFPE) samples from PDAC patients are likely to be a rich resource for linking molecular signatures to relevant clinical data. However, NGS methods on FFPE samples are severely hindered because extracting high-quality nucleic acid material from these samples is time-consuming and inefficient. We have sought to develop novel methods for improving the extraction of high-quality RNA from FFPE samples within the context of commercially-available FFPE RNA kits and protocols. Together with researchers and clinicians at Claremont BioSolutions, AntiCancer Inc. and UCSD our laboratory has developed a novel nucleic acid extraction method that significantly increases RNA yield and integrity from PDAC cell line and Patient-Derived Xenograft FFPE samples. By briefly (<5 minutes) integrating the newly designed Claremont BioSolutions’ microhomogenizer (mH) tool within the commercially available Qiagen FFPE RNA extraction protocol, RNA recovery from these samples is increased by approximately 3-fold while maintaining standard 260/280 ratios (2.03 +/- 0.02 w/ mH step) and high RNA Quality Index (RQI) values (7.3 +/- 0.6 w/ mH step). Bioanalyzer testing further demonstrated that the mH-purified FFPE RNA was longer. Previous studies have revealed that PDAC cell gene expression signatures vary significantly when cells are propagated in vitro versus in vivo as subcutaneous or orthotopic xenografts. Notably, we found that the previously published expression patterns for KRas dependency genes within these three microenvironments were most accurately reproduced when extracting PDAC FFPE RNA with our mH-based method. Finally, we used our mH-based method to test the effects of the in vivo tumor microenvironment (TME) on the expression trends of a panel of novel PDAC biomarkers. In this regard, we demonstrate that PEAK1 and MST1R expression levels are decreased and increased, respectively, by over 100 fold in the orthotopic microenvironment relative to the subcutaneous microenvironment. These results reveal the critical nature of the tumor microenvironment when evaluating the clinical relevance of new biomarkers in cell lines or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to positively impact the FFPE-RNA-NGS workflow for cancer biomarker identification/validation. Citation Format: Malachia Hoover, Yvess Adamian, Mark Brown, Ali A. Maawy, Robert Hoffman, Michael Bouvet, Robert Doebler, Jonathan A. Kelber. Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction. [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 LB-263.

Selective Depletion of Abundant RNAs to Enable Transcriptome Analysis of Low-Input and Highly Degraded Human RNA.

Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin-fixed paraffin-embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non-coding and other non-poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods. © 2016 by John Wiley & Sons, Inc.

Abstract 4901: Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring

Abstract Accurately measuring the expression of genes in formalin-fixed paraffin embedded (FFPE) tumor tissues has long been a struggle due to the inherent degradation of RNA isolated from these materials. Accurate quantification of gene expression levels in FFPE samples can enable the testing of biomarker hypotheses in the clinic and can potentially be used for patient stratification or selection in clinical trials. Platforms such as Fluidigm, Nanostring and microarrays are currently the high-throughput technologies utilized. Each of these platforms has different drawbacks and challenges such as lack of sensitivity, reproducibility or dynamic range when working with degraded FFPE samples. Currently, whole transcriptome RNA sequencing (RNA-Seq) is the only platform that offers a truly high-throughput, sensitive, and reproducible method to accurately quantify mRNA levels in RNA derived from FFPE samples. RNA-Seq, however has not yet been widely evaluated and adapted for use with degraded FFPE samples. Here we report the direct comparison of whole transcriptome RNA-Seq with two platforms that are currently compatible with FFPE derived RNA, Fluidigm and Nanostring, to determine the accuracy and feasibility of using this technology on degraded RNA. Utilizing a collection of matched fresh frozen (FF) and FFPE samples we analyzed gene expression using all three platforms to allow for a direct evaluation of each technology. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE derived RNA. By titrating down the input of FFPE RNA into the RNA-Seq library prep we were also able to define the optimal input of FFPE RNA needed to accurately and reproducibly quantify gene expression. This work allows for a systematic comparison of different gene expression platforms for their use with degraded FFPE RNA. Citation Format: Erica B. Schleifman, Maipelo Motlhabi, Craig Cummings, Rin Nakamura, Linda Bosch, Rajesh Patel, An Do, Andrew Watson, Thomas Sandmann, Walter Darbonne, Ian McCaffery, Eric Peters, Rajiv Raja. Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring. [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 4901. doi:10.1158/1538-7445.AM2015-4901

Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-κB activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-κB targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-κB target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting.

Abstract 3563: New tools for RNA-sequencing of FFPE tumor samples

Abstract Next generation RNA sequencing is an ideal platform to leverage archival formalin-fixed paraffin-embedded (FFPE) tissue samples for cancer research, as it offers extensive functional genomic information including transcriptome expression profiling, differential expression analysis and fusion detection. However, both RNA degradation from the formalin fixation process as well as limited sample material, pose major challenges for RNA sequencing of FFPE samples. Here we describe a novel method for generating whole-transcriptome RNA sequencing libraries from moderate to poor quality FFPE samples starting with as low as 20ng total RNA. This approach provides increased transcriptome coverage and robust measurement of gene expression levels compared to currently available FFPE RNA sequencing methods. Furthermore, we demonstrate that we can detect both known and novel gene fusions in clinical tumor samples that are potential drivers of disease. We anticipate this new technology will enable functional genomic studies on numerous FFPE tumor samples that would not previously be possible, providing invaluable information for developing cancer diagnostics and therapeutics. Citation Format: Lisa Watson, Smita Pathak, Stephen Gross, Irina Khrebtukova, Gary Schroth. New tools for RNA-sequencing of FFPE tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2014-3563

Methods for rapid extraction of high‐quality RNA from FFPE cancer tissue (1048.14)

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths. Because there are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have a dismal prognosis. Substantial archives of formalin‐fixed, paraffin‐embedded (FFPE) samples from PDAC tissue have been established and linked to clinical data. However, identifying biomarkers from these tumors is severely hindered because extracting high‐quality nucleic acid material from FFPE samples is time‐consuming and inefficient. Thus, developing improved methods for extracting high‐quality RNA/DNA from FFPE samples represents an unmet need in the fields of cancer biology and Next‐Generation Sequencing (NGS). We have tested Claremont BioSolutions’ microhomogenization (mH) tools in combination with commercial FFPE RNA extraction kits for their ability to improve high‐quality RNA recovery from PDAC xenograft samples. Our results demonstrate that the addition of brief (< 5min) mH steps improves total RNA yield up to 3‐fold over current methods, as well as RNA purity and length, as characterized by 260/280 ratios (2.03 +/‐ 0.02 w/ mH step) and chip‐based electrophoresis (RNA Quality Index = 7.3 w/ mH step). These results have important implications for streamlining the FFPE‐RNA‐NGS workflow as a realistic approach for cancer biomarker identification.Grant Funding Source: n/a

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Abstract 4127: Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM.

Abstract Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. We have developed methods for small RNA and whole transcriptome analysis of FFPE samples on the Ion Torrent PGM™ System using Ion total RNA-Seq Kit v2 and RiboMinus Eukaryote System v2. Paired FFPE and fresh-frozen RNA samples from lung adenocarcinoma tissue were used in this study. Small RNA libraries were prepared using the Ion Total RNA-Seq kit v2 with modified protocol, followed by sequencing on PGM™ system. Results show that small RNA extracted from FFPE sample was successfully converted to small RNA library. Although percentage of reads mapped to miRBase was slightly lower when compared to fresh-frozen sample, the overall miRBase coverage and detection sensitivity were comparable. For whole transcriptome analysis, RiboMinus Eukaryote System v2 kit was used to deplete rRNA from FFPE samples, followed by library construction using the Ion Total RNA-Seq kit v2 with a modified protocol. Significant reduction in rRNA reads was observed after sequencing. High RefSeq correlations were observed between fresh-frozen and FFPE samples. In conclusion, a full spectrum of gene expression data, from both small RNA and whole transcriptome, were generated from FFPE samples using Ion Total RNA-Seq kit v2 with modified protocols. High correlations were observed between paired fresh-frozen and FFPE samples, demonstrating the reliability of the modified workflow for gene expression profiling on FFPE samples. This study provides feasibility for systematic gene expression analysis on FFPE samples from cancer and other diseases. Citation Format: Jian Gu. Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2013-4127

Abstract 3500: Analysis of the prevalence and prognostic significance of 92 endpoints using high throughput multiplexed qPCR In archival tissue from multiple NSCLC trials.

Abstract Over one third of all cancer related deaths worldwide are caused by non-small cell lung cancer (NSCLC). Until recently, tumor histology was rarely a factor in treatment decisions. However, this paradigm is shifting with the recent emergence of molecularly targeted therapies and next-generation chemotherapies. To better define the molecular sub-types of NSCLC that associate with active therapies and to evaluate their prognostic significance in historical clinical trials, we developed a microfluidics-based multiplexed PCR platform that enables analyses of 96 gene transcripts in 96 samples using material from as little as a single biopsy slide. The multiplexed assay panel consists of Taqman assays for markers of histology, chemo-resistance, prognosis, oncogenic pathway activity, epithelial vs. mesenchymal biology, as well as NSCLC drug targets. We report here on the development and validation of this 96-gene transcript-profiling panel. Using titrations of several lung biopsy formalin-fixed paraffin embedded (FFPE) tissues, we show that as little as 10 ng of FFPE RNA is feasible and yields robust gene expression data across all assays of the profiling panel. Using this technology, we tested archival lung biopsy samples from multiple Phase II and III studies to assess the prevalence and potential prognostic significance of 92 endpoints in parallel. We identified several markers that were strongly associated with histology as defined by TTF1 or p63 expression. In addition, several of the these markers were also associated with longer survival regardless of therapy, and will be discussed. These data suggest that retrospective analyses of clinical trial samples using Fluidigm-based expression analysis can be used to better inform future trial design in NSCLC. Citation Format: An D. Do, Teiko Sumiyoshi, Yulei Wang, Erica Schleifman, Yuanyuan Xiao, Barbara Klughammer, Cheryl V. Wong, Ilma Abbas, Ling Fu, Lukas C. Amler, Rajiv Raja, David S. Shames. Analysis of the prevalence and prognostic significance of 92 endpoints using high throughput multiplexed qPCR In archival tissue from multiple NSCLC trials. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3500. doi:10.1158/1538-7445.AM2013-3500

Abstract C32: Non-uniform recovery of microRNAs from formalin-fixed paraffin-embedded tissue

Abstract Background and Objective: The availability of formalin-fixed paraffin-embedded (FFPE) tissue provides an enormous resource for the discovery of molecular markers. Several reports indicate that microRNA (also referred to as miRNA or miR, a class of small noncoding RNAs 19-24 nucleotides in length that negatively regulate translation of protein-coding mRNAs) is preserved and readily extracted from FFPE tissue. Our goal in this study was to identify a method for optimal recovery of miRNA from FFPE prostate tissue. Experimental Approach: We compared five commercially available kits that are designed to isolate miRNA from FFPE tissue: SABiosciences RT2 FFPE RNA Extraction Kit (kit 1), Applied Biosystems RecoverAll Total Nucleic Acid Isolation Kit (kit 2), Roche High Pure miRNA Isolation Kit (kit 3), Qiagen miRNeasy FFPE Kit (kit 4), and MO BIO UltraClean FFPE RNA Isolation Kit (kit 5). All kits were used to process serial sections (10 μm thickness) from the same FFPE tissue block. We assessed purity and yield of total RNA based on NanoDrop analysis of absorbance at 230, 260, and 280 nm. Equal amounts of total RNA were converted to cDNA using reverse transcriptase (RT) and miR-specific primers, and real-time PCR using TaqMan miR probes was used to quantitate 378 miRs and 3 small nucleolar RNA species (TaqMan Low Density Array). Results: The total RNA yield/section was 5.3, 1.6, 3.0, 2.5, and 3.2 μg from kits 1-5, respectively (average of two independent extractions of serial sections from the same FFPE tissue block). Nevertheless, as expected, analysis of the same amount of RNA from each kit showed that all samples had the same level (based on CT) of the small nucleolar RNA RNU48; thus, the recovery of RNU48 per unit amount of total RNA was the same for all kits. Unexpectedly, however, only a limited number of miRs were present at the same level (same CT) in all kit preparations; thus, per unit amount of total RNA, the recovery of only a limited number of miRs was independent of the method used to isolate RNA. For a substantial proportion of miRs, recovery was dependent on the kit used, and recovery differed among miRs. To quantitate differences in recovery, we normalized transcript levels to RNU48 and calculated the ratio of the level of each miR in different kits. Ratios of miRx(kit a)/miRx(kit b) = 1.0 vs. miRy(kit a)/miRy(kit b) = 2.0 would mean that the recovery of miRs x and y is not uniform. Comparing kit 4 vs. kit 1, 10% of miRs had a kit 4/kit 1 ratio &amp;lt;0.75, 17% had a ratio ∼1, 28% had a ratio between 1 and 2, 33% had a ratio between 2 and 4, and 12% had a ratio &amp;gt;4; these data indicate non-uniform recovery of different miRs. This non-uniform recovery of miRs was independent of relative abundance, affecting both high abundance and low abundance transcripts. Notably, the observation of non-uniform recovery of different miRs required the analysis of different kit preparations. Conclusions: Different miRs are not uniformly recovered from total RNA of FFPE tissue. This suggests that a miR profile in an RNA extract may not accurately represent the tissue in vivo, and that the profile observed may depend on the RNA extraction method used. We suggest that analysis of miRs in tumor relative to nontumor tissue of the same patient will provide a way to normalize for differences in miR recovery, and thus provide a reliable approach to identify miRs as markers of disease. The basis for non-uniform recovery of miRs from FFPE tissue is not known, nor whether this effect is unique to FFPE tissue. [Supported by a grant from the DOD Prostate Cancer Research Program.] Citation Format: Zizheng Hou, Evelyn R. Barrack. Non-uniform recovery of microRNAs from formalin-fixed paraffin-embedded tissue [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C32.