From Formalin-fixed Paraffin-embedded Samples Research Articles

Abstract C094: Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is a significant public health issue in Colombia, It occupies third place in incidence and ranking as the fifth leading cause of cancer-related deaths. Understanding the sociodemographic, clinicopathological, and molecular characteristics of CRC patients is crucial for prognosis, effective management, and treatment. Several molecular alterations and clinicopathological variables have been associated with the prognosis of the disease in different populations; However, we do not know if there are differentially expressed genes specific to Colombian population that are associated with CRC prognosis. Objective: Perform differential expression analysis using RNA-seq data obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples from Colombian patients with CRC. Methodology: For this preliminary analysis were included 5 patients diagnosed with CRC between 2010-2014 at Instituto Nacional de Cancerologia from Colombia. From each patient, formalin-fixed and paraffin-embedded (FFPE) samples of tumor tissue and non-tumor tissue from the surgical remnant were obtained. Extracted RNA was used to perform whole-genome RNA sequencing (RNA-seq) at a depth of 100X. Bioinformatic analysis was performed using Fastqc, Cutadapt, Ribodetector, and STAR on a Linux server. The DESeq2 package in RStudio was used to perform the differential expression analysis between tumor samples vs. non-tumorous. A significant differential expression was considered using an FDR-adjusted p ≤0.05 and a log fold change of 2. Results and discussion: A list of 18 differentially expressed genes was obtained which 5 underexpressed stand out: B3GNT6 (LFC:-10.66; padj=0.0093), ITLN1 (LFC:-6.23; padj=0.0231), NR3C2 (LFC:-3.34; padj=0.0231), NLGN1 (LFC:- 0.46; padj=0.0301), CEACAM7 (LFC:-5.91; padj= 0.0301); and one overexpressed gene: RPL9 (LFC:5.44; padj=0.0267). These genes have been previously characterized and associated with the progression and prognosis of this disease in other populations. Conclusions: 6 differentially expressed genes were found using RNA-seq in FFPE samples. These genes have been associated with CRC progression and prognosis in previous studies, but have not been evaluated in the Colombian population. From this perspective, we propose to continue with the analysis by increasing the number of samples and evaluating their association with the different clinicopathological variables and their impact on the prognosis of the disease. Citation Format: Wendy J. Montero Ovalle, Silvia J Serrano Gómez, Diego F Ballen Lozano. Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C094.

MDB-12. NANOPORE SEQUENCING AS A CUTTING-EDGE TECHNOLOGY FOR MEDULLOBLASTOMA CLASSIFICATION

Abstract INTRODUCTION Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors, divided into four distinct molecular subgroups (WNT, SHH, group 3, and group 4) with its individual prognosis. A more extensive classification of MB has recently been provided, identifying numerous subtypes, some with poor prognosis, leading to a significant number of patients succumbing to the disease while many survivors experience long-term side effects, highlighting the need for efficient subgrouping methods. Distinct genome-wide DNA methylation profiles characterize each MB subgroup, and the advent of Nanopore DNA sequencing has positioned itself as a reliable technique for comprehensive copy number and methylation profiling. MATERIAL AND METHODS We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB, on a discovery cohort of 45 frozen MB samples, benchmarked against the gold standard EPIC array, and subsequently validated on a cohort of 116 MB samples. RESULTS The majority of MB in both the discovery cohort (43/45; 95.6%) and the validation cohort (110/116; 94.8%) were accurately subgrouped by Nanopore sequencing. Flongle flow cells for 18 MB allowed for a more rapid and cost-effective analysis, with 94.4% (17/18) being correctly classified. Additionally, Nanopore sequencing allowed us to correctly subtype 24/30 (80.0%) within MB groups. DISCUSSION We provided proof of concept that Nanopore sequencing can subgroup MB using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples and cell-free DNA obtained from cerebrospinal fluid. This first large-scale study establishes the proof of concept that this modern and innovative technology is well-suited for MB classification. We confirm the use of Nanopore sequencing on circulating DNA and present an initial exploration of its application on DNA extracted from FFPE samples. Its ability to deliver quick and cost-effective results firmly establishes Nanopore sequencing as a game-changer in the field of MB classification.

Abstract 7627: First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan

Abstract Introduction The ERBB2 companion diagnostic program aims to expand the intended use of the Oncomine ™ Dx Target Test (ODxTT) to determine ERBB2 single nucleotide variants (SNVs) in exons 8, 17, 18, 19, 20, 21 and 22 and exon 20 insertions from formalin-fixed, paraffin-embedded (FFPE) tumor samples in non-small cell lung cancer (NSCLC) patients who may benefit from targeted drug therapy. Methods The ODxTT is a qualitative in vitro diagnostic test that uses targeted high throughput, parallel-sequencing technology to detect SNVs, deletions, insertions, and CNVs in genes from DNA and fusions from RNA isolated from FFPE tissue samples using the Ion PGM ™ Dx System. Analytical validation (AV) studies interrogated ODxTT’s ability to detect ERBB2 mutations. The clinical validation (CV) study involved a method comparison/concordance analysis of ERBB2 mutations between the ODxTT and the TruSight Tumor 170 (TST170) assay, as well as a series of clinical trial assays (CTAs). The CV study was conducted using clinical specimens from 91 patients enrolled in Destiny-Lung01 and 121 stage-matched commercially procured samples. The aim was to evaluate positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) for CTAs and TST170 assay. Results A total of 91 ERBB2 subjects were included in the full analysis of the Destiny-Lung01 clinical trial. 49 samples had valid ODxTT results available and 42 samples were unevaluable which included 13 unknowns and 29 test not performed. The 49 patient samples that were evaluable were used to assess the clinical efficacy based on ODxTT results. In the series of 121 samples that were used to assess clinical efficacy in the CV study, 109 had evaluable ODxTT results. The PPA, NPA and OPA of ODxTT to the CTAs when excluding unknowns were 98%, 100% and 99.4%, respectively, and based off the Clopper-Pearson method for binomial distribution a 95% lower bound confidence interval (CI) were 89.6%, 96.6% and 96.5%, respectively. There was one discordant sample with a negative ODxTT and a positive CTA result (false negative). The PPA, NPA and OPA of ODxTT to TST170 assay were 100%, 99.1% and 99.3% respectively with a 95% lower bound CI of 90.8%, 95.0% and 96.3%, respectively. There was one discordant sample with a negative TST170 and a positive ODxTT result (false positive). Conclusion AV and CV studies successfully verified and validated the ODxTT for the detection of ERBB2 mutations in NSCLC FFPE samples. The 6 AV studies met the necessary product requirements and acceptance criteria to detect clinical samples with ERBB2 mutations. Additionally, the CV study clinical accuracy and clinical efficacy results demonstrated the safety and effectiveness of the use of the ODxTT in FFPE samples as an aid to identify NSCLC patients eligible for treatment with the Daiichi Sankyo lung cancer therapeutic T-DXd. Citation Format: Thomas Ha. First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan [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 7627.

Abstract 3142: Enabling whole genome sequencing analysis from FFPE specimens in clinical oncology

Abstract Whole genome sequencing (WGS) enables the identification of all cancer associated biomarkers in a patient’s tumor genome. Whilst fresh frozen (FF) derived WGS data provides optimal data quality, the majority of clinical biospecimens are from formalin fixed paraffin embedded (FFPE) tissue which results in DNA damage and an increase in artifactual mutation calls. Development of analytical frameworks tailored to FFPE derived WGS data can unlock the potential of genome profiling in clinical oncology. We performed comprehensive WGS analysis on 58 matched FF/FFPE specimens derived from 3 cancer centers. Consensus calling detected high-confidence somatic mutations across variant classes including: single nucleotide variants (SNVs), insertions/deletions (indels), structural variants (SVs) and copy number aberrations (CNAs). For each sample, genome-wide mutational patterns including tumor mutational burden (TMB), SNV/indel signatures, and homologous recombination deficiency (HRD) scores were estimated. We developed a random forest based framework using 33 features to learn mutation patterns associated with FFPE artifacts and implemented a filtration strategy for FFPE derived WGS data within a clinical prototype analytical workflow. We identified a high degree of concordance (~91%, n=192/210) for oncogenic variants between FF/FFPE WGS data. Comparison of small mutation calls presented an average 2-fold increase in FFPE samples with a range up to 152x for SNVs and 43x for indels. However, this was not the case for SVs: -0.4x (range -0.8-1.4). We demonstrate that genome-wide mutation patterns were significantly affected, impacting estimates for TMB, HRD and signature contributions. On average, TMB was overestimated in FFPE (median=10.3, range: 1.4-94) versus FF (median=3.4, range:0.04-29.6). For 7 patients with evidence of HRD in FF, HRD scores did not reach statistical significance in FFPE. Mutational signatures in FFPE were enriched for COSMIC signatures 37 and 5. Our artifact classification model achieved ROC AUC of 97.5% and precision-recall of 98.9%. Post artifact filtration, precision in SNV/indel calling was increased from 49.3% to 93.4% and 61.8% to 82.7% respectively with no effect on driver alterations. This increased global signal concordance drastically, with comparable TMB scores (median 2.4; range .03-26.1) and improved cosine similarity for SNV/indel signatures (median 0.98; range 0.40-1). HRD was successfully detected in 7/7 patients from FFPE derived data post filtering with probability scores ranging from 0.76-1. We demonstrate that FFPE specimens harbor a variable increase in artifactual mutation burden in SNV/indels but not in SVs. We propose an effective filtering procedure which successfully removes FFPE related artifacts enabling accurate profiling of clinically relevant driver mutations and genome-wide mutation patterns from readily available FFPE-derived tumor specimens. Citation Format: Dylan Domenico, Gunes Gundem, Max F. Levine, Juanes E. Arango-Ossa, Pauline Robbe, Georgios Asimomitis, Cassidy Cobbs, Emily Stockfisch, Janine Senz, Dawn Cochrane, Neeman Mohibullah, Neerav Shukla, Sohrab P. Shah, Andrew McPherson, Anna Schuh, Andrew L. Kung, Elli Papaemmanuil. Enabling whole genome sequencing analysis from FFPE specimens in clinical oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3142.

Abstract 3728: A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types

Abstract RNA sequencing (RNA-seq) has become an invaluable tool in the study of biology, but often requires multiple days to process samples from total RNA to final libraries that can be loaded on to a sequencer. Researchers use RNA-seq to gain insight into whole tumor gene expression, and in combination with other omics data make treatment decisions. In cancer research, time taken to yield sequencing results can be critical for clinical samples. Within oncology research, libraries are often prepared from RNA sourced from formalin-fixed, paraffin-embedded (FFPE) tissues which can be particularly challenging and is not compatible with all library preparation methods. Here, we present a new protocol that allows users to easily generate robust RNA-seq libraries from a variety of sample types in a single day from 25-250 ng of total RNA. Compatible with poly(A) enrichment or ribosomal RNA depletion, this new workflow has reduced preparation time by about 40% compared to current library prep kits without sacrificing quality of libraries or any sequencing metrics, including 5’-3’ coverage, GC bias. Our new method provides equivalent transcript expression in FFPE samples when compared with existing protocols. In addition to master mixed components and reduced incubation times, the reduction in bead cleanup steps significantly reduces hands-on time for library preparation, simplifying and streamlining the process. This new library preparation method is also ideal for processing samples of various inputs with a single condition at every step throughout the protocol, without modifying cycling conditions or dilutions based on input amount. This workflow provides a great advantage in efficiently generating RNA-seq libraries while simultaneously maintaining high quality sequencing results and compatibility with both high- and low-quality RNA such as FFPE. Citation Format: Chelsea Pinegar, Dora Posfai, Gautam Naishadham, Bradley W. Langhorst, Keerthana Krishnan. A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3728.

Whole exome sequencing of FFPE samples—expanding the horizon of forensic molecular autopsies

Forensic molecular autopsies have emerged as a tool for medical examiners to establish the cause of death. It is particularly useful in sudden unexplained deaths where the cause of death cannot be determined with a regular medical autopsy. We provide the first study of exome data from formalin-fixed paraffin-embedded samples (FFPE) paired with data from high-quality blood samples in forensic applications. The approach allows exploration of the potential to use FFPE samples for molecular autopsies and identify variants in extensive exome data. We leverage the high uniformity of the hybridization capture approach provided by Twist Bioscience to target the complete exome and sequence the libraries on a NextSeq 550. Our findings suggest that exome sequencing is feasible for 24 out of a total of 35 included FFPE samples. When successful, the coverage across the exome is comparatively high (> 90% covered to 20X) and uniform (fold80 below 1.5). Detailed variant comparisons for matched FFPE and blood samples show high concordance with few false variants (positive predictive value of 0.98 and a sensitivity of 0.97) with no distinct FFPE artefacts. Ultimately, we apply carefully constructed forensic gene panels in a stepwise manner to find genetic variants associated with the clinical phenotype and with relevance to the sudden unexplained death.

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples.

Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics. Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex© Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]. The combination of FusionPlex© Solid tumor with ArcherDX® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens. Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

A comparative study of RNA yields from museum specimens, including an optimized protocol for extracting RNA from formalin-fixed specimens

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

Abstract 84: Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform

Abstract Accurate and sensitive identification of gene fusions is critical to understanding disease mechanisms and the development and selection of optimal treatment regimens for cancer patients. Typically, these fusions are detected using low-resolution karyotyping, low throughput and biased FISH assays, or RNA sequencing approaches; however, the accuracy and sensitivity of gene fusion detection can be limited by factors such as low transcript abundance, transcript length, RNA degradation from formalin fixed paraffin embedded (FFPE) tissues, or the limited availability of fresh biopsy samples for RNA extraction. To address these limitations, we developed a novel approach to identifying gene fusions from FFPE samples using the Arima-HiC platform and short-read sequencing. We performed pan-cancer analysis on 12 FFPE adult tumor biopsies, each with gene fusions known to be clinically actionable. With this newly developed Hi-C workflow we identified all the known gene fusions with 100% sensitivity in the samples including those involving ALK, NTRK3, ROS1, FGFR2, and SS18 genes. This approach also revealed that the NTRK3 gene fusion was the result of a more complex rearrangement within chr12, within chr15, and between chr12 and chr15. Additionally, we detected the presence of numerous structural variants per sample in addition to the known gene fusion in each sample. For example in a bile duct tumor, Arima-HiC detected an FGFR2-EEA1 gene fusion, as well as 25 other structural variants genome-wide including in a CPD-LASP1 gene fusion This gene fusion has not been reported to our knowledge, however, LASP1 is a reported 3’ fusion partner with MLL in leukemia. While these findings are not clinically actionable today, the unbiased, accurate, and sensitive detection of structural variants from frozen and FFPE solid tumor biopsies may facilitate further research into their relationship between disease mechanisms and clinical outcomes. Taken together, these findings demonstrate the analytical utility of Arima Hi-C sequencing technology to provide both chromosome-scale and gene-level resolution for the detection of structural variants in tumor biopsies samples. This workflow can provide improved access to critical genomic information from FFPE blocks for the identification of pathognomonic and druggable gene fusion events and other structural variants across tumor types. Citation Format: David Jacob Hermel, Kristin Sikkink, Derek Reid, Anthony Schmitt, Darren S. Sigal. Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 84.

Abstract 4104: Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology

Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of <4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4104.

Abstract 5795: Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA)

Abstract Introduction: The oncoReveal™ Dx Lung and Colon Cancer Assay (oRDx-LCCA) is an amplicon-based targeted next generation sequencing (NGS), in vitro diagnostic test that detects DNA variants across 22 genes, including single nucleotide variants (SNVs) and insertions and deletions (INDELs) in KRAS and EGFR, from DNA isolated from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) tumor tissue specimens. It is approved by the FDA as a companion diagnostic (CDx) for identifying approved therapeutic treatments for KRAS G12/G13 wild-type variants in CRC, and EGFR L858R variants and exon 19 in-frame deletions for NSCLC. oRDx-LCCA can detect non-CDx variants such as EGFR T790M, G719X and exon 20 insertions in NSCLC and KRAS Q61X and A146T and BRAF V600E in CRC. Variants are detected using Pillar Biosciences’ proprietary PiVAT® (Pillar Variant Analysis Toolkit) software. Methods: DNA input range was evaluated at 6 concentrations between 5 - 160ng in duplicate using DNA extracted from 15 clinical FFPE samples containing SNVs and INDELs from representative targeted mutations. Accuracy was determined by comparing detected variants with an externally validated comparator (CompO) across 208 samples (84 CRC, 124 NSCLC). Limit of detection (LoD) was determined by serial dilution of KRAS, EGFR, or BRAF positive FFPE samples in normal DNA to detect variant allele frequency (VAF) between 1 - 10%. Reproducibility tests were performed across 3 sites, 9 runs, 6 operators, 3 reagent lots and 3 thermocyclers, resulting in a total of 360 libraries. The non-inferiority (NI) statistical test was used to compare the oRDx-LCCA with an FDA approved CDx assay for EGFR exon 19 deletions and L858R mutations (CompQ) and for KRAS G12/G13 mutations (CompC). Synthetic FASTQ files were created at VAFs above and below the variant detection threshold for CDx mutations and used to validate quality control parameters, mean coverage, and on-target rate. Results: Effective DNA input range was determined to be between 10 - 80ng. LoD’s for KRAS G12/G13, EGFR L858 and exon 19 deletion, and BRAF V600E were established at < 4%. Positive percent agreement (PPA) and negative percent agreement (NPA) between oRDx-LCCA and CompO were > 99% each. Across all variables tested for reproducibility, the average percent positive agreement (APA) and average percent negative agreement (ANA) were each > 95%. NI margin was calculated to be < 10% between oRDx-LCCA and CompQ across all NSCLC samples and between oRDx-LCCA and CompC across all CRC samples. A total of 61 synthetic FASTQ datasets were created to validate the functionality and accuracy of PiVAT including VAF thresholds for 126 SNV and INDEL CDx variants. Conclusion: oRDx-LCCA is a highly accurate FDA approved IVD assay and PiVAT is a powerful FDA approved software tool for the detection of clinically relevant KRAS variants in CRC and EGFR variants in NSCLC and determination of approved therapy. Citation Format: Gloria Chan Johnson, Jordan Aldersley, Nicholas Lodato, Jamie Lim, Ian Flockhart, Mark Urbin, Yue Ke, Sean Polvino, Martin Zillmann, Gang Song, Zhaohui Wang. Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5795.

Evaluation of the ability of Idylla EGFR to differentiate result of ARMS EGFR in gray area and identify rare mutation variants.

e15033 Background: Molecular testing involving Epidermal growth factor receptor ( EGFR) mutations is recommended for patients with non-small cell lung cancer (NSCLC), especially with lung adenocarcinoma, and is nowadays an essential element of precision medicine. Although amplification-refractory mutation system (ARMS) is one of the most commonly used methods for EGFR mutation detection, it can only detect variants in known gene loci and give indistinct results in gray area occasionally, which may be caused by the input of poor DNA quality or low-abundance variants. Idylla EGFR assay can detect 51 EGFR mutations directly from formalin-fixed, paraffin-embedded (FFPE) samples within 2.5 hours with < 2 minutes of hands-on time, without manual data analysis need, whose accurate and robust have been proven among Caucasian and Chinese patients. But the assay’s ability to detect EGFR mutation in low abundance which may lead the result of ARMS EGFR in gray area or rare variants has never been evaluated. Methods: FFPE samples were retrospectively collected from 98 lung cancer patients followed by EGFR testing using the Idylla system, which were also previously assessed with ARMS-PCR. Forty-six of them were in gray area by ARMS-PCR of EGFR and need to be retested by ARMS-PCR or validated by the third method. In another 52 cases, no mutations were found by ARMS-PCR of EGFR, but mutations were found by next-generation sequencing (NGS). The ability of Idylla EGFR to detect cases with result in gray area and rare variants were analyzed. Results: In 46 cases with result in gray area by ARMS-PCR of EGFR, after retesting or validation by the third method, 37 of them had the same result with Idylla mutation test, with an overall concordance of 80.4%. Idylla EGFR test missed 8 mutational cases (22/30), identified 1 mutational case in addition (15/16). Except 2 cases with higher CQ value than 29, which means the poor DNA quality or low quantity input, low mutational abundance maybe the main reason for no mutation identified by Idylla EGFR test. In 52 cases with rare EGFR variants, Idylla EGFR test identified 9 mutational cases, while 4/9 mutational variants beyond the scope of Idylla EGFR test, 2 20ins, 1 L858R and 1 19del. Five of the 9 mutational cases, Idylla test detected 5 different 19dels. The variants of all the 52 cases beyond the scope of ARMS-PCR of EGFR, and ARMS-PCR of EGFR didn’t find mutation. Conclusions: The Idylla system provides a rapid and accurate ability to differentiate result of ARMS-PCR of EGFR in gray area and identify more rare mutation variants which may decrease the repeat testing or validation. But the ability of NGS to find more actionable variants is still important.

Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples

Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequenced and differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in FFPE as well as frozen tissue samples.

Identification of Novel Diagnostic Markers for Malignant Pleural Mesothelioma Using a Reverse Translational Approach Based on a Rare Synchronous Tumor.

Although the routine use of immunohistochemistry has improved the accuracy of histopathologic diagnosis in clinical practice, new methods for discovering novel diagnostic markers are still needed. We sought new diagnostic markers for malignant pleural mesothelioma (MPM) using a reverse translational approach with limited archival tissues from a very rare case. Total RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues of a synchronous collision tumor consisting of MPM and pulmonary adenocarcinoma (PAC) was employed for gene expression profiling (GEP) analysis. Among the 54 genes selected by GEP analysis, we finally identified the following two candidate MPM marker genes: PHGDH and TRIM29. Immunohistochemical analysis of 48 MM and 20 PAC cases showed that both PHGDH and TRIM29 had sensitivity and specificity almost equivalent to those of calretinin (sensitivity 50% and 46% vs. 63%, and specificity 95% and 100% vs. 100%, respectively). Importantly, of the 23 epithelioid MMs, all 3 calretinin-negative cases were positive for TRIM29. These two markers may be diagnostically useful for immunohistochemical distinction between MPMs and PACs. This successful reverse translational approach based on FFPE samples from one very rare case encourages the further use of such samples for the development of novel diagnostic markers.

Comparison of Two Illumina Whole Transcriptome RNA Sequencing Library Preparation Methods Using Human Cancer FFPE Specimens.

Objective: RNA extraction and library preparation from formalin-fixed, paraffin-embedded (FFPE) samples are crucial pre-analytical steps towards achieving optimal downstream RNA sequencing (RNASeq) results. In this study, we assessed 2 Illumina library preparation methods for RNA-Seq analysis using archived FFPE samples from human cancer indications at 2 independent vendors. Methods: Twenty-five FFPE samples from 5 indications (non-small cell lung cancer, colorectal cancer, renal carcinoma, breast cancer, and hepatocellular carcinoma) were included, covering a wide range of sample storage durations (3-25 years-old), sample qualities, and specimen types (resection vs core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20 pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry in paired-end mode with a read length of 2 × 50 bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions for each gene in each sample to compare 2 library kits at 2 different vendors. Results: Our data showed that TruSeq RNA Access libraries yield over 80% exonic reads across different quality samples, indicating higher selectivity of the exome pull down by the capture approach compared to the random priming of the TruSeq Stranded Total kit. The overall QC data for FFPE RNA extraction, library preparation, and sequencing generated by the 2 vendors are comparable, and downstream gene expression quantification results show high concordance as well. With the TruSeq Stranded Total kit, the mean Spearman correlation between vendors was 0.87 and the mean Pearson correlation was 0.76. With the TruSeq RNA Access kit, the mean Spearman correlation between vendors was 0.89 and the mean Pearson correlation was 0.73. Interestingly, examination of the cross-vendor correlations compared to various common QC statistics suggested that library concentration is better correlated with consistency between vendors than is the RNA quantity. Conclusions: Our analyses provide evidence to guide selection of sequencing methods for FFPE samples in which the sample quality may be severely compromised.

Comprehensive Analysis of the Tumoral and Microenvironmental Transcriptome of Lymph Nodes and Bone Marrow in Mantle Cell Lymphoma

The Reverse Transcription Multiplex Ligation-dependent Probe Amplification (RT-MLPA) technique is a method allowing for the semi-quantitative concomitant analysis of a number of transcripts of interest in cells or tissues. It relies on the amplification of complementary DNA (cDNA) derived from messenger ribonucleic acid (mRNA) after binding and ligation of specific probes on adjacent regions. It is thus applicable to the relatively fragmented low-quality RNA that can be derived from formalin-fixed paraffin embedded samples (FFPE), ill adapted to other methods such as RNASeq. Adequate tagging provides specific amplicon sizes for each marker, thus becoming readily identifiable after capillary electrophoresis. Signal normalization further allows to compare relative levels of transcripts in a given run.Here we report on the application of this method to explore the tumoral and microenvironmental, i.e. intrinsic and extrinsic, transcriptome of mantle cell lymphoma (MCL). Data were obtained from i)bone marrow (BM) and lymph nodes (LN) from normal individuals, ii)BM and LN from MCL patients at diagnosis (n=33) and iii)BM and LN from relapsing MCL patients (n=19). Samples were either frozen BM and frozen of FFPE LN. mRNAs were extracted and reverse-transcripted in cDNA before being processed for MLPA investigating for 17 transcripts of interest. The latter explored the intrinsic tumoral expression of CCND1, SOX11 and MKI67. The extrinsic parameters of the microenvironment investigated concerned the monocyte/macrophage compartment (CD14, CD163), T-cells (CD3E/CD3, CD8A/CD8), NK cells (CD94), the costimulation molecule CD40-ligand (CD40LG/CD40L) and immune-checkpoint molecules (CD152/CTLA4, PDC1/PD1, CD274/PDL1, PDCD1LG2/PDL2, INDO1/IDO) as well as cytokines (CSF1/MCSF , IL10, TGFB1).The samples studied were 39 MCL LN, 20 MCL BM, 10 non-tumoral LN and 9 normal BM.RT-MLPA readily differentiated MCL samples from non-tumoral samples by showing high levels of CCND1 and SOX11 transcripts in patients. Extrinsic markers expression, analyzed in principal component analysis (PCA) clearly segregated diseased and normal LN, while the difference was less clear in BM. Macrophage markers transcript levels were lower in patients' LN which also showed a down regulation of global T- and NK-cells yet increased levels of CD8 transcripts. PDL1 expression was higher in MCL BM compared to controls.LN from MCL patients with aggressive characteristics displayed lower CCND1 but higher MKI67 transcripts than other MCL patients' samples. These aggressive forms were found to be significantly associated with high CD14 transcripts in LN and high CD163 in BM. In addition, blastoid morphology was significantly associated with high CD14 (p=.005) and CD163 transcript levels in LN. Regarding T-cells, MCL aggressive forms had significantly lower amounts of CD3E transcripts, yet increased CD8 expression.LN from patients with a high proliferative index displayed higher levels of CD8 but also of CTLA-4, PD-1 and PDL1, suggesting that these T-cells are exhausted.A significant upregulation of the levels of CCND1 transcripts was observed in relapse LN. Conversely, CD163, CD3E, CD40L and CD94 levels were lower in relapsed samples compared to diagnosis, which could reflect immune depletion secondary to immuno-chemotherapy.This study confirms, in an original transcriptomic approach, the great heterogeneity of the MCL microenvironment. Besides being a useful tool at diagnosis, RT-MLPA successfully allowed for a concomitant approach of a large number of transcripts and thus a precise evaluation of intrinsic prognostic and extrinsic features of MCL. Important, yet seldom described, modulations of T-cells, NK cells, macrophages and cytokines were observed, with different profiles segregating MCL with aggressive morphology and those with resistance to treatment. DisclosuresNo relevant conflicts of interest to declare.

Prediction of tissue origin of adenocarcinomas in the esophagogastric junction by DNA methylation.

Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.

SimFFPE and FilterFFPE: improving structural variant calling in FFPE samples.

BackgroundArtifact chimeric reads are enriched in next-generation sequencing data generated from formalin-fixed paraffin-embedded (FFPE) samples. Previous work indicated that these reads are characterized by erroneous split-read support that is interpreted as evidence of structural variants. Thus, a large number of false-positive structural variants are detected. To our knowledge, no tool is currently available to specifically call or filter structural variants in FFPE samples. To overcome this gap, we developed 2 R packages: SimFFPE and FilterFFPE.ResultsSimFFPE is a read simulator, specifically designed for next-generation sequencing data from FFPE samples. A mixture of characteristic artifact chimeric reads, as well as normal reads, is generated. FilterFFPE is a filtration algorithm, removing artifact chimeric reads from sequencing data while keeping real chimeric reads. To evaluate the performance of FilterFFPE, we performed structural variant calling with 3 common tools (Delly, Lumpy, and Manta) with and without prior filtration with FilterFFPE. After applying FilterFFPE, the mean positive predictive value improved from 0.27 to 0.48 in simulated samples and from 0.11 to 0.27 in real samples, while sensitivity remained basically unchanged or even slightly increased.ConclusionsFilterFFPE improves the performance of SV calling in FFPE samples. It was validated by analysis of simulated and real data.

Sudden cardiac death due to ARVC in the young: molecular autopsy by whole exome sequencing of DNA from dried blood spots (DBS) collected at birth

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Uppsala University Hospital (ALF) and Marcus Börgström Foundation Background The post-mortem investigation of cases with sudden cardiac death (SCD) is critical for risk stratification and adequate care of surviving relatives. Genetic testing is limited by access to DNA from the proband, as blood samples are not routinely collected at the time of death, or DNA of poor quality obtained from formalin-fixed paraffin-embedded (FFPE) tissues. Whole exome sequencing (WES) on minute amounts of DNA has opened up for the possibility to use DNA from Dried Blood Spots (DBS). Purpose To evaluate the feasibility of WES on DNA from archived DBS collected at the time of newborn screening in cases of SCD. Methods Through National Health Databases and Registries, we identified all cases of SCD in the young (<35 y.o.a.) in Sweden with a post-mortem diagnosis of ARVC between 2000 and 2010 (n = 22). Clinical records, medical and family histories, and autopsy findings were collected. Surviving parents were invited to participate. DNA was extracted from samples in National Biobanks such as DBS collected at birth (n = 19), formalin-fixed paraffin-embedded (FFPE) heart tissues (n = 8) from autopsies, and frozen blood samples (n = 3). Patient and parental DNA samples (when available) underwent WES. DNA- and sequencing-quality check (QC) metrics were compared between two different sequencing platforms and specimen types. We analysed 392 cardiomyopathy and arrhythmia genes and variants were classified as pathogenic or likely pathogenic according to the criteria from the American College of Medical Genetics. Results A higher quality but a lower yield of DNA (7.3 – 140 ng) was obtained from DBS when compared to FFPE samples (515 – 3065 ng). However, 100% of DBS vs. 62,5% of FFPE samples passed QC before sequencing. The average mean target depth for DBS samples (160X) was similar to that in blood samples (155X) and higher than FFPE (82X). Furthermore, >97% of target regions were covered with read depths >30X in DNA from DBS and blood compared to 88% for FFPE samples (Min 63%; Max 97%). Analysis of WES data uncovered clinically relevant variants in 12 out of 19 families (63%), of which four were located in ARVC genes. In six cases, the molecular autopsy identified another arrhythmogenic syndrome. Additionally, we identified one SCD with hemochromatosis and one with myotonic dystrophy as a possible contributing cause. In two additional families, variants of unknown significance fulfilled criteria for further segregation analysis. The concordance of positive results between different tissue samples was 100%. Conclusions DNA obtained from DBS has a quality comparable to that of blood and is a reliable source for WES-based molecular autopsy that may improve the diagnostic yield in cases of SCD. In a proportion of cases diagnosed post-mortem with ARVC, our molecular autopsy analysis pointed to a different diagnosis, highlighting its importance for the correct medical care of surviving relatives.

Human DNA decays faster with time than viral dsDNA: an analysis on HPV16 using pathology archive samples spanning 85\xa0years

BackgroundQuality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions. We assessed the differential sensitivity of viral and human double stranded DNA (dsDNA) to degradation with storage time.MethodsWe randomly selected forty-four HPV16-positive invasive cervical cancer (ICC) FFPE samples collected between 1930 and 1935 and between 2000 and 2004. We evaluated through qPCR the amplification within the same sample of two targets of the HPV16 L1 gene (69 bp, 134 bp) compared with two targets of the human tubulin-β gene (65 bp, 149 bp).ResultsBoth viral and human, short and long targets were amplified from all samples stored for 15 years. In samples archived for 85 years, we observed a significant decrease in the ability to amplify longer targets and this difference was larger in human than in viral DNA: longer fragments were nine times (CI 95% 2.6–35.2) less likely to be recovered from human DNA compared with 1.6 times (CI 95% 1.1–2.2) for viral DNA.ConclusionsWe conclude that human and viral DNA show a differential decay kinetics in FFPE samples. The faster degradation of human DNA should be considered when assessing viral DNA prevalence in long stored samples, as HPV DNA detection remains a key biomarker of viral-associated transformation.