FFPE-derived DNA Research Articles

The mutational signatures of formalin fixation on the human genome

Clinical archives of patient material near-exclusively consist of formalin-fixed and paraffin-embedded (FFPE) blocks. The ability to precisely characterise mutational signatures from FFPE-derived DNA has tremendous translational potential. However, sequencing of DNA derived from FFPE material is known to be riddled with artefacts. Here we derive genome-wide mutational signatures caused by formalin fixation. We show that the FFPE-signature is highly similar to signature 30 (the signature of Base Excision Repair deficiency due to NTHL1 mutations), and chemical repair of DNA lesions leads to a signature highly similar to signature 1 (clock-like signature due to spontaneous deamination of methylcytosine). We demonstrate that using uncorrected mutational catalogues of FFPE samples leads to major mis-assignment of signature activities. To correct for this, we introduce FFPEsig, a computational algorithm to rectify the formalin-induced artefacts in the mutational catalogue. We demonstrate that FFPEsig enables accurate mutational signature analysis both in simulated and whole-genome sequenced FFPE cancer samples. FFPEsig thus provides an opportunity to unlock additional clinical potential of archival patient tissues.

Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5′/3′-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples.

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

1142P Improved duplex sequencing-based method suitable for detection of hypermutator phenotype in FFPE-derived tumor samples

Duplex sequencing is an extremely accurate NGS approach, which allows correcting errors caused by DNA chemical degradation, PCR amplification or wrong sequencer readings. FFPE-derived DNA, which is frequently utilized in cancer research, is significantly affected by chemical degradation. The adjustment of duplex sequencing-based methods to the requirements for the analysis FFPE tissues is of high potential importance.

Machine learning algorithm improved automated droplet classification of ddPCR for detection of BRAF V600E in paraffin-embedded samples

Somatic mutations in cancer driver genes can help diagnosis, prognosis and treatment decisions. Formalin-fixed paraffin-embedded (FFPE) specimen is the main source of DNA for somatic mutation detection. To overcome constraints of DNA isolated from FFPE, we compared pyrosequencing and ddPCR analysis for absolute quantification of BRAF V600E mutation in the DNA extracted from FFPE specimens and compared the results to the qualitative detection information obtained by Sanger Sequencing. Sanger sequencing was able to detect BRAF V600E mutation only when it was present in more than 15% total alleles. Although the sensitivity of ddPCR is higher than that observed for Sanger, it was less consistent than pyrosequencing, likely due to droplet classification bias of FFPE-derived DNA. To address the droplet allocation bias in ddPCR analysis, we have compared different algorithms for automated droplet classification and next correlated these findings with those obtained from pyrosequencing. By examining the addition of non-classifiable droplets (rain) in ddPCR, it was possible to obtain better qualitative classification of droplets and better quantitative classification compared to no rain droplets, when considering pyrosequencing results. Notable, only the Machine learning k-NN algorithm was able to automatically classify the samples, surpassing manual classification based on no-template controls, which shows promise in clinical practice.

Abstract PD2-09: The role of CYP2D6 mediated tamoxifen metabolism in the suppression of ovarian function trial (SOFT)

Abstract Tamoxifen (T) is a pro-drug that undergoes CYP2D6-mediated metabolic activation to metabolites that more potently inhibit estrogen stimulated growth compared to the parent drug. While many studies have examined the role of CYP2D6 genotype in T-treated postmenopausal women, the role of CYP2D6 metabolism in premenopausal women (pre-MW) receiving T, with or without ovarian function suppression (OFS) or exemestane (E) and OFS is unknown. Methods: SOFT randomized 3066 (pre-MW) from 2003-2011 in 27 countries, stratified according to prior receipt or nonreceipt of chemotherapy and nodal status, to receive 5 years of T, T+OFS, or E+OFS. We designed a pharmacogenetics substudy (activated October 2010) to collect blood DNA from North American (NA) patients (pts) or to extract non-tumor DNA from available formalin fixed paraffin embedded (FFPE) tissue blocks. For pts with a blood sample, CYP2D6 was genotyped beginning with the Luminex Tag-It Mutation Detection Kit and when needed, with a copy number variation assay and/or sequencing assays. For pts with FFPE-derived DNA, CYP2D6 genotyping for *3, *4, *6, *9, *10, *17 and *41 was performed using a Taqman Allelic Discrimination Assay. CYP2D6 phenotypes were called by classifying pts on the basis of a combination of poor (PM: *3, *4, *5, *6, *7, *8), slow (SM: *10), intermediate (IM: *9, *17, *29, *41) and extensive metabolizer alleles (EM; all others). Activity scores (AS) from phenotypes assigned for each allele: 0 if PM, 0.25 if SM, 0.5 if IM and 1 if EM allele, and multiplied x2 or x3 if duplicate or triplicate. With concomitant use of potent CYP2D6 inhibitor, AS=0; use of weak inhibitor subtracted 0.5. Metabolizer status was defined by CYP2D6 genotype alone or in combination with CYP2D6 inhibitor use at randomization from the AS: extensive (AS 1.25 to 3), intermediate (AS >0.5 to <1.25), slow/poor (AS 0 to 0.5) metabolizer status. The laboratory was blinded to all clinical data. The substudy primary objective was to assess the association between disease-free survival (DFS) and CYP2D6 metabolizer status in the T arm, and secondarily in the T + OFS, and E + OFS arms. A Cox model estimated hazard ratios comparing status according to treatment assignment, with prespecified prognostic factors. Results: 1200/3047 (39%) randomized pts in the intention-to-treat (ITT) population had successful CYP2D6 genotyping and 50% received prior chemotherapy. Following randomization, 435/1023 (42%) NA pts provided a blood sample and CYP2D6 genotypes were derived in 435/435. Non-tumor tissue was macrodissected from 1277 available FFPE blocks, resulting in DNA concentrations of > 0.3 ng/ml in 1053, and successfully derived CYP2D6 genotypes for 765/3047 pts (25%). 182 (15%) pts had DFS events after 8 yrs median follow-up. Metabolizer status from genotype was 57% extensive, 29% intermediate, 15% slow/poor. Metabolizer status was not associated with DFS in pts assigned T alone (P=0.60; Table), nor in pts assigned T+OFS (P=0.41) or E+OFS (P=0.30). 11% of pts used CYP2D6 inhibitors concomitantly at randomization; for 8% it changed the metabolizer status. The results using this definition were consistent. Conclusion: This retrospective-prospective SOFT pharmacogenetics substudy found no relation of CYP2D6 metabolizer status with DFS in premenopausal pts receiving T, T + OFS, or E + OFS. Given that 50% were pretreated with chemotherapy, further study is needed regarding the role of CYP2D6 metabolism in patients treated with T monotherapy. TableTreatment GroupN pts (N events)Comparison (N pts)Hazard Ratio95% CITamoxifen324 (56)Intermediate (114) vs Extensive (210)0.780.43-1.39Tamoxifen265 (52)Slow/Poor (55) vs Extensive (210)1.110.58-2.13Tamoxifen + OFS357 (46)Intermediate (122) vs Extensive (235)0.730.38-1.40Tamoxifen + OFS299 (45)Slow/Poor (64) vs Extensive (235)1.250.64-2.43Exemestane + OFS344 (46)Intermediate (107) vs Extensive (237)0.590.28-1.22Exemestane + OFS293 (47)Slow/Poor (56) vs Extensive (237)1.130.56-2.27 Citation Format: Matthew P. Goetz, Gini F. Fleming, Mary Kuffel, John R. Hawse, John L. Black, Richard Weinshilboum, James N. Ingle, Patrizia dell’Orto, Olivia Biasi, Roswitha Kammler, Sherene Loi, Marco Colleoni, Giuseppe Viale, Prudence A Francis, Meredith M Regan. The role of CYP2D6 mediated tamoxifen metabolism in the suppression of ovarian function trial (SOFT) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD2-09.

Abstract 224: Evaluation of next generation sequencing of DNA and RNA from archival formalin-fixed, paraffin-embedded pancreatic cancer tissue: A pilot study of the SEER-linked virtual tissue repository

Abstract As formalin-fixed, paraffin-embedded (FFPE) tissue is being utilized for next-generation sequencing (NGS) in research and clinical settings, we conducted a study through the Surveillance, Epidemiology and End Results (SEER)-Linked Virtual Tissue Repository (VTR) Pilot Program to determine the quality of sequencing data obtained using FFPE-derived DNA and RNA. Forty-eight pancreatic ductal adenocarcinoma (PDAC) patients, comprising 24 case-control pairs based on survival time (≥5 years [cases] vs <24 months [controls]), were selected. Participating SEER registries obtained selected diagnostic tissue blocks collected clinically and stored for 4-18 years. DNA and RNA were extracted from the FFPE specimens for 36 patients (18 pairs). Whole genome (WGS) and whole exome sequencing (WES) were performed on tumor and normal DNA from 16 patients, and a methylation array was conducted on tumor DNA from 6 of these patients. RNA-Seq was conducted on tumor RNA from 36 patients. The median coverage depths for tumor were above 300x for WES and 60x for WGS. However, the majority of sequencing reads (>60%) were duplicates. Concordant mutations (SNVs, MNVs and indels) were >50% by WGS and WES from the majority of samples (n=11, 69%), and the most common discordant mutations were C>T. On average, mutant allele frequencies (MAFs) were 20% in coding regions and 15% across the whole genome, consistent with tumor content as measured by methylation analysis for five tumor samples (18%, 22%, 28%, 42%, and 50%). WES and/or WGS revealed that specimens for five of 27 PDAC subjects tested had a high fraction of variants overlapping with germline variants in dbSNP (≥20%), indicating that tumor cellularity was low among these samples. TP53, KRAS, CDKN2A, SMAD4, and RNF43 were the most frequently mutated genes from these specimens, consistent with genes reported in studies using fresh frozen tissue. Point mutations comprised most of the gene variants, and indels were found in CDKN2A, SMAD4, and RNF43. Most of the mutation status (e.g. missense, nonsense or indels) were concordantly called by WES and WGS (e.g., 81% for TP53, 100% CDKN2A, 94% SMAD4, and 94% RNF43). Most discordant calls were mutations identified by WES but not WGS (e.g., 8 [50%] for KRAS and 3 [19%] TP53). All samples yielded RNA-Seq reads with <30% exonic mapping, 39% (14) of which had <10% exonic mapping. Our study provided important evidence for NGS applications on DNA and RNA from archival PDAC FFPE tissue specimens stored for up to 18 years. These findings demonstrate that, with sufficient tumor content and coverage depth, FFPE-derived DNA is adequate for identifying somatic driver gene mutations in PDAC patients and that the it is feasible to utilize the population-based, SEER-Linked VTR as an infrastructure for obtaining diagnostic tissue for molecular studies. Citation Format: Yao Yuan, Alison Van Dyke, Valentina Petkov, Alyssa Tuan, Aatur Singhi, Lynn Matrisian, Lola Rahib, John Pearson, Katia Nones, Nicola Waddell, Yongmei Zhao, Tsai-wei Shen, Bao Tran, Jyoti Shetty, Elizabeth Gillanders, Danielle Carrick, Rosemary Cress, Lloyd Mueller, Brenda Hernandez, Charles Lynch, Thomas Tucker, Xiao-Cheng Wu, Lynne Penberthy. Evaluation of next generation sequencing of DNA and RNA from archival formalin-fixed, paraffin-embedded pancreatic cancer tissue: A pilot study of the SEER-linked virtual tissue repository [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 224.

Evaluation of DNA and RNA quality from archival formalin-fixed paraffin-embedded tissue for next-generation sequencing - Retrospective study in Japanese single institution.

Genetic analysis on formalin-fixed paraffin-embedded (FFPE) tissue specimens has become a mainstream method, from conventional direct sequencing to comprehensive analysis using next-generation sequencing (NGS). In this study, we evaluated the quality of DNA and RNA extracted from FFPE sections, derived from surgical specimens of different tumor types. Electrophoresis was performed using a 4200 TapeStation to evaluate DNA and RNA fragmentation. DNA Ct values were higher and significantly increased over a period of 4 years compared with those from cell lines or frozen tissues. The RNA integrity number equivalent (RIN) ranged from 1 to 4.1 and DV200 ranged from 7.3 to 81%. Twelve of the 108 cases were analyzed by NGS using the AmpliSeq Cancer HotSpot Panel v2 on a Miniseq system. A sufficient number of reads and coverage were obtained in all cases. Our results revealed that NGS analysis was sufficient for FFPE-derived DNA within 4 years of preservation. Conversely, approximately 20% of the RNA derived from FFPE within 4 years from the collection could be inappropriate for gene analysis based on RIN and DV200. It was suggested that FFPE would be adequate for genetic analysis, although it is desirable to store frozen specimens for the tumor tissues to be subjected to genetic analysis.

Validation and implementation of a bespoke pan-cancer NGS panel for FFPE solid tumour analysis within an NHS setting

Abstract Background The All Wales Medical Genomics Service (AWMGS) receives around 90 FFPE solid tumour samples per week for genetic analysis. Streamlining of testing is required in order to ensure the laboratory has the capacity and capability to meet the growing needs of the precision medicine era. With this in mind, AWMGS aimed to validate and incorporate a single NGS panel into service for the detection of, in the first instance, single nucleotide variations in a range of tumour types using a minimum of 10ng FFPE-derived DNA. Methods The validation involved the evaluation of the panel for the detection of single nucleotide variations in FFPE-extracted DNA. The workflow consisted of a SeqCap EZ HyperCap (Roche) protocol, sequencing on the Nextseq 550 (Illumina) and an in-house bioinformatic analysis pipeline. The validation encompassed the determination of assay sensitivity, specificity, accuracy, reproducibility and limit of detection via a series of validation rounds whereby previously tested surplus FFPE-derived DNAs of known genotype along with 2 cell-line reference standards were used as control samples to evaluate the performance of the panel. Results The panel showed a high degree of specificity, sensitivity and reproducibility, and could reliably detect variants in samples with as little as 10ng input DNA. However, despite multiple optimisation attempts, low input samples of Conclusions AWMGS successfully validated and optimised a bespoke NGS pan-cancer panel to provide molecular characterisation of FFPE solid tumour samples, with a minimum requirement of 50ng input DNA. This panel was implemented into the laboratory in a phased approach, initially replacing existing technologies for the delivery of existing NHS services. The panel provides an increased level of genomic information compared to previous testing methods and has greater clinical utility. As the panel has been designed to detect copy number and structural variations, once validated within the laboratory, this will have an even greater impact on the streamlining of testing. Legal entity responsible for the study Cardiff and Vale University Health Board. Funding NHS England’s Estates and Technology Transformation Fund. Disclosure All authors have declared no conflicts of interest.

1717P - Clinical validation of a novel assay for the detection of diagnostic alterations in sarcomas

BackgroundSarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, diagnosis can be difficult. Approximately one third of sarcoma patients however are characterised by recurrent genetic alterations mainly chromosomal translocations, which result in fusion genes, and gene amplifications. Fusion genes are highly useful diagnostic markers and are currently identified in the clinical setting using FISH/RT-PCR. Conversely, conclusive results cannot be achieved in up to 25% of fusion positive patients using these methods. Next generation sequencing (NGS) is a promising tool that can improve soft-tissue sarcoma diagnosis. MethodsA novel sarcoma-specific NGS gene capture panel containing probes for 90 fusion genes and 7 genes with frequent copy number changes was designed, optimised and validated in order to improve fusion-positive sarcoma diagnosis. Genomic DNA was extracted from 92 well-characterised FFPE samples and libraries prepared using the KAPA Hyperplus kit (Roche) and hybridised using the SeqCap Hybridisation kit (Roche) and sequenced on a NextSeq platform (Illumina). ResultsSarcoma specific translocations or gene amplifications were identified in 92 out of 94 cases giving the targeted gene panel a sensitivity of approximately 98% and specificity of 100% (Table).Table1717P Sarcoma tumour types and number of cases with structural variants or copy number variations detectedTableSoft Tissue Tumour TypeNo. of casesSV/CNV detectedSynovial Sarcoma2525Alveolar Rhabdomyosarcoma66Well/Dedifferentiated Liposarcoma98Intimal Sarcoma11Desmoplastic Small Round Cell Tumour (DSRCT)22Ewing’s Sarcoma1111Inflammatory Myofibroblastic Tumour22Myxoid Liposarcoma1010Chondroid Lipoma11Clear Cell Sarcoma44Epithelioid Haemangioendothelioma21Low-grade Fibromyxoid Sarcoma33Nodular Fasciitis11Alveolar Soft Part Cell Sarcoma11Congenital Fibrosarcoma22Solitary Fibrous Tumour22Aneurysmal Bone Cyst11BCOR-CCNB3 Sarcoma11Extraskeletal Myxoid Chondrosarcoma55Mesenchymal Chondrosarcoma22Dermatofibrosarcoma Protuberans (DFSP)33TOTAL9492 ConclusionsTo the best of our knowledge this is the first sarcoma-specific NGS panel validated in FFPE-derived genomic DNA. It is at least as sensitive and specific as the combination of FISH and RT-PCR and can be implemented in routine clinical diagnostic workflows. We are currently investigating whether fusion genes can be detected in plasma using ctDNA from 14 patients and results will be reported at the conference. Legal entity responsible for the studyDavid Gonzalez de Castro. FundingSarcoma UK. DisclosureAll authors have declared no conflicts of interest.

Detection of gene mutations in gastric cancer tissues using a commercial sequencing panel.

Predicting malignancy is important for adequate adjuvant therapy in patients with cancer. Due to cancer being a genetic disease, the detection of gene mutations could be helpful in predicting the prognosis and efficacy of drugs. Gastric cancer is the fifth most common cancer and is the third leading cause of cancer associated mortality worldwide. Mutations in genes may correlate with clinical information in patients with gastric cancer after surgery and, therefore, may be useful for predicting the prognosis of this disease. In the present study, to assess the usefulness of a commercial sequencing panel, TruSeq® Amplicon-Cancer Panel (Illumina), using a next-generation sequencer (Illumina MiSeq), mutation analysis of fresh as well as formalin-fixed paraffin-embedded (FFPE) gastric cancer tissues was performed retrospectively. The study group comprised of 4 patients who underwent gastrectomy for gastric cancer. Cancer and normal stomach tissues were collected immediately following surgical removal. Thereafter, the specimens were fixed in 10% neutral formalin for 24–72 h. Normal and FFPE cancer tissues were histologically examined and confirmed. A total of 3 mutations were identified in the driver genes (KRAS, TP53 and APC) in cancer tissues from 2 of the 4 patients, using fresh samples. In addition, FFPE samples were analysed for the same tissues and the same results were obtained by setting the threshold for the percentage of the mutation rate to avoid detection of pseudo-positive mutations. In conclusion, the sequencing analysis using FFPE-derived DNA samples was successfully performed.

Abstract 2332: Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study

Abstract Background: Tumor infiltrating lymphocytes (TILs) are prognostic and predictive biomarkers in colorectal cancer and are associated with improved prognosis and response to immunotherapy. While TILs are routinely assessed by pathologists, a standardized technique (immunoSEQ, Adaptive Biotechnologies) that leverages targeted next-generation sequencing can also be used to quantify and characterize the T-cell receptor (TCR) repertoire of individual colorectal cancers. In a large, population-based study of incident colorectal cancer, the host immune responses were measured by an expert pathologist and ImmunoSEQ to understand the relationships between TILs, TCRs/cell and specific subgroups of colorectal cancer. Methods: Incident cases of adenocarcinoma of the colon or rectum from the Molecular Epidemiology of Colorectal Cancer (MECC) study included 1,000 cancers that were uniformly evaluated for TILs and other histopathologic features by one pathologist. FFPE-derived DNA from microdissected tumor tissue was extracted and sequenced using ImmunoSEQ analysis for the same 1,000 individuals. A resulting quantitative metric from this assay includes TCRs/cell, a measure of rearranged T cell quantity relative to all nucleated cells in a tumor sample. Gene expression in snap-frozen tissue available from 342/1,000 MECC colorectal cancers was measured with Affymetrix Human Genome U133 Arrays (U133A and U133 Plus2.0) as previously described. CMS classification was performed using the R package 3.5.1, CMS classifier, randomForest 4.6-14. Multivariate analysis assessed CMS by age, gender, TILs/HPF, TCRs/cell, MSI status, BRAF and KRAS mutational status. Results: TILs/HPF and TCRs/cell were significantly correlated among all 1000 cases (r=0.5, p<0.001). Among the 342 cases with available expression profiles, CMS1 constituted 12.0% of all CRC, with CMS2 (41.8%), CMS3 (8.5%), and CMS4 (13.7%) and unclassified (24%) representing the remaining distribution. There were statistically significant differences in the molecular and histopathologic features of colorectal cancers by CMS subgroups. MSI-H tumors were most frequently observed within CMS1 cancers (56.6% of CMS1 were MSI-H), with lower representation among CMS2 (1.5%), CMS3 (10%), CMS4 (3.5%), and unclassified CRC (9.5%) (p<0.0001). In addition, BRAF positive tumors were more frequently observed within the CMS1 group (12.2%, p =0.0065) and KRAS positive tumors within the CMS3 group (31%, p<0.0001). Consistent with prior reports, TILs/HPF were significantly higher in the CMS1 group (mean=7.7, p<0.0001). Similar statistically significant trends were observed across classes for TCRs/cell (mean=0.16, p=0.04). Conclusions: Subtypes of CRC have distinct histopathologic and molecular features that can be distinguished by expression profiles and immunoSEQ. Citation Format: Marilena Melas, Charalampos Lazaris, Stephanie L. Schmit, Asaf Maoz, Rebeca Sanz Pamplona, Chenxu Qu, Joel K. Greenson, Rork Kuick, Flavio Lejbkowicz, Hedy S. Rennert, Christopher P. Walker, Chase M. Bowen, Diane M. Da Silva, W. Martin Kast, Gregory E. Idos, Kevin J. McDonnell, Victor Moreno, Gad Rennert, Stephen B. Gruber. Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2332.

Library Preparation Using FFPE-Derived Tumor DNA for High-Throughput Hybridization-Based Targeted or Exome Sequencing.

The use of next generation sequencing (NGS) to profile tumor genomes for the presence of diagnostic, prognostic, or therapeutically targetable variants is revolutionizing the practice of oncology and is increasingly utilized in clinical laboratory settings. Beginning with the isolation of DNA of sufficient quality and quantity from a tumor specimen, the creation of a library of genomic fragments representing the portion of the genome of interest, ranging from a few genes to the entire exome, is the first step required in the sequencing process. Fixed tumor tissue in the form of a tissue block is the most commonly encountered specimen for analysis in a clinical setting. Special precautions must be employed to ensure that material isolated from these specimens is suitable for use. Once DNA is obtained, one of the most commonly used methods for library preparation involves fluid phase hybridization-based capture of the genomic regions to be interrogated. This multistep process involves fragmentation of the DNA to a uniform size distribution, ligating adapter molecules which are labeled with specific barcodes to enable downstream sequencing and sample identification, and the use of a multiplexed pool of biotinylated single stranded RNA or DNA hybridization probes to recognize and capture the targeted genomic regions. Fragments which are not specifically captured during the hybridization process are removed via a series of wash steps, and a final low cycle amplification is used to prepare the library of captured fragments for sequencing. In this chapter, we provide a step-by-step guide to the preparation of fixed tissue-derived DNA libraries for sequencing via the Illumina process and highlight some of the precautions necessary when working with these types of specimens.

Bioinformatics Basics for High-Throughput Hybridization-Based Targeted DNA Sequencing from FFPE-Derived Tumor Specimens: From Reads to Variants.

The use of next-generation sequencing and hybridization-based capture for target enrichment have enabled the interrogation of coding regions of several clinically significant cancer genes in tumor specimens using both targeted panels of a few to hundreds of genes, to whole-exome panels encompassing coding regions of all genes in the genome. Next-generation sequencing (NGS)technologies produce millions of relatively short segments of sequences or reads that require bioinformatics tools to map reads back to a reference genome using various read alignment tools, as well as to determine differences between single bases (single nucleotide variants or SNVs) or multiple bases (insertions and deletions or indels) between the aligned reads and the reference genome to call variants. In addition to single nucleotide changes or small insertions and deletions, high copy gains and losses can also be gleaned from NGS data to call gene amplifications and deletions. Throughout these processes, numerous quality control metrics can be assessed at each step to ensure that the resulting called variants are of high quality and are accurate. In this chapter we review common tools used to generate reads from Illumina-derived sequence data, align reads, and call variants from hybridization-based targeted NGS panel data generated from tumor FFPE-derived DNA specimens as well as basic quality metrics to assess for each assayed specimen.

Abstract 422: Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications

Abstract Next Generation Sequencing is increasingly used in translational cancer research and as a diagnostic test. Most tumor specimens available for testing are formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE-derived DNA is typically damaged, causing difficulties for standard library preparation methods. Frayed ends prevent blunt-ended double-stranded adapter ligation, used for whole genome sequencing (WGS) and targeted bait hybridization library preparation. Fragmented DNA and modified bases interfere with PCR, which underlies amplification based targeted sequencing. We present a single-stranded library preparation method especially forgiving for low quality FFPE-derived DNA. By excising damaged bases and using single-stranded adapter ligation, we omit repair aimed at generating blunt-ended double-stranded DNA, and eliminate the need for whole genome PCR. After ligation of the first adapter, libraries can either undergo second adapter ligation to generate WGS libraries, or be used as input to oligo-selective sequencing (OS-Seq) target capture. We show that our FFPE-derived PCR-free WGS libraries exhibit coverage depth and uniformity highly similar to double-stranded PCR-free libraries of matched cell line (non-FFPE) materials. Additionally, we present performance of our WGS library preparation on clinical FFEP samples. We further demonstrate reproducible high and uniform coverage for reference standards and clinical FFPE samples in targeted capture. We show a lack of correlation between DNA quality and sequencing metrics such as on-target rate (R2=0.005), uniformity (R2=0.0002), and coverage (R2=0.06), with successful removal of C>T FFPE artifacts comparable to standard repair methods. We demonstrate sensitive copy number alteration detection through analysis of coverage depth, feasible due to the absence of pre-capture PCR. In conclusion, our single-stranded library preparation method is forgiving for sample quality and provides uniform and high coverage as the basis for somatic variant calling. Citation Format: Anna Vilborg. Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 422.

Reference Size Matching, Whole-Genome Amplification, and Fluorescent Labeling as a Method for Chromosomal Microarray Analysis of Clinically Actionable Copy Number Alterations in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

Cancer genome copy number alterations (CNAs) assist clinicians in selecting targeted therapeutics. Solid tumor CNAs are most commonly evaluated in formalin-fixed, paraffin-embedded (FFPE) tissue by fluorescence in situ hybridization. Although fluorescence in situ hybridization is a sensitive and specific assay for interrogating preselected genomic regions, it provides no information about coexisting clinically significant copy number changes. Chromosomal microarray analysis is an alternative DNA-based method for interrogating genome-wide CNAs in solid tumors. However, DNA extracted from FFPE tumor tissue produces an essential, yet problematic, sample type. The College of American Pathologists/American Society of Clinical Oncology guidelines for optimal tumor tissue handling, published in 2007 for breast cancer and in 2016 for gastroesophageal adenocarcinomas, are lacking for other solid tumors. Thus, cold ischemia times are seldom monitored in non-breast cancer and non-gastroesophageal adenocarcinomas, and all tumor biospecimens are affected by chemical fixation. Although intended to preserve specimens for long-term storage, formalin fixation causes loss of genetic information through DNA damage. Herein, we describe a reference size matching, whole-genome amplification, and fluorescent labeling method for FFPE-derived DNA designed to improve chromosomal microarray results from suboptimal nucleic acids and salvage highly degraded samples. With this technological advance, whole-genome copy number analysis of tumor DNA can be reliably performed in the clinical laboratory for a wide variety of tissue conditions and tumor types.

Abstract A59: Impact of NGS four-gene panel in screening genes with potential therapies in NSCLC

Abstract Targetable genomic mutation detection has innovated personalized medicine in non-small cell lung cancer (NSCLC) mainly through detection of EGFR activation mutations, which predicts susceptibility to tyrosine kinase inhibitors (TKIs). Although patients render positive response to TKIs initially, resistance could eventually arise, due to the secondary mutation T790M, therefore decreasing patients' survival and limiting treatment options. NSCLC patients harbors a variety of other mutated genes, despite EGFR that could potentially be treated with drugs already approved, or in ongoing clinical trials for other types of tumor. KRAS is one of the most frequently mutated genes in NSCLC and could act as a new target for lung cancer treatment. Although no targeted therapy has yet been approved for mutated-KRAS, there are multiple potential treatment strategies under investigation. BRAF is another gene described in NSCLC that offers a rational therapeutic strategy, since its pathway plays a role in the carcinogenesis of NSCLC. In the present study the prevalence of KRAS, BRAF, and EGFR was determined among patients with next-generation sequence technology. We used a custom multigene panel for Ion Platform to investigate EGFR, KRAS, and BRAF mutations in 491 NSCLC Brazilian patients. Genomic DNA libraries were prepared from FFPE-derived DNA and sequenced in the Ion PGM platform. KRAS was the most frequently mutated gene, found in 27.6% of all samples, and thus could be an appealing target for new therapy strategies as described in other studies. 111 (82.2%) samples harbored mutation in codon 12, with G12C mutation the most common one, present in 37.8%. The second most prevalent mutated gene was EGFR with 119 (24.3%) mutated samples. The most common mutations were exon 19 deletions (most frequently E746-A750), found in 42% of the samples, and L858R, found in 32% of the samples. BRAF displayed prevalence rates of 3.7%. V600E mutation was present in 55.5% of the BRAF mutated samples, followed by mutations in codon G469 (22%). We identified 17 double and coexisting mutations. EGFR T790M was the most common secondary mutation, found in six (12%) samples concomitantly with p.E746_A750del, indicating that these patients might have a reduced response to TKIs therapy. Interesting is the fact that three of these double mutant samples had prior cobas® EGFR Test results from 2-3 previous years with absence of T790M mutation, indicating that resistance may have arrived in a 2- to 3-year life span. Although EGFR gene mutations are reported as mutually exclusive with KRAS or BRAF mutations in lung cancer, we found three samples harboring concomitant EGFR and KRAS mutations. Previous studies also reported that mutations in KRAS and BRAF are mutually exclusive, but we found one sample harboring mutations in both genes, indicating that this is a rare event. In conclusion, this study shows that in development of future therapies, targeting KRAS should be prioritized. Although BRAF-mutated samples displayed short prevalence, it could be a potential target among wild-type EGFR and KRAS cancer patients. This study shows the importance of multi-gene panel screening to reduce overall turnaround time generating results in a single test. Citation Format: Carolina Bustamante, Maíra Cristina Menezes Freire, Natália Penido Lopes, Mariano Zalis. Impact of NGS four-gene panel in screening genes with potential therapies in NSCLC [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A59.

Abstract 5347: Molecular barcoding improves confidence in variant calling with low input FFPE tissue samples

Abstract FFPE tissue archiving is the most widely used method for clinical sample preservation, and provides a valuable source of diverse genetic information for cancer biomarker discovery. NGS library preparation is often challenging, however, as FFPE-derived DNAs exhibit varying degrees of fragmentation and chemical modification depending on how tissues were handled and processed. Moreover, the presence of low allele frequency variants because of tumor heterogeneity, sample degradation, and high rate of formalin-induced mutations make SNV calling a challenging task. To improve variant calling and consistency across FFPE samples of varying quality, we developed a complete workflow solution that features DNA pre-qualification, molecular barcodes for uniquely tagging each DNA fragment, pre-capture indexing for tracking up to 192 samples, accelerated hybrid capture, and one-tube library prep, to enable the preparation of complex enriched libraries in less than 8 hours from 10-200ng of FFPE-derived DNA. We incorporate molecular barcodes at the ligation step to enable de-duplication and generation of consensus reads using SureCall software, which significantly improves confidence of variant calling (99% of calls >150 variant call quality threshold) at low allelic frequencies. Using ClearSeq Comprehensive Cancer Panel which targets 151 genes frequently mutated in solid and hematological cancers, we routinely obtain high quality libraries (with 1000X sequencing depth and after deduplication) with >99% of the targeted region covered at 20X for high quality gDNA (FF tissue), and greater than 95% and 90% covered at 20X for medium and low quality FFPE samples, respectively. Using multiplex reference samples, we demonstrate detection of low frequency variants (at 1-5%) including SNVs, small indels, and RET and ROS1 translocations. As we will further show, FFPE integrity scores determined by qPCR and TapeStation (ddCq and DIN) enable researchers to choose the appropriate quantification method, PCR cycling, and sequencing depth for optimal sequencing coverage. Citation Format: Bahram Arezi, Bilan Hsue, Keith Chen, Brenda Rogers, Christian Lecocq, Ashutosh Ashutosh, Jayati Ghosh, Gilbert Amparo, Madhuvanthi Ramaiah, Carlos Pabon, Holly Hogrefe. Molecular barcoding improves confidence in variant calling with low input FFPE tissue samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2017-5347

Accurate and precise targeted NGS with BRCA MASTR Plus Dx for BRCA1 and BRCA2 mutation detection in formalin-fixed paraffin-embedded tumor tissue-derived DNA.

e23116 Background: Targeted next-generation sequencing (NGS) has tremendous potential in clinical diagnostics as it allows oncogenetic profiling to steer therapy. Inhibitors of poly-(ADP-ribose) polymerase (PARPi) have emerged as a new class of targeted anti-cancer drugs, specifically for tumors showing homologous recombination repair deficiency, including BRCA1- and BRCA2-mutated ovarian and breast cancers. This multicentre study evaluated the performance of BRCA Tumor MASTR Plus Dx* (Multiplicom) to routinely diagnose somatic and germline BRCA mutations in formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived DNA. Methods: Three genetic centres participated in this performance evaluation study (PES) to detect single nucleotide variants (SNV) and small indels in the BRCA genes at a variant allele frequency down to 5%. The sample population comprised 54 FFPE-derived DNA extracts from 51 clinical and 3 reference samples. DNA extracts were subjected to quality control using Multiplicom’s QC plex assay. The clinical samples were characterized using an independent targeted NGS method and Integrative Genomics Viewer (IGV) analysis of the mapped raw reads. SNV calling was performed using third-party bioinformatics platforms in the BRCA coding regions +/- 2 intronic bp. Results: BRCA MASTR Plus Dx*showed a uniformity of 93.9%, i.e. the percentage of amplicons with at least 0.2x the mean amplicon coverage, and a target specificity of 99.1%. The limit of detection (LOD) proved to be as low as 1%. The diagnostic accuracy was ≥ 99.99% [95% CI ≥ 99.98%] (100% sensitivity [95% CI ≥ 99.02%] and ≥ 99.99% specificity [95% CI ≥ 99.98%]). Both repeatability and reproducibility were ≥ 99.99% [95% CI ≥ 99.98%]. Lot equivalence was 100% [95% CI ≥ 99.99%]. Conclusions: This multicentre study demonstrated that BRCA MASTR Plus Dx* can be routinely applied as an accurate and precise method with an LOD of 1%. The assay can be used to direct patients with somatic or germline BRCA mutations to PARPi therapy. Currently, a PES for BRCA MASTR Plus Dx* and Multiplicom’s MASTR Reporter software is ongoing. *Products described above are CE-IVD and not available for sale in the US

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0375-z) contains supplementary material, which is available to authorized users.