Evaluation Of Next-generation Sequencing Research Articles

Genomic profiling in advanced NSCLC in Italy: First results of NERoNE (NSCLC Emilia Romagna Next Generation Sequencing Evaluation) project.

e21077 Background: The NERoNE Project evaluates the use of Next Generation Sequencing (NGS) in molecular profiling of advanced NSCLC (aNSCLC) patients (pts) in three Oncology Units (OU) of Emilia Romagna Region aiming to clarify the impact of such extensive tool in characterizing different NSCLC subpopulations. Methods: Since January 2020 to December 2022, 970 sequential aNSCLC pts from three OU (AUSL-IRCCS of Reggio Emilia, Modena University Hospital and IRST Meldola) were profiled by NGS. Samples from Modena and Reggio Emilia were analyzed at Modena University Hospital Molecular Pathology Lab using Myriapod IL- 56G, Cancer Panel DNA, Cancer Panel RNA, Oncomine DX Target Test Assay, Oncomine Focus Assay. Samples from IRST Meldola were processed at its Molecular Diagnostic Unit by Myriapod NGS cancer panel DNA e RNA, Oncomine Focus Assay and Oncomine Comprehensive Assay v3.We considered molecular alterations with available target therapies in Italy: EGFR mutations (mut) (exon 18,19,20,21), KRAS mut, BRAF V600E mut, ALK, ROS1, RET and NTRK rearrangements, MET exon 14 skipping mut, ErbB2 mut, FGFR 1-3 mut. PDL1 expression was assessed by standard immunohistochemistry on 486 pts. Results: Among 970 pts, 501 with at least one mut (52%) were considered for analysis: 266 were female (53%), mean age was 69 ± 11 years (min-max: 33-94) and 473 pts (94%) had adenocarcinoma. Molecular features are listed in table 1. Distribution of mut among ages, categorized as < 60, 60-70 and > 70 y, was similar, EGFR activating mut were most prevalent in females; KRAS mut (G12C and non G12C) were most prevalent in males; 41 pts had co-mutations (8.2%): the most frequent co-mut were EGFR mut (27%). PDL1 expression was < 1% 129 (27%), 1-49% 211 (43,4%) and ≥50% 146 pts (30%). The majority of pts with EGFR activating mut had PDL1 < 50%. Conclusions: NERoNE evaluates data from NGS profiling of three OU mapping key actionable molecular alterations in a large population of aNSCLC also accounting for PDL1 status. This preliminary analysis will be the starting point for further evaluations in order to improve the personalized approach to aNSCLC. [Table: see text]

Race in a molecular tumor board compared to cancer registry population.

103 Background: Disparities in cancer care due to race and ethnicity are prevalent in both the care patients receive and patient outcomes. The evaluation of next generation sequencing (NGS) results from patients with advanced cancer by a molecular tumor board (MTB) has become standard practice in many institutions for the identification of additional treatment options and targeted therapies. We sought to compare the racial distribution of patients evaluated by our MTB with our institutional cancer registry (CR). Methods: We tabulated the racial distribution of 560 MTB patients chosen for presentation in a bimonthly case conference based on physician request or clinical interest from more than 2,500 NGS reports of patients with advanced cancer from 2016 through 2020. Self-identified race from patients with stage 4 cancer within our institutional CR from the same time interval was compared to the MTB population from each year using the Chi-Squared test. The Cochran-Mantel-Haenszel test was used to analyze the relationship between race and MTB/CR after controlling for year. Race categories were defined as Asian, Black/African-American (AA), White/Caucasian, and other. Results: We identified 4,151 CR patients with stage 4 cancer from 2016 through 2020, 573 of whom identified as Black/AA (13.8%). Of the 560 MTB patients, 55 were Black/AA (9.8%). When controlling for year, Black/AA patients were less frequently included in the MTB compared to the CR (p = 0.0128). Conclusions: Black/AA patients with advanced cancer are under-represented in our MTB. Larger studies are warranted to examine underlying causes of this discrepancy including implicit bias, generalizability of this finding to other minorities and institutions, and potential remedies to ensure equitable access to state-of-the-art cancer care.[Table: see text]

Evaluation of Next-Generation Sequencing and Crystal Digital PCR for Chimerism Monitoring of Post-Allogeneic Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for most hematologic diseases. To evaluate the level of donor engraftment, chimerism must be carefully monitored after HSCT. Short tandem repeats, quantitative PCR (qPCR), and, more recently, digital PCR (dPCR) are widely used to determine the proportions of donor and recipient cells after HSCT. The screening and quantification of chimerism have been evaluated by 2 new methods: a ready-to-use next-generation sequencing (NGS)-based method using the Devyser ChimerismNGS kit and an original combination of the Stilla crystal digital PCR (cdPCR) platform with 3-color multiplexing capacity using GenDX KMRtrack reagents. The genotyping of 4 HSCT pairs by cdPCR using 11 triplex mixes of the GenDX KMRtype kit was consistent at 98.8% with qPCR. Informative samples (n=20) from 6 donor-recipient pairs and 1 external proficiency test demonstrated the reliability of the results (0.1% to 50%) for the 2 methods. The methods are also highly sensitive (0.1%) and accurate. The chimerism values of the 2 methods are correlated and concordant with those of the reference methods. In addition, the ADVYSER software (Devyser) is user-friendly and well adapted to chimerism monitoring. In conclusion, these 2 innovative methods are easy to perform and user-friendly in all molecular, hematology, and immunogenetic laboratories and allow the genotyping and monitoring of chimerism with high performance and sensitivity.

Comparison of NGS and MFC Methods: Key Metrics in Multiple Myeloma MRD Assessment.

In order to meet the challenges in data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment, the goal of the current study was to provide a step-by-step evaluation of next-generation sequencing (NGS) and multicolor flow cytometry (MFC) data. Bone marrow (BM) sample pairs from 125 MM patients were analyzed by NGS and MFC MM MRD methods. Tumor load (TL) and limit of detection (LOD) and quantification (LOQ) were calculated. The best-fit MRD cut-off was chosen as 1 × 10−5, resulting in an overall 9.6% (n overall = 12 (NGS n = 2, MFC n = 10)) nonassessable cases. The overall concordance rate between NGS and MFC was 68.0% (n = 85); discordant results were found in 22.4% (11.2% (n = 14) of cases in each direction. Overall, 55.1% (n = 60/109) and 49.5% (n = 54/109) of patients with a serological response ≥ very good partial response (VGPR) showed BM MRD negativity by NGS and MFC, respectively. A good correlation in the TL assessed by both techniques was found (correlation coefficient = 0.8, n = 40, p < 0.001). Overall, our study shows good concordance between MM BM MRD status and TL when comparing NGS and MFC at a threshold of 10–5. However, a sufficient number of analyzed events and calculation of MRD key metrics are essential for the comparison of methods and evaluability of data at a specific MRD cut-off.

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 &amp;lt;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 (&amp;gt;60%) were duplicates. Concordant mutations (SNVs, MNVs and indels) were &amp;gt;50% by WGS and WES from the majority of samples (n=11, 69%), and the most common discordant mutations were C&amp;gt;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 &amp;lt;30% exonic mapping, 39% (14) of which had &amp;lt;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 next generation sequencing for detecting HER2 copy number in breast and gastric cancers.

e13004 Background: Amplicon-based next generation sequencing (NGS) approaches have been preferentially adopted by the clinical laboratories on the basis of a short turnaround time (TAT) and small DNA input needs. However, little work has been done to assess the amplicon-based NGS methods for copy number variation (CNV) detection in comparison with current standard methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The correlation between NGS based CNV detection and the later standard methods has remained unexplored. Methods: We developed an amplicon-based panel to detect human epidermal receptor growth factor (HER2) amplification in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 280 breast cancer and 50 gastric cancer patients. Assessment by IHC and FISH was conducted in parallel, and descriptive statistics were used to assess the concordance. Results: The copy number detected by NGS was correlated with either the average HER2 copy number (signals/cell) (r = 0.844; p &lt; 0.001) or the HER2/CEP17 ratio (r = 0.815; p &lt; 0.001). We determined a cut-off value for NGS to categorize HER2 amplification status by using 131 HER2 non-amplified FFPE samples. In breast cancer patients, the cut-off value was 2.910, with 95.28%, 99.23% and 97.29% sensitivity, specificity and concordance, respectively. However, this cut-off value displayed low sensitivity in gastric cancer patients (64.71%), and the following macro-dissection procedure was not effective for increasing sensitivity (57.14%). Thus indicated that HER2 copy number correlation between NGS and FISH in gastric cancer was different from breast cancer. Conclusions: Evaluation of HER2 copy number with NGS in our study was comparable with IHC and FISH in breast cancer patients, but concordance in gastric cancer was only moderate. NGS-based HER2 assessment may decrease the equivocal HER2 determinations in breast cancer patients assessed by FISH/IHC. The greater discordance in gastric cancer may reflect the underlying biological mechanisms, and further study is warranted.

Evaluation of next generation sequencing for the analysis of Eimeria communities in wildlife

Next-generation sequencing (NGS) techniques are well-established for studying bacterial communities but not yet for microbial eukaryotes. Parasite communities remain poorly studied, due in part to the lack of reliable and accessible molecular methods to analyse eukaryotic communities. We aimed to develop and evaluate a methodology to analyse communities of the protozoan parasite Eimeria from populations of the Australian marsupial Petrogale penicillata (brush-tailed rock-wallaby) using NGS. An oocyst purification method for small sample sizes and polymerase chain reaction (PCR) protocol for the 18S rRNA locus targeting Eimeria was developed and optimised prior to sequencing on the Illumina MiSeq platform. A data analysis approach was developed by modifying methods from bacterial metagenomics and utilising existing Eimeria sequences in GenBank. Operational taxonomic unit (OTU) assignment at a high similarity threshold (97%) was more accurate at assigning Eimeria contigs into Eimeria OTUs but at a lower threshold (95%) there was greater resolution between OTU consensus sequences. The assessment of two amplification PCR methods prior to Illumina MiSeq, single and nested PCR, determined that single PCR was more sensitive to Eimeria as more Eimeria OTUs were detected in single amplicons. We have developed a simple and cost-effective approach to a data analysis pipeline for community analysis of eukaryotic organisms using Eimeria communities as a model. The pipeline provides a basis for evaluation using other eukaryotic organisms and potential for diverse community analysis studies.

WS8.2 Evaluation of next generation sequencing as a diagnostic tool for CFTR rare mutation screening

Introduction: The Mayo Clinic Molecular Genetics Laboratory has completed whole CFTR gene analysis for 1,154 clinical samples. Here we report our findings for a large set of samples analyzed in an academic reference laboratory setting. Methods: All exons of the CFTR gene were sequenced using an automated, laboratory developed fluorescent Sanger sequencing workflow. Whole exon deletion/duplication testing was done using MLPA (MRC Holland). Results: 43% of samples were positive for the detection of at least one potentially disease causing allele (including 5T). 140 individuals had 2 known mutations detected, 271 were carriers of one known mutation; 57 had one mutation and one variant of uncertain significance (VUS), 100 had one VUS, and 17 had 2 or more VUS’s. Of the cases with 2 mutations, 25 had one copy of the 5T allele (13 dF508/5T); and, 41 had 2 copies of ACMG mutations (16 homozygous dF508). Homozygous results were seen for 6 additional mutations: 2954delT, G27X, I1234V, S945L, R75X, and R117H-7T in a 30 y/o female with pancreatitis. The majority of cases (125/140) with 2 known, disease causing mutations were seen in patients whose samples had a reason of referral (RFR) of possible or definite CF (or Unknown RFR) versus those with RFR’s for pancreatitis (9) or male infertility (6). Conclusions: The high positivity rate indicates that the test is being ordered appropriately. However, the high number of cases with 2 ACMG panel mutations indicates that testing first with a panel test may be more cost effective. The number of VUS’s is higher in individuals over 21 y/o and with a reason of referral of pancreatitis that in individuals being tested at under 21 years of age.