Adoption Of Next-generation Sequencing Research Articles

P223 Increase in diversity of characteristics of novel alleles discovered after adoption of next-generation sequencing

P223 Increase in diversity of characteristics of novel alleles discovered after adoption of next-generation sequencing

Genomic alterations, molecularly targeted therapy and race: Real world data from the Endometrial Cancer Molecularly Targeted Therapy Consortium (LBA 8)

<b>Objectives:</b> There has been considerable adoption of next-generation sequencing (NGS) and tumor testing to direct therapy in patients with advanced/recurrent endometrial cancer (EC), but the impact of this approach to date is unclear. We sought to confirm the proportion of patients with at least one actionable genomic alteration; assess associations between molecular alterations and race; and whether the use of molecularly targeted therapy improves response, progression-free survival (PFS) and overall survival (OS) in patients with metastatic EC. <b>Methods:</b> We formed a national multidisciplinary consortium of translational and clinical investigators to study patients with advanced/recurrent EC with tumor testing treated with systemic therapies. Tumor testing and treatment decisions were based on the treating physician's recommendation. Data were abstracted regarding age; stage; grade; histology; race; ethnicity, treatment; genomic alterations (mutations, amplifications); protein expression for Her2, p53, mismatch repair (MMR) proteins, ER and PR; Her2 FISH; microsatellite instability (MSI); treatment response; and survival. Survival outcomes and response rates will be compared to historical controls. Statistical analysis performed using SAS v9.4. <b>Results:</b> 994 patients across 12 centers were enrolled in the database. The median age was 64 years (range: 22–93). 67.2% were White; 22.7% Black; 2.4% Asian and 3.0% Hispanic. The histologic subtypes were 49.7% (n=495) endometrioid, 20.2% (n=201) serous, 10.9% (n=108) carcinosarcoma, 7.9% (n=79) mixed, and 3.7% (n=37) clear cell. 414 (41.6%) received paclitaxel/carboplatin based frontline chemotherapy, 672 (67.6%) had recurrent/persistent disease and were treated with a median of 2 (range: 0-9) therapies. 764 (76.9%) patients had tumor testing. Over 54% of patients had at least one actionable genomic alteration. NGS testing revealed the following alterations: <i>PI3K</i> (31.5%), <i>TP53</i>(30.5%), <i>PTEN</i> (25.4%), <i>CTNNB1</i> (7.2%), <i>AKT</i> (2.3%), <i>ESR</i> (1.4%), <i>mTOR</i> (1.1%), <i>TSC2</i> (0.8%), <i>POLE</i> (0.6%), and (6.1%) had high tumor mutational burden (TMB-H). Black patients were more likely to have tumors with <i>TP53</i> genomic alterations (41.6 vs 26.6%, p<0.001), and less likely to have alterations in the <i>PI3K (22.6% vs 34.1%, p<0.001) and PTEN (11.9% vs 29.9%, p<0.001) pathways</i> compared to White patients. Moreover, Black patients were less likely to have tumors with high TMB (3.1 vs 7%, p=0.001). 461 (46.4%) had additional molecular testing. IHC demonstrated 65 (6.5%) had 2-3+ Her2 overexpression, 155 (15.6%) p53 overexpression, 141 (14.2%) MMR deficiency, 336 (33.8%) ER positivity, and 261 (26.3%) PR positivity; 28 (2.8%) Her2 amplification via FISH; and 115 (11.6%) had MSI based on PCR (Table). A subset analysis of those who developed persistent or recurrent disease and received matched molecularly targeted therapy is ongoing to assess response and survival outcomes. <b>Conclusions:</b> This new multicenter consortium is facilitating the development of real-world data on patterns of genomic testing and use of molecularly targeted therapies in patients with metastatic EC, and includes a racially and geographically diverse patient population. This effort has the potential to better define the role of targeted therapies in evolving EC treatment landscape.

NGS-Based MRD Quantitation: An Alternative to qPCR Validated on a Large Consecutive Cohort of Children with ALL

Together with multicolor flow cytometry, quantitation of clonal immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements represents the current standard for the detection of minimal / measurable residual disease (MRD) in treatment protocols for pediatric acute lymphoblastic leukemia (ALL) patients. Despite the adoption of next generation sequencing (NGS) in the routine identification of clonal IG/TR gene rearrangements as markers for MRD detection, real-time quantitative (q)PCR is still the standard for MRD quantitation in follow-up samples. So far, no large-scale direct comparison of qPCR- and NGS-based MRD quantitation has been performed.We compared qPCR- and NGS-MRD evaluation in a cohort of children with B-cell precursor (BCP) ALL treated on the AIEOP-BFM ALL 2009 protocol and assessed the feasibility and relevance of this method for the stratification at day 33 (EOI).In total, 459 patients were diagnosed with BCP-ALL from 2010 to 2018, and 437 of them were included in our study based on the availability of residual DNA material isolated from day 33 bone marrow aspirates and having at least one IG/TR MRD marker detectable by standard qPCR with protocol-required sensitivity of 10 -4. Sequencing libraries were prepared according to the EuroClonality-NGS group SOP (Brüggemann et al, Leukemia 2019) with the total DNA input normalized to the equivalent of 150,000 nucleated cells to reach MRD sensitivity of 10 -5 and sequenced on Illumina NovaSeq and MiSeq instruments. In total of 780 IG/TR markers evaluated by both NGS and qPCR. Sequencing data were analyzed using the ARResT/Interrogate (Bystry et at, Bioinformatics 2017) pipeline and a custom bioinformatic analysis process and the NGS-MRD results were normalized to the EuroClonality-NGS central in-tube quality/quantification control (cIT-QC; Knecht et al, Leukemia 2019).From the total 780 IG/TR MRD markers evaluated by both methods, 629 (80.6%) were concordant with 242 markers being MRD positive and 387 negative. From 82 markers that were only positive by qPCR and not by NGS, 76 were positive below the quantitative range (positive non-quantifiable). Specificity analysis was performed for each marker by searching for the junction sequence across the dataset of all patients' NGS results. Based on these results, 22 out of 82 markers positive only by qPCR were classified as potentially unspecific (false positive) and similarly 32 unspecific markers were identified among the 69 positive only by NGS. This was also supported by unspecific amplification of the polyclonal control in 27 out of these 32 corresponding qPCR systems, in some cases leading to qPCR negative classification determined by the EuroMRD guidelines.Overall stratification of patients based only on day 33 MRD by qPCR or NGS was concordant in 76% of patients by both methods, while in 19% of patients, NGS-MRD quantitation led to the assignment to a lower-risk group, mainly due to the elimination of false-positive results. Furthermore, analysis of all positive markers across all patients' NGS libraries showed, that one out of 10 markers (mainly in the IGK, TRG and TRD loci) used for qPCR-MRD stratification did not provide satisfactory specificity, although they fully met EuroMRD criteria during the optimization of qPCR patient-specific assays.Our results show that NGS-MRD is highly concordant with traditional qPCR-based strategy and has comparable sensitivity and clinical value in the setting of a BFM-based clinical protocol, while being less laborious and providing significantly more specific results and additional information on the IG/TR repertoire (Kotrova et al, Blood 2016). Our study also emphasizes the importance of selecting MRD markers of adequate specificity at diagnosis. Currently, this selection can be assisted by these broad sequencing data on IG/TR repertoire of large number of patients. Based on these results, we propose that frontline NGS-MRD evaluation developed by the EuroClonality-NGS working group can be used as an alternative to traditional qPCR-based MRD quantitation in future MRD-based treatment protocols.Supported by grants NU20-03-00284 and NU20-07-00322 from the Czech Health Research Council and 534120 from Charles University. All methods were established through collaboration within the EuroClonality-NGS and EuroMRD groups. Disclosuresvan der Velden: Agilent: Research Funding; Navigate: Other: Service Level Agreement; Janssen: Other: Service Level Agreement; EuroFlow: Other: Service Level Agreement, Patents & Royalties: for network, not personally; BD Biosciences: Other: Service Level Agreement. Brüggemann: Incyte: Other: Advisory Board; Janssen: Speakers Bureau; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau. Langerak: Erasmus MS, University Medical Center: Current Employment; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Gilead: Research Funding; Janssen: Speakers Bureau.

Comparison of Variant Interpretation between Manual Expert Curator and Automated in-Silico Classification Using ACMG/AMP Guidelines in Myeloid Malignancies in a Routine NGS Setting

Background:In recent years, with the adoption of next generation sequencing, clinical laboratories are performing an increasing catalogue of genetic testing. This shift in paradigm was accompanied by new challenges in variant interpretation. Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories have to consider. Deciding how to weigh each type of evidence is difficult, and standards are needed. In 2015, ACMG/AMP published guidelines with 28 criteria that include evidence derived from population data, case-control analyses, functional data, computational predictions, allelic data, segregation studies, and de novo observations to assess variants with conflicting data.in different sources.Aims:We set out to evaluate in myeloid malignancies how the ACMG/AMP guidelines compare to an accredited laboratory approach to variant classification and explore the variance in the use and interpretation of the pathogenicity criteria. This may reduce variability in reporting across different labs and improve upon turn-around time of NGS results.Patient cohort and method:We have selected 50 cases with myeloid malignancies and with an interesting and complex mutational pattern, harboring variants which were difficult to classify during routine workup, analyzed either with a 26 genes myeloid panel (ThunderStorm Target Enrichment library; Raindance, Billerica, MA) or a 63 genes panel (TruSeq Custom Amplicon; Illumina, San Diego, CA). Alignment and Variant calling was performed with JSI SeqPilot (JSI Medisys, Ettenheim, Germany). Expert curators in the lab annotated each variant manually in a 3-tier system (pathogenic, uncertain significance, benign) given the lab's SOP for variant classification. Each variant was checked against the following databases: COSMIC (v76), ClinVar, dbSNP (v147) and IARC TP53 (r17). Population frequency information was extracted from ExAC (gnomAD) and 1,000 genomes. Disease-associated frequency of mutations was extracted from the lab's own database based on 235,669 variants classified between 2006 and 2017. Mutation impact prediction was performed using PolyPhen-2, SIFT and VEP.For this study, VCF files from Seqpilot were analyzed in QCI interpret software (Qiagen, Hilden, Germany), which computes variant classification based on ACMG/AMP professional guidelines without human intervention. In contrast to the lab's 3-tier system, a 5-tier system is proposed (Pathogenic, Likely pathogenic, Uncertain significance, Likely benign, Benign).Results:Among the 50 cases 747 variants were classified in our labs routine workflow according SOPs and accreditation according EN ISO15189, subsequent to the elimination of sequencing artefacts. 443 were classified as benign, 55 with variant of uncertain significance (VUS) and 248 as pathogenic by the expert curators. Using the computed classification yielded 395 benign variants, 16 likely benign, 143 with uncertain significance, 86 likely pathogenic and 107 pathogenic variants. To be able to compare, pathogenic/likely pathogenic and benign/likely benign were binned together. In 87% (651/747) of instances both approaches are concordant. Strikingly, there were no strong discordance calls, with a mismatch between pathogenic and benign (see Table 1). Manual interrogation of a subset of the discrepant calls revealed that in those instances, public data was scarce and/or in-silico mutation impact predication was unavailable. In some instances the pathogenic call in the manual approach was due to disease-specific frequency data of that particular variant in the lab's database.Conclusion:This study systematically evaluated the performance of manual curation against fully automated ACMG/AMP guideline variant classification and found a high concordance rate of 87% between both approaches. With more curated data and more comprehensive knowledge bases, the automated classification will even improve further. The automated approach seems to be more cautious, thus the bias towards more uncertain significance calls, which is preferable to miscalls. The guidelines seem to yield results sufficiently good for clinical use, especially for labs with little experience in variant classification and a big step forward regarding standardization. [Display omitted] DisclosuresNadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Survey study of barriers to evidence-based decision-making in oncology care using next-generation sequencing.

e18757 Background: Next-generation sequencing (NGS) has the potential to accelerate precision medicine in oncology by informing efficient and improved clinical treatment decision-making. However, discussions on the utility of NGS in clinical practice are ongoing. This survey study examined clinical utility barriers to adoption of NGS into oncologic care. Methods: A cross-sectional online survey was sent to a nationally representative sample of oncologists/hematologists, surgeons, and pathologists (N=201). The survey gathered information on physician demographics, practice characteristics, perceived barriers to NGS testing, and potential strategies for increasing adoption. Results: Almost all physicians in the sample (99.5%) reported using NGS. Physicians reported the following aspects of NGS as the most valuable: ability to guide decision-making (73.1%), more accurate identification of corresponding treatment (56.7%), comprehensive genomic coverage (54.2%), efficient technology with faster turnaround time (49.8%), and more accurate diagnosis or prognosis (41.3%). Physicians reported that NGS results guided treatment decisions for 63% of patients (Range: 2% - 100%). Over 85% of physicians reported that confidence in interpreting results and availability of clinical guidelines were important in undertaking NGS. Correspondingly, limited evidence of clinical utility was a top barrier to testing (80.1%), with pathologists and surgeons more likely than oncologists/hematologists to consider this. Overall, 76% of physicians shared that strategies to alleviate these clinical utility barriers included increased evidence, standardized guidelines, and interpretation support. Conclusions: Given the high uptake of NGS testing in this physician sample, but the lower rates of application of test results to guide treatment, the clinical impact of NGS may not be fully optimized. This discrepancy highlights the ongoing need for real-world evidence to better understand and further optimize the evolving role of NGS in the context of the overall management of the cancer patient. [Table: see text]

Next Generation Sequencing in Cytopathology: Focus on Non-Small Cell Lung Cancer.

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants.

Abstract 3211: Evolution of the CIViC knowledgebase for community driven curation of clinical variants in cancer

Abstract With increasing adoption of next generation sequencing into clinical practice, the problem of clinical interpretation of tumor variants arises as a bottleneck for patient care, as effective annotation of these variants draws from a constantly increasing body of largely unstructured clinical and preclinical results. One model to sustain clinical variant curation is to house variant annotation behind a paywall, using access fees to fund further curation effort. An alternate approach is to leverage public curation and expert moderation to create a free public resource to house and distribute this knowledge. The Clinical Interpretation of Variants in Cancer (CIViC, www.civicdb.org) knowledgebase employs the latter approach, and is a free and open-access public resource with an intuitive user interface and flexible public API for programmatic access to all content, which is available to the public with no restrictions on usage. All data is available for retrieval without login, while registration with a free account is required to contribute curation. The provenance of all curation and revision in CIViC is viewable through the web interface, and curators may also leave public comments on all content. Selected expert editors review and revise submitted content, which is clearly labeled as accepted once it has fully undergone moderation. All content in CIViC adheres to a structured data model which follows a published standard operating procedure for curation. This data model incorporates ontologies, standards and guidelines from across the field to promote interoperability and compatibility with other efforts. The CIViC interface also allows curators and organizations to track and display summary statistics of all their activity. CIViC currently has a community of over 190 curators and 16,000 clinical and research users around the world. CIViC continually develops and improves both new and existing features in response to user feedback as well as collaborative and internal development goals. Recently, the drugs and treatment terms used in predictive/therapeutic annotation have been normalized to the NCI Thesaurus. A conflict of interest (COI) statement is now required for all CIViC Editors, and functionality for writing and displaying the COI has been built into the interface. CIViC employs Predictive (Therapeutic), Prognostic, Diagnostic, Predisposing evidence types, and we highlight the recently introduced Functional evidence type, which has seen continued development. We will present the rationale for these changes including demonstrating how adding a Dominant Negative term better supports curation of functional genomics data sets. A focus of functional curation has been TP53, with over 50 evidence items to date. With multiple use cases for this type of data including targeted therapeutics, identification of relevant hotspots, or characterization of cancer driver mechanisms, functional evidence can be used to support conventional concepts of clinical utility and expand the CIViC data model. These developments provide a mechanism for discussion and integration of functional data into somatic variant interpretation guidelines, an area being explored but lacking expert consensus. Citation Format: Arpad Danos, Kilannin Krysiak, Erica K. Barnell, Adam C. Coffman, Joshua F. McMichael, Susanna Kiwala, Nicholas C. Spies, Lana M. Sheta, Shahil P. Pema, Lynzey Kujan, Kaitlin A. Clark, Sydney Anderson, Amber Wollam, Brian Li, Justin Guerra, Shruti Rao, Deborah I. Ritter, Cameron J. Grisdale, Gordana Raca, Alex H. Wagner, Subha Madhavan, Malachi Griffith, Obi L. Griffith. Evolution of the CIViC knowledgebase for community driven curation of clinical variants in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3211.

Abstract 741: Molecular and clinicopathological characteristics of Chinese non-small cell lung cancers with ROS1 gene fusions identified by next-generation sequencing

Abstract Background ROS1 gene rearrangements are reported at a prevalence of 1-2% in unselected cancer populations including non-small cell lung cancer (NSCLC), and notably higher in lung adenocarcinoma. More than twenty ROS1 fusion partners have been identified, which continues to increase due to the adoption of next-generation sequencing (NGS) for genetic testing. The tyrosine kinase inhibitors (TKIs) including crizotinib, lorlatinib, and entrectinib, have demonstrated favorable efficacy in the treatment of ROS1-rearranged NSCLCs. Methods A total of 17,158 Chinese non-small cell lung cancer (NSCLC) patients whose tumor specimen and/or circulating cell-free DNA (cfDNA) underwent genomic profiling by hybridization capture-based targeted NGS of exons and introns of cancer related genes were retrospectively reviewed. Clinical characteristics, treatment history, and progression-free survival (PFS) for crizotinib of a subset of 37 patients were further evaluated. Results A total of 288 ROS1-positive NSCLC patients were identified in the cohort at a frequency of 1.7% (288/17158). ROS1 fusions retaining the intact ROS1 kinase domain were confirmed in a subset of 258 patients (1.5%) with the most common fusion partner being CD74 (104/258, 40%), followed by EZR (34/258, 13%), SDC4 (28/258, 11%), SLC34A2 (27/258, 11%), and other recurrent ones at low frequency including TPM3, GOPC, MYH9, and CCDC6. Most frequent breakpoints on ROS1 are spreading in introns 33 (37%), 31 (25%), 32 (17%) and 34 (11%) with no obvious hotspots. 5.4% of cases (14/258) were fused to intergenic regions (IGR), mainly on the same chromosome of ROS1, with unknown fusion products. Notably, CD74 (63%) and EZR (50%) are more frequently fused with ROS1 intron 33, while ROS1 intron 31 was the most common breakpoint region of fusion events involving SDC4 (79%) and SLC34A2 (81%). Median PFS (mPFS) was not significantly different between fusions involving breakpoints in ROS1 introns 31-34 for crizotinib treatment, whereas non-CD74 patients (n=23) trended to demonstrate longer mPFS than that of CD74-ROS1 cases (n=14) (12.0 versus 9.9 months). Previously reported acquired resistance to crizotinib in ROS1-rearranged patients, including ROS1 G2032R and S1986F mutations, were observed in 11 out of 37 patients upon disease progression. In addition, two out of three patients who had uncharacterized fusion partners (IGR-ROS1) achieved durable clinical benefit on crizotinib. Conclusion We hereby report the prevalence of ROS1 fusions of 1.7% in a large Chinses NSCLC population detected by NGS testing and the most frequent fusion partners including CD74, EZR, SDC4, and SLC34A2. Crizotinib has demonstrated robust response in treating ROS1-rearranged NSCLC, particularly in non-CD74 ROS1-positive patients. Keywords ROS1, gene fusion, non-small cell lung cancer, crizotinib, breakpoint, next-generation sequencing Citation Format: Dongling Gao, Yuchen Han, Zhihua Zhao, Qiuxiang Ou, Xiaoling Tong, Ruiying Zhao, Nan Dong, Xue Wu, Wencai Li, Guozhong Jiang. Molecular and clinicopathological characteristics of Chinese non-small cell lung cancers with ROS1 gene fusions identified by next-generation sequencing [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 741.

Molecular Diagnostics in Non-Small Cell Lung Carcinoma.

Current clinical practice guidelines recognize EGFR, BRAF, ALK, and ROS1 as essential molecular biomarkers, and a host of other genetic alterations as emerging biomarkers for non-small cell lung carcinoma patients. The available approaches to detecting relevant alterations in these genes are diverse and often complementary. Laboratories have increasingly migrated away from a "single-gene test" approach, embracing assays that incorporate panels of genes capable of detecting a diverse set of alterations. The adoption of next generation sequencing (NGS) techniques has driven this shift; however, the approach to incorporation of NGS varies greatly between practices. Choice of molecular diagnostics assay, be it single-gene or NGS-based panel, will be driven by cost, urgency, clinical and laboratory focus, and professional considerations. Preanalytic factors including operator expertise, sample type and choice of fixative, and postanalytic factors including informatics pipeline and approaches to variant reporting have a significant impact on the quality of molecular diagnostics results. There is no real "one-size-fits-all" test for genomic profiling for lung cancer; clinicians and laboratorians must be prepared to offer a diverse set of assays in order to address turnaround time requirements and optimize detection of critical but difficult-to-detect tumor alterations such as gene fusions.

Clinical utility of comprehensive genomic pathway and integrated network analyses in personalized oncology.

e14051 Background: Adoption of next-generation sequencing (NGS) technology in routine clinical practice has enabled the detection of genetic aberrations such as single nucleotide variants, copy number alterations, and gene fusions. Pathway and network analyses (PNA) are key components for evaluation of NGS data in a clinical setting to explain findings involving thousands of altered genes and proteins with a smaller and more interpretable set of altered processes. Though PNA have been applied to identify driver genes and pathways in cohort-based analyses, its application in precision oncology remains unexplored. We investigate the potential utility of the Watson for Genomics (WfG) pathway analyses tool in interpreting complex and multiple genomic alterations in individual cancers. Methods: DNA and RNA isolated from 70 patient tumors across 30 different cancer types were processed with Illumina’s TST170 NGS platform. WfG’s feature of pathway analyses was used to identify gene variants, signaling pathways, networks, and the drugs targeting these alterations based on evidence in the clinical literature and FDA drug databases. Results: Analyses defined 5 different pathway/network models: 1) downstream therapeutic targets, 2) synthetic lethality, 3) combinatorial downstream targets + synthetic lethality, 4) two or more pathways converging to downstream targets, and 5) complex profile analyses. The five PNA models are illustrated by the following unique cases. 1) A thyroid cancer case with HRAS variant and activated RAF1 downstream pathway showed MAPK1/3 were suggestive of relevant targets. 2) An acute myeloid leukemia case with BRCA1, BRCA2 and PTEN variants, targeting a common synthetic lethal partner PARP1 was ideal for therapy. 3) A penile carcinoma case with BRAF, CDKN2A and TP53 variants, targeting the BRAF downstream pathway in combination with either CDKN2A or TP53 were the likely choice for therapy. 4) A glioma case with activated PI3K and MEK downstream pathway, targeting a common downstream marker would block both pathways. 5) A breast carcinoma case with a complex pathogenic variant profile provided relevant clinical information and levels of evidence for multiple drug targets. Conclusions: We discovered that the integrated WfG pathway analyses tool is ideal for visualization of the variants with levels of evidence from clinical literature and FDA drug databases that can help inform treatment options and provides a holistic understanding of a specific tumor profile allowing the treating clinician to select personalized targeted therapy.

Advancing precision medicine in clinical oncology: Whole exome paired tumor-normal DNA and RNA sequencing at a single-institution cancer center.

e14006 Background: Next generation sequencing (NGS) allows for reliable, comprehensive and cost-effective identification of clinically actionable genetic and genomic alterations. The increasing adoption of NGS in clinical oncology has increased our ability to identify germline alterations predisposing to cancer development as well as somatic changes enabling prescription of individualized cancer treatment and enhanced clinical trial participation. Here we summarize implementation of an NGS-based precision medicine initiative involving oncology patients from a single institution cancer center. Methods: IRB-approved NGS matched whole exome (WES) germline and solid tumor somatic tumor sequencing together with somatic tumor RNA sequencing (RNA-seq) were performed using germline DNA extracted from peripheral blood lymphocytes and nucleic acids for tumor DNA and RNA sequencing obtained from formalin-fixed, paraffin-embedded tumor specimens. Results of sequencing and analyses were presented to a multi-disciplinary tumor board to establish recommendations for management of germline pathogenic variation, therapeutic drug matching, clinical trials eligibility and molecularly informed patient prognosis. Results: A total of 1,005 patients completed sequencing. Germline and somatic WES exceeded 100X and 250X mean target coverage, respectively; somatic RNA-seq exceeded 200 million mean reads. Patients ranged in age from 17 to 90 years. The study cohort comprised comparable numbers of female (51%) and male (49%) patients. Ethnicities and races were broadly represented with 22% of participants identifying as Hispanic, 14% as Asian, 4% as Black, 55% as Non-Hispanic White and 5% as other. The most common solid tumor histological classification was colorectal (18%), followed by breast (16%), prostate (7%), head and neck (7%), sarcoma (7%), ovarian (5%), melanoma (4%) and lung (3%). Bioinformatic analyses and precision medicine tumor board review established that 12% of patients harbored a germline pathogenic variant and 43% carried clinically actionable genetic/genomic alterations; a majority of patients met molecular requirements for participation in a clinical trial. Conclusions: This study confirms the feasibility and utility of clinical NGS and precision medicine tumor board review in clinical oncology to identify germline genetic pathology, deliver personalized cancer therapeutics, increase clinical trial enrollment and clarify diagnosis and prognosis.

Clinical evaluation and performance of QIAGEN Clinical Insight Interpret (QCI-I) as a reporting tool for a comprehensive cancer panel (TSO 500).

e14048 Background: The rapid adoption of next-generation sequencing (NGS) methods into clinical diagnostic laboratories have revolutionized the field of genetic testing. To interpret the exhaustive NGS data, bioinformatics has become an inevitable component of any laboratory implementing a clinical NGS test. However, there is an unmet need for accurate, efficient and standardized variant interpretation for actionable insights. In this effort, AMP/ASCO/CAP released a joint consensus recommendation proposing a four-tiered system to categorize somatic alterations based on their clinical significance in cancer diagnosis, prognosis, or therapeutics for standardization of interpretation and reporting of results among laboratories. Thus, the aim of the study was to assess and validate QIAGEN Clinical Insight (QCI) Interpret software for clinical laboratory test interpretation. Methods: 67 malignant cases (myeloid neoplasm and lung) were analyzed using the TSO-500 NGS assay and secondary analysis platform (Illumina, San Diego, CA). The data was uploaded and analyzed using QCI Interpret. Patient case draft reports were produced using a TS0-500 customizable workflow with automated variant filtering, and variant classifications. Results: Data analysis on 67 cases resulted in a total of 251 alterations (SNVs and Indels). A total of 77 and 174 alterations were reported as pathogenic/ likely pathogenic into tier based classification by the QCI Interpret for myeloid and lung malignancies, respectively. For myeloid malignancies, the QCI Interpret was able to detect accurately all the clinically relevant variants and listed them into appropriate tier-based classification compared to the expertly reported alterations. For lung malignancies, 16 alteration were classified in tier 1, 96 alteration in tier 2, 61 alteration in tier 3, and 1 alteration in tier 4. The QCI interpret was able to detect 100% of the alterations being validated, and reported them into tier based classification. Conclusions: Systematic evaluation of an automated classification based on AMP/ASCO/CAP using an up to date knowledge base is critical to accurately classify actionable mutations and detect emerging relevant mutations. The QCI Interpret seems to be a critical bioinformatics tool that adheres to the AMP/ASCO/CAP classification system and incorporates TMB and MSI values from the TSO500 assay, which can be used across clinical laboratories for accurate, efficient and standardized variant interpretation for actionable insights.

External Quality Assessment for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Unique Requirements and Challenges.

Over the past decade, there has been an increase in the adoption of next generation sequencing (NGS) technologies for HIV drug resistance (HIVDR) testing. NGS far outweighs conventional Sanger sequencing as it has much higher throughput, lower cost when samples are batched and, most importantly, significantly higher sensitivities for variants present at low frequencies, which may have significant clinical implications. Despite the advantages of NGS, Sanger sequencing remains the gold standard for HIVDR testing, largely due to the lack of standardization of NGS-based HIVDR testing. One important aspect of standardization includes external quality assessment (EQA) strategies and programs. Current EQA for Sanger-based HIVDR testing includes proficiency testing where samples are sent to labs and the performance of the lab conducting such assays is evaluated. The current methods for Sanger-based EQA may not apply to NGS-based tests because of the fundamental differences in their technologies and outputs. Sanger-based genotyping reports drug resistance mutations (DRMs) data as dichotomous, whereas NGS-based HIVDR genotyping also reports DRMs as numerical data (percent abundance). Here we present an overview of the need to develop EQA for NGS-based HIVDR testing and some unique challenges that may be encountered.

Molecular diagnosis in recessive pediatric neurogenetic disease can help reduce disease recurrence in families

BackgroundThe causes for thousands of individually rare recessive diseases have been discovered since the adoption of next generation sequencing (NGS). Following the molecular diagnosis in older children in a family, parents could use this information to opt for fetal genotyping in subsequent pregnancies, which could inform decisions about elective termination of pregnancy. The use of NGS diagnostic sequencing in families has not been demonstrated to yield benefit in subsequent pregnancies to reduce recurrence. Here we evaluated whether genetic diagnosis in older children in families supports reduction in recurrence of recessive neurogenetic disease.MethodsRetrospective study involving families with a child with a recessive pediatric brain disease (rPBD) that underwent NGS-based molecular diagnosis. Prenatal molecular testing was offered to couples in which a molecular diagnosis was made, to help couples seeking to prevent recurrence. With this information, families made decisions about elective termination. Pregnancies that were carried to term were assessed for the health of child and mother, and compared with historic recurrence risk of recessive disease.ResultsBetween 2010 and 2016, 1172 families presented with a child a likely rPBD, 526 families received a molecular diagnosis, 91 families returned to the clinic with 101 subsequent pregnancies, and 84 opted for fetal genotyping. Sixty tested negative for recurrence for the biallelic mutation in the fetus, and all, except for one spontaneous abortion, carried to term, and were unaffected at follow-up. Of 24 that genotyped positive for the biallelic mutation, 16 were electively terminated, and 8 were carried to term and showed features of disease similar to that of the older affected sibling(s). Among the 101 pregnancies, disease recurrence in living offspring deviated from the expected 25% to the observed 12% ([95% CI 0·04 to 0·20], p = 0·011).ConclusionsMolecular diagnosis in an older child, coupled with prenatal fetal genotyping in subsequent pregnancies and genetic counselling, allows families to make informed decisions to reduce recessive neurogenetic disease recurrence.

Plant-microbe Interactions for Sustainable Agriculture in the Post-genomic Era.

Plant-microbe interactions are both symbiotic and antagonistic, and the knowledge of both these interactions is equally important for the progress of agricultural practice and produce. This review gives an insight into the recent advances that have been made in the plant-microbe interaction study in the post-genomic era and the application of those for enhancing agricultural production. Adoption of next-generation sequencing (NGS) and marker assisted selection of resistant genes in plants, equipped with cloning and recombination techniques, has progressed the techniques for the development of resistant plant varieties by leaps and bounds. Genome-wide association studies (GWAS) of both plants and microbes have made the selection of desirable traits in plants and manipulation of the genomes of both plants and microbes effortless and less time-consuming. Stress tolerance in plants has been shown to be accentuated by association of certain microorganisms with the plant, the study and application of the same have helped develop stress-resistant varieties of crops. Beneficial microbes associated with plants are being extensively used for the development of microbial consortia that can be applied directly to the plants or the soil. Next-generation sequencing approaches have made it possible to identify the function of microbes associated in the plant microbiome that are both culturable and non-culturable, thus opening up new doors and possibilities for the use of these huge resources of microbes that can have a potential impact on agriculture.

MS02.03 Achieving Effective Lung Cancer Genotyping While Balancing Constrained Resources

Modern oncologic practice for patients with non-small cell lung carcinoma (NSCLC) demands real time data for an increasing numbers of tumor biomarkers. As a result, laboratories are embracing panel-based approaches to tumor molecular profiling, a trend that is facilitated by the adoption of next generation sequencing (NGS) assays. NGS assays may be designed for highly sensitive and focused detection of hotspot mutations (typically by amplicon sequencing) or broader, more comprehensive profiling for detection of a wide variety of alterations in oncogenes and tumor suppressor genes (typically by hybrid capture technology). There are benefits and drawbacks to both approaches. In general, amplicon sequencing offers speed and sensitivity but limited scope and a propensity for PCR-related artifacts that may adversely affect detection of certain mutation types. In contrast, hybrid capture techniques offer tremendous breadth, enabling capture of 100s of genes to whole genomes, but are limited by sensitivity and longer turnaround time.1 Preanalytic steps including sample acquisition, pathologist review to confirm sample adequacy, and nucleic acid extraction often comprise the majority of the turnaround time required to complete focused tumor tissue molecular profiling. Some assays have been optimized for turnaround times of just a few hours- these assays bypass the separate nucleic acid extraction step,2 but are limited by the few number of mutational targets detected, ultimately requiring the use of multiple simultaneous or sequential focused panels.3 This practice, while perhaps the optimal current model from the standpoint of patient care, may be financially prohibitive for many laboratories. This approach additionally requires careful stewardship of patient tissues, as the use of multiple competing assays and may lead to exhaustion of tumor tissue and incomplete tumor molecular profiling. For patients with limited tissue, a considered plan incorporating input from the treating physician, surgical pathologist, and molecular laboratorian should developed early in order to guide assay priority and ensure adequate tissue is available to confirm any unexpected or contradictory results. When the turnaround time for comprehensive large panel testing is clinically acceptable, this approach may be theoretically more cost effective and in most cases will deliver information for essential and emerging biomarkers.4 It is important to keep in mind, however, that even comprehensive DNA-based assays may lack optimal sensitivity for certain structural variants (fusions, large insertion-deletion events), and it may be necessary to couple these tests with focused RNA-based panels optimized for transcript fusion detection.5 1. Rizzo JM, Buck MJ. Key principles and clinical applications of "next-generation" DNA sequencing. Cancer Prev Res (Phila). 2012;5(7):887-900. 2. Ilie M, Butori C, Lassalle S, et al. Optimization of EGFR mutation detection by the fully-automated qPCR-based Idylla system on tumor tissue from patients with non-small cell lung cancer. Oncotarget. 2017;8(61):103055-103062. 3. Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018;20(2):129-159. 4. Sireci AN. Single Genes, Panels, and Next-Generation Sequencing Platforms: A Financial Perspective. Arch Pathol Lab Med. 2018;142(7):790-791. 5. Benayed R, Offin M, Mullaney K, et al. High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden. Clin Cancer Res. 2019. amplicon sequencing, hybrid capture, turnaround time

Prenatal Testing Combined with Familial Genetic Diagnosis Reduces Disease Recurrence

Background: The causes for thousands of individually rare recessive diseases have been discovered since the adoption of next generation sequencing (NGS). Following molecular diagnosis in a child, families could use this information to make informed decisions in planning future pregnancies. However, there are no population-level studies to measure the effect of applying this knowledge on recurrence risk. Methods: In a retrospective study involving 1172 families with a recessive pediatric brain disease (rPBD) that underwent NGS-based molecular diagnosis, prenatal diagnosis was offered to families seeking to prevent recurrence through fetal genotyping. Pregnancies that were carried to term were assessed for the health of child and mother. Findings: Between 2010 and 2016, 526 families received a molecular diagnosis following NGS, 91 families returned to the clinic with 101 subsequent pregnancies, and 84 opted for fetal genotyping. Of 59 fetuses that genotyped negative and were carried to term, all were unaffected at follow-up. Of 24 that genotyped positive, 16 were terminated, reducing the disease recurrence from the expected 25% to the observed 12% ([95% CI 0·04 to 0·20], p=0·011). The remaining 8 pregnancies were carried to term, and all demonstrated disease concordant with their affected sibling(s). Interpretation: Identification of causes through NGS, coupled with prenatal fetal genotyping in subsequent pregnancies, can empower families to make informed decisions and reduce recessive disease recurrence. Funding Statement: NIH R01NS048453, R01NS052455, UL1TR001442 of CTSA and HHMI. Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: This study was approved at the host institutions of the respective study sites. The University of California San Diego IRB approved recruitment of subjects for NGS and return of research results to the referring physician and family. The National Research Centre in Cairo approved the study for confirmation of research results, fetal genotyping and genetic counseling for families. Amniocentesis and eTOP utilized standard procedural consent according to local practice.

Abstract 3277: Identification of internal tandem duplication within the FLT3 gene from AML patient next-generation sequence data

Abstract Introduction The presence of the Fms Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (FLT3-ITD) is associated with a poor prognosis and an increased rate of relapse in acute myeloid leukemia (AML). Identification of FLT3-ITD from next-generation sequencing (NGS) data is currently impeded by lack of reliable algorithms to identify large insertions. Therefore, despite the wide adoption of NGS in recent years, FLT3-ITD mutations are still being detected with PCR/electrophoresis assays which are expensive and time consuming. Algorithms that reliably identify FLT3-ITDs from NGS data are urgently required. Methods We developed a computational algorithm for FLT3-ITD calling from next-generation sequencing data. The method is based on iterative realignment of unaligned sequence read fragments. The algorithm uses sequence reads mapped to FLT3 exon 14 and unmapped sequence reads sharing similarity (at least 25bp aligned with the Smith-Waterman algorithm) with FLT3 exon 14. The procedure has two steps: (1) Smith-Waterman local pairwise alignment of each read to the reference sequence, and (2) trimming unaligned parts of each read. If a read has an unaligned part(s) identified at step 2, then these are returned to the step 1, and the whole procedure is repeated. The procedure is iterated until each read is fully-aligned to the reference sequence. A sample is considered to be ITD negative if all reads are fully-aligned after the first iteration. Otherwise, the algorithm returns fragmentized reads aligned to the reference sequence. This output can be used to discriminate between ITD positive and negative samples based on the pattern produced by aligned fragments of the reads. Reads coming from FLT3-ITD have an intrinsic property to cluster at the boundaries of the duplicated region; therefore, samples with reads ends clustering at a shared genomic position are considered ITD positive. We calculate the p-value of reads ends clustering at the same genomic position using generalized birthday-problem theory. Results We analyzed a total of 273 AML samples from 125 patients. The algorithm assigned 221 samples (~81%) as FLT3-ITD negative, forty-seven (~17%) samples as FLT3-ITD positive and 5 samples (~1.8%) were assigned as undefined (with a p-value between 0.005 and 0.1). We visually inspected the patterns produced by realigned fragments to assign a FLT3-ITD status for each of the undefined samples. We compared these results with FLT3-ITD status determined by a clinical PCR/electrophoresis assay. Our algorithm was able to recall all the 13 samples where ITD was detected by the clinical assay. Notably, FLT3-ITD mutation in two samples were detected with our method but not by the clinical assay. Conclusions 1. Our novel algorithm enables accurate identification of FLT3-ITD mutations from NGS sequence data. 2. The method was able to detect FLT3-ITD positive samples that were missed by the clinical assay. Citation Format: Yevhen Akimov, Samuli Eldfors, Disha Malani, Tero Aittokallio, Caroline Heckman. Identification of internal tandem duplication within the FLT3 gene from AML patient next-generation sequence data [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 3277.

Xome-Blender: A novel cancer genome simulator.

The adoption of next generation sequencing based methods in cancer research allowed for the investigation of the complex genetic structure of tumor samples. In the last few years, considerable importance was given to the research of somatic variants and several computational approaches were developed for this purpose. Despite continuous improvements to these programs, the validation of their results it’s a hard challenge due to multiple sources of error. To overcome this drawback different simulation approaches are used to generate synthetic samples but they are often based on the addition of artificial mutations that mimic the complexity of genomic variations. For these reasons, we developed a novel software, Xome-Blender, that generates synthetic cancer genomes with user defined features such as the number of subclones, the number of somatic variants and the presence of copy number alterations (CNAs), without the addition of any synthetic element. The singularity of our method is the “morphological approach” used to generate mutation events. To demonstrate the power of our tool we used it to address the hard challenge of evaluating the performance of nine state-of-the-art somatic variant calling methods for small and large variants (VarScan2, MuTect, Shimmer, BCFtools, Strelka, EXCAVATOR2, Control-FREEC and CopywriteR). Through these analyses we observed that by using Xome-Blender data it is possible to appraise small differences between their performance and we have designated VarScan2 and EXCAVATOR2 as best tool for this kind of applications. Xome-Blender is unix-based, licensed under the GPLv3 and freely available at https://github.com/rsemeraro/XomeBlender.

The transformation from thyroid papillary carcinoma to anaplastic carcinoma point of view

Papillary thyroid carcinoma (PTC) is the most common type of thyroid neoplasm and generally well-behaved with favorable survival rates. However, a small subset of these well-differentiated tumors can unfortunately transform into anaplastic thyroid carcinoma (ATC) with highly aggressive behavior and dismal prognosis (1-4). As widespread adoption of next generation sequencing (NGS), a number of molecular alterations involved in this transition had been delineated. Previous studies supported the hypothesis that ATC might arise from dedifferentiation of preexisting PTC tumor stepwise (2,5,6). In consideration of their totally different outcomes, it is of utmost importance to recognize the rare aggressive PTC. In addition, more precise sub-classification and prognostic evaluation system of thyroid carcinoma need be established.