NGS Assay Research Articles

An evaluation of solid versus liquid transport media for high-risk HPV detection and cervical cancer screening on self-collected specimens

BackgroundThe solid transport media is a small size card that allows fast, easy DNA extraction from a variety of biological samples. In 2016 we developed a solid media transport card; for that pilot study to control the self-collection we used a pseudo-self-collection technique. The current study expands this prior work using true self-collections and only the POI card, and aims to evaluate the solid media transport card to detect HR-HPV in self-samples compared to liquid transport media.MethodsTen thousand eight hundred eighty-five women between the ages of 30–59 with no screening for 3 years were enrolled. The self-collected sample was first applied to a new solid media transport card (Labeled as SC) then the brush placed in 6 ml ThinPrep liquid (Labeled as SL). Then a physician collected a direct endocervical specimen into ThinPrep liquid (Labeled as DL). Samples were tested with Cobas 4800 and the SeqHPV NGS assay for HR-HPV. Patients positive on any test were recalled for colposcopy and biopsy.ResultsTen thousand three hundred thirty-nine participants had complete data. The mean age was 43.9 years. CIN 2+ rates were 1.4% (142/10339). The agreement in HPV detection between the two different self-sample collection media was also good (Cobas HPV kappa = 0.86; SeqHPV kappa = 0.98). Tested with Cobas, the sensitivity of Cobas-SL and Cobas-SC for CIN 2+ was95.07 and 94.37%; and for CIN3+ was 96.30, 96.30% respectively. The specificity of Cobas-SC, and Cobas-SL for CIN2+ was 88.74 and 87.35%; for CIN3 was 88.04and 86.65% respectively. Tested with SeqHPV, the sensitivity for CIN2+ of Seq-SC and Seq-SL was 95.77 and 96.48%; for CIN3+, both the SC and SL specimens had a sensitivity of 100%. The specificity for CIN2+ of Seq-SC and Seq-SL was 89.54 and 89.53%; for CIN3+ was 88.84,88.82% respectively. For both HR-HPV assays, the sensitivities were similar for the two self-sample media (SC vs SL, p = 1.00).ConclusionsThe solid transport card for collecting vaginal self-samples as accurate as liquid transport media assayed by two different PCR based HR-HPV tests. The solid transport media is a suitable medium for collecting and storing vaginal self-samples.

Mutation profile differences in younger and older patients with advanced breast cancer using circulating tumor DNA (ctDNA).

Little is known regarding the mutation profiles of ctDNA in the older adult breast cancer population. The objective of this study is to assess differences in mutation profiles in the older adult breast cancer population using a ctDNA assay as well as assess utilization of testing results. Patients with advanced breast cancer underwent molecular profiling using a plasma-based ctDNA NGS assay (Guardant360) between 5/2015 and 10/2019 at Siteman Cancer Center. The profiling results of a multi-institutionaldatabase of patients with advanced breast cancer who had undergone molecular profiling were obtained. Associations between mutations and age group (≥ 65 vs. < 65) were examined using a Fisher's exact test. In the single-institutional cohort, 148 patients (69.2%) were < 65years old and 66 patients (30.8%) ≥ 65years old. ATM, BRAF, and PIK3CA mutations were found more frequently in older patients with ER + HER2- breast cancers (p < 0.01). In the multi-institutionalcohort, 5367 (61.1%) were < 65years old and 3417 (38.9%) ≥ 65years old. ATM, PIK3CA, and TP53 mutations were more common in the older cohort (p < 0.0001) and MYC and GATA3 mutations were less common in the older cohort (p < 0.0001). CtDNA testing influenced next-line treatment management in 40 (19.8%) patients in the single-institutional cohort. When controlling for subtype, results from a single institution were similar to themulti-institutional cohort showing that ATM and PIK3CA were more common in older adults. These data suggest there may be additional molecular differences in older adults with advanced breast cancers.

Abstract PO-003: CRESTONE – Clinical study of response to seribantumab in tumors with neuregulin-1 (NRG1) Fusions – A phase 2 study of the anti-HER3 mAb for advanced or metastatic solid tumors (NCT04383210)

Abstract Background: NRG1 (Neuregulin-1) gene fusions are rare oncogenic drivers found in 0.2% of solid tumors, including lung, pancreatic, gallbladder, breast, ovarian, colorectal, neuroendocrine, and sarcomas. NRG1 is the predominant ligand of HER3 and to a lesser extent HER4. NRG1 fusion proteins retaining an active EGF-like domain drive tumorigenesis and proliferation through aberrant HER3 activation. Importantly, NRG1 fusions are often mutually exclusive with other known driver alterations. NRG1 fusions have been correlated with worse overall and disease-free survival and poor response to treatment with standard therapies including chemotherapy, PD-(L)1 checkpoint inhibitors and combinations of these agents. Inhibition of HER3 and its dimerization partners represents a rational and novel therapeutic approach for tumors harboring an NRG1 fusion supported by case studies of clinical responses to anti-HER3 antibodies or pan-ERBB (tyrosine kinase inhibitors) TKIs like afatinib. Seribantumab is a fully human IgG2 mAb against HER3 uniquely able to inhibit NRG1-dependent activation of HER3, HER3-HER2 dimerization, and downstream signaling through the PI3K/AKT and MAPK pathways. The clinical safety profile of seribantumab has been well characterized through prior monotherapy and combination studies in over 800 patients. Methods: CRESTONE is an open label, multicenter Phase 2 basket trial of seribantumab in adult patients with NRG1 fusion-positive locally advanced or metastatic solid tumors who have progressed on or are nonresponsive to available therapies. The trial will enroll at least 75 previously treated patients across three cohorts. Cohort 1 (N=55) will include patients who have not received prior treatment with any ERBB targeted therapy. Cohort 2 (up to N=10) will include patients who have progressed after prior treatment which includes ERBB targeted therapy. Cohort 3 (up to N=10) will include patients harboring NRG1 fusions without an EGF-like binding domain. NRG1 fusion status for enrollment will be determined through a local CLIA or similarly accredited molecular assay. NRG1 fusion status for patients in Cohort 1 will be centrally confirmed using an RNA-based NGS assay. This study will evaluate a novel dosing regimen of weekly induction, biweekly consolidation, and Q3W maintenance designed to rapidly achieve steady state levels, optimize exposure, and deliver maximal NRG1 inhibition. The primary endpoint is ORR per RECIST v1.1 by independent radiologic review. Secondary endpoints include duration of response (DoR), safety, PFS, OS, and overall clinical benefit rate. An interim analysis is planned following enrollment of 20 patients in Cohort 1. CRESTONE is open and accruing patients in the United States. Clinical trial information: NCT04383210. Citation Format: Johanna C. Bendell, Kian-Huat Lim, Mark E. Burkard, Jessica J. Lin, Young Kwang Chae, Mark A. Socinski, Gazala Khan, Karen L. Reckamp, Shawn Leland, Douglas Plessinger, Lori Kunkel, Efrat Dotan, Gregory Otterson, Sai-Hong Ignatius Ou, Tejas Patil, John V. Heymach, Edward S. Kim. CRESTONE – Clinical study of response to seribantumab in tumors with neuregulin-1 (NRG1) Fusions – A phase 2 study of the anti-HER3 mAb for advanced or metastatic solid tumors (NCT04383210) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-003.

Targeted next generation sequencing panel for HPV genotyping in cervical cancer

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3+ biopsies (n = 64), CIN2 samples that progressed (n = 5), CIN2 samples that regressed (n = 3), healthy controls (n = 10), and plasma samples (n = 10) from cervical cancer patients. Sequencing was performed using a custom targeted NGS panel designed to detect all 25 high-risk and probably high-risk and two low-risk HPV genotypes. The method was validated by the SPF10 PCR-DEIA-LiPA25 assay. In the cohort, the following HPV genotypes were identified: HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59. When comparing the results from the SPF10 PCR-DEIA-LiPA25 analyses with the NGS analyses, there was close to a perfect agreement (K = 0.92) among the genotyped HPV types, while in the two cases with complete disagreement, a third assay was applied, and here the results of the NGS analyses were confirmed. Whereas multiple HPV types were detected by the SPF10 PCR-DEIA-LiPA25 assay, the NGS analysis clearly suggest that there is one predomentant HPV type. The NGS assay was capable of detecting HPV-16 in a previous false-negative sample classified by the INNO-LiPA assay, emphasizing the importance of including multiple regions of the HPV genome when genotyping. For the 10 plasma samples, our NGS analyses showed full agreement with the digital droplet PCR (ddPCR) analyses of HPV positive as well as negative plasma samples. Lastly, the custom panel was capable of detecting the integration of HPV-16 in the SiHa cell line. The HPV panel provides a highly cost-effective method for HPV detection and genotyping, as exemplified by a list price of around 75 € per sample. In conclusion, the current study demonstrates that targeted NGS is capable of detecting and genotyping HPV in both FFPE biopsies and plasma samples. This method provides for early diagnosis and prognosis of cervical cancer disease progression, thereby optimizing the potential of recovery and survival for these patients.

Gene Editing of KLF1 to Cure Sickle Cell Disease

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving &amp;gt;90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (&amp;gt;1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD.

Clinical Characteristics and Outcomes of Patients with Large Granular Lymphocytic Leukemia Treated with Methotrexate, Cyclophosphamide or Cyclosporine. a Large Single Institutional Retrospective Analysis

Introduction Large granular lymphocytic leukemia (LGLL) is a rare mature clonal lymphoproliferative disorder derived from T-cell or natural killer (NK)-cell lineages. Even though LGLL has usually an indolent course, a majority of patients (pts) require therapy due to cytopenias, recurrent infections, autoimmune disorders or symptomatic splenomegaly. There is no standard frontline therapy established for pts with LGLL based on randomized prospective studies but a single-agent immunosuppressive therapy has been used for almost four decades. In the present study, we analyzed clinical and pathological characteristics, responses to therapy and outcomes of patients diagnosed at Moffitt Cancer Center. Methods A total of 303 consecutive LGLL pts diagnosed between 2001 and 2017 were included in the study. Pts who did not meet the WHO diagnostic criteria of peripheral blood (PB) LGL count ≥2.0 k/ μL but presented with smaller, persistent LGL clonal populations along with characteristic immunophenotype and clinical features such as cytopenias, recurrent infections, autoimmune disorders, splenomegaly, and bone marrow involvement with identical clonal LGL population were considered to have LGLL. Responses to treatment were categorized as complete response (CR), partial response (PR) and no response. CR was defined as Hb&amp;gt;12g/dL, ANC≥1.5k/μL, PLT&amp;gt;150k/μL ALC&amp;lt;4 k/μL and LGL&amp;lt;0.5K/μL. PR was defined as Hb&amp;gt;8g/dL, ANC&amp;gt;0.5K/μL, PLT&amp;gt;50k/μL, and no transfusion requirement. Cox proportional hazard mode was used for multivariate survival analysis with all the covariates listed in table 3. Results The median age at diagnosis was 65 years (range: 21 - 90 years). 92.1% of pts were diagnosed with T-LGLL and 7.9% with NK-LGLL. At presentation, 50.4% of pts had anemia; 48.4% had neutropenia and 34.6% had thrombocytopenia. 21.7% of pts were transfusion dependent; 20.3% had severe neutropenia (ANC &amp;lt; 0.5K/μL) and 33% had splenomegaly. Preexisting diagnosis of autoimmune disorders was observed in 26.4% of pts (Table 1). The Foundation One Heme NGS assay (Foundation Medicine Inc, Cambridge, Massachusetts, USA) was employed in 25 pts (www.foundationmedicine.com/genomic-testing/foundation-one-heme). Nine of 25 pts were positive for STAT 3 and none for STAT5 mutations. 54.8% of pts required treatment. Table 2 summarizes the first three lines of therapy. Methotrexate (MTX), cyclophosphamide, and cyclosporine A (CSA) were the most commonly used front-line therapies. Overall response (CR + PR) for all 3 agents ranged between 55%-67% with CR rate of 11%-25%. 25% of pts receiving Cytoxan achieved CR and 11.7% on MTX achieved CR however statistical testing was not significant due to the limitation of sample size. Rates of overall response (OS) were not different among the treatment regiments. The rate of 10-year overall survival (OS) was 56.8%. Median OS was 14 years [95% CI (9.7 years, .) (Figure 1). In the multivariate Cox model, increasing age, neutropenia, thrombopenia and coexisting malignancies were independent factors associated with poor survival. Absolute number of LGLL cells in blood was not associated with OS. T-LGLL and NK-LGLL had comparable OS (Table 3). Conclusions This study is one of the largest retrospective single-institutional studies of LGLL. Consistent with prior studies, LGLL demonstrated an indolent course with a long median OS. ORR and CR rates did not significantly differ among cyclophosphamide, MTX and CSA. However, a significant percentage of patients did not respond to the frontline immunosuppressive therapy. Clinical trials with novel therapeutic agents are necessary to further improve outcomes of patients with LGLL. Disclosures Komrokji: Jazz: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Agios: Speakers Bureau; Abbvie: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria; Geron: Honoraria. Sokol:EUSA Pharma: Consultancy, Honoraria, Speakers Bureau; Kyowa/Kirin Inc.: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Cell Cultures for Virology: Usability, Advantages, and Prospects.

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

MEIRLOP: improving score-based motif enrichment by incorporating sequence bias covariates

BackgroundMotif enrichment analysis (MEA) identifies over-represented transcription factor binding (TF) motifs in the DNA sequence of regulatory regions, enabling researchers to infer which transcription factors can regulate transcriptional response to a stimulus, or identify sequence features found near a target protein in a ChIP-seq experiment. Score-based MEA determines motifs enriched in regions exhibiting extreme differences in regulatory activity, but existing methods do not control for biases in GC content or dinucleotide composition. This lack of control for sequence bias, such as those often found in CpG islands, can obscure the enrichment of biologically relevant motifs.ResultsWe developed Motif Enrichment In Ranked Lists of Peaks (MEIRLOP), a novel MEA method that determines enrichment of TF binding motifs in a list of scored regulatory regions, while controlling for sequence bias. In this study, we compare MEIRLOP against other MEA methods in identifying binding motifs found enriched in differentially active regulatory regions after interferon-beta stimulus, finding that using logistic regression and covariates improves the ability to call enrichment of ISGF3 binding motifs from differential acetylation ChIP-seq data compared to other methods. Our method achieves similar or better performance compared to other methods when quantifying the enrichment of TF binding motifs from ENCODE TF ChIP-seq datasets. We also demonstrate how MEIRLOP is broadly applicable to the analysis of numerous types of NGS assays and experimental designs.ConclusionsOur results demonstrate the importance of controlling for sequence bias when accurately identifying enriched DNA sequence motifs using score-based MEA. MEIRLOP is available for download from https://github.com/npdeloss/meirlop under the MIT license.

420P Minimal residual disease detection and tracking tumour evolution using ctDNA in stage I-III colorectal cancer patients

At diagnosis, ∼25% of colorectal cancer (CRC) patients have synchronous metastases and ∼30% develop metachronous metastases. Little is known about the heterogeneity of these tumours, which warrants better identification tools to detect recurrence. In this study, we sought to evaluate the clinical utility of circulating tumour DNA (ctDNA) in identifying minimal residual disease (MRD) and monitoring of recurrence. The prospective, multicenter cohort study recruited patients (n=196) diagnosed with stage I-III CRC. Median follow-up time was 21.9 (1.4-41.9) months. For each patient, a personalized and tumour-informed multiplex PCR (mPCR) NGS assay was used to track ctDNA in plasma samples. The relationship between ctDNA status and clinical outcomes was evaluated. The analysis of spatial heterogeneity was performed between primary and metastatic tumours, as well as between synchronous primary tumours to track clonal and subclonal mutations. Phylogenetic comparison of paired primary and metastatic mutation profiles revealed 50% overlap in mPCR targeted variants, suggesting success in targeting clonal mutations. The mutational profiles of synchronous primary tumours were non-overlapping. This led to design of unique assays for each synchronous primary tumour to reliably detect recurrence. Postoperatively and prior to ACT, 9% (14/155) of patients showed MRD-positivity, and 78.5% (11/14) relapsed (HR: 14; 95% CI: 6.1-30; P<0.001). A shift to ctDNA-positivity during longitudinal monitoring (n=120) was associated with poor clinical outcome (HR: 58, 95% CI: 17-196; P<0.001). Multivariate analysis showed longitudinal ctDNA status to be the only significant prognostic factor associated with inferior RFS (HR: 57.7, 95% CI: 16.8-196; P<0.001). Serial ctDNA analysis detected MRD up to a median of 8.08 months (0.6 - 16.6 months) ahead of radiologic relapse (sensitivity: 87.5%, specificity: 99.1%). Synchronous CRC tumours are genetically unrelated and require individual assays for ctDNA detection. Post-operative MRD status and longitudinal monitoring using ctDNA can detect patients with a high risk of recurrence and guide treatment decisions.

1966P Improved sensitivity of integrative DNA and RNA NGS assay on fusion detection

1966P Improved sensitivity of integrative DNA and RNA NGS assay on fusion detection

Clinical Application of Targeted Deep Sequencing in Metastatic Colorectal Cancer Patients: Actionable Genomic Alteration in K-MASTER Project.

PurposeNext-generation sequencing (NGS) can facilitate precision medicine approaches in metastatic colorectal cancer (mCRC) patients. We investigated the molecular profiling of Korean mCRC patients under the K-MASTER project which was initiated in June 2017 as a nationwide precision medicine oncology clinical trial platform which used NGS assay to screen actionable mutations.Materials and MethodsAs of 22 January 2020, total of 994 mCRC patients were registered in K-MASTER project. Targeted sequencing was performed using three platforms which were composed of the K-MASTER cancer panel v1.1 and the SNUH FIRST Cancer Panel v3.01. If tumor tissue was not available, cell-free DNA was extracted and the targeted sequencing was performed by Axen Cancer Panel as a liquid biopsy.ResultsIn 994 mCRC patients, we found 1,564 clinically meaningful pathogenic variants which mutated in 71 genes. Anti-EGFR therapy candidates were 467 patients (47.0%) and BRAF V600E mutation (n=47, 4.7%), deficient mismatch repair/microsatellite instability–high (n=15, 1.5%), HER2 amplifications (n=10, 1.0%) could be incorporated with recently approved drugs. The patients with high tumor mutation burden (n=101, 12.7%) and DNA damaging response and repair defect pathway alteration (n=42, 4.2%) could be enrolled clinical trials with immune checkpoint inhibitors. There were more colorectal cancer molecular alterations such as PIK3CA, KRAS G12C, atypical BRAF, and HER2 mutations and even rarer but actionable genes that approved or ongoing clinical trials in other solid tumors.ConclusionK-MASTER project provides an intriguing background to investigate new clinical trials with biomarkers and give therapeutic opportunity for mCRC patients.

Abstract 789: Development and validation of a capture based NGS assay that enables the detection of Epstein Barr virus from gastric cancer patients' tumor tissues

Abstract Background: Gastric cancer patients with positive test for Epstein Barr virus (EBV) in their tumors have been reported to benefit significantly from anti-PD-1 therapy. Current clinical testing relies on fluorescent in situ hybridization of EBV-encoded small RNA (EBER). NGS assays have been increasingly used in the clinical settings to make therapeutic recommendations to cancer patients due to its versatility in detecting various types of genomic alterations. Detecting EBV by NGS is highly desired. Method: DNA was extracted from the FFPE samples of 214 gastric cancer patients' tumors, and profiled by the comprehensive NGS assay. A classification algorithm was developed using 131 gastric cancer samples (EBV Pos, n = 31; EBV Neg, n = 100) with known EBV status derived from EBER. The algorithm and the predetermined cutoff was then applied to an independent sample set (EBV Pos, n = 30; EBV Neg, n = 53) to evaluate the test performance. Results: In the training set, we achieved a sensitivity of 93.5% at a specificity of 100%. In the validation set, we achieved a sensitivity of 93.3% at a specificity of 100%. Using the same algorithm and cutoff, in the patients with tumor content above 20%, we achieve a sensitivity of 97.9% at a specificity of 100%. Conclusion: We developed a method to detect EBV in gastric tumors' FFPE samples through a capture-based NGS assay. We also demonstrated strong concordance with an orthogonal assay (EBER) in detecting EBV. Citation Format: Yajie Hu, Bo Yang, Lei Wang, Shiyi Liu, Lei Liang, Jie Wang, Fugen Li, Yu Sun. Development and validation of a capture based NGS assay that enables the detection of Epstein Barr virus from gastric cancer patients' tumor tissues [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 789.

Abstract 5899: Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons

Abstract Introduction: SLIMamp technology allows multiplex-PCR of tiled amplicons in a single tube, which enables targeting of large exons for NGS analysis with a streamlined process. Copy number variants (CNVs) are DNA segments that are present with variation in the number of copies compared to a normal genome. CNVs have a high prevalence in the pathogenesis of cancer and their characterization is important to acquire a more comprehensive picture of the mutations present in a patient sample. Pillar developed a proprietary CNV algorithm and combined it with SLIMamp to develop an integrated multi-cancer plus CNV detection NGS panel which identifies CNVs in the ERBB2, EGFR, MET, and MYC genes. Methods: To assess the ability of the ONCO/Reveal Multi-Cancer with CNV Panel to detect ERBB2 CNV compared to DISH (dual in situ hybridization), libraries were created from 44 well-characterized FFPE breast cancer samples plus 3 Genome in a Bottle (GIAB) samples. 23 tumor samples had known ERBB2 amplification (20 with matched normal tissue), 21 were known non-amplified tumors, and 3 GIAB samples were used as negative controls. Libraries were sequenced on an Illumina MiSeq and data was analyzed by the Pillar Variant Analysis Toolkit (PiVAT). The previous DISH results were not known to Pillar. Results were provided to an independent collaborator who unblinded the study and compared the NGS results to DISH. Results: Overall the assay performed well with a mapping rate of 99.4% ± 2.1%, on-target rate of 99.1% ± 0.4%, and coverage uniformity &amp;gt;0.2x mean coverage of 93.5% ± 4.1%. With respect to CNV calling, the assay and software detected 100% (21/21) of ERBB2 amplification negative samples as confirmed by DISH. For amplification positive samples, the NGS assay detected 100% (23/23) of positive samples. The software correctly identified 91% (21/23) of amplification positive samples. The two samples that were not called by PiVAT had normalized genes counts of 1.2 and 1.4, and also had the lowest DISH scores. All samples with a normalized gene count &amp;gt;1.5 were correctly called by PiVAT. The NGS assay was able to accurately call CNV independent of tumor cellularity with tumor content of 20%-90% not impacting the CNV call. Conclusions: The ONCO/Reveal Multi-Cancer with CNV Panel is a robust assay for the detection of CNVs, SNVs, and Indels of interest across multiple solid tumor cancer types. The workflow is streamlined, with same day loading of finished libraries when starting from as little as 5ng of isolated input DNA. The assay and software demonstrate detection of low CNV with the recommended cutoff set at 1.5. With further optimization of the calling algorithm accurate calling of even lower CNVs may be possible. The detection and identification of CNVs along with other mutations gives a more comprehensive overview of the mutations present in a sample, supporting better clinical management of patients. Citation Format: Nicholas Lodato, Zonghan Liu, Akuah Kontor, Xiaoxi Wu, Lukas Hillmer, Sean Polvino, Yue Ke, Erin Petrilli, Adam Labonte, Geoffrey Richman, Zhaohui Wang. Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons [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 5899.

Abstract LB-315: Oncology research applications on an electrical impedance based, high accuracy sequencing platform

Abstract Next Generation Sequencing (NGS) technologies have revolutionized basic biological and clinical research, especially in oncology. NGS cancer assays are used to determine cancer predisposition, identify tumor mutations, monitor treatment response and develop personalized therapies. Here, GenapSys presents a scalable, low cost, and high accuracy sequencing platform based on electrical impedance detection. We demonstrate that a single run with a 16M sensor chip generates up to 2 Gb of data, with greater than 99% raw accuracy and an average read length of about 150 bp. We highlight its applications for oncology research, using diverse cancer panels on reference and clinical samples. We tested hybrid-capture and multiplex PCR-based cancer panels on a range of DNA sources, including oncology reference standards derived from cell line DNA, cfDNA standards, as well as clinical samples: FFPE, fresh frozen tumor tissue, and blood. For hybrid-capture libraries, we used the IDT xGen Pan Cancer 1.5 and Exome Research panels. We detected low frequency mutations in the range of 1%-24.5% across multiple reference standards with the cancer panel, and observed high correlation in allele frequency with expected values (R2 &amp;gt; 0.99). Similar results were obtained with an amplicon panel, the Ion AmpliSeq Cancer Hotspot Panel v2. Whole exome sequencing and pan cancer sequencing of clinical FFPE, fresh frozen and blood samples showed high concordance (F1-score &amp;gt; 95%) of SNV mutation calling with commonly used NGS technology. Thus, we demonstrate that the GenapSys Sequencing Platform is an accurate, scalable, and low cost solution for oncology research on a wide range of sample types and NGS assays. Citation Format: Saurabh Paliwal, Mohammad Fallahi, Ali Nabi, Tyson A. Clark, Bin Dong, Srijeeta Bagchi, Maryam Jouzi, Hannah Ritchie, Lydia Bonar, Anthony Thomas, Narin Tangprasertchai, Meysam R. Barmi, Xavier Gomes, Subra Sankar, Hesaam Esfandyarpour. Oncology research applications on an electrical impedance based, high accuracy sequencing platform [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 LB-315.

Abstract 215: Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research

Abstract Introduction: Next generation sequencing technologies and platforms have been evolving rapidly over the last decade with improved throughput and cost-effectiveness. The application of various NGS assays using both genome wide or targeted sequencing has been widely employed in cancer research to characterize tumor biomarker profiles including SNV/indels, CNV, and fusion in various sample types. Here we will present the performance of OncomineTM Precision Assay developed at Thermo Fisher Scientific, which is compatible with both solid tumor and liquid biopsy sample types. It is the very first automated NGS assay developed with Ion Torrent Genexus system to provide a streamlined workflow directly from sample to results with minimal hands-on time. Methods: The assay employs a novel AmpliSeq™ HD library technology to provide optimal sensitivity and specificity for challenging sample types including degraded FFPE or cell-free nucleic acid from plasma. For library preparation, 10 ng DNA or RNA was used for FFPE and 20 ng cfTNA was used for plasma samples. Ion Torrent Genexus system provides an interface to guide the user to plan the run following a defined workflow according to sample type, and performs library preparation, templating and sequencing automatically. Results: The OncomineTM Precision Assay includes focused genetic content with clinical relevance including 45 hotpot genes (SNV/Indel), 14 CNVs, and 19 structural variants (fusion and exon variants). This study evaluated the assay performance using a set of controls for FFPE or cfDNA from positive cell line material or commercial sources such as Horizon discovery and SeraCare, which contain various SNV/Indels on several important gene loci (EGFR, KRAS, NRAS, BRAR, and PIK3CA), CNV (EGFR, MET amplification and pTEN deletion), and fusion variants across ALK, ROS1, RET, and NTRKs. The results showed that all the expected SNV variants were consistently detected with sensitivity at ≥95% for FFPE with 5% AF and ≥85% for cfDNA samples down to 0.1% LOD. For fusion variants and CNV targets including gain and loss, both sensitivity and PPV at ≥90% could be achieved for FFPE and cfTNA samples. Furthermore, a cohort of positive FFPE tissue was also analyzed and detected expected variants that had been previously confirmed through an orthogonal NGS test. Lastly, a set of matched FFPE and plasma samples was also evaluated using the assay and demonstrated high concordance between FFPE and plasma from each subject. Conclusion: Overall the study demonstrates that the OncomineTM Precision Assay provides an automated and sensitive NGS solution for efficient and effective mutation assessment using both FFPE and liquid biopsy samples. Citation Format: Ru Cao, Jian Gu, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Bo Ding, Khalid Hanif, Kelli Bramlett. Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research [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 215.

Abstract 787: Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS

Abstract Background: Tumor genomic information from tissue has been the standard of practice for identifying actionable molecular alterations. The same genomic profiling is also widely available by a non-invasive blood test from circulating tumor DNA (ctDNA). We hypothesized that patients with advanced non-small cell lung cancer (NSCLC) with actionable oncogenic driver mutations with either low or high variant allelic frequency (%VAF) identified by ctDNA would have similar clinical benefits after treatment with targeted therapies. Methods: In this retrospective study (UTMDACC IRB-approved protocol PA16-0061), patients with a targetable mutation identified by ctDNA NGS assay who received an FDA-approved therapy were identified in our GEMINI database. All potential patients were individually reviewed and information confirmed with review of their electronic medical records. Blood based genomic profiling was completed by NGS of ctDNA with a panel of up to 70 cancer-related genes at a CLIA-certified lab (Guardant360, Guardant Health, Redwood City, CA). The %VAF was reported for each mutation identified. We defined low group with VAF of 0.01% to 0.99% and high group with VAF &amp;gt; 1%. Tumor measurements were assessed using RECIST V1.1. For comparisons analysis, Kaplan-Meier estimator was used to estimate progression free survival (PFS) with Mantel-Cox and un-paired t-test was used for tumor responses. Results: Eighty-nine patients with advanced NSCLC treated with an FDA approved therapy based on ctDNA genomic profiling were identified and the results are summarized in table below: LOW %VAFHIGH %VAFP valuegroup (n=39)group (n=50)Gender (Female/Male)20/1928/22Age (median, range)54(27-85)63(29-89)Targetable mutations:EGFR2246ALK114MET exon 1420ROS120BRAF20Line of therapy (median, range)2(1-7)2(1-7)ALL PatientsTumor response (mean, range)-37.23% (-100 to +38)-31.08% (-100 to +59)0.42PFS (median, range)215 (27 – 848) days280 (43 – 894) days0.70EGFR onlyTumor response (mean, range)-24.86% (-100 to +16)-23.54% (-100 to +59)0.88PFS (median, range)189 (57 – 848) days293 (43-894) days0.23 Conclusions: In this analysis of patients who were treated with an FDA approved therapy based on genomic results from ctDNA profiling, there were no differences in either tumor response (P value = 0.42) or PFS (P value = 0.70) between those patients with low and high ctDNA as assessed by %VAF of identified targetable mutation. Similar findings were also observed in the analysis of the EGFR only subgroup. This study reveals patients with low %VAF received comparable clinical benefits as those with high %VAF and no deleterious harm was observed from treating patients with low %VAF with appropriate targeted therapy. Citation Format: Hai T. Tran, Vincent K. Lam, Mayra Vasquez, Yasir Elamin, Victoria Raymond, Lingzhi Hong, George Blumenschein, Brett Carter, Richard Lanman, Mehmet Altan, Ann Tsao, Lauren Byers, Don Gibbons, Jianjun Zhang, John V. Heymach. Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS [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 787.

Abstract 3620: Actionable genomic landscape of metastatic colorectal cancer patients in Korea: Interim data from K-MASTER project

Abstract The K-MASTER cancer precision medicine diagnosis and treatment enterprise (K-MASTER project) is a precision medicine cancer treatment clinical trial platform, which uses NGS assay to screen the actionable mutations in 10,000 Korean patients with refractory solid tumors and assigns the patients to matched clinical trials. K-MASTER project was initiated at June 2017 and 52 sites, 4930 patients have participated. As of November 30 in 2019, total 889 metastatic colorectal cancer (mCRC) patients were submitted. Tumor DNA was analyzed by FIRST cancer panel (Seoul National University Hospital) and CancerSCAN (Samsung Genomic Institute) which allow to detect variations of 183 and 409 genes irrespectively. The failure rate of sequencing was 5.5% of patients with samples submitted. The mean depth was 650x (FIRST) and 701x (CancerSCAN). Average turn-around time was 3 weeks (CancerScan) and 6 weeks (FIRST). In Korean mCRC patients, variations were mainly identified in TP53 (60.9%), APC (56.9%), KRAS (41.4%), BRCA2 (19.2%), PIK3CA (14.5%), ATM (14.2%), KMT2D (13.4%), NOTCH1(13.4%), SMAD4 (11.6%), FBXW7 (10.2%), ALK (9.7%), NOTCH3 (9.2%), BRAF (8.8%). MSI-high or POLE mutant mCRC patients were enrolled in KM-01 (Avelumab). Several drugs were already approved in the other countries but not in Korea; ERBB (7.5%) with anti-HER2 agents, and MSI high such as MSH6 (3.9%), MSH2 (3.7%), MLH1 (3.4%) with immune checkpoint inhibitors. In addition, there were potentially actionable variants outside of approved label in Korea; translocation of NTRK3 (n=4), RET (n=3), ALK/EML4 (n=2). Promising drugs could be possible to incorporate in clinical trials using PIK3CA (14.5%) with PIK3CA/Akt/mTOR pathway inhibitor, DNA damage response (DDR) genes such as BRCA2 (19.2%), ATM (14.2%), FANCA (6.6%), POLE (5.8%), BRIP1(4.8%), RAD50 (4.5%), BRCA(4.0%), PALB2 (4.0%), PARP2 (3.7%), CDK12 (3.4%), MUTYH (2.7%), RAD54L (2.5%) with immune checkpoint inhibitor and/or PARP inhibitor, KRAS G12C (n=18) with AMG 510 (KRAS inhibitor), amplification of FGFR1 (n=4) and MET (n=1). There are 2 clinical trials which mCRC patients can participate in early 2020; MET alteration in KM-08 (Tepotinib), high tumor mutation burden in KM-12 (Ipilimumab and nivolumab). In conclusion, this project is efficient to detect actionable variations in mCRC. This novel knowledge provides an intriguing background to investigate new clinical trials with biomarker and give therapeutic opportunity for mCRC patients. By help of regulatory approval of innovative drugs and off-label use, biomarker-driven early phase clinical trials, it could be a huge therapeutic opportunity for refractory mCRC patients. (This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp;Welfare, Republic of Korea (Grant number: HI17C2206). Citation Format: Soohyeon Lee, Jae Sook Sung, Hee-Joon Chung, Yoon Ji Choi, Won Jin Chang, Ju Won Kim, Kyong Hwa Park, Yeul Hong Kim. Actionable genomic landscape of metastatic colorectal cancer patients in Korea: Interim data from K-MASTER project [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 3620.

Abstract 4262: Analytical validation of TruSight Oncology 500, a comprehensive genomic profiling LDT for use in routine patient care

Abstract We report the analytical performance of Illumina's TruSight™ Oncology 500 (TSO500) NGS assay for key oncologic variants/ biomarkers. A combination of commercially available reference materials and de-identified clinical FFPE samples were used as the truth set to assess accuracy, analytical sensitivity, and precision. The TSO500 assay utilizes FFPE-extracted DNA and RNA for enrichment-based, targeted next-generation sequencing to detect a comprehensive list of biomarkers relevant to solid tumors and hematological malignancies: single nucleotide variants (SNVs), copy number variants (CNVs), indels, fusions, splice variants, tumor mutational burden (TMB), and microsatellite instability (MSI). Targeted sequencing reduces sequencing data on irrelevant regions in the genome thus greatly increasing coverage on relevant genetic variants and regions. The assay covers small variants in 523 genes, fusions/spice variants in 55 genes, and CNVs in 59 genes, thus providing comprehensive coverage of the variants that play a role in tumorigenesis. The assay also provides a measure of two additional biomarkers, MSI and TMB, which are important for predicting response to cancer immunotherapy agents. De-identified, FFPE patient samples were processed and analyzed in the Illumina Lab Services (ILS, CAP-accredited laboratory). Samples that passed QC metrics were evaluated for accuracy for variants that were known to be present in the truth set. Accuracy was evaluated between the truth set and ILS for TMB using linear regression analysis and for microsatellite stability (MSS/MSI) using a cutoff of 20%. Additionally, accuracy, analytical sensitivity, and precision for the biomarkers covered by TSO500 were assessed using reference materials with known content. The assay demonstrated ≥ 99% concordance between the truth set and ILS for small variants (present at an allele frequency of ≥ 5%), fusions/splice variants, and CNVs. For the immunotherapy biomarkers, we observed high concordance between the truth set and ILS. There was 100% concordance for MSS/MSI calls with most samples being MSS. The linear regression R-squared value for TMB was 0.99 with scores ranging from very low (0) to high (370); most samples had scores between 0-20. A high level of repeatability and reproducibility was observed across three reference materials each for small variants, CNVs, and fusions/splice variants with values of ≥ 98%. For SNVs, the sensitivity of detection was 100% at the Lower Limit of Detection of 5%. Collectively we demonstrated high accuracy and precision for the variant types found in the TSO500 panel. Validation of this assay will allow the implementation of this laboratory developed test (LDT) for use in routine patient care. Citation Format: Karena Kosco, Brad Sickler, Silvia Chan, Mawadda Alnaeeli, Sue Beruti. Analytical validation of TruSight Oncology 500, a comprehensive genomic profiling LDT for use in routine patient care [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 4262.

Abstract 4893: Multi-modality sequencing of multiple HPV mediated oropharyngeal squamous cell carcinomas from single patients reveals synchrony in viral genomes yet discordant mutational profiles and signatures

Abstract Human Papilloma Virus (HPV) mediated oropharyngeal squamous cell carcinoma (HPVmOPSCC) is increasing in prevalence in the United States, as are cases of patients with multiple, spatially distinct, HPVmOPSCCs (mHPVmOPSCC). Patients with mHPVmOPSCCs present a unique opportunity to study the biology of HPV mediated tumorigenesis in the oropharynx, which currently is poorly understood. We performed whole exome sequencing and RNA-Seq on 16 paired HPVmOPSCCs (eight patients) and whole genome sequencing of the viral genome using a HPV16 NGS assay described previously. Viral sequencing revealed considerable diversity between patients, across multiple viral sublineages. However, all tumor pairs from a given patient contained the same viral genome, with at most 2 clonal SNPs distinguishing the viruses within a pair. In contrast, tumor DNA sequencing revealed striking dissimilarity between tumors from a given patient. Seven of eight tumor pairs shared no overlapping somatic mutations with one pair sharing only six variants out of 277 (2%). Mutational signature analysis demonstrated a dominant APOBEC signature in six tumors. Tumor pairs showed striking discordance of mutational signatures. In 3/8 pairs, one tumor exhibited a nearly 100% pure APOBEC+ mutational profile, while the other was totally devoid of APOBEC-signature mutations. Here, we have demonstrated that spatially independent tumors, originating within the same individual, in the same tissue type, are caused by the same virus. Interesting, despite this shared etiology, the pair of tumors within a patient develop with vastly different mutational profiles and have different mutational processes driving tumorigenesis. This suggests that despite infection by a single oncogenic virus, HPVmOPSCCs may develop via multiple, disparate pathways. Of particular interest is the tumor pairs' clear discordance in APOBEC mutational burden, which challenges existing hypotheses on the role of APOBEC mutagenesis in this disease. Citation Format: Daniel L. Faden, Adam Langenbucher, Krystle Kuhs, James S. Lewis, Michael S. Lawrence, Lisa Mirabello, Robert L. Ferris. Multi-modality sequencing of multiple HPV mediated oropharyngeal squamous cell carcinomas from single patients reveals synchrony in viral genomes yet discordant mutational profiles and signatures [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 4893.

Abstract 1978: Reference materials for measurable residual disease (MRD) monitoring

Abstract Here we describe reference materials for measurable residual disease (MRD) assay design and validation as well as initial data from testing. The analytical validation of liquid biopsy-based assays that attempt to monitor for the disappearance and reemergence of cancer can be challenging due to the need for reference materials that allow for the assessment of sensitivity and specificity at variant allele frequencies (VAFs) that can be over an order of magnitude below those that can be detected reliably by typical circulating tumor DNA (ctDNA) assays. At such low VAFs, a given plasma sample may only contain limited somatic variants - and only at a few copies - which is why some MRD assays focus on monitoring only patient-specific somatic variants and do not analyze other parts of the genome. However, this also means that validation should include the steps needed to monitor patient-specific somatic variants. Therefore, we designed our reference materials for a tumor/normal workflow as a set of three components. First, one cell line is a source of normal DNA that can be used to assess specificity. Second, a germline SNP-matched cell line provides hundreds to thousands of additional somatic variants. Third, blends of fragmented and sized DNA are used to mimic circulating cell-free DNA (ccfDNA) and serve as the input for MRD assays at VAFs from 0.1% to 0.01% (one mutant copy per 3.5 to 35 ng of ccfDNA) and at 0%. As a proof of concept, we analyzed three cell lines for somatic variants by Whole Exome Sequencing (WES). Using the resulting data, digital PCR assays were designed and two different error corrected NGS assays were customized to measure some of the identified variants. Based on past experiments, A&amp;gt;G (T&amp;gt;C) and C&amp;gt;T (G&amp;gt;A) changes had the highest background in NGS data, so these were generally avoided. The tumor and normal samples were used to assess the performance of a given customized assay to measure a variant. Digital PCR was used to verify VAFs in the ccfDNA format, which proved challenging for 0.01% given the expectation of approximately one positive droplet per assay well. NGS was then used to look for the variants using around 50 ng of input per reaction. While the detection of 0.1% VAFs appeared to be feasible with customized off-the-shelf assays, 0.01% was difficult given the need to measure a variant-containing region over 10,000 times to observe a somatic variant - if it was included in the library - and to measure each starting molecule around 10 times for error correction, which required a depth of greater than 100,000 for 20 of several hundred identified candidate variants. While we did not evaluate the reference materials with emerging clinical MRD assays and focused our initial testing on customized off-the-shelf assays, these reference materials should enable the design, validation, and evaluation of MRD assays. Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella, Russell K. Garlick. Reference materials for measurable residual disease (MRD) monitoring [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 1978.