Targeted Next-generation Sequencing Assay Research Articles

Abstract 532: Circulating tumor DNA and outcomes with lutetium-PSMA in advanced prostate cancer: Preliminary results from an Australian study

Abstract Recent clinical trials have revealed that Lutetium PSMA-617 (Lu-PSMA, a small molecular inhibitor of prostate-specific membrane antigen radiolabelled with the beta emitter 177-Lutetium) significantly improves clinical outcomes including overall survival for patients with metastatic castration resistant prostate cancer (mCRPC). Nevertheless, owing to the highly heterogeneous nature of mCRPC, responses to Lu-PSMA therapy can be variable, with resistance inevitable. Currently, there are no existing data on the genomic landscape of mCRPC in the context of this therapy, with the identification of biomarkers linked to outcomes with Lu-PSMA being a critical unmet need in order to select patients most likely to benefit from treatment and to dissect mechanisms of resistance. mCRPC patients with high PSMA expression on positron emission tomography scans and prior treatment with docetaxel chemotherapy and an androgen receptor pathway inhibitor were enrolled on a compassionate access prospective registry at Peter MacCallum Cancer Centre. Patients received Lu-PSMA every 6 weeks for 4-6 cycles and entered into follow-up. Peripheral blood samples were collected for plasma circulating tumour DNA (ctDNA) analysis at baseline and prior to cycle 2 of therapy. A highly-sensitive targeted next-generation sequencing assay was applied to patient plasma and matched leukocytes. Somatic mutations (single nucleotide variants/indels) and copy number alterations from 43 genes were reported, along with the estimated ctDNA fraction. Current patient numbers preclude formal statistical analysis of genomic aberrations, however dynamic changes in ctDNA were assessed in relation to prostate-specific antigen (PSA) response and clinical and/or radiographic progression. In total, 19 plasma samples were analysed from 13 mCRPC patients with a median age of 71. Patients had between one and four prior lines of therapy for mCRPC and had a median time on Lu-PSMA therapy of 3.7 months. ctDNA was detected in 17/19 plasma samples with a median fraction of 26% (range 0-89%). Overall, a PSA response rate of 54% was observed. Interestingly, patients with higher baseline ctDNA fractions were more likely to experience a PSA response to Lu-PSMA therapy (median ctDNA fraction of responders 49% vs 10% in non-responders, p=0.1). Additionally, no PSA responses were seen in patients who did not attain a reduction in plasma ctDNA from baseline to cycle 2. This is the first targeted assessment of serial plasma samples from patients receiving Lu-PSMA therapy. Using a highly sensitive liquid biopsy assay, ctDNA was detectable in almost all samples. In this preliminary analysis, both baseline ctDNA fraction and early dynamic changes in ctDNA fraction appear to have clinical importance in Lu-PSMA therapy. Recruitment to this registry is proceeding rapidly, with further data available by April 2022. Citation Format: Heidi Fettke, Nicole Ng, Christine Hauser, Patricia Bukczynska, Elizabeth Medhurst, Louise Kostos, James Buteau, Jason Steen, Tu Nguyen-Dumont, Michael Hofman, Arun A. Azad. Circulating tumor DNA and outcomes with lutetium-PSMA in advanced prostate cancer: Preliminary results from an Australian study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 532.

Abstract 5403: Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations

Abstract Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations Background Currently, most patients with mCRC are treated with a combination of a cytotoxic and a biologic agent. Several factors should be put into account which influencing the treatment strategy for patients with mCRC. including tumor resectability, prior systemic therapy; tumor location (left or right), and molecular characteristics such as mutation in gene Kirsten rat sarcoma viral oncogene homolog (KRAS). It has been demonstrated to be a tightly associated with poor response to anti-EGFR treatment, and even poor response to immune checkpoint inhibitors for patients with colorectal cancer. Therefore, KRAS mutational status testing has been highlighted in recent years. Methods This study queried the data from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 2,294 CRC patients which underwent a targeted next-generation sequencing assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2017 and June, 2020 in China, to obtain a comprehensive molecular profile of KRAS mutations. Co-occurring genomic alterations, tumor mutational burden (TMB), microsatellite instability (MSI) status and PD-L1 expression (Dako 22C3 or VentanaSP263, TPS score) were analyzed. High TMB was defined as TMB ≥10 muts/Mb. Postoperative follow-up data for 27 patients were collected and analyzed. Results KRAS mutation account for 48.7% Chinese CRC patients. In addition, proportions of KRAS mutation in different exons were counted. KRAS mutation in CRC mainly occurred in exon2 (84.0%), exon3 (6.1%), exon4 (7.4%), and exon5 (0.7%). KRAS mutation in exon2, the main mutations were G12D (35.4%), G12V (20.8%), G13D (21.9%), G12C (5.9%) ad G12S (5.8%). while in exon3 and exon4, the main mutations were Q61H (50.7%) and A146T (73.2%), respectively. The proportion of MSI-H in CRC patients with and without KRAS mutation were very close. While it seemed that there were more MSI-H patients in KRAS exon3 and exon4 mutation. In addition, PD-L1 expression was detected in 1220 tumor samples by means of IHC. Among these available tumor samples, level of PD-L1expression were similar among patients without KRAS mutation, KRAS mutation in different exons, but it reveled that there were very small part of patient with strong positive PD-L1 level (TPS≥50). In addition, different prognosis were found among different mutations of KRAS exon2, in detail, worse PFS was showed in CRC patients with KRAS mutation in exon2 G12D than G12V and G13D. Conclusions CRC patients with KRAS mutations in different exons and different site have different genetic and prognostic characteristics, these patients should be managed differently in clinical practice. Citation Format: Zhengqi Wen, Hushan Zhang, Ruize Zhou, Xihong Liu, Wenliang Li. Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5403.

Real-World Data and Budget Impact Analysis (BIA): Evaluation of a Targeted Next-Generation Sequencing Diagnostic Approach in Two Orthopedic Rare Diseases.

Objective: Next-generation sequencing (NGS) technology, changing the diagnostic approach, has become essential in clinical settings, and its adoption by public health laboratories is now the practice. Despite this, as technological innovations, its intake requires an evaluation of both the clinical utility and the economic investment, especially considering the rare disease scenario. This study evaluated the analytical validity and the budget impact of an NGS-Ion Torrent™ approach for the molecular germline diagnosis of two musculoskeletal rare diseases. Methods: Two cohorts of 200 and 199 patients with suspect or clinical diagnosis of multiple osteochondromas (MO) and osteogenesis imperfecta (OI) previously evaluated with a single-gene diagnostic protocol were re-analyzed using a targeted NGS assay. Analytical validity was assessed by comparing NGS and single-gene protocol. A budget impact analysis using real-world cost data-considering the healthcare perspective— was performed by applying activity-based costing (ABC). The cost considered consumables, personnel, and equipment. Additional costs not related to NGS activities were not considered. Sensitivity analysis was performed. Results: The NGS method showed a higher (for MO) and comparable (for OI) diagnostic sensitivity than the traditional techniques, apart from always reducing the time and costs of diagnosis. Overall, the cost saving per patient is € 765 for OI and € 74 for MO. Materials represented the highest cost driver of the NGS process. A time saving—proportional to the panel size—has been assessed in both cases. Conclusions: Our targeted NGS diagnostic approach decreases time to diagnosis and costs, appearing to be beneficial and recommended both for patients and from a healthcare perspective in routine diagnosis also considering very small gene panels and a low patient flow. The adequate analytical sensitivity always required the additional Sanger sequencing step of the low- and non-covered regions. A more accurate strategy evaluation is suggested in the case of ultra-rare/complex diseases, large gene-panel, or non-reference diagnostic centers.

Utility of targeted next-generation sequencing assay to detect 1p/19q co-deletion in formalin-fixed paraffin-embedded glioma specimens

Molecular classification of brain neoplasms is important for diagnosis, prognosis, and treatment outcome of histologically similar tumors. Oligodendroglioma is a glioma subtype characterized by 1p/19q co-deletion and IDH1/IDH2 mutations, which predict a good prognosis, responsiveness to therapy, and an improved overall survival compared to other adult gliomas. In a routine clinical setting, 1p/19q co-deletion is detected by interphase-FISH and SNP microarray, and somatic mutations are detected by targeted next-generation sequencing (NGS). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both 1p/19q co-deletion and somatic mutations. Among 247 consecutive patients with formalin-fixed paraffin-embedded brain tumors with various subtypes, NGS revealed 1p/19q co-deletion in 26 oligodendrogliomas and an IDH-wildtype astrocytoma, and partial loss across chromosomes 1p and 19q/whole-arm loss of 1p or 19q/copy neutral loss of heterozygosity in 11 nonoligodendrogliomas. For this 247 brain-tumor cohort, the overall sensitivity, specificity, and accuracy of detecting 1p/19q co-deletion by NGS in oligodendrogliomas were 96.2%, 99.6%, and 99.2%, respectively. The oligodendroglioma cohort had more mutations in IDH1/IDH2, CIC, FUBP1, and TERT, and fewer mutations in ATRX and TP53 than the nonoligodendroglioma cohort. This proof-of-concept study demonstrated that targeted NGS can simultaneously detect both 1p/19q co-deletion and somatic mutations, which can provide a more comprehensive genetic profiling for patients with gliomas using a single assay in a clinical setting.

Circulating tumor DNA as a predictive biomarker in Merkel cell carcinoma

Circulating tumor DNA as a predictive biomarker in Merkel cell carcinoma

Interlaboratory Comparison Study (Ring Test) of Next-Generation Sequencing-Based NTRK Fusion Detection in South Korea.

Tropomyosin receptor kinase (TRK) inhibitors are approved for the treatment of neurotrophic receptor tyrosine kinase (NTRK) fusion-positive tumors. The detection of NTRK fusion using a validated method is required before therapeutic application. An interlaboratory comparison study of next-generation sequencing (NGS)-based NTRK gene fusion detection with validated clinical samples was conducted at six major hospitals in South Korea. A total of 18 samples, including a positive standard reference and eight positive and nine negative clinical samples, were validated using the VENTANA pan-TRK (EPR17341) and TruSight Oncology 500 assays. These samples were then tested using four different NGS panels currently being used at the six participating institutions. NTRK fusions were not detected in any of the nine negative clinical samples, demonstrating 100% specificity in all six participating institutions. All assays showed 100% analytical sensitivity to identify the NTRK fusion status in formalin-fixed paraffin-embedded (FFPE) samples, although with variable clinical sensitivity. False-negative results were due to low tumor purity, poor RNA quality, and DNA-based sequencing panel. The RNA-based targeted NGS assay showed an overall high success rate of identifying NTRK fusion status in FFPE samples. This study is the first to test the proficiency of NGS-based NTRK detection in South Korea with the largest participating institutions. RNA-based NGS assays to detect NTRK fusions can accurately characterize fusion transcripts if sufficient RNA of adequate quality is available. The comparative performance data will support the implementation of targeted NGS-based sequencing assays for NTRK fusion detection in routine diagnostics.

Synchronous lung and multiple soft tissue metastases developed from osteosarcoma of tibia: a rare case report and genetic profile analysis

BackgroundOsteosarcoma is the most common primary malignant bone tumor with a highly metastatic propensity in children and young adolescents. The majority of metastases develope in the lung, while metastases to the extrapulmonary locations have rarely been discussed, especially in skeletal muscle.Case presentationWe reported a young patient with pathologically diagnosed osteosarcoma of the right tibia who was initially treated with standard chemotherapy and complete surgical resection. However, pulmonary metastases and multiple soft tissue masses in skeletal muscle developed four years after the index surgical resection. Subsequently, a targeted next-generation sequencing assay based on an 806 oncogenes and tumor suppressor genes panel was performed to analyze genetic alterations in this patient with rare metastatic pattern. The genetic analysis revealed canonical somatic mutations of RB1 and germline variants of ALK (c.862 T > C), BLM (c.1021C > T), PTCH1 (c.152_154del), MSH2 (c.14C > A), RAD51C (c.635G > A). Using silico prediction programs, the germline variants of the MSH2 and RAD51C were predicted as “Possibly Damaging” by Polymorphism Phenotyping v2 (PolyPhen-2) and “Tolerated” by Sorting Intolerant from Tolerant (SIFT); BLM was classified as “Tolerated”, while the germline variant of ALK was predicted to be pathogenic by both PolyPhen-2 and SIFT.ConclusionsOsteosarcoma with extrapulmonary metastases is rare, especially located in the skeletal muscle, which predicts a worse clinical outcome compared with lung-only metastases. The several novel variants of ALK, BLM, PTCH1 in this patient might expand the mutational spectrums of the osteosarcoma. All the results may contribute to a better understanding of the clinical course and genetic characteristics of osteosarcoma patients with metastasis.

Clinical utility of targeted next-generation sequencing assay in IDH-wildtype glioblastoma for therapy decision-making.

Targeted gene NGS testing is available through many academic institutions and commercial entities and is increasingly incorporated in practice guidelines for glioblastoma (GBM). This single-center retrospective study aimed to evaluate the clinical utility of incorporating NGS results in the management of GBM patients at a clinical trials-focused academic center. We identified 1011 consecutive adult patients with pathologically confirmed GBM (IDHwt or IDHmut) who had somatic tumor sequencing (Oncopanel, ~500 cancer gene panel) at DFCI from 2013-2019. Clinical records of all IDHwt GBM patients were reviewed to capture clinical trial enrollment and off-label targeted therapy use based on NGS results. Of the 557 IDHwt GBM patients with sequencing, 182 entered clinical trials at diagnosis (32.7%) and 213 (38.2%) entered after recurrence. Sequencing results for 130 patients (23.3%) were utilized for clinical trial enrollment for either targeted therapy indications (6.9 % upfront and 27.7% at recurrent clinical trials and 3.1% for off-label targeted therapy) or exploratory studies (55.4% upfront and 6.9% recurrent clinical trials). Median overall survival was 20.1 months with no survival difference seen between patients enrolled in clinical trials compared to those who were not, in a posthoc analysis. While NGS testing has become essential for improved molecular diagnostics, our study illustrates that targeted gene panels remain underutilized for selecting therapy in GBM-IDHwt. Targeted therapy and clinical trial design remain to be improved to help leverage the potential of NGS in clinical care.

A scalable high-throughput targeted next-generation sequencing assay for comprehensive genomic profiling of solid tumors.

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000TM sequencing run with a turnaround time of <7 days from specimen receipt to report. The results demonstrate that the scalable assay accurately and reproducibly detects small variants, copy number alterations, microsatellite instability (MSI) and tumor mutational burden (TMB) from 40ng DNA, and multiple gene fusions, including known and unknown partners and splice variants from 20ng RNA. 717 tumor samples and reference materials with previously known alterations in 96 cancer-related genes were sequenced to evaluate assay performance. All variant classes were reliably detected at consistent and reportable variant allele percentages with >99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

IntroductionB cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction.MethodsThe B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software.ResultsClonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively.ConclusionsThese results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders.For research use only. DisclosuresLowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Evaluation of a Rapid Automated Next Generation Sequencing Assay for Precision Medicine in Acute Myeloid Leukemia

Background:In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML.The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted “myeloid panel” NGS assay platform (s-NGS).Methods:DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used.Results:Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P<0.0001, Figure 1A). NPM1 mutation VAF values trended lower by s-NGS compared to NMA.We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these “edge case” variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B).Conclusions:NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. [Display omitted] DisclosuresZhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

Pan-cancer Analysis of Homologous Recombination Repair-associated Gene Alterations and Genome-wide Loss-of-Heterozygosity Score.

To study associations across tumor types between genome-wide loss of heterozygosity (gLOH) and alterations in homologous recombination repair (HRR)-associated genes beyond BRCA1 and BRCA2. Genomic profiling using a targeted next-generation sequencing assay examining 324-465 genes (FoundationOne, FoundationOne Heme, and FoundationOne CDx; Foundation Medicine, Inc.) was performed in a cohort of 160,790 samples across different tumor types. Zygosity predictions and gLOH status were calculated and linked with alterations in 18 HRR-associated genes (BRCA1, BRCA2, PALB2, BARD1, ATR, ATRX, ATM, BAP1, RAD51B, RAD51C, RAD51D, BRIP1, NBN, CHEK1, CHEK2, FANCA, FANCC, MRE11) and other genomic features, using Fisher's exact test and Mann-Whitney U tests. We identified a strong correlation between elevated gLOH and biallelic alterations in a core set of HRR-associated genes beyond BRCA1 and BRCA2, such as BARD1, PALB2, FANCC, RAD51C, and RAD51D (particularly in breast, ovarian, pancreatic, and prostate cancer). Monoallelic/heterozygous alterations in HRR-associated genes were not associated with elevated gLOH. gLOH was also independently associated with TP53 loss. Co-occurrence of TP53 loss and alterations in HRR-associated genes, and combined loss of TP53-PTEN or TP53-RB1, was associated with a higher gLOH than each of the events separately. Biallelic alterations in core HRR-associated genes are frequent, strongly associated with elevated gLOH, and enriched in breast, ovarian, pancreatic, and prostate cancer. This analysis could inform the design of the next generation of clinical trials examining DNA repair-targeting agents, including PARP inhibitors.

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.

Evaluating Targeted Next-Generation Sequencing Assays and Reference Materials for NTRK Fusion Detection

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control-validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to identify patients with NTRK fusion-positive tumors.

Evaluation of a Targeted Next-generation Sequencing Assay for BRCA Mutation Screening in Clinical Samples

Man Jin Kim, M.D., Byeong Ju Song, M.T., Si Eun Kim, M.S., Chang Seon Lee, M.S., Ji-Eun Im, M.S., Ensel Oh, Ph.D., Keunsook Lee, Ph.D., Sung Im Cho, M.T., Kwang Joong Kim, Ph.D., Sung Sup Park, M.D., Moon-Woo Seong, M.D.,. Lab Med Online 2021;11:283-9. https://doi.org/10.47429/lmo.2021.11.4.283

Clinical and biological determinants of circulating tumor DNA detection and prognostication using a next-generation sequencing panel assay

ABSTRACT Circulating tumor DNA (ctDNA) is utilized for molecular profiling of cancers, and is under investigation for a growing number of applications based on the assumption that ctDNA levels faithfully reflect disease burden. Our objective was to investigate whether patient and tumor characteristics may impact ctDNA detection or levels and the prognostic significance of ctDNA levels or mutations. We performed a retrospective cohort analysis of a comprehensively annotated cohort of 561 patients at a National Cancer Institute–designated comprehensive cancer center with advanced solid cancers who underwent ctDNA testing using a commercial targeted next-generation sequencing assay. ctDNA detection in advanced cancers was associated with older age, non-obese body mass index, and diabetes, but not with tumor diameter, volume, lesion number, or other pathological features. Regression models indicate that no more than 14.3% of the variance in ctDNA levels between patients was explained by known clinical factors and disease burden. Even after adjusting for established prognostic factors and tumor burden, ctDNA levels were associated with worse survival among patients without prior systemic therapy, while ctDNA mutations were associated with survival among patients who previously received systemic treatment. These findings uncover clinical factors that affect ctDNA detection in patients with advanced cancers and challenge the convention that ctDNA is a surrogate for tumor burden. Our study also indicates that the prognostic value of ctDNA levels and mutations are independent of tumor burden and dependent on treatment context.

Determination of COL1A1–PDGFB breakpoints by next-generation sequencing in the molecular diagnosis of dermatofibrosarcoma protuberans

ObjectiveIn most cases, dermatofibrosarcoma protuberans (DFSP) is characterized by the chromosomal translocation t (17; 22) (q22; q13) that leads to a fusion of collagen type 1 alpha 1 (COL1A1) and platelet-derived growth factor beta chain (PDGFB). Recently, next-generation sequencing (NGS) has been reported to detect fusion transcripts in some malignancies. Therefore, the present study aimed to evaluate the utility of the targeted NGS in detecting the COL1A1–PDGFB fusion in patients with DFSP. MethodsWe designed a targeted DNA capture panel to tile along the fusion regions, including exon, intron, and untranslated regions of the COL1A1 and PDGFB. A cohort of 18 DNA samples extracted from formalin-fixed, paraffin-embedded tissues was used to evaluate the targeted NGS. The results were compared with that of fluorescence in situ hybridization (FISH). ResultsThe COL1A1–PDGFB fusion was identified in 13 of 18 cases (72.2%) by targeted NGS assay. PDGFB breakpoints were constantly found in exon 2, while breakpoints in COL1A1 varied from exon 15 to 46. Of these 18 cases assayed by FISH, 12 (66.7%) exhibited COL1A1–PDGFB fusion signals. One case (P9), which was FISH-negative, was demonstrated with the fusion by targeted NGS and validated by PCR and Sanger sequencing. The targeted NGS results showed a high concordance with the results of the FISH assay (94.4%). ConclusionOur study reported a targeted NGS assay for detecting the breakpoints of the COL1A1–PDGFB fusion gene, which can be implemented in diagnosing patients with DFSP.

Abstract LB059: Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting

Abstract Genomic and epigenetic changes are known to be the underlying mechanism of cancer development. Assessing the genomic (mutation) and epigenetic (methylation) changes has become increasingly important for cancer detection and treatment selection, especially in the field of cfDNA-based liquid biopsy. Targeted Next-Generation Sequencing (NGS) enables in-depth analysis of specific genomic regions that is essential for the clinical application of liquid biopsy. However, current targeted NGS assays are complicated, lengthy, and inefficient. Two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS) from sample to sequencing ready library. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for multi-modal analyses on one sample. Thus, an assay that is capable of both methylation and mutation analysis would be optimal for clinical research and future diagnostic assays.Here, we report the performance of Point-n-Seq, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both methylation and mutation analysis without sample splitting. Custom panels of 10 to 1000s of markers can be designed with &amp;gt;99% first-pass success rate. We conducted both performance validation and multi-center, multi-operator, reproducibility studies. With spike-in titration of cancer cell-line gDNA with known mutation and methylation profile, we achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions.In a pilot study, we designed a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &amp;gt;350 hotspot mutations in 22 genes. From only 1ml of plasma from late stage CRC patients, we detected cancer-specific methylation signals in all samples tested, and hotspot mutations with clinical implications. Next, a tumor-informed mutation panel covering approximately 100 personalized mutations for each patient was compared to the tumor-independent CRC methylation panel to analyze 23 stage I and II CRC patient plasma samples. The initial results showed that tumor-independent TMS assay achieved a comparable or superior limit of detection compared to the personalized tumor-informed approach. Moreover, to further improve assay sensitivity, metrics for cfDNA size can be integrated into the analysis of the same workflow without sample splitting. The Point-n-Seq kit, with a highly streamlined workflow and dual methylation and mutation analysis, provides a highly sensitive and robust non-invasive method for cancer detection, monitoring and treatment guidance. Citation Format: Grace Q. Zhao, Yun Bao, Heng Wang, Vanita S. Natu, Jayashree Joshi, Yingmin Wang, Elena Arzumanyan, Wanping Wang, Jianmin Wang, John Coller, Shengrong Lin. Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB059.

Abstract 2179: Identification of ROS1 alterations in ctDNA from colorectal cancer patients in China

Abstract Background: ROS proto-oncogene 1 (ROS1) is an oncogenic driver involved in various cancer types for which has FDA approved therapies. ROS1 has shown to undergo genetic rearrangements in a variety of human cancers including glioblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, gastric adenocarcinoma and colorectal cancer. The rearrangements create fusion proteins in which the kinase domain of ROS1 becomes constitutively active and drives cellular proliferation. Although previous data have reported ROS1 fusions occur in 0.2% to 2.4% of colorectal cancers, the frequency and type of ROS1 alterations in Chinese CRC patients have not been fully reported. Methods: This study queried data from 1,083 colorectal cancer samples which underwent a targeted next-generation sequencing assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2017 and June, 2020 in China, to identify the incidence of ROS1 alterations detected in ctDNA in colorectal cancer patients. The detected genetic alteration types included single-nucleotide variants (SNVs), copy number variations (CNVs) and gene rearrangements/fusions. Results: ROS1 alterations were found in 114 patients among the 1,083 (10.5%) CRC patients' cohort, including 72 males and 42 females, median age was 58 years old. The ROS1 alterations included 110 (96.5%) SNVs, 3 (2.6%) CNVs and 1 (0.9%) GOPC-ROS1 fusion. Among the 110 single nucleotide variants, we identified 91 (82.7%) of ROS1 SNVs were nonsynonymous mutations. The top 5 co-occurring mutations in samples with ROS1 alterations were TP53 (71, 62.2%), CYP2C19 (48, 42.1%), APC (48, 42.1%), KRAS (46, 40.3%) and DPYD (42, 36.8%). Conclusion: Our study indicated that ctDNA can be used as a nonvasive methodology to detect cancer-specific genetic aberrations in plasma for CRC. ROS1 alterations occurred modestly in Chinese colorectal cancer patients. Co-occurring mutations may provide different biological subsets of patients with ROS1-mutant colorectal cancer to reveal patient outcomes and response to therapy. Further investigation could be focused on the effect of ROS1 nonsynonymous mutations on the ROS1 proteins in vitro to identify whether these are more actionable than currently recognized. Citation Format: Mojin Wang, Lili Zhu, Depei Huang, Xudong Shen, Hushan Zhang, Xueke She. Identification of ROS1 alterations in ctDNA from colorectal cancer patients in China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2179.