Targeted Next-generation Sequencing Panel Research Articles

Abstract 5840: The characteristics of TP53 germline mutations analyzed in a large-scale cohort of Chinese glioma patients

Abstract Backgrounds:TP53 germline mutation is well known to be the cause of Li-Fraumeni syndrome (LFS), a rare autosomal dominant cancer syndrome, characterized by predisposition of breast cancer, soft tissue carcinoma, osteosarcoma, brain cancer and etc. LFS has been reported in cases or small-scale cohorts for decades, while the characteristics of TP53 germline mutations carried by glioma patients were still less studied due to rarity. We performed large-scale retrospective research to identify TP53 germline mutations in Chinese glioma patients, which would extend the genetic knowledge of LFS related glioma. Methods:We enrolled 3325 glioma patients in this study (female=1423, male=1902). Next-generation sequencing panel targeting 825 genes was performed to identify TP53 germline mutations. Mutations were annotated pathogenic or likely pathogenic according to ACMG guidelines or referring to the literatures. Results:After annotation, 20 pathogenic or likely pathogenic germline mutations (PGMs) in TP53 were found in 3325 glioma patients. There was no statistical significance of gender ratio between all glioma patients and mutation carriers (female=6, male=14, p=0.25). The highest incidence rate of PGMs occurred in glioblastoma (n=16, 80.0%), followed by anaplastic astrocytoma (n=2, 10.0%), oligodendroglioma (n=1, 0.5%) and diffuse midline glioma (n=1, 0.5%). Significant difference of onset age between TP53 mutation carriers and non-carriers was pronounced (22.90YO vs 43.35YO avg, p<0.0001). Furthermore, the carrier rate of PGMs was prone to decrease with increasing onset age (table 1). Conclusions:This research indicated that TP53 PGMs were prone to occur in younger glioma patients, suggesting the importance of TP53 germline mutations being screened. It might be able to assist with early detection and treatment of glioma. The carrier rate of TP53 PGMs in each onset age intervals onset age glioma patient number PGM carrier number carrier rate 0-18 444 10 2.25% 19-30 263 4 1.52% 31-45 826 4 0.48% 46-55 717 1 0.14% 56-65 687 1 0.15% 66-75 337 0 0.00% 76+ 51 0 0.00% Citation Format: Xinyu Hong, Ximeng Zhao, Zeshang Guo, Xiaochen Yang, Bella Guo, Xiang Zhang, Tonghui Ma. The characteristics of TP53 germline mutations analyzed in a large-scale cohort of Chinese glioma patients [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 5840.

Novel use of ctDNA to identify muscle-invasive and non-organ-confined upper tract urothelial carcinoma.

4587 Background: Upper tract urothelial carcinoma (UTUC) is an aggressive cancer for which use of neoadjuvant chemotherapy (NAC) is limited by suboptimal clinical staging prior to nephroureterectomy. Detection of circulating tumor DNA (ctDNA) is associated with locally advanced and metastatic urothelial carcinoma of the bladder and may help identify UTUC patients who would benefit from NAC. Here we examine the feasibility and utility of plasma ctDNA in the diagnosis of non-organ confined high-risk UTUC. Methods: Patients with high-grade cTa-T2 UTUC without radiographic evidence of metastatic disease undergoing up-front radical nephroureterectomy (RNU) were prospectively accrued for pre- and post-operative plasma collection. Blood was collected preoperatively on the day of surgery, and plasma and buffy coat were processed for extraction of cell-free DNA and genomic DNA, respectively. FFPE tumor samples from RNU were used for tissue genomic DNA extraction. Targeted next-generation sequencing (NGS) was used for variant profiling. ctDNA positivity was defined as the presence of plasma cell-free DNA variants concordant with tissue-based variants. Results: NGS analyses of matched FFPE and plasma samples were successfully performed for all 19 accrued UTUC patients. Alterations in the TERT promoter (74%), TP53 (58%), FGFR3 (53%), myc amplification (53%), and ATM (42%) were demonstrated in urothelial tumor tissue. Matched plasma ctDNA showed prevalent alterations in the TERT promoter (42%), TP53 (42%), PIK3CA (37%), ATM (32%) and CD274 (26%). Nine patients (47%) had detectable plasma ctDNA mutations concordant with tumor-specific variants using the targeted NGS panel. All patients with detectable preoperative ctDNA had advanced staging (≥pT2 or ≥pN1) and lymphovascular invasion on final pathology, resulting in a 90% sensitivity. The panel was 100% specific with no patients with pTis, pTa, pT1 and pN0 having detectable concordant ctDNA mutations. Concordant plasma ctDNA was detected in four of nine patients postoperatively. Two of three (67%) who developed metastatic disease had detectable ctDNA while neither of the two who developed non-muscle-invasive bladder recurrences did. Conclusions: Prospective ctDNA analysis using a targeted NGS panel can be used to predict muscle-invasive and non-organ-confined UTUC preoperatively. Detectable postoperative ctDNA may indicate residual disease and predate clinical recurrence.

Clinical implications of next-generation sequencing on pediatric solid tumors.

e22012 Background: To assess the impact of next-generation sequencing (NGS) on Chinese pediatric cancer, we reported the first study of largest cohort in Chinese children with genomic landscape and highlighted the potential application with respect to molecular diagnosis and predictive biomarkers etc. Methods: A retrospective analysis was conducted on patients aged < 19 years diagnosed with solid tumors sequenced by targeted NGS panel. Somatic variants were analyzed following CAP/CLIA-certificated workflows, including Single Nucleotide Variant (SNV), Copy Number Variation (CNV), large insertion and deletions (Lindel) and gene rearrangements. Results: A total of 356 patients enrolled with three major cancer types including osteosarcoma (OS) (n = 78), rhabdomyosarcoma (RMS) (n = 51), Ewing sarcoma (EWS) (n = 13). Hepatocellular carcinoma (n = 9), medulloblastomas (n = 7) and colorectal adenocarcinoma (n = 5) were also included. 3487 somatic alterations were identified, TP53 (16%), EWSR1 (8.7%), CDKN2A (7.8%), BCOR (7%) and RB1 (5.6%) rank the top five reported. TP53 (21% % vs 7.9%), RB1 (10% vs 5.3%) mutations showed higher frequency in OS and BCOR (14% vs 17.6%) showed lower frequency in RMS compared to published paper (Gröbner et al 2018). We found PIK3CA mutations presented in RMS with 11.7% frequency, twice than reported (5.8%), mostly occurring at the known oncogenic codon E545. The obscure boundary of soft tissue tumor and bone tumor perplexed the pathologic diagnosis, NGS provide an important auxiliary examination. In 140 soft tissue tumor and bone tumor patients, 14.2% patients were further diagnosed with different subtype, 4.2% patients with unknown subtype were diagnosed with a specified subtype by supportive genetic alterations. Moreover, 41.5% (138/356) patients received treatment recommended . Twelve NTRK-fusions were detected and the patients were recommended therapy method upon Larotrectinib, recruitment in matched clinical trial is still on-going. Conclusions: We investigated the different genetic profiling of Chinese pediatric solid cancer and found the clinical value of NGS in pathological diagnosis and biomarker discovery.

Tissue-based next generation sequencing (NGS) testing in solid tumors: A single-center experience in Malaysia.

e15121 Background: There is an increased appreciation for precision oncology in which clinicians are utilizing NGS testing to guide treatment selection and to identify resistance mutations. This study aims to identify clinically significant biomarkers in prospectively collected FFPE tumor tissue specimens. Genomic testing of molecular alterations was conducted using an in-house 50-gene hotspot APEX panel (MiRXES). Herein, we report a single center experience with targeted NGS panel testing in solid tumors. Methods: The analyzed cohort consisted of 75 clinical samples submitted for molecular testing over a 3-month period from Beacon Hospital, Malaysia (October to December 2021). The identified variants were tiered according to AMP/ASCO/CAP guidelines, in which actionable targets are defined as being able to be matched with existing FDA-approved drugs, including off-label prescriptions. Results: Among the samples received, 85.3% of samples passed quality control requirements for SNV/Indels and amplifications. Majority of the tumor types were categorized under colorectal cancer (20.3%), NSCLC (18.7%), followed by breast cancer (13.6%). Of the samples successfully sequenced, actionable targets were identified in 76.2% of patients, with Tier I variants [such as EGFR exon19del (6), EGFR exon20insertion (1), ERBB2 amp (5), KRAS G12C (3), KRAS G12D (1), KRAS G12V(1), KRAS G12F (1) KRAS G13D (1), PIK3CA H1047R (1), MET exon 14 skipping (1) and NRAS Q61H (1)) being identified in 20 patients. When stratified across tumor types, actionability rates were found to differ, with 100.0%, 83.3% and 81.8%, of tested tissues samples yielding results informing treatment selection or resistance mutations for breast, colorectal and NSCLC respectively. Based on APEX-tissue testing, the median number of actionable genes identified was 2 (IQR: 0 – 3) while the mean time from sampling to report was 4.7 days. Conclusions: This study shows the clinical utility and feasibility of targeted panel testing in cancer types of breast, colorectal and NSCLC. With fast turnaround time of 4 working days, this ensures timely access to treatment for patients.

Clinical applicability of targeted next-generation sequencing for precision medicine in diffuse large B-cell lymphoma.

e19538 Background: Comprehensive genomic analysis based on next-generation sequencing (NGS) allows a better molecular characterization of diffuse large B-cell lymphoma (DLBCL), offering a roadmap toward precision medicine. Methods: We analyzed somatic alterations of 37 DLBCL tissue or blood samples by a hybridization capture-based NGS panel targeting 188 lymphoma-related genes. Results: Totally, 279 single-nucleotide variants and small insertions or deletions affecting 103 genes were identified, with at least 1 mutation detected in 100% (37/37) of cases. The most frequently mutated genes were those involved in epigenetic modifiers ( DNMT3A, KMT2B and KMT2D), activated signaling ( TYK2, MYD88, NOTCH2 and BCR) and transcription factors/transcriptional regulator ( RUNX1, SPEN, IRF4, KLF2 and ASXL1). Significant co-occurrences were shown between RUNX1 mutations with NOTCH2, IRF4, ARID1A and BCR ( P< 0.05, respectively), BCR and IRF4 ( P< 0.01), etc. Additionally, we analyzed the association of genomic aberrations with clinicopathological features in DLBCL. Patients aged > 60 years harbored less LRP1B mutations than those aged ≤60 years (5.3% vs. 33.3%, P< 0.05). The mutation frequency of TP53 was significantly higher in LDH-high (> 244 U/L) group than that in LDH-low (≤244 U/L) group (40% vs. 6.25%, P< 0.05). Compared with germinal center B-cell (GCB), the mutation frequency of TYK was significantly lower in non-GCB (5% vs. 36.4%, P< 0.05). New potential therapeutic targets ( CREBBP, EP300, EZH2, SMARCB1, CD79A/B, BTK, CARD11, MYD88, THFAIP3, PIK3CA, PTEN and JAK) were identified in 55% (11/20) of newly diagnosed patients and in 41.2% (7/17) of relapsed/refractory patients. Ibrutinib, as a single agent, has demonstrated limited activity in DLBCL with mutant MYD88 and wildtype CD79. In this study, we identified 3 patients with mutant MYD88 and wildtype CD79A/B (8.1%), among whom 2 harbored MYD88 L265P variants and 1 had MYD88 S219C mutation. Conclusions: The utility of mutational profiling may facilitate the identification of potential drug targets, which in turns may represent new therapeutic possibilities.

Comprehensive Development and Implementation of Good Laboratory Practice for NGS Based Targeted Panel on Solid Tumor FFPE Tissues in Diagnostics.

The speed, accuracy, and increasing affordability of next-generation sequencing (NGS) have revolutionized the advent of precision medicine. To date, standardized validation criteria for diagnostic accreditation do not exist due to variability across the multitude of NGS platforms and within NGS processes. In molecular diagnostics, it is necessary to ensure that the primary material of the FFPE sample has good quality and optimum quantity for the analysis, otherwise the laborious and expensive NGS test may result in unreliable information. Therefore, stringent quality control of DNA and RNA before, during, and after library preparation is an essential parameter. Considering the various challenges with the FFPE samples, we aimed to set a benchmark in QC metrics that can be utilized by molecular diagnostic laboratories for successful library preparation and high-quality NGS data output. In total, 144 DNA and 103 RNA samples of various cancer types with a maximum storage of 2 years were processed for 52 gene focus panels. During the making of DNA and RNA libraries, extensive QC check parameters were imposed at different checkpoints. The decision tree approach can be set as a benchmark for FFPE samples and as a guide to establishing a good clinical laboratory practice for targeted NGS panels.

Characteristics of Genomic Alterations in Pericardial Effusion of Advanced Non-small Cell Lung Cancer.

Background: The feasibility and value of pericardial effusion as a liquid biopsy sample for actionable alteration detection in patients with non-small cell lung cancer (NSCLC) has not been adequately investigated. Here, we aim to reveal genomic alterations between pericardial effusion and paired tumor tissue, plasma (plasma cfDNA), and pleural effusion supernatant (PE-cfDNA) based on second-generation sequencing technology. Material and methods: A total of 26 advanced NSCLC patients were retrospectively studied. The following samples were collected and sequenced using two targeted next-generation sequencing panels: pericardial effusion (n = 26), matched tumor tissue (n = 6), plasma (n = 16), and pleural effusion supernatant (n = 5). Results: A total of 10 actionable alterations were identified in pericardial effusion of the NSCLC patients, including MET amplification, EGFR L858R, EGFR T790M, EGFR exon 19 deletion, EGFR L861Q, KRAS G12C, EML4-ALK (exon 18: exon 20) fusion, EML4-ALK (exon 20: exon 20) fusion, EML4-ALK (exon 6: exon 20) fusion, and ERBB2 exon 20 insertion. All these actionable alterations harbored multiple drug-sensitive targets as well as several drug-resistant targets, such as EGFR T790M. Compared to plasma cfDNA of 16 patients, paired pericardial effusion had higher number of actionable alterations (p = 0.08) as well as higher percentage of the population with actionable alterations (p = 0.16). Moreover, 8 out of 10 actionable alterations with single nucleotide variations (SNVs) or insertions/deletions (indels) had a higher variant allele frequency (VAF) in pericardial effusion than plasma cfDNA. In addition, we identified two actionable alterations in paired pericardial effusion, which were absence in PE-cfDNA. Clearly, 2 out of 3 actionable alterations with SNVs/indels in pericardial effusion had a higher VAF than those in PE-cfDNA. Our finding suggested the importance of pericardial effusion in the optimal selection of patients for targeted therapy. Conclusion: Among liquid biopsy specimens from the advanced NSCLC patients, pericardial effusion may be a better candidate for genomic profiling than plasma cfDNA, while it could serve as a supplement to PE-cfDNA in detecting actionable alterations. Therefore, pericardial effusion might provide a new alternative for selection of patients for better treatment management.

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic Yield: Results from the Perth CVID Cohort Study

With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies-Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age <18 years, autoimmunity, low memory B lymphocytes, family history, and/or history of lymphoproliferation. Candidate variants were assessed by in silico predictions of deleteriousness, comparison to the literature, and classified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria. All detected genetic variants were verified independently by an external laboratory, and additional functional studies were performed if required. Pathogenic or likely pathogenic variants were detected in 6 of 22 (27%) patients. Monoallelic variants of uncertain significance were also identified in a further 4 of 22 patients (18%). Pathogenic variants, likely pathogenic variants, or variants of uncertain significance were found in TNFRSF13B, TNFRSF13C, ICOS, AICDA, IL21R, NFKB2, and CD40LG, including novel variants and variants with unexpected inheritance pattern. Targeted amplicon NGS is an effective tool to identify monogenic disease-causing variants in CVID, and is comparable or superior to other NGS methods. Moreover, targeted amplicon NGS identified patients who may benefit from targeted therapeutic strategies and had important implications for family members.

Circulating Tumor DNA in Adults With Glioma: A Systematic Review and Meta-Analysis of Biomarker Performance.

Circulating tumor DNA (ctDNA) has emerged as a promising noninvasive biomarker to capture tumor genetics in patients with brain tumors. Research into its clinical utility, however, has not been standardized because the sensitivity and specificity of ctDNA remain undefined. To (1) review the primary literature about ctDNA in adults with glioma to compare the sensitivity and specificity of ctDNA in the cerebrospinal fluid vs the plasma and (2) to evaluate the effect of tumor grade on detection of ctDNA. PRISMA-guided systematic review and meta-analysis was performed using published studies that assessed ctDNA in either plasma or cerebrospinal fluid among adult patients with confirmed glioma. Summary receiver operating characteristic curves were generated using the Rücker-Schumacher method, and area under the curve (AUC) was calculated. Meta-analysis revealed improved biomarker performance for CSF (AUC = 0.947) vs plasma (AUC = 0.741) ctDNA, although this did not reach statistical significance ( P = .141). Qualitative analysis revealed greater sensitivities among single-allele PCR and small, targeted next-generation sequencing panels compared with broader panels. It additionally demonstrated higher sensitivity of ctDNA detection in high-grade vs low-grade gliomas, although these analyses were limited by a lack of specificity reporting in many studies. ctDNA seems to be a highly sensitive and specific noninvasive biomarker among adults with gliomas. To maximize its performance, CSF should be studied with targeted genetic analysis platforms, particularly in high-grade gliomas. Further studies on ctDNA are needed to define its clinical utility in diagnosis, prognostication, glioblastoma pseudoprogression, and other scenarios wherein neoadjuvant therapies may be considered.

A Validation Framework for Somatic Copy Number Detection in Targeted Sequencing Panels

Somatic copy number alterations (SCNAs) in tumors are clinically significant diagnostic, prognostic, and predictive biomarkers. SCNA detection from targeted next-generation sequencing panels is increasingly common in clinical practice; however, detailed descriptions of optimization and validation of SCNA pipelines for small targeted panels are limited. This study describes the validation and implementation of a tumor-only SCNA pipeline using CNVkit, augmented with custom modules and optimized for clinical implementation by testing reference materials and clinical tumor samples with different classes of copy number variation (CNV; amplification, single copy loss, and biallelic loss). Using wet-bench and in silico methods, various parameters impacting CNV calling, including assay-intrinsic variables (establishment of normal reference and sequencing coverage), sample-intrinsic variables (tumor purity and sample quality), and CNV algorithm-intrinsic variables (bin size), were optimized. The pipeline was trained and tested on an optimization cohort and validated using an independent cohort with a sensitivity and specificity of 100% and 93%, respectively. Using custom modules, intragenic CNVs with breakpoints within tumor suppressor genes were uncovered. Using the validated pipeline, re-analysis of 28 pediatric solid tumors that had been previously profiled for mutations identified SCNAs in 86% (24/28) samples, with 46% (13/28) samples harboring findings of potential clinical relevance. Our report highlights the importance of rigorous establishment of performance characteristics of SCNA pipelines and presents a detailed validation framework for optimal SCNA detection in targeted sequencing panels.

Molecular Portrait of GISTs Associated With Clinicopathological Features: A Retrospective Study With Molecular Analysis by a Custom 9-Gene Targeted Next-Generation Sequencing Panel.

Objectives: The study aims to investigate genetic characterization of molecular targets and clinicopathological features with gastrointestinal stromal tumors based on targeted next-generation sequencing. Materials and Methods: We selected 106 patients with GISTs from Sir Run Run Shaw Hospital between July 2019 and March 2021. FFPE samples and paired blood samples were obtained from these patients who underwent excision of the tumor. A customized targeted-NGS panel of nine GIST-associated genes was designed to detect variants in the coding regions and the splicing sites of these genes. Results: In total, 106 patients with a GIST were included in the study which presented with various molecular driver alterations in this study. KIT mutations occurred most often in GISTs (94/106, 95.92%), followed by point mutations in PDGFRA. KIT or PDGFRA mutations were detected to be mutually exclusive in the GIST. A total of eight patients with wide-type KIT/PDGFRA were characterized as WT-GISTs, according to clinical diagnosis which included six quadruple-WT GISTs, 1 BRAF-mutant, and 1 NF1-mutant GIST. In KIT exon 11, the most common mutation type was the codon Mutation (in-frame deletion or indels), whereas the missense mutation was the dominant type in KIT exon 13 and KIT exon 17. All variations in KIT exon 11 observed in this study were concentrated at a certain position of codon 550 to codon 576. Mutation in KIT exon 9 was mostly located at codon 502–503. Two germline pathogenic mutations were detected: NF1-R681* and KRAS-T58I. NF1-L591P was a germline mutation to be identified for the first time and is not recorded in the database. The frequency of driving mutations differed between the primary anatomical site in the GIST (p = 0.0206). KIT exon 11 mutants had a lower proliferation index of Ki67 (68.66%,≤5%), while 50.00% of KIT exon 9 mutants had the Ki67 status greater than 10%. Conclusion: The occurrence and development of a GIST is driven by different molecular variations. Resistance to TKIs arises mainly with resistance mutations in KIT or PDGFRA when they are the primary drivers. Targeted NGS can simultaneously and efficiently detect nine GIST-related gene mutations and provide reference for clinicians’ individualized diagnosis and treatment. Our results have important implications for clinical management.

Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use.

PurposeThe demand for high-throughput genetic profiling of somatic mutations in cancer tissues is growing. We sought to establish a targeted next generation sequencing (NGS) panel test for clinical oncology practice.MethodsCustomized probes were designed to capture exonic regions of 141 genes selected for the panel, which was aimed for the detection of clinically actionable genetic variations in cancer, including KRAS, NRAS, BRAF, ALK, ROS1, KIT and EGFR. The size of entire targeted regions is 0.8 Mb. Library preparation used NEBNext Ultra II FS kit coupled with target enrichment. Paired-end sequencing was run on Illumina NextSeq 500 at a read length of 150 nt. A bioinformatics workflow focusing on single nucleotide variant and short insertions and deletions (SNV/indel) discovery was established using open source, in-house and commercial software tools. Standard reference DNA samples were used in testing the sensitivity and precision and limit of detection in variant calling.ResultsThe general performance of the panel was observed in pilot runs. Average total reads per sample ranged from 30 million to 48 million, 73% ~82% unique reads. All runs had more than 99% average mapping rate. Mean target coverage ranged from 727x to 879x. Depth of coverage at 50x or more reached 87% of targeted region and 60% of targeted region received 500x or more coverage depth. Using OncoSpan HD827 DNA, which bears 144 variants (SNV/indel) from 80 genes that are within the targeted region on the panel, our somatic variant calling pipeline reached 97% sensitivity and 100% precision respectively, with near 48 million reads. High concordance with orthogonal approaches in variant detection was further verified with 7 cancer cell lines and 45 clinical specimens.ConclusionWe developed a NGS panel with a focus on clinically actionable gene mutations and validated the performance in library construction, sequencing and variant calling. High concordance with reference materials and orthogonal mutation detection was observed.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Technical Validation and Clinical Utility of an NGS Targeted Panel to Improve Molecular Characterization of Pediatric Acute Leukemia.

Development of next-generation sequencing (NGS) has provided useful genetic information to redefine diagnostic, prognostic, and therapeutic strategies for the management of acute leukemia (AL). However, the application in the clinical setting is still challenging. Our aim was to validate the AmpliSeq™ for Illumina® Childhood Cancer Panel, a pediatric pan-cancer targeted NGS panel that includes the most common genes associated with childhood cancer, and assess its utility in the daily routine of AL diagnostics. In terms of sequencing metrics, the assay reached all the expected values. We obtained a mean read depth greater than 1000×. The panel demonstrated a high sensitivity for DNA (98.5% for variants with 5% variant allele frequency (VAF)) and RNA (94.4%), 100% of specificity and reproducibility for DNA and 89% of reproducibility for RNA. Regarding clinical utility, 49% of mutations and 97% of the fusions identified were demonstrated to have clinical impact. Forty-one percent of mutations refined diagnosis, while 49% of them were considered targetable. Regarding RNA, fusion genes were more clinically impactful in terms of refining diagnostic (97%). Overall, the panel found clinically relevant results in the 43% of patients tested in this cohort. To sum up, we validated a reliable and reproducible method to refine pediatric AL diagnosis, prognosis, and treatment, and demonstrated the feasibility of incorporating a targeted NGS panel into pediatric hematology practice.

Overview of SERPING1 Variations Identified in Hungarian Patients With Hereditary Angioedema

BackgroundHereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency (C1-INH-HAE) is a rare autosomal dominant disorder, characterized by recurrent, unpredictable edematous symptoms involving subcutaneous, and/or submucosal tissue. C1-INH-HAE may be caused by more than 700 different mutations in the gene encoding C1-INH (SERPING1) that may lead to decreased protein synthesis or to functional deficiency.MethodsConcentrations of C1-INH, C4, C1q, and anti-C1-INH antibodies, as well as functional C1-INH activity were determined in subjects suffering from edematous symptoms and admitted to the Hungarian Angioedema Center of Reference and Excellence. In those patients, who were diagnosed with C1-INH-HAE based on the complement measurements, SERPING1 was screened by bidirectional sequencing following PCR amplification and multiplex ligation-dependent probe amplification. For detecting large deletions, long-range PCRs covering the entire SERPING1 gene by targeting 2–7 kb long regions were applied.ResultsAltogether 197 individuals with C1-INH deficiency belonging to 68 families were identified. By applying Sanger sequencing or copy number determination of SERPING1 exons, 48 different mutations were detected in 66/68 families: 5 large and 15 small insertions/deletions/delins, 16 missense, 6 nonsense, and 6 intronic splice site mutations. Two novel variations (p.Tyr199Ser [c.596A>C] and the duplication of exon 7) were shown to cosegregate with deficient C1-inhibitor level and activity, while two other variations were detected in single patients (c.797_800delinsCTTGGAGCTCAAGAACTTGGAGCT and c.812dup). A series of long PCRs was applied in the remaining 2 families without an identified mutation and a new, 2606 bp long deletion including the last 91 bp of exon 6 (c.939_1029+2515del) was identified in all affected members of one pedigree. In the remaining one family, a deep intronic SERPING1 variation (c.1029+384A>G) was detected by a targeted next-generation sequencing panel as reported previously.ConclusionsSequencing and copy number determination of SERPING1 exons uncover most pathogenic variants in C1-INH-HAE patients, and further methods are worth to be applied in cases with unrevealed genetic background. Since knowledge of the genetic background may support the establishment of the correct and early diagnosis of C1-INH-HAE, identification of causative mutations and reporting data supporting the interpretation on the pathogenicity of these variants is of utmost importance.

Syndromes predisposing to leukemia are a major cause of inherited cytopenias in children.

Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem cell transplantation prior to the development of leukemia. We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children. The study comprises children with persistent cytopenias, myelodysplastic syndrome, aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used Sanger sequencing of commonly mutated genes and a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias; the minority subsequently underwent whole exome sequencing. In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, nine (19.1%) had inherited thrombocytopenia predisposing to leukemia, and three each (6.4%) had predisposition to myelodysplastic syndrome or congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia. In summary, almost one third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome.

Proteasomal Processing Immune Escape Mechanisms in Platinum-Treated Advanced Bladder Cancer.

In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host’s anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens’ characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased (“processing escapes”). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.

Novel use of ctDNA to identify locally advanced and metastatic upper tract urothelial carcinoma.

543 Background: Upper tract urothelial carcinoma (UTUC) is an aggressive cancer for which use of neoadjuvant chemotherapy (NAC) is limited by suboptimal clinical staging prior to nephroureterectomy. Detection of circulating tumor DNA (ctDNA) is associated with locally advanced and metastatic urothelial carcinoma of the bladder and may help identify UTUC patients who would benefit from NAC. Here we examine the feasibility and utility of plasma ctDNA in the diagnosis of non-organ confined high-risk UTUC. Methods: Patients with high-grade cTa-T2 UTUC without radiographic evidence of metastatic disease undergoing up-front radical nephroureterectomy (RNU) were prospectively accrued. Blood was collected preoperatively on the day of surgery, and plasma and buffy coat were processed for extraction of ctDNA. FFPE samples from RNU were used for tissue genomic DNA extraction. Next-generation sequencing (NGS) was used for variant profiling. Plasma and somatic tissue mutations were called by comparing with matched buffy coat samples. Detection of cancer variants with a mutation allele frequency (MAF) ≥ 0.25% and hotspot variants with a MAF down to 0.1% were reported for plasma samples targeted by a NGS panel (PredicineCARE). Variants with a MAF ≥ 5% and hotspot variants with a MAF down to 2% were reported for FFPE samples. Results: NGS analyses of matched FFPE and plasma samples were successfully performed for all 15 accrued UTUC patients. Alterations in MYC amplification (62%), TERT promoter (62%), TP53 (38%), FGFR3 (31%), ERBB2 (25%), ARID1A (19%), and PIK3CA (19%) were demonstrated in urothelial tumor tissue. Matched plasma ctDNA showed prevalent alterations in the TERT promoter (47%), TP53 (30%), ARID1A (20%), ERBB2 (20%), FGFR3 (20%), and PIK3CA (17%). Five patients (33%) had detectable plasma ctDNA mutations concordant with tumor-based genotypes using the targeted NGS panel. All patients with detectable preoperative ctDNA had advanced staging (≥pT2 or ≥pN1) and lymphovascular invasion on final pathology, resulting in a 71.4% sensitivity. The panel was 100% specific with no patients with pTis, pTa, or pT1 and pN0 having detectable concordant ctDNA mutations. Conclusions: Prospective ctDNA analysis using a targeted NGS panel is a feasible nonsurgical approach to prediction of high-risk UTUC and has the potential for identification of upper tract urothelial cancer patients that may benefit from NAC.

Do Sputnik V Vaccine-Induced Antibodies Protect Against Seasonal Coronaviruses? Case Study.

There are hundreds of coronaviruses, most of which circulate among animals, yet there are seven types that infect humans. Three of them can cause severe acute respiratory illness-SARS-CoV, SARS-CoV-2, and MERS-CoV. Other HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 usually cause only mild to moderate upper respiratory tract infections. These four coronaviruses are called seasonal, because they are continuously circulating among human population and are responsible for up to 30% of all respiratory tract infections. Genetically, these low-pathogenic types are related to SARS-CoV-2. That is why questions concerning the cross-reactivity and cross-neutralization between antibodies against different types of coronaviruses have been raised. We addressed these questions by using enzyme-linked immunosorbent assays and targeted next-generation sequencing (NGS). We established the upper respiratory infection etiology for three patients who had been vaccinated with Sputnik V and tested positive on anti-SARS-CoV-2 antibodies. The symptoms included sore throat, nasal congestion, and myalgia. Their blood serum was analyzed for anti-SARS-CoV-2 antibodies in dynamics: before vaccination, and after the first and second dose of the vaccine. After the second dose, all patients were positive for IgG antibodies against SARS-CoV-2. The targeted NGS panel sequencing data analysis showed that these patients were infected with common coronavirus HCoV-OC43. These results suggest that S protein-targeted vaccine-induced antibodies against SARS-CoV-2 are not protective against seasonal coronavirus HCoV-OC43.

Evaluating use of changing technologies for rapid next-generation sequencing in pediatrics.

Rapid next-generation sequencing (NGS) offers the potential to shorten the diagnostic process and improve the care of acutely ill children. The goal of this study was to report our findings, including benefits and limitations, of a targeted NGS panel and rapid genome sequencing (rGS) in neonatal and pediatric acute clinical care settings. Retrospective analysis of patient characteristics, diagnostic yields, turnaround time, and changes in management for infants and children receiving either RapSeq, a targeted NGS panel for 4500+ genes, or rGS, at the University of Utah Hospital and Primary Children's Hospital, from 2015 to 2020. Over a 5-year period, 142 probands underwent rapid NGS: 66 received RapSeq and 76 rGS. Overall diagnostic yield was 39%. In the majority of diagnostic cases, there were one or more changes in clinical care management. Of note, 7% of diagnoses identified by rGS would not have been identified by RapSeq. Our results indicate that rapid NGS impacts acute pediatric care in real-life clinical settings. Although affected by patient selection criteria, diagnostic yields were similar to those from clinical trial settings. Future studies are needed to determine relative advantages, including cost, turnaround time, and benefits for patients, of each approach in specific clinical circumstances. The use of comprehensive Mendelian gene panels and genome sequencing in the clinical setting allows for early diagnosis of patients in neonatal, pediatric, and cardiac intensive care units and impactful change in management. Diagnoses led to significant changes in management for several patients in lower acuity inpatient units supporting further exploration of the utility of rapid sequencing in these settings. This study reviews the limitations of comparing sequencing platforms in the clinical setting and the variables that should be considered in evaluating diagnostic rates across studies.