Targeted Next-Generation Sequencing with MultiPrime: A Reliable and Efficient Tool for Virus Detection.

Introduction Pheochromocytomas and paragangliomas (PPGLs) are highly heritable tumours, with up to 40% of cases carrying germline variants. Current guidelines recommend genetic testing for all patients with PPGLs. Next-generation sequencing (NGS) enables accurate, fast, and inexpensive genetic testing. This study aimed to compare the costs related to PPGL genetic testing between the sequential testing using the decisional algorithm proposed in the 2014 Endocrine Society guidelines and targeted NGS gene panels. Methods Patients with proven PPGLs were enrolled. A gene list covering 17 susceptibility genes related to hereditary PPGLs was developed for targeted sequencing. Validation was carried out by Sanger sequencing. We simulated the diagnostic workflow to examine the anticipated costs based on each strategy for genetic testing. Results Twenty-nine patients were included, among whom a germline variant was identified in 34.5%. A total of 22.7% with apparently sporadic PPGL carried a variant. Five genes were involved (RET, n = 3; SDHB, n = 3; SDHD, n = 2; EGLN1, n = 1; and NF1, n = 1). According to the diagnostic workflow, the average cost of the targeted NGS (534.7 US dollars per patient) is lower than that of the sequential testing (734.5 US dollars per patient). The targeted NGS can also reduce the number of hospital visits from 4.1 to 1 per person. The cost can be further reduced to 496.24 US dollars per person (32% reduction) if we apply a new syndromic-driven diagnostic algorithm to establish priorities for specific genetic testing for syndromic and selected cases, and targeted NGS for non-syndromic patients. Conclusions Targeted NGS can reduce both the cost of PPGL genetic testing and the number of hospital visits, compared with the conventional approach. Our proposed algorithm is the preferred approach due to its significant reduction of the cost of genetic testing. Key message Pheochromocytomas and paragangliomas are highly heritable neoplasms. The targeted next-generation sequencing (NGS) gene panels have proven to be fast, accurate, and inexpensive for the genetic analysis. According to this cost analysis, it is economically reasonable to use targeted NGS gene panels for genetic screening.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

ABSTRACTCurrently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method’s utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

Targeting appropriate driver genes is crucial in cancer precision medicine. To find oncogenic drivers, targeted next-generation sequencing is becoming widely used in the clinical field. Using targeted next-generation sequencing, we aimed to detect targetable genetic alterations and characterize mutational profiles of breast cancer. We analyzed breast cancer biopsy specimens using customized 50-gene panel. In total, 196 patients of primary operable breast cancer patients were selected from single institution in South Korea. Among the 196 patients, 22 kinds of genes were significantly mutated or amplified. They comprised 161 nonsynonymous missense mutations, 54 frameshift mutations, 29 missense mutations and 5 small indels; 64 gene amplifications and 2 deletions. The most frequently mutated gene was PIK3CA (n=81/196; 41.3%), followed by TP53 (n=62; 31.6%), AKT1 (n=21; 10.7%), GATA3 (n=17; 8.7%) and PTEN (n=14; 7.1%). The most frequently amplified gene was ERBB2 (n=25/196; 12.8%), followed by FGFR1 (n=11; 5.6%), MYC (n=9; 4.6%), FGFR2 (n=3; 1.5%) and FGFR3 (n=3; 1.5%). Targeted next-generation sequencing can be readily performed in daily clinical practice. The most commonly found targetable genetic alterations were mutations of PI3K/AKT/mTOR pathway genes and FGFR family gene amplifications. Despite the limited number of available targeted agents, our study shows the feasible application of precision medicine in breast cancer. Citation Format: Jung Ho Park, Seung Yeon Ko, Sang Hwa Kim, Ho Young Kim, Sung Hoon Ko, Lee Su Kim. Molecular genetic characterization of primary breast cancer via clinical next-generation sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 198.

Analysis of GATA1 Mutations in Down Syndrome Infants with Transient Abnormal Myelopoiesis and Clinical Impacts of GATA1 Mutation Types: A Report from the JPLSG TAM-10 Study

Simple SummaryThe major cause of treatment failure after allogeneic stem cell transplantation (allo-HSCT) is due to relapse of the underlying disease. Novel methods and strategies are needed to detect early relapse after allo-HSCT. The present study reports the clinical utility of monitoring measurable residual disease (MRD) and mixed chimerism (MC) by droplet-digital PCR in circulating cell-free DNA (cfDNA) in 62 patients with myeloid malignancies undergoing allo-HSCT. MC in circulating cfDNA at an optimal threshold of 18% discriminated patients with hematological relapse from patients in complete remission after allo-HSCT. Most of the mutations identified using a targeted next-generation sequencing (NGS) panel were detected in cfDNA at relapse and were suitable for the monitoring of MRD. In several cases, mutations were detected earlier in cfDNA than in peripheral blood mononuclear cells. In conclusion, longitudinal analysis of cfDNA for MRD and MC can be used as a complementary tool for early detection of relapse in patients after allo-HSCT and could be used to guide clinical interventions.Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.

Integrated diagnosis of grade 3-4 gliomas using targeted next-generation sequencing and traditional diagnostic data, and its overall implications on patient management

Targeted Next-Generation Sequencing in Molecular Subtyping of Lower-Grade Diffuse Gliomas: Application of the World Health Organization's 2016 Revised Criteria for Central Nervous System Tumors

Genetic profiling of thymic carcinoma using targeted next-generation sequencing

Several genes have been recognized to be associated with non-surgical hypoparathyroidism. Data about gene mutations in adult-onset hypoparathyroidism patients is lacking. This study was designed to screen gene mutation in adult-onset hypoparathyroidism in Chinese through the targeted next-generation sequencing (NGS). We recruited 17 patients with adult-onset hypoparathyroidism who were regularly followed or newly diagnosed at our centre during the past one year. Nine of them developed hypercalciuria during the treatment with calcium and vitamin D. Eight of them were newly diagnosed with no treatment. Targeted NGS was performed to screen 11 related genes, including AIRE, AP2S1, CASR, CLDN16, FAM111A, GATA3, GCM2, PTH, TBCE, TBX1 and TRPM6. A novel homozygosis mutation of GCMB gene[c.130G>A (p.G44S)]was identified which was predicted to be deleterious by PolyPhen2. The patient was a 36-year-old woman who suffered from paroxysmal carpopedal spasms for ten years. Before treatment, the serum calcium and phosphorus was 1.48 mmol/L and 2.29 mmol/L, respectively.Parathyroid hormonel (PTH) concentration was lower than 3.0 ng/L. Intracranial calcification and cataract were also identified. She developed hypercalciuria during treatment with calcium and vitamin D. She had no physical deformity or family history of hypoparathyroidism. In this study, the genetic defect was only identified in 1 patient (5.9%). In adult-onset hypoparathyroidism without other diagnostic clues, the gene mutation screening as the first choice to clarify the etiology was not recommended.

To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate disease-causing mutations. Sanger sequencing was performed on all subjects to confirm the candidate mutations and assess cosegregation within the family. Clinical examinations of the proband showed typical characteristics of RP. Three candidate heterozygous mutations in 3 genes associated with RP were detected in the proband by targeted NGS. The 3 mutations were confirmed by Sanger sequencing and the deletion (c.357_358delAA) in PRPF31 was shown to cosegregate with RP phenotype in 7 affected family members, but not in 3 unaffected family members. The deletion (c.357_358delAA) in PRPF31 was the disease-causing mutation for the proband and his affected family members with RP. To our knowledge, this is the second report of the deletion and the first report of the other 2 mutations in the Chinese population. Targeted NGS combined with bioinformatics analysis proved to be an effective molecular diagnostic tool for RP.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Hereditary spherocytosis (HS) is a heterogeneous genetic disorder characterized by spherocytosis on peripheral blood smear with hemolytic anemia, accompanied by signs of hemolysis. Herein, we report a 5-month-old Korean girl with HS resulting from a de novo 271Kb microdeletion of 14q23.3. She presented with hemolytic anemia and mild splenomegaly. Spherocytosis was seen on examination of peripheral blood. Eosin-5'-maleimide (EMA) test and flow cytometric osmotic fragility test were positive. She had no relevant family history of spherocytosis. No pathogenic single nucleotide variants or small insertions/deletions were detected in HS-associated genes. Array comparative genomic hybridization analysis revealed a 271Kb deletion at chromosome 14q23.3, encompassing the SPTB, CHURC1, GPX2, RAB15, FNTB, and MAX genes. We found a deletion affecting 5' UTR, exon 1, and part of intron 1 of the SPTB gene using targeted next-generation sequencing (NGS) analysis, suggesting that NGS may be able to identify disease-causing copy number variations (CNVs), as well as small point mutations in HS patients. In addition, chromosomal microarray may be useful in defining combined deleted genes. Additional evaluations should thus be considered in the diagnosis of HS, especially when CNV is revealed as disease-causing abnormality.

Background: Extraskeletal myxoid chondrosarcomas (EMCs) are rare mesenchymal neoplasms comprising approx. 3% of all soft tissue tumors. EMCs arise mainly from the deep soft tissues of the extremities, accompanied with high rates of recurrence and metastases. The molecular hallmarks of EMCs are various cytogenetic NR4A3 rearrangements, generating chimeric -NR4A3 proteins. The most common reciprocal translocation t(9;22)(q22;q12), results in a fusion of the EWS RNA-binding protein 1 gene (EWSR1) to the nuclear receptor subfamily 4, group A, member 3 gene (NR4A3 or TEC; approximately 75% of cases). Further cytogenetic t(9;17)(q22;q11) rearrangements involve TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor (TAF15; approximately 15% of cases). The less frequent reciprocal translocations t(9;15)(q22;q21) and t(9;3)(q22;q12), result in transcription factor 12 (TCF12)-NR4A3 and TRK-fused gene (TFG)-NR4A3 fusion proteins. Although the oncogenic -NR4A3 fusion transcripts seem to have a crucial role in EMC tumorigenesis and progression, the specific biological function and the mechanism of action remain to be defined. Methods: We characterized the cytogenetic rearrangements of 25 comprehensive EMC tumors by RT-PCR and/or fluorescence in situ hybridization (FISH). Next generation sequencing (NGS) was performed (Illumina MiSeq platform) to reveal additional genetic alterations besides the known chromosomal translocation. Therefore, a comprehensive cancer panel was designed, comprising 27 cancer-related genes known to be frequently mutated across various malignancies. Results: Overall, fusion transcripts were detected in 22 of 25 samples (88%). Sixteen were positive for the EWSR1-NR4A3 and six for the TAF15-NR4A3 fusion gene. The t(9;15) and t(9;3) translocations, resulting in TCF12-NR4A3 and TFG-NR4A3 fusion proteins were not identified in any EMC case. In Addition, several known oncogenic mutations were detected which have not been previously reported in EMC. Conclusions: The combination of RT-PCR and FISH on paraffin-embedded tissue is a sensitive and specific method for the molecular detection of the pathogenic translocations to be applied in the differential diagnosis of extraskeletal myxoid chondrosarcomas. Our results emphasize that cytogenetic NR4A3 rearrangements are the initiating events in the pathogenesis of EMC. Furthermore, our results indicate the occurrence of additional genetic aberrations providing a rational base for novel targeted therapeutic approaches. Citation Format: Marcel Trautmann, Magdalene Cyra, Ilka Isfort, Inga Grünewald, Konrad Steinestel, Sebastian Huss, Reinhard Büttner, Eva Wardelmann, Wolfgang Hartmann. Extraskeletal myxoid chondrosarcoma: a clinicopathologic and molecular study reveals novel genetic aberrations by targeted next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2017-2703