Next-generation Sequencing System Research Articles

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis.

Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy. This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing. The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence. The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system's sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics. This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV.

Clinicopathological features of metastatic melanoma in effusion cytology of serosal cavity

Objective: To investigate the clinical, cytomorphology, immunocytochemical and molecular features of metastatic melanoma in serosal cavity effusion. Methods: Cytological specimens of 14 patients with melanoma in the chest and abdomen were collected from 2017 to 2023, at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. SOX10, S-100 protein, PRAME, BRAF V600E, HMB45, and Melan A were detected by immunocytochemical methods. Fourteen cases were tested for routine antibody combinations, including Claudin4, HEG1, Calretinin, CD68, etc. Four of the patients had biopsy or surgical samples of metastatic solid lesions of primary sites, and further next-generation sequencing (NGS) or amplification refractory mutation system (ARMS)-PCR molecular test was performed. In addition, 30 cases of serosal effusion samples were collected as control groups (10 cases of benign mesothelial cell reactive hyperplasia, 10 cases of mesothelioma, and 10 cases of metastatic lung adenocarcinoma). Results: Among the 14 cases of melanoma, there were 7 males and 7 females, with ages ranging from 35 to 86 years, and an average age of 57 years, there 10 cases aged ≥50 years. The tumor cells in the serosal effusion varied in morphology and degree of atypia. SOX10 was positive in all 14 cases (14/14), S-100 protein was positive in 10 cases (10/14), PRAME was positive in 12 cases (12/14), BRAF V600E was positive in 10 cases (10/14), HMB45 was positive in 12 cases (12/14), and Melan A was positive in 13 cases (13/14). In 4 patients with histological correlation, the cytological and histological expression of SOX10, BRAF V600E, and PRAME was positive in all 4 cases (4/4); S-100 protein was positive in 2 cases (2/4); and HMB45 and Melan A were positive in 3 cases (3/4). Using NGS or ARMS-PCR, missense mutations of BRAF V600E were detected in all 4 patients; TERT promoter mutations was detected in 1 case; and CDKN2A terminating mutations and MSI1 deletion mutations were detected in the other case. SOX10, S-100, HMB45, Melan A, PRAME and BRAF V600E were all negative in 30 control samples of serosal cavity effusion. Conclusion: By observing the morphology of tumor cells, immunocytochemical test of several combination markers, especially the expression of SOX10, BRAF V600E and PRAME, can help to improve the positive diagnosis rate of melanoma in serous cavity effusion.

Clinicopathological significance of mutation profile detected by next generation sequencing in different metastatic organs of non-small cell lung cancers

BackgroundThe primary tumor and it’s metastases show heterogeneity in molecular studies for targeted therapies in Non-Small Cell Lung Cancer(NSCLC), the leading cause of cancer-related deaths worldwide. The study aimed to identify somatic mutations in biopsies from NSCLC patients’ metastatic organs using Next-Generation Sequencing(NGS) and examine their association with clinicopathological parameters. Materials and methodsThe study included 128 NSCLC patients and, NGS was performed on tumor biopsies from different metastatic organs at Molecular Pathology laboratory of the Department of Medical Pathology in Aydın Adnan Menderes University Faculty of Medicine. The age, gender, histopathological diagnoses, metastatic organs, smoking and mutation status were all recorded, along with the analysis results of 72 genes and 4149 primers in the panel of the NGS system. Results53.9 % of the cases had a history of smoking and patients with brain metastases had a higher smoking rate(p=0.000). The most common occurrence(39.8 %) was lymph node metastasis, followed by brain(19.5 %). There was a strong correlation between mutation presence and metastasis in the liver(p=0.012), bone(p=0.002), and pleura(p=0.008). Smokers had a higher frequency of KRAS(p=0.000) and TP53(p=0.001) mutations. Brain metastases showed a statistically significant NF1 mutation(p=0.001), while the liver exhibited a significant BRAF mutation(p=0.000). NF1-TP53, PTEN-TP53 and NF1-PTEN were the most common concomitant mutations and, the brain was the most common metastatic organ in which they occurred. ConclusionOur results suggest prizing assessing detected mutations, in the prediction, follow-up and management of metastases, especially in patients with lung adenocarcinoma. The assessment also needs to consider the tumor's mutation status in metastatic organs. New therapeutic agents targeting NF1 mutations will be available in the future to treat NSCLC, especially in metastases.

KRAS mutations in patients with AML: clinical characteristics and not reported mutations using NGS

ABSTRACT Background AML is a complex and heterogeneous disease. The KRAS gene is one of the important genes in the pathogenesis of acute myeloid leukemia (AML). Mutant RAS can promote oncogenesis via different mechanisms including oncogenic transcription, cell cycle progression, cellular survival, growth, metabolism, and cell migration. Therefore, it is important to identify the genomic landscape of AML. The aim of the study is to identify KRAS variants in AML and their association with clinic pathological criteria and possible effects on prognosis using NGS. Method Hotspot mutations in the KRAS gene were studied using Ion S5 next-generation sequencing system. Bone marrow samples of newly diagnosed AML patients were collected to identify hotspot mutations in the KRAS gene. DNA amplicons were subjected to sequencing and were analyzed using ion torrent software. Patients were classified according to the FAB classification system. Patients are also classified according to the cytogenetic groups and the ELN risk stratification system. Results KRAS mutations were detected in exon 2, 3, whereas no mutations in KRAS exon 1. Interestingly, Novel mutations were detected in KRAS in AML Egyptian patients. Also, there was no statistically significant association of RAS mutations with different clinical and prognostic parameters. However, KRAS mutant patients tended to have increased PB WBC counts, percentage of PB, and bone marrow blasts. Conclusion NGS is considered a useful tool to identify KRAS variants that could be useful for risk stratification and tailored therapy in AML patients

Performance Evaluation of a Commercial Automated Library Preparation System for Clinical Microbial Whole-Genome Sequencing Assays

Next-generation sequencing (NGS) has proven clinical utility on disease management and serves as an important tool for genomic surveillance. Currently, hurdles surrounding its implementation, namely the complex and demanding analytical workflows, have impeded its widespread use in many laboratories. To address this challenge, the UCLA Molecular Microbiology and Pathogen Genomics Laboratory evaluated the performance of the Tecan MagicPrep NGS system, a commercial automated solution for library preparation for clinical whole-genome sequencing assays, against the Illumina Nextera DNA Flex Library Prep. Using 35 unique organisms (28 bacteria and 7 fungi) for various clinical applications, including microbial identification and genomic characterization, we compared the quantity and quality of the prepared libraries and the resulting sequences, and concordance of the overall results. We also assessed the impact of its implementation on laboratory workflow. The MagicPrep NGS produced higher library concentrations with smaller sizes, and correspondingly, higher molarity. Quality metrics of the sequences, however, demonstrated no significant impact on the overall results, producing 100% concordance with the reference method. Importantly, workflow analysis showed 5 hours less hands-on time per run with more flexibility. This evaluation study indicates that performance of the MagicPrep NGS is comparable to the Nextera DNA Flex with the added benefit of improving workflow efficiency and reducing labor for performing routine clinical microbial whole-genome sequencing tests.

The performance of homopolymer detection using dichromatic and tetrachromatic fluorogenic next-generation sequencing platforms

ObjectivesHomopolymer (HP) sequencing is error-prone in next-generation sequencing (NGS) assays, and may induce false insertion/deletions and substitutions. This study aimed to evaluate the performance of dichromatic and tetrachromatic fluorogenic NGS platforms when sequencing homopolymeric regions.ResultsA HP-containing plasmid was constructed and diluted to serial frequencies (3%, 10%, 30%, 60%) to determine the performance of an MGISEQ-2000, MGISEQ-200, and NextSeq 2000 in HP sequencing. An evident negative correlation was observed between the detected frequencies of four nucleotide HPs and the HP length. Significantly decreased rates (P < 0.01) were found in all 8-mer HPs in all three NGS systems at all four expected frequencies, except in the NextSeq 2000 at 3%. With the application of a unique molecular identifier (UMI) pipeline, there were no differences between the detected frequencies of any HPs and the expected frequencies, except for poly-G 8-mers using the MGI 200 platform. UMIs improved the performance of all three NGS platforms in HP sequencing.ConclusionsWe first constructed an HP-containing plasmid based on an EGFR gene backbone to evaluate the performance of NGS platforms when sequencing homopolymeric regions. A highly comparable performance was observed between the MGISEQ-2000 and NextSeq 2000, and introducing UMIs is a promising approach to improve the performance of NGS platforms in sequencing homopolymeric regions.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Comparison of molecular analysis results determined by next-generation sequencing to immunohistochemical indicators and clinicopathological parameters in prostate adenocarcinomas.

Prostate cancer is a common cancer in males, frequently leading to mortality. Multiple genetic factors play roles in prostate cancer pathogenesis. Demonstration of pathological pathways and customised treatment options have been possible with next-generation sequencing. In this study, we aimed to evaluate the relationships of the changes in the prostate cancer pathways genes with the pathological, immunohistochemical and the clinical parameters. Retrospective cross-sectional study. Among the prostate needle biopsy materials investigated in Adnan Menderes University Faculty of Medicine, Department of Pathology, thirty-one cases, who had been analysed using the next-generation sequencing system, were included in this study. As a result of statistical analysis, a significant relationship was found between the pathogenic mutation detected in androgen receptor and Breast Cancer Gene 2 genes and tumour volume. In all cases with a pathogenic mutation in the androgen receptor gene, a pathogenic mutation in the Protein Tyrosine Phosphatase and Tensin Homolog gene was also observed and a significant relationship was found between them. Castration resistance was observed in cases with high tumour volume, and a statistically significant difference was found. A statistically significant relationship was found between tumour volume and Ki-67 expression. In addition, a significant relationship was observed between the castration resistance and Ki-67, c-erbB2 expressions. A statistically significant relationship was found between Ki-67 and c-erbB2. Regarding prognosis prediction and treatment, identifying the molecular changes in genes playing roles in prostate cancer with next-generation sequencing is very important.

Characterizing Hereditary Hemolytic Anemias in a Hispanic Cohort Using Next Generation Sequencing

Background: Hereditary hemolytic anemias (HHA) are a heterogeneous group of nonimmune disorders characterized by increased red blood cell (RBC) destruction due to abnormalities of hemoglobin stability, defects of RBC metabolism, and disorders of RBC hydration. Although uncommon, they comprise important causes of anemia in children and adults by causing chronic or episodic hemolysis. Clinical, laboratory, and genetic heterogeneity characterize this group of disorders and make recognition challenging. The advent of next generation sequencing (NGS) has made accurate genetic diagnoses attainable, with an increasing number of studies reporting clinical utility in HHA. For patients with suspected HHA, multigene testing is available incorporating an NGS panel associated genes. However, there is limited data on the use of multigene NGS to characterize HHA in the Hispanic population. Herein, we describe results from NGS testing of a pilot cohort referred for HHA investigation to hematology clinics along the US/Mexico border. Methods: Pediatric and adult hematology clinics at Texas Tech University in El Paso, TX collaborated for testing on HHA referral and clinical case review. The diagnostic panel (AnemiaID/PerkinElmer/Revvity®) included 51 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions were covered. Targeted gene capture and library construction for NGS were performed using a Whole Blood and Saliva kit and 150 base pair paired-end sequencing was done on Illumina® NGS systems at target average coverage of 80×. NGS output data were summarized descriptively for all patients and clinically correlated for diagnosis. Results: In this pilot cohort, 11 patients were tested during September 2022 to June 2023, age ranging 7-75 years, with females accounting for 64% of the group. Findings are reported in the Table below; Pathogenic variants leading to a definitive diagnosis were identified in 8 of 11 cases (73%). The most frequently mutated genes were SPTA1 followed by PIEZO1, SPTB, RHAG, GSR, SLC4A1 and TPI1. Approximately 58% of the mutations were reported as novel. Diagnoses included, most commonly, Hereditary Spherocytosis followed by Hereditary Pyropoikilocytosis and Congenital Dyserythropoietic Anemia. Compound heterozygosity leading to complex interactions was noted in 4 cases (36%), involving SPTA1 low expression alleles in trans (alpha-LELY and alpha-LEPRA) in 2 cases. Conclusion: Hereditary hemolytic anemia in the Hispanic population is characterized by complex molecular interactions and a high prevalence of novel mutations in RBC cytoskeleton/enzyme genes. NGS has clinical utility and should be considered for accurate diagnosis. Further research into the molecular basis and genetic spectrum of HHA in the Hispanic population is needed.

An Idiotype Vaccine Strategy Utilizing Targeted NGS and Cell Free Protein Synthesis System Ensures Identification and Production of Human Idiotype Antigens, and Shows Promising Results in Preclinical Models of B-Cell Lymphoma with a Novel TLR7/8 Agonist-Based Adjuvant and Anti-PD-L1 Antibody

Background: Non-Hodgkin lymphoma (NHL) is one of the most common hematological malignancies in the world. Despite advances in treatment options, such as chimeric antigen receptor T cells (CAR-T) or bispecific antibodies, many patients still recur after complete remissions (CR) or do not achieve CR. Since B cell malignancies arise from clonal expansion, the B cell receptor (BCR) expressed on the cell surface, idiotype, has been studied as a tumor-specific antigen. Several trials have evaluated idiotype vaccines in clinical setting, but failed to demonstrate efficacy. Limitations have arisen from the uncertainty of BCR sequence confirmation, long turnaround time in manufacturing idiotype antigen, and the absence of strong immune modulators. Here, we utilized targeted next generation sequencing (NGS) for BCR, cell-free protein synthesis (CFPS) system for idiotype antigen manufacturing, and a novel liposomal TLR7/8 agonist-based adjuvant to optimize idiotype vaccine. Methods: To generate the idiotype antigens as single-chain variable fragment (scFv) from A20 lymphoma cell line and biopsy samples from NHL patients, variable regions of BCR heavy and light chain were sequenced by targeted NGS. Total RNA was isolated from A20 cells and tumor tissues from patients, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using constant region-specific primers, resulting in the 1 st strand cDNA. Subsequently, the 2 nd strand cDNA was synthesized using V-gene specific primers and sequenced using an Illumina MiSeq sequencer. We applied targeted NGS to patients' tumor tissues from Asan Medical Center and Seoul National University Hospital to understand whether this method works in human tissues as well. The identified sequence was cloned into a linear DNA template and expressed by CFPS system. We optimized the CFPS system and purification process of idiotype antigen scFv from A20 cell line and human tissues. The liposomal adjuvant ProLNG-001 (TLR7/8 agonist 140 μg) and ProLNG-002 (TLR7/8 agonist 70 μg + TLR4 agonist 5 ug) were synthesized by microfluidics method. Idiotype antigen A20-scFv 50 μg was given alone or mixed with ProLNG-001 or -002. To investigate the efficacy of the idiotype vaccine, BALB/c mice were subcutaneously injected with 5×10 5 of A20 cells. Mice were subcutaneously vaccinated 4 times at 1-week intervals starting from day 2 after tumor inoculation. Atezolizumab 10 mg/kg was administered intraperitoneally 3 times a week for 3 weeks in the idiotype vaccine + immune checkpoint inhibitor combination group (Figure 1a). Results: Targeted NGS has confirmed BCR sequences from A20 cell line and dominant malignant B cell clone from patients' tumor with a success rate of over 90% when sufficient volume (more than 500 mm 3) of biopsy sample was provided for RNA extraction. Also, we were able to express the idiotype scFv antigens determined from 18 NHL patients' tumor samples using CFPS system with a 100% success rate (Figure 1b). The untreated mice (Group 1) showed a tumor growth pattern similar to that of Group 2 (vaccination with antigen only). Vaccination with idiotype antigen mixed with liposomal adjuvants, ProLNG-001 (Group 3) and ProLNG-002 (Group 4), significantly inhibited tumor growth. The combination of anti-PD-L1 antibody and idiotype antigen mixed with ProLNG-002 (Group 5) generated even better overall anti-tumor responses with 14 out of 15 mice showing no residual tumor at day 34 after tumor inoculation. Conclusion: Taken together, the idiotype vaccine strategy using targeted NGS for BCR, CFPS system for idiotype antigen manufacturing, and TLR7/8-agonist-based liposomal adjuvants showed highly effective results in the preclinical syngeneic A20 tumor model. Furthermore, the usage of immune checkpoint inhibitors combined with idiotype vaccine in B cell lymphoma may exert synergistic effects. The CFPS system can be applied to swift, robust and personalized manufacturing of idiotype antigen for individuals with different idiotype sequence in a GMP setting, which was not possible in prior studies. These results support further development of the idiotypic vaccine as a treatment option for non-Hodgkin's B cell lymphomas.

A Next-Generation Sequencing Study in a Cohort of Sicilian Patients with Parkinson's Disease.

Parkinson's disease (PD) is a multisystem and multifactorial disorder and, therefore, the application of modern genetic techniques may assist in unraveling its complex pathophysiology. We conducted a clinical-demographic evaluation of 126 patients with PD, all of whom were Caucasian and of Sicilian ancestry. DNA was extracted from the peripheral blood for each patient, followed by sequencing using a Next-Generation Sequencing system. This system was based on a custom gene panel comprising 162 genes. The sample underwent further filtering, taking into account the allele frequencies of genetic variants, their presence in the Human Gene Mutation Database, and their association in the literature with PD or other movement/neurodegenerative disorders. The largest number of variants was identified in the leucine-rich repeat kinase 2 (LRRK2) gene. However, variants in other genes, such as acid beta-glucosidase (GBA), DNA polymerase gamma catalytic subunit (POLG), and parkin RBR E3 ubiquitin protein ligase (PRKN), were also discovered. Interestingly, some of these variants had not been previously associated with PD. Enhancing our understanding of the genetic basis of PD and identifying new variants possibly linked to the disease will contribute to improved diagnostic accuracy, therapeutic developments, and prognostic insights for affected individuals.

Preliminary Information of Iranian Lizard Leishmania Promastigote Transcriptome Sequencing by Next Generation Sequencing (NGS) Method.

A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far. The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI1640 culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China). The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania. Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system.

Detection of salivary gland and sinonasal fusions by a next-generation sequencing based, ligation-dependent, multiplex RT-PCR assay.

The discovery of tumour type-specific gene fusion oncogenes in benign and malignant salivary gland and sinonasal (SGSN) tumours has significantly increased our knowledge about their molecular pathology and classification. We developed a new targeted multiplexed next-generation sequencing (NGS)-based method that utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR) to detect oncogenic fusion transcripts involving 116 genes, leading to 96 gene fusions known to be recurrently rearranged in these tumours. In all, 180 SGSN tumours (formalin-fixed, paraffin-embedded samples, 141 specimens and 39 core needle biopsies) from the REFCORpath (French network for rare head and neck cancers) with previously identified fusion genes by fluorescent in situ hybridisation (FISH), RT-PCR, or molecular immunohistochemistry were selected to test its specificity and sensitivity and validate its diagnostic use. Tested tumours encompassed 14 major tumours types, including secretory carcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, salivary gland intraductal carcinoma, clear cell carcinoma, pleomorphic adenoma, adamantinoma-like Ewing Sarcoma, EWSR1::COLCA2 sinonasal sarcoma, DEK::AFF2 sinonasal carcinoma, and biphenotypic sinonasal sarcoma. In-frame fusion transcripts were detected in 97.8% of cases (176/180). Gene fusion assay results correlated with conventional techniques (immunohistochemistry [IHC], FISH, and RT-PCR) in 176/180 tumours (97.8%). This targeted multiplexed NGS-based LD-RT-PCR method is a robust, highly sensitive method for the detection of recurrent gene fusions from routine clinical SGSN tumours. It can be easily customized to cover new fusions. These results are promising for implementing an integrated NGS system to rapidlydetect genetic aberrations, facilitating accurate, genomics-based diagnoses, and accelerate time to precision therapies in SGSN tumours.

A nation-wide genomic screening project for further development of targeted therapies in treatment-refractory non–small-cell lung cancer (LC-SCRUM-TRY).

9097 Background: Targeted therapies based on oncogenic drivers demonstrate dramatic and durable response in patients with non-small cell lung cancer (NSCLC). However, acquired resistance inevitably develop by diverse genomic resistance mechanisms. Genotype-matched targeted therapies to overcome treatment-resistance have not been established, except osimertinib against EGFR T790M mutation. Methods: We established a nation-wide genomic screening project in treatment-resistant NSCLC (LC-SCRUM-TRY; UMIN000041957) in September 2020 to identify the genetic resistant alterations, and to promote clinical therapeutic development. Enrolled patients have been screened for genetic alterations using a rapid next-generation sequencing (NGS) system, Oncomine Precision Assay (OPA) for tissue samples, and Guardant 360 (G360) or OPA for plasma samples. MET amplification was also evaluated by FISH for NSCLC post EGFR-TKI treatment. Results: As of January 2023, 129 institutions in Japan were participating, and 1252 patients had been enrolled. Sample types were tissue (84%), and plasma (16%), respectively. Turn-around-time (median [interquartile range]) of OPA and G360 was 5 (4-6), and 9 (8-10.5) days, respectively. Of these, a total of 711 (57%) were already identified to have oncogenic driver at enrollment ( EGFR, 556 [43%]; ALK, 70 [6%]; RET, 22 [2%]; ROS1, 21 [2%]; KRAS, 15 [1%]; ERBB2 exon 20 insertion [ex20ins], 8 [0.6%]; MET exon 14 skipping [ex14skip], 8 [0.6%]; BRAF V600E, 7 [0.6%]; NTRK3, 2 [0.2%]; others, 2 [0.2%]). Of 556 EGFR-mutated NSCLC, 537 (97%) were enrolled post EGFR-TKIs. EGFR mutation subtypes were exon 19 deletion (52%), L858R (41%), ex20ins (1%), others (6%), respectively. Of 537 EGFR-TKI resistant tumors, 326 (40%) had at least one genetic alteration related with drug resistance, including EGFR alterations (amplification [13%], C797S [4%], A750P [3%], E709X [2%], L792X [1%], L718Q [1%]), MET amp (16%), and other driver mutation/rearrangement (8%). Through this screening, 37 (7%) of patients resistant to EGFR-TKI were enrolled into clinical trials, with 16/102 (16%) patients with targetable alterations ( MET amp or EGFR C797S). In addition, of 541 NSCLC without oncogenic drivers at enrollment, 116 (21%) were identified to have actionable oncogenic drivers with FDA-approved drug (KRAS G12C, 23 [4%]; ERBB2 ex20ins, 23 [4%]; RET, 13 [2%]; MET ex14skip, 13 [2%]; EGFR except ex20ins, [4%]; EGFR ex20ins, 10[2%]; ALK, 6 [1%]; ROS1, 5 [1%]). Conclusions: LC-SCRUM-TRY contributes to the clinical development of precision medicine to overcome drug resistance, especially for EGFR-mutated NSCLC resistant to EGFR-TKI. This screening platform also help to practice precision medicine for patients initially diagnosed as driver-negative. Clinical trial information: UMIN000041957 .

Diagnostic utility of next generation sequencing for somatic variants in cancer: A seven year cumulative cohort.

e15060 Background: Next generation sequencing (NGS) makes it possible to identify the specific molecular biomarkers to define patient eligibility for targeted therapies via multigene panels. Since then, even though the interpretation of data is complex and not all tumors have genomic alterations within actionable genes, using somatic sequencing for cancer patients becomes a daily routine in clinical oncology. Thus, here we present the data derived from our experience in NGS testing of different cancer patients and biological samples over the course of 7 years in our genetic diagnosis center. Methods: The cohort was consisted of total 5511 cases. While 2590 (46.99%) of them were formalin fixed paraffin embedded (FFPE) tissues, 2906 (52.73%) were liquid biopsies and 15 (0.27%) were other types of biological samples. Total of 15 different cancers were classified (Table 1) and were sequenced using 3 different multigene NGS panels (additional Table 1, BRCAs’ testing for breast/ovarian cancer is not included). NGS was performed via GeneReader NGS System (Qiagen, Germany). Bioinformatics analyzes and variant classification were performed using QCI-Analyze and QCI-Interpret interfaces. Results: As a result, the cases of 93.36% (n = 5145) among all samples were successfully sequenced. The lung cancer group was the most frequent with 47.96% (n = 2643 patients), followed by 491 (%8.9) colorectal 288 (5.23%) and breast cancer patients respectively, while 781 cases (14.17%) grouped as other including many rare cancers. Overall positivity rate was 42.57% (n = 2346) and 366 samples (6.64%) were not sufficient enough to be sequenced. The distribution of positivity rates in relation to cancer type and biological samples used were listed in Table 1. Classification and diagnosis of all tested cases, specimens and Tier 1-2 variants included in this cohort are available on the Additional Table 2. Conclusions: This report is a comprehensive review of the aggregate data across the entire cohort of cancer patients referred to a genetics diagnosis center. While our findings highlight the importance of NGS testing in the somatic setting for cancer patients, also these data stress the importance of diagnosis, biological sample types tested and test results. [Table: see text]

Genetic mutations in HER2-positive breast cancer: possible association with response to trastuzumab therapy

BackgroundHER2-positive breast cancer occurs in 15–20% of breast cancer patients and is characterized by poor prognosis. Trastuzumab is considered the key drug for treatment of HER2-positive breast cancer patients. It improves patient survival; however, resistance to trastuzumab remains a challenge in HER2-positive breast cancer patients. Therefore, the prediction of response to trastuzumab is crucial to choose optimal treatment regimens. The aim of the study was to identify genetic variants that could predict response to anti-HER2-targeted therapy (trastuzumab) using next-generation sequencing.MethodGenetic variants in the hotspot regions of 17 genes were studied in 24 Formalin-Fixed Paraffin-Embedded (FFPE) samples using Ion S5 next-generation sequencing system. FFPE samples were collected from HER2‑positive breast cancer patients previously treated with anti‑HER2‑targeted treatment (Trastuzumab). Patients were divided into two groups; trastuzumab-sensitive group and trastuzumab-resistant group based on their response to targeted therapy.ResultsWe identified 29 genetic variants in nine genes that only occurred in trastuzumab-resistant patients and could be associated with resistance to targeted therapy including TP53, ATM, RB1, MLH1, SMARCB1, SMO, GNAS, CDH1, and VHL. Four variants out of these 29 variants were repeated in more than one patient; two variants in TP53, one variant in ATM gene, and the last variant in RB1 gene. In addition, three genes were found to be mutated only in resistant patients; MLH1, SMARCB1 and SMO genes. Moreover, one novel allele (c.407A > G, p. Gln136Arg) was detected within exon 4 of TP53 gene in one resistant patient.ConclusionNGS sequencing is a useful tool to detect genetic variants that could predict response to trastuzumab therapy.

Impact of assigning 2-field HLA alleles from real-time PCR on deceased donor assessments and conformance with high resolution alleles.

Waitlisted sensitised transplant recipients with HLA allele level antibodies to their own HLA antigen family are disadvantaged by current deficiencies in HLA typing for deceased donors. This is primarily because at time of organ allocation, HLA typing is provided at antigen level whereas solid phase assays provide allele level antibody definition. The gold standard for HLA allele typing is next generation sequencing (NGS), however time limitations with established NGS systems prevent NGS use for deceased donors. Instead, many labs use a real-time PCR (qPCR) antigen level result for deceased donors, which can disadvantage sensitised patients. Here, we compared assigning qPCR 2-field alleles to qPCR antigen level to determine the impact on virtual crossmatch (VXM) and discuss impact on donor-specific antibody (DSA) assignments. 244 consecutive deceased donors were HLA typed to allelic level by qPCR (LinkSeq SABR) and subsequently by NGS (One Lambda Alltype). The impact of qPCR allele assignments on potential DSA identification was investigated, by retrospectively investigating all 3904 VXMs, where recipient DSA assessments were assessed against donor HLA, was performed within the cohort. There was 96.3% concordance between qPCR and NGS for all allele level loci, with HLA-A; DQB1; and DPB1 having best agreement (99.4%, 98.4% and 99.4% respectively). Of the 3904 VXMs with qPCR allele assignment, there were 13 (<1%) occasions where the potential DSA assignment was impacted, with DQA1 having the most impact. Assigning alleles derived from qPCR to define unacceptable antigens for VXMs, can allow improved access to donor offers for sensitised patients by better defining alleles.

Abstract 5547: Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations

Abstract Introduction: The use of blood-based molecular diagnostics is increasingly becoming routine in clinical oncology practice. The purpose of this study was to evaluate variant resulting concordance for the four major somatic variant classes using two clinical next-generation sequencing (NGS) systems for conducting highly sensitive blood-based analyses. All donor specimens were de-identified remnants from patients previously diagnosed with advanced NSCLC. We utilized two independent panels, systems and bioinformatic pipelines that are focused on clinical testing for key actionable variants. The fifty-gene panel Oncomine Precision Assay (OPA) GX was performed on the Genexus Integrated Sequencer and the 52 gene GeneStrat NGS (GSNGS) test was run as a reference on the Ion GeneStudio S5 PRIME system. The four major classes of mutations evaluated included Single Nucleotide Variants (SNV), Insertions and Deletions (INDEL), copy number amplifications (CNAs) and gene fusions. Methods: For this study, de-identified reference GSNGS cell-free nucleic acid (cfNA) remnant specimens (n=33 variants) that passed all validated QC bioinformatics thresholds were blinded and tested on the Genexus. DNA concentration was measured by fluorometry, and specimens were diluted to an input of 6.6 ng to 53.4 ng for library preparation. The Genexus system is pre-programmed to process specimens to result generation. Variant calls for all mutation classes were conducted using the on-board bioinformatics pipelines, and results were compared to those generated by the GeneStrat NGS Test. We included specimens harboring somatic variant mutations in KRAS, NRAS, BRAF, EGFR, ERBB2, and FGFR; CNAs; EML4-ALK fusions; and MET exon 14 skipping. Results: All samples passed the OPA (Genexus) bioinformatic quality control criteria and mapped reads were highly consistent, demonstrating accuracy of calls between both the Ion Torrent platforms, informatic pipelines and panels. We observed 100% concordance in variant calls for SNVs, INDELs and fusions between the two platforms. There was one CNA variant observed on the S5 that was not called by the Genexus workflow, yielding overall concordance of 97% (32 of 33). Of note, 44 additional somatic variants (SNV and INDELs) and 1 additional fusion were detected using the Genexus workflow. This was due to the lower threshold for variant calling on this system. Conclusion: The high concordance of the independent workflows for the detection of nucleic acid variants in circulation demonstrates the capability of both systems to be used for testing of clinical specimens. Specifically, actionable variants in the four major mutation classes were successfully detected in the reference and test specimen set. As a part of clinical validation, orthogonal testing of variants will need to be conducted. Citation Format: Sarah Kreston, Claire Gould, Kylie Blair, Leisa Jackson, Janice Riley, Gary A. Pestano. Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5547.

Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma

Oncogenic mutations in the EGFR gene are targets of tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LC) patients, and their search is mandatory to make decisions on treatment strategies. Liquid biopsy of circulating tumour DNA (ctDNA) is increasingly used to detect EGFR mutations, including main activating alterations (exon 19 deletions and exon 21 L858R mutation) and T790M mutation, which is the most common mechanism of acquired resistance to first- and second-generation TKIs. In this study, we prospectively compared three different techniques for EGFR mutation detection in liquid biopsies of such patients. Fifty-four ctDNA samples from 48 consecutive advanced LC patients treated with TKIs were tested for relevant EGFR mutations with Therascreen® EGFR Plasma RGQ-PCR Kit (Qiagen). Samples were subsequently tested with two different technologies, with the aim to compare the EGFR detection rates: real-time PCR based Idylla™ ctEGFR mutation assay (Biocartis) and next-generation sequencing (NGS) system with Ion AmpliSeq Cancer Hotspot panel (ThermoFisher). A high concordance rate for main druggable EGFR alterations was observed with the two real-time PCR-based assays, ranging from 100% for T790M mutation to 94% for L858R variant and 85% for exon 19 deletions. Conversely, lower concordance rates were found between real-time PCR approaches and the NGS method (L858R: 88%; exon19-dels: 74%; T790M: 37.5%). Our results evidenced an equivalent detection ability between PCR-based techniques for circulating EGFR mutations. The NGS assay allowed detection of a wider range of EGFR mutations but showed a poor ability to detect T790M.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.