Applicability Of Next-generation Sequencing Research Articles

B-245 Applicability of metagenomic Next-Generation Sequencing (mNGS) in challenging infectious diseases in a tertiary hospital

Abstract Background Infectious diseases caused by rare pathogens or with unusual presentations pose a diagnostic challenge. Traditional microbiologic tests can be time-consuming, labor-intensive and have limited sensitivity for some organisms. Plasma metagenomic Next-Generation Sequencing (mNGS) detects whole microbial genetic material and detects even unsuspected organisms. We present three cases of challenging infectious diseases and discuss the applicability of mNGS in different scenarios. Methods Plasma NGS was performed at Karius, Inc. (Redwood City, CA, USA). Patient data was collected from hospital and laboratory electronic records. Results Case 1: An 8-year-old female with sickle-cell anemia developed a persistent daily fever two weeks after recovery from sepsis due to Streptococcus pyogenes. Clinical exam was normal and investigation was negative (blood cultures, echocardiogram, thoracic and abdominal CT scans and bone scintigraphy). Two weeks later mNGS was performed, which came out positive for Cytomegalovirus. As patient became afebrile in 48 h and the viral burden was low, she could be safely discharged. Case 2: A 43-year-old male with prosthetic aortic valve and an intraluminal aortic ring had a year-long history of fatigue, weight loss and hypoalbuminemia. CT scans showed ground-glass infiltrates and enlarged mediastinal lymph nodes. Empirical treatment for tuberculosis was ineffective. A year later, he developed hepatosplenomegaly, fever and pancytopenia. Blood cultures, bone marrow biopsy and serologies (HIV, hepatitis B/C, syphilis, brucellosis, bartonellosis, leishmaniasis) were negative. Cardiac surgery found no apparent signs of endocarditis and the prosthetic aortic valve biopsy revealed only an inflammatory infiltrate. A new CT scan showed thickened intraluminal aortic ring with aortic pseudoaneurysms and splenomegaly with splenic infarction. A blood sample was then collected for mNGS and liver biopsy and splenectomy were performed. However, surgery complicated with hemodynamic instability, renal and liver failure and the patient died in less than 24 h. Two days later, mNGS came out positive for Coxiella burnetti. Case 3: A 45-year-old female with relapsed AML treating a chronic disseminated candidiasis with micafungin for 3 months had persistent liver and splenic nodules and new pulmonary nodules on the CT scan. In the following three months after chemotherapy (Gilteritinib), she developed multiple episodes of febrile neutropenia, some without microbiological documentation, leading to multiple courses of antibiotic treatment and a shift to liposomal amphotericin B. After clinical improvement and bone marrow recovery, liposomal amphotericin B was replaced by isavuconazole. A negative mNGS result allowed antifungal treatment to be safely discontinued, avoiding six months of unnecessary antifungal treatment. Conclusion We summarize three different scenarios of mNGS applications. In common, patients were submitted to multiple laboratory and image testing and received large doses of empirical antimicrobials due to lack of a precise diagnosis. In two cases, mNGS was diagnostic, shortened hospital stay and reduced overall costs. In case 2, the patient’s death might have been prevented if mNGS had been performed earlier. Vulnerable hospitalized patients with a broad spectrum of diagnostic possibilities, in whom sequential diagnostic tests could delay adequate therapy and cause significant adverse effects, could greatly benefit from mNGS when performed in a timely manner.

Characterization of pharmacogenomic variants in a Brazilian admixed cohort of elderly individuals based on whole-genome sequencing data.

Introduction: Research in the field of pharmacogenomics (PGx) aims to identify genetic variants that modulate response to drugs, through alterations in their pharmacokinetics (PK) or pharmacodynamics (PD). The distribution of PGx variants differs considerably among populations, and whole-genome sequencing (WGS) plays a major role as a comprehensive approach to detect both common and rare variants. This study evaluated the frequency of PGx markers in the context of the Brazilian population, using data from a population-based admixed cohort from Sao Paulo, Brazil, which includes variants from WGS of 1,171 unrelated, elderly individuals. Methods: The Stargazer tool was used to call star alleles and structural variants (SVs) from 38 pharmacogenes. Clinically relevant variants were investigated, and the predicted drug response phenotype was analyzed in combination with the medication record to assess individuals potentially at high-risk of gene-drug interaction. Results: In total, 352 unique star alleles or haplotypes were observed, of which 255 and 199 had a frequency < 0.05 and < 0.01, respectively. For star alleles with frequency > 5% (n = 97), decreased, loss-of-function and unknown function accounted for 13.4%, 8.2% and 27.8% of alleles or haplotypes, respectively. Structural variants (SVs) were identified in 35 genes for at least one individual, and occurred with frequencies >5% for CYP2D6, CYP2A6, GSTM1, and UGT2B17. Overall 98.0% of the individuals carried at least one high risk genotype-predicted phenotype in pharmacogenes with PharmGKB level of evidence 1A for drug interaction. The Electronic Health Record (EHR) Priority Result Notation and the cohort medication registry were combined to assess high-risk gene-drug interactions. In general, 42.0% of the cohort used at least one PharmGKB evidence level 1A drug, and 18.9% of individuals who used PharmGKB evidence level 1A drugs had a genotype-predicted phenotype of high-risk gene-drug interaction. Conclusion: This study described the applicability of next-generation sequencing (NGS) techniques for translating PGx variants into clinically relevant phenotypes on a large scale in the Brazilian population and explores the feasibility of systematic adoption of PGx testing in Brazil.

Applicability of Next-Generation Sequencing for Analysis of Stomach Contents in Fish

Applicability of Next-Generation Sequencing for Analysis of Stomach Contents in Fish

Abstract 5232: Next-generation sequencing revealed clonal architecture and faciliated diagnosis of patients with multiple lung tumors: An analysis of 12 cases

Abstract Diagnosing multiple lung tumors as multiple primary lung cancer (MPLC) or intrapulmonary metastasis (IPM) has important therapeutic implications. The clinical applicability of next-generation sequencing (NGS) has been explored, although there is currently no consensus regarding how to integrate its findings for cancer staging. We performed a retrospective analysis of the mutational landscape of 13 patients with multiple tumors and underwent surgery at Renmin Hospital of Wuhan University from Sep. 2018 to Dec. 2020.The cohort consisted mostly of women (11/13, 84.6%) and non-smokers (12/13, 92.3%). Median age was 58 years (range 26-71). No patient manifested relapse after a median follow-up duration of 11.2 months (range 8.2-36.0). NGS was performed with a large panel (≥ 350 genes) in most cases (11/13, 84.6%). Most nodules were invasive adenocarcinomas (25/41, 61.0%), followed by adenocarcinomas in situ (13/41, 31.7%), and minimally invasive adenocarcinomas (3/41, 7.3%). Four patients were clinically diagnosed with IPM, of whom 1 had only one nodule sequenced. Of the 12 patients with genomic profiles from multiple nodules, 10 (83.3%) had consistent NGS inference and clinical diagnosis of MPLC or IPM. Of the remaining two, P1 was clinically diagnosed with IPM but NGS did not detect any genomic aberrations from one nodule, which may have missed evidence that could have led to inference of clonal relationship. P8 was diagnosed with MPLC but had two nodules that harbored EGFR L858R mutation. NGS revealed clonal relationships that enabled high-confidence differential diagnosis in the concordant cases. For instance, the 3 nodules in P10 each harbored an oncogenic driver: EGFR exon 19 deletion, EGFR L858R, and ALK fusion, which were suggestive of tumor clones of separate origins. Similar mutual exclusion was also observed in P6 (EGFR exon 19 deletion vs L858R), P2 (EGFR exon 19 deletion vs MAP2K1), and P7 (2 EGFR exon 18 mutations G719A and E709 in one nodule vs EGFR E746_A750del in another). On the other hand, P9 illustrates a case of complex clonal architecture, in which 8 nodules were derived from 5 origins. Further analysis found that alterations in KRAS were associated with smaller nodule size, whereas those in TP53, CDKN2A, NKX2-1, or GNAS were associated with greater size.In summary, NGS achieved highly concordant MPLC/IPM differentiation with clinical diagnosis based on histopathologic and radiographic evidence. However, caution is warranted when NGS detects no aberration from any of the multiple sequenced nodules. Examination of the mutational landscape found that altered proto-oncogene KRAS was associated with smaller tumors whereas altered tumor suppressors TP53 and CDKN2A associated with smaller ones, suggesting different roles in these molecules play in tumor growth. Citation Format: Wenyang Jiang, Wei Wang, Luting Qiu, Xiao Zou, Zhiqiu Chen, Qing Geng. Next-generation sequencing revealed clonal architecture and faciliated diagnosis of patients with multiple lung tumors: An analysis of 12 cases [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 5232.

Concordance Between Tissue ALK Detection by Immunohistochemistry and Plasma ALK Detection by Next-Generation Sequencing in the Randomized Phase 3 ALEX Study in Patients With Treatment-Naive Advanced ALK-Positive NSCLC

IntroductionThe Blood First Assay Screening Trial revealed the clinical applicability of blood-based next-generation sequencing to identify patients with ALK-positive NSCLC for alectinib treatment. To understand the relationship between tissue-based versus blood-based testing, we retrospectively investigated concordance between VENTANA ALK (D5F3) CDx immunohistochemistry and the FoundationACT (FACT; Foundation Medicine, Inc.) plasma assay, and compared clinical efficacy between phase 3 ALEX study subpopulations. MethodsPatients with advanced ALK-positive (by immunohistochemistry) NSCLC were randomized 1:1 to alectinib 600 mg or crizotinib 250 mg, twice daily. Assessable baseline plasma samples were analyzed for ALK positivity by FACT; positive percent agreement with immunohistochemistry was evaluated. Progression-free survival (PFS), duration of response, and objective response rate were compared between intention-to-treat (ITT) and biomarker-evaluable populations, and plasma ALK-positive and plasma ALK-negative subpopulations. ResultsIn the ITT population (303 patients; alectinib, 152; crizotinib, 151), all patients had ALK-positive tumors by immunohistochemistry. In the biomarker-evaluable population (149 patients; alectinib, 76; crizotinib, 73), 105 had plasma ALK-positive and 44 had plasma ALK-negative tumors. Positive percent agreement between immunohistochemistry and FACT was 70.5% (105 of 149; 95% confidence interval: 62.5–77.7). Baseline characteristics were generally balanced, with some exceptions, notably tumor burden. Median PFS in plasma ALK-positive and ALK-negative patients was 22.4 months and not estimable with alectinib and 7.3 months and 12.9 months with crizotinib, respectively; median duration of response was 25.9 months and not estimable with alectinib and 5.6 months and 11.5 months with crizotinib, respectively. ConclusionsReasonable concordance between FACT and immunohistochemistry was observed; both methods are valuable in identifying ALK-positive patients, separately or concurrently. Alectinib was found to have superior PFS in the plasma ALK-positive population, as in the ITT population.

Comparison of ear canal microbiome in rabbits with and without otitis externa using next generation DNA sequencing

Contemporary research has increasingly explored the clinical applicability of next-generation DNA sequencing (NGS) technologies in veterinary medicine, which has provided new and practical opportunities for rabbit otitis externa clinical interventions. The objectives of this study were to characterize the normal external ear canal microbiome of clinically healthy rabbits compared to otitis externa presenting rabbits, and to assess the diagnostic viability of NGS in aural veterinary medicine. Swabs from the external ear canal of 34 clinically healthy rabbits and 16 rabbits diagnosed with otitis externa were collected. Alongside bioinformatic analysis, library preparation was performed targeting the V1-V3 region of the 16S rRNA bacterial gene and the ITS-2 region for fungal DNA analysis. In the clinically healthy group, the bacterial species with the highest relative abundances were an uncharacterized Phytoplasma from the family Acholeplasmataceae (8.74%) and Staphylococcus epidermidis (5.56%), while in the otitis group the species with the highest relative abundances were Staphylococcus aureus (12.59%), Corynebacterium lactis (9.27%), and Corynebacterium mastitidis (7.92%). Fungal species with the highest relative abundances in the healthy group were a species from the genus Cladosporium (14.46%), while in the otitis externa group the fungal species with the highest relative abundances were a species from the genus Cladosporium (9.89%) and Malassezia restricta (4.76%). Additionally, there was a significantly higher number of different bacterial and fungal species in the clinically healthy group compared to the otitis group ( P = 0.00 and P = 0.00, respectively). This study provided evidence that the rabbit aural microbiome profile is distinctly different between a clinically healthy and an otitis state. It also highlighted new bacterial and fungal organisms of note in rabbits diagnosed with otitis externa compared to those previously thought to be the primary disease-causing organisms. Understanding the microbial population dynamics in the rabbit aural microbiome is particularly critical for helping clinicians recognize, prevent, or revert the progression of otitis.

Molecular profiling of gene fusions in soft tissue sarcomas by Ion AmpliSeqTM: a study of 35 cases.

BackgroundThe accurate diagnosis of sarcoma can be difficult as there are over 70 different subtypes. While molecular profiling in soft tissue sarcoma (STS) has gradually been incorporated into routine diagnostics, conventional methods such as fluorescence in situ hybridization (FISH), reverse transcriptase-PCR (RT-PCR), and Sanger sequencing have several drawbacks. By allowing simultaneous analysis of multiple targets and increasing sequencing depth to achieve ultra-sensitivity, next-generation sequencing (NGS) can not only detect common genetic abnormalities without prior assumptions but also identify uncommon or even new variants.MethodsIn this study, the applicability of NGS in assessing STS using the Ion Torrent Proton was evaluated and compared with other methods. A cohort of 35 tissue specimens from STS patients, including alveolar soft-part sarcoma (ASPS), Ewing’s sarcoma (ES), synovial sarcoma (SS), dermatofibrosarcoma protuberans (DFSP), and myxoid liposarcoma (MLPS) patients, were subjected to NGS by an Ion AmpliSeqTM Custom panel.ResultsA proportion of 97.14% (34/35) were successfully conducted to detect gene fusion positive events and met all criteria for good quality. The concordance between NGS and conventional techniques was 94.12% (32/34). NGS also showed superior results, as Sanger sequencing and FISH in two cases were false negatives, demonstrating the excellent diagnostic utility of NGS for translocation detection in STS.ConclusionsThe results in this study show the potential for NGS to aid in diagnosis and clinical monitoring of STS and warrant additional studies in larger cohorts.

Next Generation Sequencing (NGS)-Based Post-Transplant Monitoring in Acute Myeloid Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

▪ObjectiveTo assess applicability of serial next generation sequencing (NGS) in monitoring acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HCT).IntroductionClinical application of NGS in allogeneic HCT is a topic of interest. Post-HCT mutation dynamics using longitudinal NGS have not been thoroughly examined. We hypothesized that serial sequencing of pre- and post-HCT using NGS could provide a clinically relevant value in AML patients receiving HCT. The present study aimed to evaluate mutation dynamics in AML using serial samples from pre- and post-HCT with respect to transplant outcomes.Patients and Methods104 AML patients were enrolled and sequenced using an Illumina HiSeq 2000 sequencer (84 myeloid custom gene panel) on 529 bone marrow/peripheral blood samples at diagnosis (n=104), pre-HCT (n=104), 21 days post-HCT (n=104), at 6 (n= 38), 12 (n= 20), beyond 12 months (n=4) and at relapse (n=20). Two patients relapsed by day 21. T-cell (n=80) and donor samples (n=57) were also sequenced. All computational and statistical analyses were performed using Python and R.ResultsThe average on-target coverage in 529 samples was 1725.6x. In total, we detected 256 mutations at diagnosis in 90/104 patients (86.5%, median 3 mutations per patient). DNMT3A ( n=26, 25.0%), NPM1 ( n=23, 22.1%), CEBPA ( n=15, 14.4%), IDH2 ( n=15, 14.4%), FLT3 (n=14, 13.5%), PTPN11 (n=11, 10.6%) , and NRAS (n=11, 10.6%) were commonly mutated at diagnosis (Figure A). When grouped by 8 defined biological pathways, activated signaling (n=45, 43.2%), DNA methylation (n=44, 42.3%), NPM1 (n=23, 22.1%), and myeloid transcription factors (n=21, 20.2%) were mutated in more than 20% of the cohort (Figure B). Assessment of mutation dynamics in serial samples revealed that mutations from non-relapsed patients show rapid clearance after HCT without acquisition of somatic mutations and that allelic burden is maintained at very low level in longer follow-up period (less than 0.1%) (Figure C). On the other hand, relapsed patients tend to carry higher allelic burden even at day 21 after HCT (2.9% as opposed to 0.04% in non-relapsed patients), which suggests that allelic burden throughout the course of the disease and treatment can possess predictive power for detecting early relapse and predicting long-term overall survival (Figure D). We then assessed whether the mutation status at pre- and post-HCT has any impact on overall survival (OS) and relapse after HCT. With median follow-up of 6.3 years (range 0.7 - 12.3 years), patients with overall variant allele frequency (VAF) ≥ 0.2% at day 21 after HCT showed worse OS (Hazard Ratio (HR) 2.62 [1.28-5.35], p= 0.008) as well as increased risk of relapse (HR 6.16 [2.57-14.73], p < 0.001) (Figure E and F). Multivariate analyses verified that VAF ≥ 0.2% at day 21 was an adverse prognostic factor independent of age and cytogenetics risk group for OS (HR 3.06, [1.46-6.39], p = 0.003). Non-relapse mortality did not show significant difference regardless of VAFs ≥ 0.2% at day 21 (p = 0.21). Overall, 23 mutations from 16 patients were detected at day 21 after HCT (VAFs ≥ 0.2%) where 22 mutations were detected either at diagnosis or relapse, or both. In addition, mutations in DNMT3A (n=10) and NPM1 (n=3) were found in one than one patients at day 21 after HCT (13/23 mutations, 56%).Conclusion and summaryThis study revealed that the allelic burden immediately after HCT, within 21 days, with low VAF (0.2%) can be used to predict relapse after HCT as well as maintenance of nearly cleared allelic burden was observed in samples taken at longer follow-up period (mostly 6 and 12 months) from non-relapsed patients (less than 0.1%). Altogether, our data and analyses illustrated the value of longitudinal NGS-based monitoring strategies for AML patients after allogeneic HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Standardized IGH-Based Next-Generation Sequencing for MRD Detection in Follicular Lymphoma

Introduction: MRD monitoring is of great value to determine the risk of relapse in patients with follicular lymphoma (FL).However, MRD assessment is mainly restricted to those 50-65% of patients harbouring a PCR-detectable t(14;18) rearrangement as clonal marker for RQ-PCR. Next generation sequencing (NGS) of immunoglobulin (IG) gene rearrangements might overcome limitations of current PCR-based approaches. Our aim was to test the stability and reproducibility of a standardized amplicon-based NGS approach developed by the EuroClonality-NGS Consortium to a): detect clonally rearranged IG clonotypes and b) to use IG-NGS for MRD quantitation in FL.Methods: Peripheral blood (PB) and bone marrow (BM) samples were collected within randomized trials of the German Low-Grade Lymphoma Study Group (GLSG) and Fondazione Italiana Linfomi treating first-line stage III/IV FL with combined immunotherapy regimen. At first we tested the applicability of NGS for MRD marker screening in randomly selected diagnostic samples from 100 patients (50 PB, 50 BM). Next, NGS-MRD was assessed by sequencing IGH clonotypes in 113 FL patients selected according to the presence of a clonal IGH rearrangement by consensus PCR at diagnosis and the availability of post induction follow-up samples for MRD. Paired-end libraries were prepared and sequenced on Illumina MiSeq (2 x 250bp). FU samples were spiked with predefined copy numbers of internal reference DNA to enable assessment of MRD by normalisation of NGS reads after sequencing. Identification of IGH clonotypes and quantitation of MRD was done with the bioinformatic analysis pipeline ARResT/Interrogate (Bystry, Bioinformatics, 2016). For clonotype identification we used a 5% threshold. For validation NGS results were compared to classical RQ-PCR with either t(14;18) RQ-PCR (n=38 pats.) or IGH ASO RQ-PCR (n= 32 pats.).Results: Marker screening by NGS detected clonal IGH-FR2 sequences with comparable frequency of 26/50 in PB and 24/50 BM samples. Additionally, IGK light chain clonotypes were detected with similar frequencies, thus leading to an IG clonotype being detected in 64% of samples.In the patient cohort analysed for MRD NGS identified an IGH clonotype in 105/113 diagnostic samples. MRD results by RQ-PCR were available in parallel from 70 patients. In 5/8 NGS-neg. diagnostic samples RQ-PCR demonstrated circulating FL cells above the quantitative range underlining the role of somatic hypermutation potentially resulting in false-negativity. At follow-up, 43/123 samples were MRD+ and 42 were MRD- by both methods. 32 were MRD+ by RQ-PCR only, 6 by NGS only. 16/32 NGS-/RQ-PCR+ samples showed low-level MRD below <1E-04 by RQ-PCR. MRD results determined by NGS were well correlated to RQ-PCR values (r2=0.599, p<0.00001).Correlation of NGS-MRD response with outcome in 43 patients from the GLSG cohort demonstrated that achievement of MRD response in PB and/or BM at end of induction is associated with a significantly longer median progression free survival of 11 years compared to 2.2 years for MRD+ patients (median follow-up 6.2 years, Figure 1).Conclusions: The standardized EuroClonality NGS approach shows robust identification of IGH clonotypes in FL patients allowing reliable MRD monitoring. Lymphoma infiltration of diagnostic samples and somatic mutation of IGH are critical points for clonotype identification by NGS. IG-NGS can complement current MRD methods allowing MRD assessment in the majority of FL patients. [Display omitted] DisclosuresPott:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Kneba:AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding. Hoster:Roche: Other: Travel support, Research Funding.

Immunoglobulin gene rearrangement in Koreans with multiple myeloma: Clonality assessment and repertoire analysis using next-generation sequencing.

IntroductionWe assessed the applicability of next-generation sequencing (NGS)-based IGH/IGK clonality testing and analyzed the repertoire of immunoglobulin heavy chain (IGH) or immunoglobulin kappa light chain (IGK) gene usage in Korean patients with multiple myeloma (MM) for the first time.MethodsFifty-nine bone marrow samples from 57 Korean patients with MM were analyzed, and NGS-based clonality testing that targeted the IGH and IGK genes was performed using IGH FR1 and IGK primer sets.ResultsClonal IGH and IGK rearrangements were observed in 74.2% and 67.7% of samples from Korean patients with kappa-restricted MM, respectively (90.3% had one or both), and in 60.7% and 95.5% of samples from those with lambda-restricted MM, respectively (85.7% had one or both). In total, 88.1% of samples from Koreans with MM had clonal IGH and/or IGK rearrangement. Clonal rearrangement was not significantly associated with the bone marrow plasma cells as a proportion of all BM lymphoid cells. IGHV3-9 (11.63%) and IGHV4-31 (9.30%) were the most frequently reported IGHV genes and were more common in Koreans with MM than in Western counterparts. IGHD3-10 and IGHD3-3 (13.95% each) were the most frequent IGHD genes; IGHD3-3 was more common in Koreans with MM. No IGK rearrangement was particularly prevalent, but single IGKV-J rearrangements were less common in Koreans with kappa-restricted MM than in Western counterparts. IGKV4-1 was less frequent in Koreans regardless of light chain type. Otherwise, the usages of the IGH V, D, and J genes and of the IGK gene were like those observed in previous Western studies.ConclusionNGS-based IGH/IGK clonality testing ought to be applicable to most Koreans with MM. The overrepresentation of IGHV3-9, IGHV4-31, and IGHD3-3 along with the underrepresentation of IGKV4-1 and the differences in IGK gene rearrangement types suggest the existence of ethnicity-specific variations in this disease.

Minimal Residual Disease Detection by Next-Generation Sequencing in Multiple Myeloma: A Comparison With Real-Time Quantitative PCR.

Here we compared clonotype identification by allele-specific oligonucleotide real-time quantitative-PCR (ASO RQ-PCR) and next-generation sequencing (NGS) in 80 multiple myeloma patients. ASO RQ-PCR was applicable in 49/55 (89%) and NGS in 62/78 (80%). Clonotypes identified by both methods were identical in 33/35 (94%). Sensitivity of 10−5 was confirmed in 28/29 (96%) by NGS while sensitivity of RQ-PCR was 10−5 in 7 (24%), 5 × 10−5 in 15 (52%), and 10−4 in 7 (24%). Among 14 samples quantifiable by ASO RQ-PCR, NGS yielded comparable results in 12 (86%). Applicability of NGS can be improved if immunoglobulin heavy-chain incomplete DJ primers are included.

Metagenomic next-generation sequencing technology for detection of pathogens in blood of critically ill patients

ObjectiveTo explore the applicability of metagenomic next-generation sequencing (mNGS) technology for the detection of blood pathogens in intensive care unit patients. MethodsThe clinical data of 63 critically ill patients who could not be diagnosed with blood culture (BC) and who underwent mNGS blood sample testing were retrospectively analyzed. The diagnostic efficacy of mNGS was compared with that of traditional detection methods; the distribution of the pathogens identified by mNGS was analyzed; and the differences in laboratory tests, comorbidities, treatment, and prognosis between the mNGS-positive and mNGS-negative groups were compared. ResultsThe positive rate of mNGS was 41.3% (26/63), and 16 patients were found to have mixed infections. However, the positive rate of BCs performed simultaneously with mNGS was only 7.9% (5/63). The results of univariate analysis showed that the average length of intensive care unit stay (β, −8.689 [95% CI, −16.176, −1.202]; P = 0.026) and the time from onset to sequencing (β, −5.816 [95% CI,−9.936, −1.696]; P = 0.007) of the mNGS-positive group were significantly shorter than those of the mNGS-negative group. More patients in the positive group were adjusted for anti-infective treatment after mNGS (OR, 3.789 [95% CI,1.176, 12.211]; P < 0.001). ConclusionsDetection of blood pathogens by mNGS has good applicability for critically ill patients who cannot be diagnosed by BC in the early stages of infection, and mNGS should be performed as early as possible to obtain higher pathogen detection rates.

Barley chloroplast and mitochondrial genomes diversity evaluation by ngs of the organelle DNA mixtures

In this study, complete chloroplast and mitochondrial genomes of wild ( Hordeum vulgare subsp. spontaneum ) and cultivated ( Hordeum vulgare subsp. vulgare ) barley forms were compared to evaluate the diversity of these genomes within the species. NGS (next generation sequencing) was conducted for mixtures of chloroplast and mitochondrial DNA of 17 barley samples. The chloroplast DNA was obtained from the fraction of these organelles, isolated by differential centrifugation. Such fraction contained an admixture of mitochondrial DNA, which made it possible to conduct sequencing of both genomes simultaneously. The NGS data processing algorithm included some features to prevent mistakes during the alignment of reads from the homology regions within and between the genomes. Five sequences of barley chloroplast genome and two sequences of mitochondrial genome accessible in NCBI GenBank were also involved in the full-genome comparative analysis. Comparison of 22 complete barley chloroplast genome sequences revealed a high level of diversity of these genomes within Hordeum vulgare . There were 107 polymorphic loci: 9 INDELs, 79 SNPs and 19 polymorphisms of SSR-regions (simple sequence repeats). Twenty from 79 found SNPs were located in coding sequences. Analysis of 19 complete sequences of mitochondrial genomes (8 H. vulgare subsp. vulgare and 11 H. vulgare subsp. spontaneum ) showed a lower level of variability of these organelles: 1 INDEL and 22 SNP (4 in coding sequences). Two SNPs were found in one of the genes of the small subunit of ribosomes and two were located in the exon of a pseudogene. The results of the study proved the applicability of NGS of chloroplast and mitochondrial DNA mixtures to the obtaining of complete sequences of these genomes. Revealed polymorphic loci could be used for barley intraspecific identification and phylogeny.

Usefulness and applicability of next generation sequencing in neuroendocrine neoplasms.

626 Background: Neuroendocrine neoplasms (NENs) are rare tumors that can arise anywhere in the body and treatment options are limited due to their rarity. Knowledge of their mutational status might allow for tumor agnostic treatments or aid in enrollment in clinical trials, especially in the metastatic setting. Methods: Eligible NEN patients on an institutional, IRB approved protocol, who had NGS as standard of care and were treated in the past 24 months were included. Tumors were categorized by location and histologic grade. We explored the actual and theoretical eligibility for tumor agnostic treatments and enrollment in clinical trials as available on clinicaltrials.gov. Results: Between August 2017 and July 2019 a total of 107 patients were eligible. Globally 102 clinical trials included patients with NEN and specific mutations.NGS detected one (1%) case of MSI high and one (1) TRK fusion positive tumor, eligible for checkpoint inhibitor and TRK inhibitor therapy respectively. Moreover, NGS identified 16 (15%) cases of MEN1, 1 (1%) of RET, 2 (2%) of NF1 and 3 (2.8%) of MUTYH, 2 (2%) TSC or TSC2, BRCA in 1(1%). These patients were appropriately referred to genetic counseling. About 51.5% of patients would in theory be eligible for an investigational treatment based on mutations and clinical trial availability. Fifty two of 107 patients (48.5%) would not have been eligible for a clinical trial with reasons varying between no mutations (24%), sample failure (8.4%) or nonactionable mutations (15.9%). Conclusions: NGS can point to clinical trial eligibility and guide genetic counseling and should probably be a standard approach in the evaluation of new metastatic NEN patients.

625P - Clinical relevance of circulating tumour (ct)DNA genotyping for first-line cetuximab-based treatment monitoring in metastatic colorectal cancer (mCRC): A prospective multicentric study

BackgroundGenotyping of ctDNA characterizes the molecular profile and monitors tumor molecular dynamics, but the clinical applicability of next generation sequencing (NGS) DNA sequencing has not been assessed in a large prospective cohort of mCRC patients (pts). MethodsmCRC pts with RAS wt tumors treated in first line with chemotherapy (CT) + cetuximab in 16 Spanish hospitals were included. Plasma samples were collected baseline (BL) and every 2 cycles until progression. ctDNA was isolated and sequenced with NGS platform Oncomine Colon cfDNA Assay which identifies hotspot mutations (mut) in AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53 and APC. RAS ctDNA mut were also assessed by dPCR (BEAMing) at BL. Detection of any mut in plasma was considered as ctDNA+. Mut in BRAF/RAS/MAP2K1/PIK3CA were considered as anti-EGFR resistance. Results101 pts were enrolled (65.3% male; median age 64.5y; 77.6% left colon). RAS mut were detected in 11 pts BL by BEAMing. After quality assessment, 84 samples were sequenced by NGS BL and at least one mut was detected in 65 samples (77.3%; median 1, range 0-5) and 10 RAS mut were detected. Plasma samples from 47 pts were also analyzed after 4 cycles of treatment (C4) and mut were detected in 30 pts (63.8%; median 1, range 0-3). Median mutant allele fraction for paired samples was 24.9 at BL and 0.56 at C4 (p=0.0045). Treatment clinical benefit (CB: PR, CR and SD≥16weeks) was observed in 72 out of 85 pts. BL ctDNA+ or C4 ctDNA change were not associated with CB (p=0.722). Assessment of RAS BL mut was not associated with CB (p=0.243 by NGS and p=0.712 by BEAMing), while the absence of RAS mut at C4 predicted treatment CB (93.2% vs. 6.8% RAS mut p=0.02). Median PFS for all pts was 10.13 month. ctDNA+ at BL or C4 were not correlated with PFS (HR 0.8; p=0.73). No differences in PFS were found in ctDNA RAS mut vs RAS wt BL (BEAMing+ HR=1.2 p=0.79 and NGS+ HR=2.6 p=0.052). Among all anti-EGFR resistant mut, only ctDNA RAS mut were detected at C4 (N=4). Persistence or emergence of RAS mut at C4 were strongly associated with PFS (12.9m vs. 4.2. HR 8.8 p=0.0003). ConclusionsEarly RAS ctDNA dynamics predicts benefit to first line CT+cetuximab in mCRC pts. Legal entity responsible for the studyGrupo de Tratamiento de los Tumores Digestivos (TTD), Spain. FundingMerck-Serono. DisclosureJ. Vidal Barrull: Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Amgen; Honoraria (self): Sysmex-Inostics. E. Elez Fernández: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Sanofi. F. Losa: Honoraria (self): Amgen; Honoraria (self): Merck-Serono. R. Salazar: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Guardant-Helth; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Ferrer; Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Novartis; Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: MSD. J. Tabernero: Honoraria (self), Advisory / Consultancy: Array Biopharma; Honoraria (self), Advisory / Consultancy: Merck-Serono; Honoraria (self), Advisory / Consultancy: Molecular Partners; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (institution), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Halio DX SAS. E. Aranda Aguilar: Honoraria (self): Celgene; Honoraria (self): Merck-Serono; Honoraria (self): Roche; Honoraria (self): Sanofi. B. Bellosillo: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Biocartis; Advisory / Consultancy: Novartis; Advisory / Consultancy: Thermofisher. C. Montagut Viladot: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Advisory / Consultancy: Sysmex-Inostics; Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Biocartis; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Guardant-Helth; Advisory / Consultancy: Symphogen. All other authors have declared no conflicts of interest.

Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained fromFFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Unmasking the identity of toxigenic cyanobacteria driving a multi-toxin bloom by high-throughput sequencing of cyanotoxins genes and 16S rRNA metabarcoding

Cyanobacterial harmful algal blooms (CyanoHABs) are complex communities that include coexisting toxic and non-toxic strains only distinguishable by genetic methods. This study shows a water-management oriented use of next generation sequencing (NGS) to specifically pinpoint toxigenic cyanobacteria within a bloom simultaneously containing three of the most widespread cyanotoxins (the hepatotoxins microcystins, MCs; and the neurotoxins anatoxin-a, ATX, and saxitoxins, STXs). The 2013 summer bloom in Rosarito reservoir (Spain) comprised 33 cyanobacterial OTUs based on 16S rRNA metabarcoding, 7 of which accounted for as much as 96.6% of the community. Cyanotoxins and their respective biosynthesis genes were concurrently present throughout the entire bloom event including: MCs and mcyE gene; ATX and anaF gene; and STXs and sxtI gene. NGS applied to amplicons of cyanotoxin-biosynthesis genes unveiled 6 toxigenic OTUs, comprising 3 involved in MCs production (Planktothrix agardhii and 2 Microcystis spp.), 2 in ATX production (Cuspidothrix issatschenkoi and Phormidium/Tychonema spp.) and 1 in STXs production (Aphanizomenon gracile). These toxigenic taxa were also present in 16S rRNA OTUs list and their relative abundance was positively correlated with the respective toxin concentrations. Our results point at MC-producing P. agardhii and ATX-producing C. issatschenkoi as the main contributors to the moderate toxin concentrations observed, and suggest that their distribution in Southern Europe is broader than previously thought. Our findings also stress the need for monitoring low-abundance cyanobacteria (<1% relative abundance) in cyanotoxicity studies, and provide novel data on the presence of picocyanobacteria and potentially ATX-producing benthic taxa (e.g., Phormidium) in deep thermally-stratified water bodies. This study showcases a straightforward use of amplicon metagenomics of cyanotoxin biosynthesis genes in a multi-toxin bloom thus illustrating the broad applicability of NGS for water management in risk-oriented monitoring of CyanoHABs.

NGS-based amplicon sequencing approach; towards a new era in GMO screening and detection

The development and commercialization of Genetically Modified Organisms (GMOs) and its related products have been increasing in the last two decades. This challenges the currently applied time-consuming and expensive qPCR screening procedure from a practical perspective, due to the necessity to develop and validate additional targets at a regular pace and the increasing number of targets included in a single screening. In this study we developed a next generation sequencing (NGS)-based GMO screening approach covering 96 GMO targets and compared it to the two-step qPCR GMO screening approach; the two approaches were evaluated with five feed samples known to contain GMOs. The amplicons obtained from the feed samples were analyzed using 150-bp Paired-End sequencing, Illumina HiSeq 4000 platform. A dedicated data analysis pipeline was developed, which allows automated identification of GMOs and associated genetic elements and constructs. The result of the NGS-based screening were compared with the qPCR approach, indicating that 92% of the targets were commonly identified between the qPCR and NGS-based screening. The remaining 8% of the targets had discrepancies in detection between the two methods. This was mainly observed for targets that were detected in qPCR with high Cq values (above 36), which could not be detected in NGS-based screening. Additionally, due to the more extensive screening in the NGS-based strategy, in total 43 additional GMOs and related targets were identified compared to the standard qPCR screening. From the commonly identified targets in both approaches, 8 targets could not be associated with the detected GMOs. These targets had late Cq values (above 36) and could indicate traces of unknown GMOs in the samples. The current study shows the applicability of NGS as a novel, broad and reliable screening strategy for GMOs and its potential to improve current screening methods.

Evaluation of preimplantation genetic testing for chromosomal structural rearrangement by a commonly used next generation sequencing workflow

ObjectivesTo evaluate the applicability of a commonly used next generation sequencing workflow in detecting unbalanced meiotic segregation products for reciprocal translocation and inversion carriers. Study designAll preimplantation genetic testing treatment cycles performed for reciprocal translocation or inversion carriers from 2012 to April 2017 were included. Three hundreds and forty-two archived whole genome amplified DNA, which had previously analyzed by array comparative genomic hybridization (aCGH), were retrospectively analyzed by next generation sequencing (NGS). Concordance on overall diagnosis and segmental aneuploidies related to the translocation/inversion breakpoints between aCGH and NGS were determined. ResultsRetrospective analysis of 287 blastomere biopsies and 55 trophectoderm (TE) biopsies showed that the concordance rate on the overall diagnosis between aCGH and NGS on abnormal samples was 100% (266/266), irrespective to the type of biopsy. The concordance rates of normal biopsies were 98.4% (61/62) on blastomere and 78.6% (11/14) on TE biopsies. NGS detected a de novo segmental aneuploidy on one blastomere biopsy and three possible low level mosaic aneuploidies on 3 TE biopsies, which were previously concluded as euploid by aCGH. Using the karyotype of reciprocal translocation/inversion carriers, size of anticipated segmental aneuploidies could be calculated and be used to predict the applicability of NGS before proceeding to treatment. ConclusionThis is the first report to evaluate the applicability of a commercial NGS-based workflow for preimplantation testing for reciprocal translocations/inversions. Our study demonstrated that NGS can diagnose unbalanced translocation/inversion products with the same efficiency as aCGH. The applicability of NGS, with respect to individual karyotype, can be predicted before proceeding to treatment.

Evaluation of Quality Assessment Protocols for High Throughput Genome Resequencing Data.

Large-scale initiatives aiming to recover the complete sequence of thousands of human genomes are currently being undertaken worldwide, concurring to the generation of a comprehensive catalog of human genetic variation. The ultimate and most ambitious goal of human population scale genomics is the characterization of the so-called human “variome,” through the identification of causal mutations or haplotypes. Several research institutions worldwide currently use genotyping assays based on Next-Generation Sequencing (NGS) for diagnostics and clinical screenings, and the widespread application of such technologies promises major revolutions in medical science. Bioinformatic analysis of human resequencing data is one of the main factors limiting the effectiveness and general applicability of NGS for clinical studies. The requirement for multiple tools, to be combined in dedicated protocols in order to accommodate different types of data (gene panels, exomes, or whole genomes) and the high variability of the data makes difficult the establishment of a ultimate strategy of general use. While there already exist several studies comparing sensitivity and accuracy of bioinformatic pipelines for the identification of single nucleotide variants from resequencing data, little is known about the impact of quality assessment and reads pre-processing strategies. In this work we discuss major strengths and limitations of the various genome resequencing protocols are currently used in molecular diagnostics and for the discovery of novel disease-causing mutations. By taking advantage of publicly available data we devise and suggest a series of best practices for the pre-processing of the data that consistently improve the outcome of genotyping with minimal impacts on computational costs.