Resistance-associated Variants Research Articles

P670: ABSENCE OF BTK, BCL2, AND PLCG2 MUTATIONS IN RELAPSING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER FIRST-LINE TREATMENT WITH FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V)

Background: CAPTIVATE (PCYC-1142; NCT02910583) is a multicenter phase 2 study of first-line I+V in patients (pts) with CLL in 2 cohorts: Minimal Residual Disease (MRD) and Fixed Duration (FD). We previously reported that fixed-duration I+V provides deep, durable responses (Ghia et al, ASCO 2021; Wierda et al, J Clin Oncol 2021). Clinically-relevant mutations in BTK, PLCG2, and BCL2 have previously been reported in pts with progressive disease (PD) following single agent BTK or BCL2 inhibitor therapies. Such resistance-associated mutations may occur with increasing frequency after prolonged drug exposure. Fixed duration I+V is hypothesized to decrease the likelihood of developing resistance mutations and facilitate extended clinical benefit. Aims: We sought to ascertain presence or absence of resistance mutations at the time of PD in samples from pts treated with fixed-duration I+V in CAPTIVATE. Baseline genomic risk features and potentially prognostic mutations in CLL-related genes of interest were also evaluated in pts with and without subsequent PD. Methods: The analysis was conducted in 191 pts aged ≤70 y with previously untreated CLL who completed fixed-duration I+V, defined as 3 x 28d cycles of I then 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally); this included pts from the FD cohort, and pts from the MRD cohort with confirmed undetectable MRD who received an additional cycle of I+V and were randomized to placebo. Samples from pts at the time of PD were evaluated for resistance mutations by targeted next-generation sequencing (NGS) using a custom-designed high-read-depth 100-gene panel (limit of detection 1% VAF, negative predictive value ≥95% assuming population prevalences <20%, for BTK, PLCG2, or BCL2 variants) in CD19-enriched peripheral blood (PB) or bone marrow. Mutations in additional genes of interest (Table) were evaluated by NGS using the Personalis ACE panel in baseline PB samples. Genomic risk features (Table) were also assessed at baseline. Mutations and genomic risk features evident at baseline were compared across PD and non-PD pts. Results: Baseline mutations in genes of interest and genomic risk features in 190 pts who completed fixed-duration I+V and with available baseline data are shown in the Table. Rates of mutations were similar between pts with (n=30) and without (n=160) subsequent PD. Of 30 pts with PD events (occurring between 16 and 53 mos after start of treatment), 22 (73%) had baseline mutations in genes of interest and 27 (90%) had ≥1 baseline genomic risk feature. Pts with PD generally had more baseline genomic risk features than pts without PD (median 3 vs 2; Wilcoxon P=0.02; analysis restricted to FD cohort pts to avoid bias). To date, 26 of 30 PD patient samples have been evaluated for mutations; no resistance-associated variants in BTK, PLCG2, or BCL2 were detected. Image:Summary/Conclusion: Earlier work has demonstrated a promising safety profile and efficacy of fixed-duration I+V in first-line CLL. Importantly, the current results show that no resistance-associated mutations in BTK, PLCG2, or BCL2 were identified in the pts with PD evaluated to date after first-line, fixed-duration I+V. The absence of such mutations at PD suggests that fixed-duration I+V treatment may offer the possibility of effective subsequent retreatment options with I and/or V and extend clinical benefit.

Evaluation of the Safety and Resistance Associated Variants of Sofosbuvir/Daclatasvir among Egyptian Patients with Hepatitis C Virus: A Prospective Study

Evaluation of the Safety and Resistance Associated Variants of Sofosbuvir/Daclatasvir among Egyptian Patients with Hepatitis C Virus: A Prospective Study

Distribution of Primary Resistance Mutations in Saint Petersburg in Patients with Chronic Hepatitis C.

The advent of direct-acting antiviral drugs (DAAs) was a breakthrough in the treatment of patients with chronic hepatitis C, yet high viral replication errors can lead to the development of resistance associated variants (RAVs). Thus, assessment of RAV in infected patients is necessary to monitor treatment effectiveness. The aim of our study was to investigate the presence of primary resistance mutations in the NS3 and NS5 regions of HCV in treatment-naive patients. Samples were taken from 42 patients with HCV who had not previously received DAA treatment. In the present study, we used the method for determining drug resistance mutations based on direct sequencing of the NS3, NS5A, and NS5B genes developed by the Saint Petersburg Pasteur Institute. Primary mutations associated with resistance were detected in 5 patients (12%). According to the Geno2pheno [hcv] 0.92 database, nucleotide substitutions were identified in various viral genes conferring resistance or decreased sensitivity to the respective inhibitors. This study has shown different mutations in the analyzed genes in patients with HCV who had not previously received DAA treatment. These mutations may increase the likelihood of treatment failure in the future.

Acquired bedaquiline resistance during the treatment of drug-resistant tuberculosis: a systematic review.

BackgroundDrug-resistant tuberculosis (DR-TB) is considered to be a public health threat and is difficult to cure, requiring a lengthy treatment with potent, potentially toxic drugs. The novel antimicrobial agent bedaquiline has shown promising results for patients with DR-TB, improving the rate of culture conversion and reducing TB-related mortality. However, increasing numbers of cases with acquired bedaquiline resistance (ABR) have been reported in recent years.MethodsThis systematic review aimed to assess the frequency of ABR and characteristics of patients acquiring it. Studies showing data on sequential bedaquiline drug-susceptibility testing in patients treated with a bedaquiline-containing regimen were included. The databases CENTRAL, PubMed and Embase were manually searched, and 866 unique records identified, eventually leading to the inclusion of 13 studies. Phenotypic ABR was assessed based on predefined MIC thresholds and genotypic ABR based on the emergence of resistance-associated variants.ResultsThe median (IQR) frequency of phenotypic ABR was 2.2% (1.1%–4.6%) and 4.4% (1.8%–5.8%) for genotypic ABR. Among the studies reporting individual data of patients with ABR, the median number of likely effective drugs in a treatment regimen was five, in accordance with WHO recommendations. In regard to the utilization of important companion drugs with high and early bactericidal activity, linezolid was included in the regimen of most ABR patients, whereas the usage of other group A (fluoroquinolones) and former group B drugs (second-line injectable drugs) was rare.ConclusionsOur findings suggest a relevant frequency of ABR, urging for a better protection against it. Therefore, treatment regimens should include drugs with high resistance-preventing capacity through high and early bactericidal activity.

Emergence of resistance against direct acting antivirals in chronic HCV patients: A real-world study

Interferon/Ribavirin therapy has been replaced by Direct Acting Antivirals (DAAs) due to emergence of Resistance Associated Variants (RAVs) and decrease Sustain Virologic Response (SVR). Current study investigated treatment response of Sofosbuvir and Ribavirin in chronic HCV patients. Total 256 HCV patients with genotype 1a, 2 and 3a received sofosbuvir/ribavirin according to international standards. HCV RNA presence in serum was used as marker for end treatment response (ETR) and sustain virologic response after 24 weeks of treatment (SVR24) in each case. Response to treatment with SOF + RBV was found statistically significant among different HCV genotypes (GT) as out of 47 HCV GT1 patients 42(89.36%) resulted into good ETR but 4(9.52%) of these relapsed and 5(10.63%) led into virologic failure. 5(100%) HCV GT2 patients resulted into SVR24 whereas, out of 204 HCV GT3 patients 194(95.69%) achieved good ETR however, 8(4.12%) of these relapsed and 10(4.90%) resulted in to virologic failure. Efficacy of therapy was found non-significant in treatment naïve and treatment experienced patients as in this study out of 145 treatment naïve patients 139(95.86%) achieved good ETR where 4(2.87%) relapsed while 6(4.13%) led into virologic break through on the other hand among 111 treatment experienced patients 102(91.89%) resulted into good ETR but 8(7.84%) relapsed whereas 9(8.10%) lead into virologic failure. Current study also propose that various liver and spleen complications/liver cirrhosis are related to response of HCV patients to SOF + RBV therapy whereas, variables like old age, gender is not compromising treatment response to DAAs therapy. Various mild side effects encountered by patients during treatment were fatigue, insomnia, headache, nausea, burning body, diarrhea, cough. Overall, this study reported 89.45% efficacy of SOF + RBV regime in chronic HCV Pakistani patients. Current study suggests hunting for possible reasons of resistance so that SOF + RBV therapy may not share the same fortune as previous therapies in near future.

Frequency distribution of HCV resistance-associated variants in infected patients treated with direct-acting antivirals

Hepatitis C virus (HCV) is a global public health problem. Second-generation direct-acting antivirals targeting non-structural regions on the viral genome are the cornerstone for treatment of chronic infection. However, resistance-associated variants (RAVs) have been reported to be associated with therapeutic failure. The aim of this study was to assess the frequency of variants, including RAVs, in the NS3, NS5A and NS5B regions at baseline in Brazilian patients with chronic hepatitis C with HCV genotypes 1a, 1b and 3a. Serum samples from 13 patients were used to obtain viral RNA. Massively parallel sequencing was performed using genotype-specific amplicons and a panel of Ampliseq technology for all genotypes. Several non-synonymous substitutions were detected at baseline for 11 responders and pre-/post-treatment for two non-responders. HCV genotype 3a was found to have significantly more non-synonymous substitutions than HCV genotype 1 in the NS3 and NS5A regions. Analyses were conducted using quantitative and qualitative inter- and intrapatient comparisons. Variants that confer resistance to the treatment used by the patients were found in both responders and non-responders. A wide frequency distribution of RAVs was found at baseline, and this did not interfere with the achievement of a sustained response. Evaluation of the presence of RAVs requires additional study in order to determine clinical relevance.

Cost-Effectiveness of Testing for NS5A Resistance to Optimize Treatment of Elbasvir/Grazoprevir for Chronic Hepatitis C in China.

Objectives: Baseline presence of nonstructural protein 5A (NS5A) resistance-associated variants can attenuate the efficacy of new direct-acting antivirals. A potential method to attain the higher efficacy would be to screen for NS5A polymorphisms prior to the initiation of therapy and to adjust the treatment length based on the test results. However, baseline testing adds additional costs and it is unclear whether this would represent a high value strategy for chronic hepatitis C in China. Methods: A hybrid model compared 1) standard 12-weeks treatment (no testing), 2) shortened 8-weeks treatment (no testing), and 3) baseline testing with 12-/8-weeks treatment for those with/without NS5A polymorphisms from a lifetime Chinese health care payer perspective. All model inputs were retrieved from clinical trials and publically available literature. And sensitivity analyses were also conducted to assess the impact of uncertainty. Results: Baseline testing was associated with overall increase in total health care cost of USD 13.50 and in QALYs of 0.002 compared with standard 12-weeks treatment (no testing), yielded in an ICER of USD 6750/QALY gained. Scenario analyses suggested that shortened 8-weeks treatment (no testing) was found to be lower costs and great QALYs compared with other two strategies when the sustained virologic response (SVR) rate increased to 95%. Sensitivity analyses indicated that the results were robust. Conclusions: Our results suggest prior assessment of NS5A sensitivity followed by optimizing treatment duration was an economic strategy. In addition, shortened 8-weeks treatment (no testing) was shown to be dominant with the SVR rate increased to 95%.

MiR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

Viral hepatitis update: Progress and perspectives.

Viral hepatitis, secondary to infection with hepatitis A, B, C, D, and E viruses, are a major public health problem and an important cause of morbidity and mortality. Despite the huge medical advances achieved in recent years, there are still points of conflict concerning the pathogenesis, immune response, development of new and more effective vaccines, therapies, and treatment. This review focuses on the most important research topics that deal with issues that are currently being solved, those that remain to be solved, and future research directions. For hepatitis A virus we will address epidemiology, molecular surveillance, new susceptible populations as well as environmental and food detections. In the case of hepatitis B virus, we will discuss host factors related to disease, diagnosis, therapy, and vaccine improvement. On hepatitis C virus, we will focus on pathogenesis, immune response, direct action antivirals treatment in the context of solid organ transplantation, issues related to hepatocellular carcinoma development, direct action antivirals resistance due to selection of resistance-associated variants, and vaccination. Regarding hepatitis D virus, we describe diagnostic methodology, pathogenesis, and therapy. Finally, for hepatitis E virus, we will address epidemiology (including new emerging species), diagnosis, clinical aspects, treatment, the development of a vaccine, and environmental surveillance.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System for Detecting Gene Fusions in Hematologic Malignancies

Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest with a high resolution and throughput. However, RNA-seq has not been widely performed in clinical molecular laboratories because of the complexity of data processing and interpretation. We developed and validated a customized RNA-seq panel and data processing protocol for fusion detection using 4 analytical validation samples and 51 clinical samples, covering seven types of hematologic malignancies. Analytical validation showed that the results for target gene coverage and between- and within-run precision and linearity tests were reliable. Using clinical samples, RNA-seq based on filtering and prioritization strategies detected all 25 known fusions previously found by multiplex reverse transcriptase-PCR and fluorescence in situ hybridization. It also detected nine novel fusions. Known fusions detected by RNA-seq included two IGH rearrangements supported by expression analysis. Novel fusions included six that targeted just one partner gene. In addition, 18 disease- and drug resistance-associated transcript variants in ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were designated simultaneously. Expression analysis showed distinct clustering according to subtype and lineage. In conclusion, this study showed that our customized RNA-seq system had a reliable and stable performance for fusion detection, with enhanced diagnostic yield for hematologic malignancies in a clinical diagnostic setting.

Factors influencing the failure of interferon-free therapy for chronic hepatitis C: Data from the Polish EpiTer-2 cohort study.

BACKGROUNDThe introduction of direct-acting antiviral drugs into clinical practice has revolutionized the treatment of chronic hepatitis C, making it highly effective and safe for patients. However, few researchers have analyzed the factors causing therapy failure in some patients.AIMTo analyze factors influencing the failure of direct antiviral drugs in the large, multicenter EpiTer-2 cohort in a real-world setting.METHODSThe study cohort consisted of patients with chronic hepatitis C treated at 22 Polish centers from 2016-2020. Data collected from the online EpiTer-2 database included the following: hepatitis C virus (HCV) genotype, stage of fibrosis, hematology and liver function parameters, Child-Turcotte-Pugh and Model for End-stage Liver Disease scores, prior antiviral therapy, concomitant diseases, and drugs used in relation to hepatitis B virus (HBV) and/or human immunodeficiency virus (HIV) coinfections. Adverse events observed during the treatment and follow-up period were reported. Both standard and machine learning methods were used for statistical analysis.RESULTSDuring analysis, 12614 patients with chronic hepatitis C were registered, of which 11938 (mean age: 52 years) had available sustained virologic response (SVR) data [11629 (97%) achieved SVR and 309 (3%) did not]. Most patients (78.1%) were infected with HCV genotype 1b. Liver cirrhosis was diagnosed in 2974 patients, while advanced fibrosis (F3) was diagnosed in 1717 patients. We included patients with features of hepatic failure at baseline [ascites in 142 (1.2%) and encephalopathy in 68 (0.6%) patients]. The most important host factors negatively influencing treatment efficacy were liver cirrhosis, clinical and laboratory features of liver failure, history of hepatocellular carcinoma, and higher body mass index. Among viral factors, genotype 3 and viral load also exerted an influence on treatment efficacy. Classical statistical analysis revealed that treatment ineffectiveness seemed to be influenced by the male sex, which was not confirmed by the multivariate analysis using the machine learning algorithm (random forest). Coinfection with HBV (including patients with on-treatment reactivation of HBV infection) or HIV, extrahepatic manifestations, and renal failure did not significantly affect the treatment efficacy.CONCLUSIONIn patients with advanced liver disease, individualized therapy (testing for resistance-associated variants and response-guided treatment) should be considered to maximize the chance of achieving SVR.

Dynamic changes of HCV genomes under selective pressure from DAAs therapy in relapsed patients

Currently, direct-acting antiviral drugs (DAAs) are widely used as therapeutic methods for hepatitis C virus (HCV)-positive patients, however, patients may experience treatment failure, and the dynamic changes of HCV genomes in these patients are unknown. In this study, three real-world DAAs cohorts were enrolled to observe clinical efficacy. In addition, serum samples from treatment failure patients at baseline and relapse were used to analyze changes of the HCV genomes at near full-length genome level, including resistance-associated variants (RAVs), viral quasispecies diversity and selection analysis. Next-generation sequencing was used as the detection method. The overall sustained virological response at 12 w after the end of treatment was achieved in 91.9% (57/62) of HCV patients, and 3 paired samples obtained from relapsed patients. All the 3 patients harbored baseline NS5A RAVs, the frequency of NS5A RAVs increased in 2 patients and a new NS5A RAV emerged in 1 patient at relapse, and almost all the viral strains existed with NS5A RAVs at relapse. The results of the viral quasispecies diversity analysis revealed that viral quasispecies diversity decreased at relapse compared to baseline, and the results of selection analysis indicated that the virus population experienced a bottleneck phenomenon, recent selective sweep and population expansion or was under purification selection after DAAs treatment. This study indicated that the clinical efficacy was excellent in real-world DAAs cohorts, and the viral strains existed at relapse were selective by DAAs therapy.

Comparative Analysis of Within-Host Mutation Patterns and Diversity of Hepatitis C Virus Subtypes 1a, 1b, and 3a.

Understanding within-host evolution is critical for predicting viral evolutionary outcomes, yet such studies are currently lacking due to difficulty involving human subjects. Hepatitis C virus (HCV) is an RNA virus with high mutation rates. Its complex evolutionary dynamics and extensive genetic diversity are demonstrated in over 67 known subtypes. In this study, we analyzed within-host mutation frequency patterns of three HCV subtypes, using a large number of samples obtained from treatment-naïve participants by next-generation sequencing. We report that overall mutation frequency patterns are similar among subtypes, yet subtype 3a consistently had lower mutation frequencies and nucleotide diversity, while subtype 1a had the highest. We found that about 50% of genomic sites are highly conserved across subtypes, which are likely under strong purifying selection. We also compared within-host and between-host selective pressures, which revealed that Hyper Variable Region 1 within hosts was under positive selection, but was under slightly negative selection between hosts, which indicates that many mutations created within hosts are removed during the transmission bottleneck. Examining the natural prevalence of known resistance-associated variants showed their consistent existence in the treatment-naïve participants. These results provide insights into the differences and similarities among HCV subtypes that may be used to develop and improve HCV therapies.

Real-World Outcomes of Direct-Acting Antiviral Treatment and Retreatment in United Kingdom-Based Patients Infected With Hepatitis C Virus Genotypes/Subtypes Endemic in Africa.

BackgroundChronic hepatitis C virus (HCV) infection affects 71 million individuals, mostly residing in low- and middle-income countries (LMICs). Direct-acting antivirals (DAAs) give high rates of sustained virological response (SVR) in high-income countries where a restricted range of HCV genotypes/subtypes circulate.MethodsWe studied United Kingdom–resident patients born in Africa to examine DAA effectiveness in LMICs where there is far greater breadth of HCV genotypes/subtypes. Viral genome sequences were determined from 233 patients.ResultsFull-length viral genomic sequences for 26 known subtypes and 5 previously unidentified isolates covering 5 HCV genotypes were determined. From 149 patients who received DAA treatment/retreatment, the overall SVR was 93%. Treatment failure was associated primarily with 2 subtypes, gt1l and gt4r, using sofosbuvir/ledipasvir. These subtypes contain natural resistance-associated variants that likely contribute to poor efficacy with this drug combination. Treatment failure was also significantly associated with hepatocellular carcinoma.ConclusionsDAA combinations give high SVR rates despite the high HCV diversity across the African continent except for subtypes gt1l and gt4r, which respond poorly to sofosbuvir/ledipasvir. These subtypes are widely distributed across Western, Central, and Eastern Africa. Thus, in circumstances where accurate genotyping is absent, ledipasvir and its generic compounds should not be considered as a recommended treatment option.

High frequency of bedaquiline resistance in programmatically treated drug-resistant TB patients with sustained culture-positivity in Cape Town, South Africa

Aims &amp; objectives: Potentially transformative new tuberculosis (TB) drugs like bedaquiline are undergoing roll-out, however, this is largely in the absence of programmatic drug susceptibility testing (DST). We lack information on how susceptibility changes during treatment in patients on bedaquiline-containing regimens, especially in at risk patients who have complex treatment histories, are in programmatic rather than trial environments, and have a delayed treatment response (defined here as sustained culture-positivity). Methods: Serial isolates from 51 patients with drug resistant (DR-)TB who were culture-positive after ≥4 months of a programmatically administered bedaquiline-containing regimen, were collected. Bedaquiline phenotypic DST in MGIT 960 (1μg/ml), targeted deep sequencing (Rv0678, atpE, pepQ) and whole genome sequencing was done on paired isolates (pre-bedaquiline initiation, post-four-month). Results: 24/51 (47%) patients were phenotypically and genotypically resistant (39% acquired resistance). Excluding one patient with an unknown history, prior clofazimine exposure was associated with bedaquiline-resistance [21/24 (88%) bedaquiline resistant cases had prior clofazimine vs. 12/26 (46%) susceptibles; p=0.002]. Diverse combinations of single nucleotide polymorphisms (SNPs) and indels were in the Rv0678 promoter region and the Rv0678 and atpE genes. Examples of newly described resistance associated variants (RAVs) include Rv0678 -8 T/G and atpE 223 C/T. RAVs were not in defined hotspots and sometimes occurred concurrently with atpE RAVs. Discussion: The rate of bedaquiline resistance acquisition in this population is alarmingly high and associated with prior clofazimine exposure. The diverse RAVs pose challenges to molecular test development. This study highlights the existence of a potentially infectious pool of bedaquiline-resistant patients present under programmatic conditions in a resource-constrained setting and illustrates the danger of starting patients with complex histories on a novel drug without routinely available DST.

Robust detection of point mutations involved in multidrug-resistant Mycobacterium tuberculosis in the presence of co-occurrent resistance markers.

Tuberculosis disease is a major global public health concern and the growing prevalence of drug-resistant Mycobacterium tuberculosis is making disease control more difficult. However, the increasing application of whole-genome sequencing as a diagnostic tool is leading to the profiling of drug resistance to inform clinical practice and treatment decision making. Computational approaches for identifying established and novel resistance-conferring mutations in genomic data include genome-wide association study (GWAS) methodologies, tests for convergent evolution and machine learning techniques. These methods may be confounded by extensive co-occurrent resistance, where statistical models for a drug include unrelated mutations known to be causing resistance to other drugs. Here, we introduce a novel ‘cannibalistic’ elimination algorithm (“Hungry, Hungry SNPos”) that attempts to remove these co-occurrent resistant variants. Using an M. tuberculosis genomic dataset for the virulent Beijing strain-type (n = 3,574) with phenotypic resistance data across five drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin), we demonstrate that this new approach is considerably more robust than traditional methods and detects resistance-associated variants too rare to be likely picked up by correlation-based techniques like GWAS.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System As a Comprehensive Assay for Hematologic Malignancies

Introduction Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest and can overcome limitations regarding resolution, throughput, and multistep workflow. However, RNA-seq has not been widely performed in clinical molecular laboratories due to the complexity of data processing and interpretation. We developed a customized targeted RNA-seq panel with a data processing protocol and validated its analytical performance for gene fusion detection using a subset of samples with different hematologic malignancies. Additionally, we investigated its applicability for identifying transcript variants and expression analysis using the targeted panel. Methods The target panel and customized oligonucleotide probes were designed to capture 84 genes associated with hematologic malignancies. Libraries were prepared from 800 to 1,500 ng of total RNA using GeneMediKit NGS-Leukemia-RNA kit (GeneMedica, Gwangju, Korea) and sequenced using Miseq reagent kit v3 (300 cycles) and MiseqDx (Illumina, San Diego, CA, USA). The diagnostic samples included one reference DNA (NA12878), one reference RNA (Cat no. 740000, Agilent Technologies), 14 normal peripheral blood (PB) samples, four validation bone marrow (BM) samples with known gene fusions, and 30 clinical BM or PB samples from seven categories of hematologic malignancies. The clinical samples included 27 BM aspirates and three PB samples composed of six acute myeloid leukemia, nine B-lymphoblastic leukemia/lymphoma, four T-lymphoblastic leukemia/lymphoma, three mature B-cell neoplasms, six MPN, one myelodysplastic/myeloproliferative neoplasm, and one myeloid/lymphoid neoplasm with eosinophilia and gene rearrangement. For the analytical validation of fusion detection, target gene coverage, between-run and within-run repeatability, and dilution tests (1:2 to 1:8 dilution) were performed. For the comparative analysis of fusion detection, the RNA-seq data were analyzed by STAR-Fusion and FusionCatcher and processed with stepwise filtering and prioritization strategy (Figure 1), and the result was compared to those of multiplex RT-PCR (HemaVision kit; DNA Technology, Aarhus, Denmark) or FISH (MetaSystems Gmbh, Althusseim, Germany) using 30 clinical samples. The RNA-seq data from clinical samples were additionally analyzed by FreeBayes for variant detection and by StringTie for expression profiling (Figure 1). Results First, the analytical validation showed reliable results in target gene coverage, between-run and within-run repeatability, and linearity tests. The uniformity of coverage (% of base pairs higher than 0.2 × total average depth) was calculated to be 99.8%, which revealed even coverage for the target genes in the panel using the reference DNA. Both in the within-run and between-run tests, the read counts and FFPM (fusion fragments per million) of all replicates showed reliable repeatability (r2 = 0.9655 and 0.9874, respectively). The FFPM of the diluted analytical samples including BCR-ABL1 and PML-RARA showed linear log2-fold-changes (r2 = 0.9852 and 0.9447, respectively). Second, compared to multiplex RT-PCR and FISH using 30 clinical samples, targeted RNA-seq combined with filtering and prioritization strategies detected all 13 known fusions and newly detected 17 fusions. Finally, 16 disease- and drug resistance-associated variants on the expressed transcripts of ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were simultaneously designated and expression analysis showed distinct four clusters of clinical samples according to the cancer subtypes and lineages. Conclusions Our customized targeted RNA-seq system provided a stable analytical performance and a more sensitive identification of gene fusions than conventional molecular methods in various clinical samples. In addition, clinically significant variants in the transcripts and expression profiling could be simultaneously identified directly from the RNA-seq data without the need for additional parallel testing. Our study identified the advantages of the clinical laboratory-oriented targeted RNA-seq system to enhance the diagnostic yield for gene fusion detection and to simplify the diagnostic steps as providing a comprehensive tool for analyzing hematologic malignancies in the clinical laboratory. Figure 1 Disclosures Lee: National Research Foundation of Korea: Research Funding.

The incidence of resistance-associated variants to NS5A in HCV subtypes 1a and 1b in Taiwan

BackgroundResistance-associated variants (RAVs) to direct-antiviral agents (DAAs) may hamper treatment. There was a lack of data on the natural prevalence of RAVs in Taiwanese HCV-infected patients. We investigated the real-life presence of RAVs in the nonstructural 5A (NS5A) region in HCV genotype 1a and 1b in chronically infected individuals in Taiwan. MethodsIn this single-center cohort study, nested polymerase chain reaction and direct sequencing analysis was used to determine the frequency of RAVs in the HCV NS5A region in patients with HCV genotype 1a (n = 55) and 1b (n = 525). ResultsIn genotype 1a strains, the incidence of RAVs was 16.4% (9/55) in the NS5A region (M28V/T, n = 6, 10.9%; Q30L, n = 1, 1.8%; Y93N/H, n = 3, 5.5%). In genotype 1b, the incidence of RAVs was 17.5% (92/525) in the NS5A region (L31I/M/V, n = 7, 1.3%; Y93 H/S, n = 87, 16.5%). Patients with RAVs had significantly higher HCV RNA levels (6.1 ± 0.7 vs 5.9 ± 0.8 log IU/mL, p = 0.001) and lower rGT levels (28.9 ± 18.9 vs. 42.9 ± 57.0 U/L, p = 0.001) compared to those without RAVs. Multivariate analysis identified HCV RNA levels (odds ratio = 1.145, 95% CI: 1.060–1.237, p = 0.001) and rGT (OR = 0.989, 95% CI: 0.978–0.999, p = 0.035) as risk factors that are associated with the presence of RAVs. Importantly, there is no association between the presence of RAVs and no SVR (3.8% in patients with RAVs, 15.9% in patients without RAVs, p = 0.32). ConclusionRAVs, especially M28V and Y93H in the NS5A region, were highly prevalent in patients with genotype 1a and 1b HCV, respectively, in Taiwan, and they were linked to high HCV RNA levels and low rGT levels. Before using the NS5A inhibitors, the presence of mutated HCV variants should be taken into consideration.

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis

SummaryBackgroundBedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa.MethodsIn this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates.FindingsWe sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12–0·5 μg/mL, and in isolates with RAVs from 0·25–4·0 μg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance.InterpretationBedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge.FundingWellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health.

THU411 - Resistance-associated variants conferring quite a high level of resistance to all approved NS5A inhibitors in genotype 2 hepatitis C virus

THU411 - Resistance-associated variants conferring quite a high level of resistance to all approved NS5A inhibitors in genotype 2 hepatitis C virus