Using Unique Molecular Identifiers Research Articles

Combining PET/CT and Ctdna Assessments at 6 Months from Induction Treatment Better Predicts Outcome in Previously Untreated Patients with Follicular Lymphoma: A Relevance Ancillary Lysa Study

Background Most patients with follicular lymphoma (FL) have long-term disease control resulting in a prolonged overall survival (OS), beyond 20 years. However, approximately 20% of patients experience disease progression within two years after treatment initiation (POD24) which is associated with a shorter OS but early prediction of POD24 remains challenging. Positron emission tomography computed tomography (PET/CT) positivity after induction therapy has been associated with shortened progression-free survival (PFS). Also, minimal residual disease (MRD) positivity in peripheral blood using BCL2-JH translocation predicts outcome but barely 50% of patients are informative with this tumor marker. Circulating tumor DNA (ctDNA) may increase the proportion of MRD informative patients up to 80%. Our objective was to evaluate the predictive value of ctDNA for PFS and POD24, either alone or coupled with PET/CT response in first-line patients enrolled in the RELEVANCE trial. Methods This ancillary study was carried out on patients included by French LYSA centers in RELEVANCE, a randomized phase III trial comparing the chemotherapy-free regimen R2 versus standard R-chemo followed by Rituximab maintenance in previously untreated patients with FL. All patients who had a PET/CT at diagnosis and at week 24 (W24, end of induction treatment) and for whom serum was stored at these 2 time points were eligible. Cell-free DNA (cfDNA) was extracted from a median of 2.8 mL of serum (range 0.35 to 4.1). Libraries were generated from 129kb captured-DNA custom panel (Agilent, XTHS2). Variant calling and phased variant annotation were performed using Unique Molecular Identifiers (UMI). Libraries were sequenced with a median coverage of 3257 UMI. Concentrations of ctDNA were calculated from TapeStation migration system (Agilent) using 50 bp to 600 bp limits and expressed as log hGE/mL. To ensure maximum specificity in a context of non-optimal serum samples, only patients with phased variants in ctDNA at diagnosis were studied for MRD at W24. Total metabolic tumor volume (TMTV) was computed using the 41% SUVmax method and PET/CT response assessed with Lugano criteria. Results As PET/CT at W24 was optional in RELEVANCE, only 141 out of 581 patients enrolled were eligible. Compared to the overall RELEVANCE population, baseline characteristics were similar except for FLIPI (7.1% FLIPI 0-1 versus 15.9% respectively, p=0.013) and PFS event (29.3% versus 39.5% respectively, p=0.035). At time of diagnosis, with a median of 81 variants per patient (range 1 to 659), a mutational profile could be identified from cfDNA in 99% (140/141) of patients. A significant correlation was found between ctDNA load (median 2.39 log hGE/mL (range 1.01 to 4.20) and TMTV (median 298 mL, range 5 to 3100) (p=0.023). Phased variants were detected in 124 patients (87.9%) at diagnosis and were used for MRD analyses. At W24, ctDNA remained detectable (ctDNA+) in 15 patients, 9/69 (13%) in the R2 arm and 6/55 (11%) in the R-chemo arm (p=0.787). PET/CT remained positive (Deauville 4 and 5) in 20 patients, 11/72 (15.2%) in the R2 arm and 9/56 (16.1%) in the R-chemo arm. With a median follow-up of 6.4 years, ctDNA+ patients exhibited a median PFS of 37 months versus not reached for ctDNA- patients, p=0.0096. Similarly, PET/CT+ patients had a median PFS of 34 months versus not reached for PET/CT- patients (p<0.001). When adjusting on FLIPI in a multivariate Cox model, ctDNA remains statistically significant (HR=2.63 [1.20; 5.77], p=0.0163). Among PET/CT+ patients, 13/20 (65%) patients showed discrepant PET/CT and ctDNA responses and double positive patients (7/20, 35%) presented a shorter median PFS of 8.6 months (versus not reached, p=0.0147; Figure 1). Regarding POD24, negative predictive value (NPV) and positive predictive value (PPV) were respectively 91.7% and 46.7% for ctDNA MRD and 93.3% and 45.0% for PET/CT. When combined, PET+/MRD+ identified POD24 patients with a NPV and PPV of 91.5% and 85.7% respectively. Conclusion Combining the results of ctDNA and PET/CT at W24 post induction improves early prediction of POD24 in previously untreated patients with follicular lymphoma.

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization.

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediatedLong-readSequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.

EARLY CTDNA CLEARANCE AFTER CAR T‐CELL INFUSION PREDICTS OUTCOME IN PATIENTS WITH LARGE B‐CELL LYMPHOMA : RESULTS FROM ALYCANTE, A PHASE 2 LYSA STUDY

Introduction: CAR T-cells have significantly improved the outcome of patients with relapsed or refractory (R/R) large B-cell lymphoma (LBCL). However, more than half of the patients fail to obtain a prolonged remission, resulting in a poor prognosis. Early identification of these patients would allow early intervention to improve their outcome. Circulating tumor DNA (ctDNA) assessment is a promising tool to monitor early response in LBCL, but few data are available regarding ctDNA monitoring after treatment with CAR T-cells. The aim of our study was to monitor ctDNA before and after CAR T-cell infusion and correlate the results with clinical outcome in the ALYCANTE trial. Methods: Patients’ samples were prospectively collected in the ALYCANTE trial, an open-label, phase 2 study (NCT04531046), designed to evaluate the efficacy and safety of axi-cel in 62 patients with R/R LBCL not intended for autologous stem cell transplantation after 1 prior line of therapy. DNA was extracted from 2 to 4 mL of plasma. Libraries were generated from 129 kb captured-DNA custom panel (Agilent, XTHS2). Variant calling and phased variant annotation were performed using Unique Molecular Identifiers (UMI). Concentrations of ctDNA were expressed as hGE/mL and were measured at leukapheresis, after bridging/before lymphodepletion, at time of CAR T-cell infusion (D0) and at day 14 (D14), month 1 (M1), 3, 6, 9, and 12 after infusion. These results were correlated with clinical outcome. Results: With a median of 85 variants (range 1–707), a mutational profile could be identified from ctDNA in 88% (55/62) of patients at time of leukapheresis (n = 54) or after bridging (n = 1). The median ctDNA load was 77 hGE/mL (range 0–6803) at leukapheresis and 39 hGE/mL (range 2–17,354) after bridging therapy. Only 2/55 (4%) patients experienced ctDNA clearance after bridging. To date, we report the clinicobiological correlations for the first 40 patients included in the ALYCANTE trial. Early ctDNA evaluation was performed in 38/40 (95%) patients: at D14 and M1 (n = 34), at D14 only (n = 2), or at M1 only (n = 2). For patients with D14 and M1 evaluation, the results were concordant in 32/34 (94%) patients, either both positive (n = 19) or both negative (n = 13). A negative ctDNA at D14 (n = 14/36, 38.9%) or at M1 (n = 16/36, 44.4%) was associated with a complete metabolic response at 3 months in 93% (p = 0.03) and 94% (p = 0.009) of cases, respectively. After a median follow-up of 10 months, the median progression-free survival was not reached in patients with negative ctDNA at D14 and M1 versus 7.6 months and 5.8 months for patients with positive ctDNA at D14 and M1, respectively (Figure 1). Encore Abstract—previously submitted to EHA 2023 The research was funded by: Kite-Gilead, as a prespecified exploratory analysis Keywords: cellular therapies, liquid biopsy, minimal residual disease Conflicts of interests pertinent to the abstract M. Delfau-Larue Other remuneration: Education, symposia: TAKEDA, AMGEN, ROCHE, GILEAD, ABBVIE G. Cartron Consultant or advisory role: Advisory Board: Ownards Therapeutics, MAbQi. Consultancy: Roche, BMS, Abbvie. Honoraria: Jansen, Gilead, Takeda, Novartis, Milteny. F. Morschhauser Consultant or advisory role: Roche, Gilead, Genmab, Novartis, Abbvie Honoraria: Chugai (scientific lectures) L. Oberic Consultant or advisory role: Roche, Kite Gilead, Incyte Honoraria: Speaking: Roche, Kite Gilead, AstraZeneca, Jansen, BMS E. Bachy Consultant or advisory role: Kite, a Gilead Company, Bristol Myers Squibb, Novartis, Pfizer; Honoraria: Kite, a Gilead Company, Bristol Myers Squibb, Novartis, Pfizer, Incyte, ADC Therapeutics Research funding: Amgen, BMS R. Dulery Honoraria: Takeda, Novartis, and Biotest C. Laurent Educational grants: Janssen R. Houot Consultant or advisory role: Kite/Gilead, Novartis, Bristol-Myers Squibb/Celgene, ADC Therapeutics, Incyte and Miltenyi. Honoraria: Kite/Gilead, Novartis, Incyte, Janssen, MSD, Takeda and Roche

P037: High breadth whole exome sequencing of circulating tumor DNA identifies novel recurrent genetic alterations in Hodgkin lymphoma

Hodgkin lymphoma (HL) is a B-cell lymphoma with a generally favorable prognosis. However, side effects of aggressive front-line treatment and difficult-to-treat relapsed/refractory disease are still a clinical challenge. A better understanding of the biological diversity of HL at baseline might help to further individualize the treatment of patients and thus ultimately improve outcome. A broad characterization of the genetic drivers of HL is highly important to improve our understanding of the biological diversity of HL. Here, we present the results of our study using whole-exome sequencing of circulating tumor (ct)DNA to broadly characterize the HL oncogenome in 165 patients. After extraction of germline and cell-free DNA, WES was performed, targeting 37 Mb of the human genome. An in-house customized bioinformatic pipeline including twofold error reduction using unique molecular identifiers (UMIs) and digital error suppression was used as previously described by us (Sobesky et. al., Med, 2021). The median ctDNA concentration per patient was 2.26 log haploid genome equivalents per milliliter (log hGE/ml) (range: 1.41–4.18 log hGE/ml) without significant correlation with clinical parameters such as sex, international prognostic score (IPS) or clinical stage. In addition to the detection of several genetic aberrations involved in the pathogenesis of HL (such as B2M, CSF2RB, GNA13, SOCS1), we were able to detect several novel, recurrently mutated potential oncogenes and tumor suppressor genes. Among the most frequently mutated genes not previously described in HL were olfactory receptor family 10 subfamily G member 7 (OR10G7) (11.5%), Apolipoprotein B (APOB) (10.3%) and Filaggrin 2 (FLG2) (9.7%). Furthermore, several previously undescribed copy number variations (CNV) were detected (losses: 13q13.1, 1p36.13a, 6p21.33; gains: 19q13.42, 9p24, 12q12). To decipher the detected genes’ and CNVs’ contribution to the biological diversity of HL, we performed non-negative matrix factorization. Furthermore, neoantigen load was assessed through human-leukocyte antigen (HLA) sequencing. Detailed results will be presented at the meeting. In conclusion, our ctDNA based WES approach provides sufficient sensitivity to both perform genotyping in HL and detecting novel genomic aberrations potentially involved in the pathogenesis of HL.

UMIErrorCorrect and UMIAnalyzer: Software for Consensus Read Generation, Error Correction, and Visualization Using Unique Molecular Identifiers.

Targeted sequencing using unique molecular identifiers (UMIs) enables detection of rare variant alleles in challenging applications, such as cell-free DNA analysis from liquid biopsies. Standard bioinformatics pipelines for data processing and variant calling are not adapted for deep-sequencing data containing UMIs, are inflexible, and require multistep workflows or dedicated computing resources. We developed a bioinformatics pipeline using Python and an R package for data analysis and visualization. To validate our pipeline, we analyzed cell-free DNA reference material with known mutant allele frequencies (0%, 0.125%, 0.25%, and 1%) and public data sets. We developed UMIErrorCorrect, a bioinformatics pipeline for analyzing sequencing data containing UMIs. UMIErrorCorrect only requires fastq files as inputs and performs alignment, UMI clustering, error correction, and variant calling. We also provide UMIAnalyzer, a graphical user interface, for data mining, visualization, variant interpretation, and report generation. UMIAnalyzer allows the user to adjust analysis parameters and study their effect on variant calling. We demonstrated the flexibility of UMIErrorCorrect by analyzing data from 4 different targeted sequencing protocols. We also show its ability to detect different mutant allele frequencies in standardized cell-free DNA reference material. UMIErrorCorrect outperformed existing pipelines for targeted UMI sequencing data in terms of variant detection sensitivity. UMIErrorCorrect and UMIAnalyzer are comprehensive and customizable bioinformatics tools that can be applied to any type of library preparation protocol and enrichment chemistry using UMIs. Access to simple, generic, and open-source bioinformatics tools will facilitate the implementation of UMI-based sequencing approaches in basic research and clinical applications.

Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.

The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.

Application of the 3\u2032 mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue

BackgroundArchival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation.ResultsThe total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67).ConclusionIn this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

Targeted Next Generation Sequencing of Circulating Tumor DNA in Multiple Myeloma

Introduction: Immunophenotypic and chromosomal analysis among patients with multiple myeloma (MM) has traditionally been performed on plasma cells enriched from bone marrow aspirates. However, due to the patchy nature of MM, a bone marrow aspirate may not accurately represent the total body burden of disease nor does it reflect the true heterogeneity of the cancer cells. Additionally, since it is an invasive procedure, frequent resampling to monitor clonal evolution is infeasible. Next generation sequencing of circulating tumor DNA is a promising new approach that may address these shortcomings. Circulating tumor DNA, either from cell-free DNA or circulating tumor cells, originates from multiple different sites of disease and may be obtained from a single blood sample. This non-invasive approach may enable clinicians to detect early signs of resistance, monitor minimal residual disease, identify therapeutic targets, and provide prognostic information to help guide treatment decisions.Methods: Bone marrow mononuclear cells (BMMC), peripheral blood mononuclear cells (PBMC), and plasma samples were obtained from 45 patients treated at our center at the time of MM diagnosis, remission, or disease relapse. All samples were obtained through an Institutional Review Board approved protocol. DNA was extracted and library preparation for next generation sequencing was performed using unique molecular identifiers (UMI). The incorporation of UMIs allows for the identification and removal of errant base calls introduced during PCR amplification and Illumina sequencing thereby increasing the sensitivity of the assay to detect rare mutations. Target enrichment was performed using a panel of primers specific to exons of genes known to be significantly, recurrently mutated in MM as well as genes with predictive or prognostic value. Illumina sequencing was performed to a depth of 10,000X. Comparison of single nucleotide variants (SNV) and indels between matched BMMC, PBMC and plasma samples was determined. Correlation of clinical outcomes with mutation status and variant allele frequency was performed.Results: While good concordance was found between BMMC, PBMC, and plasma samples among the SNVs and indels detected, distinct variants unique to each tissue compartment were also identified. Indicating the assay may have utility for disease monitoring, variants detected in the blood pre-treatment were no longer detectable in post-treatment remission samples. Additionally, new variants in blood samples not present prior to initiation of therapy were detected at later time points providing evidence for clonal evolution. Finally, driver mutations were found in genes for which known targeted therapy is available or in genes that may theoretically confer drug resistance such as CUL4A that binds to Cereblon, a target of the immunomodulatory drug lenalidomide.Conclusions: While bone marrow biopsy represents the standard of care for diagnosis and disease monitoring in patients with multiple myeloma, the procedure is subject to sampling bias and frequent serial biopsies are impractical. A noninvasive approach using targeted next generation sequencing of circulating tumor cells is an appealing alternative. We demonstrate the feasibility of this technique in MM PBMC and plasma samples and find evidence that the assay may support clinical decision making. DisclosuresNo relevant conflicts of interest to declare.

Ultra-Deep Sequencing As a Predictive Tool for MDS Progression after Allogeneic Hematopoietic Cell Transplant

Background:Allogeneic hematopoietic cell transplantation (alloHCT) is the only curative treatment for patients with myelodysplastic syndromes (MDS). Up to 40% of MDS patients will die within the first year post-alloHCT due to progressive disease or treatment-related mortality. Predictors of post-alloHCT progression are needed. While recent data have shown that gene mutations in pre-transplant samples, including TP53, are associated with worse outcomes post-alloHCT, it is not known whether persistence of patient-specific mutations after alloHCT are predictive of progression. In this study we identified somatic mutations in MDS patients prior to alloHCT and then used ultra-deep sequencing of day 30 and day 100 post-alloHCT bone marrows to determine whether the extent of mutation clearance was associated with outcome.Methods:We selected 55 MDS patients who underwent alloHCT with a mean age of 55.6 years (range 23-71) and IPSS-R scores of very low (1), low (12), intermediate (6), high (23), and very high (13). Transplants included myeloablative and non-myeloablative conditioning regimens with related and unrelated donors. Of the 55 patients, 19 relapsed post-alloHCT and 36 did not with a mean of 242 days to relapse (range 27-1015) and a mean follow-up of 883 days (range 103-2106), respectively. Initial pre-alloHCT bone marrows and paired normal skin (as a source of control DNA) were enhanced exome sequenced (EES) to yield higher coverage in known recurrently mutated AML/MDS genes. To provide high sensitivity at post-alloHCT time points, an ultra-sensitive, error-corrected sequencing approach using unique molecular identifiers (UMIs) was combined with high-coverage depths. Probes were designed to target mutations detected by EES in the pre-alloHCT samples. Initial diagnostic marrow, skin, and day 30 post-alloHCT marrow samples were sequenced in all patients and day 100 post-alloHCT marrows were available in 37 patients. Variant allele fractions (VAFs) of somatic single nucleotide variant (SNV) mutations at day 30 and day 100 post-alloHCT were detected using a custom analysis pipeline employing read-level UMI-based error correction and base-level background error rate corrections. SNV calls were correlated with post-alloHCT progression-free survival and overall survival using Kaplan-Meier and Cox proportional hazards models, respectively.Results:EES was performed to a mean of 230x depth (1310x for recurrently mutated genes). A mean of 40.3 (range 17-151) somatic variants per case were identified with a mean VAF of 29.6%. Of these, a mean of 23 (range 1-70) SNVs per case were targeted and analyzed. Day 30 post-alloHCT marrows were sequenced to a mean depth of 23,062x corresponding to 2,753x unique coverage; day 100 samples were sequenced to 27,936x corresponding to 3,430x unique coverage. A SNV was included in the analysis if the error-corrected pre-transplant VAF was >10% and had >100x unique coverage at day 30. Nineteen of 55 (35%) and 18 of 37 (49%) cases had no mutations detected on post-alloHCT days 30 and 100, respectively. For patients with detectable mutations, the mean VAF per patient at day 30 was 2.35% (range of detected mutations 0.001-25.81%) and 3.44% (range 0.001-21.45%) at day 100. For patients with detectable mutations, the mean maximum VAF per patient at day 30 was 7.21% (range 0.020-88.19%) and 3.44% (range 0.023-38.98%) at day 100.The presence of at least 1 mutation with a VAF greater than 0.5% (equivalent to 1 heterozygous mutant cell in 100) at day 30 post-alloHCT was associated with a worse progression-free survival (median 3.8 vs. 21.9 months; p=0.031). Similar results were obtained using VAF cutoffs of 1% and 2.5% (p= 0.018, p=0.0002, respectively). In contrast, day 100 post-alloHCT marrows did not show a similar correlation. The effect of persistent mutations on overall survival was assessed with a Cox proportional hazards model. A higher mean VAF at day 30 post-alloHCT was associated with worse overall survival (p=0.028); for each mean VAF increase of 1% at day 30, the risk of death increased by about 10% [HR 1.10, 95% CI (1.01,1.20)].Conclusions:Detection of persistent mutations in the bone marrow by ultra-sensitive, error-corrected sequencing at day 30 post-alloHCT is associated with an increased risk of progression. These findings suggest that sequencing could be used as an individualized risk-assessment marker in MDS patients who have undergone alloHCT. DisclosuresUy:Novartis: Consultancy, Other: Travel Suppport; Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy.

A unique molecular identifier-based and clonal hematopoiesis-aware approach for accurate mutation calling in cell-free DNA assays.

e22515 Background: Given peripheral blood cells (PBCs) matched cell-free DNA (cfDNA), accurate mutation calling in next generation sequencing (NGS)-based assays relies on discriminating artifacts and clonal hematopoiesis mutations from tissue originated somatic mutations. Although clonal hematopoiesis has been considered in previous overall error modeling, it has not been adapted to PBCs without using unique molecular identifiers (UMIs). Moreover, previous studies on background error profiling were mainly based on healthy controls without matched PBC gDNA, which may lead to potential overestimation of the error rates on those sites with clonal hematopoiesis mutations. Additionally, the fraction of tissue cells is also an important influencing factor but is usually ignored. Methods: We performed UMI-assisted capture-based DNA assays on cfDNA samples, matched PBCs, and oral epithelium cells from 150 healthy donors. A site-specific and subtype-specific background error model was first built for PBCs using the SNVs called from PBCs with matched oral epithelium cells to exclude potential clonal hematopoiesis mutations. Then a similar background model was established for cfDNA with the SNVs inferred from cfDNA to exclude clonal hematopoiesis. The SNVs identified in cfDNA and matched PBCs were separately filtered with the cfDNA and PBC background error models. In this study, we used the ultrasensitive liquid biopsy approach to evaluate paired with tissue and blood samples from 56 early-stage NSCLC patients. All samples are sequenced using NGS target-capture panels covering 29 genes. Results: The mutations were detected in 91.1% of tissue and 67.9% were discovered in plasma. The coincidence rate between tissue and plasma of the 56 early-stage NSCLC patients was 66.1%. Conclusions: We have developed a novel method tailored for UMI-assisted capture-based targeted DNA assays, which outperforms currently available methods in terms of modeling background errors and filtering clonal hematopoietic mutations.

MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction.

Sequencing and PCR errors are a major challenge when characterizing genetic diversity using high-throughput amplicon sequencing (HTAS). We have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. Erroneous sequences are recognized because, across the data set, they are over-represented among the minor sequences associated with UMIs. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming dada2 and unoise3, and the ability to confidently recognize genuine alleles that are present at low abundance or resemble chimeras. The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

Circulating exosomal small RNAs are promising non-invasive diagnostic biomarkers for gastric cancer.

Dysregulation of small non‐coding RNA (ncRNA) is associated with various human diseases including cancer. This study aimed to evaluate the circulating exosomal small RNAs including microRNAs (miRNAs) and P‑element‑induced wimpy testis (PIWI)‐interacting RNAs (piRNAs) as sensitive and specific non‐invasive biomarkers for gastric cancer (GC) diagnosis. Serum exosomal small RNA transcriptome was examined using unique molecular identifiers (UMI) small RNA sequencing. Dysregulated miRNAs and piRNAs were verified in 70 GC patients and 60 healthy controls (HC) by reverse transcription quantitative PCR. The expressions of miR‐1307‐3p, piR‐019308, piR‐004918 and piR‐018569 in serum exosomes were significantly increased in GC group as compared to those in HC group. Moreover, GC patients with metastasis had significantly higher expression levels of piR‐004918 and piR‐019308 than GC patients without metastasis. The area under the curve (AUC) for miR‐1307‐3p, piR‐019308, piR‐004918 and piR‐018569 in the GC group was 0.845, 0.820, 0.754 and 0.732, respectively. The combination of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 199 can improve the AUC of miR‐1307‐3p to 0.902 and piR‐019308 to 0.914 for GC diagnosis. In conclusion, our findings indicate that serum exosomal piRNAs are promising non‐invasive diagnostic biomarkers for GC patients and potential markers for monitoring metastasis.

Detection of genomic alterations in breast cancer with circulating tumour DNA sequencing

Analysis of circulating cell-free DNA (cfDNA) has opened new opportunities for characterizing tumour mutational landscapes with many applications in genomic-driven oncology. We developed a customized targeted cfDNA sequencing approach for breast cancer (BC) using unique molecular identifiers (UMIs) for error correction. Our assay, spanning a 284.5 kb target region, is combined with a novel freely-licensed bioinformatics pipeline that provides detection of low-frequency variants, and reliable identification of copy number variations (CNVs) directly from plasma DNA. We first evaluated our pipeline on reference samples. Then in a cohort of 35 BC patients our approach detected actionable driver and clonal variants at low variant frequency levels in cfDNA that were concordant (77%) with sequencing of primary and/or metastatic solid tumour sites. We also detected ERRB2 gene CNVs used for HER2 subtype classification with 80% precision compared to immunohistochemistry. Further, we evaluated fragmentation profiles of cfDNA in BC and observed distinct differences compared to data from healthy individuals. Our results show that the developed assay addresses the majority of tumour associated aberrations directly from plasma DNA, and thus may be used to elucidate genomic alterations in liquid biopsy studies.

Sequencing-Based Measurable Residual Disease Testing in Acute Myeloid Leukemia.

Next generation sequencing (NGS) methods have allowed for unprecedented genomic characterization of acute myeloid leukemia (AML) over the last several years. Further advances in NGS-based methods including error correction using unique molecular identifiers (UMIs) have more recently enabled the use of NGS-based measurable residual disease (MRD) detection. This review focuses on the use of NGS-based MRD detection in AML, including basic methodologies and clinical applications.

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids

Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability invitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) toadjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight apowerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

BackgroundRNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases.ResultsAll methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency.ConclusionsOur data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

Development and analytical validation of a 523-gene clinical assay for cell-free DNA.

3039 Background: Liquid biopsies are emerging as a powerful complement to tumor biopsies for the clinical management of cancer patients. A large gene panel with robust analytical performance that accurately assesses variants, tumor mutational burden (TMB), and microsatellite instability in plasma would be of high value for immunotherapy studies, monitoring minimal residual disease and early cancer detection. To this end, we have completed the initial validation for the cell-free DNA (cfDNA) assay, TruSight Oncology 500 (TSO500), which interrogates the full coding region of 523 genes plus selected intronic regions for fusion detection in 23 driver genes. Methods: Cell-free DNA was extracted from plasma collected from Streck or EDTA blood tubes and quantitated to achieve an assay input of ≥10 ng. Libraries were constructed using unique molecular identifiers (UMIs) and duplex barcodes for error correction, then enriched by target capture and sequenced on a NovaSeq 6000. Healthy donor (HD) specificity assessment used matched white blood cell results to filter germline and clonal hematopoiesis variants. Contrived specimens were used to evaluate sensitivity. Single nucleotide variants (SNVs) (n = 36), insertion/deletions (indels) (n = 19), copy number variants (CNVs) (n = 6), and fusions (n = 5) were tested in 2 multi-site replicates. Results: Sensitivity of detection at 0.5% variant allele fraction (VAF) was > 95% and > 97% for SNVs and indels, respectively. All expected CNVs were identified at the targeted threshold of ≥1.3X change and showed strong correlation with matched digital PCR results. All fusions were identified at ≥0.4% VAF. Specificity in HD was > 99.99%. In 22 temporally matched tumor and blood samples from late-stage patients, 58% of all reportable mutations in tumor were identified in cfDNA. Preliminary TMB analysis identified one TMB high case with POLE p.P286R observed in both tissue and cfDNA. Conclusions: In this initial validation study the TSO500 cfDNA assay exhibited high sensitivity and specificity consistent with requirements for clinical applications. Ongoing studies will further evaluate TSO500 as a complement or potential alternative to tissue biopsy for the genomic profiling of cancer patients.

Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI

For specific detection of somatic variants at very low levels, artifacts from the NGS workflow have to be eliminated. Various approaches using unique molecular identifiers (UMI) to analytically remove NGS artifacts have been described. Among them, Duplex-seq was shown to be highly effective, by leveraging the sequence complementarity of two DNA strands. However, all of the published Duplex-seq implementations so far required pair-end sequencing and in the case of combining duplex sequencing with target enrichment, lengthy hybridization enrichment was required. We developed a simple protocol, which enabled the retrieval of duplex UMI in multiplex PCR based enrichment and sequencing. Using this protocol and reference materials, we demonstrated the accurate detection of known SNVs at 0.1–0.2% allele fractions, aided by duplex UMI. We also observed that low level base substitution artifacts could be introduced when preparing in vitro DNA reference materials, which could limit their utility as a benchmarking tool for variant detection at very low levels. Our new targeted sequencing method offers the benefit of using duplex UMI to remove NGS artifacts in a much more simplified workflow than existing targeted duplex sequencing methods.

Targeted Next-Generation Sequencing of Cell-Free DNA in Diffuse Large B-Cell Lymphoma

Background: Minimal residual disease (MRD) has an emerging diagnostic and prognostic role in Diffuse Large B-cell Lymphoma (DLBCL). However, repetitious bone marrow aspirations or lymph node biopsies would bring great pains to patients. At present, deep next-generation sequencing (NGS) based on circulating tumor DNA (ctDNA), allows quantitative mutational analysis with high sensitivity simultaneously, thereby providing us a promising non-invasive and radiation-free approach to monitor MRD and track sophisticated dynamic evolution of tumor clones.Methods: A total of 21 patients with denovo DLBCL and 6 patients with transformed DLBCL treated at our center were enrolled in this study. Formalin-fixed paraffin-embedded (FFPE) tissues at diagnosis were collected. Peripheral blood samples were obtained at the time of DLBCL diagnosed, remission, relapse, or other specific points in follow-up duration. Genomic DNA (gDNA) and cell-free DNA (cfDNA) were extracted from FFPE specimens and plasma samples, respectively. Targeted sequencing based on Illumina HiSeq 2000 were performed in all samples, which covering the whole coding sequences or hotspot regions in 61 genes known to be significantly associated with DLBCL. Library was prepared using unique molecular identifiers (UMI), which enabled the reduction of most synthesis-based errors. Sequencing depth was performed up to 2000× for FFPE specimens and 25000× for plasma samples, respectively. All mutations detected by cfDNA were validated by droplet digital PCR using Bio-Rad QX200TM. Concordance of filtered variants was determined between FFPE specimens and plasma samples at diagnosis. Correlation between clinical conditions and variant allele frequency (VAF) was also analyzed. This study was approved by Institutional Review Board of Tongji Hospital.Results: The median sequencing depth of FFPE specimens and cfDNA samples was 2312× and 28211×, respectively. Compared sequencing results of FFPE specimens with paired plasma samples at diagnosis, the number of cases with complete concordance, partial concordance, and complete discordance were 7, 12, and 8. For ultimate filtered variants detected in FFPE specimens and plasma at diagnosis, the consistency was 70% in transformed DLBCL and 55% in de novo DLBCL. Taking International Prognostic Index (IPI) score into consideration, the consistency of detection of denovo DLBCL patients with IPI score ≤ 2 was significant lower than that of patients with IPI score ≥ 3 (37% vs. 74%, P < 0.001). Besides, the presence of mutations in cfDNA also predicted MRD-positivity even though the patients achieved complete remission (CR), and the dynamic change of variant allele frequency is also associated with the evolution of tumor clones in most cases. Finally, long-last negativity of variants in cfDNA after treatment was significantly associated with superior progression-free survival.Conclusions: Our study indicated that targeted NGS of cfDNA in DLBCL is a promising test that facilitate disease monitoring and individualized therapy. A high level of concordance between FFPE specimens and plasma samples is associated with progression or evolvement that underlies clinical conditions, and ctDNA analysis may be a practical testing method to assess the prognosis of patients, although further investigations based on an extended sequencing panel and study cohort are warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TRUmiCount: correctly counting absolute numbers of molecules using unique molecular identifiers.

MotivationCounting molecules using next-generation sequencing (NGS) suffers from PCR amplification bias, which reduces the accuracy of many quantitative NGS-based experimental methods such as RNA-Seq. This is true even if molecules are made distinguishable using unique molecular identifiers (UMIs) before PCR amplification, and distinct UMIs are counted instead of reads: Molecules that are lost entirely during the sequencing process will still cause underestimation of the molecule count, and amplification artifacts like PCR chimeras create phantom UMIs and thus cause over-estimation.ResultsWe introduce the TRUmiCount algorithm to correct for both types of errors. The TRUmiCount algorithm is based on a mechanistic model of PCR amplification and sequencing, whose two parameters have an immediate physical interpretation as PCR efficiency and sequencing depth and can be estimated from experimental data without requiring calibration experiments or spike-ins. We show that our model captures the main stochastic properties of amplification and sequencing, and that it allows us to filter out phantom UMIs and to estimate the number of molecules lost during the sequencing process. Finally, we demonstrate that the phantom-filtered and loss-corrected molecule counts computed by TRUmiCount measure the true number of molecules with considerably higher accuracy than the raw number of distinct UMIs, even if most UMIs are sequenced only once as is typical for single-cell RNA-Seq.Availability and implementationTRUmiCount is available at http://www.cibiv.at/software/trumicount and through Bioconda (http://bioconda.github.io).Supplementary information Supplementary information is available at Bioinformatics online.