PURPOSE Patients with acute myeloid leukemia (AML) in remission remain at risk for relapse even after allogeneic hematopoietic cell transplantation (alloHCT). AML measurable residual disease (MRD) status before alloHCT has been shown to be prognostic. Whether modulation of the intensity of the alloHCT conditioning regimen in patients with AML who test positive for MRD can prevent relapse and improve survival is unknown. METHODS Ultra-deep, error-corrected sequencing for 13 commonly mutated genes in AML was performed on preconditioning blood from patients treated in a phase III clinical trial that randomly assigned adult patients with myeloid malignancy in morphologic complete remission to myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC). RESULTS No mutations were detected in 32% of MAC and 37% of RIC recipients; these groups had similar survival (3-year overall survival [OS], 56% v 63%; P = .96). In patients with a detectable mutation (next-generation sequencing [NGS] positive), relapse (3-year cumulative incidence, 19% v 67%; P < .001) and survival (3-year OS, 61% v 43%; P = .02) was significantly different between the MAC and RIC arms, respectively. In multivariable analysis for NGS-positive patients, adjusting for disease risk and donor group, RIC was significantly associated with increased relapse (hazard ratio [HR], 6.38; 95% CI, 3.37 to 12.10; P < .001), decreased relapse-free survival (HR, 2.94; 95% CI, 1.84 to 4.69; P < .001), and decreased OS (HR, 1.97; 95% CI, 1.17 to 3.30; P = .01) compared with MAC. Models of AML MRD also showed benefit for MAC over RIC for those who tested positive. CONCLUSION This study provides evidence that MAC rather than RIC in patients with AML with genomic evidence of MRD before alloHCT can result in improved survival.

RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ∼10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.

Introduction Minimal residual disease (MRD) detection in solid tumors describes isolation of circulating tumor DNA (ctDNA) molecules in plasma following definitive treatment of a cancer. Detection of MRD following surgical tumor excision categorizes patients as high risk for disease recurrence. Establishing an MRD approach to treating early-stage NSCLC will facilitate escalation of standard of care (SoC) treatment only in patients destined to relapse from their cancer and overcome challenges associated with conventional adjuvant drug-trial design. Here, we present data from the lung TRACERx study where patients with early-stage NSCLC underwent phylogenetic ctDNA profiling following resection. Methods Patient specific anchored-multiplex PCR (AMP) enrichment panels were generated for 78 lung TRACERx patients who underwent surgery for stage I-III NSCLC; 608 plasma samples were analyzed. Extensive patient-specific cfDNA enrichment panels targeted a median of 196 (range 72 to 482) clonal and subclonal variants detected in primary tumor tissue by multi-region exome sequencing. A novel MRD-caller controlled and estimated background sequencing error to maximize ctDNA detection at low mutant allele frequencies (MAFs). Analytical validation experiments benchmarked assay performance. Results Analytical validation of a 50-variant AMP-MRD assay demonstrated a sensitivity of 89% for mutant DNA at a MAF of 0.008% (with 25ng of DNA input into the assay), specificity was 100% experimentally and 99.9% (95% CI: 99.67 to 99.99%) modelled in-silico. 45 patients suffered relapse of their primary NSCLC; ctDNA was detected at or before clinical relapse in 37 of 45 patients. In these 37 patients the median ctDNA lead-time (time from ctDNA detection to clinical relapse) was 151 days (range 0 to 984 days) and the median time to relapse from surgery was 413 days (range 41 to 1242 days). In 10 of 10 patients who developed second primary cancers during follow-up no ctDNA was detected, reflecting specificity of the MRD assay toward the primary tumor. In 23 patients who remained relapse-free during a median of 1184 days of study follow-up, ctDNA was detected in 1 of 199 time-points analyzed. Analysis of SoC adjuvant surveillance imaging (CT, PET-CT or MRI, 220 encounters) revealed examples of MRD positive patients where SoC radiological surveillance was negative for impending relapse. Through application of large cfDNA enrichment panels targeting up to 483 variants per patient we observed dynamic changes in clonal composition and copy-number status prior to NSCLC relapse, categorized relapse as monoclonal or polyclonal and identified distinct subclonal dynamics during systemic intervention for disease recurrence. Conclusions ctDNA is an adjuvant biomarker capable of both detecting MRD following surgery and defining the clonality of relapsing disease. These data pave the way for clinical trials predicated on escalation of adjuvant standard of care in NSCLC patients who exhibit MRD positive status following surgery. Citation Format: Chris Abbosh, Alexander Frankell, Aaron Garnett, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel Cooke, Clare Puttick, Maise Al Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Ariana Huebner, Nicolas Carey, Joan Riley, Paula Roberts, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Nicolai Birkbak, Josh Stahl, Charles Swanton, Lung TRACERx consortium. Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT023.

Pseudoknots have been recognized to be an important type of RNA secondary structures responsible for many biological functions. PseudoBase, a widely used database of pseudoknot secondary structures developed at Leiden University, contains over 250 records of pseudoknots obtained in the past 25 years through crystallography, NMR, mutational experiments and sequence comparisons. To promptly address the growing analysis requests of the researchers on RNA structures and bring together information from multiple sources across the Internet to a single platform, we designed and implemented PseudoBase++, an extension of PseudoBase for easy searching, formatting and visualization of pseudoknots. PseudoBase++ (http://pseudobaseplusplus.utep.edu) maps the PseudoBase dataset into a searchable relational database including additional functionalities such as pseudoknot type. PseudoBase++ links each pseudoknot in PseudoBase to the GenBank record of the corresponding nucleotide sequence and allows scientists to automatically visualize RNA secondary structures with PseudoViewer. It also includes the capabilities of fine-grained reference searching and collecting new pseudoknot information.

This is a repository copy of Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3′-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC.

Abstract

PURPOSE Patients with myelodysplastic syndrome (MDS) are at risk of relapse after allogeneic hematopoietic cell transplantation. The utility of ultra-deep genomic testing to predict and the impact of conditioning intensity to prevent MDS relapse are unknown. METHODS Targeted error-corrected DNA sequencing was performed on preconditioning blood samples from patients with MDS (n = 48) from the Blood and Marrow Transplant Clinical Trials Network 0901 phase III randomized clinical trial, which compared outcomes by allogeneic hematopoietic cell transplantation conditioning intensity in adult patients with < 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment. Clinical end points (53-month median follow-up) included transplant-related mortality (TRM), relapse, relapse-free survival (RFS), and overall survival (OS). Of the 48 patients examined, 14 experienced TRM, 23 are relapse-free, and 11 relapsed, of which 7 died. RESULTS Using a previously described set of 10 gene regions, 42% of patients (n = 20) had mutations detectable before random assignment to reduced intensity conditioning (RIC) or myeloablative conditioning (MAC). Testing positive was associated with increased rates of relapse (3-year relapse, 40% v 11%; P = .022) and decreased OS (3-year OS, 55% v 79%, P = .045). In those testing positive, relapse rates were higher (3-year relapse, 75% v 17%; P = .003) and RFS was lower (3-year RFS, 13% v 49%; P = .003) in RIC versus MAC arms. Testing additional genes, including those associated with MDS, did not improve prognostication. CONCLUSION This study provides evidence that targeted DNA sequencing in patients with MDS before transplant can identify those with highest post-transplant relapse rates. In those testing positive, random assignment to MAC lowered but did not eliminate relapse risk.