Noah C. Welker

Fragment Length of Circulating Tumor DNA

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Novel specific microRNA biomarkers in idiopathic inflammatory bowel disease unrelated to disease activity.

The diagnosis of idiopathic inflammatory bowel disease can be challenging. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein synthesis through post-transcriptional suppression. This study is to identify new miRNA markers in inflammatory bowel disease, and to examine whether miRNA biomarkers might assist in the diagnosis of inflammatory bowel disease. Illumina small RNA sequencing was performed on non-dysplastic fresh-frozen colonic mucosa samples of the distalmost colectomy tissue from 19 patients with inflammatory bowel disease (10 ulcerative colitis and 9 Crohn disease) and 18 patients with diverticular disease serving as controls. To determine differentially expressed miRNAs, the USeq software package identified 44 miRNAs with altered expression (fold change ≥2 and false discovery rate ≤0.10) compared with the controls. Among them, a panel of nine miRNAs was aberrantly expressed in both ulcerative colitis and Crohn disease. Validation assays performed using quantitative reverse transcription PCR (qRT-PCR) on additional frozen tissue from ulcerative colitis, Crohn disease, and control groups confirmed specific differential expression in inflammatory bowel disease for miR-31, miR-206, miR-424, and miR-146a (P<0.05). The expression of these four miRNAs was further evaluated on formalin-fixed, paraffin-embedded tissue of the distalmost colectomy mucosa from cohorts of diverticular disease controls (n=29), ulcerative colitis (n=36), Crohn disease (n=26), and the other diseases mimicking inflammatory bowel disease including infectious colitis (n=12) and chronic ischemic colitis (n=19), again confirming increased expression specific to inflammatory bowel disease (P<0.05). In summary, we demonstrate that miR-31, miR-206, miR-424, and miR-146a are novel specific biomarkers of inflammatory bowel disease. Furthermore, miR-31 is universally expressed in both ulcerative colitis and Crohn disease not only in fresh-frozen but also in formalin-fixed, paraffin-embedded tissues.

Dynamic Linear Model for the Identification of miRNAs in Next-Generation Sequencing Data

Next-generation sequencing technologies are poised to revolutionize the field of biomedical research. The increased resolution of these data promise to provide a greater understanding of the molecular processes that control the morphology and behavior of a cell. However, the increased amounts of data require innovative statistical procedures that are powerful while still being computationally feasible. In this article, we present a method for identifying small RNA molecules, called miRNAs, which regulate genes by targeting their mRNAs for degradation or translational repression. In the first step of our modeling procedure, we apply an innovative dynamic linear model that identifies candidate miRNA genes in high-throughput sequencing data. The model is flexible and can accurately identify interesting biological features while accounting for both the read count, read spacing, and sequencing depth. Additionally, miRNA candidates are also processed using a modified Smith-Waterman sequence alignment that scores the regions for potential RNA hairpins, one of the defining features of miRNAs. We illustrate our method on simulated datasets as well as on a small RNA Caenorhabditis elegans dataset from the Illumina sequencing platform. These examples show that our method is highly sensitive for identifying known and novel miRNA genes.

Novel MicroRNA Signature to Differentiate Ulcerative Colitis from Crohn Disease: A Genome-Wide Study Using Next Generation Sequencing

BACKGROUND The diagnosis of ulcerative colitis (UC) or Crohn disease (CD) can be challenging given the overlapping features. Knowledge of microRNAs in IBD has expanded recently and supports that microRNAs play an important role. This study aimed to identify novel microRNA biomarkers through comprehensive genome-wide sequencing to distinguish UC from CD. DESIGN Illumina next generation sequencing was performed on nondysplastic fresh-frozen colonic mucosa of the distal-most colectomy from 19 patients (10 UC and 9 CD) and 18 patients with diverticular disease serving as controls. RESULTS USeq software package identified 44 microRNAs with altered expression (fold change ≥2 and false discovery rate ≤0.10) compared to controls. Among them, a panel of 11 microRNAs was aberrantly expressed between UC and CD. qRT-PCR validation assays performed on frozen tissue from additional samples of UC (n=20) and CD (n=10) confirmed specific differential expression of miR-147b, miR-194-2, miR-383, miR-615 and miR-1826 (P<0.05). In addition, pathway analysis identified target genes of epithelial adhesion junction, integrin, glycolysis and cell cycle that involve in signaling pathways of TGF-β, STAT3, IL-8 and PI3L/AKT/mTOR. CONCLUSION Identification of differentially expressed microRNAs in UC and CD supports the hypothesis that UC and CD are regulated by distinct pathophysiologic mechanisms. MicroRNA panels show promise as diagnostic biomarkers for the subtyping of inflammatory bowel disease.

Abstract 5690: Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA)

The evaluation of cfDNA allows novel approaches to noninvasive detection of actionable alterations, resistance mechanisms, and tumor monitoring in patients with cancer. Importantly, tumor-specific DNA fragments represent a small minority of the cfDNA and can be obscured by false positive (FP) variants introduced by chemical damage and sequencer error. To address this, we improved key processes in the design of NGS libraries, including a new molecular barcoding approach, that maximize molecular recovery while eliminating spurious variants. We engineered a set of Illumina sequencing chemistry compatible adaptors incorporating unique molecular identifiers (barcodes) enabling reconstruction of the sequence of both strands of the original DNA molecule. These barcodes incorporate a number of key design improvements as compared to published methodologies, which enhance sequencer cluster density, thereby increasing library diversity and molecular recovery. Our new design identified both chemical and sequencer errors, reducing incorrect base calls to rates below 5e-7. We validated our methodology for use in cfDNA using both dilution experiments and patient blood samples with known oncogenic alterations via a custom capture panel targeting actionable genomic alterations in a 55kb region. By identifying the molecular origin of each read, we found that the sensitivity of detection obtained from barcoded libraries followed ideal binomial sampling expectations. We obtained an average molecular depth of 1,000 molecules per site from the plasma extracted from a single blood collection tube, which corresponded to an 80% sensitivity of detection of known oncogenic single-nucleotide and indel mutations at 0.15% mutant allele frequency (MAF) in cfDNA with no FP calls. Furthermore, we successfully detected known gene-fusions at 0.5%, and amplifications (>10 copies) down to 1% MAF. We designed and validated a custom-engineered error-correcting sequencing adapters, ideal for broad range of applications requiring high accuracy detection of ultra-low frequency alterations. Note: This abstract was not presented at the meeting. Citation Format: Carlo G. Artieri, Kyle A. Beauchamp, Valentina S. Vysotskaia, Noah C. Welker, Eric A. Evans, Clement Chu, Haluk Tezcan, Imran S. Haque. Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5690. doi:10.1158/1538-7445.AM2017-5690

Abstract 4891: Comprehensive genetic profiling of chromosomal translocations in lung cancer tumors: development and validation of a next-generation sequencing panel in an international multicenter study

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. While tumors harboring ALK fusions are highly sensitive to crizotinib, emerging data have demonstrated that individuals with ROS1 or RET fusions may also benefit from inhibitors targeting these two kinases. A new oncogenic fusion involving NTRK1 that can be targeted by kinase inhibitors has been recently described. The two main methods to identify translocations, fluorescent in situ hybridization (FISH) and Immunohistochemistry (IHC) are labor intensive and sensitivity is rather low. Common sample types for lung cancer analysis are biopsies making the study of more than one fusion gene rather difficult. In this study we aimed to develop and validate a workflow based on AmpliSeq™ technology to comprehensively profile ALK, ROS1, RET and NTRK1 chromosomal translocations in lung tumors. Two hundred lung cancer retrospective samples, including tumor biopsy, were collected from 12 laboratories - the OncoNetwork Consortium. All the samples were previously characterized by orthologous techniques (FISH, IHC, RT-PCR and/or MassArray). More than 30 ALK positive samples were selected. The panel targets over 70 fusion transcripts associated with ALK, RET, ROS1, and NTRK1 genes. The panel also includes 5′ and 3′ gene expression assays for each gene as indicators of a translocation event and assays for 5 internal control genes. The workflow is compatible with formalin fixed paraffin embedded (FFPE) tissue, requiring only 10ng of total RNA and able to multiplex up to 16 libraries on a single Ion 318™ chip. A dedicated data analysis pipeline using the Ion Reporter software 4.2 was evaluated. Serial dilutions of cell lines RNA harboring known fusion events, in normal RNA showed that the methodology had 1% mutant RNA limit of detection. A second dilution experiment using RNA extracted from FFPE lung fusion positive samples diluted in a negative FFPE sample showed a detection limit of 15%. The use of a common control fusion positive RNA samples among all the consortia laboratories showed 100% reproducibility. Concordance study was performed using 200 FFPE samples previously characterized with standard method. A concordance of >98% was obtained between the methodologies. The discordant results are currently under study. A negative FFPE samples was run as single test on an Ion 318™ chip on to evaluate the possibility of false positive results due to high number of reads. No false positive was detected in this experiment. In this study we present a workflow that provides a robust, reproducible and accurate comprehensive genetic screening tool well suited for FFPE lung tumor biopsies, in a fast and cost-efficient manner. This may provide a valuable tool for reducing turn-around-time and expense in lung cancer analysis. Citation Format: Pierre Laurent - Puig, Jose Louis Costa, Orla Sheils, Bastiaan Tops, Andrea Mafficini, Delphine Le Corre, Henriette Kurth, Anna Maria Rachiglio, Helene Blons, Eliana Amato, Christoph Noppen, Renato Franco, Anne Reiman, Roy Bastien, Noah Welker, Jose Carlos Machado, Ian Cree, Harriet Feilotter, Marjolijn Ligtenberg, Aldo Scarpa, Nicola Normanno, Kazuto Nishio, Cecily Vaughn. Comprehensive genetic profiling of chromosomal translocations in lung cancer tumors: development and validation of a next-generation sequencing panel in an international multicenter study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2015-4891