Christina Wood-Bouwens

Linked read sequencing resolves complex genomic rearrangements in gastric cancer metastases

BackgroundGenome rearrangements are critical oncogenic driver events in many malignancies. However, the identification and resolution of the structure of cancer genomic rearrangements remain challenging even with whole genome sequencing.MethodsTo identify oncogenic genomic rearrangements and resolve their structure, we analyzed linked read sequencing. This approach relies on a microfluidic droplet technology to produce libraries derived from single, high molecular weight DNA molecules, 50 kb in size or greater. After sequencing, the barcoded sequence reads provide long range genomic information, identify individual high molecular weight DNA molecules, determine the haplotype context of genetic variants that occur across contiguous megabase-length segments of the genome and delineate the structure of complex rearrangements. We applied linked read sequencing of whole genomes to the analysis of a set of synchronous metastatic diffuse gastric cancers that occurred in the same individual.ResultsWhen comparing metastatic sites, our analysis implicated a complex somatic rearrangement that was present in the metastatic tumor. The oncogenic event associated with the identified complex rearrangement resulted in an amplification of the known cancer driver gene FGFR2. With further investigation using these linked read data, the FGFR2 copy number alteration was determined to be a deletion-inversion motif that underwent tandem duplication, with unique breakpoints in each metastasis. Using a three-dimensional organoid tissue model, we functionally validated the metastatic potential of an FGFR2 amplification in gastric cancer.ConclusionsOur study demonstrates that linked read sequencing is useful in characterizing oncogenic rearrangements in cancer metastasis.

Chromosome-scale mega-haplotypes enable digital karyotyping of cancer aneuploidy

Abstract Genomic instability is a frequently occurring feature of cancer that involves large-scale structural alterations. These somatic changes in chromosome structure include duplication of entire chromosome arms and aneuploidy where chromosomes are duplicated beyond normal diploid content. However, the accurate determination of aneuploidy events in cancer genomes is a challenge. Recent advances in sequencing technology allow the characterization of haplotypes that extend megabases along the human genome using high molecular weight (HMW) DNA. For this study, we employed a library preparation method in which sequence reads have barcodes linked to single HMW DNA molecules. Barcode-linked reads are used to generate extended haplotypes on the order of megabases. We developed a method that leverages haplotypes to identify chromosomal segmental alterations in cancer and uses this information to join haplotypes together, thus extending the range of phased variants. With this approach, we identified mega-haplotypes that encompass entire chromosome arms. We characterized the chromosomal arm changes and aneuploidy events in a manner that offers similar information as a traditional karyotype but with the benefit of DNA sequence resolution. We applied this approach to characterize aneuploidy and chromosomal alterations from a series of primary colorectal cancers.

Identification of large rearrangements in cancer genomes with barcode linked reads

Abstract Large genomic rearrangements involve inversions, deletions and other structural changes that span Megabase segments of the human genome. This category of genetic aberration is the cause of many hereditary genetic disorders and contributes to pathogenesis of diseases like cancer. We developed a new algorithm called ZoomX for analysing barcode-linked sequence reads—these sequences can be traced to individual high molecular weight DNA molecules (>50 kb). To generate barcode linked sequence reads, we employ a library preparation technology (10X Genomics) that uses droplets to partition and barcode DNA molecules. Using linked read data from whole genome sequencing, we identify large genomic rearrangements, typically greater than 200kb, even when they are only present in low allelic fractions. Our algorithm uses a Poisson scan statistic to identify genomic rearrangement junctions, determine counts of junction-spanning molecules and calculate a Fishers exact test for determining statistical significance for somatic aberrations. Utilizing a well-characterized human genome, we benchmarked this approach to accurately identify large rearrangement. Subsequently, we demonstrated that our algorithm identifies somatic rearrangements when present in lower allelic fractions as occurs in tumors. We characterized a set of complex cancer rearrangements with multiple classes of structural aberrations and with possible roles in oncogenesis.

Single-cell transcriptome analysis identifies distinct cell types and intercellular niche signaling in a primary gastric organoid model

The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. Here we applied an air-liquid interface method to culture primary gastric organoids that contains epithelium with endogenous stroma. To characterize the microenvironment and intercellular signaling in this model, we analyzed the transcriptomes of over 5,000 individual cells from primary gastric organoids cultured at different time points. We identified epithelial cells, fibroblasts and macrophages at the early stage of organoid formation, and revealed that macrophages were polarized towards wound healing and tumor promotion. The organoids maintained both epithelial and fibroblast lineages during the course of time, and a subset of cells in both lineages expressed the stem cell marker Lgr5. We identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that air-liquid-interface-derived organoids provide a novel platform to study intercellular signaling and immune response in vitro.

Abstract 2715: Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA

Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients. Citation Format: Christina M. Wood-Bouwens, Christine Handy, Billy Lau, Hanlee Ji. Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA [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 2715. doi:10.1158/1538-7445.AM2017-2715