Low-pass Sequencing Research Articles

Abstract IA42: Detecting and quantifying mutations associated with treatment resistance in aggressive lymphomas using ctDNA

Abstract A significant proportion of diffuse large B-cell lymphoma (DLBCL) patients treated with immunochemotherapy containing rituximab (R-CHOP) exhibit either primary or acquired treatment resistance. The advancement of therapeutics in the relapse setting has likely been encumbered by our limited understanding of the molecular features that underlie resistance to R-CHOP. Unfortunately, our knowledge of DLBCL genetics is mostly limited to analyses conducted on diagnostic tissue biopsies, which have not been exposed to the selective pressures imposed by therapy. Identifying genetic alterations that contribute to treatment resistance may reveal additional treatment options and lead to biomarkers allowing patients to be paired with appropriate treatments. Genetic subgroups are gaining popularity as a new strategy to implement precision medicine in DLBCL (1). The relevance of these and other biomarkers in the relapse setting remains unclear due to limited genetic exploration of relapsed and refractory DLBCL (rrDLBCL). Progress has been limited, in part, by the requirement of tissue biopsies collected after relapse. It is well established that quantitative genomic techniques such as digital PCR and targeted sequencing can be used to determine the proportion of tumor DNA in plasma from lymphoma patients (2). With a sufficiently broad panel, sequencing affords additional opportunities including the ability to identify subclonal structure and population dynamics over time. This presentation will discuss our recent analysis of a large collection of ctDNA primarily comprising DLBCL patients on various clinical trials (3). Targeted sequencing of these samples and comparison to exome data from a meta-cohort of previously characterized untreated DLBCL biopsies revealed six genes significantly enriched for mutations upon relapse. We found both TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations persisted in the dominant clone following relapse, suggesting a role in primary treatment resistance. By inferring subclonal dynamics, we observed recurrent patterns of clonal expansion and contraction following rituximab-based therapy, with MS4A1 mutations representing the only example of consistent clonal expansion. MS4A1 missense mutations within the transmembrane domains led to loss of CD20 expression in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. Our analysis nominates TP53 and KMT2D mutation status as novel prognostic factors that may facilitate the identification of high-risk patients prior to therapy. Moreover, we have demonstrated the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens. In certain scenarios whole-exome sequencing (WES) or whole-genome sequencing (WGS) can be successfully applied to ctDNA, thereby allowing the identification of mutations, structural variation, and copy number changes. Low-pass sequencing of shotgun libraries can also be used to ascertain course estimates of ctDNA levels as well as the copy number landscape (4). Given the importance of copy number and structural alterations in the inference of genetic subgroups, these methods may allow the exploration of these groups and their stability over time. Through a series of illustrative examples, this presentation will explore the benefits of each of these techniques in the study of tumor evolution and acquired treatment resistance in DLBCL.

Data from: Assessment of imputation from low-pass sequencing to predict merit of beef steers

Data from: Assessment of imputation from low-pass sequencing to predict merit of beef steers

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Targeted Sequencing of Sorted Esophageal Adenocarcinoma Cells Unveils Known and Novel Mutations in the Separated Subpopulations.

INTRODUCTION:Our study aimed at investigating tumor heterogeneity in esophageal adenocarcinoma (EAC) cells regarding clinical outcomes.METHODS:Thirty-eight surgical EAC cases who underwent gastroesophageal resection with lymph node dissection in 3 university centers were included. Archival material was analyzed via high-throughput cell sorting technology and targeted sequencing of 63 cancer-related genes. Low-pass sequencing and immunohistochemistry (IHC) were used to validate the results.ResultsThirty-five of 38 EACs carried at least one somatic mutation that was absent in the stromal cells; 73.7%, 10.5%, and 10.5% carried mutations in tumor protein 53, cyclin dependent kinase inhibitor 2A, and SMAD family member 4, respectively. In addition, 2 novel mutations were found for hepatocyte nuclear factor-1 alpha in 2 of 38 cases. Tumor protein 53 gene abnormalities were more informative than p53 IHC. Conversely, loss of SMAD4 was more frequently noted with IHC (53%) and was associated with a higher recurrence rate (P = 0.015). Only through cell sorting we were able to detect the presence of hyperdiploid and pseudodiploid subclones in 7 EACs that exhibited different mutational loads and/or additional copy number amplifications, indicating the high genetic heterogeneity of these cancers.DISCUSSION:Selective cell sorting allowed the characterization of multiple molecular defects in EAC subclones that were missed in a significant number of cases when whole-tumor samples were analyzed. Therefore, this approach can reveal subtle differences in cancer cell subpopulations. Future studies are required to investigate whether these subclones are responsible for treatment response and disease recurrence.

Abstract 1327: Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells

Abstract Background: Genome-wide analysis of Loss-of-heterozygosity (LoH) has been shown to be important in several contexts, including the assessment of BRCAness signature, a marker of homologous repair deficiency in several cancer types associated with efficacy of platinum therapy and PARP inhibitors. Methods to infer genome-wide LoH status require high-coverage whole-genome sequencing or low-pass sequencing of a large number of single cells. We present a method to infer genome-wide LoH status of as low as one single-cell from low-pass sequencing of libraries produced by Ampli1™ LowPass kit, at a fraction of the cost of current NGS-based methods. Methods: We used low-pass sequencing data generated from 294 single Circulating Tumor Cells (CTCs) from prostate, multiple myeloma and lung cancer patients, and from 24 single cells from 8 cell lines. DNA amplified with Ampli1™ WGA kit (based on ligation-mediated PCR) combined with Ampli1 LowPass kit for Illumina generates a reduced genome representation, whereby a significant fraction of fragments is expected to be covered by more than one read. We reasoned that, even at a mean coverage insufficient for variant calling (≤0.05x), a statistically significant decrease in loci showing 2 alleles should be expected in LoH regions. A pileup of mapped sequences at dbSNP loci was obtained (minor allele frequency ≥5%; read depth ≥2). Mono- and bi-allelic loci (pileup containing 1 or 2 different alleles, respectively), were counted in each uniform copy-number (CN) genome segment within a single chromosome arm. Regions with CN equal to cell ploidy were used as internal reference. A one-sided Fisher test on biallelic sites depletion (multiplicity corrected p-value threshold of 0.05) was used to call LoH on each region. Results: Compared with libraries obtained using random shearing, Ampli1 LowPass single-cell libraries showed an increase in mean coverage of covered genome from 1.1 to 1.9 starting from the same number of reads (1M) thus increasing the number of loci for LoH calling. Analysis of 10 patients' single cells with known LoH events, corresponding to regions at copy number 1, detected 1006 events out of 1142. Several (492) copy neutral LoH events or LoHs in copy gained regions were also detected. Representatives of such events were verified successfully by high pass sequencing of the same library. Performance was confirmed by analysis of 24 single cells from cell lines whose LoH status was inferred from CCLE database. Conclusions: This innovative method leverages reduced genome representation inherent in Ampli1 LowPass libraries, to compute genome-wide LoH along with copy number (CN) status, meeting the need for LoH/CN analysis at single-cell level on rare-cells such as CTCs. Citation Format: Alberto Ferrarini, Claudio Forcato, Marianna Garonzi, Genny Buson, Massimiliano Pellicano, Francesca Fontana, Nicolò Manaresi. Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells [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 1327.

Abstract 2702: Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells

Abstract Background: Clonal evolution and heterogeneity are among the factors that make treatment of Multiple Myeloma (MM) challenging, and disease monitoring requires invasive bone marrow biopsies. Tumor heterogeneity has previously been demonstrated at copy-number level, showing patterns of recurring Copy-Number Alterations (CNA). Conversely, Loss-of-Heterozygosity (LoH) profiling has been investigated less extensively. Presence of genomic patterns of LoH has been associated with Homologous Recombination Deficiency (HRD) in MM, suggesting a role for therapy with PARP inhibitors. Here we report the genome-wide LoH profiling in single-CMMCs isolated from enriched peripheral blood of four different MM patients using a non-invasive approach that combines CellSearch® and DEPArray™ technology. Methods: CMMCs were obtained from peripheral blood of four MM patients using CellSearch for enrichment and DEPArray NxT for isolation. CMMCs enrichment was obtained using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. After single cell isolation, Ampli1™ Whole Genome Amplification (WGA) kit was used to amplify single-cell genomic DNA. Whole genome sequencing libraries were prepared from WGA products using Ampli1™ LowPass kit and low-pass sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBiosuite. Single-cell genome-wide LoH profiles were obtained by a custom algorithm based on the ratio between mono- and bi-allelic loci in non-overlapping segments of uniform copy-number. Results: Out of 315 cells analyzed from the four MM patients, 244 (77%) showed LoH events. Extensive LoH regions were detected in all patients, with 231 cells showing copy-neutral LoH events and 121 showing LoH events in copy-gain regions. Globally, 460 copy-neutral LoHs, and 292 LoHs in copy-gain regions were detected. Two highly conserved copy-neutral events (9p; 16q) were detected in one of the patients analyzed, suggesting they are early events. Interestingly, the first of the 2 events includes CDKN2A, a tumor suppressor which has been implicated in predisposition to MM. Notably, in a second patient, chromosome 11 is associated both to a copy-neutral LoH and to an LoH event in a copy-gain segment including the CCND1 gene, whose amplification in MM has been associated with multidrug resistance expression. Conclusions: Here we present a non-invasive workflow for the molecular characterization of single CMMCs isolated from peripheral blood. Specifically, we reported the presence of chromosomal or sub-chromosomal LoH events in all MM patients, suggesting a pervasive phenomenon in MM which deserves to be further explored as potential therapy target. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Paola Tononi, Marianna Garonzi, Valentina del Monaco, Andrea Raspadori, Steven Gross, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells [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 2702.

Phylogenetic double placement of mixed samples.

MotivationConsider a simple computational problem. The inputs are (i) the set of mixed reads generated from a sample that combines two organisms and (ii) separate sets of reads for several reference genomes of known origins. The goal is to find the two organisms that constitute the mixed sample. When constituents are absent from the reference set, we seek to phylogenetically position them with respect to the underlying tree of the reference species. This simple yet fundamental problem (which we call phylogenetic double-placement) has enjoyed surprisingly little attention in the literature. As genome skimming (low-pass sequencing of genomes at low coverage, precluding assembly) becomes more prevalent, this problem finds wide-ranging applications in areas as varied as biodiversity research, food production and provenance, and evolutionary reconstruction.ResultsWe introduce a model that relates distances between a mixed sample and reference species to the distances between constituents and reference species. Our model is based on Jaccard indices computed between each sample represented as k-mer sets. The model, built on several assumptions and approximations, allows us to formalize the phylogenetic double-placement problem as a non-convex optimization problem that decomposes mixture distances and performs phylogenetic placement simultaneously. Using a variety of techniques, we are able to solve this optimization problem numerically. We test the resulting method, called MIxed Sample Analysis tool (MISA), on a varied set of simulated and biological datasets. Despite all the assumptions used, the method performs remarkably well in practice.Availability and implementationThe software and data are available at https://github.com/balabanmetin/misa and https://github.com/balabanmetin/misa-data.Supplementary information Supplementary data are available at Bioinformatics online.

Abstract IA10: Liquid biopsies as biomarkers of disease progression in multiple myeloma

Abstract Multiple myeloma (MM) is a plasma cell dyscrasia that is almost always preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Some patients rapidly progress from MGUS/SMM to overt MM while others remain indolent for many years. Disease evolution also depends on the surrounding microenvironment, which can be either permissive or inhibitory of clonal expansion. The use of liquid biopsies for cancer detection and monitoring is rapidly gaining prominence. Current methods for the detection of circulating tumor cell-free DNA (cfDNA) include low-pass genome sequencing for copy number profiling and deep targeted sequencing for detection of recurrent somatic mutations. However, the sensitivity of these methods is limited among patients with minimal disease, such as MGUS or the minimal residual disease (MRD) state, on account of low tumor burden and a highly variable genomic landscape, whereby most patients do not carry any of the recurrent alterations. I will discuss our current data on detecting cfDNA and circulating tumor cells in the peripheral blood of patients with MGUS, SMM, and overt MM as well as novel personalized, sensitive methods to detect cfDNA with deep UMI-based sequencing for potential use to detect MRD. There is an urgent need for a liquid biopsy assay that can be integrated in the clinical diagnosis of patients for more sensitive tumor detection along with better characterization of their genomic and epigenetic alterations leading to much-needed solutions for integrative staging of MM precursor and MRD patients, with potential implications for patient monitoring and treatment.

Abstract A094: MSIClass - Identification and classification of microsatellite unstable tumors using cell-free DNA from ultra low pass sequencing

Abstract The acquisition of mismatch repair deficiency is common in many tumor types. A unique subtype of mismatch repair deficient tumors, called tumors with microsatellite instability (MSI), has been recently approved for treatment with PD-1\PD-L1 blockade immunotherapy. Therefore, developing a quick, inexpensive, and easy-to-implement technique for classifying tumors as MSI is of utmost importance. Here, we describe such a method using next-generation sequencing data, and show that it can detect MSI from cell-free DNA (cfDNA), even when there is low fraction of tumor DNA in the cfDNA. We first analyzed the unique mutational features of MSI vs. microsatellite stable (MSS) tumors. Using our SignatureAnalyzer tool, we analyzed the signatures of the different insertions and deletions within microsatellite loci (MS-indels), and found that MSI tumors harbor elevated MS-indels of certain types, which we further validated using whole genome sequencing data from The Cancer Genome Atlas. We next used this information to develop a method (MSIClass) that analyzes the WGS data of a given sample by capturing the features unique to MSI tumors. This method assigns each sample a score based on aggregating information from all MS loci simultaneously; since MSIClass is a sample-based tool, it can call the MSI status of tumor samples that have no patient-matched normal sample. We further demonstrate that MSIClass requires minimal genomic information; indeed, sequencing coverage as low as 0.05x (current sequencing cost of ~$2/sample) is sufficient for accurate classification by MSIClass. Using in silico and in vitro DNA mixing we show that MSIClass can accurately identify MSI cases in very low purity cases, at 0.1% tumor DNA (using 0.5x sequencing coverage). We show that MSIClass, applied on cfDNA, could accurately classify 29 colon cases -- 22 MSS and 7 MSI cases, 3 of which are early stage disease and with low tumor content. We then applied MSIClass to ~2000 cfDNA samples from various tumor types and found MSI cases in tumors that are not associated with MSI such as breast. These results demonstrate that MSIClass is an inexpensive MSI test that can be applied in various cases: bulk sequencing where a matched normal is not available, cases of very low purity tumors, and even using cfDNA from blood in cases a biopsy is not available or possible. Finally, this methodology can be the basis for monitoring minimal residual disease, and potential be used for early detection of MSI tumors. Citation Format: Yosef E. Maruvka, Ruslana Frazer, Jonna Grimsby, Emily E Van Seventer, Carrie Cibulskis, Viktor Adalsteinsson, Ryan Corcoran, Gad Getz. MSIClass - Identification and classification of microsatellite unstable tumors using cell-free DNA from ultra low pass sequencing [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A094. doi:10.1158/1535-7163.TARG-19-A094

Abstract 431: Semi-automated detection, isolation and molecular analysis of single disseminated melanoma cells from lymph nodes

Abstract We recently showed that lymph node disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCCs) and demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology (Ulmer et al., PLoS Medicine, 2014). Moreover, genetic profiling of single melanoma DCCs identified a colonization signature consisting of specific copy number alterations and point mutations that identify patients with high risk of progression (Werner-Klein et al., Nature Communications 2018). Here, we present the adaptation of this method to a semi-automated workflow for detection, isolation and molecular analysis of single melanoma DCCs. The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells, immunofluorescence staining against melanoma-associated markers gp100 and MCSP and depletion of CD45-positive cells. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis. In total, we processed 20 lymph nodes of melanoma patients and detected melanoma DCCs in 11/20 samples (55%). The quality of isolated cells was checked by Ampli1TM QC and 174 isolated single cells were further analyzed by Sanger sequencing for specific point mutations (BRAF, NRAS and cKIT) and Ampli1TM low pass kit for Illumina for copy number variation (CNV). Successful molecular analysis was correlated with genome integrity score (GII) as determined by Ampli1TM QC, with more than 95% of cells with GII 3-4 showing good performance in low pass sequencing. In conclusion, a new DepArrayTM based application for marker-dependent single cell isolation from malignant melanoma lymph nodes was successfully established and tested on a cohort of 20 melanoma patients. Molecular analyses of isolated single cells confirmed the tumor origin by CNV profiling and mutational analysis of melanoma-associated mutations. In the future, this approach could help to select individualized therapies for melanoma patients. Citation Format: Barbara Alberter, Sebastian Scheitler, Giancarlo Feliciello, Alberto Ferrarini, Melanie Werner-Klein, Sebastian Haferkamp, Christoph A. Klein, Bernhard Polzer. Semi-automated detection, isolation and molecular analysis of single disseminated melanoma cells from lymph nodes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 431.

Skmer: assembly-free and alignment-free sample identification using genome skims

The ability to inexpensively describe taxonomic diversity is critical in this era of rapid climate and biodiversity changes. The recent genome-skimming approach extends current barcoding practices beyond short markers by applying low-pass sequencing and recovering whole organelle genomes computationally. This approach discards the nuclear DNA, which constitutes the vast majority of the data. In contrast, we suggest using all unassembled reads. We introduce an assembly-free and alignment-free tool, Skmer, to compute genomic distances between the query and reference genome skims. Skmer shows excellent accuracy in estimating distances and identifying the closest match in reference datasets.

Abstract 4579: The genetic heterogeneity and the molecular evolution of systemic metastatic castration resistant prostate cancer during therapy

Abstract Background: Circulating tumour cells (CTCs) have a significant prognostic impact in metastatic castration-resistant prostate cancer (mCRPC) and provide direct access to the systemic disease. Here, we performed genome-wide copy number analysis of CellSearchTM detected CTCs to follow the genomic evolution of mCRPC during systemic therapy. Methods: Blood samples were collected from 13 patients with mCRPC before, during and after two different targeted therapy regimes. Single CTCs were isolated from CellSearchTM cartridges using the DEPArray™ system and the MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using Ampli1™ WGA kit. Amplification products were analysed by array-based comparative genomic hybridization (aCGH) and low-pass sequencing (LPS) using Ampli1™ LowPass kit to detect somatic chromosomal copy number aberrations (CNAs) and explore the degree of genomic heterogeneity. Results: We analysed >300 CTCs for CNAs by aCGH and/or LPS. Although most CTCs displayed CNAs typical for mCRPC, we identified three genomic CTC-groups across all cells with the EpCAMpos/CKpos/DAPIpos/CD45neg phenotype: CTCs with typical mCRPC CNAs (Type A; 80%), extremely aberrant CTCs (Type B; 11%), CNA-negative/low CTCs (Type C; 9%). The occurrence of Type B and C was almost mutually exclusive. At baseline, we noted different levels of CTC-heterogeneity and different CTC-aberration levels between the different patients. Interestingly, CTCs of patients with a relevant decrease in CTC-count under PARP-inhibition (CTC-responder) displayed significantly elevated CNAs at baseline compared to CTC-non-responders. During therapy, we could observe clonal changes among all patients. Conclusions: Our data show that CTC-analysis enables the dissection of clinical relevant intra-patient heterogeneity and clonal evolution under therapy in mCRPC patients. Comprehensive genomic monitoring during therapy might help to tailor therapies more effectively and may pinpoint to molecular mechanism of therapeutic resistance. Citation Format: Rui PL Neves, Streit LRF Anna, Katharina Raba, Elina-Katharina Bongers, Bianca Behrens, Guus van Dalum, Penny Flohr, Joaquin Mateo, Semini Sumanasuriya, Mateus Crespo, Berni Ebbs, Gemma Fowler, Pasquale Rescigno, Suzanne Carreira, Maryou Lambros, Edoardo Petrini, Marianna Garonzi, Nicolò Manaresi, Johann de Bono, Nikolas H. Stoecklein. The genetic heterogeneity and the molecular evolution of systemic metastatic castration resistant prostate cancer during therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4579.

Abstract 4588: Prognostic association of plasma cell free DNA (cfDNA) copy number variation based algorithmic score with survival in metastatic castration resistant prostate cancer (mCRPC)

Abstract Introduction: Survival in mCRPC stage is heterogeneous and in this stage no molecular predictors of survival are known. Elevated circulating tumor cell (CTC) counts (>5/7.5 ml blood CellSearch® assay) is the only FDA cleared prognostic biomarker in mCRPC stage that predicts poor prognosis but is not routinely performed in clinical practice. We evaluated a novel plasma cfDNA based molecular algorithmic score which is based on copy number variations (CNVs) in multiple candidate genes as a prognostic predictor of survival and also compared it to CTCs. Methods: We obtained biospecimens in a prospective cohort study (NCT#01953640) conducted in mCRPC patients who demonstrated progression on androgen deprivation therapy and were candidates to initiate pre-chemotherapy abiraterone acetate/prednisone (AA/P). Biospecimen collection was performed prior to initiating AA/P and included plasma and CTCs, both obtained at the same time. All samples underwent uniform processing. Cellsearch® assay was used for enumerating CTC counts. Plasma cfDNA was extracted using Qiagen whole blood kit and low pass sequencing was performed in the extracted plasma cfDNA and germline DNA. An algorithmic score based on CNVs in 13 candidate genes was developed after calculating an Absolute Log2 Ratio (ALR) for each pre-selected gene. The sum of ALRs for these 13 genes were combined to make a “Plasma Genome Abnormality” (PGA) score which was evaluated for association with the primary endpoint of overall survival (OS) using Hazard ratio (HR) with significance set at p ≤ 0.05. Association of concurrently collected CTC counts with OS was also determined. The PGA score based survival was adjusted for CTC counts in the final analysis. Results: Plasma and CTCs were available for analysis in 72/90 enrolled patients between 05/2013 and 09/2015 with the last enrolled patient having completed 23.5 months follow-up. 45/72 patients have died of cancer progression and the median OS of the cohort is 26.5 months (IQR: 19-46). The median plasma cfDNA amount obtained was 7.2 (Range: 1.6-70.4)ng/ml plasma and the median CTC count is 1/7.5ml blood (range: 0-308). The median PGA score was 1.108 (Range: 0.56-6.96) and the CNV based PGA score was observed to be associated with OS (HR: 1.63; 95% C.I: 1.144, 2.308); p-value= 0.0066). Association of elevated CTC counts (≥ 5/7.5 ml blood) with OS showed a statistically significant trend (HR: 1.92; CI: 0.99 - 3.7; p-value 0.051). After adjusting for CTC counts, an increased PGA score remained predictive of poor survival (HR: 1.52; 95% CI: (1.04, 2.21); p-value 0.028). Conclusions: In this cohort the PGA score, a molecular algorithm predictor based on plasma cfDNA copy number variations in candidate genes was a better prognostic marker of survival than CTC counts and potentially offers a biomarker tool which could be adopted for wider clinical application. Citation Format: Manish Kohli, Meijun Du, Liang Wang, Chiang-Ching Huang. Prognostic association of plasma cell free DNA (cfDNA) copy number variation based algorithmic score with survival in metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4588.

Abstract 4331: High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients

Abstract Formalin-fixed, paraffin-embedded (FFPE) storage is a universally-adopted, cost-effective and long-term solution for tissue storage. Given the abundance of FFPE clinical samples, they provide a good source of tissue for genome sequencing studies. However, the extraction of nucleic acids from FFPE tissues yield highly fragmented DNA of variable quality. The poor quality and low yield make it difficult to generate adequate sequencing libraries for genomic studies. For a population cancer genome study, we optimized a procedure for whole-genome sequencing on FFPE tumor and normal tissue of colorectal cancer patients. This pilot study aimed to first identify changes in DNA copy number using low-pass sequencing, with the additional goal of later adding structural variant and gene fusion detection and single nucleotide variant analysis with deeper sequencing. We processed, sequenced and analyzed 193 patients. These FFPE samples had varying storage characteristics. We optimized the initial extraction quality thresholds, DNA extraction and library preparation to generate high quality sequencing data. We identified an initial sample quality and library threshold to hold the best correlation for sequencing quality. Optimal yield and DNA size from extracted FFPE was achieved by using Promega's LEV DNA FFPE Extraction Kit. Library prep optimization involved using mechanical shearing of DNA to increase overall library size for structural variant detection as well as using unique dual index adapters to identify index swapping artifacts during sequencing. An additional cleanup was also performed post-fragmentation to increase overall library size. Overall, we successfully developed a robust protocol for conducting whole genome sequencing on FFPE tumor samples. Citation Format: Matthew Kubit, Christina Wood-Bouwens, Sue Grimes, John Bell, GiWon Shin, Billy Lau, Mickey Miller, Kenneth Day, Helaman Escobar, Hanlee Ji, Lincoln Nadauld, Paul Van Hummelen. High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4331.

Abstract 2519: Identification of novel MET fusion transcript amplification in glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary CNS malignant tumor with a 15.2% 2 year survival rate for tumors diagnosed between 2008 and 2012. Amplification of MET proto-oncogene has been identified in 4% of glioblastomas, leading to high expression and ligand independent activation in some cases. Our goal was to develop MET-amplified GBM models to study signaling and investigate response to MET inhibitors as a therapy for GBMs. We identified 2 GBM patients (HF3035 and HF3077) with MET gene amplifications after low-pass whole genome DNA sequencing of 13 cases. Fluorescent in situ hybridization (FISH) analyses confirmed heterogeneous MET amplification in HF3035 and HF3077 tumors, in 63.5% and 83.0% of nuclei, respectively. In vitro neurosphere cultures derived from these tumors showed drastic depletion of MET amplicons, to 15.5% MET in HF3035 (P7) and 1.5% in HF3077 (P11). FISH in the metaphase neurosphere spreads showed that MET amplification was extrachromosomal. Interestingly, MET-amplified neurospheres were strongly selected for after intracranial (IC) implant in immunocompromised mice. HF3035 and HF3077 PDX presented MET amplified in high frequency: 79.5-86.5% for HF3035 and 47-65% for HF3077. Met expression levels by RNAseq were congruent with the oscillating gene amplification pattern. In depth RNA sequencing analysis using PRADA has revealed genomic rearrangements involving MET, yielding three novel MET-CAPZA2 fusion transcripts. For both cell lines exon 1 of CAPZA2 was fused to exon 2 of MET, resulting in full length MET coding region, with altered 5' cis-regulatory sequences. For HF3035 samples, we observed an additional in frame fusion of exon 1 of CAPZA2 to exon 6 of MET, resulting in a truncated MET transcript with 13 codons from CAPZA2. Co-expression of the wild-type and fusion MET transcripts in the tumors and PDXs were validated using PCR. MET and p-MET levels were high thoughout the parental and PDX tumors. Capmatinib, which is a selective c-MET inhibitor was administered to the PDXs orally 5days/week. The treatment was effective in improving survival of HF3077 IC PDXs (p=0.028) and decreasing subcutaneous tumor size to 30% of the controls after 2 week treatment (t-test, p=0.017). However, treatment of HF3035 IC PDXs did not significantly improve survival (p=0.313). Kaplan-Meier survival curves were compared by log-rank (Mantel-Cox) test, sig. set at p<0.05. MET and p-MET detection by IHC of control and capmatinib treated xenografts show complete inhibition of p-MET, but did not affect MET overexpression in HF3035 PDX. Our results show that highly amplified regions are susceptible to genomic arrangements and the formation of fusion genes. Under investigation, is the basis for the strong selection for MET expressing cells in vivo and potential novel roles for MET in tumor progression. Citation Format: Oluwademilade Nuga, Ana deCarvalho, Yuling Meng, Laura Hasselbach, Kevin Nelson, Susan Irtenkauf, Andrea Transou. Identification of novel MET fusion transcript amplification in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2519.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Abstract P4-03-09: Chromosomal instability predicts taxane sensitivity in breast cancer

Abstract Taxanes remain among the most effective agents for the treatment of breast cancer. However, ˜50% of patients who receive this therapy do not have a clinical response. Previously, we identified the mechanistic effects of taxane therapy by sampling primary human breast tumors 20 hours after infusion (Sci Trans Med 26:229ra43, 2014). We determined that paclitaxel caused cells to transit mitosis on multipolar spindles, resulting in increased chromosome gains and losses. Preclinical models suggest that tumors have enhanced sensitivity if they have baseline chromosome gains/losses, known as chromosome instability (CIN). The aim of this study was to compare CIN against other predictive biomarkers in a retrospective cohort of advanced breast cancer patients who receive single-agent taxane therapy. Methods: We identified 36 subjects with metastatic breast cancer and measurable disease who received taxane therapy and had available archived tissue. Responses were determined by RECIST 1.1 criteria. We evaluated chromosome number on a cell-by-cell basis using 6 centromeric FISH probes. Additionally we measured rates of proliferation by phospho-histone H3 (p-HH3) and Ki67, previously reported biomarkers, β-tubulin III, P-gp1 and MAD1 by quantitative immunofluorescence. CIN was estimated as the fraction of cells with non-modal chromosome numbers across chromosomes 3, 4, 7, 9, 10, and 17. Results: Of the 36 subjects, 19 had ER/PR+HER2- disease (53%), 9 had HER2-positive disease (25%), and 8 TNBC (22%). Single-agent chemotherapy was used for all including paclitaxel in 16, nab-paclitaxel in 17, and docetaxel in 3 (HER2+ patients received concurrent trastuzumab). RECIST responses ranged from disease progression (8%, n=3), stable disease (50%, n=18), and partial response (42%, n=15). No complete responses were observed. Time on therapy ranged from 1.4 months to 28 months. No statistically significant correlations were found between tumor type or prior chemotherapy and response to taxanes. Archived metastatic samples were available for 21 subjects. Analysis showed large variations in Ki67, pHH3, β-tubulin III, P-gp1, MAD1, and CIN amongst samples. The strongest correlation was found between tumor response and high levels of CIN, with a Spearman's correlation coefficient of 0.38 (p=0.04). Surprisingly, there was an inverse correlation between Ki67 and taxane response, although this did not reach statistical significance. Biomarkers correlated with response to taxaneN=21Ki67 (%)pHH3 (H)βtub3 (H)P-gp1 (H)MAD1 (H)CIN (%)min0.850.000.673.002.8324.2max45.14.8915129923165.8Spearman rho-0.3650.242-0.1650.232-0.08960.382p (1-tailed)0.05190.1400.2370.1550.3500.0425H = H-score; CIN is estimated as % of cells with non-modal chromosomes Conclusions: Chromosomal instability is a promising biomarker for predicting sensitivity to taxane therapies. Additional studies will be necessary to validate CIN as a biomarker and to determine whether 6-chromosome FISH can be supplanted by low-pass single-cell DNA sequencing. Citation Format: Esbona K, Jin N, Correia-Staudt KL, Lager AM, Heidke T, Laffin J, Weaver BA, Burkard ME. Chromosomal instability predicts taxane sensitivity in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-09.

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.

Abstract IA08: Analysis of a histiocytic carcinoma in the dog: The canine's utility in fetching disease genes

Abstract Histiocytic sarcoma (HS) is a rare human hematopoietic neoplasm that accounts for less than 1% of hematolymphoid cancers and occurs either concurrently with or subsequently to B- or T-lymphoblastic lymphoma and other B-cell cancers. As such, its genetic etiology has been difficult to establish. HS in dogs, by comparison, shows strong breed specificity with 20% of flat-coated retrievers and 25% of Bernese mountain dogs (BMD) estimated to develop the disease in their lifetime. These two affected breeds share no recent common ancestors and display differences in disease presentation, offering a unique system in which to investigate the genetic predisposition and progression of HS. Our lab has previously undertaken genome wide association study (GWAS) on both BMD and FCR collected in North America and Europe and identified four significantly associated loci, two in BMD and two in FCR. In each breed, only one locus was segregating with disease in the American population while both were present in the European populations. There were no loci in common between the two breeds. Fine mapping of the common BMD HS locus on canine chromosome (CFA) CFA11 revealed a single haplotype spanning the MTAP (methylthioadenosine phosphorylase) gene and part of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which was present in 96% of BMD with HS. The European BMD also showed a significant locus on CFA14 that may be approaching fixation in the American dogs based on allele frequency calculations. We have done targeted resequencing of the CFA14 locus in a subset of European BMD and are in the process of filtering the identified mutations. Promising candidate genes in this region include GRM8, which inhibits the cyclic AMP cascade in the central nervous system and has shown to be involved in several of cancers; POT1, which is involved in telomere length maintenance and protection; and HYAL4, a hyaluronidase potentially involved in extracellular matrix degradation. In addition, the laboratory has generated 30x sequence from two HS cases and a control BMD, two FCR cases, and a control and low pass sequence from 16 dogs (8 cases and controls) in order to identify the likely causative mutations in each of the associated loci as well as breed specific mutations that may predispose the dogs to aggressive cancers. Citation Format: Heidi G. Parker, Edouard Cadieu, Abigail Shearin, Gerard Rutteman, Elaine A. Ostrander. Analysis of a histiocytic carcinoma in the dog: The canine's utility in fetching disease genes [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA08.

A recurrence-based approach for validating structural variation using long-read sequencing technology.

Although numerous algorithms have been developed to identify structural variations (SVs) in genomic sequences, there is a dearth of approaches that can be used to evaluate their results. This is significant as the accurate identification of structural variation is still an outstanding but important problem in genomics. The emergence of new sequencing technologies that generate longer sequence reads can, in theory, provide direct evidence for all types of SVs regardless of the length of the region through which it spans. However, current efforts to use these data in this manner require the use of large computational resources to assemble these sequences as well as visual inspection of each region. Here we present VaPoR, a highly efficient algorithm that autonomously validates large SV sets using long-read sequencing data. We assessed the performance of VaPoR on SVs in both simulated and real genomes and report a high-fidelity rate for overall accuracy across different levels of sequence depths. We show that VaPoR can interrogate a much larger range of SVs while still matching existing methods in terms of false positive validations and providing additional features considering breakpoint precision and predicted genotype. We further show that VaPoR can run quickly and efficiency without requiring a large processing or assembly pipeline. VaPoR provides a long read–based validation approach for genomic SVs that requires relatively low read depth and computing resources and thus will provide utility with targeted or low-pass sequencing coverage for accurate SV assessment. The VaPoR Software is available at: https://github.com/mills-lab/vapor.