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XLSX file - 45K, Genes containing breakpoint from translocation in mTNBC

XLSX file - 273K, Somatic genic focal copy number changes in mTNBC

XLSX file - 598K, edgeR differential expression results in mTNBC for select cancer related genes

XLSX file - 607K, Outlier differential expression results in mTNBC for select cancer related genes

XLSX file - 61K, List of somatic point mutations in mTNBC detected by NGS sequencing analysis

XLSX file - 42K, Ingenuity Canonical Pathways results for mTNBC based on edgeR differential expression results

XLSX file - 23K, Mapping statistics for genome sequencing of tumor and germline DNA from mTNBC

XLSX file - 40K, Characteristics of mTNBC study population

PDF file - 1635K, PET/CT images of mTNBC2 breast lesion before and after treatment on combination MEK and AKT inhibitors

XLSX file - 48K, Transcriptome sequencing (RNA-seq) run statistics for mTNBC

PDF file - 135K, Validation of RB1 39bp homozygous deletion in mTNBC1

Background Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888) assessed the performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity. Participants and methods The 6689 participants [2482 cancer (>50 cancer types), 4207 non-cancer] were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization. Results Performance was consistent in training and validation sets. In validation, specificity was 99.3% [95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR)]. Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ∼63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%. Conclusions cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC).Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers.Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies).The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.

Background: Triple-negative breast cancer (TNBC) has been plagued by the absence of targeted therapies. Discovery of therapeutic targets in TNBC has in part, been hampered by an inadequate understanding of the transcriptional biology of the normal breast as an optimal comparator. Using next-generation sequencing, we embarked on a study to compare the transcriptomes of TNBC and normal breast to comprehensively identify novel targets by analyzing all full length transcripts expressed in these tissues. Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, normal tissues were laser capture microdissected for ductal epithelium. cDNA libraries from 10 TNBC tumors and 10 normal breast tissues were sequenced on an Applied Biosystems (AB) SOLiD3 sequencer using 50bp fragment runs. For gene expression, mapping of reads to the genome was performed using the AB BioScope 1.2 Pipeline and outputs imported into Partek Genomics Suite for analysis. In Partek, mapped reads were cross-referenced against known genes from the UCSC database followed by statistical comparison of RPKM values for each gene between TNBC and normal. Dimensionality reduction analyses (PCA & Hierarchical clustering) and identification of Novel Transcribed Regions were also performed in Partek, whereas construction of gene networks was performed using Ingenuity Pathway Analysis. To identify gene fusions, partially mapped reads were interrogated utilizing a novel algorithm that searched for reads spanning exons from two different genes. Fusions that were supported by at least 3 reads (of which 2 had to be unique) were considered candidates and were subsequently validated. Results/Discussion: Sequencing produced 1.1 billion reads equaling 57.3GB of data of which 36.0GB (63%) mapped to the human genome. In comparing RPKM values between TNBC and Normal, we report 7140 RefSeq Genes, 22 pre-miRNAs, 109 lincRNA exons, and 15 ultraconserved regions that were differentially expressed between these tissues (FDR Conclusion: We report an extensive comparison of the transcriptomes of TNBC and normal ductal epithelium. We identified numerous genes previously unknown to be dysregulated in TNBC that can be utilized for therapeutic discovery. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-08.

CCGA [NCT02889978] is the largest study of cfDNA-based early cancer detection; the first CCGA learnings from multiple cfDNA assays are reported here. This prospective, multi-center, observational study has enrolled 10,012 of 15,000 demographically-balanced participants at 141 sites. Blood was collected from participants with newly diagnosed therapy-naive cancer (C, case) and participants without a diagnosis of cancer (noncancer [NC], control) as defined at enrollment. This preplanned substudy included 878 cases, 580 controls, and 169 assay controls (n=1627) across 20 tumor types and all clinical stages. All samples were analyzed by: 1) Paired cfDNA and white blood cell (WBC)-targeted sequencing (60,000X, 507 gene panel); a joint caller removed WBC-derived somatic variants and residual technical noise; 2) Paired cfDNA and WBC whole-genome sequencing (WGS; 35X); a novel machine learning algorithm generated cancer-related signal scores; joint analysis identified shared events; and 3) cfDNA whole-genome bisulfite sequencing (WGBS; 34X); normalized scores were generated using abnormally methylated fragments. In the targeted assay, non-tumor WBC-matched cfDNA somatic variants (SNVs/indels) accounted for 76% of all variants in NC and 65% in C. Consistent with somatic mosaicism (i.e., clonal hematopoiesis), WBC-matched variants increased with age; several were non-canonical loss-of-function mutations not previously reported. After WBC variant removal, canonical driver somatic variants were highly specific to C (e.g., in EGFR and PIK3CA, 0 NC had variants vs 11 and 30, respectively, of C). Similarly, of 8 NC with somatic copy number alterations (SCNAs) detected with WGS, 4 were derived from WBCs. WGBS data revealed informative hyper- and hypo-fragment level CpGs (1:2 ratio); a subset was used to calculate methylation scores. A consistent “cancer-like” signal was observed in 99% specificity for invasive cancer, and support the promise of cfDNA assay for early cancer detection. Additional data will be presented on detected plasma:tissue variant concordance and on multi-assay modeling. Citation Format: Alexander A. Aravanis, Geoffrey R. Oxnard, Tara Maddala, Earl Hubbell, Oliver Venn, Arash Jamshidi, Ling Shen, Hamed Amini, John A. Beausang, Craig Betts, Daniel Civello, Konstantin Davydov, Saniya Fazullina, Darya Filippova, Sante Gnerre, Samuel Gross, Chenlu Hou, Roger Jiang, Byoungsok Jung, Kathryn Kurtzman, Collin Melton, Shivani Nautiyal, Jonathan Newman, Joshua Newman, Cosmos Nicolaou, Richard Rava, Onur Sakarya, Ravi Vijaya Satya, Seyedmehdi Shojaee, Kristan Steffen, Anton Valouev, Hui Xu, Jeanne Yue, Nan Zhang, Jose Baselga, Rosanna Lapham, Daron G. Davis, David Smith, Donald Richards, Michael V. Seiden, Charles Swanton, Timothy J. Yeatman, Robert Tibshirani, Christina Curtis, Sylvia K. Plevritis, Richard Williams, Eric Klein, Anne-Renee Hartman, Minetta C. Liu. Development of plasma cell-free DNA (cfDNA) assays for early cancer detection: first insights from the Circulating Cell-Free Genome Atlas Study (CCGA) [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 LB-343.

SUMMARY Ion channels establish and regulate membrane potentials in excitable and non-excitable cells. How functional diversification of ion channels contributed to the evolution of nervous systems may be understood by studying organisms at key positions in the evolution of animal multicellularity. We have carried out the first analysis of ion channels cloned from a marine sponge, Amphimedon queenslandica. Phylogenetic comparison of sequences encoding for poriferan inward-rectifier K+ (Kir) channels suggests that Kir channels from sponges, cnidarians and triploblastic metazoans each arose from a single channel and that duplications arose independently in the different groups. In Xenopus oocytes, AmqKirA and AmqKirB produced K+ currents with strong inward rectification,as seen in the mammalian Kir2 channels, which are found in excitable cells. The pore properties of AmqKir channels demonstrated strong K+selectivity and block by Cs+ and Ba2+. We present an original analysis of sponge ion channel physiology and an examination of the phylogenetic relationships of this channel with other cloned Kir channels.

Motivation: Gene duplications and losses (GDLs) are important events in genome evolution. They result in expansion or contraction of gene families, with a likely role in phenotypic evolution. As more genomes become available and their annotations are improved, software programs capable of rapidly and accurately identifying the content of ancestral genomes and the timings of GDLs become necessary to understand the unique evolution of each lineage. Results: We report EvolMAP, a new algorithm and software that utilizes a species tree-based gene clustering method to join all-to-all symmetrical similarity comparisons of multiple gene sets in order to infer the gene composition of multiple ancestral genomes. The algorithm further uses Dollo parsimony-based comparison of the inferred ancestral genes to pinpoint the timings of GDLs onto evolutionary intervals marked by speciation events. Using EvolMAP, first we analyzed the expansion of four families of G-protein coupled receptors (GPCRs) within animal lineages. Additional to demonstrating the unique expansion tree for each family, results also show that the ancestral eumetazoan genome contained many fewer GPCRs than modern animals, and these families expanded through concurrent lineage-specific duplications. Second, we analyzed the history of GDLs in mammalian genomes by comparing seven proteomes. In agreement with previous studies, we report that the mammalian gene family sizes have changed drastically through their evolution. Interestingly, although we identified a potential source of duplication for 75% of the gained genes, remaining 25% did not have clear-cut sources, revealing thousands of genes that have likely gained their distinct sequence identities within the descent of mammals. Availability: Query server, source code and executable are available at http://kosik-web.mcdb.ucsb.edu/evolmap/index.htm Contact: kosik@lifesci.ucsb.edu, oakley@lifesci.ucsb.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90% of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Horizontal gene transfer (HGT) is common between prokaryotes and phagotrophic eukaryotes. In metazoans, the scale and significance of HGT remains largely unexplored but is usually linked to a close association with parasites and endosymbionts. Marine sponges (Porifera), which host many microorganisms in their tissues and lack an isolated germ line, are potential carriers of genes transferred from prokaryotes. In this study, we identified a number of potential horizontally transferred genes within the genome of the sponge, Amphimedon queenslandica. We further identified homologs of some of these genes in other sponges. The transferred genes, most of which possess catalytic activity for carbohydrate or protein metabolism, have assimilated host genome characteristics and are actively expressed. The diversity of functions contributed by the horizontally transferred genes is likely an important factor in the adaptation and evolution of A. queenslandica. These findings highlight the potential importance of HGT on the success of sponges in diverse ecological niches.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer. Mol Cancer Ther; 12(1); 104–16. ©2012 AACR.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions–particularly those expressed with low abundance– is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample., Author Summary Advances in sequencing technology are enabling detailed characterization of RNA transcripts from biological samples. The fundamental challenge of accurately mapping the reads on transcripts and gleaning biological meaning from the data remains. One class of transcripts, gene fusions, is particularly important in cancer. Some gene fusions are prominent markers in leukemia, prostate, and other cancers and putatively causative in certain tumor types. We present a set of new RNA-Seq analysis techniques to map reads, and count expression of genes, exons and splicing junctions, especially those that give evidence of gene fusions. These tools are available in a software package with a straightforward graphical user interface. Using this software, we called and validated several gene fusions in a breast cancer cell line. By testing the presence of these fusions in a larger population of tumor cell lines and clinical samples, we found that two of them were expressed recurrently.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we built a mapping pipeline with features including filter and junction mapping, annotation specific pairing rescue and accurate mapping quality values. We combined this pipeline with a suffix array spliced read alignment algorithm to sensitively detect RNA splicing and gene fusion events. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions, of which 25 were expressed specifically in MCF-7. We further explore possible extension of suffix arrays, and other alignment strategies for longer Ion Torrent reads.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

PDZ domains are protein-protein interaction modules widely used to assemble membranous signaling complexes including those found in the neuronal synapse. PDZ-containing genes encoded in metazoan genomes vastly outnumber those in prokaryotes, plants, and fungi. By comparing 40 proteomes to track the evolutionary history of the PDZ domain, we observed that the variety of associations between PDZ and other domains expands greatly along the stem leading to metazoans and choanoflagellates. We asked whether the expansion of PDZ domains was due to random or specific sequence changes. Studying the sequence signatures of 58 PDZ lineages that are common to bilaterian animals, we showed that six common amino acid residues are able to classify 96% of PDZ domains to their correct evolutionary lineage. In PDZ domain-ligand cocrystals, four of these "classifying positions" lie in direct contact with the -1 and -3 residues of the ligand. This suggests coevolution of the more flexible regions of the binding interaction as a central mechanism of specialization inherent within the PDZ domain. To identify these positions, we devised two independent algorithms--a metric termed within-clade entropy (WCE) and an average mutual information (AvgMI) score--that both reached similar results. Extending these tools to the choanoflagellate, Monosiga brevicollis, we compared its PDZ domains with their putative metazoan orthologs. Interestingly, the M. brevicollis genes lack conservation at the classifying positions suggesting dissociation between domain organization in multidomain proteins and specific changes within the PDZ domain.

Background The evolution of complex sub-cellular structures such as the synapse requires the assembly of multiple proteins, each conferring added functionality to the integrated structure. Tracking the early evolution of synapses has not been possible without genomic information from the earliest branching animals. As the closest extant relatives to the Eumetazoa, Porifera (sponges) represent a pivotal group for understanding the evolution of nervous systems, because sponges lack neurons with clearly recognizable synapses, in contrast to eumetazoan animals. Methodology/Principal Findings We show that the genome of the demosponge Amphimedon queenslandica possesses a nearly complete set of post-synaptic protein homologs whose conserved interaction motifs suggest assembly into a complex structure. In the critical synaptic scaffold gene, dlg, residues that make hydrogen bonds and van der Waals interactions with the PDZ ligand are 100% conserved between sponge and human, as is the motif organization of the scaffolds. Expression in Amphimedon of multiple post-synaptic gene homologs in larval flask cells further supports the existence of an assembled structure. Among the few post-synaptic genes absent from Amphimedon, but present in Eumetazoa, are receptor genes including the entire ionotropic glutamate receptor family. Conclusions/Significance Highly conserved protein interaction motifs and co-expression in sponges of multiple proteins whose homologs interact in eumetazoan synapses indicate that a complex protein scaffold was present at the origin of animals, perhaps predating nervous systems. A relatively small number of crucial innovations to this pre-existing structure may represent the founding changes that led to a post-synaptic element.

Genomic instability, the hallmark of cancer, presents with a variety of mutation types, most commonly single nucleotide variations (SNVs) and copy number variations (CNVs), which traditionally have required different methods for identification. It has proven challenging to simultaneously achieve sufficient breadth to detect CNVs and depth to detect SNVs on samples of limited input amount. The objective of this study was to validate a new methodology for detection of SNVs and CNVs in a single assay. We used a massively multiplex PCR/NGS approach combining an SNV panel covering 585 point mutation hotspots in breast cancer (Cosmic) and a CNV panel targeting 28,000 SNPs designed to detect copy number at chromosomes 1, 2, 13, 18, 21, and X, and focal regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13. We applied these panels to breast cancer cell lines and fresh frozen (FF) breast tumor samples; the presence of CNVs in circulating cell-free tumor DNA (ctDNA) in the plasma of breast cancer patients was also investigated. The CNV assay methodology was validated using genomic DNA isolated from 96 human samples with known karyotype; sensitivity to single region deletions or duplications was 100% (71/71) and specificity was 100% for normal regions in the same samples. Single-molecule sensitivity for the detection of CNVs was established by analyzing isolated single cells. Performance of the mutation assay was demonstrated with the analysis of 5 matched tumor and normal cell lines, with 24 out of 27 SNVs known to be present in these cell lines detected. The 3 undetected SNVs were determined to be a result of assay design failure. Also, multiple somatic CNVs (median: 13) were detected in all 5 tumor cell lines. Analysis of the normal cell lines found no cancer related SNVs or CNVs. In 32 FF tumor samples, 78.1% (25/32) had SNVs detected; of samples with SNVs, 88% (22/25) had SNVs in TP53 or PIK3CA. Of the same 32 FF breast tumor samples, 96.9% (31/32) showed full or partial CNVs in at least 1 and up to 15 regions; of the 31 samples with detected CNVs, 93.5% had a CNV of either 1q or 17p, two of the three most prevalent breast cancer CNVs (the 16q region was not represented in this panel). Overall, a combination of SNV and CNV testing allowed identification of genetic changes in 100% of the breast tumor samples, a significant improvement in diagnostic yield than using SNV detection alone. Of the 12 breast cancer patients with matched tumor tissue and plasma samples, 83.3% (10/12) had CNVs detected in tissue. The CNVs present in each primary tumor sample were identified in corresponding plasma ctDNA samples (1 stage IIa, 7 stage IIb, and 2 stage III). The ctDNA fractions in these samples ranged from 0.58 to 4.33%; detection required as few as 86 heterozygous SNPs per CNV. Analysis of ctDNA for cancer-associated mutations may allow earlier, safer and more accurate profiling and monitoring of breast cancer. Thus, this targeted PCR approach offers the promise of an assay able to detect both cancer-associated SNVs and CNVs in the same sample with good sensitivity and specificity, and improved detection rates compared to assays that only detect SNVs. Citation Format: Robert J Pelham, Bernhard G Zimmermann, Eser Kirkizlar, Ryan K Swenerton, Bin Hoang, Onur Sakarya, Joshua E Babiarz, Nicholas Wayham, Tudor Constantin, Styrmir Sigurjonsson, Matthew Rabinowitz, Matthew Hill. Detection of single nucleotide variations and copy number variations in breast cancer tissue and ctDNA samples using single-nucleotide polymorphism-targeted massively multiplexed PCR [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-03.