DLC-001 Study Design (CONSORT diagram)

Minimum Technical Data Elements for Liquid Biopsy Data Submitted to Public Databases Phillip G. Febbo, Anne-Marie Martin, Howard I. Scher, J. Carl Barrett, Julia A. Beaver, Paul J. Beresford, Gideon M. Blumenthal, Kelli Bramlett, Carolyn Compton, Ryan Dittamore, David A. Eberhard, Daniel Edelstein, James Godsey, Andrew Gruen, Sean E. Hanlon, James Hicks, Daniel Hovelson, Melanie Hullings, Donald Johann, Justin Johnson, Anand Kolatkar, Peter Kuhn, Rebecca Levine, Jean-Francois Martini, Daniel P. Miller, Carissa Moore, Bryan Moy, Anand Pathak, Reena Philip, David Reese, Wendy Royalty, Matthew Ryder, Hakan Sakul, Lea M. Salvatore, Andrew Schade, Angela Silvestro, John K. Simmons, Jonathan Simons, Seema Singh Bhan, Matthew D. Smalley, Stella B. Somiari, AmirAli Talasaz, Muneesh Tewari, Hsian-Rong Tseng, Jake Vinson, Walt Wells, Allison Welsh, Robert L. Grossman*,, Jerry S. H. Lee and Lauren C. Leiman

Neoplastic cellularity contributes to the analytic sensitivity of most present technologies for mutation detection, such that they underperform when stroma and inflammatory cells dilute a cancer specimen’s variant fraction. Thus, tumor purity assessment by light microscopy is used to determine sample adequacy before sequencing and to interpret the significance of negative results and mutant allele fraction afterwards. However, pathologist estimates of tumor purity are imprecise and have limited reproducibility. With the advent of massively parallel sequencing, large amounts of molecular data can be analyzed by computational purity algorithms. We retrospectively compared tumor purity of 3 computational algorithms with neoplastic cellularity using hematoxylin and eosin light microscopy to determine which was best for clinical evaluation of molecular profiling. Data were analyzed from 881 cancer patients from a clinical trial cohort, LCCC1108 (UNCseq), whose tumors had targeted massively parallel sequencing. Concordance among algorithms was poor, and the specimens analyzed had high rates of algorithm failure partially due to variable tumor purity. Computational tumor purity estimates did not add value beyond the pathologist’s estimate of neoplastic cellularity microscopy. To improve present methods, we propose a semiquantitative, clinically applicable strategy based on mutant allele fraction and copy number changes present within a given specimen, which when combined with the morphologic tumor purity estimate, guide the interpretation of next-generation sequencing results in cancer patients.

Background: Metastases play a role in about 90% of cancer deaths. Markers of epithelial-mesenchymal transition (EMT) measured in primary tumor cancer cells might provide diagnostic information about the likelihood that cancer cells have detached from the primary tumor. Used together with established diagnostic tests of detachment-lymph node evaluation and radiologic imaging-EMT marker measurements might improve the ability of clinicians to assess the patient's risk of metastatic disease. Translation of EMT markers to clinical use has been hampered by a lack of valid analyses of clinically-informative parameters. Here, we demonstrate a rigorous approach to estimating the sensitivity, specificity, and prediction increment of an EMT marker to assess cancer cell detachment from primary tumors. Methods: We illustrate the approach using immunohistochemical measurements of the EMT marker E-cadherin in a set of colorectal primary tumors from a population-based prospective cohort in North Carolina. Bayesian latent class analysis was used to estimate sensitivity and specificity in a setting of multiple imperfect diagnostic tests and no gold standard. Risk reclassification analysis was used to assess the extent to which addition of the marker to the panel of established diagnostic tests would improve mortality prediction. We explored how changing the latent class conditional dependence assumptions and definition of marker positivity would impact the results. Results: All diagnostic accuracy and prediction increment statistics varied with the choice of cut point to define marker positivity. When comparing different definitions of marker positivity to each other, numerous trade-offs were observed in terms of sensitivity, specificity, predictive discrimination, and prediction model calibration. We then discussed several implementation considerations and the plausibility of analytic assumptions. Conclusions: The approaches presented here can be extended to any EMT marker, to most forms of cancer, and to different kinds of EMT marker measurements, such as RNA or gene methylation data. These methods provide valid, clinically-informative assessment of whether and how to use a given EMT marker to refine tumor staging and consequent treatment decisions.

Background: The Oncotype SEQ® Liquid Select™ assay is a next-generation sequencing gene panel assay performed as a laboratory-developed test in a central CLIA-certified laboratory by Genomic Health, Inc. Oncotype SEQ® detects clinically relevant genomic alterations in 17 genes using ctDNA isolated from blood plasma. Here we summarize genomic findings from patients (pts) tested after commercial launch of Oncotype SEQ® in June 2016. Methods: 126 NSCLC pts from 17 community cancer centers in the US were tested with Oncotype SEQ® in routine clinical care. Proprietary technologies and bioinformatics tools were used to identify actionable genomic variants, which were reported to pts' medical records. Results: Tested NSCLC pts were stages IV (124), III (1) and II (1). Diagnoses were adenocarcinoma (94), squamous cell carcinoma (19), other (11), or unknown primary (2). Mean age (range) was 69 y (44–90 y), 64% were ≥65 y, 16% were >80 y. 56% were female. Results were reported for 112 (89%) pts. 135 gene variants were detected in 72/112 (64%) pts across 12 genes. 66% of identified variants were SNV, 5% indels, 27% CNV, and 2% fusions. 39 pts had 1 variant, 19 had 2, and 14 had ≥3. In nonsquamous NSCLC, 91/117 (78%) reported variants were SNV/indel/fusions and 26/117 (22%) were CNV; in squamous tumors, these were 7/16 (44%) and 9/16 (56%), respectively. Clinically actionable variants specified in FDA drug labels or in NCCN guidelines for NSCLC were reported for 42/112 (38%) pts in EGFR (13 variants), ALK (3), KRAS (21), MET (7), BRAF (2), and ERBB2 (1). These were observed at allele fractions as low as 0.12% for KRAS G12D and ranging from 0.26% to 22% for EGFR E746_A750del (n = 4). Other reported variants were potentially actionable, either specified in FDA labels of drugs approved for other tumor types, associated with active therapeutic clinical trials in NSCLC, or represented possible germline variants warranting genetic counseling. Conclusions: Oncotype SEQ® sensitively identifies gene variants that are important to inform optimal management decisions for pts with advanced-stage NSCLC. A retrospective review of medical records to examine concurrent tissue testing, treatments received, and clinical outcomes is ongoing and will be presented. Legal entity responsible for the study: Genomic Health, Inc. Funding: Genomic Health, Inc. Disclosure: D.A. Eberhard, J. Bennett, D. Davison, C. Hammond, A. Petty, J. Pluenneke, A. Dei Rossi, G. Alexander, D. Paragas, M. Lopatin: Employment and stock ownership: Genomic Health.

Background Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human “molecular tumor boards” (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. Materials and Methods One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. Results Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. Conclusion These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. Implications for Practice The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.

Abstract Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator's choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints. Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n = 54; GCB, n = 48) received ≥1 dose of lenalidomide or IC. Hematologic treatment-emergent adverse events with lenalidomide versus IC included neutropenia (42.6%; 36.4%), anemia (33.3%; 47.3%), thrombocytopenia (24.1%; 43.6%), and leukopenia (5.6%; 12.7%), respectively. Overall, lenalidomide-treated patients had an ORR of 27.5% versus 11.8% in IC (ORRs were similar regardless of IHC-defined DLBCL subtype). Median PFS was increased in patients receiving lenalidomide (13.6 weeks) versus IC (7.9 weeks; P = 0.041), with greater improvements in non-GCB patients (15.1 vs. 7.1 weeks, respectively; P = 0.021) compared with GCB (10.1 vs. 9.0 weeks, respectively; P = 0.550). Conclusions: The clinical benefit of lenalidomide monotherapy in DLBCL patients was more evident in the non-GCB subtype. Exploratory analyses suggest that this preferential benefit was more pronounced in the GEP-defined ABC population, demonstrating a need for additional studies of lenalidomide in DLBCL using GEP subtyping. Clin Cancer Res; 23(15); 4127–37. ©2017 AACR.

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator9s choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype (germinal center B-cell [GCB] vs non-GCB; determined by immunohistochemistry [IHC]), then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis (GCB vs activated B-cell [ABC]) using gene expression profiling (GEP) were exploratory endpoints. Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n=54 …

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

Abstract Background: Targeted therapies have the potential to revolutionize cancer treatment in older adults as they are often oral, convenient, may be better tolerated than cytotoxic chemotherapy, and can be tailored to an individual's biomarker profile. We explore the frequency and distribution of potentially actionable genomic alterations among older (≥65) and younger (<65) patients (pts) with metastatic breast cancer (MBC). Method: Next generation genetic sequencing (UNCseq™) of a dynamic panel of target genes was prospectively offered to pts with MBC treated at the University of North Carolina at Chapel Hill (UNC). DNA libraries were prepared separately from a retrieved archival FFPE tumor sample and a matched normal sample from each pt. Relevant targets were enriched by custom Agilent SureSelect hybrid capture baits using standard protocols. Samples were sequenced on Illumina HiSeq 2000/2500 platforms. Mutational findings were reviewed by a molecular tumor board; variants identified to be potentially actionable underwent confirmatory testing in a CLIA approved laboratory. Confirmed findings were inserted into the pt's EMR accessible by both the pt and the treating oncologist. Two-sided Fisher's exact test was used to compare percentages between age-specific groups. Results: As of 3/31/16, results were available for 140 pts. 19% were 65 years or older. Breast cancer clinical subtypes were: HR+/HER2- 49%, HER2+ (HR any) 17%, TN 34% and metastatic location was: bone only 5%, visceral only 44%, bone & visceral 51%; no significant differences were observed between older and younger age groups. Older pts were more likely to be Caucasian compared to younger patients (92% v 75%, p=0.06). Overall, older patients had a higher total number of mutations compared to younger patients (see Table) (p=0.04). Mutation types were similar between age groups, although a trend for more PIK3CA mutations among older patients was seen (37% v 20%, p=0.07). Observed Mutations by Age. ≥ 65 years (%) N=27< 65 years (%) N=113pNumber of Mutations 01127.0414849.0423320.04374.04Type of mutation PIK3CA3720.07CCND179.99NF-1115.37FGFR144.99PTEN49.69EGFR04.99 Conclusion: Genomic alterations may allow therapeutic tailoring in both older and younger patients with breast cancer. In this cohort with metastatic disease, older patients had significantly more mutations, but no clear difference in mutational types was seen by age. The relative small number of older pts in this cohort limits generalization, but supports the need for more extensive characterization of molecular aberrations among older pts with metastatic breast cancer in the new era of targeted therapy. Research support by the University Cancer Research Fund, NCI Breast Cancer SPORE grant (CA58223), John A. Hartford Foundation and Susan G. Komen Foundation. Citation Format: Jolly TA, Grilley-Olson JE, Deal AM, Ivanova A, Hayward MC, Benbow JM, Parker JS, Patel NM, Eberhard DA, Weck KE, Mieczkowski P, Dees EC, Muss HD, Reeder-Hayes KE, Earp HS, Sharpless NE, Carey LA, Hayes DN, Anders CK. Comparing the frequency and types of genetic aberrations between older and younger women with metastatic breast cancer at the University of North Carolina at Chapel Hill [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-20.

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator9s choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL).Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints.Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n …

Human papillomaviruses (HPV) are oncogenic DNA viruses implicated in squamous cell carcinomas of several anatomic sites, as well as endocervical adenocarcinomas. Identification of HPV is an actionable finding in some carcinomas, potentially influencing tumor classification, prognosis, and management. We incorporated capture probes for oncogenic HPV strains 16 and 18 into a broader next-generation sequencing (NGS) panel designed to identify actionable mutations in solid malignancies. A total of 21 head and neck, genitourinary, and gynecologic squamous cell carcinomas and endocervical adenocarcinomas were sequenced as part of the UNCSeq project. Using p16 immunohistochemical results as the gold standard, we set a cutoff for proportion of aligned HPV reads that maximized performance of our NGS assay (92% sensitive, 100% specific for HPV). These results suggest that sequencing of oncogenic pathogens can be incorporated into targeted NGS panels, extending the clinical utility of genomic assays.

Abstract Background: Targeted sequencing has become common in the care of some patients with cancer, both for the detection of standard of care clinical mutations as well as in the care of patients with advanced disease who are looking for clinical trials or other non-standard therapies. Many patients have mutations detected, although many variants are of unknown significance, and many patients have no mutations at all. We added RNA targeted sequencing along with DNA targeted sequencing to add utility of targeted sequencing strategy in cancer genomics and assessed the performances of RNA sequencing analysis. Methods: Genomic sequencing of investigative biomarkers was prospectively offered to selected patients. DNA and RNA libraries were prepared separately from a retrieved archival FFPE tumor sample or a fresh frozen tumor sample from each patient. Relevant targets were enriched by custom designed Agilent SureSelect hybrid capture baits using standard protocols. Samples were sequenced on Illumina HiSeq 2000/2500 platforms. We compared somatic variants detected by DNA alone to those detected by DNA plus RNA using the UNCeqR algorithm and software. Results: From a population of 2200 patients consented as part of LCCC1108 (NCT01457196), a subset of 300 patients was selected at random for RNA profiling. Selected patients were 7 to 83 years old (median, 54) and the cases included more than 20 cancer types including uterus, breast, ovary, and thyroid etc. And stages of cancers were as follows; I, 34.6%, II, 16.8%, III, 25.2%, and IV, 23.4% respectively. Sequencing of RNA was successful in 90% of specimens using 2.5 ug of RNA. RNA sequencing could detect 97.5% of sequence variants called by DNA sequencing and detect 20% more variants than DNA sequencing. Also 97.9% of variants showed higher mutant allele fraction (MAF) in RNA sequencing than DNA sequencing. We further interrogated the impact of certain classes of mutations on transcript structure and abundance. Specifically, we observed that splice site mutations and indels were associated with detectable alterations in the full length transcripts of their respective genes as well as overall gene abundance, with nonsense and frameshift mutations associated with decreased relative expression of the transcript relative to controls. We also observed that gene amplification of EGFR and ERBB2 was reflected in the RNA sequencing as increased gene expression with statistical significance by Fishers’ exact test (P < 0.05), which suggests that RNA can be used as a surrogate for known protein and nucleic quantitative assays. Conclusion: Using clinical samples, including relatively small quantities, we were able to confirm that nucleotide variants detected at DNA level leads to significant alteration at the transcription level and to have additional information potentially helpful for better management of cancer patients. Citation Format: Woochang Lee, Heejoon Jo, Xiaoying Yin, David A. Eberhard, Nirali M. Patel, Michele C. Hayward, Ashley H. Salazar, Joel S. Parker, William Y. Kim, Henry S. Earp, Norman E. Sharpless, David N. Hayes. Integration of targeted RNA sequencing to targeted DNA sequencing for the characterization of clinically relevant variants in a population of thousands of patients treated on clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4499.

Most cancer deaths are due to metastases. Markers of epithelial-mesenchymal transition (EMT) measured in primary tumor cancer cells could be helpful to assess patient risk of metastatic disease, even among those otherwise diagnosed with local disease. Previous studies of EMT markers and patient outcomes used inconsistent methods and did not compare the clinical impact of different expression cut points for the same marker. Using digital image analysis, we measured the EMT markers Snail and E-cadherin in primary tumor specimens from 190 subjects in tissue microarrays from a population-based prospective cohort of colorectal cancer patients and estimated their associations with time-to-death. After measuring continuous marker expression data, we performed a systematic search for the cut point for each marker with the best model fit between dichotomous marker expression and time-to-death. We also assessed the potential clinical impact of different cut points for the same marker. After dichotomizing expression status at the statistically-optimal cut point, we found that Snail expression was not associated with time-to-death. When measured as a weighted average of tumor cores, low E-cadherin expression was associated with a greater risk of dying within 5 years of surgery than high expression (risk difference = 33 %, 95 % confidence interval 3–62 %). Identifying a clinically-optimal cut point for an EMT marker requires trade-offs between strength and precision of the association with patient outcomes, as well as consideration of the number of patients whose treatments might change based on using the marker at a given cut point.

The histopathological evaluation of morphological features in breast tumours provides prognostic information to guide therapy. Adjunct molecular analyses provide further diagnostic, prognostic and predictive information. However, there is limited knowledge of the molecular basis of morphological phenotypes in invasive breast cancer. This study integrated genomic, transcriptomic and protein data to provide a comprehensive molecular profiling of morphological features in breast cancer. Fifteen pathologists assessed 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). Morphological features were significantly associated with genomic alteration, DNA methylation subtype, PAM50 and microRNA subtypes, proliferation scores, gene expression and/or reverse-phase protein assay subtype. Marked nuclear pleomorphism, necrosis, inflammation and a high mitotic count were associated with the basal-like subtype, and had a similar molecular basis. Omics-based signatures were constructed to predict morphological features. The association of morphology transcriptome signatures with overall survival in oestrogen receptor (ER)-positive and ER-negative breast cancer was first assessed by use of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset; signatures that remained prognostic in the METABRIC multivariate analysis were further evaluated in five additional datasets. The transcriptomic signature of poorly differentiated epithelial tubules was prognostic in ER-positive breast cancer. No signature was prognostic in ER-negative breast cancer. This study provided new insights into the molecular basis of breast cancer morphological phenotypes. The integration of morphological with molecular data has the potential to refine breast cancer classification, predict response to therapy, enhance our understanding of breast cancer biology, and improve clinical management. This work is publicly accessible at www.dx.ai/tcga_breast. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Background: An increasing number of molecularly-targeted therapies for metastatic breast cancer (MBC) are clinically-available (approved and investigational). These anti-cancer agents target specific molecular abnormalities such as mutated, amplified, deleted, or rearranged genes. Reporting of unique tumor genetic alterations is not included in routine clinical/diagnostic panels. In MBC, knowledge of mutational status may foster efficient transitions in clinical care and trial enrollment at disease progression. We describe the development and implementation of a clinically-integrated genomic sequencing program and report how information regarding targetable genomic aberrations in MBC patients (pts) is used to improve clinical practice in an academic setting. Methods: Genomic sequencing of investigative biomarkers was prospectively offered to pts with MBC. DNA libraries were prepared separately from a retrieved archival FFPE tumor sample and a matched normal sample from each pt. Relevant targets were enriched by custom Agilent SureSelect hybrid capture baits using standard protocols. Samples were sequenced on Illumina HiSeq 2000/2500 platforms. Mutational findings were reviewed by a molecular tumor board (MTB); variants identified to be potentially actionable underwent confirmatory testing in a CLIA-approved laboratory. Confirmed findings were inserted into the pt's EMR accessible by both the pt and the treating oncologist. Results: Of the 725 MBC pts seen at UNC since 1/1/2012, 194 (27%) contributed samples for genomic sequencing. Of those whose tumors were sequenced, average age at MBC diagnosis was 54 (25 - 91); 73% were Caucasian, 16% African American. De novo MBC accounted for 39 (20%) sequenced pts. Of sequenced patients, sites of metastatic disease included bone only (7%), visceral only (46%), and both bone and visceral (47%). Approximately 1/3 of pts were consented for sequencing at time of initial MBC diagnosis, 1/4 after 1st line therapy for MBC, and the remaining at or beyond their 2nd line. In total, 131 (68%) pts have sequencing results available of which 43% of pts had reportable mutations deemed actionable by the MTB. Specific mutations and observed frequency by subtype are shown below. Pts (19%) whose tumors were sequenced were more commonly enrolled in a therapeutic clinical trial for MBC, a higher rate than seen in the non-sequenced group (7%) (p<0.001). To date, 27% of pts' tumors harbored an alteration that is an eligibility requirement for a molecularly-targeted therapeutic trial accruing pts at UNC. Observed Mutation by Clinical Subype Genes Total # (56 pts)HR+/HER2- (25 pts)HER2+ (13 pts)TNBC (18pts)PIK3CA15933TP5315456CCND19531NF-14103FGFR13300PTEN3012KRAS2011MDM22110PIK3R12002ROS12011TSC12011Other*14518TOTAL73281728*Mutations observed only once Conclusion: Preemptive genomic sequencing can be integrated into the clinical and operational practice of a comprehensive cancer center. Currently this research tool and program provides valuable information that has the potential to foster both clinical trial eligibility and/or enrollment. With longer follow-up, we hope such an approach ultimately will improve patient outcomes. Citation Format: Grilley-Olsen J, Keith KC, Hayward M, Dees EC, Deal A, Ivanova A, Benbow JM, Parker J, Patel NM, Eberhard D, Mieczkowski P, Weck KE, Hayes DN, Muss H, Jolly T, Reeder-Hayes K, Earp HS, Sharpless N, Carey L, Anders CK. Genomic sequencing in metastatic breast cancer patients to inform clinical practice at the University of North Carolina at Chapel Hill. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD6-07.

Assessing genomic alterations in circulating tumor DNA (ctDNA) from liquid biopsies may better reflect tumor heterogeneity, facilitate monitoring of tumor evolution and overcome the challenges of obtaining tissue biopsies. Analytic performance of such assays should be established on a per sample basis, using clinically relevant variants at levels representative of ctDNA. Here we report the analytic performance of a 17-gene panel (Oncotype SEQ™, Liquid Select) and illustrate its ability to detect ctDNA in cancer patients. Analytical specificity and sensitivity were characterized through determination of Limit of Blank (LoB) and Limit of Detection (LoD) respectively. 73 cell-free DNA (cfDNA) samples from 60 healthy donors were used to determine the LoB and set detection thresholds. A model system using cell line DNA harboring clinically actionable variants was then used to determine the allele fraction (AF)/copy number (CN) required for a 95% rate of detection (LoD), using 30-50ng DNA input. Repeatability and reproducibility was assessed using pools of cfDNA from cancer patients. Finally, liquid biopsies from 15 stage II-IV cancer patients (on or after therapy) were assayed for genomic alterations. Detection thresholds were set above the LoB corresponding to >99% per sample specificity. LoD was calculated using 105 samples for each variant tested. Mean LoDs were as follows; single nucleotide variants (SNVs), 0.56% AF; insertions/deletions (indels), 0.19% AF; fusions, 0.37% AF, and CN gain, 2.7 copies. Accuracy was verified using additional variant positive and variant negative standards. In the repeatability and reproducibility study using cfDNA pools, on average >95% of expected variants were detected in each run. 10 SNVs and 2 indels were found in the 15 patient plasma samples, ranging from <0.1-32% AF. The 17-gene panel (Oncotype SEQ™, Liquid Select) provides high sensitivity, detecting ctDNA at <0.1% in stage III or later disease. In addition, its high specificity and reproducibility ensures reliable patient reporting.