Supplemental Figure 1. Distribution of EGFR extracellular domain (ECD) mutations in ctDNA samples from patients with CRC. Supplemental Figure 2. Response to crizotinib in a patient with small bowel adenocarcinoma and GOPC-ROS1 fusion. Supplemental Figure 3. Detection of copy number amplification is associated with increased ctDNA fraction. Supplemental Table 1. List of 62 genes sequenced in this study using the FoundationACT ctDNA assay. Supplemental Table 2. Genomic alterations detected in temporally matched ctDNA and tissue samples.

This supplementary file contains additional Matierials and Methods detailing the VERSA process. Figure S1. Longitudinal analysis of capture efficiency of Epcam antibody. Figure S2. Multi-parametric analysis of gene expression, genomic profiling and AR protein analysis from captured CTCs. Table S1. Catalog numbers of primers used for quantitative qPCR. Table S2. Patients characteristics for patients 1-17. Table S3. Patient characteristics from patients 18-43

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

Background: Although both seminomatous and nonseminomatous testicular germ cell tumors (TGCTs) have favorable outcomes with chemotherapy, a subset is chemorefractory, and novel therapeutic options are needed. Objective: To molecularly characterize chemotherapy-refractory TGCTs. Design, setting, and participants: Archival tissues from 107 chemotherapy-treated and relapsed TGCT patients (23 seminomas; 84 nonseminomas) underwent hybrid-capture-based genomic profiling to evaluate four classes of genomic alterations (GAs). Tumor mutational burden (TMB) and microsatellite instability (MSI) were also measured. Intervention: Genomic profiling on tumor samples from chemotherapy-refractory TGCTs. Outcome measurements and statistical analysis: Descriptive analyses and differences between seminoma and nonseminoma subgroups were reported. Results and limitations: The mean GA/tumor was 2.9 for seminomas and 4.0 for non-seminomas (p = 0.04). KRAS alterations (mainly amplifications) were the most common GAs at the single-gene level (47.8% of seminomas and 51.2% of nonseminomas). RAS-RAF pathway (56.5% vs 52.3%) and cell-cycle pathway (52.2% vs 56.0%) were the most common GA classes in seminomas and nonseminomas, respectively. Receptor tyrosine kinase pathway and PI3K pathway GAs were more frequent in seminomas (p = 0.02). Median TMB was 1.8 mutations/Mb for seminomas and 2.7 mutations/Mb for non-seminomas (p = 0.098), and MSI-high status was found in one nonseminoma only (1.2%). A lack of clinical outcome correlation is a limitation of the present analyses. Conclusions: In chemotherapy-refractory TGCTs, trials with agents targeting the KRAS pathway may be pursued due to the high frequency of KRAS GAs. Overall, the GAs found in refractory seminomas and nonseminomas differ significantly. Considering the frequency of high TMB or MSI-high status, immunotherapy may benefit a small subset of nonseminomas. Patient summary: Testicular cancers that are resistant to or relapse after standard chemotherapy may harbor genomic alterations that are potentially druggable, particularly in the clinical trial setting, and genomic profiling can guide clinical research and disclose therapeutic opportunities for these patients. (C) 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Abstract Purpose: Genomic profiling of tumor biopsies from advanced gastrointestinal and anal cancers is increasingly used to inform treatment. In some cases, tissue biopsy can be prohibitive, and we sought to investigate whether analysis of blood-derived circulating tumor DNA (ctDNA) may provide a minimally invasive alternative. Experimental Design: Hybrid capture–based genomic profiling of 62 genes was performed on blood-based ctDNA from 417 patients with gastrointestinal carcinomas to assess the presence of genomic alterations (GA) and compare with matched tissue samples. Results: Evidence of ctDNA was detected in 344 of 417 samples (82%), and of these, ≥1 reportable GA was detected in 89% (306/344) of samples. Frequently altered genes were TP53 (72%), KRAS (35%), PIK3CA (14%), BRAF (8%), and EGFR (7%). In temporally matched ctDNA and tissue samples available from 25 patients, 86% of alterations detected in tissue were also detected in ctDNA, including 95% of short variants, but only 50% of amplifications. Conversely, 63% of alterations detected in ctDNA were also detected in matched tissue. Examples demonstrating clinical utility are presented. Conclusions: Genomic profiling of ctDNA detected potentially clinically relevant GAs in a significant subset of patients with gastrointestinal carcinomas. In these tumor types, most alterations detected in matched tissue were also detected in ctDNA, and with the exception of amplifications, ctDNA sequencing routinely detected additional alterations not found in matched tissue, consistent with tumor heterogeneity. These results suggest feasibility and utility of ctDNA testing in advanced gastrointestinal cancers as a complementary approach to tissue testing, and further investigation is warranted. Clin Cancer Res; 24(8); 1881–90. ©2018 AACR.

Tumor genome sequencing has become an invaluable resource in determining targets for new therapies. In this report, we describe the case of a patient with metastatic urothelial carcinoma with sarcomatoid features. Sarcomatoid differentiation is a rare histologic subtype that confers a more aggressive course. The first-line treatment for patients with urothelial carcinoma is platinum-based chemotherapy. Next generation tumor sequencing performed using the FoundationOne assay revealed loss of one NF2 allele and an unbalanced der(22)t(10;22)(p11.22;q12.2) chromosomal rearrangement involving the other NF2 allele, resulting in truncation and predicted loss of function. Fluorescence in situ hybridization (FISH) analysis confirmed the presence of one NF2 signal. NF2 mutations have been found in a variety of cancers and result in activation of the mTOR pathway. As such, the use of mTOR inhibitors, such as everolimus are thought to be particularly effective in the case of NF2 loss. Our patient had a dramatic response to first-line chemotherapy, but unfortunately experienced subsequent progression of his cancer and could not tolerate everolimus. Although our patient's tumor demonstrated unique acquired genetic features including both loss of heterozygosity and truncation of the NF2 locus, he still achieved a meaningful response to platinum-based chemotherapy.

Introduction: Genomic profiling informs selection of matched targeted therapies as part of routine clinical care in NSCLC. Tissue biopsy is the criterion standard; however, genomic profiling of blood-derived circulating tumor DNA (ctDNA) has emerged as a minimally invasive alternative. Methods: Hybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 1552 patients with NSCLC. Results: Evidence of ctDNA was detected in 80% of samples, and in 86% of these cases, at least one reportable genomic alteration (GA) was detected. Frequently altered genes were tumor protein p53 gene (TP53) (59%), EGFR (25%), and KRAS (17%). Comparative analysis with a tissue genomic database (N = 21,500) showed similar frequencies of GAs per gene, although KRAS mutation and EGFR T790M were more frequent in tissue and ctDNA, respectively (both p < 0.0001), likely reflecting the use of liquid versus tissue biopsy after relapse during targeted therapy. In temporally matched ctDNA and tissue samples from 33 patients with evidence of ctDNA in their blood, 64% of GAs detected in tissue were also detected in ctDNA, including 78% of short variants (58 of 74) and 100% of rearrangements (four of four), but only 16% of amplifications (four of 25). Conclusions: Genomic profiling of ctDNA detected clinically relevant GAs in a significant subset of NSCLC cases. Most alterations detected in matched tissue were also detected in ctDNA. These results suggest the utility of ctDNA testing in advanced NSCLC as a complementary approach to tissue testing. Blood-based ctDNA testing may be particularly useful at the time of progression during targeted therapy. (C) 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

Abstract Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

Purpose With increased use of targeted therapeutics, the ability to perform a ‘liquid biopsy’ on circulating tumor cells (CTCs) holds potential to address the need for a more representative and longitudinal means to monitor tumor burden in patients. However, the analytic and clinical validity of such an assay has proved difficult to establish, as the cell population in circulation is complex, and the CTC portion is heterogeneous, evolving, and highly dependent on clinical factors as well as CTC isolation methods. No studies to date have characterized CTCs for the full range of relevant genomic alterations in the context of patient-matched primary and metastatic tissue biopsies. We set out to develop a method to perform comprehensive NGS-based genomic profiling on phenotypically-isolated CTCs, and to compare CTC analysis with a comprehensive tissue-based assessment of paired primary and metastatic tissue in patients. Methods An integrated platform (VERSA) was employed for capture of EpCAM CTCs, enumeration and DNA extraction. Extracted DNA was amplified using a modified version of MDA-based whole genome amplification (WGA), followed by standard adaptor-ligated library construction and solution hybridization using a custom DNA baitset before sequencing on Illumina HiSeq. Data was analyzed using a computational workflow developed to detect base substitutions, indels, high-level copy number alterations, and select gene rearrangements. We performed extensive analytic validation of the assay using over 65 cell line mixtures, constructed to contain all classes of known variants across all allele frequencies (0-100%). We used the assay to analyze CTC DNA from 20 lung cancer patients, paired with analysis of temporally-matched primary and/or metastatic FFPE samples analyzed by FoundationOne, an FFPE tissue-based assay. Results Validation experiments showed that for samples meeting minimum acceptance criteria (50 cells at 50% purity), assay sensitivity exceeded 95% for detections of base substitutions, 85% for indels and rearrangements, and 80% for high-level copy number alterations, while maintaining tight specificity control. WGA-induced coverage bias limited sensitivity to low-level copy number alterations. Comparison of driver alterations across CTC samples and paired primary/metastatic tissue for 20 patients with lung cancer is ongoing. Conclusions A comprehensive NGS-based approach to genomic profiling is feasible on CTCs isolated from lung cancer patients, and demonstrated accuracy sufficient to characterize actionable targets for clinical decision-making. However, more extensive tumor-type specific studies with a similar matched-tissue approach must be performed in order to establish full analytic and clinical validation, and assay specifications will need to be optimized to enable use on broader patient populations. Citation Format: Allison Welsh, Garrett M. Frampton, Zachary R. Chalmers, Jamie Sperger, Roman Yelensky, Doron Lipson, Geoff Otto, Lindsay Strotman, Scott Berry, Hannah Pezzi, Anne Traynor, David J. Beebe, Vincent A. Miller, Joshua M. Lang, Philip J. Stephens. Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2015-1602

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from >0.92 (nearly homogeneous) to <0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

The insulin-like growth factor I receptor (IGF1R) interacts with estrogen receptor-α (ERα) and HER2. We examined the effect of combinations of IGF1R antagonists (α-IR3, AG1024) and anti-estrogens (4-hydroxy tamoxifen, fulvestrant) in two human ER+ breast cancer cell lines: BT474 (HER2 overexpressing, IGF1R low) and MCF7 (HER2 non-overexpressing, IGF1R high). In BT474 cells, growth was inhibited by anti-estrogens, but not by IGF1R antagonists; however, adding IGF1R inhibitors to anti-estrogens enhanced growth inhibition. In MCF7 cells, growth was inhibited by IGF1R and ER antagonists and more so by their combination. In both cell lines, no single agents could induce apoptosis, but combining IGF1R inhibitors with anti-estrogens induced dramatic levels of apoptosis. IGF1R antagonists enhanced the ability of the anti-estrogens to inhibit ER transcriptional activity in BT474 cells, but not in MCF7 cells. The drug combination synergistically inhibited ER and IGF1R activity. Such combinations may be useful therapy for breast cancer.

Abstract The large number and elusive nature of analytical and pre-analytic variables associated with immunohistochemistry (IHC) have relegated the technique to a semi-quantitative or qualitative status. However, as companion diagnostics become more critical to patient management, there is new motivation to move IHC to a fully quantitative assay. Toward this goal, we have developed the AQUA® method of quantitative immunofluorescence (QIF) to measure critical analytes in breast tissue. Using a series of cell lines which are standardized to recombinant protein quantified on western blot, we have measured estrogen receptor in three separate cohorts of breast cancer cases. The quantitative approach reveals a cutpoint of 50pg/ug total protein and suggests that conventional IHC may have a misclassification rate of around 15%. Furthermore, when comparing the misclassified cases, it appears that QIF classification predicts outcome more accurately than the conventional methods. As accurate as QIF is, it does not address preanalytical variables, most significantly including cold ischemic time, or the time between surgical removal of the tissue sample and fixation. A series of QIF methods for antibody validation may allow neutralization of this variable in assessment of tumor tissue. In summary, measurement of protein on a pathology slide can now be achieved with accuracy and reproducibility of nucleic acid assays or ELISA assays. Combining QIF with rigorous methods of standardization and antibody validation allows companion diagnostic tests to be done on very small tissue fragments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):ED01-04.