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Background Circulating cell‐free tumor DNA (ctDNA)‐based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next‐generation sequencing platform in a large series of patients with non–small cell lung cancer (NSCLC). Methods Plasma‐based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co‐occurrence, and mutual exclusivity. Results Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%‐97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment‐induced resistance mutations were common in this cohort, including driver‐dependent and driver‐independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard‐of‐care, tissue‐based testing at diagnosis. A pooled data analysis on this plasma‐based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis. Conclusions Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies., Circulating cell‐free tumor DNA‐based liquid biopsy using next‐generation sequencing detects a spectrum of targetable alterations at frequencies expected in patients with advanced non–small cell lung cancer. These findings support the concept of a plasma‐first algorithm at the time of progression on targeted therapy for this population.

Purpose:To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing.Experimental Design:Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated.Results:cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit.Conclusions:cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887

Background Because cell-free DNA (cfDNA) analysis facilitates the noninvasive genomic profiling of metastatic castration-resistant prostate cancer (mCRPC), the authors evaluated the association between cfDNA alterations and outcomes and evolution with therapy. Methods Patients with mCRPC underwent cfDNA genomic profiling using Guardant360, which examines major cancer-associated genes. Clinical factors, therapy information, failure-free survival, and overall survival (OS) were obtained for select patients. The association between genomic alterations and outcomes was investigated. Results Of 514 men with mCRPC, 482 (94%) had ≥1 circulating tumor DNA (ctDNA) alteration. The most common recurrent somatic mutations were in TP53 (36%), androgen receptor (AR) (22%), adenomatous polyposis coli (APC) (10%), neurofibromin 1 (NF1) (9%), epidermal growth factor receptor (EGFR), catenin beta-1 (CTNNB1), and AT-rich interactive domain-containing protein 1A (ARID1A) (6% each); and BRCA1, BRCA2, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (5% each) The most common genes with increased copy numbers were AR (30%), MYC (20%), and BRAF (18%). Clinical outcomes were available for 163 patients, 46 of whom (28.8%) were untreated for mCRPC. A higher number of ctDNA alterations, AR alterations, and amplifications of MYC and BRAF were associated with worse failure-free survival and/or OS. On multivariable analysis, MYC amplification remained significantly associated with OS. Prior therapy and serial profiling demonstrated the evolution of alterations in AR and other genes. Conclusions ctDNA frequently was detected in this large cohort of "real-world" patients with mCRPC, and the alterations appeared to be similar to previously reported tumor tissue alterations. A higher number of alterations, and AR and MYC alterations, appear to compromise clinical outcomes, suggesting a role for immune checkpoint inhibitors and novel AR and BET inhibitors in selected patients.

Clinical studies support the efficacy of programmed cell death 1 (PD-1) targeted therapy in a subset of patients with metastatic gastric cancer (mGC). With the goal of identifying determinants of response, we performed molecular characterization of tissues and circulating tumor DNA (ctDNA) from 61 patients with mGC who were treated with pembrolizumab as salvage treatment in a prospective phase 2 clinical trial. In patients with microsatellite instability-high and Epstein–Barr virus-positive tumors, which are mutually exclusive, dramatic responses to pembrolizumab were observed (overall response rate (ORR) 85.7% in microsatellite instability-high mGC and ORR 100% in Epstein–Barr virus-positive mGC). For the 55 patients for whom programmed death-ligand 1 (PD-L1) combined positive score positivity was available (combined positive score cut-off value ≥1%), ORR was significantly higher in PD-L1(+) gastric cancer when compared to PD-L1(−) tumors (50.0% versus 0.0%, P value

Noninvasive genomic profiling of tumors may be possible with next-generation sequencing (NGS) of blood-derived circulating tumor DNA (ctDNA), but proof of concept in a large cohort of patients with diverse cancers has yet to be reported. Here we report the results of an analysis of plasma-derived ctDNA from 670 patients with diverse cancers. The tumors represented in the patient cohort were mainly gastrointestinal (31.8%), brain (22.7%), or lung (20.7%). ctDNA obtained from most patients [N = 423 (63%)] displayed at least one alteration. The most frequent alterations seen, as characterized mutations or variants of unknown significance, occurred in TP53 (32.5% of patients), EGFR (13%), KRAS (12.5%), and PIK3CA (9.1%); for characterized alterations, 30.7% (TP53), 7.6% (EGFR), 12.2% (KRAS), and 7.7% (PIK3CA). We found that 32% of brain tumors had at least one ctDNA alteration. Head and neck tumors were independently associated with a higher number of alterations in a multivariable analysis (P = 0.019). Notably, 320/670 (48%) of patients displayed potentially actionable alterations, with 241 patients possible candidates for on-label or off-label treatment with an FDA-approved drug. Several illustrations of the clinical utility of the information obtained for improving treatment of specific patients is provided. Our findings demonstrate the feasibility and impact of genomic profiling of tumors by ctDNA NGS, greatly encouraging broader investigations of the application of this technology for precision medicine in cancer management. Cancer Res; 77(19); 5419–27. ©2017 AACR.

Purpose: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies. Experimental Design: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA). Results: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18–91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1–13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%–55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20–38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04). Conclusions: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

Background: Inactivating mutations in homologous recombination repair (HRR) genes are associated with a response to PARP inhibitors and platinum-based therapies. However, secondary reversion mutations in HRR genes remain a major mechanism of resistance. We sought to characterize the prevalence and spectrum of these reversions in advanced cancer patients in BRCA1/2 and other HRR genes, such as ATM and CDK12. Methods: We analyzed cfDNA from >75,000 late-stage cancer patients previously tested as part of routine clinical care using Guardant360®, a 73- to 74-gene panel. This assay detects single-nucleotide variants (SNVs), indels, fusions, and copy-number alterations in cfDNA with a reportable range of ≥0.04%, 0.02%, 0.04%, and ≥2.12 copies, respectively. Secondary reversion mutations were identified by re-analysis using a bespoke HRR deficiency (HRD) Module, which annotates SNV/indels and calls multi-exon deletions and copy-number loss with a 95% limit of detection of ≥ 0.1% and 10 to 25% tumor fraction, respectively. Results: Among >20,000 breast, ovarian, prostate and pancreatic cancer patients, deleterious germline or somatic mutations were detected in BRCA1 (2.7%), BRCA2 (4.8%), ATM (2.9%) and CDK12 (1.7%). The majority of reversions occurred in BRCA1/2-mut (8.7%), compared to 0.07% of CDK12-mut and none in ATM. Reversions showed selective gene and allele representation. In breast cancer, reversions in BRCA2-mut patients were twice as frequent as those in BRCA1-mut (12.9% vs 5.5%, p-value=0.0046), while the near opposite was true for ovarian (7.6% vs 12.8%, p-value=0.73). In pancreatic cancer (>7,500 patients), reversions presented exclusively in BRCA2-mut (8.4%, n=131 vs 0 in BRCA1-mut, n=63). CDK12 reversions were relatively rare: 1 detected in >5,000 prostate cancer patients. Across all genes, the majority of reversions were small indels while 18% of reversions removed the exon entirely. Pathogenic hotspots BRCA2 codons A938 and S1982 - accounted for ≥10% of reversions and were more likely to revert compared to other deleterious sites (p-value Conclusions: cfDNA has been previously shown to detect BRCA1/2 germline, somatic and polyclonal reversion mutations acquired over PARP and platinum-based therapy. We demonstrate that the Guardant HRD Module can robustly identify these reversions in the cfDNA of metastatic patients. We emphasize that reversions were frequent among BRCA1/2-mut breast, ovarian, prostate and pancreatic cancers, with preferential enrichment based on gene and tumor type. Understanding resistance patterns may aid in the treatment selection for progressing patients. Further work is needed to define these ideal treatment options. Citation Format: Yu Fu, Jennifer Yen, Alexander Aartyomenko, Sante Gnerre, Caroline Weiport, Elena Helman, Arielle Yablonovitch, Stephen Pettitt, Christopher Llord, Andrew Tutt, Rebecca Nagy, Justin Odegaard, Darya Chudova, AmirAli Talasaz. Reversion patterns in homologous recombination repair genes in metastatic cancer patients profiled by cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 25.

Background: Estrogen receptor (ER)-α is expressed in about 70% of breast cancers and drugs that target the receptor function, selective estrogen receptor modulators (SERM) and aromatase inhibitors (AIs) represent the standard of care for patients (pts) with ER+ breast cancer. Nevertheless, prolonged exposure to endocrine therapy may result in acquired resistance and subsequent progression of disease. Recent evidence showed that activating mutations (muts) in the ligand-binding domain of ER-α occur in approximately 20% of pts exposed to endocrine therapies and those genomic abnormalities may represent the driver of endocrine resistance. In this context, ctDNA provides a non-invasive source for real-time next generation sequencing (NGS) studies, in order to understand the biology of ABC and guide and monitor treatment. Methods: We conducted a retrospective review of 91 pts with ABC, including 57 pts with ER+ tumor, who had longitudinal assessment of their disease by ctDNA analysis. At the time of baseline sampling, 50/57 pts had stage IV cancer. The total number of blood samples collected was 184. 38 (67%) pts had serial samples. The average number of samples for each pt was 3 (range 1-7). The plasma-based assay was performed utilizing Guardant360 (Guardant Health, CA), a digital NGS technology to sequence a panel of > 50 cancer genes. Results: Among the ER+ subgroup (57 pts), we identified 11 pts (19%) harboring ESR1 muts in ctDNA. All 11 pts had metastatic disease: 2 (18%) had bone metastases, 2 (18%) had visceral metastases, 7 (64%) had both sites of disease. The median age was 55 years (range 33-73). 5 pts had inflammatory breast cancer. The most common ESR1 muts were: Y537S (6/11, 55%), D538G (4/11, 36%) and Y537N (3/11, 27%). 7 pts carried polyclonal muts. At the time of testing, 10 pts had already failed at least 1 line of endocrine therapy (average 2, range 1-5), including 6 pts that had received a fulvestrant-containing regimen, 8 pts ≥ 1 line of AIs. After the mut detection, 5 pt were on endocrine therapy and 4 pts were started on/continued chemotherapy. ESR1 muts disappeared in 2 pts (fulvestrant-palbociclib and chemotherapy respectively) who achieved stable disease as best response. Three pts continued to harbour muts and then progressed (one died). 2 pts had tissue NGS and ESR1 mut was not identified. Progression free survival and overall survival were 8 months (ms) and 21.5 ms in ESR1+ subpopulation versus 6.2 ms and 22.2 ms in the ESR1- pts (p = 0.78 and p = 0.97, respectively). At the time of analysis 5 pts were dead, 6 were currently alive. ESR1+ (n. pts) ESR1- (n. pts) Pts (total n.)1146 Previous chemotherapies11 (100%)31 (67%) Previous fulvestrant-containing regimens6 (54%)20 (43%) Previous AIs ± targeted therapy8 (73%)27 (59%) Conclusions: We observed that ESR1 muts, a known driver of endocrine resistance, occurs at a high frequency in heavily pre-treated estrogen receptor positive ABC. Blood-based diagnostics can be used to identify ESR1 muts sometimes not detected by tissue-based sequencing of the metastatic lesions indicating tumor heterogeneity and allowing dynamic monitoring of ABC. Citation Format: Rossi G, Lima Barros Costa R, Nagy RJ, Rademaker AW, Gradishar WJ, Santa-Maria CA, Curry-Edwards RL, Jain S, Flaum LE, Zagonel V, Platanias LC, Giles FJ, Talasaz A, Cristofanilli M. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA): A guide to the management of advanced breast cancer (ABC) [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 P1-05-06.

// Victoria Villaflor 1 , Brian Won 2 , Rebecca Nagy 3 , Kimberly Banks 3 , Richard B. Lanman 3 , AmirAli Talasaz 3 , Ravi Salgia 4 1 Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 2 University of Chicago School of Medicine, MC 2115, Chicago, IL 60637, USA 3 Guardant Health, Redwood City, CA 94063, USA 4 City of Hope, Duarte, CA 91010, USA Correspondence to: Victoria Villaflor, email: victoria.villaflor@nm.org Ravi Salgia, email: rsalgia@coh.org Keywords: circulating tumor DNA, cell-free DNA, non-small cell lung cancer, next-generation sequencing Received: April 17, 2016 Accepted: August 13, 2016 Published: September 01, 2016 ABSTRACT Introduction: The potential of oncogene-driven targeted therapy is perhaps most fully realized in non-small cell lung cancer (NSCLC), given the number of genomic targets and approved matched therapies. However, invasive tissue biopsy at the time of each disease progression may not be possible and is associated with high morbidity and cost. Use of newly available “liquid biopsies” can circumvent these issues. Results: 83% of subjects had at least one genomic alteration identified in plasma. Most commonly mutated genes were TP53 , KRAS and EGFR . Subjects with no detectable ctDNA were more likely to have small volume disease, lepidic growth pattern, mucinous tumors or isolated leptomeningeal disease. Methods: Subjects were individuals with NSCLC undergoing analysis of cell-free circulating tumor DNA using a validated, commercially-available next-generation sequencing assay at a single institution. Demographic, clinicopathologic information and results from tissue and plasma-based genomic testing were reviewed for each subject. Conclusions: This is the first clinic-based series of NSCLC patients assessing outcomes of targeted therapies using a commercially available ctDNA assay. Over 80% of patients had detectable ctDNA, concordance between paired tissue and blood for truncal oncogenic drivers was high and patients with biomarkers identified in plasma had PFS in the expected range. These data suggest that biopsy-free ctDNA analysis is a viable first choice when the diagnostic tissue biopsy is insufficient for genotyping or at the time of progression when a repeated invasive tissue biopsy is not possible/preferred.

Purpose: On the basis of preclinical evidence of synergistic activity between MEK and EGFR inhibitors in pancreatic ductal adenocarcinoma (PDAC), we evaluated the safety and efficacy of selumetinib, a MEK1/2 inhibitor, plus erlotinib in patients with previously treated advanced PDAC. Experimental Design: In this single-arm phase II trial, eligible patients received the combination of erlotinib 100 mg plus selumetinib 100 mg daily in 3-week cycles. Study assessments included measurement of clinical outcomes, with a primary endpoint of overall survival, and exploration of potential molecular predictors of treatment benefit. Results: Forty-six patients were enrolled and received a median of two cycles (range, 1–7). Although no objective responses were observed, 19 patients (41%) showed evidence of stable disease for ≥6 weeks, and 13 of 34 patients (38%) had a CA19-9 decline ≥50%. Median progression-free survival was 1.9 months [95% confidence interval (CI), 1.4–3.3 months], with a median overall survival of 7.3 months (95% CI, 5.2–8.0 months). Common adverse events included rash, diarrhea, and nausea/vomiting. Patients with tumors exhibiting an epithelial phenotype (demonstrated by a high level of E-cadherin expression) were more likely to be sensitive to study treatment. Tumor-derived DNA was detectable in plasma from the majority of patients using next-generation digital DNA sequencing, and its relative abundance correlated with tumor burden. Conclusions: A therapeutic strategy of dual targeted inhibition of the MEK and EGFR pathways shows modest antitumor activity in pancreatic cancer. Specific molecular subtypes may derive greatest benefit from this combination. Further exploration, both with more potent MEK inhibitors and in molecularly enriched patient subsets, is warranted. Clin Cancer Res; 22(1); 61–68. ©2015 AACR.

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

Background Major guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise. Patients and Methods We retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cell-free circulating DNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel. Results In the overall cohort, 467 patients (94.9%) had a diagnosis of LUSC, and 25 patients (5.1%) had mixed histology with a squamous component. A total of 10.5% of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8%), ALK/ROS1 (1.3%), BRAF (1.5%), and MET amplification or exon 14 skipping (5.1%). Sixteen percent of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response. Conclusion In this large, real-world LUSC cohort, we observed a clinically significant prevalence of actionable alterations. Accurate local histopathologic assessment in advanced-stage LUSC can be challenging. Further evaluation of the genomic landscape in this setting is warranted to potentially identify underappreciated treatment options.

“Liquid biopsy” approaches analyzing cell-free DNA (cfDNA) from the blood of patients with cancer are increasingly utilized in clinical practice. However, it is not yet known whether cfDNA sequencing from large cohorts of patients with cancer can detect genomic alterations at frequencies similar to those observed by direct tumor sequencing, and whether this approach can generate novel insights. Here, we report next-generation sequencing data from cfDNA of 1,397 patients with colorectal cancer. Overall, frequencies of genomic alterations detected in cfDNA were comparable to those observed in three independent tissue-based colorectal cancer sequencing compendia. Our analysis also identified a novel cluster of extracellular domain (ECD) mutations in EGFR, mediating resistance by blocking binding of anti-EGFR antibodies. Patients with EGFR ECD mutations displayed striking tumor heterogeneity, with 91% harboring multiple distinct resistance alterations (range, 1–13; median, 4). These results suggest that cfDNA profiling can effectively define the genomic landscape of cancer and yield important biological insights. Significance: This study provides one of the first examples of how large-scale genomic profiling of cfDNA from patients with colorectal cancer can detect genomic alterations at frequencies comparable to those observed by direct tumor sequencing. Sequencing of cfDNA also generated insights into tumor heterogeneity and therapeutic resistance and identified novel EGFR ectodomain mutations. Cancer Discov; 8(2); 164–73. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 127

Purpose Retrospective studies have demonstrated that cell-free circulating tumor DNA (ctDNA) hotspot testing predicts matched therapy response to first- and second-line therapies in patients with advanced non–small-cell lung cancer (NSCLC). However, no prospective outcomes studies have evaluated ctDNA-guided matched therapy decision making on the basis of comprehensive plasma genomic testing including all four major classes of alterations. Here, we report the clinical utility of this approach in advanced solid tumor cancers. Patients and Methods We conducted a multiple parallel cohort, open-label, clinical trial using ctDNA-guided matched therapy when tissue was insufficient or unobtainable for next-generation sequencing. Plasma-based digital sequencing identified point mutations in 70 genes and indels, fusions, and copy number amplifications in selected genes. Patients with prespecified targetable alterations in metastatic NSCLC, gastric cancer (GC), and other cancers were matched to several independent targeted agent trials at a tertiary academic center. Results Somatic alterations were detected in 59 patients with GC (78%), and 25 patients (33%) had targetable alterations ( ERBB2, n = 11; MET, n = 5; FGFR2, n = 3; PIK3CA, n = 6). In NSCLC, 62 patients (85%) had somatic alterations, and 34 (47%) had targetable alterations ( EGFR, n = 29; ALK, n = 2; RET, n = 1; ERBB2, n = 2). After confirmation of ctDNA findings on tissue (to meet trial eligibility criteria), 10 patients with GC and 17 patients with NSCLC received molecularly matched therapy. Response rate and disease control rate were 67% and 100%, respectively, in GC and 87% and 100%, respectively, in NSCLC. Response was independent of targeted alteration variant allele fraction in NSCLC ( P = .63). Conclusion To our knowledge, this is the first prospective feasibility study of comprehensive ctDNA-guided treatment in advanced GC and lung cancers. Response rates in this interim analysis are similar to those in tissue-based targeted therapy studies.

Background About 65% of breast cancers express the estrogen receptor α and the mainstay of treatment are therapies that result in estrogen receptor modulation (selective estrogen receptor modulators, SERMs) or estrogen deprivation (aromatase inhibitors, AIs). Even though endocrine therapy has resulted in reduced recurrence and mortality, a significant portion of patients relapse with metastatic disease and subsequently progress while on therapy for advanced disease (endocrine resistance). Recent evidence showed that activating hot spot mutations in the ligand binding domain of the ERα (ESR1) are acquired on treatment (frequency of 20%) and can drive resistance to endocrine therapy, especially AIs. ESR1 mutations can be detected by evaluation of circulating tumor DNA (ctDNA), a method where circulating DNA fragments with tumor-specific sequence alterations are identified in the blood of patients. Methods This is a retrospective evaluation of 9 patients with hormone receptor positive (HR+) metastatic breast cancer (MBC) who had progressed on multiple lines of endocrine therapy (ET) and were found to have ESR1 mutations in ctDNA. Patients had blood drawn for ctDNA analysis either at progression to serve as a baseline before starting a new regimen or to monitor response to ongoing treatment. Guardant360™(Guardant Health) involves ctDNA isolation from plasma using a Qiagen circulating nucleic acid kit, then a panel of 68 gene mutations associated with solid tumors as reported in the COSMIC database sequenced using single-molecule digital sequencing technology. Results All of the patients had MBC and were luminal subtype except for one HER2+, and most had invasive ductal carcinoma although 2 patients were invasive lobular carcinoma (22%). Most patients had both bone and visceral involvement (78%), only two patients had bone only metastasis. The patients were generally heavily pretreated with an average of 3 lines of ETs and 6 lines of therapy altogether (chemotherapy + ET). Duration on endocrine therapy ranged from 23 months to 7 years (mean 4.3 years). All patients were found to have ESR1 mutations on ctDNA, the range of percentage of mutant allele was 0.28-23.76%. Three patients had tissue sent for NGS and none of the tissue samples had an ESR1 mutation detected although they were biopsied at various time points in treatment. One of those patients had two ESR1 mutations in ctDNA, which were not detected on tissue sent for NGS one year prior, and had not been on ET for several years. One patient with abdominal carcinomatosis from lobular carcinoma who had been on ETs therapies for 6 years was found to have 4 distinct ESR1 mutations in a single blood draw, suggesting sub-clonal evolution of resistance. One patient also had 5 circulating tumor cells, all of which had ESR1 mutations detected when circulating tumor cells were individually sequenced. Conclusions ctDNA is a sensitive test for detection of ESR1 in HR+ MBC patients, with the advantage of being a blood based assay which lends itself to serial analysis. In this patient population ctDNA can be a helpful tool to predict response to ET and predict treatment failure. Citation Format: Austin L, Rodriguez A, Jaslow R, Fortina P, Nagy R, Zill O, Talasaz A, Cristofanilli M. Detection of activating estrogen receptor 1 (ESR1) in circulating tumor DNA (ctDNA) in hormone-receptor positive metastatic breast cancer (MBC). [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 P3-05-02.

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.

Background: Molecular diagnostic, in particular next-generation sequencing (NGS) technologies, improved the detection of actionable mutations (muts) in MBC at baseline and recurrence. We evaluated the ability of ctDNA to detect molecular abnormalities, monitor disease progression and predict outcome. Methods: We conducted a retrospective study of 91 patients (pts) with locally advanced and MBC, who had longitudinal assessment of their disease by ctDNA analysis. The plasma-based assay was performed utilizing Guardant360 (Guardant Health, CA), a digital NGS technology to sequence a panel of > 50 cancer genes. After tabulating number of muts and quantification of overall ctDNA detected for every patient at baseline, a receiver operating characteristic (ROC) analysis was performed to identify the best cut-offs that separated the pts who had a disease progression from those who hadn't, and the patients who died from those still alive. The overall survival (OS) analysis has been performed using Kaplan-Meier curves. Results: 84 pts (92%) had stage IV cancer. 63% cases were ER+, 27% HER2+, 29% TNBC. 277 blood samples were collected and 84% had muts. 65% of the pts had serial samples. The average number of alterations detected in each sample was 3 (0-27) and the average ctDNA fraction detected was 4.5% (0-88.2%). The most common alterations were: TP53 (52%), PIK3CA (40%), ERBB2 (20%), NOTCH1 (15.5%), APC (14%), MET (13%). 16 pts (19%) were initiated on a targeted therapy based on ctDNA test results. At the time of analysis 36 pts (39.6%) were dead, 55 (60.4%) were currently alive. PFS was 5.2 months (ms) and OS was 21.5 ms. A statistically significant difference in PFS and OS by log rank test was found between % ctDNA at baseline < 0.5 versus ≥ 0.5 (p = 0.003 and p = 0.012, respectively) and number of muts at baseline < 2 versus ≥ 2 (p = 0.059 borderline and p = 0.0015). Moreover, a statistically significant association by Fisher's exact test was found between the number of alterations and the % ctDNA detected in the baseline sample (% of pts with muts ≥ 2 was 19% when % ctDNA < 0.5%, versus 85% when % ctDNA ≥ 0.5%; p < 0.0001). PFS (ms) p = 0.059 (log rank test)Muts < 2 (n = 32)Muts ≥ 2 (n = 58)658%40%1230%13%1821%6%24--PFS(ms) p = 0.003 (log rank test)% ctDNA < 0.5(n = 27)% ctDNA ≥ 0.5(n = 60)665%39%1241%10%1823%6%24-- OS(ms) p = 0.002 (log rank test)Muts < 2(n = 32)Muts ≥ 2(n = 57)697%66%1288%51%1888%42%24-29%OS(ms) p = 0.012 (log rank test)% ctDNA < 0.5(n = 27)% ctDNA ≥ 0.5(n = 59)696%69%1290%55%1885%48%24-35% Conclusions: ctDNA liquid biopsy provides a real-time, quantitative NGS-based assessment of MBC which is useful for treatment planning, disease monitoring and prognostic evaluation. Future prospective studies should consider the use of ctDNA for molecular and prognostic stratification. Citation Format: Rossi G, Austin LK, Nagy RJ, Rademaker AW, Gradishar WJ, Santa-Maria CA, Curry-Edwards RL, Jain S, Flaum LE, Lima Barros Costa R, Zagonel V, Platanias LC, Giles FJ, Talasaz A, Cristofanilli M. Circulating tumor DNA (ctDNA): A real-time application of precision medicine to the management of metastatic breast cancer (MBC) [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 PD1-02.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Background Circulating cell-free DNA (cfDNA) isolated from the plasma of cancer patients (pts) has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC) has not been previously described. Methods Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC]), with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE) tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility. Results 128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128) of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30) of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents. Conclusions cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.

s / 60 (2016) e1ee19 e5 AOS11. CIRCULATING CELL-FREE DNA PROFILING OF PATIENTS WITH ADVANCED UROTHELIAL CARCINOMA G. Sonpavde*, R.J. Nagy, S. Varambally , R. Lanman, A. Talasaz. University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL, USA. Guardant Health, Inc, Redwood City, CA, USA Background: Circulating cell-free DNA (cfDNA) is obtained noninvasively from peripheral blood and appears to be detectable in most patients with advanced urothelial carcinoma. We report cfDNA profiling of patients with advanced urothelial carcinoma. Methods: 50 patients with urothelial carcinoma that underwent cfDNA analysis using Guardant360 were identified. The 68-gene cfDNA next generation sequencing (NGS) panel from Guardant360 offers complete sequencing for all exons in 29 genes and critical exons in 39 other genes, and copy number amplifications (16), fusions (four), and indels (one) in selected potentially actionable genes harvested from 20 ml of peripheral blood. Findings: Of 50 patients with urothelial carcinoma, cfDNA was detectable in 44 patients (88%). Of these, 39 patients had at least muscle-invasive disease (21 of these had reported metastatic disease) and 11 had no stage information. The most common genes with recurrent somatic mutations in the 44 patients with at least one alteration were TP53 (n Z 26), ARID1A (n Z 10), BRCA2, FGFR2, NF1 (n Z 8 each), BRCA1, CDKN2A, EGFR, PDGFR (n Z 5 each), FGFR3, APC, MET, B-RAF (n Z 4 each), PTEN, ERBB2, K-RAS (n Z 3 each), ALK, GNAS, AR, PIK3CA, NOTCH1, ROS1, ATM, and IDH2 (n Z 2 each). The most common genes with recurrent amplifications were EGFR and ERBB2 (n Z 2 each). Analysis of additional patients is ongoing in this continuously expanding dataset. Interpretation: cfDNA is frequently detected in patients with muscle-invasive and advanced urothelial carcinoma, and alterations appear similar to those reported from urothelial carcinoma tumour tissue. Given that cfDNA offers a non-invasive means of profiling tumour DNA, further development of this promising modality is warranted to guide precision medicine. Additionally, serial cfDNA monitoring may provide insights regarding tumour biology. http://dx.doi.org/10.1016/j.ejca.2016.03.024 AOS12. FUNCTIONAL ACTIVITY OF KINASES IN MUSCLE-INVASIVE BLADDER CANCER TO IDENTIFY POTENTIALLY ACTIONABLE THERAPEUTIC TARGETS G. Sonpavde*, J.C. Anderson, G. Naik, W.E. Grizzle , C.D. Willey. University of Alabama at Birmingham (UAB) Comprehensive Cancer Center, Birmingham, AL, USA. UAB Department of Radiation Oncology, Birmingham, AL, USA. UAB Department of Pathology, Birmingham, AL, USA Background: The Cancer Genome Atlas (TCGA) provided insights into muscle-invasive bladder cancer (MIBC) biology by studying mutations and amplifications in genes, gene expression, andproteins.However, the functional significance of the identified alterations in kinase genes such as those encoding FGFR3, PI3K/AKT/mTOR, and MAPK is unclear. We studied protein kinase activity inMIBC tissue using a novel platform. Methods: We measured kinase activity level using the multiplex substrate microarray PamGene platform (Netherlands) in 24 fresh frozen tissue samples of MIBC and 24 matched adjacent normal bladder tissue samples in patients undergoing radical cystectomy. The PamGene assay measures the ability of lysates from fresh frozen tissue to phosphorylate substrate peptidesandcalculates theupstreamkinases that are functionally active. Kinomic profiling was analysed using software including Evolve (PamGene), and BioNavigator (PamGene) for raw data transformation into kinetic and steady state values. The upstream kinases were identified by using the UpKin PamApp (PamGene) based on the Kinexus database. Clustering of kinases was done in both unpaired and paired manner in cancerous versus healthy tissue specimens. The tumour specimens were compared with matched healthy bladder tissue specimens using the t test. Findings: 23 of 24 patients’ tissue specimens were evaluable since they had acceptable kinase activity. The three kinases with the highest tumour tissue activity (YES1, HCK, and BLK) were all members of the SRC family of tyrosine kinases. Other kinases such as EGFR, PDGFR, CDK1, and PKC also had increased kinase signalling in tumours compared with normal tissue. Among the key functionally activated pathways were RAS, CXCR4, JAK-STAT, CTLA-4, and hyaluronic acid/CD44. Interpretation: In this first innovative study to measure protein kinase activity in MIBC, multiple potential drivers and therapeutic targets were identified. Our data complement studies that measure genomic and transcriptomic kinase alterations and warrant further confirmation to yield therapeutic advances. http://dx.doi.org/10.1016/j.ejca.2016.03.025 AOS13. ANALYSES OF COMBINED PROSTATE CANCER IN RESECTED SPECIMENS BY TOTAL CYSTECTOMY DUE TO URINARY BLADDER CANCER Y. Adachi *, T. Magaribuchi, Y. Nakano, N. Takao, J. Watanabe, T. Shirahase, T. Sakatani , Y. Taki , H. Takeuchi, M. Li, S. Ikehara. Toyooka Hospital, Toyooka, Hyogo, Japan. Kansai Medical University, Hirakata, Osaka, Japan. Hirakata Hospital, Kansai Medical University, Hirakata, Osaka, Japan Background: Presently, malignant tumours are the number one cause of death in Japan, and the number of patients with various cancers is increasing. Prostate cancer is the sixth leading cause of death in men in Japan, with a death rate of 18.9 per 100,000 men. It has been reported that rates in latent prostate cancer are 21.7%, 34.7%, and 50% for men in their 60s, 70s, and 80s or older, respectively. Methods: In this study, we analysed specimens of total cystectomy for evidence of prostate cancer. We focused on 40 cases of total cystectomy at our hospital between 2010 and 2015. The ages of the patients from whom the specimens were obtained ranged from 52 to 92 years (mean 73.5 9.27 years). Findings: 28 (70%) of the 40 cases of total cystectomy showed evidence of prostate cancer. The proportions of patients that underwent cystectomy that were found to also have prostate cancer in the defined age groups (50s, 60s, 70s and 80s and older) were 40% (2/5), 50% (3/6), 75% (15/20), and 89% (8/9), respectively. Interpretation: To our knowledge, the proportion of patients with bladder cancer who also had prostate cancer was high in comparison with any previous data. http://dx.doi.org/10.1016/j.ejca.2016.03.026

Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Background Metastatic breast cancer (BC) is an incurable condition and treated with palliative intent. Standard diagnostic imaging and serum markers have limited sensitivity and are not recommended in clinical practice. Micrometastatic disease in the bone marrow (DTC) and peripheral blood (CTCs) is a recognized prognostic marker but with limited clinical utility. The detection of asymptomatic disease using a sensitive, reproducible and robust blood-based molecular test, or molecular residual disease (MoRD) could potentially represents a tool with the capability to design early therapeutic interventions and improve outcome. Circulating tumor DNA (ctDNA) has the potential to reflect residual tumor burden with higher diagnostic accuracy. We performed a pilot study in patients with high-risk primary BC. Methods This is a prospective study of 30 patients with either locally advanced BC who had completed primary therapy (21 patients) and had no evidence of disease (NED), or were metastatic but treated with curative intent and currently NED or stable (9 patients). Plasma was analyzed for ctDNA either after completing neo-adjuvant therapy (NAT), for recurrence monitoring after surgery, or when there was a clinical suspicion of recurrence. Guardant360™(Guardant Health) is a ctDNA next generation sequencing panel which produces a quantitative measurement of the mutant allele fraction for single nucleotide variants in 54 genes and copy number variants in 3 genes (panel was expanded to 68 genes in Feb '15) using digital sequencing technology. Results Baseline ctDNA analysis was done for 30 patients and 25 (83%) had serial draws for a total of 76 samples. All patients were stage 3-4 except for two stage 2 patients. ctDNA or MoRD was detected in 17 (57%) of patients and in 39 (51%) of the samples. Of the 18 patients treated with NAT, 11 achieved pCR or had minimal residual disease. Of these 11, six had no ctDNA detected after surgery, 3 had mixed results of no ctDNA and low volume ctDNA alterations on different draws, and 2 had persistent mutations on 2 draws. Ten of these 11 patients remain NED with median follow up of 24 months, while the one patient who recurred had persistent low volume missense ctDNA alterations on serial draws, first detected 6 months before clinically evident recurrence. Of the 7 patients with significant residual disease (less than PR) after NAT, 6 had post-surgical ctDNA detected and 5 have recurred at a median of 13 months after surgery. Four of those patients had ctDNA tested prior to recurrence and all had alterations detected in the blood prior to clinical recurrence. Lastly, one HER2+ metastatic patient treated with curative intent with a subsequent negative PET scan and no ctDNA detected after HER2-targeted therapy progressed on CT 2 months later and repeat ctDNA revealed EGFR mutant allele fraction of 51% and ERBB2 amplification. Conclusions The evaluation of ctDNA in high-risk BC patients can identify MoRD and predict for clinical recurrence. Patients with no or low volume ctDNA after primary treatment remained NED longer than those with multiple or high volume alterations. Future studies will validate these early observations and aid in selecting patients for additional systemic therapy with the hope of improving outcome. Citation Format: Austin L, Jaslow R, Fortina P, Nagy R, Zill O, Talasaz A, Cristofanilli M. Circulating tumor DNA (ctDNA) for detection of molecular residual disease (MoRD) in breast cancer. [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 P6-03-06.

We have developed a novel sample preparation technology for complete purification of circulating tumor cells (CTCs). The target cells are magnetically labeled with anti-EpCAM particles and then immunostained with anti-EpCAM antibodies. CTCs have been selectively captured by applying absorption magnetic forces and desorbing fluidic shear force to the cells (MagSweeper technology). CTCs, defined as EpCAM+ CD45- cells are then picked individually for nucleic acid analysis. We have utilized this platform for isolation of CTCs from prostate cancer patients. From each case, 7.5ml blood sample is processed within 24hr of collection. The purified CTCs have been validated to be EpCAM+ CD45- by immunostaining and qPCR analysis (91%± 6% specificity). So far we have isolated CTCs from 14 out of 35 cases. The CTC counts isolated by MagSweeper have correlated well with the counts obtained from the same cases using FDA-cleared CellSearch system. We are also developing new genomic technologies for whole-genome expression profiling of single CTCs. The purified CTCs are lysed under control conditions, and then processed with our whole transcriptome amplification (WTA) protocol. The amplified cDNA are used for SBS sequencing library preparation. Our WTA protocol has been validated through mRNA-sequencing of cultured single prostate LNCaP and bladder T24 cells. We found that more than 66% of all genes are reproducibly sequenced and the expression profile of single LNCaP and T24 cells correlated well within each cell line (R⁁2=0.75 for LNCaP and R⁁2=0.87 for T24 cells). We have sequenced the whole transcriptome of 7 CTCs and one leukocyte cell isolated from blood sample of a metastatic prostate cancer patient. We found that 7/7 CTC cells express KLK3, AR and EpCAM, 6/7 express CD24 and AMACR, 5/7 express MYC and 0/7 express CD45. On the other hand, the WBC does not express any of these genes except CD45. At present we are analyzing the data for the presence of fusion genes. So far we have not detected the TMPRSS2-ERG fusion gene in any of the CTCs analyzed; however, the search for looking into other known fusion genes is currently under progress. In addition, we clustered CTCs with LNCaP and T24 single cells. We found that the 5/7 CTC cells cluster together, and are different from LNcap or T24 clusters. 2/7 CTCs show different expression profile, demonstrating expected heterogeneity between CTCs. We have introduced a new end-to-end integrated workflow for CTC genomic analysis. The workflow has been successfully tested in isolation and mRNA-seq profiling of CTCs from prostate cancer patient samples. This novel workflow will open up new opportunities in studying CTC biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4856. doi:10.1158/1538-7445.AM2011-4856

We are developing novel sample preparation technologies for genomic analysis of circulating tumor cells (CTCs): a new cell-sorting device for purification of single CTCs from a tube of blood; and highly sensitive and quantitative genomic technologies for single cell analysis using Illumina's microarray and next-generation sequencing platforms. We have prototyped a new ultra-rare cell enrichment device that produces cell samples compatible with Illumina's nucleic-acid analysis platforms. Sorting is performed on whole blood samples with no pre-fractionation, to minimize the genetic alteration of CTCs. To validate the performance of our cell sorting device, we have monitored the recovery of LNCaP prostate cancer cells spiked into 7.5ml normal blood samples at concentration of 10 cells/7.5ml. For ease of analysis, the cancer cells were labeled with cell tracker dye and leukocyte nuclei in blood were labeled with Hoechst 33342 dye. Cancer cells were immunomagnetically labeled in blood with EpCAM beads and then isolated by our cell sorter. We found that the capturing efficiency of our platform was 60% ± 24% for 10 cells/7.5ml samples. The purity of cancer cells among contaminating white blood cells was 91% ± 6%, after a second round of extraction of individual cancer cells. The entire purification protocol of CTCs from 7.5ml blood samples takes 2 hours. This platform has been earlier validated for CTC isolation from blood samples of breast cancer patients and is currently under evaluation for prostate and ovarian cancer patients. We are also developing new genomic technologies for whole-genome expression profiling, somatic mutation analysis, and transcriptome sequencing of purified CTCs: a) using a Multiple Displacement Amplification (MDA)-based protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 and 5 LNCaP cells, respectively. We also detected the chromosomal amplification and loss-of-heterozygosity; b) with our current RNA amplification protocol, we were able to generate reproducible expression profiles, R2 = 0.37 and 0.75, from 1 and 10 cell inputs, respectively. In addition, the expression profiles correlated well with those obtained with standard 100 ng total RNA input, R2 = 0.36 and 0.72, respectively; c) we have also developed next-generation sequencing protocols to profile single-cell transciptomes. We have introduced a new platform for isolation of small numbers of CTCs from patient blood samples that are compatible with Illumina genomic assays. Our gDNA and RNA amplification protocols work with direct cell lysates; no need to extract DNA or RNA from single cells. The protocols are compatible with Illumina microarray and next-gen sequencing platforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1144.

We are developing highly sensitive and quantitative genomic technologies for genetic and expression analysis of single cells or trace amount of DNA/RNA materials, using microarray and next-generation sequencing platforms. We have tested various Multiple Displacement Amplification (MDA)-based protocols under a variety of reaction conditions. With our current protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 cell and 5 cells, respectively. We are also developing RNA amplification methods for high-throughput expression profiling of single cells. With our current protocol, we were able to generate reproducible expression profiles, R2 = 0.73 and 0.77, using 10 pg and 50 pg total RNA input, respectively. In addition, the profiles correlated well with those obtained with standard 100 ng total RNA input, R2 = 0.61 and 0.77, respectively. Our data show that sequencing of single-cell transcriptomes can clearly distinguish embryonic stem cells from embryonic fibroblasts and tumor cells. We are currently using these technologies to study medical specimens such as circulating tumor cells and cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1149.