Introduction: Diagnosis of leptomeningeal metastasis (LM) is limited by low sensitivity of cerebrospinal fluid (CSF) cytopathology. Detecting circulating tumor cells (CTCs) in CSF might be more sensitive. We evaluated if TargetSelector™ (TS), a novel assay that captures CTCs in peripheral blood, detects CTCs in CSF. Methods: We enrolled adults with metastatic solid tumors or primary CNS malignancies and clinical suspicion for LM to undergo lumbar puncture (LP) for CSF cytopathology and TS. TS captured CTCs using a primary 10-antibody mixture, streptavidin-coated microfluidic channel, and biotinylated secondary antibodies. CTCs from patients with metastatic breast cancer (MBC) were assessed for estrogen receptor (ER) expression by fluorescent antibody and HER2 amplification by fluorescent in situ hybridization (FISH). CSF cell-free DNA (cfDNA) was extracted for next-generation sequencing (NGS). Results: Fourteen patients, median age 56 years (range, 32-75), underwent diagnostic LP. Primary malignancies were breast (n=10), lung (n=1), colon (n=1), CNS lymphoma (n=1), and glioma (n=1). Among thirteen patients who underwent CSF evaluation by TS, TS had sensitivity of 83% (95% Confidence Interval [CI], 33-100%) and specificity of 86% (95% CI, 42-100%) for LM, defined as positive CSF cytology and/or unequivocal MRI findings. Among MBC patients, concordance of ER and HER2 status between CTCs and metastatic biopsy were 60% and 75%, respectively. NGS of CSF cfDNA identified somatic mutations in three MBC patients, including one with PIK3CA p.H1047L in blood and CSF. Conclusions: TargetSelector™ is a viable platform to detect CSF CTCs, with potential use as a diagnostic tool for LM. Additional, larger studies are warranted.

Abstract INTRODUCTION: Despite improvements in early detection, 1 in 8 women in the US (12%) will develop invasive breast cancer over the course of her lifetime. Approximately 20% of breast cancer is HER2 positive. During treatment and at disease progression, HER2 receptor conversion may occur. Once metastatic, it may be difficult to access multiple metastatic sites or perform serial biopsies. Therefore, accuracy of results may be sub-optimal as tissue biopsy is a single time point collection and limited by sampling (inter-tumoral and intra-tumoral heterogeneity). A liquid biopsy is a contemporaneous non-invasive and cost-effective method that allows for collection and analysis of tumor material and includes circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA). We compared prospectively the expression of HER2 in metastatic tumors to HER2 amplification in CTCs. METHODS: We enrolled patients with metastatic breast cancer in the Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE) II Study (NCT02965755). All patients regardless of subtype, had at least one line of therapy (chemotherapy, hormone therapy, or anti-HER2 therapy as appropriate). We analyzed HER2 status on tumor biopsies obtained 0-43 months (mean 7.3 months) prior to enrolling in IMAGE, and CTCs isolated from peripheral blood (PB) drawn ideally before starting a new treatment, 1-2 weeks after starting a new treatment and at the time of first restaging. CTCs were captured by Target Selector TM (Biocept) and analyzed for HER2 amplification by FISH. The biomarker expression profile on the metastatic tumor and CTCs were compared for each patient. Concordance of HER2 expression between CTCs and the metastatic tumor tissue was analyzed using McNemar’s test. RESULTS: For 36 evaluable patients, the specificity of HER2 on CTCs to tissue was 92.9% for PB samples collected within 5 weeks of the tumor biopsy and 100% at for PB samples collected between 5-10 weeks post biopsy, with overall concordance of 65% (independent of CTC collection time point), accuracy of 76.5% and specificity of 79.7%. A change in HER2 in amplification between the metastatic tumor and CTCs was noted in 36% (13/36) of patients with 7 patients HER2+ in tissue, HER2- on CTCs and 8 patients HER2- on tissue, HER2+ on CTCs. CONCLUSION: These data demonstrate high accuracy of HER2 amplification on CTCs at baseline and within 10 weeks of treatment and provide a sensitive and specific mechanism to monitor for changes in HER2 status that may be due to either tissue heterogeneity or receptor switch, a well-established phenomenon. This ability to effectively and contemporaneously monitor HER2 status on CTCs has the potential to identify patients who may benefit from the addition of anti-HER2 therapy and those are on anti-HER2 therapy who may not benefit optimally and for whom additional therapeutic options may warrant consideration. Citation Format: Vered Stearns, Jennifer Lehman, Christine Mitchell, Barbara Blouw, Lan Huynh, Veena Singh. Her2 expression in matched metastatic tumor and circulating tumor cells (ctcs) in breast cancer: Implications for profiling and monitoring of her2 status to help guide anti-her2 therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-14.

e14502 Background: Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with surgical biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal metastases (LM). Here we report the analytical and clinical validation of Target Selector CSF assays. Validation testing included pre-analytical and analytical steps. Methods: CSF was collected prospectively from pts with a prior solid tumor diagnosis and suspected BM or LM. CTCs were captured utilizing a primary ten antibody cocktail followed by biotinylated secondary antibodies that bind selectively to CTCs followed by staining with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs were captured in a microfluidic channel,classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) lung and breast multi-gene testing to assess for molecular alterations. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software. Results: The Target Selector CTC platform assays performed on clinical samples (n = 89) resulted in clinical accuracy = 85.4%, clinical precision (intra-assay, inter-assay, inter-operator, and inter-instrument) = 100% for each measure, clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0%, and analytical specificity = 96.0% (acceptance criteria was 95%) at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical accuracy = 87.4%, clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%. Conclusions: Target Selector is a viable, sensitive, reproducible platform for CTC detection and molecular analysis of CSF samples from patients with breast or lung cancer with CNS metastases especially as the sensitivity of CSF cytology is low and MRI findings can be equivocal. Identifying CTCs and molecular alterations can help characterize both tumor genomic evolution as well as guide treatment following cancer metastasis to the CNS.

Abstract BACKGROUND Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal disease (LMD). Here we report the analytical and clinical validation of Target Selector™ CSF assays. METHODS CSF was collected prospectively from pts with a prior solid tumor diagnosis and confirmed or clinical/ radiological suspicion of BM or LMD. CTCs were captured in microfluidic channel and classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) NSCLC and breast multi-gene testing. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software. RESULTS The Target Selector™ CTC platform assays performed on clinical samples (n = 89) resulted in clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0% at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%. CONCLUSIONS Target Selector™ is a viable and sensitive platform for CTC detection and molecular analysis of CSF samples from patients with NSCLC or breast cancer with CNS metastases compared to the current standard of care (CSF cytology) and when imaging findings are equivocal. Identifying CTCs and actionable biomarkers can help to confirm CNS involvement when clinically suspected, guide targeted therapy selection and potentially monitor treatment response.

Aims The identification of actionable DNA mutations associated with a patient's tumour is critical for devising a targeted, personalised cancer treatment strategy. However, these molecular analyses are typically performed using tissue obtained via biopsy, which involves substantial risk and is often not feasible. In addition, biopsied tissue does not always reflect tumour heterogeneity, and sequential biopsies to track disease progression (eg, emergence of drug resistance mutations) are not well tolerated. To overcome these and other biopsy-associated limitations, we have developed non-invasive 'liquid biopsy' technologies to enable the molecular characterisation of a patient's cancer using peripheral blood samples. Methods The Target Selector ctDNA platform uses a real-time PCR-based approach, coupled with DNA sequencing, to identify cancer-associated genetic mutations within circulating tumour DNA. This is accomplished via a patented blocking approach suppressing wild-type DNA amplification, while allowing specific amplification of mutant alleles. Results To promote the clinical uptake of liquid biopsy technologies, it is first critical to demonstrate concordance between results obtained via liquid and traditional biopsy procedures. Here, we focused on three genes frequently mutated in cancer:EGFR(Del19, L858, and T790),BRAF(V600) andKRAS(G12/G13). For each Target Selector assay, we demonstrated extremely high accuracy, sensitivity and specificity compared with results obtained from tissue biopsies. Overall, we found between 93% and 96% concordance to blinded tissue samples across 127 clinical assays. Conclusions The switch-blocker technology reported here offers a highly effective method for non-invasively determining the molecular signatures of patients with cancer.

3567 Background: Diagnosis of LM from solid tumors can be challenging. The TargetSelector (TS) CTC detection assay has demonstrated highly specific and sensitive CTC capture both for epithelial (CK+) and non-epithelial (CK-) subsets. The assay utilizes a ten-antibody (ab) capture cocktail followed by biotinylated secondary abs that bind to CTCs, enriched in a microfluidic device. TS targeted next-generation sequencing (NGS) assay detects somatic mutations in 12 breast cancer-related genes. The aim was to determine whether TS can improve sensitivity in the diagnosis of LM compared to CSF cytology by lumbar puncture (LP). Methods: CSF was collected prospectively from patients (pts) with a prior solid tumor diagnosis and suspicion of LM. CTCs were isolated from CSF using the TS platform. Cells were stained with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs captured in a microchannel were classified as CK + or -. Peripheral blood samples obtained at time of LP underwent similar CTC analysis. Cell-free total nucleic acids (cfTNA) were extracted from plasma and CSF followed by NGS. Data analysis used the Ion Torrent Suite with annotation and report curation by Ion Reporter and Oncomine Knowledgebase Reporter software respectively. Results: There were 14 pts (13 women and 1 man), median age 56 years (range 32-75) with cancers of the breast (10), lung (1), colon (1), CNS lymphoma (1) or glioma (1). Pts had received a median of 2.5 lines of systemic metastatic therapy (range 0-8). CSF cytology was not sent for 1 pt and TS was not performed for 1 pt. TS and standard cytology had 89% agreement in pts with metastatic breast cancer (MBC, 8/9). Of the 6 pts for whom CTCs were detected in CSF by TS, 3 pts had + cytology (all MBC), 2 pts had - cytology and 1 pt with MBC was not tested by cytology. Of the 3 pts with + CSF by cytology (all MBC), all were detected by TS (Table). Among 5 MBC pts with CTCs present in CSF, ER status was concordant in 2 of 5 (40%). HER2 status was concordant in 3 of 4 (75%) evaluable pts and not determined in 1 pt. Analysis of cfDNA from CSF identified somatic mutations in 3 pts (TP53, PIK3CA, CCND1, respectively). In 1 of 3 pts, the mutation identified in the CSF (PIK3CA) in HR+/HER2- MBC was also identified in the blood. Conclusions: TargetSelector is a viable platform for the detection of breast cancer CTCs in the CSF. NGS performed on CSF samples can identify potentially actionable mutations. [Table: see text]

Clinical Data in Over 1,500 Patients Across All Stages of Breast Cancer with Target Selector Circulating Tumor Cell Technology Deanna M Fisher B.S., Lan Huynh B.S., Edgar V. Sales B.S., Julie Ann Mayer Ph.D., and Veena M. Singh M.D. Introduction: The detection and molecular characterization of circulating tumor cells (CTCs) in patients with breast cancer affords the ability to profile and monitor patients in real time for progression, risk stratification, recurrence, identification of potentially actionable therapeutic targets, and monitoring of treatment efficacy and emergence of resistance mechanisms. To harness the promise of CTC analysis a highly sensitive, robust, reproducible and clinically validated technology is required. Information acquired from a single tissue biopsy has temporal and spatial limitations; additionally, in patients with progressive/metastatic disease, a single biopsy may not be informative or in some instances difficult to perform and might fail to reflect inherent tumoral heterogeneity. CTCs on the other hand can provide a contemporaneous landscape of all cancerous lesions (primary and metastases) as well as the opportunity to track the evolving tumor genetic mechanisms. Methodology:Samples were collected in CEE Sure blood collection tubes and buffy coat isolation was performed using a percoll density gradient separation. Capture in the microchannel was performed after incubation with a ten-antibody capture cocktail followed by the addition of a biotinylated secondary antibody. The microchannels were stained with pan-cytokeratin cocktail, CD45, pan-CTC stain, DAPI and the following protein biomarkers (AR, ER, PR, and PDL1). Following enumeration of the CTCs, the microchannels were subsequently sent for multi-color FISH biomarkers such as HER2. Results: 1,687 patients across all stages of breast cancer and treatment time points (pre- treatment, post-treatment, on treatment) were analyzed. CTCs were detected up to 70% of the time across early to late stage patients. The dynamic range of CTC detection and enumeration in a single microfluidic channel (8mL blood) ranged from 1 to 38,419 CTCs. CTC detection has been previously validated to a 1 CTC limit of detection. CTCs identified varied in both size and antigen profile. Target Selector CTC platform technology identifies both cytokeratin positive and cytokeratin negative phenotypes of CTCs. Biomarker interrogation was undertaken in all CTC subtypes by FISH probes (Fluorescent in situ hybridization) and ICC (Immunocytochemical) antibody clones similar to those used on tissue sections. Genomic alterations commonly detected in breast cancer (i.e. HER2 amplification, ER) were detected in all CTC subtypes. HER2 amplification was identified in 15% of cases with CTCs detected and ER in 24 %. Conclusions: This data demonstrates the ability of the Target Selector CTC Platform to accurately detect, enumerate, and interrogate for genomic alteration across a broad spectrum of CTC phenotypes spanning various clinical and treatment stages. This enables clinicians and patients a more real time option to profile and monitor disease. The Target Selector CTC technology allows for the analysis of a broader spectrum of CTC phenotypes thus increasing the likelihood of identifying actionable biomarkers for risk stratification, recurrence, potential treatment selection and monitoring for emergence of resistance. Citation Format: Deanna M Fisher, Lan Huynh, Edgar V Sales, Julie Ann Mayer, Veena M Singh. Clinical data in over 1,500 breast cancer patients across all stages of breast cancer with target selector circulating tumor cell technology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-13.

Background Personalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection. Methods The Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days. Results A total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence. Conclusions Highly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management.

*Correspondence: Veena M. Singh, Biocept, Inc., 5810 Nancy Ridge Drive, San Diego, CA, USA, Tel: 858-320-8203; Fax: 858-3208261; E-mail: vsingh@biocept.com Received Date: 10 Dec 2018 Accepted Date: 02 Jan 2019 Published Date: 08 Jan 2019 Citation: Erlich R, Vibat CRT, Singh VM. Demonstrated Clinical Utility of Target SelectorTM ctDNA Testing: Liquid Biopsy EGFR Mutation Detection Enabled Targeted Therapy Selection for Three Advanced NSCLC Patients. Clin Oncol. 2019; 4: 1567. Copyright © 2019 Veena M. Singh. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Article Published: 08 Jan, 2019

Abstract Background: Liquid biopsy has recently emerged as a minimally invasive and cost effective means to assess cancer biomarkers without the risk of surgical biopsy complications. Using a simple blood draw, circulating tumor cell (CTC) analysis is capable of providing information with implications for diagnostics, prognosis, and treatment decisions of various carcinomas. Serial CTC measurements can aid monitoring disease progression or response to therapy. Biocept's proprietary TargetSelector™ technology captures CTCs and provides assessment of enumeration, protein expression by immunofluorescence, and gene amplifications or translocations via FISH. There is limited data on clinical utility of CTCs in germ cell tumors (GCT). We aim to evaluate the prognostic and predictive significance of CTCs in refractory GCT in an ongoing clinical trial of brentuximab vedontin and bevacizumab in refractory CD 30+ GCT (NCT02988843). CTC enumeration will be performed at baseline, prior to cycle 3 of treatment, and at progression to monitor treatment response in blood from patients with refractory testicular cancer. Methods: Peripheral whole blood samples from refractory testicular cancer patients are collected into Biocept CEE-Sure™ blood collection tubes that are validated to preserve CTCs for up to 96 hours. Biocept's TargetSelector™ platform utilizes a proprietary antibody capture cocktail and microchannel enabling enrichment, enumeration, and CTC analyses which were performed at Biocept's CLIA-certified and CAP accredited laboratory. Results: From a patient with heavily pretreated refractory testicular cancer, serial blood collections were obtained at baseline and prior to cycle 3 (approximately six week interval). Two CTCs were detected at baseline. Prior to cycle 3, there was 1 CTC (corresponding to tumor marker response and CT scan showing stable disease). At progression after cycle 4, the CTC count increased to 4, corresponding with disease progression in tumor markers and CT scan. In this patient, the CTC capture utilizing the TargetSelector™ CTC technology was very predictive of clinical response and progression. Conclusions: Clinical application of Biocept's TargetSelector™ CTC technology enables the sensitive detection of CTCs in testicular cancer. Longitudinal CTC assessment implemented in the clinical setting can be used to assess drug responsiveness and follow disease progression in this rare disease, as well as in more common solid tumor cancers. The TargetSelector™ liquid biopsy platform provides an economical, non-invasive, and reliable means to arm physicians with valuable information for disease management and patient care. Ongoing collection of blood samples and data analysis from patients being enrolled on the study will provide us a better understanding of the potential clinical use of this novel technology in GCT patients. Citation Format: Shilpa Gupta, Benjamin L. Maughan, Cecile Rose T. Vibat, Veena M. Singh. TargetSelector™ CTC technology demonstrates clinical utility in monitoring treatment response in germ cell tumors (testicular cancer) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5597.

Initial diagnostic biopsy procedures often yield insufficient tissue for molecular testing, and invasive surgical biopsies can be associated with significant cost as well as risk to the patient. Liquid biopsy offers an alternative and economical means for molecular characterization of tumors via a simple peripheral blood draw. This case report describes the ability of liquid biopsy to detect an ALK translocation where tissue analysis by fluorescence in situ hybridization was negative for the genetic alteration. Identification of an ALK rearrangement in circulating tumor cells from a blood specimen led to sequential targeted therapies that included crizotinib followed by alectinib. The patient demonstrated outstanding clinical response during treatment with each of the prescribed ALK inhibitors. This case demonstrates the clinical utility of Biocept’s liquid biopsy to detect actionable biomarkers by surveying the systemic landscape of a patient’s disease where identification of the same genetic drivers may be missed in analyses of heterogeneous tumor tissue.

ROS1 is a receptor tyrosine kinase of the insulin receptor family, and ROS1 gene fusions are uncommon oncogenic drivers of NSCLC. Liquid biopsy represents a valuable alternative for molecular analyses when a traditional biopsy of the primary tumor yields insufficient tissue.1Vendrell J.A. Mau-Them F.T. Béganton B. Godreuil S. Coopman P. Solassol J. Circulating cell free tumor DNA detection as a routine tool for lung cancer patient management.Int J Mol Sci. 2017; 18: 264Crossref Scopus (71) Google Scholar Moreover, liquid biopsies can detect aberrations missed in tissue testing of heterogeneous tumors. Here, we report the pioneering detection of ROS1 rearrangements in circulating tumor cells (CTCs) in cases in which next-generation sequencing (NGS) of plasma failed to identify a genetic alteration. Lung adenocarcinoma in a right pleural effusion was diagnosed a 44-year-old male Hispanic nonsmoker. Molecular testing of collected fluid failed to reveal a genetic aberration. Palliative chemotherapy was initiated; it consisted of carboplatin/pemetrexed/bevacizumab for six cycles, followed by maintenance chemotherapy with pemetrexed/bevacizumab for 23 cycles. At the time of disease progression, the patient’s tumor was insufficient for further molecular tests. Blood analysis was performed using the VeriStrat test (Biodesix, Boulder, CO). The patient began second-line erlotinib therapy, which was continued for 22 months until disease progression with peritoneal carcinomatosis. NGS done on plasma failed to reveal actionable gene aberrations; a biopsy was done, and a ROS1 gene translocation was identified in tissue and concordant with the results of subsequent fluorescence in situ hybridization analysis of blood CTCs (Fig. 1) . The patient began crizotinib therapy with disease stabilization. Brain metastases were detected 21 and 34 months later, and both were treated with stereotactic brain radiation. Because of the emergence of resistance, the patient was switched to ceritinib and has been stable for 6 months. ROS1 rearrangements have been detected in CTCs from four patients known to harbor ROS1 translocations in tumor tissue.2Pailler E. Auger N. Lindsay C.R. et al.High level of chromosomal instability in circulating tumor cells of ROS1-rearranged non-small-cell lung cancer.Ann Oncol. 2015; 26: 1408-1415Crossref PubMed Scopus (99) Google Scholar However, our case is the first in which ROS1 rearrangements were detected in CTC analysis of a peripheral blood sample when NGS evaluation of plasma failed to reveal genetic alterations. Thanks to confirmation of the ROS1 rearrangement, the patient has been alive for 40 months while being treated with anaplastic lymphoma kinase inhibitors (criztonib first and cetinib later). Moreover, detection of a ROS1 rearrangement in CTCs but not by NGS analysis of plasma suggests that CTC analysis may improve detection of this alteration, as we have seen with other genetic aberrations. We therefore encourage future comparisons of ROS1 detection techniques. Whether tumor tissue is truly the criterion standard for molecular analysis is currently disputed, as blood tests can reveal alterations not discovered in tumor tissue.3Wang Q. Yang X. He Y. et al.Droplet digital PCR for absolute quantification of EML4-ALK gene rearrangement in lung adenocarcinoma.J Mol Diagn. 2015; 17: 515-520Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Our study underscores the need to define an analytical criterion standard for identifying the largest possible amount of druggable alterations. In conclusion, we report that CTC analysis can identify ROS1 rearrangements. This and other liquid biopsies can improve patient clinical outcomes (i.e., expand therapeutic options) compared with tissue testing alone.

Abstract Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, “liquid biopsy” offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelectorTM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelectorTM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer. Methods: The TargetSelectorTM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelectorTM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-SureTM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony. Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelectorTM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed >99% analytical sensitivity and >99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r2 >0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity >99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelectorTM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT. Conclusions: TargetSelectorTM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelectorTM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4534.

*Correspondence: Katherine H. R. Tkaczuk, University of Maryland Greenebaum Comprehensive Cancer Center, 22 South Greene Street, Baltimore, MD 21201, USA, Tel: 410-328-7394; Fax: 410-328-6896; E-mail: ktkaczuk@umm.edu Received Date: 17 Apr 2018 Accepted Date: 02 May 2018 Published Date: 11 May 2018 Citation: Seale K, Singh VM, Huynh L, Rosenblatt P, Vibat CRT, Tkaczuk KHR. Clinical Utility of Target SelectorTM Circulating Tumor Cell (CTC) Testing in Tumor Marker Gene Amplification and Protein Expression in Metastatic Breast Cancer Management. Clin Oncol. 2018; 3: 1466.

Abstract Background: Liquid biopsies represent a noninvasive alternative to traditional tissue biopsies, enabling the detection and tracking of cancer driver mutations from a simple blood draw. Biocept’s Target SelectorTM test platform offers the unique ability to analyze biomarkers from both circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Here we performed a clinical concordance study, comparing the ABI 7900 to the newer ABI QuantStudio 5 (QS5) for integration of the QS5 into Biocept’s CAP/CLIA certified laboratory. TargetSelectorTM ctDNA mutation tests were validated for five targets, including EGFR (Del19, L858R, or T790M), BRAF, and KRAS, all markers integral to devising personalized therapies for non-small cell lung cancer (NSCLC) patients. Methods: Prior to assessing clinical samples, extensive analytical validation was performed with DNA extracted from cancer cell lines containing the relevant EGFR, BRAF or KRAS mutations. TargetSelectorTM ctDNA mutant assays procedurally incorporate real-time PCR and DNA sequencing. The analytical validation was conducted to compare the performance of the ABI 7900 vs QS5 instruments within Biocept’s ctDNA testing platform. Evaluation of 3000 samples across the five TargetSelectorTM assays demonstrated single mutant copy detection sensitivity on the QS5 platform, with >99% sensitivity and >99% specificity for each of the ctDNA mutant assays. Following analytical evaluation, synchronized aliquots of 13 patient ctDNA samples, extracted from whole blood collected in Biocept CEE-SureTM Blood Collection tubes, were used to test the performance of both the ABI 7900 and QS5 instruments in the TargetSelectorTM ctDNA assays. Results: EGFR, BRAF and KRAS TargetSelectorTM assays that incorporate the ABI QS5 vs the ABI 7900 enable more sensitive ctDNA testing, as demonstrated by analytical validation and subsequent analyses of clinical samples. In patient samples, TargetSelectorTM tests using the QS5 identified all of the mutations detected by same assays on the ABI 7900 platform. Utilization of the QS5 instrument within the TargetSelectorTM also enabled identification of additional mutations not detected in the assays where the ABI 7900 was used. Conclusions: Implementation of the QuantStudio 5 real-time PCR instrument into Biocept’s TargetSelectorTM ctDNA assays has improved performance over the older TargetSelectorTM platform that utilized the ABI 7900. The more sensitive QS5-based TargetSelectorTM assays increase the likelihood of identifying molecular drivers linked to a patient’s cancer. Liquid biopsy detection of EGFR, BRAF and KRAS mutant ctDNA provides a minimally invasive means to gain valuable information towards developing personalized treatment strategies, monitoring therapeutic response, and identifying potential resistance mechanisms, all of which are vital for disease management and NSCLC patient care. Citation Format: Shan Fu Wu, Jason C. Poole, Tim T. Lu, Lyle J. Arnold, Jeffrey Chen, Anh Pham, Veena M. Singh. Validation of the QuantStudio5 instrument for use in Biocept’s TargetSelectorTM ctDNA lung cancer assays [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A20.

Abstract Biocept’s Target Selector technology is a targeted hotspot mutation panel designed to enrich for mutant targets in a large excess of WT DNA. The test is specific to small regions of interest, is highly sensitive, validated down to 7 mutant copies in a background of 14,000 WT at >98% sensitivity. The ArcherDX Reveal ctDNA test is a 28 gene NGS panel that targets key oncogene activating mutations, drug resistance mutations, in addition to full coverage of TP53. The Reveal ctDNA assay utilizes Anchored Multiplex PCR to enrich, tag, and efficiently capture short ctDNA fragments. Both technologies are designed specifically for use with plasma associated ctDNA. We undertook to evaluate the feasibility of using the ArcherDX Reveal ctDNA 28 NGS panel with the DNA extracted from plasma collected with Biocept’s patented blood collection tube which has been validated at room temperature for 4 days for circulating tumor cells and 8 days for ctDNA. The ability to build complex, targeted libraries, free of chemically induced mutations is of major importance for the detection of the vanishingly rare mutations that can be present in the plasma of cancer patients. We found that high quality NGS libraries were produced from plasma collected and stored in the Biocept blood tube, indicating that little damage occurred to the DNA during preservation. In addition, the two methods were found to be highly concordant and complementary when using both mutation positive and negative patient samples. The Target-Selector assay was the more sensitive, however, the ArcherDX panel revealed several additional mutations not targeted by the Biocept Target Selector assay. Citation Format: Jason C. Poole, Brian Kudlow, Jill Stefanelli, Skyler Mishkin, Anh Pham, Jeff Chen, Veena M. Singh, Josh Stahl, Lyle J. Arnold. A concordance study of the ArcherDX RevealTM ctDNA 28 NGS panel and Biocept’s Target SelectorTM mutation assay using ctDNA collected in Biocept CEE-sureTM blood collection tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2017-762

e23036 Background: Liquid biopsy is a minimally invasive and cost effective way to assess cancer biomarkers without the risk of surgical biopsy complications. Circulating tumor cell (CTC) analysis from body fluids can provide critical information towards early detection, prognosis and treatment decisions. Accurate CTC evaluations require optimal cell preservation. Cell lysis, DNA degradation, or membrane alterations compromise CTC analyses and accurate diagnoses. This work compares Biocept’s proprietary CEE-Sure BCT and Saccomanno's Cytology Fixative largely used for sputum collection. Methods: One million BT474 (HER2 amplified) or H3112 (ALK re-arranged) cells were spiked into 500 µl medium; 500 µl of CEE-Sure or Saccomanno fixative was added. Tubes were stored at 4°C for 1 day, 1 week, or 1 month. Cells were centrifuged, resuspended, and counted (Celigo). Around 150 cells in 15 µl of RPMI medium were flowed into Biocept's microfludic system for cell capture; recovery (%) was calculated. Captured cells were subjected to fluorescent in situ hybridization (FISH) analyses for qualitative signal evaluation. Results: As similar results were observed for both cell lines and all time points, combined data will be shown. Median cell recovery after CEE-Sure incubation was 14.1% (range 1.7–44%, n = 12) vs 5.4% (range 0.07–26.9%, n = 12) in Saccomanno's fixative. Median cell capture of ~150 cells fed into Biocept’s microchannel was 96% (range 72-98%) for CEE-Sure vs 82% (range 21-96%) for Saccomanno. Paired t-tests showed significant differences for both recovery and capture. FISH signals from CEE-Sure samples were qualitatively rated Fair to Good, while Saccomanno samples had Poor to Fair, grainy, non-specific signals. Conclusions: This preliminary work shows consistently higher cell recovery, better cell membrane maintenance, and higher quality FISH signals for samples stored in Biocept's CEE-Sure vs Saccomano’s fixative. With liquid biopsy testing gaining rapid traction, maximal cell stability during the transport and storage are crucial. Additional fixative comparison is ongoing in various patient specimen types. These results support expansion of molecular analyses in sputum samples enriched for lung epithelial cells.

e23037 Background: EGFR mutations are the most frequent targetable genomic alterations in non-small cell lung cancer (NSCLC) patients (pts). While tissue biopsy remains the standard for assessing of EGFR mutation status, it is invasive and not always feasible. Liquid biopsy is a minimally invasive alternative. Biocept’s proprietary TargetSelector system evaluates circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in blood. We aimed to clinically validate the accuracy of EGFR-specific TargetSelector in NSCLC pts. Methods: At three time points (T0: baseline before TKI, T1: during EGFR-TKI therapy, T2: after progression), blood samples were collected in Biocept OncoCEE BCT validated to preserve DNA up to 8 days. These samples were interrogated for three EGFR mutations: exon 19 deletions (Del 19), L858R, and T790M. The objectives are to assess detection sensitivity of liquid biopsy using EGFR mutation status vs the tissue as gold standard and to evaluate whether the detection sensitivity changes with EGFR-TKI therapy. Results: A total of 53 study pts were enrolled (male, 21; female, 32). The mean age was 70.6 (range: 46 – 90). Most pts had stage IV disease (43, 81.1%) and lung adenocarcinoma (48, 90.6%). 26 (49.1%) pts had EGFR mutations in tumor tissue: Del 19, 13; L858R, 8; T790M, 6; other, 8. Detection sensitivity for sensitizing EGFR mutations (Del 19 and L858R) at T0, T1, and T2 was 60.0% (6/10), 33.3% (5/15), and 33.3% (1/3), respectively. There was no statistical difference in CTC counts between activating EGFR mutation-positive and -negative pts (mean CTC count: 10.5 vs 20.1; p = 0.11 by two-sided t-test). Detection sensitivity for T790M was 33.3% (2/6) and 5 of 6 pts were receiving T790M directed therapy (3, rociletinib; 2, osimertinib) at the time of blood draw. Two pts – one patient before initiation of EGFR-TKI and the other during treatment with erlotinib – were found to have T790M mutations only in blood and not in tissue. Conclusions: Activating EGFR mutation detection may decrease during the course of TKI therapy, possibly due to treatment response. Further research with an expanded sample size and serial collections are needed to evaluate this finding, and to investigate possible implications of the presence of T790M only in blood.

e23033 Background: Targeted cancer therapy relies on identifying specific DNA mutations from a patient’s tumor. Tyrosine kinase inhibitors (TKIs) tend to be effective for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations, of which exon 19 deletions (Del19) and L858R are most common. Acquired resistance to TKI therapy is associated with a T790M mutation. Standard biomarker analyses may not reflect tumor heterogeneity; they entail tissue biopsies often with surgical complications. To address these limitations, Biocept developed a minimally invasive method to characterize cancer biomarkers in blood. Biocept's proprietary TargetSelector assays selectively amplify relevant mutations from circulating tumor DNA (ctDNA). Clinical validations demonstrated high concordances between molecular tests in blood vs tissue. As further validation, EGFR mutation detection frequencies were compared to US averages (mycancergenome.org). Here we analyze 2000 blood samples received at Biocept from 1Mar 2016 to 4Jan 2017 from late stage NSCLC patients. Methods: Blood was collected in Biocept OncoCEE BCT validated to preserve DNA ≤ 8 days. TargetSelector was used to detect ctDNA L858R, Del19 and T790M.EGFR allele copy numbers for wild type and each mutant were calculated. The prevalence of each mutation was compared to US averages. Results: Del19, L858R, and T790M mutations were detected in 12.9%, 8.5%, and 9.9% of the analyzed blood samples, respectively. This is concordant with US averages, which are 10% for each mutation. Median copy numbers/ml of blood were 30 for Del19 (range: 1 – 91974), 15 for L858R (range: 1 – 91200), and 10 for T790M (range: 1 – 137360). The median wild type EGFR copy number detected/ml blood was 2304 (range: 8 – 2498725). In ~80% of T790M cases, ≥ 1 concomitant activating mutation was detected. Conclusions: Biocept's TargetSelector detects EGFR mutations (Del19, L585R, and T790M) at a very high level of sensitivity down to 1 mutant copy/ml in advanced NSCLC patients at frequencies consistent with cited US rates. Moreover, the underlying activating mutation was detected in ~80% of T790M cases.