Publications, Studies, Posters and Abstracts

Advanced NSCLC, Peer Review

Background Over half of lung cancer patients present with advanced stage disease at diagnosis. Liquid biopsy is emerging as a minimally invasive and cost-effective means of cancer biomarker evaluation to select appropriate treatment and monitor disease burden. CTC enumeration may have prognostic and predictive potential for patients receiving chemotherapy. Here we describe interim results of a prospective study analyzing blood CTCs from treatment-naïve patients with histologically confirmed metastatic non-small cell lung cancer (NSCLC) who are candidates for systemic cytotoxic chemotherapy.

Method Venous blood samples were obtained for CTC analysis before administration of chemotherapy on treatment days (D) 1, 8, 22 and 43, and at disease progression. Blood was collected in Biocept CEE-Sure™ blood collection tubes, and samples were processed at Biocept’s CLIA-certified, CAP-accredited laboratory. Biocept’s Target Selector™ CTC platform uses an antibody capture cocktail and microfluidic channel for CTC capture and biomarker analysis. Study objectives include determining the proportion of patients with D1 CTC detection, and CTC correlation to time to progression and overall survival (OS).

Result Twenty-three patients have been enrolled so far in this ongoing study: 16 (69.5%) adenocarcinoma, 7 (30.4%) squamous cell carcinoma. Fifteen patients progressed on initial therapy to date, with a median time to progression of 110 days. Of 22 patients with blood collections at D1 (treatment start), 14 (63.6%; 10 adenocarcinoma, 4 squamous) had detectable CTCs. For patients with detectable D1 CTCs, CTC count at D8 decreased in 13/14 (92.9%; 9 adenocarcinoma, 4 squamous) subjects, and was unchanged in 1 individual (7.1%; adenocarcinoma). Patients with 1-5 CTCs at D1 had a mean time to progression of 140.75 days; patients with >5 CTCs at D1 had a mean time to progression of 101.83 days (p=0.17). Among patients with undetectable CTCs at D1, CTCs remained undetectable in 4/8 (50%; all adenocarcinoma) and increased in 2/8 (25%; one each of adenocarcinoma and squamous) of subjects; CTCs were not collected at D8 for 2 individuals.

Conclusion Implementation of Biocept’s TargetSelector™ enables the highly sensitive detection of CTCs in the clinical setting. CTCs are detectable in the majority of patients with metastatic NSCLC. In subjects with detectable CTCs prior to chemotherapy, CTC count declines within a week after starting chemotherapy in >90% patients. Preliminary data suggest that CTC enumeration may have prognostic and predictive potential for patients receiving chemotherapy. Further data from this ongoing study may provide additional insight into the role of CTC analysis applied to clinical practice.

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Lara Kujtan1, Cecile Rose T. Vibat2, Veena M. Singh2, Kevin F. Kennedy3, Anuj Shrestha1, Sheshadri Madhusudhana1, Jacob Smeltzer3, Ashiq Masood4, Janakiraman Subramanian5

1University of Missouri at Kansas City, Kc/United States of America, 2Biocept, Inc., San Diego, CA/United States of America, 3St. Luke’s Hospital, Kansas City/United States of America, 4University of Missouri at Kansas City, Kansas City/United States of America, 5Saint Luke’s Cancer Institute, Kansas City, MO/United States of America

Background: Liquid biopsy using blood has emerged as a non-invasive and economical method to assess cancer biomarkers. Applying liquid biopsy methods to evaluate cerebrospinal fluid (CSF) is less well documented and provides key information to supplement routine cytology in patients with brain metastases or leptomeningeal disease (LM). Current first line tyrosine kinase inhibitor (TKI) therapy of (non-small cell lung cancer) NSCLC patients with activating EGFR mutations, exhibits poor penetration to the central nervous system (CNS). About 28% of patients treated with erlotinib or gefitinib progress with CNS involvement. Here we present Biocept’s Target Selectortechnology to evaluate circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in the CSF of NSCLC patients with LM, who were treated with tesevatinib in Kadmon’s KD019-206 study.

Methods: CSF samples were collected in Biocept CEE-Sure™ blood collection tubes that are validated to preserve CTCs and ctDNA at room temperature for up to 96 hours from collection. The Target Selector™ CTC platform uses an antibody capture cocktail and microfluidic channel for CTC capture and biomarker analysis; the quantitative ctDNA platform incorporates switch-blockers, real time PCR, and sequencing to detect a mutant allele frequency down to 0.05% against wildtype.

Results: CSF collections from 6 NSCLC patients with LM were obtained at baseline, C1D14, and C3D1 of tesevatinib therapy. In all 6 patients, EGFR mutations detected in CSF were concordant to the original tissue mutations. Baseline or emergent T790M ctDNA that was detected in CSF, paralleled progression in 3 patients. CTC enumeration mirrored response to therapy, decrease of symptoms, or progression in 5 of 6 patients; T790M emerged at C1D14 in the patient whose CTCs decreased at progression. Serial CSF CTC and ctDNA analyses were consistent with the overall clinical course of disease.

Conclusion: Biocept’s Target Selector™ technology in CSF analysis demonstrated both highly sensitive detection of CTCs and mutant ctDNA in NSCLC patients with LM as well as concordance with tissue and clinical course. Hence, serial monitoring of CSF with CTCs and ctDNA can be utilized to evaluate drug response and disease progression, providing pertinent vital information for disease management and care of LM patients.

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Veena M. Singh1, Cecile Rose T. Vibat1, Masha V. Poyurovsky2, Laurie S. Green2, Beth Mac Gillivray2, and David A. Eiznhamer2

Biocept, Inc.1, Kadmon Corporation, LLC2

For breast cancer patients, molecular tumor characterization (including protein expression levels and genetic alterations or amplifications) identifies features that predict drug responsiveness. This information can be used to design personalized therapeutic strategies. Approximately 75% of breast tumors express hormonal receptors for Estrogen (ER) and/or Progesterone (PR) [1,2]; these patients typically respond well to endocrine therapy with or without CD4/6 kinase inhibitors [3]. Another major tumor growth driver of breast cancer is HER2 gene amplification, which is seen in ~20% of breast cancers. These patients can be treated successfully with monoclonal antibodies that block HER2 function [4,5]. Despite benefits to patient survival, molecular data is often difficult to obtain in metastatic settings when patients’ health or refusals preclude biopsy, or the tumor metastasizes to a difficult-to-sample region of the body. Additionally, tissue molecular analyses may be inconclusive due to insufficient tissue amounts from biopsies or interference of bone tissue decalcification procedures with immunohistochemical (IHC) stains. Without understanding the molecular drivers of patients’ disease, treatment regimens based on tumor characteristics can neither be appropriately prescribed nor adjusted to tackle evolving properties of progressive disease.

The recent development of “liquid biopsy” technologies that analyze circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) allows physicians to obtain molecular data for guiding individual patient’s treatment approaches [6]. CTCs are cells that have shed into the bloodstream from a primary or metastatic tumor, representing an alternative source of tumor material for non-invasive disease assessment [7,8]. Importantly, liquid biopsies provide a systemic representation of existing tumor clones, giving insight into tumor heterogeneity, emergence of new drivers, and the divergence between primary and metastatic tumors [6,9].

Here we describe a patient with recurrent breast cancer, who at one point declined an additional bone biopsy. Biocept’s Target Selector™ liquid biopsy [10] (Figure 1) revealed ER expression and HER2 gene amplification in CTCs (Figure 2). Based on these data, the patient was able to receive anti-HER2 therapy earlier, providing a clinical example of the utility of liquid biopsy testing to gather molecular data that was unsuccessful by standard image-guided biopsy. In this case, CTC results of newly found HER2 amplification were paramount towards altering treatment strategies and inclusion of HER2 targeted therapies, which ultimately extended patient survival and quality of life.

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Conclusions:

Each Biocept’s TargetSelector™ ctDNA assay shows >99% analytical sensitivity and >99% analytical specificity.

TargetSelector™ ctDNA assays show single mutant copy detection based on experimental data compared to theoretical estimates, with sensitivity at 0.02% MAF or better in a background of excess WT DNA.

Biocept’s ctDNA assays detected no false positives from 20 healthy donors, and showed >99% clinical specificity.

Implementation of the QuantStudio 5 qPCR platform into Biocept’sTargetSelector™ ctDNA assays ranslated into high clinical sensitivity and fast turnaround time for patients in Biocept’s CLIA certified and CAP accredited laboratory.

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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.

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To the Editor:
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.1 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 diag-nosed a 44-year-old male Hispanic nonsmoker. Molecu-lar 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 per-formed 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.2 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.3 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.

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