Biopeptide (United States)

CNSide is a platform that detects and characterizes tumor cells in the cerebrospinal fluid (CSF) of patients with leptomeningeal disease (LMD). The platform was validated per College of American Pathologists (CAP) and Clinical Laboratories Improvement Amendment (CLIA) guidelines and run as a commercial Laboratory Developed Test (LDT) at Biocept in San Diego, CA. The platform allows CSF tumor cell (CSF-TC) enumeration and biomarker characterization by fluorescent in situ hybridization (FISH). We performed a multicenter retrospective chart review of HER2 FISH CNSide test results that were commercially ordered on 26 patients by physicians for LMD breast cancer patients between April 2020 and October 2022. We show that HER2 is amplified on CSF tumor cells in 62% (16/26) of LMD breast cancer patients. 10/26 (38%) patients had discordant HER2-positivity between the primary tumor tissue and CSF-TC; of these, 35% (9/26) of the patients displayed HER2 amplification on the CSF-TCs, however were categorized as HER2 negative on the primary tumor. Of the 27% (7/26) patients with a HER2 positive primary tumor, one patient showed a HER2 negative LMD tumor. Two patients, 8% (2/26) had a HER2 equivocal primary tumor; of these, one demonstrated a HER2 negative, and one a HER2 positive LMD tumor. Serial analysis (at least 4 longitudinal tests) of HER2 status of the CSF-TC throughout therapy was available for 14 patients and demonstrated that HER2 status of the LMD changed in 29% (4/14) during their treatment course and impacted care decisions. Our data suggests that CSF-TC HER2 FISH analysis in LMD breast cancer patients may be discordant to the primary tumor sample and the discovery of HER2 positivity in the CSF may open doors to anti-HER2 targeted therapy options for LMD patients.

BackgroundLeptomeningeal disease (LMD) is a devastating complication for patients with advanced cancer. Diagnosis and monitoring the response to therapy remains challenging due to limited sensitivity and specificity of standard-of-care (SOC) diagnostic modalities, including cerebrospinal fluid (CSF) cytology, MRI, and clinical evaluation. These hindrances contribute to the poor survival of LMD patients. CNSide is a CLIA-validated test that detects and characterizes CSF-derived tumor cells and cell-free (cf) DNA. We performed a retrospective analysis on the utility of CNSide to analyze CSF obtained from advanced non–small cell lung cancer (aNSCLC) patients with suspected LMD treated at the Huntsman Cancer Institute in Salt Lake City, UT.MethodsCNSide was used to evaluate CSF from 15 patients with aNSCLC. CSF tumor cell quantification was performed throughout treatment for 5 patients. CSF tumor cells and cfDNA were characterized for actionable mutations.ResultsIn LMD-positive patients, CNSide detected CSF tumor cells in 88% (22/25) samples versus 40% (10/25) for cytology (matched samples). CSF tumor cell numbers tracked response to therapy in 5 patients where CNSide was used to quantify tumor cells throughout treatment. In 75% (9/12) of the patients, genetic alterations were detected in CSF, with the majority representing gene mutations and amplifications with therapeutic potential. The median survival for LMD patients was 16.1 m (5.2-NR).ConclusionsWe show that CNSide can supplement the management of LMD in conjunction with SOC methods for the diagnosis, monitoring response to therapy, and identifying actionable mutations unique to the CSF in patients with LMD.

Abstract BACKGROUND Pineal parenchymal tumors of intermediate differentiation (PPTID) account for 21% to 54% of pineal parenchymal tumors and may be complicated by cerebrospinal dissemination (most commonly at time of reoccurrence; up to 4-10 years post resection). The integral membrane protein synaptophysin is expressed in neuroendocrine cells and virtually all neuraxial neurons that contribute to synaptic transmission. PPTID frequently demonstrates synaptophysin independently of other neural differentiation markers. Cerebrospinal fluid (CSF) cell capture assays have demonstrated utility for assessing disseminated intracranial neoplasms, thus we explore this technology for monitoring a patient with PPTID. METHODS A 30-year-old female patient with PPTID diagnosed two years prior was suspected for intracranial dissemination and underwent five CSF collections over the course of 64 days. Per collection, approximately 7 mL of CSF was submitted for each cytology and cell capture. Cytology was performed at Providence Saint John’s Health Center. The CNSide™ platform (Biocept, Inc.) was used to capture and stain cells for DAPI, synaptophysin, and CD45. Multiple capture antibody cocktails (termed 1986, 1822, and gTP1—originally developed for use in carcinoma, melanoma, and glioma) were tested. Cells of interest (COI) were defined by positive immunoreactivity for both DAPI and synaptophysin and no immunoreactivity for CD45. Results were quantified and compared longitudinally. RESULTS CSF analysis on days 0, 9, 21, 36, and 64 demonstrated negative results by cytology and 51, 96, 170, 130, and 43 COI/mL by the CNSide platform, respectively. Cumulative mean capture rates for cocktails 1986, 1822, and gTP1 were 47, 14, and 36 COI/mL/sample, respectively. DISCUSSION A significant amount of PPTID patients will develop cerebrospinal dissemination. The importance of craniospinal control in this setting has been previously demonstrated. Our work suggests that cell capture assays paired with immunocytochemistry can be used as sensitive means to monitor disseminated PPTID and may impact treatment decisions.

Abstract INTRODUCTION Patients with leptomeningeal metastasis (LM) have limited treatment options and a poor outcome. However, they may benefit from targeted therapy. LM patients with HER2-positive primary breast cancer treated with IT trastuzumab demonstrated clinical benefit (Malani, 2020), and improved PFS (Figura, 2019). HER2 amplification in the primary and metastatic tumors can be divergent. Biocept’s CNSideTM is a CLIA validated test that can detect CSF tumor cells (CSF-TCs) and interrogate those cells with FISH and NGS. We analyzed HER2 amplification on CSF-TCs in LM patients with breast cancer, non-small cell lung cancer (NSCLC) and upper GI cancer. METHODS CSF was collected from patients with suspected or confirmed LM with breast cancer (N =134 patients), NSCLC (Nf28 patients) or upper GI cancer (Nf2 patients). CSF TCs were tested for HER2 amplification by FISH using CNSide. RESULTS HER2 amplification in CSF-TCs was detected in 46% (76/164) of all patients. Of the breast cancer patients, 37% (49/134) flipped HER2 status in the LM tumor, 38% (41/108) switched from a HER2 negative or equivocal primary tumor to HER2 positive CSF-TCs, and 27% (7/26) from a HER2 positive primary tumor to HER2 negative CSF-TCs. For the NSCLC patients, 50% (14/28) showed HER2 amplification on the CSF-TCs. For the upper GI cancer patients, HER2 amplification in CSF-TCs was detected in both patients, one patient had an equivocal HER2 primary tumor and HER2 positive CSF-TCs. CONCLUSION HER2 amplification was detected in a substantial fraction of CSF-TCs from patients with LM from breast, upper GI, and NSCL cancers. This finding may have important therapeutic (justifying the use of intra-CSF trastuzumab), prognostic (HER2 positivity in NSCLC is associated with poorer prognosis and a higher frequency of CNS metastases) and pathophysiologic implications (a potential role of HER2 amplification in the genesis of CNS metastases). Additional investigations are underway.

Lyme disease is the most common vector-borne disease in the northern hemisphere. Current serodiagnostics are insensitive in early infection. Sensitivity in these seroassays is compromised by the necessity to preserve specificity in the presence of cross-reactive epitopes in Borrelia burgdorferi target antigens. We evaluated the efficacy of using synthetic peptides containing epitopes unique to B. burgdorferi as antigen targets in a Lyme disease seroassay. We performed linear B cell epitope mapping of the proteins p35 (BBH32) and ErpP to identify unique epitopes. We generated peptides containing these newly identified linear epitope sequences along with previously identified epitopes from the antigens FlaB and VlsE and evaluated their diagnostic capabilities via ELISA using large serum sets. Single-epitope peptides, while specific, demonstrated insufficient sensitivity. However, when epitopes from FlaB, ErpP, or p35 were combined in tandem with an epitope from VlsE, the sensitivity of the assay was significantly increased without compromising specificity. The identification of additional unique epitopes from other B. burgdorferi antigens and the further development of a combined multi-peptide-based assay for the laboratory diagnosis of Lyme disease offers a way to address the poor specificity associated with the use of whole protein antigen targets and thus significantly improve the laboratory diagnosis of Lyme disease.

Formaldehyde was used as a versatile C1 building block to forge either acyclic or heterocyclic molecules via multicomponent reactions with the potential to be more sustainable than lengthier alternatives.

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.

Background: Acquired resistance to anti-hormonal therapy is a common reason for treatment failure in advanced and metastatic breast cancer (mBRC). Mutations in the ESR1 gene, encoding the estrogen receptor (ER) protein, are often associated with this drug resistance. Accurate and timely ESR1 mutation detection may enable early identification of progression and prediction of treatment failure. In contrast to using difficult and painful tissue biopsies, “liquid biopsy” offers a non-invasive and systemic approach to identify solid tumor mutations in peripheral blood by assessing circulating tumor DNA (ctDNA). Biocept has developed the highly sensitive TargetSelectorTM NGS assay to detect low frequency mutant alleles in ctDNA using a patented blocker that suppresses excess WT alleles, while still allowing amplification of tumor derived mutant sequences. Here, we focus on a five-codon ESR1 mutation hotspot that imparts tumor cell resistance to anti-hormonal therapy in mBRC.Methods: The TargetSelectorTM assay applies a specific blocker on a short stretch of target DNA (up to 13 bp). The ESR1 assay consists of two blockers in the region of codons 534-538 of the ESR1 gene. We validated the TargetSelectorTM assays by spiking low level g-block DNA fragments (IDT Inc.) carrying point mutations for each of the targeted ESR1 sites, into human placental DNA. Mutant sequences were enriched via PCR and mutations were subsequently confirmed on the Illumina MiSeq down to a limit of detection (LOD) of 0.01-0.05% minor allele frequency (MAF). For ctDNA testing, whole blood was collected in Biocept CEE-SureTM Blood Collection tubes, and total nucleic acid extraction from plasma was performed on the QIAsymphony.Results: In total, we tested >600 samples for ESR1 TargetSelectorTM ctDNA assays, with ESR1 WT as the background reference. The assay shows >99% analytical sensitivity (at 0.05% MAF) and >97% analytical specificity. Based on practical and theoretical estimates and using ddPCR as an orthogonal verification method, the ESR1assays demonstrate single mutant copy detection for most tested positions, and in all cases showed an LOD of at least 0.05% (5 mutant copies in a background of 10,000 WT copies). Samples tested from 20 healthy donors (40 tests in total) showed high clinical specificity, indicating the test is competent for use in a CLIA certified, CAP accredited laboratory setting.Conclusions: Biocept’s TargetSelectorTM ctDNA assays are validated both analytically and clinically, showing single mutant copy detection and LOD at 0.05% or better in a background of excess WT DNA. Implementation of the Illumina MiSeq platform into our TargetSelectorTM assays leads to higher sample throughput, greater sensitivity and a faster turnaround time than Sanger sequencing. These factors enable a sensitive and efficient test crucial for guiding treatment decisions and patient care.Citation Format: Shan-Fu Wu, Lyle J. Arnold, Jolly Shrivastava, Jason C. Poole. Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelectorTM enrichment technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2300.

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.

- To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. - The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. - Twenty-one guideline statements were established. - Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.