BackgroundContemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.Methodology/Principal FindingsRecent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.Conclusions/SignificanceThe LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.
11040 Background: Recently a succession of new hormonal therapies has emerged, highlighting the need for biomarkers to guide the management of HSPC. Biomarker development in HSPC has been hampered by the absence of primary tumor tissue in men who undergo radiation or present with metastatic disease. CTCs can address this challenge by providing real-time cancer tissue for biomarker analysis in HSPC. To test this approach we conducted a pilot of CTC capture and targeted NGS in HSPC. Methods: Under IRB approval, blood samples from patients with HSPC were labeled with EpCAM ferrofluid and placed into the LiquidBiopsy platform (Cynvenio Biosystems, Inc.), an immunoaffinity-based microfluidic device tailored to query genomic events. CTCs were identified by CK, CD45 and DAPI expression. A matched WBC pellet served as a control representing germline sequence. Amplicon libraries were generated using Life Technologies AmpliSeq 2.0 and sequenced on an Ion Torrent platform. Somatic single nucleotide variants (SNV) present in CTCs but not in WBC were identified. Results: CTCs were detected in all 8 patients with HSPC (CTC median 64.5, range 17-217). Germline variants were consistently detected in matched CTC and WBC samples. Significant SNVs (occurring in > 1% of DNA in a sample) were found in 4 of 8 CTC samples (range 1-5 SNVs/sample, frequency 1.2%-11.9% with 620X-14,422X coverage). Notably, 3 patients had biochemical recurrence only (no clinical metastases) yet still yielded CTCs associated with SNVs in KIT, APC, RET, SMAD4 and PTEN. One patient who had untreated metastatic disease had the highest number of CTCs which harbored 4 SNVs. Conclusions: This pilot demonstrates the feasibility of using CTCs as real-time disease relevant substrate for NGS to identify personalized genomic targets in HSPC. A high number of CTCs were detectable in all patients and CTC germline variants correlated with matched WBC controls. Encouragingly, even with a relatively narrow, primary tumor-derived AmpliSeq platform, cancer relevant SNVs were detected in half of the patients including those with only biochemical recurrence, making targeted NGS of CTCs a promising approach for biomarker discovery and validation in HSPC.
Estimates of the prevalence of long COVID vary widely. This retrospective cohort study describes the incidence of long COVID symptoms 12-20 weeks post-diagnosis in a US ambulatory care setting and identifies potential risk factors. We identified patients with and without a diagnosis of or positive test for COVID-19 between 1/1/2020 and 3/13/2022 in the Veradigm EHR database. We captured patient demographics, clinical characteristics, and COVID-19 comorbidities in the 12-month baseline period. We compared long COVID symptoms between matched cases and controls 12-20 weeks post-index (COVID-19 diagnosis date [cases] or median visit date [controls]). Multivariable logistic regression was used to examine associations between baseline COVID-19 comorbidities and long COVID symptoms. Among 916,894 patients with COVID-19, 14.8% had at least one long COVID symptom in the 12-20 weeks post-index compared to 2.9% of patients without documented COVID-19. Commonly reported symptoms were joint stiffness (4.5%), cough (3.0%), and fatigue (2.7%). Among patients with COVID-19, the adjusted odds of long COVID symptoms were significantly higher among patients with a baseline COVID-19 comorbidity (odds ratio: 1.91 [95% confidence interval: 1.88-1.95]). In particular, prior diagnosis of cognitive disorders, transient ischemic attack, hypertension, and obesity were associated with higher odds of long COVID symptoms.
Both cell free DNA (cfDNA) and circulating tumor cells (CTC) represent important possible templates for mutation analysis of clinical samples with different theoretical advantages for a clinical test. cfDNA is easy to access and isolate, while CTC can provide both DNA as well as RNA for clinical testing. We have tested matched cfDNA and CTC DNA in a Next Generation sequencing test of clinical samples. Both cfDNA and CTC samples provided sufficient quantity for a direct sequencing clinical test. No whole genome amplification was required. The test consisted of coupling these purification technologies to an amplicon re-sequencing panel of 50 cancer-associated genes using a CLIA validated sequencing pipeline for single nucleotide variant (SNV) mutations with a sensitivity of 1%. Typically using this pipeline, blood borne cancer cells are isolated, extracted, sequenced and analyzed in 7 days. In the CLIA setting, the validated DNA sequencing process applied to breast cancer patient samples, has demonstrated useful and actionable clinical data. Results of SNV detection from known clinical positive control samples were quite definitive. We observed perfect concordance in True Positive (TP) SNV detection between the cfDNA and CTC templates. The only discordant results observed were in the detection of False Positives (FP). Using the identical software for SNV detection and variant calls, CTC templates exhibited ∼5x greater sensitivity in our sample set. cfDNA samples showed a much greater noise spectrum of FP. As blood samples are routinely fixed at collection, we evaluated whether fixation might contribute to the cfDNA observed high FP frequency. Our experimental results conclusively show that fixation did not contribute to increased FP observed with cfDNA. Whereas CTC samples showed zero FP over the reportable range, the cfDNA samples exhibited variable FP output ranging from 0 to >10 FP per sample. To extend the capabilities of sequencing rare cells, several RNA sequencing panels have been developed that allow for both SNV, expression, and fusion transcript detection. Although these panels have not been successfully used with cell free templates, they have been successfully applied to rare cell templates and promise to be an important capability for clinical use of CTC in the future. Looking at both lung and breast cancer derived samples we have measured both SNV, CNV (expression based), and fusion transcripts. We describe simultaneous detection of SNV mutations (KRAS: G12S, STK11:Q37*, PIK3CA:E545K, EGFR:L858R, EGFR:T790M, TP53:R273H) as well as fusion transcripts events EML4-ALK.E6aA20.AB374361, and EML4-ALK.E13A20.AB462411. In summary, both cfDNA and CTC derived DNA provided concordant results in SNV mutation detection. cfDNA exhibited a much higher FP rate as compared to CTC DNA. We further demonstrate that rare cells can provide useful template for RNA sequencing. Citation Format: William M. Strauss, Erich Klem, Maria Cuellar, Jill Simmons, Jessamine Winer-Jones, Paul W. Dempsey. Molecular platforms for mutation analysis from whole blood derived clinical samples by nextgen sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2014-2846
580 Background: The treatment of advanced breast cancer demands systemic therapies that can address disease heterogeneity and the development of treatment resistance without a “real-time” molecular window into disease biology. New technologies have focused on increased capture and molecular analysis of circulating tumor cells (CTCs) including cells undergoing epithelial mesenchymal transition (EMT). We conducted a pilot experiment to test the efficiency of capture and cytokeratin (CK) detection and the presence of single point variants (SNV) to determine the best utility of scoring alternatives for CTC. Methods: EpCAM expressing CTC were recovered from breast cancer patients using CellSearch (Veridex) and LiquidBiopsy (Cynvenio Biosystems). EpCAM recovery and CK scoring were indexed in spiked samples and in 12 inflammatory breast cancer (IBC) patient samples using antibodies against CKs 7, 8 or CKs 1-8, 10, 13-16, 18, 19. Additionally, LiquidBiopsy template was analyzed using an Ampliseq 1.0 panel on the IonTorrent PGM. SNV present in the CTC but not white blood cell (WBC) negative controls were identified and where possible, compared to tissue biopsy SNV analyzed using Foundation One (Foundation Medicine). Results: CTCs were detected using CellSearch 10/12 (83%) (range 0-2502 CTC/7.5ml) and LiquidBiopsy 12/12 (100%) (range 6-2800 CTC/7.5mL). More CK positive events were scored using CKs 1-8, 10, 13-16, 18, 19 than CKs 7, 8 in patient samples. Upon sequencing, shared germline polymorphisms were observed in CTC and WBC. Conversely, 1 or 2 SNV were detected in the Epcam selected population but not WBC controls from 6/12 patients (frequency 1.1%-2.1% with 520-5160x coverage) with SNV observed in TP53, MPL, PIK3ca, MET and IDH1. All but one of the PIK3ca mutations were absent in evaluable tissue biopsy. Conclusions: CTC recovery and scoring are two separate events. Altered CK detection emphasized the need to tailor CTC classification to specific disease settings. Sequence analysis showed one correlated SNV among 6 evaluable comparisons to tissue reflecting variable analysis as well as the biologic disparity of metastatic disease. This pilot demonstrates the feasibility of using CTC for molecular analysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.