Consistency evaluation of lung adenocarcinoma tissue and circulating tumor cells related gene mutation detection based on multi-site immunomagnetic beads

2022 ◽  
pp. 088532822110658
Author(s):  
Keying Xue ◽  
Bingqing Luo ◽  
Xiaoqing Li ◽  
Weixian Deng ◽  
Chunyan Zeng ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Targeted Therapies ◽  
Sanger Sequencing ◽  
Gene Mutations ◽  
Genetic Mutations ◽  
Separation System ◽  
Tumor Tissues

This study was designed to investigate the feasibility of genetic testing using circulating tumor cells (CTCs) instead of tumor tissues in lung adenocarcinoma to break through its limitation. Separation system for epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and Vimentin expressing CTCs was constructed and used to capture CTCs in the blood samples of 57 patients with lung adenocarcinoma. Genetic mutations of genes involved in targeted therapies were detected by next-generation sequencing (NGS) in tissues from these patients. Blood CTC samples with the gene mutations identified by tissue-NGS were selected and corresponding gene mutations were detected by Sanger sequencing. The specificity of the CTC separation system was 95.48% and the sensitivity was 90.85%. The average number of CTCs in the blood of patients with lung adenocarcinoma was 12.47/7.5 mL. Comparison of the tissue-NGS with the CTC-Sanger sequencing showed that the consistencies of genetic mutations of EGFR ( n = 24), KRAS ( n = 9), TP53 ( n = 19), BRAF ( n = 1), ERBB2 ( n = 2), and PIK3CA ( n = 3) were 92.31%, 75.00%, 86.36%, 50.00%, 66.67%, and 75.00%, with an overall consistency of 84.06%. The CTC separation system established in this study shows high specificity and sensitivity. CTCs can be used as a suitable alternative to tumor tissues that are difficult to obtain in clinical practice and overcome the difficulties in tumor tissue collection, which is of significance in guiding clinical medication and individualized treatment with significant clinical application value in terms of genetic testing for targeted therapies in tumor treatment.

EML4-ALK-gene rearrangement comparative analysis in circulating tumor cells and in tumor tissue of patients with lung adenocarcinoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7591-7591
Author(s):  
Marius Ilie ◽  
Elodie Long ◽  
Catherine Butori ◽  
Veronique Hofman ◽  
Celine Coelle ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Gene Rearrangement ◽  
Alk Rearrangement ◽  
Molecular Subgroup ◽  
Non Invasive ◽  
Tumor Tissues ◽  
Gene Status

7591 Background: The implementation of new theranostic biomarkers in Oncology is leading to impressive therapeutic improvements. In patients with lung cancer, the possibility to use Circulating Tumor Cells (CTCs) as a non-invasive theranostic approach is a clinically appealing challenge. Adenocarcinomas with EML4-ALK rearrangement are a new molecular subgroup of lung tumors with very good response to Crizotinib, an ALK inhibitor. We have thus aimed at developing an informative assay characterizing the ALK-gene status in CTCs isolated from patients with lung cancer. Methods: CTCs were isolated preoperatively using Isolation by Size of Epithelial Tumor cells method (ISET) from 65 patients with lung adenocarcinoma and blindly screened for ALK-gene status. ALK break-apart fluorescence in situ hybridization (FISH) (LSI ALK dual colour probes set) and immunochemistry using an anti-ALK antibody (5A4 clone) were blindly performed on CTCs and corresponding tumor tissues and results were compared. Results: Two patients consistently showed ALK-gene rearrangement and strong ALK protein expression in CTCs and corresponding tumor samples. Negative results (both ALK FISH and ALK immunochemistry) were found in CTCs and corresponding tumor samples from the other 63 patients. Conclusion: We have developed an approach allowing to characterize ALK-gene status in CTCs from patients with lung cancer and shown consistent results in CTC and tumor tissues. These preliminary results encourage larger studies and open new avenues for non-invasive, real-time, theranostic monitoring of cancer patients.

scholarly journals Effect of Napsin A and Circulating Tumor Cells with Mesenchymal Phenotype in Lung Adenocarcinoma

2021 ◽  
Author(s):  
Yu Hong Wei ◽  
yi Zhi He
Keyword(s):  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Tumor Stage ◽  
Free Survival ◽  
Mesenchymal Phenotype ◽  
Napsin A ◽  
Tumor Tissues ◽  
Positive Rate ◽  
Degree Of Differentiation

Abstract Objective: To evaluate the clinical significance of Napsin A and circulating tumor cells（CTCs）with mesenchymal phenotype (M-CTC) in lung adenocarcinoma(LUAD).Materials and Methods: Clinical data of 97 LUAD patients were retrieved. The CanPatrolTM CTC enrichment platform was used to isolate CTCs from the peripheral blood of LUAD patients. The protein expression of Napsin A in the tumor tissues was analyzed by immunohistochemistry. Results: 20 of the 97 patients (20.62%) were negative expression of Napsin A （Napsin A-）and 60 (61.86%) were M-CTC positive（M-CTC+). Both Napsin A expression (P=0.004) and M-CTC+ (P<0.001) showed significant correlation to lymphatic metastasis, and M-CTC+ was also significantly correlated with the tumor stage (P=0.009) but was not correlated with gender, age, smoking, tumor size and degree of differentiation. Furthermore, the Napsin A- patients had a higher positive rate of M-CTC. In addition, the recurrence-free survival (RFS; Log-rank P <0.001) and overall survival (OS; Log-rank P <0.001) of the M-CTC+ LUAD patients were significantly worse. Likewise, Napsin A- was also associated with poor RFS (Log-rank P <0.001) and OS (Log-rank P = 0.0003).Conclusion: LUAD patients with Napsin A- have a higher frequency of M-CTC+, and the Napsin A- and M-CTC+ status portends poor prognosis.

scholarly journals A Modified Method to Isolate Circulating Tumor Cells and Identify by a Panel of Gene Mutations in Lung Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382199527
Author(s):  
Helin Wang ◽  
Jieqing Wu ◽  
Qi Zhang ◽  
Jianqing Hao ◽  
Ying Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Detection Rate ◽  
Target Genes ◽  
White Blood Cells ◽  
Gene Mutations ◽  
Copy Number Variations ◽  
Immunomagnetic Beads ◽  
Membrane Rupture

The CellSearch system is the only FDA approved and successful used detection technology for circulating tumor cells(CTCs). However, the process for identification of CTCs by CellSearch appear to damage the cells, which may adversely affects subsequent molecular biology assays. We aimed to explore and establish a membrane-preserving method for immunofluorescence identification of CTCs that keeping the isolated cells intact. 98 patients with lung cancer were enrolled, and the efficacy of clinical detection of CTCs was examined. Based on the CellSearch principle, we optimized an anti-EpCAM antibody and improved cell membrane rupture. A 5 ml peripheral blood sample was used to enrich CTCs with EpCAM immunomagnetic beads. Fluorescence signals were amplified with secondary antibodies against anti-EpCAM antibody attached on immunomagnetic beads. After identifying CTCs, single CTCs were isolated by micromanipulation. To confirm CTCs, genomic DNA was extracted and amplified at the single cell level to sequence 72 target genes of lung cancer and analyze the mutation copy number variations (CNVs) and gene mutations. A goat anti-mouse polyclonal antibody conjugated with Dylight 488 was selected to stain tumor cells. We identified CTCs based on EpCAM+ and CD45+ cells to exclude white blood cells. In the 98 lung cancer patients, the detection rate of CTCs (≥1 CTC) per 5 ml blood was 87.76%, the number of detections was 1–36, and the median was 2. By sequencing 72 lung cancer-associated genes, we found a high level of CNVs and gene mutations characteristic of tumor cells. We established a new CTCs staining scheme that significantly improves the detection rate and allows further analysis of CTCs characteristics at the genetic level.

scholarly journals The Discordance of Gene Mutations between Circulating Tumor Cells and Primary/Metastatic Tumor

2019 ◽  
Vol 15 ◽  
pp. 21-29 ◽  
Author(s):  
Qi Wang ◽  
Lanbo Zhao ◽  
Lu Han ◽  
Xiaoqian Tuo ◽  
Sijia Ma ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Gene Mutations ◽  
Metastatic Tumor

scholarly journals P2.01-060 Comparative Analysis of PD-L1 Expression between Circulating Tumor Cells and Tumor Tissues in Patients with Lung Cancer

2017 ◽  
Vol 12 (1) ◽  
pp. S822-S823
Author(s):  
Yasuhiro Koh ◽  
Satomi Yagi ◽  
Hiroaki Akamatsu ◽  
Ayaka Tanaka ◽  
Kuninobu Kanai ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Comparative Analysis ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Tumor Tissues

Antibody-independent ApoStream technology isolates folate receptor alpha (FRA)–positive circulating tumor cells from blood of non-small cell lung adenocarcinoma patients.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21028-e21028
Author(s):  
Darren W. Davis ◽  
Christopher Neal ◽  
Vishal Gupta ◽  
Vladislava O. Melnikova ◽  
Jacky Woo ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Folate Receptor ◽  
Small Cell ◽  
Normal Donor ◽  
Small Cell Lung ◽  
Folate Receptor Alpha ◽  
Receptor Alpha ◽  
Nsclc Patients

e21028 Background: The ability to enrich and interrogate circulating tumor cells (CTCs) from blood may allow for the analysis of metastatic dissemination and potential use of CTCs as surrogates for monitoring drug efficacy. We developed ApoStream, a dieletrophoresis field-flow fractionation based platform, for antibody-independent CTC isolation. We demonstrated that ApoStream successfully isolates non-small cell lung adenocarcinoma (NSCLC) CTCs that express Folate Receptor alpha (FRA), a GPI-anchored receptor, which has emerged as a cancer biomarker and potential therapeutic target in multiple cancer types. Methods: ApoStream technology was used to enrich CTCs from NSCLC patients’ blood. CTC enrichment by ApoStream was compared to that of the FDA cleared CellSearch CTC kit. A multiplexed immunofluorescent assay was developed to enable CTC enumeration (Cytokeratin+/CD45-/DAPI+ cells) and analysis of FRA expression (detection by murine antibody clone 26B3) using single cell quantitative laser scanning cytometry (LSC). Results: In a side-by-side comparison with the CellSearch CTC kit, ApoStream isolated significantly higher numbers of CTCs in 9 metastatic adenocarcinoma NSCLC patients (Apostream: mean =139, range 3-487 per 7.5 mL of blood, versus CellSearch kit: mean =2, range 0-8 per 7.5 mL of blood, n= 9, p=0.041). All patients were found to be CTC-positive by ApoStream, while only 3 of 9 (33%) patients were CTC-positive based on the CellSearch kit. LSC analysis demonstrated that 8-33% of all CTCs isolated expressed FRA and that FRA expression was confined to CTCs only. No false positive CTCs and no FRA-expressing cells were isolated by ApoStream from normal donor blood (n=15). Conclusions: All NSCLC adenocarcinoma patients analyzed had FRA-positive CTCs, suggesting that FRA may play a key role in metastasis and that screening of patients with the ApoStream CTC isolation system may identify patients who could benefit from FRA-targeted therapy.

ARv567es as detected in circulating tumor cells: An innovative methodology for the detection of a prognostic and therapeutic biomarker.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 112-112
Author(s):  
Avani Atul Shah ◽  
Zhigang Kang ◽  
Ravi Amrit Madan ◽  
Jennifer L. Marte ◽  
James L. Gulley ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Ligand Binding ◽  
Tumor Cells ◽  
Molecular Characterization ◽  
Circulating Tumor Cells ◽  
Sanger Sequencing ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Tumor Evolution ◽  
Blood Samples

112 Background: Increased androgen receptor (AR) expression and signaling is associated with the progression of prostate cancer. Constitutively active AR splice variants (ARVs) are thought to arise following castration and play a key role in progression. The most well studied variant, ARv567es, lacks portions of the ligand-binding domain, increasing expression of full-length AR and transcriptional activity of AR in human prostate cancer cell lines. Tumor-derived tissue can be difficult to obtain for molecular studies on patients (pts) with castration-resistant prostate cancer (CRPC), with circulating tumor cells (CTCs) representing a unique avenue to investigate changes at the molecular level. Methods: Forty-seven whole blood samples drawn from 36 pts with metastatic CRPC (mCRPC) were processed for CTC isolation followed by molecular characterization of cDNA using epithelial tumor markers and prostate cancer specific markers. Microfluidics devices used in this study for CTCs isolation and retrieval is a surface marker independent, label-free cell trap system developed to isolate CTCs based on cell size and deformability (Clearbridge Biomedics). The ARv567eswas detected by multiplex digital PCR System (BioMark HD, Fluidigm) and confirmed by Sanger sequencing. Results: Four out of 36 pts and 6 out of 47 samples had the ARv567es.. The ARv567esamplified from three pts was confirmed by Sanger sequencing in which the ligand binding domain of the receptor was absent. Metastatic disease on presentation was seen in three out of four pts with the ARVs. The ARVs were detected late in the disease course, with the survival after assessment being brief (49, 106, and 200 days) for three out of four pts. One patient is still at 343 days. Due to the nature of this analysis it is unclear when these ARVs developed since baselines samples were not available. Conclusions: This is the first report of the ARv567es found in CTC-enriched peripheral blood samples from CRPC pts. This methodology can also be employed to find other ARVs, which could serve as potential biomarkers in pts, allowing for more appropriate treatment selection based on tumor evolution. This noninvasive method could significantly impact the molecular characterization and potentially the treatment science of CRPC.

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