Detection of circulating tumor cells using manually performed immunocytochemistry (MICC) does not correlate with outcome in patients with early breast cancer - Results of the German SUCCESS-A- trial.
Jueckstock Julia,Rack Brigitte,Friedl Thomas W P,Scholz Christoph,Steidl Julia,Trapp Elisabeth,Tesch Hans,Forstbauer Helmut,Lorenz Ralf,Rezai Mahdi,Häberle Lothar,Alunni-Fabbroni Marianna,Schneeweiss Andreas,Beckmann Matthias W,Lichtenegger Werner,Fasching Peter A,Pantel Klaus,Janni Wolfgang,
BACKGROUND:Recently, the prognostic significance of circulating tumor cells (CTCs) in primary breast cancer as assessed using the Food-and-Drug-Administration-approved CellSearch® system has been demonstrated. Here, we evaluated the prognostic relevance of CTCs, as determined using manually performed immunocytochemistry (MICC) in peripheral blood at primary diagnosis, in patients from the prospectively randomized multicenter SUCCESS-A trial (EudraCT2005000490-21). METHODS:We analyzed 23 ml of blood from 1221 patients with node-positive or high risk node-negative breast cancer before adjuvant taxane-based chemotherapy. Cells were separated using a density gradient followed by epithelial cell labeling with the anti-cytokeratin-antibody A45-B/B3, immunohistochemical staining with new fuchsin, and cytospin preparation. All cytospins were screened for CTCs, and the cutoff for positivity was at least one CTC. The prognostic value of CTCs with regard to disease-free survival (DFS), distant disease-free survival (DDFS), breast-cancer-specific survival (BCSS), and overall survival (OS) was assessed using both univariate analyses applying the Kaplan-Meier method and log-rank tests, and using multivariate Cox regressions adjusted for other predictive factors. RESULTS:In 20.6 % of all patients (n = 251) a median of 1 (range, 1-256) CTC was detected, while 79.4 % of the patients (n = 970) were negative for CTCs before adjuvant chemotherapy. A pT1 tumor was present in 40.0 % of patients, 4.8 % had G1 grading and 34.6 % were node-negative. There was no association between CTC positivity and tumor stage, nodal status, grading, histological type, hormone receptor status, Her2 status, menopausal status or treatment. Univariate survival analyses based on a median follow-up of 64 months revealed no significant differences between CTC-positive and CTC-negative patients with regard to DFS, DDFS, BCSS, or OS. This was confirmed by fully adjusted multivariate Cox regressions, showing that the presence of CTCs (yes/no) as assessed by MICC did not predict DFS, DDFS, BCSS or OS. CONCLUSIONS:We could not demonstrate prognostic relevance regarding CTCs that were quantified using the MICC method at the time of primary diagnosis in our cohort of early breast cancer patients. Further studies are necessary to evaluate if the presence of CTCs assessed using MICC has prognostic relevance, or can be used for risk stratification and treatment monitoring in adjuvant breast cancer. TRIAL REGISTRATION:The ClinicalTrial.gov registration ID of this prospectively randomized trial is NCT02181101 ; the (retrospective) registration date was June 2014 (study start date September 2005).
Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy.
Kasimir-Bauer Sabine,Bittner Ann-Kathrin,König Lisa,Reiter Katharina,Keller Thomas,Kimmig Rainer,Hoffmann Oliver
Breast cancer research : BCR
BACKGROUND:Patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT) may experience metastatic relapse despite achieving a pathologic complete response. We analyzed patients with BC before and after NACT for disseminated tumor cells (DTCs) in the bone marrow(BM); comprehensively characterized circulating tumor cells (CTCs), including stem cell-like CTCs (slCTCs), in blood to prove the effectiveness of treatment on these cells; and correlated these findings with response to therapy, progression-free survival (PFS), and overall survival (OS). METHODS:CTCs (n = 135) and slCTCs (n = 91) before and after NACT were analyzed using the AdnaTest BreastCancer, AdnaTest TumorStemCell, and epithelial-mesenchymal transition (QIAGEN Hannover GmbH Germany). The expression of estrogen receptor, progesterone receptor, and the resistance marker excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), nuclease were studied in separate single-plex reverse transcription polymerase chain reaction experiments. DTCs were evaluated in 142 patients before and 165 patients after NACT using the pan-cytokeratin antibody A45-B/B3 for immunocytochemistry. RESULTS:The positivity rates for DTCs, CTCs, and slCTCs were 27 %, 24 %, and 51 % before and 20 %, 8 %, and 20 % after NACT, respectively. Interestingly, 72 % of CTCs present after therapy were positive for ERCC1, and CTCs before (p = 0.005) and after NACT (p = 0.05) were significantly associated with the presence of slCTCs. Whereas no significant associations with clinical parameters were found for CTCs and slCTCs, DTCs were significantly associated with nodal status (p = 0.03) and histology (0.046) before NACT and with the immunohistochemical subtype (p = 0.02) after NACT. Univariable Cox regression analysis revealed that age (p = 0.0065), tumor size before NACT (p = 0.0473), nodal status after NACT (p = 0.0137), and response to NACT (p = 0.0136) were significantly correlated with PFS, whereas age (p = 0.0162) and nodal status after NACT (p = 0.0243) were significantly associated with OS. No significant correlations were found for DTCs or any CTCs before and after therapy with regard to PFS and OS. CONCLUSIONS:Although CTCs were eradicated more effectively than DTCs, CTCs detected after treatment seemed to be associated with tumor cells showing tumor stem cell characteristics as well as with resistant tumor cell populations that might indicate a worse outcome in the future. Thus, these patients might benefit from additional second-line treatment protocols including bisphosphonates for the eradication of DTCs.
Is it feasible to detect epidermal growth factor receptor mutations in circulating tumor cells in nonsmall cell lung cancer?: A meta-analysis.
Liu Yafang,Xing Ze,Zhan Ping,Liu Hongbing,Ye Wei,Lv Tangfeng,Song Yong
BACKGROUND:The value of circulating tumor cells (CTCs) in detecting epidermal growth factor receptor (EGFR) mutations in patients with nonsmall cell lung cancer (NSCLC) is controversial. We performed a meta-analysis to investigate the diagnostic significance of CTCs with tumor tissues as the standard control. METHODS:A systematic literature search, including papers published until November 26, 2015, was performed using PubMed, Medline, Embase, Web of Science, and the China National Knowledge Infrastructure, and the references of retrieved articles were screened. The pooled sensitivity, specificity, and diagnostic odds ratio (DOR) were calculated according to the data selection from the included studies. The evaluation indexes of the diagnostic performance were the summary receiver operating characteristic curve (SROC) and area under the SROC (AUSROC). RESULTS:Eight eligible articles with a total of 170 participants were identified in our meta-analysis. The pooled sensitivity and specificity were 0.91 [95% CI: 0.55-0.99] and 0.99 [95% CI: 0.59-1.00]. The positive likelihood ratio and negative likelihood ratio were 68 [95% CI: 1.4-3364] and 0.09 [95% CI: 0.01-0.64], respectively. The DOR was 788 [95% CI: 9-71884]. The high diagnostic performance of CTCs in detecting EGFR mutations was indicated by the AUSROC of 0.99 [95% CI: 0.98-1.00]. CONCLUSIONS:CTCs are a feasible and highly specific biomarker for detecting the EGFR mutation status in NSCLC patients.
Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.
Sakurai Fuminori,Narii Nobuhiro,Tomita Kyoko,Togo Shinsaku,Takahashi Kazuhisa,Machitani Mitsuhiro,Tachibana Masashi,Ouchi Masaaki,Katagiri Nobuyoshi,Urata Yasuo,Fujiwara Toshiyoshi,Mizuguchi Hiroyuki
Molecular therapy. Methods & clinical development
Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.
Prognostic and predictive value of circulating tumor cells and CXCR4 expression as biomarkers for a CXCR4 peptide antagonist in combination with carboplatin-etoposide in small cell lung cancer: exploratory analysis of a phase II study.
Salgia Ravi,Weaver R Waide,McCleod Michael,Stille John R,Yan S Betty,Roberson Stephanie,Polzer John,Flynt Amy,Raddad Eyas,Peek Victoria L,Wijayawardana Sameera R,Um Suzane L,Gross Steve,Connelly Mark C,Morano Carrie,Repollet Madeline,Sanders Renouard,Baeten Kurt,D'Haese David,Spigel David R
Investigational new drugs
Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4 tumor, ≥7% CTCs with CXCR4 expression (CXCR4 CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4 tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4 CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4 CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.
Circulating tumor cells predict prognosis following secondline AZD 9291 treatment in EGFR-T790M mutant non-small cell lung cancer patients.
Yang Baohong,Zheng Dejie,Zeng Υanyan,Qin Aiying,Gao Jianfeng,Yu Guohua
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
PURPOSE:AZD9291 has been developed as third-generation epithermal growth factor receptor (EGFR)- tyrosine kinase inhibitor (TKI) with activities against T790M mutation. This study aimed to isolate and quantify the circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients after first-line EGFR TKIs and investigate their role in providing prognostic information. METHODS:Enrolled patients confirmed with EGFR T790M mutation received AZD9291 80 mg orally once daily as second-line treatment. Serial blood samples were taken at baseline (CTC-d0) and on day 28 (CTC-d28) following the initiation of AZD9291 for detection of CTCs using the Cell-Search system. RESULTS:The CTC measurements were dichotomized as favorable (<5 CTCs) and unfavorable (≥5 CTCs) groups. Patients in the favorable group at baseline exhibited significantly longer median progression-free survival (PFS) compared with patients in the unfavorable group (9.3 vs. 6.5 months; p=0.0002). The PFS interval for patients in the favorable group on day 28 was 9.7 months, significantly longer than the median PFS time of 6.2 months achieved by patients in the unfavorable group (p=0.011). In univariate and multivariate analysis, CTC-d0 ≥5 vs CTC-d0=0-4 was significantly associated with poor PFS. CONCLUSIONS:This is the first report over the presence of CTCs and their prognostic role in patients with EGFR T790M-positive NSCLC following disease progression on an EGFR TKI. The use of serial CTC evaluation as a surrogate biomarker needs further validation in larger samples of patients.
Detection of circulating tumor cells in patients with non-small cell lung cancer using a size-based platform.
Sonn Chung-Hee,Cho Jong Ho,Kim Jae-Won,Kang Moon Sung,Lee Jinseon,Kim Jhingook
The detection of circulating tumor cells (CTCs) is limited by the rarity of these cells in the peripheral blood of patients with cancer. Understanding tumor biology may be useful in the development of novel therapeutic strategies for patients with lung cancer. The present study evaluated a novel size-based filtration platform for enriching CTCs from patients with lung cancer. Blood samples were obtained from 82 patients with lung cancer for CTC analysis. CTC enrichment by size-based filtration was performed on 5-ml blood samples. The collected cells were detected by immunofluorescence using monoclonal anti-human antibodies against protein tyrosine phosphatase, receptor type C (CD45) and epithelial cell adhesion molecule (EpCAM; an epithelial cell marker), as well as a DAPI nucleic acid stain. CTCs were detected in 57 patients (69.5%) using the size-based filtration platform. The mean CTC counts, defined as the number of cells with DAPI-positive, CD45-negative and EpCAM-positive staining, were 1.48±1.71 per 5 ml blood for the 66 stage I-III patients and 8.00±9.95 per 5 ml blood for the 16 stage IV patients. The presence of ≥1 CTCs per 5-ml blood sample was significantly associated with pathological stage (stage IV vs. stage I-III, P=0.009), but not with patient age or gender, tumor histology, tumor size or lymphovascular invasion. The mean CTC count of healthy donors was 0.25±0.55 per 5 ml blood. In summary, CTCs from the blood of patients with lung cancer were enriched using a size-based filtration platform and immunofluorescent staining with DAPI, CD45 and EpCAM. The CTC counts of patients with stage IV cancer were higher than those of patients with stages I-III cancer. These results suggest that this novel platform may be a useful tool for determining the prognosis of patients with lung cancer.
Construction of Epidermal Growth Factor Receptor Peptide Magnetic Nanovesicles with Lipid Bilayers for Enhanced Capture of Liver Cancer Circulating Tumor Cells.
Ding Jian,Wang Kai,Tang Wen-Jie,Li Dan,Wei You-Zhen,Lu Ying,Li Zong-Hai,Liang Xiao-Fei
Highly effective targeted tumor recognition via vectors is crucial for cancer detection. In contrast to antibodies and proteins, peptides are direct targeting ligands with a low molecular weight. In the present study, a peptide magnetic nanovector platform containing a lipid bilayer was designed using a peptide amphiphile (PA) as a skeleton material in a controlled manner without surface modification. Fluorescein isothiocyanate-labeled epidermal growth factor receptor (EGFR) peptide nanoparticles (NPs) could specifically bind to EGFR-positive liver tumor cells. EGFR peptide magnetic vesicles (EPMVs) could efficiently recognize and separate hepatoma carcinoma cells from cell solutions and treated blood samples (ratio of magnetic EPMVs versus anti-EpCAM NPs: 3.5 ± 0.29). Analysis of the circulating tumor cell (CTC) count in blood samples from 32 patients with liver cancer showed that EPMVs could be effectively applied for CTC capture. Thus, this nanoscale, targeted cargo-packaging technology may be useful for designing cancer diagnostic systems.
(64)Cu- and (68)Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA-Folate.
Farkas Renáta,Siwowska Klaudia,Ametamey Simon M,Schibli Roger,van der Meulen Nicholas P,Müller Cristina
A number of folate-based radioconjugates have been synthesized and evaluated for nuclear imaging purposes of folate receptor (FR)-positive tumors and potential therapeutic application. A common shortcoming of radiofolates is, however, a significant accumulation of radioactivity in the kidneys. This situation has been faced by modifying the folate conjugate with an albumin-binding entity to increase the circulation time of the radiofolate, which led to significantly improved tumor-to-kidney ratios. The aim of this study was to develop an albumin-binding folate conjugate with a NODAGA-chelator (rf42) for labeling with (64)Cu and (68)Ga, allowing application for PET imaging. The folate conjugate rf42 was synthesized in 8 steps, with an overall yield of 5%. Radiolabeling with (64)Cu and (68)Ga was carried out at room temperature within 10 min resulting in (64)Cu-rf42 and (68)Ga-rf42 with >95% radiochemical purity. (64)Cu-rf42 and (68)Ga-rf42 were stable (>95% intact) in phosphate-buffered saline over more than 4 half-lives of the corresponding radionuclide. In vitro, the plasma protein-bound fraction of (64)Cu-rf42 and (68)Ga-rf42 was determined to be >96%. Cell experiments proved FR-specific uptake of both radiofolates, as it was reduced to <1% when KB tumor cells were coincubated with excess folic acid. In vivo, high accumulation of (64)Cu-rf42 and (68)Ga-rf42 was found in KB tumors of mice (14.52 ± 0.99% IA/g and 11.92 ± 1.68% IA/g, respectively) at 4 h after injection. The tumor-to-kidney ratios were in the range of 0.43-0.55 over the first 4 h of investigation. At later time points (up to 72 h p.i. of (64)Cu-rf42) the tumor-to-kidney ratio increased to 0.73. High-quality PET/CT images were obtained 2 h after injection of (64)Cu-rf42 and (68)Ga-rf42, respectively, allowing distinct visualization of tumors and kidneys. Comparison of PET/CT images obtained with (64)Cu-rf42 and a (64)Cu-labeled DOTA-folate conjugate (cm10) clearly proved the superiority of NODAGA for stable coordination of (64)Cu. (64)Cu-cm10 showed high liver uptake, most probably as a consequence of released (64)Cu(2+). The data reported in this study clearly proved the promising features of (64)Cu-rf42, particularly in terms of favorable tumor-to-kidney ratios. The relatively long half-life of (64)Cu (T1/2 = 12.7 h) matches well with the enhanced circulation time of the albumin-binding NODAGA-folate, allowing PET imaging at longer time points after injection than is possible when using (68)Ga (T1/2 = 68 min).
[Relationship between FGFR1 Gene Regulation of Circulating Tumor Cells and Clinical Features of Non-small Cell Lung Cancer].
Liu Lei,Huang Cheng,Li Li,Liang Naixin,Li Shanqing
Zhongguo fei ai za zhi = Chinese journal of lung cancer
BACKGROUND:The methods of detection for recurrence and metastasis in patients with non-small cell lung cancer (NSCLC) have hysteresis and one-sidedness. This study summarizes the relationship between the circulating tumor cell (CTC) in peripheral blood, expression of fibroblast growth factor receptor 1 (FGFR1) and clinic pathological features in 30 patients with NSCLC so as to provide new ideas for the detection of tumor recurrence and metastasis. METHODS:To analyze the clinical data and CTC detection data of 30 cases of NSCLC in Department of Thoracic Surgery, Peking Union Medical College Hospital from November 2016 to June 2017. RESULTS:Data analysis showed that the positive rate of CTC in peripheral blood was remarkably correlated with the smoking history (P=0.016). There was no significant correlation among the pathological type and CTC positive rate and the expression of FGFR1 (P=0.202, P=0.806). There was no significant difference in the expression of FGFR1 in different type CTC cells (P=0.094). CONCLUSIONS:The positive rate of CTC was significantly correlated with the smoking history of patients with NSCLC. There was no significant difference in CTC classification and FGFR1 expression in different pathological types of NSCLC. There was no significant difference in the expression of FGFR1 between different types of CTCs. We look forward to a larger sample size and inclusion of follow-up data to arrive at more clinically relevant conclusions about CTC and FGFR1 gene expression.
A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma.
Court Colin M,Hou Shuang,Winograd Paul,Segel Nicholas H,Li Qingyu Wilda,Zhu Yazhen,Sadeghi Saeed,Finn Richard S,Ganapathy Ekambaram,Song Min,French Samuel W,Naini Bita V,Sho Shonan,Kaldas Fady M,Busuttil Ronald W,Tomlinson James S,Tseng Hsian-Rong,Agopian Vatche G
Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society
Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel "liquid-biopsy" assay capable of detecting HCC circulating tumor cells (CTCs) and characterizing phenotypic subpopulations with prognostic significance. Using HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), glypican-3, and epithelial cell adhesion molecule was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (P < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive CTCs accurately discriminated early-stage, LT eligible patients (median, 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median, 6 CTCs; AUROC = 0.89; P = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [HR], 2.21; P = 0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early-stage HCC (HR, 3.14; P = 0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC CTCs with high efficiency and accuracy. A phenotypic subpopulation of VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.
Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in future application in mimicking circulating tumor cells.
Garcia Jessica,Wozny Anne-Sophie,Geiguer Florence,Delherme Aurélia,Barthelemy David,Merle Patrick,Tissot Claire,Jones Frederick S,Johnson Chassidy,Xing Xiaobin,Xu Zhenyu,Edelstein Daniel L,Brevet Marie,Souquet Pierre-Jean,Rodriguez-Lafrasse Claire,Payen Léa,Couraud Sébastien
Cell-free plasma DNA (cfDNA) and mimicking circulating tumor cells (mCTCs) have demonstrated tremendous potential for molecular diagnosis of cancer and have been rapidly implemented in specific settings. However, widespread clinical adoption still faces some obstacles. The purpose was to compare the performance of a BEAMing (beads, emulsion, amplification, and magnetics) assay (OncoBEAM™-epidermal growth factor receptor [EGFR] [Sysmex Inostics]) and a next-generation sequencing assay (NGS; 56G Oncology panel kit, Swift Bioscience) to detect the p.T790M EGFR mutation in cfDNA of non-small cell lung cancer (NSCLC) patients. CfDNA samples (n = 183) were collected within our hospital from patients having a known EGFR sensitizing mutation, and presenting disease progression while under first-line therapy. EGFR mutations were detected using NGS in 42.1% of samples during progression in cfDNA. Testing using the OncoBEAM™-EGFR assay enabled detection of the p.T790M EGFR mutation in 40/183 NSCLC patients (21.8%) versus 20/183 (10.9%), using the NGS assay. Samples that were only positive with the OncoBEAM™-EGFR assay had lower mutant allelic fractions (Mean = 0.1304%; SD ± 0.1463%). In addition, we investigated the detection of p.T790M in mCTCs using H1975 cells. These cells spiked into whole blood were enriched using the ClearCellFX1 microfluidic device. Using the OncoBEAM™-EGFR assay, p.T790M was detected in as few as 1.33 tumoral cells/mL. Overall, these findings highlight the value of using the OncoBEAM™-EGFR to optimize detection of the p.T790M mutation, as well as the complementary clinical value that each of the mutation detection assay offers: NGS enabled the detection of mutations in other oncogenes that may be relevant to secondary resistance mechanisms, whereas the OncoBEAM™-EGFR assay achieved higher sensitivity for detection of clinically actionable mutations.
Isolation of circulating tumor cells and detection of EGFR mutations in patients with non-small-cell lung cancer.
Zhang Qi,Nong Jingying,Wang Jinghui,Yan Zhuohong,Yi Ling,Gao Xin,Liu Zhidong,Zhang Hongtao,Zhang Shucai
The aim of the present study was to develop a procedure for the isolation of circulating tumor cells (CTCs), and to evaluate its application in the detection of epidermal growth factor receptor (EGFR) mutations, and potential heterogeneity in patients with non-small-cell lung cancer (NSCLC). Peripheral blood samples were collected from 91 patients with lung cancer, 10 patients with benign disease and 10 healthy volunteers. CTCs were enriched by positive immunomagnetic separation, detected by immunocytochemistry, and processed for single-cell capture. Pure CTC DNA was amplified, and the EGFR gene was analyzed using the amplification refractory mutation system (ARMS) and digital polymerase chain reaction (dPCR). The CTC capture rate in patients with lung cancer was 61.5% (56/91), whereas no CTCs were detected in patients with benign lung disease or in healthy volunteers. The CTC-positive detection rates were 69.3% (52/75) and 25.0% (4/16) in patients with TNM stage III and IV disease, respectively. Markedly more CTCs were captured from patients with small-cell lung cancer compared with patients with other types of cancer. In patients who were positive for EGFR mutations, the detection rate of these mutations was low (16.67%, 2/12), at the single CTC level. The sensitivity increased as the number of CTCs per sample increased. A total of four patients displayed consistent detection of EGFR mutations at the 10-cell level, and one patient exhibited a clear, inconsistent and rare mutation (G719×) between CTCs. A simplified technique for isolating CTCs from blood was established, though multiple CTCs were required to sensitively detect mutations in these cells. The detection of EGFR mutations in CTCs and tissue specimens was generally homogeneous, and therefore, the CTC-level mutation analysis may potentially contribute to the discovery of heterogeneous mutations.
The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.
Cristofanilli Massimo,Pierga Jean-Yves,Reuben James,Rademaker Alfred,Davis Andrew A,Peeters Dieter J,Fehm Tanja,Nolé Franco,Gisbert-Criado Rafael,Mavroudis Dimitrios,Grisanti Salvatore,Giuliano Mario,Garcia-Saenz Jose A,Stebbing Justin,Caldas Carlos,Gazzaniga Paola,Manso Luis,Zamarchi Rita,de Lascoiti Angela Fernandez,De Mattos-Arruda Leticia,Ignatiadis Michail,Cabel Luc,van Laere Steven J,Meier-Stiegen Franziska,Sandri Maria-Teresa,Vidal-Martinez Jose,Politaki Eleni,Consoli Francesca,Generali Daniele,Cappelletti Maria Rosa,Diaz-Rubio Eduardo,Krell Jonathan,Dawson Sarah-Jane,Raimondi Cristina,Rutten Annemie,Janni Wolfgang,Munzone Elisabetta,Carañana Vicente,Agelaki Sofia,Almici Camillo,Dirix Luc,Solomayer Erich-Franz,Zorzino Laura,Darrigues Lauren,Reis-Filho Jorge S,Gerratana Lorenzo,Michiels Stefan,Bidard François-Clément,Pantel Klaus
Critical reviews in oncology/hematology
BACKGROUND:The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. METHODS:In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with ≥ 5 CTCs were classified as Stage IV, those with < 5 CTCs as Stage IV Survival was analyzed using Kaplan-Meier curves and the log rank test. RESULTS:For all patients, Stage IV patients had longer median overall survival than those with Stage IV (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IV vs. 18.7 months Stage IV, p < 0.0001). Moreover, patients with Stage IV disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P < 0.0001), HER2-positive (36.7 months vs. 20.4 months, P < 0.0001), and triple negative (23.8 months vs. 9.0 months, P < 0.0001). Similar results were obtained regardless of prior treatment or disease location. CONCLUSIONS:We confirm the identification of two subgroups of MBC, Stage IV and Stage IV, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.
Peptide-Functionalized Nanomaterials for the Efficient Isolation of HER2-Positive Circulating Tumor Cells.
Peng Jiaxi,Zhao Qiong,Zheng Wangshu,Li Wenzhe,Li Ping,Zhu Ling,Liu Xiaoran,Shao Bin,Li Huiping,Wang Chen,Yang Yanlian
ACS applied materials & interfaces
The detection of circulating tumor cells (CTCs) with a specific antigen expression is necessary in therapeutic response monitoring and targeted therapy guidance. The existence of human epidermal growth factor receptor 2 (HER2) and the concentration of HER2-positive CTCs are strong indicators for patient diagnosis, prognosis, and therapeutic monitoring. Herein we report the direct isolation of HER2-positive CTCs by peptide-functionalized nanomaterials. We designed and screened out a peptide as an HER2 antibody alternative demonstrating high HER2 affinity and selectivity. This HER2 recognition peptide bound efficiently with HER2 at the ligand-binding domain. Efficient HER2-positive CTC capture and detection were demonstrated using magnetic nanoparticles functionalized with the HER2 recognition peptide.
Characterization of circulating tumor cells in newly diagnosed breast cancer.
Xu Lu,Jia Songlin,Li Hengyu,Yu Yue,Liu Guoping,Wu Yanmei,Liu Xishui,Liu Chaoqian,Zhou Yue,Zhang Zhenzhen,Sheng Yuan
Identification of circulating tumor cells (CTCs) by surface marker expression and ploidy analysis [immunostaining-fluorescence hybridization (iFISH)] has been shown to be a highly sensitive method in the identification of certain solid cancers. In the present study, iFISH analysis was performed to identify CTCs in 184 patients with newly diagnosed non-metastatic breast cancer, and the distribution of CTC subtypes was characterized based on cytokeratin (CK) expression and ploidy status. It was revealed that CTCs of non-metastatic, aneuploid breast cancers, independent of CK expression profile, can be detected with high sensitivity (90.76%) by the iFISH system. Higher CTC counts and sensitivity were observed in patients with increased tumor size burden and unfavorable hormone receptor status. Investigation of CTC subtypes based on ploidy analysis indicated that triploid CTCs constituted the majority of CTCs evaluated. While CK-positive CTCs were detected in a small cohort of patients, an overall low rate of CK expression was observed in the 18 patient samples evaluated. Of note, CK expression was rare in CTCs detected in patients with Herceptin 2 (Her2)-positive or triple-negative [estrogen receptor (ER)-, progesterone receptor (PR)- and Her2-negative], indicating that lack of ER and PR may result in promotion of epithelial-mesenchymal transition and enhancement of tumor aggression.
[Correlation between circulating tumor cells and clinicopathological features of early breast cancer].
Gong Jia,Xu Feng,Zhou Meirong,Wu Yufang,Xie Pingfang
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
OBJECTIVE:To investigate the correlation between the number of peripheral blood circulating tumor cells (CTCs) and clinicopathological features of early breast cancer.
Methods: The clinical and pathological data from 100 patients with early breast cancer treated by a breast cancer treatment team in the Department of Breast Surgery, Second Xiangya Hospital, Central South University, were collected from January 2017 to December 2018. For these patients, their peripheral blood CTCs were detected, enumerated and typed by CanpatrolTM CTC assay.
Results: The positive rate of CTCs was 90% in peripheral blood of patients with early breast cancer, and the majority of molecular phenotypes was hybrid CTCs (55.6%). The positive rate of CTCs was only related to the pathological type of tumor (P<0.05), but not to other clinicopathological features. No correlation between clinicopathological features and the total number of CTCs, the number of epithelial CTCs or the number of hybrid CTCs was found. However, the number of mesenchymal CTCs was significantly correlated with the expression of hormone receptors and Ki-67 (r=0.200, P<0.05), and there was a significant correlation between the proportion of mesenchymal CTCs and the expression level of Ki-67 (r=0.213, P<0.05).
Conclusion: The number of CTCs is not correlated with all clinicopathological features, but patients with negative hormone receptor and high expression of Ki-67 probably have more hybrid CTCs.
Sorting and gene mutation verification of circulating tumor cells of lung cancer with epidermal growth factor receptor peptide lipid magnetic spheres.
Wang Sheng-Guang,Zhang Bin,Li Chen-Guang,Zhu Jian-Quan,Sun Bing-Sheng,Wang Chang-Li
BACKGROUND:This study aimed to identify an efficient, simple, and specific method of detecting mutations in the epidermal growth factor receptor (EGFR) gene in isolated lung cancer circulating tumor cells (CTCs) and to improve the ability to obtain tumor tissue clinically. METHODS:EGFR peptide lipid magnetic spheres (EG-P-LMB) were prepared by reverse evaporation, and characterization and cell capture efficiency assessed. The peripheral blood samples of 30 lung cancer patients were isolated and identified with the EG-P-LMB using 20 healthy volunteers as controls. Finally, the isolated CTCs were tested for EGFR gene mutations, and the tissue samples selected for comparison. RESULTS:The prepared magnetic spheres had a smaller particle size and higher stability according to the particle size potential test. Their morphology was homogeneous by atomic force observation, and the UV test showed that there were peptides on the surface. The separation efficiency of EG-P-LMB was greater than 90% in PBS and greater than 80% in the blood simulation system. Compared with the tissue sample results, the positive rate of EGFR gene mutations was 94%. The CTC test results of 27 patients were consistent with the tissue test results of the corresponding patients, and the consistency with the tissue comparison test results was 90% (27/30). CONCLUSIONS:EG-P-LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice. KEY POINTS:SIGNIFICANT FINDINGS OF THE STUDY: EG-P-LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice. WHAT THIS STUDY ADDS:This study added EGFR peptide lipid magnetic spheres to capture CTCs in the blood. Genetic testing was performed and compared with tissues. It solves the problem of clinically difficult tumor tissue sampling.
Interleukin 15 and Eotaxin correlate with the outcome of breast cancer patients vice versa independent of CTC status.
Vilsmaier Theresa,Heidegger Helene Hildegard,Schröder Lennard,Trapp Elisabeth,Zehni Alaleh Zati,Rack Brigitte,Janni Wolfgang,Mahner Sven,Weissenbacher Tobias,Jeschke Udo,Mumm Jan-Niclas,
Archives of gynecology and obstetrics
BACKGROUND:Circulating tumor cells (CTC) in the peripheral blood in women with breast cancer has been found to be an indicator of prognosis before the start of systemic treatment. The aim of this study is the assessment of specific cytokine profiles as markers for CTC involvement that could act as independent prognostic markers in terms of survival outcome for breast cancer patients. METHODS:Patients selected for this study were defined as women with breast cancer of the SUCCESS study. A total of 200 patients' sera were included in this study, 100 patients being positive for circulating tumor cells (CTC) and 100 patients being CTC negative. The matching criteria were histo-pathological grading, lymph node metastasis, hormone receptor status, TNM classification, and patient survival. Commercial ELISA with a multi cytokine/chemokine array was used to screen the sera for Interleukin 15 (IL-15) and eotaxin. RESULTS:Statistically significant concentrations were exposed for IL-15 levels regardless of the CTC-Status, lymph node involvement, or hormone receptor status. Significantly enhanced serum IL-15 concentrations were observed in those patients with worse overall survival (OS) and disease-free survival (DFS). Elevated serum concentrations of IL-15 significantly correlate with patients diagnosed with Grade 3 tumor and worse OS. In contrast, patients with a Grade 3 tumor with a favourable OS and DFS demonstrated significantly decreased IL-15 values. The CTC negative patient subgroup with a favourable OS and DFS, showed statistically significant elevated eotaxin values. CONCLUSION:These findings suggest a potential functional interaction of increased IL-15 concentrations in the peripheral blood of patients with a worse OS and DFS, regardless of prognostic factors at primary diagnosis. The increased levels of the chemokine eotaxin in CTC negative patients and a favourable OS and DFS, on the other hand, suggest that the overexpression inhibits CTCs entering the peripheral blood, thus emphasizing a significant inhibition of circulation specific metastasis. To sum up, IL-15 could be used as an independent prognostic marker in terms of survival outcome for breast cancer patients and used as an early indicator to highlight high-risk patients and consequently the adjustment of cancer therapy strategies.
Circulating tumor cell levels and carcinoembryonic antigen: An improved diagnostic method for lung adenocarcinoma.
Ding Cheng,Zhou Xiaofei,Xu Chun,Chen Jun,Ju Shen,Chen Tengfei,Liang Zhipan,Cui Zihan,Li Chang,Zhao Jun
BACKGROUND:The aim of this study was to determine a correlation between benign and malignant lung solitary pulmonary nodules (SPN), and analyze the association between circulating tumor cell (CTC) levels and different subtypes of lung adenocarcinoma. METHODS:A total of 200 patients (80 with SPNs and 120 diagnosed with lung cancer) were included in the study. The CTC levels were quantified by identifying the folate receptor on the surface of tumor cells; clinical tumor specific markers were detected by biochemical immunization. The content of peripheral blood CTCs in benign and malignant lung SPN patients was detected and the differences in preoperative CTC levels in different pathological subtypes were analyzed. Based on the collected data, receiver operating characteristic curves were calculated and the rate of lung cancer was predicted. RESULTS:The peripheral blood CTC levels in patients with malignant lung SPNs were higher than in patients with benign SPNs. The maximum nodule diameter, carcinoembryonic antigen, and CTC levels were independent risk factors for malignant lung SPNs. The peripheral blood CTC levels in patients with stage III-IV lung adenocarcinoma were higher than in stage I-II patients. The peripheral blood CTC levels in patients with microinvasive and invasive adenocarcinoma were higher than in adenocarcinoma in situ patients. The CTC levels in the peripheral blood of patients with maximum tumor diameter > 2 cm were higher than in patients with tumors < 2 cm. CONCLUSION:The detection of CTCs can be used as a biomarker for screening SPNs and diagnosing early-stage lung cancer. Using the combination of CTC levels and CEA significantly improves the efficacy of lung adenocarcinoma diagnosis.
Prognostic and Predictive Value of Circulating and Disseminated Tumor Cells in Breast Cancer: A National Cancer Database (NCDB) Analysis.
Bilani Nadeem,Elson Leah,Liang Hong,Elimimian Elizabeth Blessing,Arteta-Bulos Rafael,Nahleh Zeina
Technology in cancer research & treatment
IMPORTANCE:Our understanding of the utility of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) as clinical biomarkers continues to evolve. OBJECTIVE:This study evaluated (1) clinicopathologic factors associated with the presence of CTCs or DTCs, (2) the prognostic value of CTCs or DTCs by disease stage, 3), the value of these biomarkers in predicting the benefit of chemotherapy. DESIGN:This is a retrospective analysis of patients with breast cancer (BC) diagnosed between 2004 and 2016 using the National Cancer Database (NCDB). To evaluate variables associated with the presence of CTCs or DTCs at the univariate level, we used chi-squared and Wilcoxon rank-sum tests. Multivariate logistic regression models were then constructed using significant variables. Consequently, we included CTC or DTC status (i.e. positive or negative) in multivariate, stage-by-stage Cox regression analyses for overall survival (OS). After stratifying by receptor status and staging, we used the Kaplan-Meier method to explore chemotherapy efficacy in CTC- or DTC-positive vs. CTC- or DTC-negative subsets. RESULTS:Factors significantly associated with CTCs were race, progesterone receptor status, HER2 status, histology and AJCC N- and M-staging. Factors associated with DTCs were race, HER2 status, histology and AJCC N-staging. CTCs were associated with poor OS in late-stage (III and IV) but not early-stage (0-II) BC. DTCs were not significantly associated with OS in either context. In hormone receptor (HR)-positive disease, chemotherapy was associated with better OS when CTC status was positive, both in early-stage and late-stage disease. In a subset of patients without CTCs, however, chemotherapy conferred no survival benefit. DTC status was not a significant predictor of chemotherapy efficacy in early or late-stage, HR+ disease. CONCLUSIONS:This study suggests that CTC-status is a significant prognostic factor at later stages of BC; yet it can also help guide management of early-stage disease as it appears predictive for chemotherapy benefit.
The Value of Monitoring the Behavior of Circulating Tumor Cells at the End of Endocrine Therapy in Breast Cancer Patients.
Pachmann Katharina,Schuster Stefan
After five years of endocrine therapy, patients with ER+ (estrogen receptor positive) breast cancer face the question of the benefit of further treatment. Ten years of endocrine therapy has been demonstrated to improve survival compared to five years. However, the individual benefit of continuation remains unclear. Therefore, markers for predicting benefit from endocrine treatment and extended endocrine treatment are desperately needed. In this study the dynamics over time of the tumor cells circulating in peripheral blood of patients, circulating tumor cells/ circulating epithelial tumor cells (CTC/CETC), as the systemic part of the tumor were investigated in 36 patients with ER+ primary breast cancer. CTC/CETCs were monitored serially during and after endocrine therapy. After termination of endocrine therapy 12 patients showed an increase in CTC/CETCs, with 8 of 12 suffering relapse. No change or a reduction was observed in 24 patients, with 2 of 24 suffering relapse. Initial tumor size was marginally prognostic ( = 0.053) but not nodal status nor the mere number of CTC/CETCs. Only the trajectory of CTC/CETCs was a statistically significant predictor of relapse free survival (increasing cell numbers: mean = 940 days vs. stable/decreasing cell numbers mean not reached). Individual cases demonstrated that an increase of CTC/CETCs after discontinuation of tamoxifen therapy could be stopped by resuming the endocrine therapy.
Combined use of EpCAM and FRα enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer.
Chen Luojun,Peng Min,Li Na,Song Qibin,Yao Yi,Xu Bin,Liu Huali,Ruan Peng
Circulating tumor cells (CTCs) provide a new approach for auxiliary diagnosis, therapeutic effect evaluation, and prognosis prediction for cancer patients. The epithelial cell adhesion molecule (EpCAM)-based separation method (CellSearch) showed good clinical use in multiple types of cancer. Nevertheless, some non-small cell lung cancer (NSCLC) tumor cells have a lower expression of EpCAM and are less frequently detected by CellSearch. Here, we present a highly sensitive immunomagnetic separation method to capture CTCs based on two cell surface markers for NSCLC, EpCAM and Folate receptor alpha (FRα). Our method has been demonstrated to be more efficient in capturing NSCLC cells (P < 0.01) by enriching three types of CTCs: EpCAM/FRα, EpCAM/FRα, and EPCAM/FRα. In 41 NSCLC patients, a significantly higher CTC capture rate (48.78% vs. 73.17%) was obtained, and by using a cutoff value of 0 CTC per 2 ml of blood, the sensitivities were 53.66% and 75.61% and the specificities were 100% and 90% for anti-EpCAM-MNs or a combination of anti-EpCAM-MNs and anti-FRα-MNs, respectively. Compared with the tumor-specific LT-PCR based on FRα, our method can isolate intact FRα CTCs, and it is advantageous for additional CTC-related downstream analysis. Our results provide a new method to increase the CTC capture efficiency of NSCLC.
Detection of AXL expression in circulating tumor cells of lung cancer patients using an automated microcavity array system.
Ikeda Mio,Koh Yasuhiro,Teraoka Shunsuke,Sato Koichi,Kanai Kuninobu,Hayata Atsushi,Tokudome Nahomi,Akamatsu Hiroaki,Ozawa Yuichi,Akamatsu Keiichiro,Endo Katsuya,Higuchi Masayuki,Nakanishi Masanori,Ueda Hiroki,Yamamoto Nobuyuki
Noninvasive diagnostics using circulating tumor cells (CTCs) are expected to be useful for decision making in precision cancer therapy. AXL, a receptor tyrosine kinase is associated with tumor progression, epithelial-to-mesenchymal transition (EMT), and drug resistance, and is a potential therapeutic target. However, the epithelial markers generally used for CTC detection may be not enough to detect AXL-expressing CTCs due to EMT. Here, we evaluated the detection of AXL-expressing CTCs using the mesenchymal marker vimentin with a microcavity array system. To evaluate the recovery of cancer cells, spike-in experiments were performed using cell lines with varying cytokeratin (CK) or vimentin (VM) expression levels. With high CK and low VM-expressing cell lines, PC-9 and HCC827, the recovery rate of AXL-expressing cancer cells was 1%-17% using either CK or VM as markers. Whereas, with low CK and high VM-expressing cell lines, MDA-MB231 and H1299, it was 52%-75% using CK and 72%-88% using VM as a marker. For clinical evaluation, peripheral blood was collected from 20 non-small cell lung cancer patients and CTCs were detected using CK or VM as markers in parallel. Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive CTCs (P < .001). Furthermore, CTC clusters were identified only among VM-positive CTCs in 20% of patients. Patients with one or more prior treatments harbored significantly more VM-positive AXL-expressing CTCs, suggesting the involvement of these CTCs in drug resistance. These results indicate the necessity of integrating mesenchymal markers with CTC detection and this should be further evaluated clinically.
Effect of Vein-First vs Artery-First Surgical Technique on Circulating Tumor Cells and Survival in Patients With Non-Small Cell Lung Cancer: A Randomized Clinical Trial and Registry-Based Propensity Score Matching Analysis.
Wei Shiyou,Guo Chenglin,He Jintao,Tan Qunyou,Mei Jiandong,Yang Zhenyu,Liu Chengwu,Pu Qiang,Ma Lin,Yuan Yong,Lin Feng,Zhu Yunke,Liao Hu,Wang Wenping,Liu Zheng,Li Qiang,Jiang Bin,Li Chuan,Xia Liang,Zhao Kejia,Gan Fanyi,Cheng Jiahan,Wu Zhu,Wang Yun,Lin Yidan,Kou Yingli,Che Guowei,Chen Longqi,Li Jing,Liu Lunxu
Importance:It is important to develop a surgical technique to reduce dissemination of tumor cells into the blood during surgery. Objective:To compare the outcomes of different sequences of vessel ligation during surgery on the dissemination of tumor cells and survival in patients with non-small cell lung cancer. Design, Setting, and Participants:This multicenter, randomized clinical trial was conducted from December 2016 to March 2018 with patients with non-small cell lung cancer who received thoracoscopic lobectomy in West China Hospital, Daping Hospital, and Sichuan Cancer Hospital. To further compare survival outcomes of the 2 procedures, we reviewed the Western China Lung Cancer database (2005-2017) using the same inclusion criteria. Interventions:Vein-first procedure vs artery-first procedure. Main Outcomes and Measures:Changes in folate receptor-positive circulating tumor cells (FR+CTCs) after surgery and 5-year overall, disease-free, and lung cancer-specific survival. Results:A total of 86 individuals were randomized; 22 patients (25.6%) were younger and 64 (74.4%) older than 60 years. Of these, 78 patients were analyzed. After surgery, an incremental change in FR+CTCs was observed in 26 of 40 patients (65.0%) in the artery-first group and 12 of 38 (31.6%) in the vein-first group (P = .003) (median change, 0.73 [interquartile range (IQR), -0.86 to 1.58] FU per 3 mL vs -0.50 [IQR, -2.53 to 0.79] FU per 3 mL; P = .006). Multivariate analysis confirmed that the artery-first procedure was a risk factor for FR+CTC increase during surgery (hazard ratio [HR], 4.03 [95% CI, 1.53-10.63]; P = .005). The propensity-matched analysis included 420 patients (210 with vein-first procedures and 210 with artery-first procedures). The vein-first group had significantly better outcomes than the artery-first group for 5-year overall survival (73.6% [95% CI, 64.4%-82.8%] vs 57.6% [95% CI, 48.4%-66.8%]; P = .002), disease-free survival (63.6% [95% CI, 55.4%-73.8%] vs 48.4% [95% CI, 40.0%-56.8%]; P = .001), and lung cancer-specific survival (76.4% [95% CI, 67.6%-85.2%] vs 59.9% [95% CI, 50.5%-69.3%]; P = .002). Multivariate analyses revealed that the artery-first procedure was a prognostic factor of poorer 5-year overall survival (HR, 1.65 [95% CI, 1.07-2.56]; P = .03), disease-free survival (HR, 1.43 [95% CI, 1.01-2.04]; P = .05) and lung cancer-specific survival (HR = 1.65 [95% CI, 1.04-2.61]; P = .03). Conclusions and Relevance:Ligating effluent veins first during surgery may reduce tumor cell dissemination and improve survival outcomes in patients with non-small cell lung cancer. Trial Registration:ClinicalTrials.gov identifier: NCT03436329.
A Novel System to Detect Circulating Tumor Cells Using Two Different Size-selective Microfilters.
Sonoda Tomoaki,Yanagitani Noriko,Suga Kanako,Yoshizawa Takahiro,Nishikawa Shingo,Kitazono Satoru,Horiike Atsushi,Shiba Kiyotaka,Ishizuka Tamotsu,Nishio Makoto,Matsusaka Satoshi
BACKGROUND/AIM:Clusters of circulating tumor cells (CTCs) increase metastatic potential compared to single CTC. However, conventional technologies have been unable to generate an accurate analysis of single and cluster CTCs in the peripheral blood. We propose an effective strategy to detect and isolate both single and cluster CTCs using two size-selective microfilters. MATERIALS AND METHODS:Five ml of whole blood were collected from 10 patients with epidermal growth factor receptor mutation-positive non-small cell lung cancer. Single and cluster CTCs were identified using precision microfiltration membranes with two distinct pore sizes together with anti-EpCAM antibody labeling. RESULTS:Single and cluster CTCs were detected by simultaneously using two size-selective microfilters. The EGFR-L858R mutation was detected in the DNA from cells captured using both microfilters. CONCLUSION:Our method can be used to detect and isolate single and cluster CTCs in the whole blood and may facilitate the development of a liquid biopsy strategy.
Reliability of using circulating tumor cells for detecting epidermal growth factor receptor mutation status in advanced non-small-cell lung cancer patients: a meta-analysis and systematic review.
Hu Fang,Mao Xiaowei,Zhang Yujun,Zheng Xiaoxuan,Gu Ping,Wang Huimin,Zhang Xueyan
OncoTargets and therapy
Purpose:To evaluate the clinical value of circulating tumor cells as a surrogate to detect epidermal growth factor receptor mutation in advanced non-small-cell lung cancer (NSCLC) patients. Methods:We searched the electronic databases, and all articles meeting predetermined selection criteria were included in this study. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were calculated. The evaluation indexes of the diagnostic performance were the summary receiver operating characteristic curve and area under the summary receiver operating characteristic curve. Results:Eight eligible publications with 255 advanced NSCLC patients were included in this meta-analysis. Taking tumor tissues as reference, the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of circulating tumor cells for detecting the epidermal growth factor receptor mutation status were found to be 0.82 (95% confidence interval [CI]: 0.50-0.95), 0.95 (95% CI: 0.24-1.00), 16.81 (95% CI: 0.33-848.62), 0.19 (95% CI: 0.06-0.64), and 86.81 (95% CI: 1.22-6,154.15), respectively. The area under the summary receiver operating characteristic curve was 0.92 (95% CI: 0.89-0.94). The subgroup analysis showed that the factors of blood volume, histological type, EGFR-tyrosine kinase inhibitor therapy, and circulating tumor cell and tissue test methods for EGFR accounted for the significant difference of the pooled specificity. No significant difference was found between the pooled sensitivity of the subgroup. Conclusion:Our meta-analysis confirmed that circulating tumor cells are a good surrogate for detecting epidermal growth factor receptor mutation when tumor tissue is unavailable in advanced NSCLC patients, but more precise techniques are needed to improve their clinical efficiency.
Assessment of folate receptor alpha and beta expression in selection of lung and pancreatic cancer patients for receptor targeted therapies.
Shen Jiayin,Hu Yingwen,Putt Karson S,Singhal Sunil,Han Haiyong,Visscher Daniel W,Murphy Linda M,Low Philip S
A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-β. Logically, if the FR positive cell population in a tumor mass is dominated by FR-β positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-β, no study to date has investigated expression of both FR-α and FR-β in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-β (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-β combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-β while pancreatic cancers express primarily FR-β. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-β should be obtained to predict the potential efficacy of a folate-targeted drug.
Simultaneous Single Cell Gene Expression and EGFR Mutation Analysis of Circulating Tumor Cells Reveals Distinct Phenotypes in NSCLC.
Owen Sarah,Lo Ting-Wen,Fouladdel Shamileh,Zeinali Mina,Keller Evan,Azizi Ebrahim,Ramnath Nithya,Nagrath Sunitha
While cancer cell populations are known to be highly heterogeneous within a tumor, the current gold standard of tumor profiling is through a tumor biopsy. These biopsies are invasive and prone to missing these clones due to spatial heterogeneity, and this bulk analysis approach can miss information from rare subpopulations. To noninvasively investigate tumor cell heterogeneity, a streamlined workflow is developed to scrutinize rare cells, such as circulating tumor cells (CTCs), for simultaneous analysis of mutations and gene expression profiles at the single cell level. This powerful workflow overcomes low-input limitations of single cell analysis techniques. The utility of this multiplexed workflow to unravel inter- and intra-patient heterogeneity is demonstrated using non-small-cell lung cancer (NSCLC) CTCs (n = 58) from six epidermal growth factor receptor (EGFR) mutant positive NSCLC patients. CTCs are isolated using a high-throughput microfluidic technology, the Labyrinth, and their EGFR mutation status and gene expression profiles are characterized.
Dynamic Monitoring and Predictive Value of Circulating Tumor Cells in EGFR-Mutated Advanced Non-Small-Cell Lung Cancer Patients Treated With First-Line EGFR Tyrosine Kinase Inhibitors.
Jiang Tao,Zhao Jing,Zhao Chao,Li Xuefei,Shen Jiqiao,Zhou Juan,Ren Shengxiang,Su Chunxia,Zhou Caicun,O'Brien Mary
Clinical lung cancer
BACKGROUND:There is an urgent need to develop a convenient and less invasive technique to monitor the efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in patients with EGFR-mutated non-small-cell lung cancer (NSCLC). We proposed folate receptor-based assay to count circulating tumor cells (CTCs) to predict and dynamically monitor the therapeutic response to first-line EGFR-TKIs in patients with EGFR-mutated NSCLC. PATIENTS AND METHODS:Eligible patients were enrolled, and 3 mL of blood was obtained before initial treatment, 1 month after treatment, and every 2 months thereafter. CTCs were isolated on the basis of negative enrichment by immunomagnetic beads and detected by a ligand-targeted PCR method. RESULTS:A total of 232 patients with EGFR-mutated NSCLC and treated with first-line EGFR-TKIs were included. Patients with low baseline CTC count had a markedly longer progression-free survival (hazard ratio = 0.48; P < .001) and overall survival (hazard ratio = 0.52; P = .002) than those with high count. This difference remained significant in multivariate analysis. Dynamic change of CTC count was significantly associated with partial response (P = .042) and stable disease/progressive disease (P = .032). Notably, dynamic monitoring of CTC provided evidence of resistance to EGFR-TKIs before computed tomographic scanning with a median lead time of 113 days (range, 45-169 days). CONCLUSION:The current evidence suggests that folate receptor-positive CTC counts can be used for both the dynamic monitoring and prediction of outcome in EGFR-mutated NSCLC patients treated with EGFR-TKIs, which could serve as an alternative or supplement to computed tomographic scanning.
Isolation of Circulating Tumor Cells from Multiple Epithelial Cancers with ApoStream(®) for Detecting (or Monitoring) the Expression of Folate Receptor Alpha.
O'Shannessy Daniel J,Davis Darren W,Anderes Kenna,Somers Elizabeth B
This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream(®) technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα(+) CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα(+) CTCs. We believe that the developed methodology will have applications in both the diagnosis and the monitoring of FRα-expressing cancers. Folate receptor alpha (FRα) expression may have utility as a potential diagnostic and therapeutic target in solid tumors. As tissue samples are not always available for patient screening, this study evaluated a noninvasive assay in CTCs from blood samples to detect FRα expression. The presence of FRα(+) CTCs enriched using ApoStream(®) and detected using laser capture cytometry was evaluated in blood samples from cancer patients [NSCLC adenocarcinoma (n = 14), breast cancer (n = 20), ovarian cancer (n = 6), and squamous lung cancer patients (n = 6)] and healthy subjects (n = 20). The data demonstrated that FRα(+) CTCs were detected in blood from NSCLC adenocarcinoma, breast, and ovarian cancer patients, whereas squamous cell lung cancer patients and normal healthy controls lacked FRα(+) CTCs as previously known. We demonstrate that CTCs captured using ApoStream(®) can be used to detect FRα(+) CTCs and may have clinical utility as a real-time liquid biopsy for assessing FRα levels in cancer patients.
Clinical Significance of Folate Receptor-positive Circulating Tumor Cells Detected by Ligand-targeted Polymerase Chain Reaction in Lung Cancer.
Wang Lin,Wu Chuanyong,Qiao Lihua,Yu Wenjun,Guo Qiaomei,Zhao Mingna,Yang Guohua,Zhao Hang,Lou Jiatao
Journal of Cancer
: As the heterogeneity of CTCs is becoming increasingly better understood, it is clear that identifying particular subtypes of CTCs would be more relevant. : We detected folate receptor (FR)-positive circulating tumor cells (FR-CTCs) by a novel ligand-targeted polymerase chain reaction (LT-PCR) detection technique. : In the none-dynamic study, FR-CTC levels of patients with lung cancer were significantly higher than controls (patients with benign lung diseases and healthy controls). With a threshold of 8.7 CTC units, FR-CTC showed a sensitivity of 77.7% and specificity of 89.5% in the diagnosis of lung cancer. When compared with established clinical biomarkers including carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and neuron-specific enolase (NSE), FR-CTC showed the highest diagnostic efficiency. Notably, the combination of FR-CTC, CEA, NSE, and CYFRA21-1 could significantly improve the diagnostic efficacy in differentiating patients with lung cancer from benign lung disease. In our dynamic surveillance study, the CTC levels of 62 non-small cell lung cancer (NSCLC) patients decreased significantly after tumor resection. : We established a LT-PCR-based FR-CTC detection platform for patients with lung cancer that exhibits high sensitivity and specificity. This platform would be clinical useful in lung cancer diagnosis and treatment response assessment.
[Diagnostic Value of Folate Receptor-positive Circulating Tumor Cell in Lung Cancer: A Pilot Study].
Lian Huanhuan,Ding Zhidan,Yuan Dongfeng,Ma Jie,Qin Jianjun
Zhongguo fei ai za zhi = Chinese journal of lung cancer
BACKGROUND:The aim of this study is to determine the efficacy and feasibility of a novel folate receptor (FR)-based circulating tumor cell (CTC) detection method in the diagnosis of lung cancer. CTCs were collected from 3 mL of blood based on negative enrichment by immunomagnetic beads and then labeled by a conjugate of a tumor-specific ligand folate and an oligonucleotide. METHODS:After washing off redundant conjugates, the bound conjugates were removed and analyzed by quantitative polymerase chain reaction. RESULTS:The CTC levels of 97 patients with lung cancer were significantly higher than that of the controls (18 patients with benign lung diseases; P<0.001). With a threshold of 8.7 Folate units, the method showed a sensitivity of 82.5% and a specificity of 72.2% in the diagnosis of lung cancer, especially a sensitivity of 86.8% in stage I disease. Compared with the existing clinical biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and CYFRA21-1, the method showed the highest diagnostic efficiency in lung cancer (area under the curve, 0.859; 95%CI: 0.779-0.939) and stage I lung cancer (area under the curve, 0.912; 95%CI: 0.829-0.994). For future work, the CTC levels of 5 lung cancer patients higher than 8.7 Folate units/3 mL in their postoperation. CONCLUSIONS:FR-positive CTCs were feasible diagnostic biomarkers in patients with lung cancer, as well as in early-stage tumors.
Predictive and prognostic value of folate receptor-positive circulating tumor cells in small cell lung cancer patients treated with first-line chemotherapy.
Shen Jiqiao,Zhao Jing,Jiang Tao,Li Xuefei,Zhao Chao,Su Chunxia,Zhou Caicun
To assess the predictive and prognostic significance of folate receptor (FR)-positive circulating tumor cells (CTCs) in patients with small cell lung cancer (SCLC) received first-line chemotherapy. Eligible patients with chemotherapy-naïve, unresectable SCLC were enrolled and blood samples were collected. CTCs were enumerated using ligand-targeted polymerase chain reaction (LT-PCR) at baseline, after two cycles of chemotherapy regimen and on disease progression. In total, 80 patients were enrolled and 67 (83.8%) had positive CTC count at baseline (CTCs ≥ 8.7 FU/3mL). The baseline CTC counts in patients with partial response (PR) were significantly higher than those with progression disease (PD) (P = 0.0365). An obvious reduction of CTC enumeration after two cycles of chemotherapy was significantly correlated with PR (P = 0.0380), instead of SD (P = 0.4934). Among positive CTC count group, patients with relative low CTC level had significantly longer progression-free survival (PFS) and overall survival (OS) than those with high CTC level (PFS: 9.1 vs 6.9 months, P = 0.0458; OS: 11.1 vs 8.6 months, P = 0.056). In multivariate analysis, distant metastases (HR = 1.466, P = 0.021) and relative low CTC level (HR = 0.656, P = 0.049) were the independent predictive factors for patients with SCLC received first-line chemotherapy. The present results demonstrated that baseline CTC counts could be the valuable predictive and prognostic biomarker for patients with SCLC received first-line chemotherapy. The reduction of CTC enumeration after two cycles of chemotherapy was a potential predictor of chemotherapeutic response in SCLC.
Clinical Significance of Circulating Tumor Cells in Hormone Receptor-positive Metastatic Breast Cancer Patients who Received Letrozole with or Without Bevacizumab.
Magbanua Mark Jesus M,Savenkov Oleksandr,Asmus Erik J,Ballman Karla V,Scott Janet H,Park John W,Dickler Maura,Partridge Ann,Carey Lisa A,Winer Eric P,Rugo Hope S
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:We evaluated the prognostic and predictive value of circulating tumor cells (CTCs) hormone receptor-positive (HR) metastatic breast cancer (MBC) patients randomized to letrozole alone or letrozole plus bevacizumab in the first-line setting (CALGB 40503). EXPERIMENTAL DESIGN:Blood samples were collected at pretreatment and three additional time points during therapy. The presence of ≥5 CTCs per 7.5 mL of blood was considered CTC positive. Association of CTCs with progression-free survival (PFS) and overall survival (OS) was assessed using Cox regression models. RESULTS:Of 343 patients treated, 294 had CTC data and were included in this analysis. Median follow-up was 39 months. In multivariable analysis, CTC-positive patients at baseline (31%) had significantly reduced PFS [HR, 1.49; 95% confidence interval (CI), 1.12-1.97] and OS (HR, 2.08; 95% CI, 1.49-2.93) compared with CTC negative. Failure to clear CTCs during treatment was associated with significantly increased risk of progression (HR, 2.2; 95% CI, 1.58-3.07) and death (HR, 3.4; 95% CI, 2.36-4.88). CTC-positive patients who received only letrozole had the worse PFS (HR, 2.3; 95% CI, 1.54-3.47) and OS (HR, 2.6; 95% CI, 1.59-4.40). Median PFS in CTC-positive patients was significantly longer (18.0 vs. 7.0 months) in letrozole plus bevacizumab versus letrozole arm ( = 0.0009). Restricted mean survival time analysis further revealed that addition of bevacizumab was associated with PFS benefit in both CTC-positive and CTC-negative patients, but OS benefit was only observed in CTC-positive patients. CONCLUSIONS:CTCs were highly prognostic for the addition of bevacizumab to first-line letrozole in patients with HR+ MBC in CALGB 40503. Further research to determine the potential predictive value of CTCs in this setting is warranted.
Folate-receptor-positive circulating tumor cells as an efficacious biomarker for the diagnosis of small pulmonary nodules.
Xue Yang,Cong Wei,Xie Shenglong,Shu Jun,Feng Gang,Gao Hong
Journal of cancer research and therapeutics
Objective:The objective of this study is to investigate the clinical significance of folate-receptor-positive circulating tumor cells (FR+CTC) for the diagnosis of lung cancer, especially in early-stage patients. Materials and Methods:A total of 72 lung cancer patients, including 31 with stage I diseases and two with stage 0 diseases, were enrolled in this study. Twenty-four patients with benign lung diseases and two healthy volunteers served as the control group. Three milliliters of peripheral blood were collected from each participant for FR+CTC analysis on enrollment. FR+CTC enumeration was performed using immunomagnetic leukocyte depletion and ligand-targeted polymerase chain reaction techniques. Results:The study results revealed that using a cutoff value of 8.7 CTC Units/3 mL, the sensitivity, and specificity of FR+CTC for diagnosis of lung cancer were 81.94% and 73.08%, respectively. Such high sensitivity (74.19%) and specificity (73.08%) persisted even if only stage I lung cancer patients were retained in the analysis. In receiver operating characteristic analysis, the performance of FR+CTC (area under the curve = 0.8153) was superior to other clinical biomarkers such as carcinoembryonic antigen, neuron-specific enolase, and cytokeratin 19 fragments. In a subgroup analysis, patients with nodule size of >3 cm showed an improved sensitivity (88.46%); although, the specificity appeared to decrease (40%). All five patients with benign diseases in this subgroup had inflammatory diseases, indicating that large inflammatory nodules may also release FR -expressing cells into the circulatory system. Conclusion:FR+CTC is a reliable biomarker for the early diagnosis of small-sized lung cancer. Further study with larger sample size is required to assess the diagnostic efficiency of FR+CTC in patients with large nodule sizes.
Folate receptor-positive circulating tumor cells as a predictive biomarker for the efficacy of first-line pemetrexed-based chemotherapy in patients with non-squamous non-small cell lung cancer.
Chen Xiaoxia,Zhou Fei,Li Xuefei,Yang Guohua,Zhao Chao,Li Wei,Wu Fenying,Yu Jia,Gao Guanghui,Li Jiayu,Li Aiwu,Ren Shengxiang,Zhou Caicun
Annals of translational medicine
Background:There is a lack of well-established biomarkers to predict the efficacy of pemetrexed-based chemotherapy. In this prospective phase II study, we investigated the correlation of folate receptor (FR)-positive circulating tumor cell (CTC) level with the clinical outcomes of patients with advanced non-squamous non-small cell lung cancer (nsNSCLC) when treated with pemetrexed-based chemotherapy. Methods:A total of 98 nsNSCLC patients were enrolled. Peripheral blood was collected from each patient prior to initiation of treatment. FR-positive CTCs were enriched by immunomagnetic leukocyte depletion and quantified using ligand-targeted polymerase chain reaction (LT-PCR) method. Results:Patients with relatively low CTC level (11-16 FU/3 mL, n=32) showed a significantly shorter progression-free survival (PFS) and overall survival (OS) compared with those in the "high CTC level group" (>16 FU/3mL, n=28; median PFS, 133 versus 320 days, P<0.001; median OS, 632 days versus "not reached", P=0.003). Patients in the "high CTC level group" also achieved superior objective response rate (ORR) and disease control rate (DCR) over those in the "low CTC level group" (ORR, 40.9% versus 9.5%, P=0.0339; DCR, 100% versus 81.0%, P=0.0485). The clinical outcomes of pemetrexed in the "negative-CTC group" (<11 FU/3mL, n=38) fell between the "high CTC level group" and the "low CTC level group" (median PFS, 290 days; median OS, 1,122 days; ORR: 21.2%, DCR: 93.9%). Further multivariate Cox proportional hazards regression analysis demonstrated that "high CTC level" was an independent factor that was significantly associated with better PFS [hazard ratio (HR) =0.26, 95% confidence interval (CI), 0.12-0.58, P=0.001] and OS (HR =0.23, 95% CI, 0.06-0.92, P=0.037). Conclusions:Our results implied that FR-positive CTC is a promising biomarker to predict the clinical outcome of pemetrexed-based chemotherapy in patients with advanced nsNSCLC.
The utility of folate receptor-positive circulating tumor cell in cancer diagnosis in the elderly population.
Li Na,Zhong Dingrong,Chen Huang,Huang Tiequn,Hou Pihua,Zhang Yinan,Chen Fangling,Wang Xiaoping,Zhang Hongchun
Cancer management and research
Cancer mortality is relatively high in the elderly population. Folate receptor-positive circulating tumor cell (FR+CTC) has proven an effective biomarker for diagnosis of lung cancer and bladder cancer and may be suitable for other cancer types accompanied with a high expression of FR. To date, the diagnostic efficiency of FR+CTC in the elderly population has not been systematically studied. Herein, we sought to investigate the utility of FR+CTC in cancer diagnosis in the elderly population and the influence of comorbidities on FR+CTC levels in such a population. A total of 35 cancer patients (including 23 lung cancers, 8 colorectal cancers, and 4 other cancers) and 40 noncancer participants, aged between 80 and 110, were recruited in this study. Three milliliters of pretreatment peripheral blood was collected from each participant for FR+CTC analysis. Compared to previous studies, the FR+CTC level was slightly higher in the elderly population (median FR+CTC levels in cancer patients versus noncancer participants were 14.3 versus 9.2 CTC U/3 mL, respectively, =0.0002). With 10.0 CTC U/3 mL as the cut-off value, the sensitivity and specificity of FR+CTC were 85.7% and 65.0%, respectively. In combination with established serum tumor biomarkers, the diagnostic efficiency of FR+CTC further improved (sensitivity=87.9%, specificity=71.8%). Clinical factors including diabetes, cardiovascular diseases, respiratory diseases, cerebral infarction, and cardiac, liver, and kidney function were not associated with the FR+CTC level (>0.05). In this exploratory study, we showed that FR+CTC is an effective biomarker for cancer diagnosis in the elderly population. The presence of comorbidities did not affect the diagnostic efficiency of FR+CTC.
Diagnostic accuracy of folate receptor-positive circulating tumor cells detected by ligand-targeted polymerase chain reaction in patients with non-small-cell lung cancer: A meta-analysis.
Illahi Y,Siddiqui N,Nadeem M
Hematology/oncology and stem cell therapy
BACKGROUND:Circulating tumor cells (CTCs) serve as predictive and prognostic markers for numerous cancers including non-small-cell lung cancer (NSCLC). CTCs are extremely rare in the blood with a frequency of one CTC per 10-10 leukocytes, therefore, highly sensitive methods are required to detect them. Recently, a new method using the detection of folate receptor-positive CTCs showed promising results in NSCLC patients. We conducted a meta-analysis of the diagnostic accuracy of the ligand-targeted polymerase chain reaction (LT-PCR) in the detection of folate-positive CTCs and thus, the diagnosis of NSCLC. METHODS:We conducted a systematic search of Medline (PubMed) and the Cochrane. Central Register of Controlled Trials for abstracts and fully published studies (from inception through May 2019). Only fully published studies and abstracts measuring the diagnostic accuracy of LT-PCR in the detection of folate-positive CTCs in patients with NSCLC were included in the meta-analysis. A total of 71,116 studies were identified. Studies which were duplicate, systematic reviews or meta-analysis, or studies based on other techniques to detect CTCs were excluded.Data were abstracted by two independent reviewers (YI, NS). Pooled sensitivities, pooled specificities and summary receiver operating curves (SROC) were obtained. Meta Disc version 1.4 software was used for statistical analyses. RESULTS:Four fully published studies and one abstract were used for the meta-analysis. Pooled sensitivity and specificity for LT-PCR to detect folate-positive CTCs were 0.77 (confidence interval: 0.75-0.79) and 0.87 (confidence interval: 0.85-0.89), respectively. Heterogeneity for both pooled sensitivity and specificity was not significant. The SROC obtained in the meta-analysis showed an area under the curve (AUC) of 0.84. CONCLUSION:Detection of folate-positive CTCs with LT-PCR showed very high accuracy in our meta-analysis.
Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.
Patil Roshani A,Srinivasarao Madduri,Amiji Mansoor M,Low Philip S,Niedre Mark
Molecular imaging and biology
PURPOSE:We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. PROCEDURES:We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. RESULTS:EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. CONCLUSIONS:This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.
Exploiting the folate receptor α in oncology.
Scaranti Mariana,Cojocaru Elena,Banerjee Susana,Banerji Udai
Nature reviews. Clinical oncology
Folate receptor α (FRα) came into focus as an anticancer target many decades after the successful development of drugs targeting intracellular folate metabolism, such as methotrexate and pemetrexed. Binding to FRα is one of several methods by which folate is taken up by cells; however, this receptor is an attractive anticancer drug target owing to the overexpression of FRα in a range of solid tumours, including ovarian, lung and breast cancers. Furthermore, using FRα to better localize effective anticancer therapies to their target tumours using platforms such as antibody-drug conjugates, small-molecule drug conjugates, radioimmunoconjugates and, more recently, chimeric antigen receptor T cells could further improve the outcomes of patients with FRα-overexpressing cancers. FRα can also be harnessed for predictive biomarker research. Moreover, imaging FRα radiologically or in real time during surgery can lead to improved functional imaging and surgical outcomes, respectively. In this Review, we describe the current status of research into FRα in cancer, including data from several late-phase clinical trials involving FRα-targeted therapies, and the use of new technologies to develop FRα-targeted agents with improved therapeutic indices.
Overcoming Obstacles in Pathological Diagnosis of Pulmonary Nodules through Circulating Tumor Cell Enrichment.
Yin Wei,Zhu Junjie,Ma Benting,Jiang Gening,Zhu Yuming,He Wei,Yang Yang,Zhang Zhemin
Small (Weinheim an der Bergstrasse, Germany)
With the popularity of low-dose computed tomography (LDCT) in clinical examination of the lung, the prevalence of pulmonary nodules has significantly increased, thus significantly improving the early diagnosis of lung cancer, but also potentially contributing to overtreatment. This study aims to develop a noninvasive method to assist in diagnosing the pulmonary nodules. To do so, 3798 patients are recruited from the Department of Thoracic Surgery at Shanghai Pulmonary Hospital and peripheral blood samples are collected from them before surgery. From these samples, circulating tumor cells (CTC) are isolated using folate receptor (FR) positivity, and then enriched and analyzed in relation to cancer gene expression, stage, and level of invasion. The average CTC concentration of patients with lung disease is 11.97 functional unit (FU) in a 3 mL sample of blood. FR-positive CTC levels correlate with the expression of lung cancer driver genes tumor-node-matastasis (TNM) stage, and pleura invasion. The sensitivity of CTC levels to lung cancer diagnosis is 87.05%. Results from this study demonstrate that the determination of FR-positive CTC concentration is a convenient and time-saving strategy to improve the pathological diagnosis of pulmonary nodules.
Value of folate receptor-positive circulating tumour cells in the clinical management of indeterminate lung nodules: A non-invasive biomarker for predicting malignancy and tumour invasiveness.
Zhou Qianjun,Geng Qing,Wang Lin,Huang Jia,Liao Meilin,Li Yan,Ding Zhengping,Yang Shentu,Zhao Hang,Shen Qiang,Pan Changqing,Lou Jiatao,Lu Shun,Chen Chang,Luo Qingquan
BACKGROUND:Non-invasive lung adenocarcinoma could benefit from limited resection, nonetheless, there is a lack of method to determine preoperative tumour invasiveness. We aimed to investigate whether folate receptor-positive circulating tumour cells (FR-CTCs) in combination with maximum tumour diameter (MTD) determines, before surgery, the invasiveness of small-sized, indeterminate solitary pulmonary nodules (SPNs). METHODS:A total of 382 patients with suspicious lung adenocarcinoma on computed tomography who were expected to undergo lung resection were enrolled in this study at three participating institutes and randomly assigned into training and validation cohorts. Before surgery, 3 mL peripheral blood was collected from all participants. FR-CTCs were analyzed using immunomagnetic leukocyte depletion and quantitated by ligand-targeted PCR method. After surgery, the resected tissues were diagnosed by pathologists according to IASLC/ATS/ERS classification. FINDINGS:FR-CTC levels in the peripheral blood can differentiate benign from malignant nodules with a sensitivity of 78·6%-82·7% and a specificity of 68·8%-78·4%. Both FR-CTC and MTD are independent predictive indices of invasive tumours for lung adenocarcinoma ≤2 cm based on multivariate analyses. Further, FR-CTC count in combination with MTD can differentiate non-invasive cancers from invasive cancers with a sensitivity of 63·6%-81·8% and a specificity of 71·4%-89·7%. INTERPRETATION:Detection of FR-CTC is a reliable method to differentiate malignancy of indeterminate SPNs. Combining of FR-CTC count and MTD could possibly enhance preoperative determination of the invasiveness of lung nodules and guide surgeons to select limited lung resection and avoid overtreatment for patients with non-invasive lesions. FUND: None.