Use of circulating tumor cells in prospective clinical trials for NSCLC patients – standardization of the pre-analytical conditions

Abstract Background: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. Methods: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. Results: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. Conclusions: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.

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Circulating tumor cells and T790M in metastatic non-small cell lung cancer patients with EGFR mutations and acquired resistance to TKI.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e18127 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e18127-e18127

Author(s):

Kazutoshi Isobe ◽

Yoshinobu Hata ◽

Keita Sato ◽

Keishi Sugino ◽

Go Sano ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Acquired Resistance ◽

Stage Iv ◽

Distant Metastases ◽

Egfr Mutations ◽

Cellsearch System ◽

Metastatic Nsclc ◽

Nsclc Patients ◽

Egfr Tki

e18127 Background: This study assessed correlations between the presence of circulating tumor cells (CTCs), detection of T790M in organs with metastases or circulating-free DNA (cfDNA), and prognosis in metastatic NSCLC patients with acquired resistance to EGFR-TKI. Methods: Metastatic NSCLC patients with activating EGFR mutations, who initially responded but subsequently experienced disease progression while on EGFR-TKI treatment, were defined as having ‘acquired resistance’. Blood samples were collected after development of such acquired resistance and CTCs were counted using the CellSearch system (Veridex). At the same time, T790M in affected organs or cfDNA was analyzed with cycleave real-time PCR assay and fragment analysis. Results: : Six men and 14 women with a mean age of 63.5 yrs (22-84) were enrolled. Histological subtypes were adenocarcinoma in 19 and squamous cell carcinoma in the remaining one. Clinical stages were stage IV in 14 and recurrence with distant metastases after surgical resection in 6. EGFR mutations in tumors at the primary site were G719C in 1, exon 19 deletion in 7, L858R in 10, and G791C + L858R in 2. CTCs were detected in 8 (40%). Numbers of CTCs (per 7.5 ml blood) were 1 in 4 cases, and 3, 4, 8, and 24 in 1 case each. Patients without CTCs survived significantly longer than those with CTCs (≥1 per 7.5 ml). Mean survival time from first detection of CTCs was 3.0 months in patients with CTCs and not reached in patients without CTCs (p < 0.001). T790M was detected in 6 cases (30%). T790M was found in 75% (n = 6/8) of patients without CTCs but in 0% (n = 0/12) of those with CTCs (p < 0.05). Conclusions: The presence of CTCs was correlated with poorly prognosis and lack of T790M in affected organs or cfDNA. The presence of CTCs was informative for distinguishing patients with or without T790M.

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Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients

Cancers ◽

10.3390/cancers12020442 ◽

2020 ◽

Vol 12 (2) ◽

pp. 442 ◽

Cited By ~ 3

Author(s):

Claudia Koch ◽

Simon A. Joosse ◽

Svenja Schneegans ◽

Okka J. W. Wilken ◽

Melanie Janning ◽

...

Keyword(s):

Clinical Trials ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Tumor Cell Line ◽

Response To Therapy ◽

Clinical Samples ◽

Blood Collection ◽

Multiple Parameters ◽

Healthy Donors

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers expressed on CTCs, we tested the Parsortix® system (ANGLE plc) which enables label-independent CTC enrichment from whole blood based on increased size and deformability of these tumor cells compared to leukocytes. We performed extensive comparisons both with spiked-in blood models (i.e., MDA-MB-468 tumor cell line cells spiked at very low concentration into blood from healthy donors) and validated the protocol on actual clinical samples from breast, lung, and gastrointestinal cancer patients to define optimal conditions for CTC enrichment. Multiple parameters including cassette gap, separation pressure, and cell fixatives were compared in parallel. Also, the compatibility of blood collection tubes with whole genome amplification of isolated tumor cells was demonstrated and we furthermore established a workflow for semi-automated CTC detection using a quantitative cell imager. The established workflow will contribute to supporting the use of size-based CTC enrichment platforms in clinical trials testing the clinical validity and utility of CTCs for personalized medicine.

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Predictive impact of PD-L1-expressing circulating tumor cells in NSCLC patients treated with nivolumab.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.11541 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 11541-11541

Author(s):

Ryota Shibaki ◽

Yasuhiro Koh ◽

Hiroaki Akamatsu ◽

Kazuki Kurita ◽

Satomi Yagi ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Stage Iv ◽

University Hospital ◽

Diagnostic Modality ◽

Prior Chemotherapy Regimen ◽

Tumor Tissues ◽

Nsclc Patients ◽

Medical University ◽

Serial Evaluation

11541 Background: PD-L1 expression on tumor tissue is associated with response to PD-1 blockade in NSCLC. Here, we conducted a serial evaluation of PD-1-expressing circulating tumor cells (CTCs) as a potential real-time diagnostic modality in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected for CTC evaluation at baseline and at week 4. CTCs were detected using microcavity array system (Hitachi Chemical Co., Ltd, Chikusei, Japan). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs. This study was registered at UMIN (ID: 000024414). Results: Thirty patients were registered in the study between January 2016 and September 2016 at Wakayama Medical University Hospital and 29 were included in the analysis. Demographics of the patients were as follows: median age 70 (range, 49 to 86); male 73 %; stage IV, 100 %; squamous/non-squamous, 27/73 %. At baseline, CTCs were detected in all patients (median, 15; range, 1 to 90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs ranged from 6% to 100%, indicating intrapatient heterogeneity. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on TPS (R2 = 0.0035). Overall response rate was 25% (7/29), and disease control rate was 54% (15/29). Total CTC count was significantly decreased after nivolumab treatment at week 4 (p < 0.05), but no significant change was observed in PD-L1 TPS on CTC. Patients harboring CTCs with PD-L1 TPS 50% or more at baseline were significantly more likely to achieve non-PD than those harboring CTCs with TPS less than 50% (p < 0.05). Conclusions: This is the first report on a serial monitoring of PD-L1 expression on CTCs in patients treated with nivolumab. PD-L1-expressing CTCs are suggested to hold potential for predicting clinical benefit. Clinical trial information: 000024414.

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Assessment of the Mutational Status of NSCLC Using Hypermetabolic Circulating Tumor Cells

Cancers ◽

10.3390/cancers10080270 ◽

2018 ◽

Vol 10 (8) ◽

pp. 270 ◽

Cited By ~ 5

Author(s):

Matteo Turetta ◽

Michela Bulfoni ◽

Giulia Brisotto ◽

Gianpiero Fasola ◽

Andrea Zanello ◽

...

Keyword(s):

Glucose Uptake ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Primary Tumor ◽

Stage Iv ◽

Kras Mutations ◽

Mutational Status ◽

Downstream Analysis ◽

Nsclc Patients ◽

New Mutations

Molecular characterization is currently a key step in NSCLC therapy selection. Circulating tumor cells (CTC) are excellent candidates for downstream analysis, but technology is still lagging behind. In this work, we show that the mutational status of NSCLC can be assessed on hypermetabolic CTC, detected by their increased glucose uptake. We validated the method in 30 Stage IV NSCLC patients: peripheral blood samples were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry. Cells with the highest glucose uptake were sorted out. EGFR and KRAS mutations were detected by ddPCR. In sorted cells, mutated DNA was found in 85% of patients, finding an exact match with primary tumor in 70% of cases. Interestingly, in two patients multiple KRAS mutations were detected. Two patients displayed different mutations with respect to the primary tumor, and in two out of the four patients with a wild type primary tumor, new mutations were highlighted: EGFR p.746_750del and KRAS p.G12V. Hypermetabolic CTC can be enriched without the need of dedicated equipment and their mutational status can successfully be assessed by ddPCR. Finally, the finding of new mutations supports the possibility of probing tumor heterogeneity.

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Significance of Circulating Tumor Cells in Nonsmall-Cell Lung Cancer Patients: Prognosis, Chemotherapy Efficacy, and Survival

Journal of Healthcare Engineering ◽

10.1155/2021/2680526 ◽

2021 ◽

Vol 2021 ◽

pp. 1-5

Author(s):

Jianpeng Li

Keyword(s):

Overall Survival ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Stage Iv ◽

Positive Association ◽

Nonsmall Cell Lung Cancer ◽

Stage Iii ◽

Chinese Patients ◽

Prognostic Information ◽

Nsclc Patients

Introduction. We aimed to evaluate whether circulating tumor cells (CTCs) were the prognostic indicator responsible for chemotherapy and survival of NSCLC patients. Methods. Between January 2013 and September 2017, CTCs in the peripheral blood of histologically confirmed stages III and IV NSCLC patients were collected. Blood specimens were obtained on the first day of treatment, chemotherapy 2 and 4 cycles, or targeted therapy 1 and 2 months for CTCs detection. The positive CTC status was defined as one or more CTCs per 7.5 ml. Results. 100 patients were enrolled, of which 48 patients (48%) were identified to be CTC positive at baseline. A higher CTC-positive rate was observed in stage IV NSCLC patients than stage III patients (69% vs. 40%, P = 0.015 ). CTC cluster was significantly correlated with disease control rate. Based on the baseline CTC number, patients were divided into low CTC levels (<4 CTCs, LL) and high CTC levels (≥4 CTCs, HL). There was clinically significant shorter median OS and OS (overall survival) and PFS (progression-free survival) in HL group patients ( P < 0.001 ). Conclusions. The positive association between the CTC number and survival suggested that the baseline CTC number and changes during treatment might be the prognostic information of response rate and overall survival in Chinese patients suffering stage III/IV NSCLC.

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Circulating tumor cells: a valuable marker of poor prognosis for advanced nasopharyngeal carcinoma

Molecular Medicine ◽

10.1186/s10020-019-0112-3 ◽

2019 ◽

Vol 25 (1) ◽

Cited By ~ 1

Author(s):

Guoping Ou ◽

Shan Xing ◽

Jianpei Li ◽

Lin Zhang ◽

Shulin Chen

Keyword(s):

Nasopharyngeal Carcinoma ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Roc Curve ◽

Poor Prognosis ◽

Regression Models ◽

Prognostic Value ◽

Stage Iv ◽

Stage Iii ◽

Ebv Dna

Abstract Purpose To evaluate the prognostic value of circulating tumor cells (CTCs) in nasopharyngeal carcinoma (NPC). Methods Cox’s proportional hazards regression models were used to identify whether CTCs was a poor prognostic factor for NPC. Chi-square tests were used to analyze and compare the distribution characteristics of CTCs in NPC. ROC curve was used to estimate the cut-off point of CTCs. Kaplan-Meier survival analyses were used to observe the prognostic value of CTCs alone and in combined with Epstein-Barr Virus DNA (EBV-DNA). Results CTCs was confirmed to be an independent risk factor for poor prognosis of NPC by Cox’s regression models that enrolled 370 NPC cases and took age, gender, EBV-DNA and CTCs as variables. The proportion of CTCs in stage IV NPC was statistically different from that in stage III; the cut-off point of CTCs between stage IV (288 cases) and stage III (70 cases) NPC estimated by ROC curve was 0.5. The prognosis of advanced NPC patients became worse with the increase of CTCs count. The combined detection of CTCs and EBV-DNA could better predict the prognosis of NPC compared with the single detection of EBV-DNA.

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