Journal List > J Lung Cancer > v.10(1) > 1050624

Yang and Yang: Circulating Tumor Cells in Lung Cancer

Abstract

Circulating Tumour Cells (CTCs) can be released from the primary lung tumour into the bloodstream and they may colonize distant organs and give rise to metastasis. The presence of CTCs in the blood has been documented more than a century ago, and ultrasensitive methods have been recently developed to detect circulating tumour cells (CTCs) in the peripheral blood of lung cancer patients. Most CTCs require an initial enrichment step, since CTCs are a very rare event. The different technologies and also the differences among the screened populations make the clinical significance of detecting CTCs difficult to interpret. Peripheral blood analyses are more convenient for patients than invasive BM sampling and many research groups are currently assessing the clinical utility of CTCs for assessing the prognosis and monitoring the response to systemic therapies in lung cancer patients. Here we will review the different assays that are currently available for CTC detection and analysis of lung cancer. Moreover, molecular analyses of CTCs have provided new insights into the biology of metastasis of lung cancer with important implications for the clinical management of lung cancer patients. (J Lung Cancer 2011;10(1): 13 ? 25)

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Fig. 1.
Putative roles of EMT and mesenchymal-to-epithelial transition (MET) in tumour cell dissemination. In the primary tumour, a subpopulation of tumour cells (red) acquires a mesenchymal-like migratory phenotype during tumour progression. They lose their epithelial properties (ie, downregulate EpCAM and CK) through the EMT process and enter the bloodstream. These specific CTCs are thought to have stem cell properties. After extravasation into distant organs, these tumour cells (DTCs) have to re-express their epithelial properties through MET to form tumour cell clusters (micrometastases). Another subpopulation of CTCs, which is not able to undergo EMT (blue), can also disseminate through the bloodstream into distant organs but lacks cancer stem cell properties and, therefore, does not form (micro)metastases. These cells are detected by current CTC technologies, whereas the EMT-induced CTCs (red) are missed. This figure is reprinted from the article by Pantel K, et al. (42).
jlc-10-13f1.tif
Fig. 2.
Epithelial (E-cadherin) and mesenchymal (vimentin) markers in circulating tumor microemboli (CTM) and circulating tumor cells (CTCs) profiled by dual-color IHC. Blood samples from lung cancer patients were subjected to ISET filtration and CTM/CTCs were profiled for both epithelial (E-cadherin) and mesenchymal (vimentin) markers by dual-color IHC. (A) Vimentin-positive CTM (cytoplasm stained in red) with negative staining for E-cadherin (brown). (B) E-cadherin positive CTM (brown) without vimentin expression. (C) Displays CTC (black arrow), which is positively stained for E-cadherin in cytosol and negative for vimentin. White blood cells (white arrow in C and cells in D) serve as control and demonstrate positive staining for vimentin, negative staining for E-cadherin, and typical neutrophil nuclear morphology. This picture is reprinted from the article by Hou JM, et al. (17).
jlc-10-13f2.tif
Fig. 3.
Illustrative cartoon demonstrating a patient with non-small cell lung cancer (NSCLC) donating a tube of peripheral blood which is then processed in the circulating tumor cell (CTC)-chip immediately, without any required preprocessing. CTCs are captured against the sides of the anti-Ep-CAM-coated posts (epithelial cell adhesion molecule), and then can be stained with fluore-scently-labeled markers for enumeration or undergo genomic DNA extraction for epidermal growth factor receptor mutation or other molecular analysis. This picture is reprinted from the article by Sequist LV, et al. (28).
jlc-10-13f3.tif
Fig. 4.
Deposition of a single CTC on the AmpliGrid chip. (A) AmpliGrid chip with 48 reaction sites for single cell deposition and PCR analysis. (B) A single CTC is deposited with a capillary on the reaction site of the Ampligrid chip. CTCs are normally resuspended in physiological buffer and the volume of deposition is approxi-mately 20 nl. (C) A single CTC can be easily visualised on the reaction site when the DNA is stained with Hoechst dye. This pricture is reprinted from the article by Alunni-Fabbroni M, et al. (43).
jlc-10-13f4.tif
Fig. 5.
(A) Distribution of CTC count in primary lung cancer patients without distant metastasis and in patients with distant metastasis. (B) ROC curves for CTC count and serum CEA level to predict the absence or presence of distant metastasis among primary lung cancer patients. CTC: Circulating tumor cell, CEA: Carcinoembryonic Antigen. This picture is reprinted from the article by Tanaka F, et al. (36).
jlc-10-13f5.tif
Fig. 6.
Disease progression and progression-free survival in surgically resected NSCLC patients according to paired presurgery and postsurgery TTF-1 (+) CTC status. When pairing presurgery and postsurgery TTF-1 (+) CTC status and allocating patients to four groups, the Pre (−) Post (+) group (TTF-1 (+) CTCs were not present at presurgery, but detected at postsurgery time) most frequently developed disease progression (A), and showed shortest progression-free survival (B) as compared to other three groups. This picture is reprinted from the article by Yoon SO, et al. (37).
jlc-10-13f6.tif
Fig. 7.
Serial Analyses of Circulating Tumor Cells during Therapy. (A) Shows serial analyses of the numbers of circulating tumor cells (CTCs) per milliliter and the radiographic tumor burden in centimeters in four patients with non-mall-cell lung cancer with EGFR mutations, as measured at multiple time points during the course of treatment with gefitinib, another chemotherapy agent (chemo), or an experimental agent (exp). The duration of each therapy is indicated by the gray bars. The genotypes of circulating tumor cells are shown for various time points. Mutations in brackets are those that were present at low allele frequencies. (B) SARMS analysis of EGFR genotypes in Patient 9 shows an increased abundance of the T790M drug-resistance allele during disease progression. Arrows denote the cycle of threshold for amplification cycles (Ct) required for detection of the primary mutation (Del or Deletion, referring to the grouped exon 19 deletions) and the T790M mutation, as compared with the exon 2 control. Δ Ct reflects the difference in allele frequency between the primary mutation and T790M in the tumor-biopsy specimen, the circulating tumor cells that were isolated at the time of response to gefitinib therapy, and the circulating tumor cells that were isolated at the time of disease progression. This picture is reprinted from the article by Maheswaran S, et al. (38).
jlc-10-13f7.tif
Fig. 8.
Correlation of circulating tumor cell (CTC) counting and computed tomography (CT) scanning with a follow-up study. Correlation of CT scanning and CTC counting was evaluated on a small scale of follow-up study. CTC was enumerated from each patient before chemotherapy started. After two courses of first line chemotherapy, patient CTC was measured, followed by CT examination. Eight of 12 patients (patient 1-) including 5 adenocarcinoma (ADC), 2 squamous cell carcinoma (SCC) and 1 small cell lung cancer (SCLC) were classified by CT as stable disease. Among those 8 patients, 5 patients dropped their CTC to 0, 1 patient dropped from 21 to 3, 2 patients remained 0, and in 1 patient the count increased from 2 to 3. Three patients had a partial radiologic response: For partial response (PR) patients (patients 9-1), one was noted to have a CTC drop from 52 to 0, while the CTC in the other 2 patients remained below 2. Only one patient was noted to have an increasing CTC, from 2 to 12 after treatment, and this patient was confirmed to have progressive disease (PD). Clinical responses are classified by CT scanning according to the Response Evaluation Criteria in Solid Tumors (RECIST). This pricture is reprinted from the article by Wu C, et al. (39).
jlc-10-13f8.tif
Table 1.
Summary of Different CTC Enrichment Approaches
Enrichment method Advantages Disadvantages
Size based ISET Easy and cheap Low specificity
    Feasible with EpCam positive and negative tumour cells Loss of small CTC which can pass through the pores
      Enrichment of large leukocytes
  Density gradient Easy and cheap Low specificity
    Feasible for EpCAM positive and negative tumour cells Cross contamination with blood mononuclear cells possible
    Feasible for negative selection  
  OncoQuick Density gradient based Low specificity
    Feasible for EpCAM positive and negative tumour cells  
    Cross contamination reduced because of additional barrier  
  Rosette Sept Good clean up of unwanted hemapoietic cells Cross contamination possible
Immunomagnetic based MACS/Dynal Flexible False positive due to the expression of the same antigens on non-tumour cells
  Magnetic Beads/Easy Sep Cell integrity preserved  
      False negative due to loss of antigens on CTCs
  AdnaTest Recognition of .xed markers (EpCAM, MUC1) No fiexibility
    Downstream analysis (RT-qPCR of MUC1, HER2 and GA73.3.2) False positive due to the expression of the same antigens on non-tumour cells
    Possibility to characterize for stem cell and Epithelial
    False negative due to loss of antigens on CTCs
    Mesenchimal Transition
  CellSearch Semi-automated Only EpCAM positive CTCs detected
    Combination of positive (anti-EpCAM) and negative (anti-CD45) selection False positive due to the expression of the same antigens on non-tumour cells
    FDA approved False negative due to loss of antigens on CTCs
  CTC-Chip Good enrichment grade 98% cell viability Only EpCAM positive CTCs detected
    Further analysis possible Clinical validation not yet available
      Not yet on the market

This Table is reprinted from the article by Mostert B, et al. (44).

Table 2.
Summary of Different CTC Identification Approaches
Identification process Advantages Disadvantages
PCR-based analysis RT-PCR High sensitivity RNA degradation
      False positive results due to unspecific amplification, contaminations, pseudogenes
      No distinction between viable and non-viable cells
      False negative results due to low expression level
  RT-qPCR High sensitivity No visualisation of CTCs
    Quantitative No further analysis possible
Cytometric analysis FAST Scan analysis of large Lack of validation studies in clinical settings
    volume of sample  
    Cell loss minimised  
    Quick analysis  
    (up to 300,000 cells/s)  
  LSC Fast Technically challenging
    High specificity Low sensitivity
  Flow cytometry High specificity Low sensitivity
  CellSearch Multiple parameters Semi-automated Only EpCAM/CK/CD45 CTCs detected
    High sensitivity Subjective images interpretation
    CTC quantification No further analysis possible
    Reproducible  
    Recognition of .xed marker  
    (EpCAM, CKs, CD45)  
    FDA approved  
  CTC-chip 98% Cell viability Only EpCam positive CTCs detected
    High detection rate Not commercially available
    Further analysis possible Lack of validation studies in clinical settings
  EPISPOT Analysis only on viable cells CTC isolation not possible, therefore further analysis not possible
    High sensitivity
      Need of active protein secretion
      Technically challenging
  FISH Genetic analysis Further analysis not possible

This Table is reprinted from the article by Alunni-Fabbroni M, et al. (43).

Table 3.
Clinical Relevance of CTC Detection in Breast Cancer Patients
Study Tumour stage Method Number of patients CTC detection rate (%) Clinical results
Cristofanilli et al. (23) Metastatic breast cancer CellSearch 177 49 CTCs P 5/7.5 ml associated with reduced PFS (p<0.001) and OS (p<0.001)
Nolé et al. (45) Metastatic breast cancer CellSearch 80 61 CTCs P 5/7.5 ml associated with reduced PFS (p=0.002)
Budd et al. (46) Metastatic breast cancer CellSearch 138 25.4 CTC evaluation after 4 weeks from the start of chemotherapy are better predictor of OS compared to radiologic response evaluation performed at 10 weeks
De Giorgi et al. (47) Metastatic breast cancer CellSearch 115 21 Univariate analysis: CTCs levels at 9.12 weeks (p<0.0001) and FDG-PET/CT (p≤0.001) associated with OS Multivariate analysis: only CTCs levels at 9.12 weeks (p<0.001) associated with OS
Pierga et al. (48) Locally advanced breast cancer CellSearch 118 27 CTCs P 1/22.5 ml associated with reduced DFS (0.017)
Stathopoulou et al. (49) Early breast cancer (stage I-II) RT-PCR (CK19) 148 29.7 Reduced DFS (p=0.0007) and OS (p=0.01)
Xenidis et al. (50) Early breast cancer (stage I-II) RT-PCR (CK19) 167 21.6 Reduced DFS (p<0.00001) and OS (p=0.008)
Ignatiadis et al. (51) Early breast cancer (stage I-III) RT-PCR (CK19) 444 40.8 Reduced DFS (p<0.001) and OS (p=0.001)
Apostolaki et al. (52) Early breast cancer (stage I-II) RT-PCR (HER2) 214 21 Reduced DFI (p=0.006), no association with OS (p=0.2)
Xenidis et al. (53) Early breast cancer (stage I-III) RT-PCR (CK19) 437 32.7 Reduced DFS (p<0.001) and OS (p=0.001)
Rack et al. (54) Early breast cancer (stage I-III) CellSearch 1,500 9 CTCs>1/22.5 mL associated with reduced DFS (p=0.04) and OS (p=0.03)

This Table is reprinted from the article by Alunni-Fabbroni M, et al. (43).

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