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Detection of circulating tumor cell subpopulations in patients with head and neck squamous cell carcinoma (HNSCC).

Weller P, Nel I, Hassenkamp P, Gauler T, Schlueter A, Lang S, Dountsop P, Hoffmann AC, Lehnerdt G - PLoS ONE (2014)

Bottom Line: Individual cell type profiles were analyzed.We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-.We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods: 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results: We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-. We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions: This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

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Related in: MedlinePlus

Association between CTC subtypes and tumor staging.A) Mann-Whitney test showed that he total amount of CTC was elevated in T3 patients compared to T2, but the difference did not reach level of significance. B–C) Prior to resection, the amount of N-cadherin-positive (mesenchymal) and CD133-positive (stem cell-like) CTC was significantly higher in stage T3 compared to T2 patients (both p = 0.05). D) Stem cell-like CK−/CD133+/CD45− cells were only present in stage T3 patients.
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pone-0113706-g005: Association between CTC subtypes and tumor staging.A) Mann-Whitney test showed that he total amount of CTC was elevated in T3 patients compared to T2, but the difference did not reach level of significance. B–C) Prior to resection, the amount of N-cadherin-positive (mesenchymal) and CD133-positive (stem cell-like) CTC was significantly higher in stage T3 compared to T2 patients (both p = 0.05). D) Stem cell-like CK−/CD133+/CD45− cells were only present in stage T3 patients.

Mentions: Spearman's rank correlation revealed that the survival time was significantly associated to the presence of N-cadherin+ (p = 0.04) and CK+ cells (p = 0.06) and the total amount of N-cadherin+ cells (p = 0.04) and CK+ cells (p = 0.05). Survival was also significantly correlated with the number of CK+/N-cadherin+/CD45− (p = 0.04), CK+/N-cadherin+/CD45+ (0.03) and CK−/CD133+/CD45+ cells (p = 0.03). Furthermore, Spearman test showed statistical association between survival and the number of N-cadherin+/CK−/CD45− (p = 0.17), N-cadherin+/CK+/CD45+ (p = 0.13) and N-cadherin−/CK+/CD45+ (p = 0.14) cells as well as the ratio of mesenchymal to stem cell-like cells (N-cadherin/CD133; p = 0.15). Interestingly, tumor staging was significantly associated to the number of stem cell-like CD133+/CK−/CD45− (p = 0.004), CD133+/CK+/CD45+ (p = 0.01) cells and the ratio of epithelial to mesenchymal cells (CK/N-cadherin; p = 0.01). The total number of CD133+ cells was significantly correlated with epithelial CK+/N-cadherin−/CD45− (p = 0.01), CK+/CD133−/CD45− (p = 0.02) and mesenchymal N-cadherin+/CK−/CD45− cells (p = 0.03). Wilcoxon test for paired samples showed that the total number of CTC was significantly decreased after resection compared to CTC prior to resection (p = 0.002). The subtype analysis using Wilcoxon test revealed a significant decrease of epithelial CK+ (P = 0.02), mesenchymal N-cadherin+ (p = 0.02) and stem cell-like CD133+ cells (p = 0.01) after resection (Figure 3 A–D; Table 3). Kaplan Meier test indicated a significantly shortened survival time when N-cadherin+ cells were present after resection (p = 0.04; 474 vs 235 days; CI: 0.8856–10.8339; [HR] = 3.1; Figure 4). To investigate the association between CTC subtypes and tumor staging we used Mann-Whitney test and revealed that the total number of CTC and the number of epithelial CK+ CTC was elevated in stage III compared to stage II patients, but did not differ significantly (both p = 0.3; Fig. 5 A). Interestingly, the number of mesenchymal N-Cadherin+ (p = 0.05) and stem cell-like CD133+ (p = 0.05) cells as well as CK−/CD133+/CD45− cells (p = 0.05) was significantly increased in Stage III compared to stage II patients (Figure 5 B–D; Table 4).


Detection of circulating tumor cell subpopulations in patients with head and neck squamous cell carcinoma (HNSCC).

Weller P, Nel I, Hassenkamp P, Gauler T, Schlueter A, Lang S, Dountsop P, Hoffmann AC, Lehnerdt G - PLoS ONE (2014)

Association between CTC subtypes and tumor staging.A) Mann-Whitney test showed that he total amount of CTC was elevated in T3 patients compared to T2, but the difference did not reach level of significance. B–C) Prior to resection, the amount of N-cadherin-positive (mesenchymal) and CD133-positive (stem cell-like) CTC was significantly higher in stage T3 compared to T2 patients (both p = 0.05). D) Stem cell-like CK−/CD133+/CD45− cells were only present in stage T3 patients.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4257624&req=5

pone-0113706-g005: Association between CTC subtypes and tumor staging.A) Mann-Whitney test showed that he total amount of CTC was elevated in T3 patients compared to T2, but the difference did not reach level of significance. B–C) Prior to resection, the amount of N-cadherin-positive (mesenchymal) and CD133-positive (stem cell-like) CTC was significantly higher in stage T3 compared to T2 patients (both p = 0.05). D) Stem cell-like CK−/CD133+/CD45− cells were only present in stage T3 patients.
Mentions: Spearman's rank correlation revealed that the survival time was significantly associated to the presence of N-cadherin+ (p = 0.04) and CK+ cells (p = 0.06) and the total amount of N-cadherin+ cells (p = 0.04) and CK+ cells (p = 0.05). Survival was also significantly correlated with the number of CK+/N-cadherin+/CD45− (p = 0.04), CK+/N-cadherin+/CD45+ (0.03) and CK−/CD133+/CD45+ cells (p = 0.03). Furthermore, Spearman test showed statistical association between survival and the number of N-cadherin+/CK−/CD45− (p = 0.17), N-cadherin+/CK+/CD45+ (p = 0.13) and N-cadherin−/CK+/CD45+ (p = 0.14) cells as well as the ratio of mesenchymal to stem cell-like cells (N-cadherin/CD133; p = 0.15). Interestingly, tumor staging was significantly associated to the number of stem cell-like CD133+/CK−/CD45− (p = 0.004), CD133+/CK+/CD45+ (p = 0.01) cells and the ratio of epithelial to mesenchymal cells (CK/N-cadherin; p = 0.01). The total number of CD133+ cells was significantly correlated with epithelial CK+/N-cadherin−/CD45− (p = 0.01), CK+/CD133−/CD45− (p = 0.02) and mesenchymal N-cadherin+/CK−/CD45− cells (p = 0.03). Wilcoxon test for paired samples showed that the total number of CTC was significantly decreased after resection compared to CTC prior to resection (p = 0.002). The subtype analysis using Wilcoxon test revealed a significant decrease of epithelial CK+ (P = 0.02), mesenchymal N-cadherin+ (p = 0.02) and stem cell-like CD133+ cells (p = 0.01) after resection (Figure 3 A–D; Table 3). Kaplan Meier test indicated a significantly shortened survival time when N-cadherin+ cells were present after resection (p = 0.04; 474 vs 235 days; CI: 0.8856–10.8339; [HR] = 3.1; Figure 4). To investigate the association between CTC subtypes and tumor staging we used Mann-Whitney test and revealed that the total number of CTC and the number of epithelial CK+ CTC was elevated in stage III compared to stage II patients, but did not differ significantly (both p = 0.3; Fig. 5 A). Interestingly, the number of mesenchymal N-Cadherin+ (p = 0.05) and stem cell-like CD133+ (p = 0.05) cells as well as CK−/CD133+/CD45− cells (p = 0.05) was significantly increased in Stage III compared to stage II patients (Figure 5 B–D; Table 4).

Bottom Line: Individual cell type profiles were analyzed.We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-.We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods: 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results: We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-. We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions: This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

Show MeSH
Related in: MedlinePlus