Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer - PubMed (original) (raw)

Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer

L M Flores et al. Br J Cancer. 2010.

Abstract

Background: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods.

Methods: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer.

Results: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60-70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs.

Conclusion: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour.

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Figures

Figure 1

Figure 1

CPK method improves cell yields over CEK method. The blood samples from patients with (A) breast cancer (_n_=75) or (B) NSCLC (_n_=71) processed in parallel by the CEK method with semi-automated quantification (open columns) or the CPK method with manual quantification (closed columns).

Figure 2

Figure 2

The CPK method isolates a highly enriched population of CTCs. Percentage of total cells captured by the CPK method from patients with NSCLC, staining for cytokeratin (AE1/AE3), CD45, or DAPI nuclear stain alone by (A) immunofluorescence or (B) FACS. Similar results were seen with samples from patients with breast cancer. (C) Representative immunofluorescence image of CPK-captured cells from patient with NSCLC, labelled with cytokeratin (green), DAPI (blue), and CD45 (red).

Figure 3

Figure 3

FISH analysis confirms that samples processed by the CPK method have a low percentage of contaminating normal cells. Representative FISH images of cells processed by CPK method, (A) lymphocyte with two copies of CEP7 (green), EGFR (red), and MET (blue), (B) CTC with amplified EGFR and MET. (C) Percentage of total cells captured by the CPK method from patients with NSCLC with ⩾4 copies of EGFR, CEP7, and MET per nucleus. (D) Percentage of total cells captured by the CPK method from patients with clinically defined HER2-positive (patient number 1–24) or HER2-negative (patient number 25–30) breast cancer, with the indicated copies of HER2 per nucleus.

Figure 4

Figure 4

Apoptotic CTCs are less effectively captured by CEK or CPK methods. SKBR3 (HER2+ breast cancer) or HCC827 (EGFR mutant NSCLC) cells treated with vehicle or tyrosine kinase inhibitor (SKBR3: 1 _μ_M lapatinib and HCC827; 1 _μ_M gefitinib) for 24 h and processed with (A) CEK method. (B) CPK method or (C) smeared directly on slide without processing. Plots depict percentage of cells (±1 s.d.) staining for Ki67 (open bars, proliferation marker) or TUNEL (closed bars, apoptosis marker) by immunohistochemistry. (D) Percentage of CPK-processed CTCs from patients with NSCLC staining positive for KI67 and TUNEL. (E) Comparison of KI67 expression in CTCs recovered by CEK or CPK methods.

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