[show abstract][hide abstract] ABSTRACT: BACKGROUND: Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay. METHODS: We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array". RESULTS: Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT). CONCLUSIONS: The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.
BMC Cancer 05/2012; 12(1):178. · 3.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: The numbers of circulating tumor cells (CTCs) and their expression/activation of epidermal growth factor receptor (EGFR) during the course of combined chemo- or bioradiotherapy regimens as potential biomarkers of treatment efficacy in squamous cell carcinoma of the head and neck (SCCHN) were determined.
Peripheral blood samples from SCCHN patients with locally advanced stage IVA/B disease who were treated with concurrent radiochemotherapy or induction chemotherapy followed by bioradiation with cetuximab were included in this study. Using flow cytometry, the absolute number of CTCs per defined blood volume as well as their expression of EGFR and its phosphorylated form (pEGFR) during the course of treatment were assessed.
Before treatment, we detected ≥1 CTC per 3.75 mL blood in 9 of 31 patients (29%). Basal expression of EGFR was detected in 100% and pEGFR in 55% of the CTC+ cases. The frequency of CTC detection was not influenced by induction chemotherapy. However, the number of CTC+ samples significantly increased after radiotherapy. This radiation-induced increase in CTC numbers was less pronounced when radiotherapy was combined with cetuximab compared to its combination with cisplatin/5-fluorouracil. The former treatment regimen was also more effective in reducing pEGFR expression in CTCs.
Definitive radiotherapy regimens of locally advanced SCCHN can increase the number of CTCs and might thus contribute to a systemic spread of tumor cells. Further studies are needed to evaluate the predictive value of the radiation-induced increase in CTC numbers and the persistent activation of the EGFR signalling pathway in individual CTC+ cases.
International journal of radiation oncology, biology, physics 05/2012; 83(5):e685-90. · 4.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.
Cytometry Part A 03/2012; 81(6):489-95. · 3.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: The role of epithelial-mesenchymal transition (EMT) in the poor outcome of EGFR-overexpressing SCCHN was evaluated.
SCCHN cell lines were characterized for their cell morphology and expression of EGFR and the EMT-associated factors E-cadherin, vimentin and Snail1. The migratory potential of cells was assessed in motility assays. Response to irradiation and cetuximab was determined using clonogenic survival assays.
High basal expression of E-cadherin but low to absent vimentin expression could be observed in all SCCHN cell lines. Although E-cadherin expression levels did not change after treatment with EGF we observed a significant change in cell morphology resembling EMT. SCCHN cells with high basal levels of Snail1 resulting from constitutive EGFR activation were characterized by mesenchymal-like morphology, elevated migratory potential, reduced sensitivity to irradiation and cetuximab but increased sensitivity to the combined treatment.
Autocrine activation of EGFR leading to EMT is associated with a metastatic phenotype and reduced sensitivity of SCCHN cells to single-modality treatment with cetuximab or irradiation. The potential of Snail1 as biomarker for selection of patients who will mostly benefit from a combination of cetuximab and radiotherapy has to be evaluated in future clinical studies.
Radiotherapy and Oncology 06/2011; 101(1):158-64. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Constitutive activation of epidermal growth factor receptor (EGFR) as a result of gene amplification, mutation, or overexpression of its ligands has been associated with response to EGFR targeting strategies. The role of these molecular mechanisms for the responsiveness of squamous cell carcinoma of the head and neck (SCCHN) to cetuximab-containing regimens remains unknown.
Tumor biopsies from 47 patients, enrolled in a single-arm phase II multicenter study for second-line treatment of recurrent or metastatic SCCHN with cetuximab and docetaxel, were analyzed by immunohistochemistry for expression of EGFR, its deletion variant III (EGFRvIII) and its ligand amphiregulin (AREG). The relation between expression levels and disease control rate (DCR) was evaluated by logistic regression. Association between expression levels, progression-free survival (PFS), and overall survival (OS) was determined by Kaplan-Meier analysis, log-rank test, and uni- and multivariate Cox regression analysis.
High expression of EGFR, EGFRvIII, and AREG was detected in 73%, 17%, and 45% of SCCHN cases, respectively. Expression levels of EGFR had no impact on PFS or OS. High expression levels of EGFRvIII were significantly associated with reduced DCR and shortened PFS (HR: 3.3, P = 0.005) but not with OS. Patients with high AREG expression in tumor cells had significantly shortened OS (HR: 2.2, P = 0.002) and PFS (HR 2.2, P = 0.019) compared with patients with low expression score. Multivariate Cox analysis revealed an independent association of AREG and EGFRvIII with PFS but only AREG was an independent prognosticator of OS.
High EGFRvIII and AREG expression levels identify SCCHN patients who are less likely to benefit from combination treatment with cetuximab and docetaxel.
Clinical Cancer Research 06/2011; 17(15):5197-204. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.
The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.
CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).
Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.
Journal of Translational Medicine 01/2011; 9:70. · 3.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: For breast cancer patients, treatment decisions based on the molecular profile of the primary tumor have been used for many years. In Her2-overexpressing tumors, trastuzumab is a key component of therapy. However, despite persistent expression of Her2, most tumors eventually become resistant to trastuzumab. When this happens, the patients benefit from a regime containing lapatinib, a dual EGFR and Her2 tyrosine kinase inhibitor. We report on a patient affected by chemo-refractory metastatic Her2-positive breast cancer enrolled in a translational research program for the detection and characterization of circulating tumor cells (CTCs). Depletion of the EGFR-positive CTC pool in the blood was associated with tumor response, whereas disease progression was related to a recurrence in CTCs, which were both EGFR and Her2 negative. Although a proof for the clinical significance of EGFR-positive circulating tumor cells is currently lacking, expression of EGFR may predict response to lapatinib-based treatments as in the case presented.
Cancer biology & therapy 11/2010; 10(9):860-4. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-α (PFT-α), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and -proficient glioma. We observed significant interaction of PFT-α with radiation-induced G(1) checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-α-mediated inhibition of radiation-induced expression of the p53 target gene p21(Waf1). Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-α in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-α significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-α treatment. In conclusion, PFT-α might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and -deficient tumors.
Radiation Research 11/2010; 174(5):601-10. · 2.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has shown clinical efficacy in squamous cell carcinoma of the head and neck with prolonged progression-free (PFS) and overall survival (OS). In this study, we analyzed whether cetuximab-induced skin rash was correlated with distinct polymorphisms within the EGFR gene known to modulate EGFR expression, ligand binding, or signaling activity.
Fifty-one patients enrolled in a single-arm phase II multicenter study for second-line treatment of recurrent or metastatic squamous cell carcinoma of the head and neck with cetuximab/docetaxel were genotyped for two genetic variations in the EGFR gene, a point substitution G-->A in exon 13 resulting in an amino acid substitution in position 521 (EGFR-R521K) and a CA repeat (CA-SSR) polymorphism in intron 1. Association between genotypes and incidence/grade of skin rash was determined by Fisher's exact test. The predictive value of genotypes for PFS and OS was determined using the log-rank test.
Overall, 21 patients (41%) developed skin rash with grade >1 within 6 weeks of treatment. The common EGFR-R521K genotype (G/G) was significantly associated with increased skin toxicity (P = 0.024) and showed a trend toward reduced risk of tumor progression (hazard ratio, 0.55; 95% confidence interval, 0.27-1.08; P = 0.08), whereas no correlation of the EGFR-R521K genotype with OS could be observed (P = 0.20). No significant interaction between CA-SSR polymorphism and skin toxicity, PFS, or OS could be detected.
Our study revealed an influence of the EGFR-R521K genotype on skin toxicity and suggested its relation to clinical activity of cetuximab/docetaxel treatment.
Clinical Cancer Research 12/2009; 16(1):304-10. · 7.84 Impact Factor