Monitoring of Circulating Tumor Cells and Their Expression of EGFR/Phospho-EGFR During Combined Radiotherapy Regimens in Locally Advanced Squamous Cell Carcinoma of the Head and Neck

doi:10.1016/j.ijrobp.2012.02.009

International Journal of Radiation OncologyBiologyPhysics

Volume 83, Issue 5, 1 August 2012, Pages e685-e690

https://doi.org/10.1016/j.ijrobp.2012.02.009 Get rights and content

Purpose

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.

Methods and Materials

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.

Results

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.

Conclusions

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.

Introduction

The presence of circulating tumor cells (CTCs) in peripheral blood of patients with solid tumors has previously been associated with a more aggressive disease, an increased risk of local or distant metastasis and reduced disease-free and overall survival 1, 2, 3. In a recent study using a multicolor flow cytometry protocol for detection and molecular characterization of CTCs their presence was detected in 43% of locally advanced, inoperable squamous cell carcinoma of the head and neck (SCCHN) cases (4). Their detection was independent from T stage and tumor volume but was significantly associated with nodal disease (4).

Beside the prognostic value of CTC detection before treatment, monitoring of CTCs during and after therapy has also been proposed as a potential diagnostic tool allowing adjustment of treatment in cases of inadequate treatment and nonresponding tumors. Indeed, a significant correlation between treatment-related changes in CTC numbers or their persistence after treatment with PFS and OS in metastatic colorectal (3) and prostate cancer (5) and with relapse-free survival in nonmetastatic breast cancer (6) has been demonstrated. The previous analysis of blood samples from SCCHN patients collected before treatment and during the course of concurrent chemoradiation revealed that despite a reduction in their frequency, CTCs persisted during treatment in 20% of cases (4). We extended the analysis of CTCs in SCCHN in the current study and evaluated their response to different treatment schedules: induction chemotherapy followed by radiotherapy plus cetuximab treatment or concurrent chemoradiation. The integration of the CTC analysis in a multicenter clinical trial performed under the leadership of our clinical department allowed us to collect blood samples at predefined time points: before therapy and after completion of the individual treatment regimens (induction chemotherapy, radiotherapy plus cetuximab, concurrent chemoradiation).

Taking into account the important role of EGFR signaling in metastasis and for treatment efficacy (7) and its relevance as therapeutic target for increasing the efficacy of radiotherapy and chemotherapy in SCCHN 8, 9, we assessed the influence of treatment on the detection rate of CTCs and also determined the changes in expression of EGFR and its phosphorylated form (pEGFR) in CTCs during the course of treatment.

Section snippets

Patients

This study was approved by the local ethics committee. Patients with locally advanced stage IVA/B SCCHN (n=38) participating in an ongoing multicenter randomized phase II clinical trial were included in this translational research program. The clinical trial is evaluating the feasibility and efficacy of induction chemotherapy with docetaxel, cisplatin, and 5-fluorouracil (TPF) followed by radiotherapy plus cetuximab compared with concurrent chemoradiation with cisplatin and 5-FU. Of the 38

Basal CTC numbers and their changes during treatment

For detection of CTCs in blood samples from SCCHN patients the previously described multicolor flow cytometry protocol for detection of EpCAM+cytokeratin+CD45− cells was used (4). Before treatment, ≥1 CTC per 3.75 mL blood were detected in 9 of 31 samples (29%, Figure 1A). The frequency of CTC detection was not dependent on tumor localization (data not shown). The gating strategy for detection of EpCAM+cytokeratin+CD45− CTCs and a representative result for a CTC+ sample with 2 CTCs detected in

Discussion

Although the benefit of radiotherapy in tumor therapy is well established, a relevant number of previous studies also suggest that radiation of the primary tumor might not only lead to growth inhibition and tumor cell killing but also increase the migratory and invasive potential of surviving radioresistant tumor cell subclones 10, 11. Indeed, a significant increase in the formation of lung metastases has been reported for mice bearing lung carcinoma xenografts that were treated with

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Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2–2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4–332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens.
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Potentially malignant disorders are precursor lesions of oral squamous cell carcinoma with a moderate to high risk of transformation into malignancy of many of these disorders. A bidirectional role of autophagy is also noted in oral squamous cell carcinoma. The tumor microenvironment influences the proliferation of cancer cells and progression of this malignancy including its metastasis to distant locations.
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Circulating tumour cells (CTCs) are cancer cells released by cancer into the peripheral circulation. Haematogenous tumour spread is a hallmark of metastatic malignancy and a key factor in cancer recurrence and prognosis. CTCs have diagnostic and prognostic significance for a number of adenocarcinomas and melanoma. A review of the published peer-reviewed literature was performed to determine the clinical relevance of CTCs as a biomarker in the management of oral squamous cell carcinoma (OSCC). Fourteen studies met the eligibility criteria. With regard to patients with OSCC, this review found the following: (1) CTCs have been detected using multiple techniques; (2) the presence of CTCs does not appear to be related to tumour differentiation or size; (3) CTCs may be detected without lymph node involvement; (4) the detection of CTCs may be prognostic for both disease-free survival and overall survival; (5) quantification of CTCs may reflect the efficacy of therapy; (6) CTCs may be of value for ongoing patient monitoring. Preliminary evidence suggests that CTCs have diagnostic and prognostic potential as a biomarker for oral cancer management and warrant further investigation to determine their appropriate place in the management of OSCC patients.
Clinicopathological and prognostic significance of circulating tumor cells in head and neck squamous cell carcinoma: A systematic review and meta-analysis
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Wang et al also pointed that CTCs count changes could be an independent prognostic factor in patients with locally advanced HNSCC during concurrent chemoradiotherapy [47]. Moreover, the increased number of CTCs and the increased expression of epidermal growth factor receptor (EGFR)/ phosphorylated EGFR (pEGFR) can promote the systemic spread of tumor cells in HNSCC patients [48]. There also are some limitations in our meta-analysis.
The prognostic significance of circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC) is still unclear. The objective of this study was to estimate its correlation with clinicopathological and prognostic significance in HNSCC.
Two authors systematically searched the studies independently with keywords in PubMed, Web of science, Embase, the Cochrane database, the CNKI database, the Science citation index and the references of relevant studies (up to February 2019). Odds ratio (OR), risk ratio (RR), pooled hazard ratio (HR) and 95% confidence intervals (95%CI) were calculated as effect values.
Twenty studies containing 1054 patients with HNSCC were included in this meta-analysis. The CTC-positive rate was higher in the T3-T4 group (RR = 1.29, 95% CI [1.11, 1.49], I² = 47.3%), the N1-N3 group (RR = 1.18, 95% CI [1.02, 1.36], I² = 12.4%) and the III-IV group (RR = 1.13, 95% CI [1.02, 1.25], I² = 0%). Positive CTCs were significant associated with overall survival (HR = 1.37, 95% CI [0.59, 2.15], I² = 9.7%), progression-free survival (HR = 3.40, 95%CI [1.47, 5.32], I² = 0%), and disease-free survival (HR = 3.57, 95%CI [1.06, 6.08], I² = 81%).
Our meta-analysis results indicated that CTCs are significant associated with prognosis of patients with HNSCC. The presence of CTCs can be used as a monitoring tool for survival prognosis of HNSCC patients in the future.
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The majority of cancer-related deaths result from metastasis, the process by which cancer cells escape the primary tumor site and enter into the blood circulation in order to disseminate to secondary locations throughout the body. Tumor cells found within the circulation are referred to as circulating tumor cells (CTCs), and their detection and enumeration correlate with poor prognosis. The epithelial-to-mesenchymal transition (EMT) is a dynamic process that imparts epithelial cells with mesenchymal-like properties, thus facilitating tumor cell dissemination and contributing to metastasis. However, EMT also results in the downregulation of various epithelial proteins typically utilized by CTC technologies for enrichment and detection of these rare cells, resulting in reduced detection of some CTCs, potentially those with a more metastatic phenotype. In addition to the current clinical role of CTCs as a prognostic biomarker, they also have potential as a predictive biomarker via CTC characterization. However, CTC characterization is complicated by the unknown biological significance of CTCs possessing an EMT-like phenotype, and the ability to capture and understand this CTC subpopulation is an essential step in the utilization of CTCs for patient management. This chapter will review the process of EMT and its contribution to metastasis; discusses current and future clinical applications of CTCs; and describes both traditional and novel methods for CTC enrichment, detection, and characterization with a specific focus on CTCs with an EMT phenotype.
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: This study was supported by a research grant from Merck Pharma GmbH, Germany (to IT and UK) and the Berliner Krebsgesellschaft (to IT).

: Merck Serono has reviewed the publication. The views and opinions described in the publication do not necessarily reflect those of Merck Serono.

: Conflict of interest: Dr Tinhofer has received a research grant and support for travel to meetings from Merck Pharma GmbH. Dr Keilholz has received a research grant and support for travel to meetings, for consultancy and lectures from Merck Serono and Sanofi-Aventis. Dr Stromberger was supported for travels to meetings by Merck Pharma GmbH. Dr Budach has received honorarium and support for travel to meetings from Merck Serono and Sanofi-Aventis.

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Biology ContributionMonitoring of Circulating Tumor Cells and Their Expression of EGFR/Phospho-EGFR During Combined Radiotherapy Regimens in Locally Advanced Squamous Cell Carcinoma of the Head and Neck

Purpose

Methods and Materials

Results

Conclusions

Introduction

Section snippets

Patients

Basal CTC numbers and their changes during treatment

Discussion

Circulating tumor cells, disease progression, and survival in metastatic breast cancer

N Engl J Med

Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer

Clin Cancer Res

The presence of circulating tumor cells (CTCs) correlates with lymph node metastasis in nonresectable squamous cell carcinoma of the head and neck region (SCCHN)

Ann Oncol

Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data

Lancet Oncol

An increase in cell number at completion of therapy may develop as an indicator of early relapse: quantification of circulating epithelial tumor cells (CETC) for monitoring of adjuvant therapy in breast cancer

J Cancer Res Clin Oncol

Targeting epidermal growth factor receptor and SRC pathways in head and neck cancer

Semin Oncol

Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck

N Engl J Med

Platinum-based chemotherapy plus cetuximab in head and neck cancer

N Engl J Med

Radiation therapy to a primary tumor accelerates metastatic growth in mice

Cancer Res

Biology Contribution
Monitoring of Circulating Tumor Cells and Their Expression of EGFR/Phospho-EGFR During Combined Radiotherapy Regimens in Locally Advanced Squamous Cell Carcinoma of the Head and Neck