Circulating Tumor Cell Detection during Neoadjuvant Chemotherapy to Predict Early Response in Locally Advanced Oropharyngeal Cancers: A Prospective Pilot Study.

Patients with locally advanced oropharyngeal carcinoma treated with neoadjuvant chemotherapy are reassessed both radiologically and clinically to adapt their treatment after the first cycle. However, some responders show early tumor progression after adjuvant radiotherapy. This cohort study evaluated circulating tumor cells (CTCs) from a population of locally advanced oropharyngeal carcinoma patients treated with docetaxel, cisplatin, and 5-fluorouracil (DCF) induction chemotherapy or DCF with a modified dose and fractioned administration. The counts and phenotypes of CTCs were assessed at baseline and at day 21 of treatment, after isolation using the RosetteSepTM technique based on negative enrichment. At baseline, 6 out of 21 patients had CTCs (28.6%). On day 21, 5 out of 11 patients had CTCs (41.6%). There was no significant difference in the overall and progression-free survival between patients with or without CTCs at baseline (p = 0.44 and 0.78) or day 21 (p = 0.88 and 0.5). Out of the 11 patients tested at day 21, 4 had a positive variation of CTCs (33%). Patients with a positive variation of CTCs display a lower overall survival. Our findings suggest that the variation in the number of CTCs would be a better guide to the management of treatment, with possible early changes in treatment strategy.

CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma.

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells.

We investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects using head and neck squamous cell carcinoma with opposite radiosensitivities. In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. We then demonstrated in SCC61 cells that nontargeted cell response was driven by the formation of the radiation-induced ceramide-enriched domain. By contrast, the existence of these platforms in SQ20B cells confers a permissive region for phosphatidylinositol-3-kinase (PI3K)/AKT activation. The disruption of lipid raft results in strong inhibition of PI3K/AKT signaling, leading to radiosensitization and apparition of nontargeted effects. These results suggest that ceramide-enriched platforms play a significant role in targeted and nontargeted effects during radiotherapy and that drugs modulating cholesterol levels may be a good alternative for improving radiotherapy effectiveness.

ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and Carbon Ion Irradiation on Cancer Stem Cell Migration and Invasion.

Although conventional radiotherapy promotes the migration/invasion of cancer stem cells (CSCs) under normoxia, carbon ion (C-ion) irradiation actually decreases these processes. Unraveling the mechanisms of this discrepancy, particularly under the hypoxic conditions that pertain in niches where CSCs are preferentially localized, would provide a better understanding of the origins of metastases. Invasion/migration, proteins involved in epithelial-to-mesenchymal transition (EMT), and expression of MMP-2 and HIF-1α were quantified in the CSC subpopulations of two head-and-neck squamous cell carcinoma (HNSCC) cell lines irradiated with X-rays or C-ions. X-rays triggered HNSCC-CSC migration/invasion under normoxia, however this effect was significantly attenuated under hypoxia. C-ions induced fewer of these processes in both oxygenation conditions. The differential response to C-ions was associated with a lack of HIF-1α stabilization, MMP-2 expression, or activation of kinases of the main EMT signaling pathways. Furthermore,we demonstrated a major role of reactive oxygen species (ROS) in the triggering of invasion/migration in response to X-rays. Monte-Carlo simulations demonstrated that HO● radicals are quantitatively higher after C-ions than after X-rays, however they are very differently distributed within cells. We postulate that the uniform distribution of ROS after X-rays induces the mechanisms leading to invasion/migration, which ROS concentrated in C-ion tracks are unable to trigger.

Dual "mAb" HER family blockade in head and neck cancer human cell lines combined with photon therapy.

Head and neck cancer stem cells (CSCs) are highly resistant to treatment. When EGFR is overexpressed in head and neck squamous cell carcinoma (HNSCC), HER2 and HER3 are also expressed. The aim of the present study was to investigate the effect of HER1/2/3 blockade through a combination of cetuximab and pertuzumab, with or without photon irradiation, on the proliferation and migration/invasion capabilities of an HNSCC chemo- and radioresistant human cell line (SQ20B) and its corresponding stem cell subpopulation. Cell proliferation, migration and invasion were studied after treatment with cetuximab +/- pertuzumab +/- 10 Gy photon irradiation. EGFR, phospho-EGFR, HER2 and HER3 protein expression levels were studied. Activation or inhibition of the RAS/MAPK and AKT-mTOR downstream signalling cascades was investigated through phospho-AKT and phospho-MEK1/2 expression. Cetuximab strongly inhibited SQ20B and FaDu cell proliferation, migration and invasion, whereas it had little effect on SQ20B-CSCs. Cetuximab-pertuzumab combined with radiation significantly inhibited SQ20B and FaDu cell and SQ20B-CSC proliferation, migration and invasion. Cetuximab-pertuzumab with 10 Gy photon irradiation switched off both phospho-AKT and phospho-MEK1/2 expression in the three populations. The triple therapy is therefore thought to inhibit SQ20B cells, SQ20B-CSCs and FaDu cells through an AKT-mTOR and Ras-MAPK downstream signalling blockade.

Differential pattern of HIF-1α expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1α (HIF-1α) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. METHODS: Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1α. RESULTS: Under hypoxia, compared to non-CSCs, HIF-1α is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1α expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1α expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1α. Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1α inhibition. CONCLUSIONS: Hypoxia-inducible factor-1α plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1α targeting an attractive therapeutic challenge.