Background and Purpose:Helium ion therapy offers favorable dose distributions and potential biological advantages over photon and proton therapies, yet data on cellular response in clinically relevant Spread-Out Bragg Peak (SOBP) configurations remain limited. This study measured the surviving fraction of radioresistant human laryngeal squamous cell carcinoma (SQ20B) cells irradiated in a helium SOBP and compared experimental results with predictions from NanOx, a nanodosimetry and oxidative stress model implemented in Monte Carlo simulations. Materials and Methods:SQ20B cells were irradiated at three positions along a helium SOBP using a passive beamline. Survival fractions were measured for doses between 1 and 4 Gy. The setup was simulated in GATE, and survival was predicted using the NanOx model via the Biodose Actor. Relative biological effectiveness (RBE) was derived from linear quadratic fits using photon reference parameters. Results:NanOx predicted decreasing survival towards the distal edge, with surviving fractions at 4 Gy of 11%, 7%, and 4% at the proximal edge, plateau, and distal edge, respectively. Experimentally, fitted surviving fractions at 4 Gy were 10%, 12%, and 8%, showing no spatial trend. Deviations between model predictions and experimental averages ranged from 20 to 37%. Experimental RBE values ranged from 2.1 to 2.4, in reasonable agreement with NanOx predictions (2.1-2.9). Conclusions:In this SQ20B cell model under the present experimental conditions, NanOx predictions differed from experimental data by 20-37% depending on position. These results support further validation of the model in a broader range of cell lines and helium ion irradiation configurations.

Objectives: Non-HPV-induced squamous cell carcinomas (SCC) of the oropharynx have a poorer prognosis than HPV-induced cancers because they are more radio- and chemoresistant. Currently, no predictive biomarker exists for survival outcome in this type of tumor. NRF2 is a nuclear transcription factor that regulates the expression of cytoprotective genes, and peroxiredoxin 6 is one of the proteins regulated by NRF2. Both are potentially involved in the resistance to treatments as they play a central role in the response to oxidative stress. Material and Methods: In a retrospective cohort study of 53 patients with advanced HPV-negative squamous cell carcinomas of the oropharynx, we studied the correlation between clinical characteristics, response to treatment (surgery + radiotherapy), patient survival, and the expression of NRF2 and peroxiredoxin 6 in pre-treatment tumor biopsies. We prepared Tissue Micro Arrays (TMA) before performing immunohistochemistry analyses. Results: For a given patient, prior to any treatment, NRF2 and peroxiredoxin 6 are overexpressed in tumor tissue compared with healthy tissue. Moreover, patients with high NRF2 expression tended to have poorer progression-free survival (p = 0,09). Patients with high expression of peroxiredoxin 6 had significantly worse progression-free survival and overall survival (p = 0.015 and p < 0.01, respectively). The combination of high NRF2 and peroxiredoxin 6 expression was associated with even worse survival outcomes (p < 0.01and p = 0.01, respectively). Conclusion: Elevated expression levels of peroxiredoxin 6, either alone or in combination with NRF2, are poor prognostic indicators for patient survival.

PURPOSE:Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive. METHODS AND MATERIALS:The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells. RESULTS:Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells. CONCLUSIONS:SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.

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.

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.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide. It is often associated with a history of smoking/alcohol consumption or exposure to the human papilloma virus (HPV). Beyond surgery, treatment usually includes DNA damaging treatments i.e. cisplatin/5FU combined or not with radiation therapy. Treatment failure rates are still high due to intrinsic and acquired mechanisms of resistance, largely involving DNA repair mechanisms. Here we precisely examined the main DNA repair mechanisms susceptible to drive tumor progression and resistance to treatment, using a miniaturized comprehensive functional approach on biochip. Enzymatic DNA repair profiles were compared for lymphocytes and tumor biopsies taken before treatment from 38 patients in a prospective clinical study. We simultaneously investigated double strand break repair pathways (HR, NHEJ, SSA, alt-EJ), excision/synthesis repair mechanisms (BER, NER, ICLR) and several glycosylases/AP endonuclease activities, using lysates prepared from the samples. Results were correlated with physiologic and lifestyle/risk factors, TNM tumor classification, treatment-induced adverse effects, and disease progression or death at 18 months. Analysis of blood cells and biopsies provided different and complementary information. Indeed, the prediction of treatment-induced severe toxicity was effective on blood cells. Several risk factors significantly affected specific repair activities of tumor cells. HPV positive and negative tumors displayed distinct DNA repair profiles and, cancer progression and tumor staging correlated with deregulated repair activities in tumors. Interestingly, the most affected DNA repair activities concerned double strand break repair, repair of cisplatin adducts, and repair of oxidative damage. This accurate DNA repair profiling represents an innovative strategy to reveal tumor diversity and better understand the impact of risk factors. It improved our understanding of the role of DNA repair in the development and progression of cancer. In addition, the use of a panel of DNA repair-based enzymatic biomarkers is more accurate than the single parameter approach in stratifying patients into different groups, thus allowing for more effective therapeutic strategies. Citation Format: Sylvie Sauvaigo, Giovanna Muggiolu, Sarah Libert, Bertrand Treillard, Gersende Alphonse, Christian A. Righini, Philippe Ceruse, Pierre Philouze, Claire Rodriguez-Lafrasse. Comprehensive analysis of the DNA repair enzyme signature in tumor and blood cells from head and neck cancer patients and correlation with clinical data from a 18-months follow-up study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3493.

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

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.

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Background: Hadrontherapy is an alternative to radiotherapy in the treatment of Head-and-Neck cancers (HNSCC), because of accurate ballistic and high biological efficiency, even in hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrence related to the presence of cancer stem cells (CSCs) located in hypoxic niches. Aim of this work was to determine the molecular specificities of the response to carbon-ion irradiation versus photons in HNSCC cancer cell lines and their CSCs’ subpopulation, under hypoxic and normoxic conditions. Methods : SQ20B, FaDu cells, and their CSCs were irradiated with photons or carbon-ion (290MeV/n,NIRS) in normoxia or hypoxia (1%O2). Cell survival curves, expression of HIF-1a reactive oxygen species (ROS) and Migration/Invasion processes were quantified. Results/Conclusions: For CSCs and non-CSCs, an oxygen-enhancement-ratio (OER) upper than 1.2 was measured in response to photons, associated with stabilization of HIF-1α. This stabilization, depending on the ROS production, appears earlier in CSCs. Inhibition of HIF-1α expression results in decreased survival in HNSCC-CSCs after both type of radiation under hypoxia associated with a significant increase in residual DNA-DSBs. Furthermore, a relationship is demonstrated between HIF-1α expression and the DSBs’ detection and repair by the Homologous-Recombination. Finally, the dense and homogeneous ROS production induced by photons, essential for HIF-1α stabilization, leads to the activation of the 3 major epithelio-mesenchymal transition (EMT) signaling pathways (STAT3,MEK/p38/JNK,Akt/mTOR). At the opposite, the ROS concentrated into the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways. All these results, supported by Monte-Carlo simulations, converge towards the central role of spatial ROS distribution at the nanometric scale to explain the specificities of the molecular response to carbon ions. Their therapeutic advantage may result both from unrepaired complex-DNA lesions and the non-activation of ROS-dependent-signaling pathways involved in tumor cell defense. Supported by Labex PRIMES-ANR-11-LABX-0063;ANR-11-IDEX-0007;ITMO-Cancer-AVIESAN

The relative biological effectiveness (RBE) in particle therapy is currently estimated using biophysical models. We compared experimental measurements to the α curves as function of linear energy transfer computed by the Local Effect Model (LEM I-IV), the Microdosimetric Kinetic Model (MKM) and the NanOx model for HSG, V79 and CHO-K1 cells in response to monoenergetic irradiations. Although the LEM IV and the MKM predictions accurately reproduced the trend observed in the data, NanOx yielded a better agreement than the other models for more irradiation configurations. Its χ 2 estimator was indeed the lowest for three over seven considered cases.

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.

S1263ESTRO 37Meanwhile, recurrent patients tended to exhibit increased post-RT peripheral neutrophil count (4.2×10 9 /L vs. 3.6×10 9 /L, p=0.09) and NLR (8.5 vs. 6.5, p=0.08) in comparison with non-recurrent patients.Multivariate analysis (MVA) identified patient age >50 years old (HR=3.4,p=0.02), weight loss during RT> 7.5% (HR=3.2,p=0.03), and post-RT peripheral NLR >7.05 (HR=2.5, p=0.04, 5-year OS 71.4% vs. 87.8%)as unfavorable prognostic factors for OS.There was also a nonsignificant trend in the MVA that patients with post-RT peripheral NLR >7.05 showed worse PFS (HR=1.9,p=0.06, 5-year PFS 64.1% vs. 81.8%). ConclusionPost-RT NLR predicted OS and PFS in LANPC patients treated with sequential chemoradiotherapy.The dynamic change of the routinely tested immune factors could help make appropriate treatment options and follow-up strategies.

Introduction : La survie a 5 ans des patients ayant un cancer des VADS n’excede pas 40% en raison d’un taux eleve de recidives loco-regionales et a distance. Le phenomene metastatique est gouverne par une serie complexe d’evenements incluant la migration des cellules tumorales a partir du site primaire, leur passage dans la circulation sanguine et l’invasion du tissu cible. Les cellules, appelees cellules tumorales circulantes (CTCs), sont le reflet de l’agressivite et de la progression tumorale. Ce travail s’interesse a la numeration et caracterisation des CTCs chez des patients atteints de cancer des VADS en regard de l’analyse de biopsies tumorales. Methodes : Des prelevements sanguins et biopsiques sont realises avant et pendant le traitement (chimiotherapie/radiotherapie) et en cas de recidive. Les CTCs sont isolees a partir du sang (Rosette et ISET). Un marquage immunohistochimique de proteines specifiques de cellules souches cancereuses et de la transition epithelio-mesenchymateuse est ensuite realise afin de les compter et les caracteriser. L’analyse transcriptomique des voies de progression tumorale et des systemes de reparation des lesions de l’ADN est realisee par la technologie Nanostring sur les biopsies tumorales et les CTCs (apres amplification). Resultats : Dix-huit patients ont ete inclus dans l’etude a ce jour. Les premiers resultats de comptage montrent une grande heterogeneite au niveau du nombre de CTCs. Differents phenotypes (mesenchymateux ou epitheliaux) de CTCs sont mis en evidence selon les patients. L’analyse transcriptomique des biopsies et CTCs est en cours. Conclusion : La numeration et la caracterisation (phenotype et profil transcriptomique) des CTCs et leur comparaison aux donnees des biopsies devraient permettre de definir des parametres biologiques predictifs de la reponse therapeutique des cancers des VADS. Soutenu par le Canceropole CLARA, Grenoble-Alpes Metropole, Conseil Regional Rhone-Alpes, Conseil General du Rhone et LabEx PRIMES