Radiotherapy For Head And Neck Squamous Cell Carcinoma Research Articles

Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1β leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Glutamine inhibition combined with CD47 blockade enhances radiotherapy-induced ferroptosis in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a formidable cancer type that poses significant treatment challenges, including radiotherapy (RT) resistance. The metabolic characteristics of tumors present substantial obstacles to cancer therapy, and the relationship between RT and tumor metabolism in HNSCC remains elusive. Ferroptosis is a type of iron-dependent regulated cell death, representing an emerging disease-modulatory mechanism. Here, we report that after RT, glutamine levels rise in HNSCC, and the glutamine transporter protein SLC1A5 is upregulated. Notably, blocking glutamine significantly enhances the therapeutic efficacy of RT in HNSCC. Furthermore, inhibition of glutamine combined with RT triggers immunogenic tumor ferroptosis, a form of nonapoptotic regulated cell death. Mechanistically, RT increases interferon regulatory factor (IRF) 1 expression by activating the interferon signaling pathway, and glutamine blockade augments this efficacy. IRF1 drives transferrin receptor expression, elevating intracellular Fe2+ concentration, disrupting iron homeostasis, and inducing cancer cell ferroptosis. Importantly, the combination treatment-induced ferroptosis is dependent on IRF1 expression. Additionally, blocking glutamine combined with RT boosts CD47 expression and hinders macrophage phagocytosis, attenuating the treatment effect. Dual-blocking glutamine and CD47 promote tumor remission and enhance RT-induced ferroptosis, thereby ameliorating the tumor microenvironment. Our work provides valuable insights into the metabolic and immunological mechanisms underlying RT-induced ferroptosis, highlighting a promising strategy to augment RT efficacy in HNSCC.

The value of subcutaneous xenografts for individualised radiotherapy in HNSCC: Robust gene signature correlates with radiotherapy outcome in patients and xenografts

PurposeTo assess the robustness of prognostic biomarkers and molecular tumour subtypes developed for patients with head and neck squamous cell carcinoma (HNSCC) on cell-line derived HNSCC xenograft models, and to develop a novel biomarker signature by combining xenograft and patient datasets. Materials and methodsMice bearing xenografts (n = 59) of ten HNSCC cell lines and a retrospective, multicentre patient cohort (n = 242) of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) were included. All patients received postoperative radiochemotherapy (PORT-C). Gene expression analysis was conducted using GeneChip Human Transcriptome Arrays. Xenografts were stratified based on their molecular subtypes and previously established gene classifiers. The dose to control 50 % of tumours (TCD50) was compared between these groups. Using differential gene expression analyses combining xenograft and patient data, a gene signature was developed to define risk groups for the primary endpoint loco-regional control (LRC). ResultsTumours of mesenchymal subtype were characterized by a higher TCD50 (xenografts, p < 0.001) and lower LRC (patients, p < 0.001) compared to the other subtypes. Similar to previously published patient data, hypoxia- and radioresistance-related gene signatures were associated with high TCD50 values. A 2-gene signature (FN1, SERPINE1) was developed that was prognostic for TCD50 (xenografts, p < 0.001) and for patient outcome in independent validation (LRC: p = 0.007). ConclusionGenetic prognosticators of outcome for patients after PORT-C and subcutaneous xenografts after primary clinically relevant irradiation show similarity. The identified robust 2-gene signature may help to guide patient stratification, after prospective validation. Thus, xenografts remain a valuable resource for translational research towards the development of individualized radiotherapy.

HYPORT: A Phase I Study of Hypofractionated Post-Operative Radiation Therapy for Head and Neck Squamous Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Shortening the overall radiotherapy (RT) treatment time decreases the burden and costs of treatment while minimizing the effects of accelerated repopulation in head and neck squamous cell carcinoma (HNSCC). However, data on hypofractionated RT in HNSCC are limited with no published prospective studies in the post-operative setting. This phase I study was conducted to assess the toxicity and tolerability of moderately hypofractionated post-operative RT and to select the dose/fractionation for the phase II study. <h3>Materials/Methods</h3> Patients with completely resected stage I-IVB squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx with intermediate risk factor(s) including T3/4 disease, positive lymph node(s), close margin(s), perineural invasion, and/or lymphovascular invasion were enrolled on a rolling 6 design dose escalation phase I study from 2020-2021. Biologically effective dose (BED) approximately equivalent to 60 Gy in 30 fractions (for alpha/beta ratio of 3 and 10 accounting for repopulation) were chosen with level 0 consisting of 46.5 Gy in 15 fractions (3.1 Gy/fraction) delivered 5 days a week and level 1 consisting of 44.4 Gy in 12 fractions (3.7 Gy/fraction) delivered 4 days a week. The primary endpoint was maximum tolerated dose/fraction (MTD) of moderately hypofractionated RT. The MTD was defined as highest dose at which ≤ 1 of 6 patients experience a dose limiting toxicity (DLT), defined as persistent grade 3 mucositis at 3 months post-treatment or any grade 4 toxicity. <h3>Results</h3> A total of 12 patients were enrolled with 6 each on level 0 and level 1. Primary site included oral cavity, oropharynx, and larynx in 6, 5 (all p16+), and 1 patient, respectively. No patient experienced a DLT or grade 4-5 toxicity. Acute grade 3 toxicity occurred in 2 patients on level 0 (weight loss/anorexia, neck abscess) and 3 patients on level 1 (oral mucositis, all resolved by 3 months post-treatment). Overall, toxicity appeared more favorable on level 0 vs level 1 (Table). With a median follow-up of 8.7 months from end of RT (interquartile range 5.4-12.2), one patient had a regional recurrence in the undissected, unirradiated contralateral neck from a well-lateralized tonsil primary and one patient had an in-field local recurrence of oral tongue primary, both on level 1. The MTD was determined to be 44.4 Gy in 12 fractions, but given the favorable tolerability with equivalent BED, 46.5 Gy in 15 fractions was chosen as the recommended phase II dose/fractionation. <h3>Conclusion</h3> Moderately hypofractionated RT delivered over 3 weeks is well-tolerated in patients with HNSCC following surgical resection. A randomized phase II trial of 46.5 Gy in 15 fractions vs 60 Gy in 30 fractions is ongoing. Table. Cumulative grade 2+ acute toxicity

Effect of low-level light therapy before radiotherapy in oral squamous cell carcinoma: An in vitro study.

Radiation therapy for head and neck squamous cell carcinoma (HNSCC) is associated with several complications. Although photobiomodulation (PBM) has radioprotective effects in normal tissue, it could also enhance the growth of neoplastic cells. Thus, the present study aimed to investigate the cellular response of oral squamous cell carcinoma with pre-exposure to low-level phototherapy before radiotherapy. SCC9, Cal-27, A431, and HaCaT cell lines were subjected to low-level light therapy and radiotherapy. The cells were treated with a single energy density (300 J/cm2) of a light-emitting diode (660 nm) prior to ionizing radiation at different doses (0, 2, 4, and 6 Gy). After 24 h, wound scratch, proliferation, clonogenic cell survival, cell death, and reactive oxygen species (ROS) analyses were performed to evaluate cell response. The cell lines pre-exposed to PBM at the analyzed dosage were radiosensitive. The treatment significantly reduced cell proliferation and clonogenic cell survival. Migration and cell death assays also revealed positive results, with the treatment group showing lower rate of migration and higher cell death than did the control group. Moreover, PBM effectively increased the intracellular levels of ROS. PBM at 300 J/cm2 is a promising radiosensitizing modality to reduce the radiation dose and avoid the intolerable side effects of radiotherapy for HNSCC, thus increasing the probability of successful treatment. However, further studies are needed to support and confirm the results.

Target Definition in MR-Guided Adaptive Radiotherapy for Head and Neck Cancer.

Simple SummaryAdaptive radiotherapy for head and neck cancer has become more routine due to an increase in imaging quality and improvement in radiation techniques. With the availability of faster adaptive workflows, it is possible to adapt more easily to (daily) changes. MRI offers besides great anatomical imaging, also functional information about the tumor and surrounding tissue. The aim of this review is to provide current state of evidence about target definition on MRI for adaptive strategies in the treatment of head and neck cancer.In recent years, MRI-guided radiotherapy (MRgRT) has taken an increasingly important position in image-guided radiotherapy (IGRT). Magnetic resonance imaging (MRI) offers superior soft tissue contrast in anatomical imaging compared to computed tomography (CT), but also provides functional and dynamic information with selected sequences. Due to these benefits, in current clinical practice, MRI is already used for target delineation and response assessment in patients with head and neck squamous cell carcinoma (HNSCC). Because of the close proximity of target areas and radiosensitive organs at risk (OARs) during HNSCC treatment, MRgRT could provide a more accurate treatment in which OARs receive less radiation dose. With the introduction of several new radiotherapy techniques (i.e., adaptive MRgRT, proton therapy, adaptive cone beam computed tomography (CBCT) RT, (daily) adaptive radiotherapy ensures radiation dose is accurately delivered to the target areas. With the integration of a daily adaptive workflow, interfraction changes have become visible, which allows regular and fast adaptation of target areas. In proton therapy, adaptation is even more important in order to obtain high quality dosimetry, due to its susceptibility for density differences in relation to the range uncertainty of the protons. The question is which adaptations during radiotherapy treatment are oncology safe and at the same time provide better sparing of OARs. For an optimal use of all these new tools there is an urgent need for an update of the target definitions in case of adaptive treatment for HNSCC. This review will provide current state of evidence regarding adaptive target definition using MR during radiotherapy for HNSCC. Additionally, future perspectives for adaptive MR-guided radiotherapy will be discussed.

CENPM upregulation by E5 oncoprotein of human papillomavirus promotes radiosensitivity in head and neck squamous cell carcinoma

ObjectivesThis study aims to investigate how human papillomavirus (HPV) affects the key gene in the biological behaviors of head and neck squamous cell carcinoma (HNSCC) that leads to better response to radiotherapy. Materials and MethodsThe expression of key gene CENPM was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data and HPV positive and HPV negative HNSCC tumors and cells. Assays with siRNAs, CRISPR/Cas9-based models, Western blot, qRT-PCR, ChIP, etc., were used to explore how HPV affects CENPM and response to radiotherapy for HNSCC. ResultsCENPM occupies the hub in the HPV-related gene network. HPV-positive HNSCC showed higher level of CENPM expression comparing with HPV-negative HNSCC. HPV E5 has the most pronounced impact on CENPM (R = 0.44, p = 0.00081). This might result from the binding of transcription factor E2F1 to CENPM. We further found that inhibition of CENPM expression in HPV-positive HNSCC cell line SCC47 increased resistance to X-ray radiation by approximately 59% under 2 Gy irradiation, which may be resulted from a reduced proportion of mitotic cells. ConclusionHPV E5 enhances CENPM expression by transcription factor E2F1 in HNSCC, which results in a radiosensitive profile in cell cycle redistribution of HNSCC. Thus, HPV infection in HNSCC provides profound evidence that underscores the magnitude of E2F1 control of CENPM expression illustrating the potential clinical benefit of CENPM examination for difficult-to-treat HPV-negative cancers.

Hypoxia induces radioresistance, epithelial‑mesenchymal transition, cancer stem cell‑like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma.

Hypoxia has been linked with increased resistance to treatment in various solid tumors, including head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to identify genes involved in hypoxia-mediated responses to radiotherapy in HNSCC. A total of three HNSCC cell lines with an epithelial phenotype were selected for this study and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. The sensitivity of the HNSCC cells to radiotherapy was assessed by a crystal violet assay. Western blotting (for protein expression), cDNA microarrays and reverse transcription-quantitative PCR (for gene expression) were also applied. Small interfering RNA silencing was used to knock down target genes. The results revealed that hypoxia negatively affected the response of HNSCC cells to radiotherapy. Of note, increased levels of N-cadherin, vimentin and fibronectin, as well as stem cell-associated transcription factors, were observed under hypoxia. The microarray analysis revealed a number of hypoxia-regulated genes that were involved in multiple biological functions. However, downregulation of hypoxia-regulated genes did not affect sensitivity to radiotherapy of the investigated cell lines. Taken together, the present findings indicated several important pathways and genes that were involved in hypoxia and radiotherapy resistance. It is hypothesized that panels of reported hypoxia-regulated genes may be useful for the prediction of radiotherapy responses in patients with HNSCC.

Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy

PurposeProviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threonine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients.MethodsWe determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, including patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15).ResultsDuring the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence-free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009).ConclusionPatients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recurrence after radiotherapy.

Hypofractionated Versus Conventional Radiotherapy with Chemotherapy in Head and Neck Cancer: A Comparative Study

Head and neck cancers are a group of malignancies that arise from common anatomic sites which include the oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, sinonasal cavities and from the salivary glands. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and has a five-year survival rate of less than 50%. It has high recurrence rates and metastasis. It is a Prospective Observational, and Comparative Study carried out in 30 patients of Loco-regionally advanced carcinoma of head and neck. The diagnosis of loco-regionally advanced cancers of head and neck was made following AJCC staging. The subjects were divided into two groups with 15 in each arm -Accelerated Hypo Fractionated Arm and Conventional Normal Fractionated Arm. All the patients were systematically interviewed, and clinical details of all the subjects were recorded. Among the 30 patients, the highest performance status of (83.3%) ECOG-1 was observed in both the groups when compared to ECOG-2, which is not statistically significant. Hypofractionated radiotherapy can achieve similar tumour response to conventionally fractionated radiotherapy in HNSCC, although with some increase in toxicity. However, to draw some reasonable conclusion, a study with a broader sample and longer follow-up needs to be performed.

Abstract 6161: PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models

Abstract Purpose: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study if SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). Experimental Design: We analyzed HNSCC TCGA data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a Phase I trial investigating olaparib were analyzed. Results: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4 mutant but not SMAD4 positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4 deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared to vehicle treated tumors. In vitro, olaparib or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death and less proliferation in SMAD4 deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a Phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. Conclusion: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs. Citation Format: Christian D. Young, Ariel L. Hernandez, Li Bian, Sana D. Karam, David Raben, Xiao-Jing Wang. PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6161.

Abstract B23: The role of bone marrow transplantation in patients with Fanconi anemia and head and neck squamous cell carcinoma receiving radiotherapy: An examination of the International Fanconi Anemia (IFAR) Registry

Abstract Introduction: Fanconi anemia (FA) patients have a high predilection for developing aggressive head and neck squamous cell carcinoma (HNSCC) at an early age. Because of their aggressive HNSCC, these patients frequently require multiple modality treatment, including radiation therapy. However, their underlying chromosomal instability makes giving radiation to these patients difficult because of increased radiation-induced complications. In order to better understand this problem, we evaluated a large population of FA patients with HNSCC who received radiation therapy for HNSCC. Hypothesis: FA patients who have undergone prior bone marrow transplant (BMT) and present with head and neck squamous cell carcinoma (HNSCC) demonstrate decreased relative risk of radiation-induced complications compared to FA patients who have not received BMT. Study Design: Retrospective Cohort Study Methods: Demographic information, genotypic data, prognostic factors, therapeutic management, and survival outcomes for FA patients enrolled in the International Fanconi Anemia Registry (IFAR) who developed HNSCC from 1981-2017 were analyzed. Results: Data were collected from the IFAR registry for n=50 patients. The mean age at time of HNSCC diagnosis was 30.5 + 9.0 years, and 36% of total patients were male. 42% of the patients received adjuvant or neoadjuvant radiotherapy for HNSCC, with the most common subsites involving the oral tongue (n=8), followed by alveolus (n=3) and supraglottis (n=2). The average dose given was 48.8 + 16.9 Gy over a 28- to 70-day period. Among patients receiving radiotherapy, 24% had received a bone marrow transplant. No patients who had a prior BMT were noted to develop pancytopenia as a radiation-related complication, while 68% of those without prior BMT developed pancytopenia from radiation. 6/16 patients who did not undergo transplant completed the full course of radiation, while 4/5 BMT patients were able to complete treatment. Both mucositis and sepsis were secondary complications more common in the non-BMT group (56% vs. 40%, and 12% vs. 0%, respectively). The relative risk of pancytopenia among patients receiving BMT and adjuvant radiation was 0.15 (95% CI 0.01-2.2); however, this was not statistically significant given the small BMT-group sample size (p=0.17). Interestingly, 80% of patients in the BMT group exhibited genetic mutations of the Fanconi complementation group C. Among patients who received a BMT and adjuvant radiotherapy, 5 patients died of XRT-related complications, while only 1 patient died from these complications in the BMT group. Conclusion: While it is well known that FA patients have a high predilection for developing aggressive HNSCC at an early age, among the patients who received preradiation bone marrow transplant the incidence of radiation-induced pancytopenia was reduced. This reduction suggests that FA patients undergoing radiotherapy for HNSCC may exhibit less adverse side effects of radiation treatment if they received a prior BMT. Citation Format: Ade Obayemi, K.R. Patel, O. Rosti, A. Smogorzewska, D. Kutler. The role of bone marrow transplantation in patients with Fanconi anemia and head and neck squamous cell carcinoma receiving radiotherapy: An examination of the International Fanconi Anemia (IFAR) Registry [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B23.

PARP Inhibition Enhances Radiotherapy of SMAD4-Deficient Human Head and Neck Squamous Cell Carcinomas in Experimental Models.

SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

Incidence of hypothyroidism in head and neck squamous cell carcinoma patients receiving radiotherapy with and without immune checkpoint inhibitors.

e18551 Background: Treatment for locally advanced head and neck squamous cell carcinoma (HNSCC) involves a combination of surgery, chemotherapy, and radiotherapy (RT). RT for HNSCC is a known risk factor for the development of hypothyroidism. Recently, anti-PD1 therapies have been approved for recurrent and metastatic HNSCC and are moving to the forefront of HNSCC care. Similarly, thyroid dysfunction is a common immune-related adverse event following anti-PD1 therapy. Whether the addition of anti-PD1 to RT increases the likelihood of developing hypothyroidism remains unknown. Methods: The rate of hypothyroidism in HNSCC patients receiving RT (+/- chemotherapy and surgery) was compared to HNSCC patients receiving RT + anti-PD1 therapy either concurrently or after RT. Exclusion criteria were preexisting thyroid dysfunction, RT dose &lt; 45 Gy and patients with incomplete treatment records. We defined clinical hypothyroidism as an elevation of TSH with low T3, T4 or elevation of TSH with symptoms requiring levothyroxine initiation. Hypothyroidism incidence was compared using Fisher’s exact test. Results: 153 patients were evaluated. In the RT group (N = 103), patients received RT +/- surgery or chemotherapy. 82/103 (80%) were male, median age was 57 and primary tumor groups included oropharynx 62/103 (60%), larynx 29/103 (28%), oral cavity 9/103 (9%) and other 3/103 (3%). In the RT + anti-PD1 group (N = 50), 36/50 (72%) were males, median age was 57 and primary tumor groups included oral cavity 19/50 (38%), oropharynx 17/50 (34%), larynx 8/50 (16%), and other 6/50 (12%). In the RT group, median follow up after RT was 801 days. In the RT+ anti-PD1 group, median follow up was 595 days from RT and 388 days from anti-PD1. The rate of hypothyroidism was significantly higher in the RT group 22.3% (23/103) versus 6% (3/50)after anti-PD1 therapy (p = 0.011). Multinomial logistical regression found no significant difference in hypothyroidism based on age, sex, or BMI. Larynx as primary tumor location was an independent risk factor for development of hypothyroidism (OR 4.74, p = 0.002). Conclusions: The addition of anti-PD1 therapy to standard HNSCC treatments does not significantly increase the risk of developing hypothyroidism. In fact, this study finds a lower incidence of hypothyroidism in HNSCC patient receiving RT + PD1 therapy which may be due to shorter duration of follow up and lower proportion of laryngeal cancer patients who are at relatively higher risk for surgical hypothyroidism.

Neuregulin Signaling Is a Mechanism of Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma.

EGFR signaling confers resistance to radiotherapy and is a validated target in head and neck squamous cell carcinoma (HNSCC). The inhibition of EGFR in combination with radiotherapy improves local control and overall survival in these patients; however, therapeutic resistance limits the efficacy of this approach. We therefore sought to identify cellular mechanisms that cause resistance to EGFR inhibition and radiotherapy in HNSCC. Though clonal isolation of carcinoma cells exposed to increasing concentrations of cetuximab, we found that resistant cells upregulate prosurvival ErbB3 and AKT signaling. Using EFM-19 cells and confirmatory analysis of protein levels, we demonstrate that cetuximab resistance is characterized by enhanced neuregulin expression identifying a novel adaptive mechanism of therapeutic resistance. Inhibition of this autocrine loop with CDX-3379 (an ErbB3 specific antibody) was sufficient to block ErbB3/AKT signaling in cetuximab resistant cells. The combination of CDX-3379 and cetuximab reduced proliferation and survival after radiotherapy in several HNSCC cell lines. These in vitro findings were confirmed in xenograft tumor growth experiments including an approach using growth factor-supplemented Matrigel. In vivo, the delivery of EGFR and ErbB3 antibodies significantly reduced tumor growth in cetuximab-resistant FaDu and CAL27 xenografts. In summary, this work demonstrates that autocrine NRG ligand secretion is a mechanism for therapeutic resistance to cetuximab and radiotherapy. This cross-resistance to both therapeutic modalities identifies NRG as an actionable therapeutic target for improving treatment regimens in HNSCC.

Value of Serial Multi-Parametric Functional MRI and FDG-PET during Radiation Therapy in Predicting Clinical Outcomes in Patients with Head and Neck Squamous Cell Carcinoma

To assess the utility of mid-treatment FDG-PET, DWI and R2*-MR functional imaging in predicting clinical response in patients receiving definitive radiotherapy (RT) for mucosal head and neck squamous cell carcinoma (HNSCC). Patients undergoing definitive radiotherapy with/without systemic therapy were recruited in a prospective imaging biomarker study. FDG-PET/CT was performed at baseline, during (week 3) and post RT (3 months). Diffusion-weighted imaging (DWI) and susceptibility-weighted MRI were performed using 3T scanner at baseline and during RT (week 2, 3, 5 and 6). The mean apparent diffusion coefficient (ADCmean) from DWI, mean R2* values derived from T2*-MRI and PET metabolic parameters including maximum standardized uptake (SUVmax), mean standardized uptake (SUVmean), metabolic tumor volume (MTV) and total lesional glycolysis (TLG) were measured for the primary tumor. Absolute and relative percentage change (%Δ) in serial PET and MR parameters were correlated to local and distant recurrence and survival using the Mann-Whitney U test. Poor imaging response was dichotomized in relation to; 1) mean values of ADCmean and; 2) 50% reduction in PET parameters for Kaplan-Meier survival analysis. Pre-treatment scans were performed in 33 patients and analyzable mid-treatment scans were available for 21 patients. Median follow-up was 43 months and 2-year local recurrence-free survival (LRFS), distant metastatic relapse-free survival (DMFS) and overall survival (OS) rates were 81%, 90% and 62%, respectively. Patients with local recurrence (LR) had a significantly lower %Δ in SUVmax (p=0.05), SUVmean (p=0.04) and TLG (p=0.05) at week 3 compared to baseline. There was trend towards higher pre-treatment ADCmean in patients with LR (1.09 vs 1.27 x 10-3mm2s-1) without reaching statistical significance potentially due to sample size. A significant rise in ADCmean was found during the first half (p < 0.001) of RT but not in the second half of the treatment. Patients with poor week 2 %Δ ADCmean rise during RT had worse LRFS (100% vs 63%, p<0.04). %Δ ADCmean at subsequent weeks were not associated with LRFS, DMFS, or OS. No absolute or %Δ R2* values were associated with clinical outcomes. Using combination of poor week 2 %Δ ADCmean and SUVmax response at week 3 was associated with improved LRFS (100% vs 43%, p=0.005). Early changes in FDG-PET and ADC can potentially predict clinical response to radiotherapy in HNSCC and aid in future risk-adaptation studies. Combination of functional imaging parameters show a promising avenue to enhance prediction of response. Based on our results week 2 appears to be a promising time period for mid-treatment DWI assessment of tumor response.

Organ Function Preservation Failure after (Chemo)Radiotherapy in Head and Neck Cancer: A Retrospective Cohort Analysis.

The aim of the current study was to determine the incidence of organ function preservation failure (OFPF) in patients with head and neck squamous cell carcinoma (HNSCC) treated by (chemo)radiotherapy and to identify its risk factors. Retrospective cohort analysis. Tertiary cancer care center. A single-center retrospective cohort analysis was done (n = 703) in which OFPF after (chemo)radiotherapy was assessed. OFPF was defined as local failure or pure functional failure in the absence of local failure because of major surgical intervention (total laryngectomy, commando resection, permanent tracheostomy) or feeding tube dependence >2 years. OFPF occurred in 153 patients (21.8%). Reasons for OFPF were local failure in 103 patients (14.6%) and functional failure in 50 patients (7.2%). Evidence of functional failure included need for total laryngectomy (n = 9, 1.3%), commando resection (n = 2, 0.3%), permanent tracheostomy (n = 16, 2.3%), and/or long-term feeding tube for functional reasons (n = 23, 3.3%). In a Cox proportional hazards model, OFPF was worse for patients with T4 tumors (hazard ratio [HR] <0.5 and P < .001 for all other stages), for laryngeal vs oropharyngeal cancer (HR, 1.83; 95% confidence interval [CI], 1.20-2.79, P = .005, hypopharyngeal not significant), and for smokers (HR, 1.68; 95% CI, 1.10-2.56, P = .015). Exploratory multivariate analysis by tumor site showed that T4 tumor and pretreatment tracheostomy were the strongest predictive factors for OFPF in laryngeal and hypopharyngeal carcinoma while T4 tumor and smoking were predictive for poor OFPF in oropharyngeal carcinoma. This work shows a detrimental effect of smoking on functional outcomes after (chemo-)radiotherapy for HNSCC. Moreover, T4 tumor, laryngeal subsite, and pretreatment tracheostomy are strong predictors of OFPF.

CDK4/6 Inhibitor LEE011 Is a Potential Radiation-sensitizer in Head and Neck Squamous Cell Carcinoma: An In Vitro Study.

Radiotherapy (RT) combined with a radiosensitizer represents an important treatment for head and neck squamous cell carcinoma (HNSCC). Only few chemotherapy agents are currently approved as radiosensitizers for targeted therapy. In this study, the potent cyclin-dependent kinase 4/6 (CDK4/6) inhibitor LEE011 was tested for potential to act as a radiosensitizer during RT. RT enhancement by LEE011 was assessed by in vitro clonogenic assay, flow cytometry, and western blot in a variety of HNSCC cell lines. The HNSCC cell line OML1 and its radiation-resistant clone OML1-R were used. LEE011 induced cell-cycle arrest in SCC4/SCC25 cells during the G1/M phase through inhibition of retinoblastoma protein phosphorylation. LEE011 enhanced the effects of radiation in OML1 cells and overcame radiation resistance in OML1-R cells. LEE011 is a potential radiosensitizer that can enhance the cytotoxic effects of RT. Clinical trials including LEE011 during RT for HNSCC should be considered.

Randomized double‐blind trial of amifostine versus placebo for radiation‐induced xerostomia in patients with head and neck cancer

The role of the radioprotector amifostine in ameliorating radiotherapy side effects in head and neck squamous cell carcinoma (HNSCC) is controversial. This trial aimed to determine whether pretreatment with amifostine reduced the incidence of Radiation Therapy Oncology Group grade ≥2 acute and late xerostomia in patients receiving definitive or adjuvant radiotherapy for HNSCC, without reducing tumour control or survival. Between 14 September 2001 and 8 November 2004, 44 Royal Adelaide Hospital patients were randomized double-blind to receive amifostine (200mg/m2 IV) or placebo (normal saline IV) 5days/week, prior to standard radiotherapy (60-70Gy), each having ≥75% of the parotids treated to ≥40Gy. Side effects were assessed weekly during treatment, at 3 and 5months after radiotherapy, then every 6 months until disease progression or death. The accrual target was 200 patients over 4-5years, but the trial closed prematurely when only 44 patients had been randomized after 3years. Of 41 evaluable patients, 80% (16/20) in the amifostine arm had grade ≥2 acute radiation salivary toxicity versus 76% (16/21) in the placebo arm (P=1.00). The rate of grade ≥2 late radiation salivary toxicity at 12months was 66% in the amifostine arm and 82% in the placebo arm (estimated hazard ratio 1.61, 95% confidence interval 0.74-3.49, P=0.22). Other toxicities tended to be worse in the amifostine arm: acute grade 3-4 skin 35% vs 5% and mucous membrane 40% vs 5%; grade ≥2 vomiting 35% vs 5%, hypocalcaemia 25% vs 5% and fatigue 85% vs 33%, with only the latter retaining statistical significance after adjusting for multiple comparisons. There were no significant differences in failure-free (P=0.70) or overall survival (P=0.86), with estimated 4-year rates of 48% vs 54% and 49% vs 59% for the amifostine vs placebo arms respectively. There was no clear evidence that pretreatment with amifostine made any difference to the incidence of grade ≥2 acute or late xerostomia. Other toxicity tended to be more severe with amifostine. There was no effect on failure-free or overall survival. Acknowledging the low statistical power, these results do not support the use of IV amifostine pre-radiotherapy in HNSCC.

Characterization of FaDu-R, a radioresistant head and neck cancer cell line, and cancer stem cells

ObjectivesThe aim of this study was to evaluate the impact of CSC on insensitivity to radiotherapy in HNSCC. MethodsA radioresistant cell line, FaDu-R, was established using fractionated ionizing radiation. Cells with high and low CD44/ALDH activity were isolated. ResultsFaDu-R cells demonstrated significantly increased cell viability after radiation exposure compared with parental cells. CD44high/ALDHhigh FaDu-R cells demonstrated significantly faster wound closure (p<0.05) and more efficient invasion (p<0.05) compared to the CD44high/ALDHhigh FaDu cells or the CD44low/ALDHlow FaDu-R cells. There was a significant difference in tumor volume between the CD44high/ALDHhigh FaDu-R cells and the CD44high/ALDHhigh FaDu cells (p<0.05) as well as the CD44low/ALDHlow FaDu-R cells (p<0.05). ConclusionCancer stem cells (CSC) were associated with invasion and tumorigenesis in a radioresistant head and neck squamous cell carcinoma (HNSCC) cell line. This concept might help to improve the understanding of these mechanisms and to develop drugs that can overcome radioresistance during radiotherapy.