Correlation and colocalization of HIF-1a and pimonidazole staining for hypoxia in laryngeal squamous cell carcinomas:A digital, single-cell-based analysis

OBJECTIVE: Tumor hypoxia results in worse local control and patient survival. We performed a digital, single-cell-based analysis to compare two biomarkers for hypoxia (hypoxia-inducible factor 1-alpha [HIF-1α] and pimonidazole [PIMO]) and their effect on outcome in laryngeal cancer patients treated with accelerated radiotherapy with or without carbogen breathing and nicotinamide (AR versus ARCON). MATERIALS AND METHODS: Immunohistochemical staining was performed for HIF-1α and PIMO in consecutive sections of 44 laryngeal cancer patients randomized between AR and ARCON. HIF-1α expression and PIMO-binding were correlated using digital image analysis in QuPath. High-density areas for each biomarker were automatically annotated and staining overlap was analyzed. Kaplan-Meier survival analyses for local control, regional control and disease-free survival were performed to predict a response benefit of ARCON over AR alone for each biomarker. RESULTS: 106 Tissue fragments of 44 patients were analyzed. A weak, significant positive correlation was observed between HIF-1α and PIMO positivity on fragment level, but not on patient level. A moderate strength correlation (r = 0.705, p < 0.001) was observed between the number of high-density staining areas for both biomarkers. Staining overlap was poor. HIF-1α expression, PIMO-binding or a combination could not predict a response benefit of ARCON over AR. CONCLUSION: Digital image analysis to compare positive cell fractions and staining overlap between two hypoxia biomarkers using open-source software is feasible. Our results highlight that there are distinct differences between HIF-1α and PIMO as hypoxia biomarkers and therefore suggest co-existence of different forms of hypoxia within a single tumor

18F-FDG-PET/CT-guided radiotherapy of cervical lymph nodes in head and neck squamous cell carcinoma

The use of positron emission tomography with fluor-18-fluorodeoxyglucose (FDG-PET) in clinical practice for patients with head and neck squamous cell carcinoma (HNSCC) has expanded rapidly, with implications for diagnostic staging, radiotherapy planning, adaptive radiotherapy, and post-therapy evaluation. The implementation of FDG-PET/CT in radiation treatment planning not only has consequences for target volume definition and dose prescription but is also associated with an increased overall survival in patients with HNSCC. FDG-PET/CT-guided gradient dose prescription provides a window of opportunity for treatment de-intensification of the neck in order to decrease treatment-related toxicity without compromising oncological outcome. Further, interim FDG-PET/CT during radiotherapy can be useful to assess metabolic tumor response and enables opportunities for adaptive treatment strategies. The goals are to increase treatment effectivity in poor responders and reduce unnecessary toxicity in patients with good early tumor response. Further prospective trials investigating adaptive radiotherapy based on interim PET-evaluation are needed, especially regarding human papilloma virus-negative HNSCC and patients treated with primary radiotherapy

Expression of EGFR under tumor hypoxia: identification of a subpopulation of tumor cells responsible for aggressiveness and treatment resistance

Item does not contain fulltextPURPOSE: Overexpression of epidermal growth factor receptor (EGFR) and tumor hypoxia have been shown to correlate with worse outcome in several types of cancer including head-and-neck squamous cell carcinoma. Little is known about the combination and possible interactions between the two phenomena. METHODS AND MATERIALS: In this study, 45 cases of histologically confirmed squamous cell carcinomas of the head and neck were analyzed. All patients received intravenous infusions of the exogenous hypoxia marker pimonidazole prior to biopsy. Presence of EGFR, pimonidazole binding, and colocalization between EGFR and tumor hypoxia were examined using immunohistochemistry. RESULTS: Of all biopsies examined, respectively, 91% and 60% demonstrated EGFR- and pimonidazole-positive areas. A weak but significant association was found between the hypoxic fractions of pimonidazole (HFpimo) and EGFR fractions (F-EGFR) and between F-EGFR and relative vascular area. Various degrees of colocalization between hypoxia and EGFR were found, increasing with distance from the vasculature. A high fraction of EGFR was correlated with better disease-free and metastasis-free survival, whereas a high degree of colocalization correlated with poor outcome. CONCLUSIONS: Colocalization of hypoxia and EGFR was demonstrated in head-and-neck squamous cell carcinomas, predominantly at longer distances from vessels. A large amount of colocalization was associated with poor outcome, which points to a survival advantage of hypoxic cells that are also able to express EGFR. This subpopulation of tumor cells might be indicative of tumor aggressiveness and be partly responsible for treatment resistance

Implications of improved diagnostic imaging of small nodal metastases in head and neck cancer : Radiotherapy target volume transformation and dose de-escalation

Diagnostic imaging continues to evolve, and now has unprecedented accuracy for detecting small nodal metastasis. This influences the tumor load in elective target volumes and subsequently has consequences for the radiotherapy dose required to control disease in these volumes. Small metastases that used to remain subclinical and were included in elective volumes, will nowadays be detected and included in high-dose volumes. Consequentially, high-dose volumes will more often contain low-volume disease. These target volume transformations lead to changes in the tumor burden in elective and “gross” tumor volumes with implications for the radiotherapy dose prescribed to these volumes. For head and neck tumors, nodal staging has evolved from mere palpation to combinations of high-resolution imaging modalities. A traditional nodal gross tumor volume in the neck typically had a minimum diameter of 10–15 mm, while nowadays much smaller tumor deposits are detected in lymph nodes. However, the current dose levels for elective nodal irradiation were empirically determined in the 1950s, and have not changed since. In this report the radiobiological consequences of target volume transformation caused by modern imaging of the neck are evaluated, and theoretically derived reductions of dose in radiotherapy for head and neck cancer are proposed. The concept of target volume transformation and subsequent strategies for dose adaptation applies to many other tumor types as well. Awareness of this concept may result in new strategies for target definition and selection of dose levels with the aim to provide optimal tumor control with less toxicity

Implications of improved diagnostic imaging of small nodal metastases in head and neck cancer : Radiotherapy target volume transformation and dose de-escalation

Diagnostic imaging continues to evolve, and now has unprecedented accuracy for detecting small nodal metastasis. This influences the tumor load in elective target volumes and subsequently has consequences for the radiotherapy dose required to control disease in these volumes. Small metastases that used to remain subclinical and were included in elective volumes, will nowadays be detected and included in high-dose volumes. Consequentially, high-dose volumes will more often contain low-volume disease. These target volume transformations lead to changes in the tumor burden in elective and “gross” tumor volumes with implications for the radiotherapy dose prescribed to these volumes. For head and neck tumors, nodal staging has evolved from mere palpation to combinations of high-resolution imaging modalities. A traditional nodal gross tumor volume in the neck typically had a minimum diameter of 10–15 mm, while nowadays much smaller tumor deposits are detected in lymph nodes. However, the current dose levels for elective nodal irradiation were empirically determined in the 1950s, and have not changed since. In this report the radiobiological consequences of target volume transformation caused by modern imaging of the neck are evaluated, and theoretically derived reductions of dose in radiotherapy for head and neck cancer are proposed. The concept of target volume transformation and subsequent strategies for dose adaptation applies to many other tumor types as well. Awareness of this concept may result in new strategies for target definition and selection of dose levels with the aim to provide optimal tumor control with less toxicity

Targeting Hypoxia, HIF-1, and Tumor Glucose Metabolism to Improve Radiotherapy Efficacy

Item does not contain fulltextRadiotherapy, an important treatment modality in oncology, kills cells through induction of oxidative stress. However, malignant tumors vary in their response to irradiation as a consequence of resistance mechanisms taking place at the molecular level. It is important to understand these mechanisms of radioresistance, as counteracting them may improve the efficacy of radiotherapy. In this review, we describe how the hypoxia-inducible factor 1 (HIF-1) pathway has a profound effect on the response to radiotherapy. The main focus will be on HIF-1-controlled protection of the vasculature postirradiation and on HIF-1 regulation of glycolysis and the pentose phosphate pathway. This aberrant cellular metabolism increases the antioxidant capacity of tumors, thereby countering the oxidative stress caused by irradiation. From the results of translational studies and the first clinical phase I/II trials, it can be concluded that targeting HIF-1 and tumor glucose metabolism at several levels reduces the antioxidant capacity of tumors, affects the tumor microenvironment, and sensitizes various solid tumors to irradiation. Clin Cancer Res; 18(20); 5585-94. (c)2012 AACR

Time patterns of recurrence and second primary tumors in a large cohort of patients treated for oral cavity cancer

Introduction: Routine follow-up after curative treatment of patients with oral squamous cell carcinoma (OSCC) is common practice considering the high risk of second primaries and recurrences (ie second events). Current guidelines advocate a follow-up period of at least 5 years. The recommendations are not evidence-based and benefits are unclear. This is even more so for follow-up after a second event. To facilitate the development of an evidence- and personalized follow-up program for OSCC, we investigated the course of time until the second and subsequent events and studied the risk factors related to these events. Materials and methods: We retrospectively studied 594 OSCC patients treated with curative intent at the Head and Neck Cancer Unit of the Radboud University Medical Centre from 2000 to 2012. Risk of recurrence was calculated addressing death from intercurrent diseases as competing event. Results: The 1-, 5- and 10-year cumulative risks of a second event were 17% (95% CI:14%;20%), 30% (95% CI:26%;33%), and 37% (95% CI:32%;41%). Almost all locoregional recurrences occurred in the first 2 years after treatment. The incidence of second primary tumors was relatively stable over the years. The time pattern of presentation of third events was similar. Discussion: Our findings support a follow-up time of 2 years after curative treatment for OSCC. Based on the risk of recurrence there is no indication for a different follow-up protocol after first and second events. After 2 years, follow-up should be tailored to the individual needs of patients for supportive care, and monitoring of late side-effects of treatment

Assessing radiation-induced carotid vasculopathy using ultrasound after unilateral irradiation: a cross-sectional study

Background: Increased head and neck cancer (HNC) survival requires attention to long-term treatment sequelae. Irradiated HNC survivors have a higher ischemic stroke risk. However, the pathophysiology of radiation-induced vasculopathy is unclear. Arterial stiffness could be a biomarker. This study examined alterations in intima-media thickness (IMT) and stiffness-related parameters, shear wave (SWV) and pulse wave velocity (PWV), in irradiated compared to control carotids in unilateral irradiated patients. Methods: Twenty-six patients, median 40.5 years, 5–15 years after unilateral irradiation for head and neck neoplasms underwent a bilateral carotid ultrasound using an Aixplorer system with SL18-5 and SL10-2 probes. IMT, SWV, and PWV were assessed in the proximal, mid, and distal common (CCA) and internal carotid artery (ICA). Plaques were characterized with magnetic resonance imaging. Measurements were compared between irradiated and control sides, and radiation dose effects were explored. Results: CCA-IMT was higher in irradiated than control carotids (0.54 [0.50–0.61] vs. 0.50 [0.44–0.54] mm, p = 0.001). For stiffness, only anterior mid-CCA and posterior ICA SWV were significantly higher in the irradiated side. A radiation dose–effect was only (weakly) apparent for PWV (R2: end-systolic = 0.067, begin-systolic = 0.155). Ultrasound measurements had good–excellent intra- and interobserver reproducibility. Plaques had similar characteristics but were more diffuse in the irradiated side. Conclusions: Increased CCA-IMT and SWV in some segments were seen in irradiated carotids. These alterations, even in young patients, mark the need for surveillance of radiation-induced vasculopathy. Trial registration: clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT04257968)