Brachial Plexus Tolerance to Single-Session SABR in a Pig Model.

PURPOSE: The single-session dose tolerance of the spinal nerves has been observed to be similar to that of the spinal cord in pigs, counter to the perception that peripheral nerves are more tolerant to radiation. This pilot study aims to obtain a first impression of the single-session dose-response of the brachial plexus using pigs as a model. METHODS AND MATERIALS: Ten Yucatan minipigs underwent computed tomography and magnetic resonance imaging for treatment planning, followed by single-session stereotactic ablative radiotherapy. A 2.5-cm length of the left-sided brachial plexus cords was irradiated. Pigs were distributed in 3 groups with prescription doses of 16 (n = 3), 19 (n = 4), and 22 Gy (n = 3). Neurologic status was assessed by observation for changes in gait and electrodiagnostic examination. Histopathologic examination was performed with light microscopy of paraffin-embedded sections stained with Luxol fast blue/periodic acid-Schiff and Masson's trichrome. RESULTS: Seven of the 10 pigs developed motor deficit to the front limb of the irradiated side, with a latency from 5 to 8 weeks after irradiation. Probit analysis of the maximum nerve dose yields an estimated ED50 of 19.3 Gy for neurologic deficit, but the number of animals was insufficient to estimate 95% confidence intervals. No motor deficits were observed at a maximum dose of 17.6 Gy for any pig. Nerve conduction studies showed an absence of sensory response in all responders and absent or low motor response in most of the responders (71%). All symptomatic pigs showed histologic lesions to the left-sided plexus consistent with radiation-induced neuropathy. CONCLUSIONS: The single-session ED50 for symptomatic plexopathy in Yucatan minipigs after irradiation of a 2.5-cm length of the brachial plexus cords was determined to be 19.3 Gy. The dose-response curve overlaps that of the spinal nerves and the spinal cord in the same animal model. The relationship between the brachial plexus tolerance in pigs and humans is unknown, and caution is warranted when extrapolating for clinical use.

In Silico Single-Fraction Stereotactic Ablative Radiation Therapy for the Treatment of Thoracic and Abdominal Oligometastatic Disease With Online Adaptive Magnetic Resonance Guidance.

PURPOSE: Although SABR can improve oncologic outcomes for patients with oligometastatic disease, treatment of metastases near critical organs remains challenging. The purpose of this study is to determine the dosimetric feasibility of delivering magnetic resonance imaging (MRI)-guided adaptive SABR in a single fraction for abdominal and thoracic metastases. METHODS AND MATERIALS: Previously delivered MRI-guided radiation therapy plans for 20 patients with oligometastatic disease in the thorax or abdomen, with 70% (14/20) of the lesions within 8 mm from dose-limiting organs at risk (OARs), were used to simulate the delivery of 24 Gy in a single fraction. Planning objectives included planning target volume (PTV) V95% >90%, optimized PTV (PTVopt) V95% >90%, and PTVopt D99% >20 Gy with no OAR dose violations, where PTVopt removed overlap with nearby planning organ at risk volume (PRV). Single-fraction plans were simulated on the first 5 daily setup breath-hold MRI scans, and the plans were reoptimized to consider variations in setup position and anatomy. RESULTS: The mean PTV V95% for single-fraction SABR plans was lower compared with multifraction plans (mean 85.4% vs 92.6%, P = .02), but mean PTVopt V95% was not different (95.3% vs 98.2%, P = .62). After reoptimization of the single-fraction plan to the treatment day MRI, there was an increase in mean PTV V95% (85.0% vs 88.1%, P = .05), increase in mean PTVopt V95% (92.7% vs 96.3%, P = .02), increase in mean PTVopt D99% (19.7 Gy vs 23.8 Gy, P < .01), increase in mean frequency of meeting PTV D99% >20 Gy (52% vs 87%, P < .01), and increase in mean gross tumor volume minimum dose (17.5 Gy vs 19.3 Gy, P < .01). Reoptimization decreased mean frequency of OAR dose constraint violation (48% vs 0%, P < .01). CONCLUSIONS: Single-fraction MRI-guided SABR is a dosimetrically feasible treatment for oligometastases that allows for on-table adaptation to avoid OAR dose constraint violations, but this method requires clinical validation.

Existence of a Dose-Length Effect in Spinal Nerves Receiving Single-Session Stereotactic Ablative Radiation Therapy.

PURPOSE: The spinal nerves have been observed to have a similar single-session dose tolerance to that of the spinal cord in pigs. Small-animal studies have shown that spinal cord dose tolerance depends on the length irradiated. This work aims to determine whether a dose-length effect exists for spinal nerves. METHODS AND MATERIALS: Twenty-seven Yucatan minipigs underwent computed tomography and magnetic resonance imaging for treatment planning, followed by single-session stereotactic ablative radiation therapy. A 0.5 cm length of the left-sided C6, C7, and C8 spinal nerves was targeted. The pigs were distributed into 6 groups with prescription doses of 16 Gy (n = 5), 18 Gy (n = 5), 20 Gy (n = 5), 22 Gy (n = 5), 24 Gy (n = 5), or 36 Gy (n = 2) and corresponding maximum doses of 16.7, 19.1, 21.3, 23.1, 25.5, and 38.6 Gy, respectively. Neurologic status was assessed with a serial electrodiagnostic examination and daily observation of gait for approximately 52 weeks. A histopathologic examination of paraffin-embedded sections with Luxol fast blue/periodic acid-Schiff's staining was also performed. RESULTS: Marked gait change was observed in 8 of 27 irradiated pigs. The latency for responding pigs was 11 to 16 weeks after irradiation. The affected animals presented with a limp in the left front limb, and 62.5% of these pigs had electrodiagnostic evidence of denervation in the C6 and C7 innervated muscles. A probit analysis showed the dose associated with a 50% incidence of gait change is 23.9 Gy (95% confidence interval, 22.5-25.8 Gy), which is 20% higher than that reported in a companion study where a 1.5 cm length was irradiated. All symptomatic pigs had demyelination and fibrosis in the irradiated nerves, but the contralateral nerves and spinal cord were normal. CONCLUSIONS: A dose-length effect was observed for single-session irradiation of the spinal nerves in a Yucatan minipig model.

Spinal Nerve Tolerance to Single-Session Stereotactic Ablative Radiation Therapy.

PURPOSE: This study was performed to determine the dose-related incidence of neuropathy from single-session irradiation of the C6-C8 spinal nerves using a pig model and to test the hypothesis that the spinal nerves and spinal cord have the same tolerance to full cross-sectional irradiation. METHODS AND MATERIALS: Twenty-five Yucatan minipigs received study treatment. Each animal underwent computed tomography and magnetic resonance imaging for treatment planning, followed by single-session stereotactic ablative radiation therapy. A 1.5-cm length of the left-sided C6, C7, and C8 spinal nerves was targeted. Pigs were distributed into 5 groups with prescription doses of 16 (n = 7), 18 (5), 20 (5), 22 (5), or 24 (3) Gy with corresponding maximum nerve doses of 17.3, 19.5, 21.6, 24.1, and 26.2 Gy. The neurologic status of all animals was followed for approximately 52 weeks by serial electrodiagnostic examination and daily observation of gait. Histopathologic examination of paraffin-embedded sections with Luxol fast blue/periodic acid-Schiff staining was performed on bilateral spinal nerves and the spinal cord. RESULTS: Marked gait change was observed in 15 of the 25 irradiated pigs. Affected animals presented with a limp in their left front limb, and electromyography demonstrated evidence of denervation in C6 and C7 innervated muscles. Probit analysis showed the ED50 for gait change after irradiation of the spinal nerves to be 19.7 Gy (95% confidence interval, 18.5-21.1). The latency for all responding pigs was 9 to 15 weeks after irradiation. All symptomatic pigs had demyelination and fibrosis in their irradiated nerves, whereas contralateral nerves and spinal cord were normal. CONCLUSIONS: The ED50 for symptomatic neuropathy after full cross-sectional irradiation of the spinal nerves was found to be 19.7 Gy. The dose response of the C6-C8 spinal nerves is not significantly different from that of full cross-sectional irradiation of the spinal cord as observed in companion studies.

Corrigendum: Potential role of the glycolytic oscillator in acute hypoxia in tumors (2015 Phys. Med. Biol. 60 9215).

At the time of publication, our group had performed short tandem repeat (STR) testing on the SCC22B cell line and believed that had been correctly identified. As part of a recent comprehensive process to confirm the identity of cell lines in use in our lab, we repeated STR testing on all cell lines. These results were compared to the ExPASy Cellosaurus database (http://web.expasy.org/cellosaurus/). One cell line used in this manuscript was a near perfect match for T24 (CVCL_0554), a bladder carcinoma cell line commonly found as a cellular contaminant. Although we are unable to test the exact cells used in this manuscript, we believe that the cells labeled as SCC22B are most likely to actually be T24. The authors believe that neither the results nor the conclusions have been significantly changed on the basis of the specific cell line utilized.

Potential role of the glycolytic oscillator in acute hypoxia in tumors.

Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation.

Prognostic value of the proliferation marker Ki-67 in laryngeal carcinoma: results of the accelerated radiotherapy with carbogen breathing and nicotinamide phase III randomized trial.

BACKGROUND: The prognostic and predictive value of the proliferation marker Ki-67 was investigated in a randomized trial comparing accelerated radiotherapy with carbogen breathing and nicotinamide (ARCON) to accelerated radiotherapy in laryngeal carcinoma. METHODS: Labeling index of Ki-67 (Li Ki-67) in immunohistochemically stained biopsies and the colocalization with carbonic anhydrase IX (CAIX) were related to tumor control and patient survival. RESULTS: On average, node-positive patients had a higher Li Ki-67 (median 14% vs 8%; p < .01). In patients with a high Li Ki-67, the 5-year regional control and metastases-free survival were 79% versus 96% (p < .01) and 71% versus 88% (p = .05) for accelerated radiotherapy and ARCON, respectively. The 5-year local control and disease-specific survival were not significantly different. Patients with low Ki-67 expression had an excellent outcome with accelerated radiotherapy alone. CONCLUSION: Patients with laryngeal carcinomas with high proliferative activity are at increased risk of regional and distant metastases formation. This risk can be reduced by treatment with ARCON.

Hypoxia, metabolism, and growth factor signaling in head and neck squamous cell carcinoma: correlation between primary and xenograft tumors.

BACKGROUND: Hypoxia, metabolism, and growth factor signaling are important prognostic features in most solid tumors. The purpose of this study was to determine whether head and neck squamous cell carcinoma (HNSCC) xenografts show similar biological and molecular characteristics as the primary tumor they originate from. METHODS: Eighteen HNSCC primary tumor-xenograft pairs were immunofluorescently stained for pimonidazole (hypoxia), carbonic anhydrase IX (CAIX), glucose transporter-1 (GLUT-1), monocarboxylate transporter-1 (MCT-1), monocarboxylate transporter-4 (MCT-4), epidermal growth factor receptor (EGFR), and phosphorylated protein kinase B (pAKT). RESULTS: Although no correlation was found for the amount of hypoxia, significant correlations between primary tumors and xenografts were observed for both the percentage of cells positive for expression and the hypoxia-related expression pattern of CAIX, GLUT-1, and MCT-1. For EGFR and MCT-4, the intensity of expression was correlated. No correlation was observed for pAKT. CONCLUSION: Xenografts did not always resemble the primary tumor they originate from, but the xenografts did represent the variability in expression levels and patterns observed in the primary tumors.

Systematic analysis of 18F-FDG PET and metabolism, proliferation and hypoxia markers for classification of head and neck tumors.

BACKGROUND: Quantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors. METHODS: Several imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters. RESULTS: We found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2×10-32) than the performance of the IHC parameter/feature based model. CONCLUSIONS: With a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes.

Combining radiotherapy with MEK1/2, STAT5 or STAT6 inhibition reduces survival of head and neck cancer lines.

BACKGROUND: Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients. METHODS: An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines. RESULTS: The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition. CONCLUSIONS: Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.

Paralysis following stereotactic spinal irradiation in pigs suggests a tolerance constraint for single-session irradiation of the spinal nerve.

BACKGROUND AND PURPOSE: Paralysis observed during a study of vertebral bone tolerance to single-session irradiation led to further study of the dose-related incidence of motor peripheral neuropathy. MATERIALS AND METHODS: During a bone tolerance study, cervical spinal nerves of 15 minipigs received bilateral irradiation to levels C5-C8 distributed into three dose groups with mean maximum spinal nerve doses of 16.9 ± 0.3 Gy (n=5), 18.7 ± 0.5 Gy (n=5), and 24.3 ± 0.8 Gy (n=5). Changes developing in the gait of the group of pigs receiving a mean maximum dose of 24.3 Gy after 10-15 weeks led to the irradiation of two additional animals. They received mean maximum dose of 24.9 ± 0.2 Gy (n=2), targeted to the left spinal nerves of C5-C8. The followup period was one year. Histologic sections from spinal cords and available spinal nerves were evaluated. MR imaging was performed on pigs in the 24.9 Gy group. RESULTS: No pig that received a maximum spinal nerve point dose ≤19.0 Gy experienced a change in gait while all pigs that received ≥24.1 Gy experienced paralysis. Extensive degeneration and fibrosis were observed in irradiated spinal nerves of the 24.9 Gy animals. All spinal cord sections were normal. Irradiated spinal nerve regions showed increased thickness and hypointensity on MR imaging. CONCLUSION: The single-session tolerance dose of the cervical spinal nerves lies between 19.0 and 24.1 Gy for this model.

Dasatinib Inhibits DNA Repair after Radiotherapy Specifically in pSFK-Expressing Tumor Areas in Head and Neck Xenograft Tumors.

Src family kinases (SFKs) have been implicated in resistance to both radiation and epidermal growth factor receptor (EGFR) inhibition. Therefore, we investigated whether inhibition of SFK through dasatinib (DSB) can enhance the effect of radiotherapy in two in vivo human head and neck squamous cell carcinoma (HNSCC) models. Response to DSB and/or radiotherapy was assessed with tumor growth delay assays in two HNSCC xenograft models, SCCNij153 and SCCNij202. Effects on EGFR signaling were evaluated with Western blot analysis, and effects on DNA repair, hypoxia, and proliferation were investigated with immunohistochemistry. DSB and radiotherapy induced a significant growth delay in both HNSCC xenograft models, although to a lesser extent in SCCNij202. DSB did not inhibit phosphorylated protein kinase B (pAKT) or phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) but did inhibit (phosphorylated) DNA-dependent protein kinase. Moreover, DSB reduced repair of radiation-induced DNA double-strand breaks as shown by an increase of p53-binding protein 1 (53BP1) staining 24 hours after radiation. This effect on DNA repair was only observed in the cell compartment where phosphorylated SFK (pSFK) was expressed: for SCCNij153 tumors in both normoxic and hypoxic areas and for SCCNij202 tumors only in hypoxic areas. No consistent effects of DSB on hypoxia or proliferation were observed. In conclusion, DSB enhances the effect of radiotherapy in vivo by inhibition of radiation-induced DNA repair and is a promising way to improve outcome in HNSCC patients.

Epidermal growth factor receptor expression in laryngeal cancer predicts the effect of hypoxia modification as an additive to accelerated radiotherapy in a randomised controlled trial.

Accelerated radiotherapy (AR) improves the poor prognosis associated with epidermal growth factor receptor (EGFR) overexpression frequently seen in head and neck carcinomas. Combining AR with carbogen and nicotinamide (ARCON) counteracts enhanced tumour cell proliferation- and hypoxia-related radioresistance. The purpose of this study was to investigate if EGFR expression levels are associated with response to ARCON in patients with carcinoma of the larynx. Patients (N=272) with advanced stage larynx carcinoma were randomised between AR alone and ARCON. Paraffin-embedded biopsies from these patients were processed for immunohistochemical staining of EGFR. EGFR fraction was quantitated by automated image analysis and related to clinical outcome. A large variation was observed in EGFR fraction between tumours with expression levels ranging from 0 to 0.93 (median fraction 0.4). No difference in 5-year locoregional control was found between low and high EGFR expressing tumours in the AR arm (69% versus 75%), which is in line with the established effect of AR in EGFR overexpressing tumours. There was, however, a significant association in the ARCON arm: patients with low EGFR levels had a better 5-year locoregional control (88% versus 72% p=0.02) and disease-specific survival (92% versus 77% p=0.01). ARCON improved locoregional control relative to AR only in patients with low EGFR expression (hazard ratio (HR) 0.34 p=0.009). In conclusion, only in tumours with a low EGFR fraction, adding hypoxia modification to AR has an additive beneficial effect on outcome. EGFR expression is a predictive biomarker for the selection of patients that will or will not respond to ARCON.

Evaluation and immunohistochemical qualification of carbogen-induced ΔR2 as a noninvasive imaging biomarker of improved tumor oxygenation.

PURPOSE: To evaluate and histologically qualify carbogen-induced ΔR2 as a noninvasive magnetic resonance imaging biomarker of improved tumor oxygenation using a double 2-nitroimidazole hypoxia marker approach. METHODS AND MATERIALS: Multigradient echo images were acquired from mice bearing GH3 prolactinomas, preadministered with the hypoxia marker CCI-103F, to quantify tumor R2 during air breathing. With the mouse remaining positioned within the magnet bore, the gas supply was switched to carbogen (95% O2, 5% CO2), during which a second hypoxia marker, pimonidazole, was administered via an intraperitoneal line, and an additional set of identical multigradient echo images acquired to quantify any changes in tumor R2. Hypoxic fraction was quantified histologically using immunofluorescence detection of CCI-103F and pimonidazole adduct formation from the same whole tumor section. Carbogen-induced changes in tumor pO2 were further validated using the Oxylite fiberoptic probe. RESULTS: Carbogen challenge significantly reduced mean tumor R2 from 116 ± 13 s(-1) to 97 ± 9 s(-1) (P<.05). This was associated with a significantly lower pimonidazole adduct area (2.3 ± 1%), compared with CCI-103F (6.3 ± 2%) (P<.05). A significant correlation was observed between ΔR2 and Δhypoxic fraction (r=0.55, P<.01). Mean tumor pO2 during carbogen breathing significantly increased from 6.3 ± 2.2 mm Hg to 36.0 ± 7.5 mm Hg (P<.01). CONCLUSIONS: The combined use of intrinsic susceptibility magnetic resonance imaging with a double hypoxia marker approach corroborates carbogen-induced ΔR2 as a noninvasive imaging biomarker of increased tumor oxygenation.