The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations. The ethical approval for clinical investigation was in accordance with relevant guidelines and regulations by the ethical committee of the Sun Yat-sen University, China
We retrospectively reviewed the clinical data of 48 NPC patients who were diagnosed with radio-induced brain necrosis at Jiangmen Central Hospital and Sun Yat-sen University Cancer Center between January 2008 and December 2016. All of these patients underwent craniotomy for their radio-induced brain necrosis because conservative treatments were confirmed unsuccessful with obviously neurological symptoms. The clinicopathological characteristics, including age at craniotomy, sex, pathological classification of NPC, tumor stage at initial diagnosis (according to 8th American Joint Committee on Cancer (AJCC) classification), radiation dosage, as well as latency period, initial symptoms, imaging features, pathologic findings, outcomes of radio-induced brain necrosis after radiotherapy, were collected.
This study was approved by the ethics committee of Sun Yat-sen University Cancer Center. Written informed consent was obtained from all the included patients. The dateset supporting the conclusions of this article is available in the public Research Data Deposit (RDD) platform (www.researchdata.org.cn) with approval (RDD Number RDDA2017000203).
MRI data collection
We retrospectively reviewed all the MRI data of the 48 NPC patients. The images were obtained on a head and neck MRI scanner from diagnosis of radio-induced brain necrosis to the end of follow-up after brain necrosis resection. We exported all the images and chose T1-weighted images (WIs), T2WIs, contrast-enhanced T1WIs, and diffusion-weighted images (DWIs) of every patient’s brain and nasopharynx region. We clearly mapped the dynamic changes in the features of radio-induced brain necrosis.
Radiation dose calculation
The radiation plans of 11 patients were obtained and reanalyzed successfully. The planning target volume (PTV) that was contoured in the radiotherapy program included the gross target volume of the nasopharynx (GTVnx), clinical target volume (CTV1), and clinical target of preventive radiation (CTV2). After delineation of the above target areas, the corresponding planning target areas (PTVnx, PTV1, and PTV2) were generated by expansion using the treatment planning system (TPS) (Eclipse Version 15.5, Varian, Palo Alto, California, USA) with setup errors. The organs at risk (OARs) that were contoured included the brain stem, spinal cord, lens, optic nerve, optic chiasm, pituitary gland, parotid gland, temporal lobe, temporomandibular joint (TMJ), and mandible. Nine dynamic intensity-modulated radiotherapy (dIMRT) synchronous doseplans were created using the TPS. The prescription dose and OAR dose constraints were applied to the treatment plans, with 6 MV X-ray radiationas the prescription doses for PTVnx, PTV1, and PTV2. The calculation grid of the planned dose was 3 mm × 3 mm × 3 mm. We delineated the areas of radio-induced brain necrosis according to the MRI images and summed the dose per unit volume (Figure 1).
Immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay
Formalin-fixed, paraffin-embedded radio-induced brain necrosis specimens were sectioned at 5 µm. After being baked at 65 °C for 1 hours, the samples were deparaffinized in xylene and rehydrated in a series of graded ethanol. For antigen retrieval, the sections were microwaved for 30 minutes in a repair solution (pH 9.0). Then, the samples were incubated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were incubated with the primary antibodies CD3 (1:200, ZA-0503-1.5, ZSGB-BIO, Beijing, China), CD31 (1:200, ZM-0044-1.5, ZSGB-BIO, Beijing, China), CD68 (1:200, ZM-0060-1.5, ZSGB-BIO, Beijing, China), CD11b (SAB, #46806, 1:150 dilution) and Ki-67 (1:200, ZM-0167-1.5, ZSGB-BIO, Beijing, China) in a 37°C incubator for 50 min. Subsequently, the samples were incubated with secondary antibodies and ABC reagent (#PK-7200, VECTOR LABORATORIES, Burlingame, CA, USA) at room temperature for 30 minutes. Immunoperoxidase staining was performed using an ImmPACT DAB Peroxidase Substrate (#SK-4105, VECTOR LABORATORIES, Burlingame, CA, USA) according to the manufacturer’s recommendations. Apoptotic cells were identified using the Click-iT™ TUNEL Colorimetric IHC Detection Kit (C10625, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instruction.
Descriptive statistics were used to analyze the clinical records and radiation doses, and the data are presented as the mean ± standard error (SE). The analyses were performed using the Statistical Package for the Social Science (SPSS version 16.0; SPSS Inc., Chicago, IL, USA).