Patients
All consecutive newly diagnosed NPC patients referred to the Department of Radiotherapy at Hainan General Hospital (The Affiliated Hainan Hospital of Hainan Medical University) between May 2018 and October 2020 were included in this prospective study approved by the hospital IRB (NO.2018025). All participating subjects were formally informed about the purpose of this study and a letter of consent was signed by every subject involved. The T, N, M, and clinical staging was performed according to the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for NPC (8th edition, 2016).14 Patients met the inclusion criteria by (1) having Karnofsky performance status ≥70%, (2) having NPC as confirmed by biopsy and histopathology, (3) performed PTEN immunohistochemical staining before treatment, and (4) having undergone DCE-MRI before treatment in our hospital. Patients were excluded if they (1) had MRI (eg, artificial cochlea, cardiac pacemaker implantation) or radiotherapy contraindications, (2) Failure to complete treatment or loss of follow-up less than 3 months, (3) had previously received radiotherapy in the neck region, (4) had any other malignant tumors in the prior 5 years, or (5) suffered from severe neurological or psychiatric diseases.
Clinical endpoints were progression-free survival (PFS) for follow-up status assessed on 1 Jan 2021, using the last MR follow-up as the endpoint. For PFS, time from diagnosis to disease-related death, the first event of relapse, or clinical follow-up endpoint was used. Patients suffering from disease-related death or relapse were categorized under “poor prognosis”, others categorized under “good prognosis”.
Treatment regimen
The choice of treatment regimen according to National Comprehensive Cancer Network (NCCN) Guidelines (https://www.nccn.org/).15 All patients were treated by radical three-dimensional conformal and intensity-modulated radiation therapy (IMRT). Radiotherapy was completed using the following equipment: three-dimensional treatment planning system (Eclipse, American), 3000A mobile laser positioning system (Gammex A), Trilogy medical linear accelerator 6MVX line (Varian), 16-slice spiral CT (Siemens), supporting vacuum bag fixing device, and three-dimensional fixing frame. The patients were in a supine position and fixed with a thermoplastic memory membrane in the head and neck. Positioning CT was performed using a thin-layer continuous scanning scheme with 3 mm thickness, tube voltage 140kV, current 280mAs. The images were input into Eclipse three-dimensional treatment planning system. The range of tumors was determined by MR images, and the target area was delineated and planned by fused MR and positioning CT images. The planning target volume (PTV) formed by expanding 3-5mm from each target area was administered a prescription dose: nasopharyngeal tumor volume (GTVnx) and cervical lymph node volume (GTVnd) at 66-73 Gy and 63-70 Gy, respectively; high-risk area of primary focus (CTV1) at 62-64 Gy; low-risk area of primary focus and cervical lymph node drainage area (CTV2) at 54-58 Gy; PTV1 and PTV2 at 54 Gy and 50 Gy, respectively. The number of segmentations was 31-35 times, 5 times per week.
Induction chemotherapy consisted of Docetaxel and Cisplatin and were given at 75 mg/m2 intravenously on day 1, or 25 mg/m2 intravenously on days 1-3, once per 3 weeks lasting for 2 to 4 rounds. Concurrent Chemotherapy consisted of Cisplatin 75 mg/m2 on day 1 (or 25 mg/m2 days 1-3) once per 3 weeks.
Follow-up assessments were performed every 3 months for the first year, every 6 months for years 2-5, then every year subsequently. Routine follow-up assessments included assessment of vital signs and any adverse events. MR scans were done at every follow-up time point.
MRI acquisition
MRI examinations were performed on a 3.0-T scanner (Skyra, Siemens Medical Solutions, Erlangen, Germany), equipped with a 20-channel sensitivity-encoding combined head and neck coil. Patients underwent routine MRI, including T1-weighted spin-echo sequence (field of view [FOV], 180×180 mm2; section thickness, 4 mm; repetition time [TR]/echo time [TE], 625/9.0 milliseconds), T2-weighted fast spin-echo sequence (FOV, 180×180 mm2; section thickness, 4 mm; TR/TE, 4070/30 milliseconds). Baseline T1-mapping was obtained before DCE-MRI with 5 different flip angles, including 3°, 6°, 9°, 12°, and 15°, using a fast low angle shot (FLASH)/vibe sequence. All other parameters were held constant as the following - DCE-MRI was performed with a (FLASH)/vibe sequence, 50 dynamic acquisitions, 4.9 seconds per dynamic acquisition, with the following parameters: TR/TE, 4.09/1.47 milliseconds; flip angle, 9; matrix, 192×144; FOV, 180×180 mm2; section thickness, 4 mm; phase, 75%; bandwidth, 400 Hz. Contrast enhancement was performed with a standard 0.1 mmol/kg body weight dose of gadolinium-based contrast agent of Gadodiamide (Omniscan, GE Medical Systems, Amersham, Ireland). The gadolinium-based contrast agent was administered through a catheter in the antecubital vein by an automatic power injector (Medrad, Pittsburgh, Pennsylvania, USA) at a rate of 2 mL/second and was followed by a double bolus injection of isotonic saline. The dynamic acquisition was performed with a temporal resolution of 4.9 seconds, and the contrast agent was administered after 5 baseline dynamics. After the DCE-MRI, contrast-enhanced T1-weighted spin-echo sequences were conducted on axial, coronal, and sagittal planes.
MRI post-processing and analysis.
DCE-MRI data were analysed using the OmniKinetics (OK) software (GE Pharmaceutical). The DCE parameters (Ktrans, reflux rate [Kep], the volume fraction of the extravascular extracellular matrix [Ve], and blood plasma volume [Vp]) were estimated using the extended Toft’s two-compartment model16 and population-averaged AIF.17
Two independent radiologists, (readers I (Weiyuan) and II (Wenzhu); 12 and 5 years of experience in MRI, respectively), who were blinded to clinical information, performed tumor segmentation to encompass whole tumor volume on anatomic reference images; among axial T1WI, PdWI, and post-contrast T1WI. Reference image and DCE parameter maps were loaded into a multimodality tumor tracking application ITK-SNAP (version 3.4.0, USA, http://www.itksnap.org). 18 The regions of interest (ROIs) drawn on the anatomic reference images were simultaneously mapped to the corresponding location on the DCE parameter maps. The mean values of Ktrans, Kep, Ve, and Vp from the volumetric ROIs were recorded for correlations and analysis.
PTEN protein expression detecting by immunohistochemistry
In every patient, the diagnosis was confirmed by tumor biopsy. The pathological type was classified according to the cell type and degree of differentiation: undifferentiated non-keratinized carcinoma, differentiated non-keratinized carcinoma, or keratinizing squamous cell carcinoma.19 Immunohistochemistry for PTEN protein was performed for all obtained biopsies for all cases used in this study, and the loss of protein expression was defined as an absence of cytoplasmic staining around a counterstained nucleus. Positive controls were obtained on a per biopsy basis from surrounding unaffected stroma and vasculature. After tissue sections were deparaffinized and rehydrated, tissue was sectioned into 4 μm thick slices were treated with antigen retrieval buffer (S1699, Dako) in a steamer for 20 minutes. Anti-PTEN antibody (100 μL [product number 9188, Cell Signaling Technology]) was applied to tissue sections for overnight incubation at 4°C in a humidity chamber. After a Tris-buffered saline (PBS) wash, tissue sections were incubated with biotinylated monoclonal anti-rabbit IgG (100 μL [product number ZA-0635, ZSGB-BIO]) for 20 minutes at 37℃. The antigen-antibody binding was detected by a kit (Vectastain Elite ABC kit, product number PK-6100, Vector Laboratories) and a DAB (3, 3-diaminobenzidine) system (product number K3468, Dako). Tissue sections were briefly immersed in hematoxylin for counterstaining and were covered with cover glasses. PTEN expression was scored by the pathologist (Y.W.) based on the percentage of positive staining tumor cells (Nucleus/cytoplasm). PTEN staining in the adjacent stroma and blood vessels served as a positive internal control, due to heterogeneity within the tumor focus. To account for this heterogeneity, foci were scored as PTEN-positive (≥95% cancer cells positive), and PTEN-negative (<5% cancer cells positive) (Figure 1).
Statistical analysis.
Patients’ characteristics were compared between the PTEN positive and PTEN negative groups. Continuous variables were analyzed using the independent sample t-test or Mann-Whitney U test, and categorical variables were analyzed using a chi-square test or Fisher’s exact test according to the data distribution, respectively. For continuous variables with a p-value less than 0.20, a receiver operating characteristic (ROC) curve was constructed and the area under the curve (AUC) was calculated. Optimal cutoff points were determined based on the maximum Youden index. Spearman correlation analyses (correlation coefficient, ρ) were performed to assess the correlation between PTEN positive or negative and clinical or radiographic features. Cox survival analyses were performed to evaluate the prognostic significance of PTEN expression in NPC patients. DCE parameters’ values, gender, age, EBV, T, N, M stage, PFS, and prognosis status were used as covariates to evaluate the independent detecting value of the classifier using multiple variables logistic regression.
Interobserver agreement between reader I and II was assessed using the intraclass correlation coefficient (ICC). An ICC of 1.0 was considered to represent perfect agreement; 0.81-0.99, almost perfect agreement; 0.61-0.80, substantial agreement; 0.41-0.60, moderate agreement; 0.21-0.40, fair agreement; and 0.20 or less, slight agreement.28
All statistical analyses and graphing were performed using Prism GraphPad Software version 9.0 (GraphPad Software, La Jolla, CA, USA. https://www.graphpad.com/scientific-software/prism/), and a p-value less than 0.05 was considered statistically significant.