Patients
We conducted a retrospective analysis of prospectively collected data. The study cohort consisted of 256 consecutive, previously untreated patients with primary head and neck tumors enrolled between November 2011 and October 2019. Complete data on both 11C-4DST and 18F-FDG PET/CT studies before therapy, patients with OPSCC, and p16 status were available for 34 patients from July 2013 to October 2019. Six of them were excluded because they did not exhibit sufficient 11C-4DST and/or 18F-FDG tumor uptake to perform textural analysis. Finally, 28 patients (25 males, 3 females; mean age, 66.5 years; age range, 52-87 years) were available for the study. Their clinical data are summarized in Table 1. The study protocol was approved by our institutional ethics review committee. The requirement for informed consent was waived due to its retrospective nature.
Radiotracer synthesis and PET/CT imaging
11C-4DST and 18F-FDG were produced using an automated synthesis system with HM-18 cyclotron (QUPID; Sumitomo Heavy Industries Ltd, Tokyo, Japan). The 11C-4DST was synthesized using the method mentioned by Toyohara et al. (Toyohara et al., 2011).
All acquisitions were performed using a Biograph mCT 64-slice PET/CT scanner (Siemens Medical Solutions USA Inc., Knoxville, TN, USA), which has an axial field of view of 21.6 cm. The mean time interval between 11C-4DST and 18F-FDG PET/CT scans was 6 days (range 0–24 days).
Patients were instructed to fast for at least 5 h before 18F-FDG administration. A normal glucose level in the peripheral blood was confirmed before the injection. PET emission scanning (2 min per bed position) was performed 15 min after intravenous injection of 11C-4DST (7.4 MBq/kg) and 90 min after intravenous injection of 18F-FDG (3.7 MBq/kg) from the midcranium to the proximal thighs, and co-registered with an unenhanced CT of the same region (Quality Reference mAs: 100 mAs [using CARE Dose4D]; reconstructed slice thickness: 5 mm). The PET data were reconstructed with a baseline ordered-subset expectation maximization algorithm, incorporating correction with point-spread function and time-of-flight model (2 iterations, 21 subsets). A Gaussian filter with a full-width at half-maximum of 5 mm was used as a post-smoothing filter.
Image analyses
The LIFEx software was used to extract the texture indices of PET images from the volume of interest (VOI) of the primary tumor (Nioche et al., 2018). The patients’ PET images in DICOM format were imported into this software. A board-certified nuclear medicine physician used the 40 % threshold of SUVmax to semi-automatically set the primary tumor. If non-tumoral areas of activity were incorrectly included within the VOI, adjustments were performed by the operator.
The SUVmax was calculated using the following formula: SUV = cdc/(di/w), where cdc is the decay-corrected tracer tissue concentration (Bq/g); di, the injected dose (Bq); and w, the patient’s body weight (g).
Six texture indices (homogeneity, entropy, short-run emphasis (SRE), long-run emphasis (LRE), low gray-level zone emphasis (LGZE), and high gray-level zone emphasis (HGZE) were calculated according to a report by Orlhac et al. (Orlhac et al., 2017; Orlhac et al., 2014).
Immunohistochemistry
Paraffin-embedded samples of the primary tumor obtained by surgical resection (n = 6) and biopsy (n = 22) were immunostained for p16. Staining was performed using the labeled streptavidin biotinylated antibody method with an autostaining system (Ventana Benchmark System, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s protocol. Mouse monoclonal antibody against p16/INK4a (Ventana Medical Systems, Tucson, AZ, USA) was used as the primary antibody. Staining of p16 was considered positive when strong nuclear and cytoplasmic staining was present in 75 % or more of the tumor cells (Lydiatt et al., 2017).
Statistical analyses
All statistical analyses were performed using a software package (SPSS Statistics, version 26; IBM). The 11C-4DST and 18F-FDG SUVmax values were compared using the paired t-test. The differences between PET parameters and p16 status were compared using the Mann-Whitney U test, and were considered statistically significant at P values less than 0.05.