Ethics approval and consent to participate:
The Institutional Review Board at Eye, Ear, Nose, and Throat Hospital of Fudan University approved this study. Written informed consent was obtained from the parents, caretakers, or guardians on behalf of all of the children.
Patients
The retrospective study was conducted at the Department of Ophthalmology, Eye, Ear, Nose and Throat Hospital of Fudan University in Shanghai, China, from January to December 2020. Consecutive patients with RB treated with IVC and enucleation performed earlier in life and currently between the ages of 7 and 15 years were enrolled in this study. The authors divided the eyes into three groups: bilateral RB patients that had a preserved eye with extramacular tumours, with the staging as Group A-C at diagnosis (group I, n = 10), unilateral RB treated with IVC that had a normal fellow study eye (group II, n = 10), and age-matched healthy controls (control group, n = 10).
Subjects with any of the following were excluded: newly diagnosed RB cases and those with less than one year follow-up time following the complete stoppage of systemic chemotherapy and/or local therapy; patients who had received external beam radiotherapy (brachytherapy) and subconjunctival chemotherapy, intraarterial or intravitreal chemotherapy; with tumours affecting the macular and optic disc region (edge of tumour was less than 3 mm from fovea and optic disc boundary); or with macular subretinal fluid, cystoid macular edema, vitreous haemorrhage or cataract.
Ophthalmological examination
All the children had a full history taken which included their age at the time of the present study, sex, age at the time of first presentation of RB, duration of follow-up, family history, modality of treatment used, number of IVC cycles, international classification of retinoblastoma (ICRB), number of tumours, distance to optic disc (mm) and to foveal (mm). All patients underwent comprehensive ophthalmologic examinations, which included best-corrected visual acuity (BCVA), refraction measurement using auto refraction, spherical equivalence (SE) calculation, slit-lamp biomicroscopy, dilated fundus examination and axial length (AL) measured with an optical biometry device (IOLMaster; Carl Zeiss AG, Jena, Germany).
Optical coherence tomography angiography
OCTA was employed using a spectral domain system RTVue-XR Avanti (Optovue, Fremont, CA, USA). This system has an A-scan rate of 70 kHz per second with a light source centred at a wavelength of 840 nm and a bandwidth of 45 nm[12-14]. Two volumetric raster scans, including one horizontal priority (x-fast) and one vertical priority (y-fast), were obtained consecutively for each area and repeated again. The SSADA algorithm was employed to improve the signal-to-noise ratio by splitting the spectrum to generate multiple repeat OCT frames from the two original repeat OCT frames[15], and any motion artefacts were removed with 3-D orthogonal registration and the merging of the two scans.
Macular (6 × 6 mm) and optic disc (4.5 × 4.5 mm) OCT angiography scans were acquired. The vessel densities in the foveal, parafoveal and peripapillary areas were assessed as follows. The foveal area was a 1 mm diameter circle centred on the fovea. The parafoveal area was defined as an annulus with an outer diameter of 3 mm and an inner diameter of 1 mm centred on the fovea. The peripapillary area was defined as a 700 μm wide elliptical annulus extending outward from the optic disc boundary. The retinal vessel densities of the specific areas were defined as the percentage area occupied by vessels in the corresponding segmented areas[13].
At the same time, the FAZ area and macular thicknesses were measured using the same OCTA system. The FAZ was automatically outlined and measured by built-in ImageJ software. The macular thicknesses, including the foveal and parafoveal area, were obtained using the retinal map protocol. The full retinal thickness was measured from the internal limiting membrane to the middle of the retinal pigment epithelium and the inner retinal thickness from the internal limiting membrane to the outer boundary of the inner plexiform layer. The retinal thicknesses of each area were automatically determined using the system’s software and were defined as the mean thicknesses of each area.
Colour Doppler imaging measurements
The HDI 5000 CDI (Philips Ultrasound, Bothell, Washington, USA) with a 7.5 MHz linear probe was used to evaluate the blood flow of retrobulbar vessels posterior ciliary artery (PCA), central retinal artery (CRA) and ophthalmic artery (OA)). The patients were placed in the supine position and examined by the same experienced sonographer. All processes were done in accordance with the CDI measurements protocol, as described previously [16]. The peak systolic velocity (PSV) and end diastolic velocity (EDV) in each vessel were measured. The pulsatility (PI) and resistance (RI) indices were calculated automatically by the scanner.
Statistical analyses
All statistical analyses, clinical, OCTA and CDI parameters, were tabulated using Microsoft Excel 2017 (Version 15.32; Microsoft Corporation, Redmond, WA) and its built-in statistical functions (Analysis Toolpak). The comparison between the two groups was evaluated using the unpaired Student’s t-test, or Fischer’s exact test, where appropriate. P < 0.05 was considered statistically significant.