Study Design
FABULAS was a three-centre study (Barts Health NHS Trust, Queen Mary University of London; Addenbrooke’s Hospital, University of Cambridge; and University College London Hospitals NHS Foundation Trust, University College London). It was modelled on phase 1b/2a trials of novel drugs. The primary aim was to assess the safety of EUS-RFA, while the secondary objective was to determine the feasibility and efficacy of the procedure. The trial was performed in accordance with the ethical principles of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Council for Harmonisation. The protocol was approved by the London - Bloomsbury Research Ethics Committee (United Kingdom, REC ref: 17/LO/0948, IRAS ID 222446) and by the local research and development department at each participating centre. The trial registration was submitted to ClinicalTrials.gov on 20th January 2018 (NCT03405025). Informed, written consent was obtained from all study participants before any trial-related activity.
Participants
The trial enrolled participants >18 years of age with a diagnosis of PA meeting the Endocrine Society’s criteria,1 and evidence of a probable or definite left-sided APA. This was determined by adrenal vein sampling (AVS) or molecular imaging ([11C]-metomidate PET-CT, MTO, or para-chloro-2-[18F]fluoroethyletomidate PET-CT, [18F]CETO).2,3 The inclusion criteria for each of the three sequential, overlapping groups of 10 participants is detailed below.
Group 1:
- Left-sided APA proven on either AVS or PET-CT.
- Participants wishing to take fewer drugs for their hypertension.
- Participants meeting Endocrine Society criteria for considering curative treatment of unilateral disease, but who were often not referred for surgery because the benefit: risk was considered too low.
- Participants aged ≥60 whose BP was at or near target (BP140/90 mmHg for most participants, or BP 130/80 mmHg in those with co-morbidities listed in the Hypertension guidelines) on treatment with tolerated antihypertensive medication.
- Participants with identified macroadenomas (APAs > 1 cm in diameter), who had probably at least 1 cm of peri-adrenal fat on axial and coronal projections.
Group 2:
Participants with either:
(i) a definite unilateral left-sided APA, but did not want surgery; or
(ii) probable but not unequivocal evidence of a unilateral left adrenal APA.
Group 3:
Participants >18 years of age meeting criteria for surgery but consented to undergo endoscopic ablation instead.
Individuals were excluded from the study if they were unable to give informed consent, were unable to discontinue beta blockers or direct renin blockers, were pregnant or unable/unwilling to take secure contraceptive precautions, or had any illness, condition or drug regimen considered a contraindication by the Principal Investigator or Chief Investigator.
AVS
Adrenal Vein Sampling (AVS) was performed under cosyntropin stimulation. An infusion of 50 ug/h of intravenous cosyntropin was commenced one hour prior to the procedure and continued throughout. Cannulation was considered successful if the cortisol level in each adrenal vein was ≥3× greater than that in the iliac and/or infrarenal inferior vena cava. A high probability of unilateral left sided PA was diagnosed if the aldosterone/cortisol ratio in left adrenal vein was ≥4 times that in the right adrenal vein.4,5 A 'probable’ left sided PA was diagnosed if the aldosterone/cortisol ratio was three to four.
[11C]metomidate and [18F]CETO PET-CT
[11C]metomidate/ [18F]CETO PET-CT was performed to confirm the presence of a focal, left sided-APA which could be targeted for ablation. All participants were pre-treated with 0.5 mg dexamethasone orally four times a day for 72 h before scanning. PET-CT imaging was performed on a GE Discovery PET-CT 690 scanner (GE Medical Systems). Non-contrast CT images were acquired over the adrenals (140 kV; 30 mA; slice thickness = 3.75 mm). Following an intravenous injection of [11C]metomidate or [18F]CETO (mean: 215 mBq; range: 86–289 MBq), dynamic PET images were acquired for 30 minutes at 30 minutes after administration. The images were reconstructed with iterative reconstruction (ordered subset expectation maximization) using two iterations, 24 subsets and a Gaussian filter of 6.4 mm. The reconstruction included time-of-flight, attenuation, scatter and decay corrections. The images were converted to standardized uptake values (SUVs; g ml−1) by dividing the activity concentration in the image voxels (Bq ml−1) by the injected activity per patient weight (Bq g−1).
A left-sided APA was diagnosed if it fulfilled the following three criteria: a focal adrenal nodule with Hounsfield Units in keeping with a benign adrenocortical adenoma, evidence of high [11C]metomidate or [18F]CETO uptake into the identified nodule, and a calculated left/right maximum SUV (SUVmax) ratio of >1.256. A ‘probable’ left-sided APA was diagnosed if 2/3 of the above criteria were fulfilled. Pre- and post-ablation PET-CTs were performed to allow comparison of the tracer-avid nodule(s) and determine whether ablation was successful from a radiological perspective.
EUS-RFA technique and protocol
As a preventive measure against the consequences of possible adrenomedullary stimulation during RFA, all participants received combined alpha and beta-blockade for two weeks prior to ablation. If required, other drugs were reduced in dose to maintain stable BP. The protocol allowed for RFA to be performed under general anaesthesia (GA) or deep sedation, and the latter was adopted by the last of the three sites to undertake EUS-RFA.
On the day of the procedure participants were examined and clinic BPs and blood tests (full blood count, urea and electrolytes, liver function tests, amylase/lipase and c-reactive protein) were taken as a baseline. Baseline home BP readings taken over the 4 days prior to ablation (three readings taken twice daily, 24 in total) were also recorded. After induction of GA or deep sedation, a linear-array EUS endoscope (Olympus, Keymed UK Ltd.; Pentax, Hitachi Medical Systems UK Ltd.) was advanced into the stomach, and the left adrenal gland identified using EUS. The nodule, presumed APA, can be identified as an echo-poor region (Supplementary Figure 1). The pre-ablation PET-CT was also used to help confirm the target lesion. Prior to advancement of the RFA needle into the targeted lesion, surrounding vascular structures were carefully visualized, aided by colour Doppler, in order to avoid inadvertent thermal injury during ablation of the APA. A 25-gauge needle was then passed transgastrically into the nodule enabling a fine needle biopsy (FNB) to be drawn into formalin or RNAlater© (Thermofisher, USA) and stored for subsequent immunohistochemistry (IHC) and molecular analyses respectively. A 19-gauge monopolar needle electrode with a non-insulated 5mm/10mm tip (EUSRATM, STARmed, Taewoong, Korea) and Viva Combo RF Generator System was used to deliver the radiofrequency (RF) current.
The tip of the RFA catheter was positioned at the distal margin of the targeted lesion under sonographic guidance. The RF current was delivered with the tip of the electrode maintained within the target mass and controlled by the endo-sonographer. Short bursts of treatment/burtns up to 25 seconds at 15-30W were applied, and stopped when hyperechoic bubbles were observed at the site of ablation, or impedance exceeded 100 Ohms. The acoustic scattering from formed gas bubbles was used as an estimate of the induced area of tissue necrosis. Depending on size, repeated treatments were performed by catheter withdrawal and repositioning in different planes in a fanned movement. Each treatment lasted 5-20 seconds before the catheter was repositioned within the nodule. The number of treatments during each ablation procedure increased during the study, as operators modified their approach based on the findings from post-RFA PET-CTs from the initial cases.
For participants who underwent a second procedure, the extent of viable adenoma was assessed intra-procedurally with or without the administration of contrast medium (SonoVue, Bracco UK Ltd) to areas of untreated tissue, and repeated treatment cycles applied as deemed appropriate by the operator in order to attempt complete ablation of the targeted lesion.
Post-procedure Monitoring
All participants had clinical examination and blood tests (full blood count, urea and electrolytes, liver function tests, amylase/lipase and c-reactive protein) at baseline (morning of procedure) and at 24-48 hours post-procedure, to identify any procedure-related complications. In addition, an abdominal CT was also performed at 24-48 hours to look for radiological evidence of any of the pre-specified major hazards.
Follow-up Visits
Participants were followed up at one, three and six months post-EUS-RFA. Home BP readings (taken during the 4 days prior to each visit) were recorded. Clinic BPs and blood tests (full blood count, urea and electrolytes, creatinine, bicarbonate, liver function tests, amylase/lipase, c-reactive protein, renin and aldosterone) were also collected to allow for assessment of efficacy (biochemical and clinical cure). A repeat PET-CT scan was performed at three months post-ablation to assess evidence of successful ablation of APA, or ‘radiological cure’.
MDT Decision Regarding a Second Ablation Procedure
The protocol allowed a second ablation procedure to be offered to participants who did not achieve complete biochemical success and who also had a residual radiological target on molecular imaging. It was considered that this would be necessary in individuals with large APAs. The post-ablation PET-CT scan was reviewed, along with biochemical and clinical data, at a monthly multidisciplinary team (MDT) meeting. A decision for offering a second ablation was based on a number of factors, including reductions in isotope uptake, change in SUVmax ratio between PET-CT scans before and after the first ablation, changes in clinic and home blood pressures, as well as participant wishes for a second procedure. The ablation procedure and follow-up visits in those who underwent a second ablation were the same as per the first ablation.
Safety Committee
The independent Safety Committee comprised of an expert adrenal radiologist (Anju Sahdev, FRCR), an endocrinologist (Scott Akker, PhD MRCP), and a hepatobiliary-pancreatic surgeon with experience of RFA (Satyajit Bhattacharya, FRCS). Their role was to independently assess the safety of EUS-RFA. They initially convened after the first two participants had undergone EUS-RFA, and then once again following the subsequent two participants. Recruitment into each subsequent group could only commence if the Safety Committee deemed the procedure to be safe in the first four participants of each prior group.
Primary Outcomes
The primary endpoint was safety, defined as occurrence of any of the three pre-specified major hazards: perforation, haemorrhage and infarction of major visceral organs. These were sought radiologically on the 24/48-hour post-procedure CT and supported by clinical examination, and comparison of serum amylase (and/or lipase) and full blood count pre- and 24/48 hours post-RFA. This assessment was made by the independent Safety Committee, who also reviewed the severity of all documented serious adverse events (SAEs) and their likelihood in relation to EUS-RFA.
Reporting of Events
We recorded all SAEs. A SAE is officially defined as any untoward medical occurrence that results in:
• Death
• A life-threatening event
• Inpatient hospitalisation or prolongation of hospitalisation.
• Severe or permanent disability
• Cancer (other than cancers diagnosed prior to enrolment in studies involving patients with cancer)
• Congenital anomaly
• Any grade four toxicity
We also recorded events related to GA or sedation (e,g: drowsiness, confusion and respiratory depression). Events related to the gastric puncture and delivery of the RFA (e.g: bowel perforation, haemorrhage and infarction of major organs) and finally events related to complications of accidental adrenomedullary stimulation (e.g: severe hypertension and rise in plasma metanephrines).
Serious Adverse Events (SAEs) were reported to and independently reviewed by the Sponsor. Decisions on further reporting to the regulatory body (Medicines and Healthcare products Regulatory Agency) were made by the Sponsor, according to local protocol.
Extended Safety Meeting
At the end of the study an extended safety meeting was held at which all unexpected events / SAEs were discussed and scored by the Safety Committee. The below scoring system was drawn up and applied.
Safety committee scoring system: Level of significant event:
a. Low: asymptomatic or mild symptoms, minimal or no clinical intervention indicated.
b. Medium: symptomatic, minimal invasive intervention indicated e.g. intravenous fluids.
c. High: severe to life-threatening medically significant event requiring urgent intervention, hospitalisation, or prolongation of hospitalisation.
Likelihood of significant event being associated with intervention:
1. Low: no association.
2. Medium: minimum to moderate association.
3. High: as a direct consequence of.
Statistical Analysis
The sample size of 30 was estimated on the basis of a 95% probability of at least one serious complication occurring, whose true risk was greater than one-in-ten ((1-p)^30=0.05), and 80% probability of at least one serious complication occurring if risk-per-participant was >one-in-twenty ((1- p)^30=0.2). The study was not powered for efficacy.
No hypothesis testing was planned for the primary endpoint (safety), or for the categorical secondary biochemical and/or clinical success endpoints. In order to avoid adjustment for multiplicity, and so maximise the likelihood of detecting a significant efficacy endpoint in 30 patients, there was a single pre-specified hypothesis, for the comparison of pre- and post-ablation aldosterone/renin ratios. A Wilcoxon signed rank test was used. For other continuous variables, descriptive statistics with 95% confidence intervals were reported. Efficacy was estimated after all ablations in each participant, and then only after first ablations.
Secondary Outcomes
The secondary outcomes were evidence of clinical and biochemical success at six months post-RFA (as defined by the international PASO Consensus, Supplementary Table 1),7.
Successful ablation of the APA, or ‘radiological success’ was quantified by comparing PET-CT images from before and after EUS-RFA. Visual assessment of the PET-positive nodule(s) in all planes was reviewed by a radiologist (H.C.) without prior knowledge of the biochemical and clinical outcomes for each participant. For quantification, the reduction in isotope activity was graded by as a percentage of the original PET-positive nodule, and grouped into quartiles of reduction (<25%, 25-50%, 50-75% or 75-100% reduction).
Exploratory Analysis
Exploratory outcomes included immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) for CYP11B2(aldosterone synthase) expression were performed on the FNB samples to confirm whether the targeted nodule was an APA.
Immunohistochemistry (IHC)
IHC was performed on 3-μm sections cut from paraffin blocks using a fully automated system (BOND III IHC and ISH stainer from Leica Biosystems). The CYP11B1 primary antibody clone RAT-87 (MABS502, Merck) was used at a dilution of 1:100 after heat-induced epitope retrieval at pH 9.0 (BOND Epitope Retrieval Solution 2, AR9640, Leica) for 20 minutes; and the CYP11B2 primary antibody clone EPR10494 (ab168388, Abcam) was used at a dilution of 1:200 after proteolytic-induced epitope retrieval with Proteinase K (BOND Enzyme Pre-treatment kit, AR9551, Leica) for 10 min. The primary antibody binding to tissue sections was visualized using BOND Polymer Refine Detection system (DS9800, Leica). Semi quantitative analysis of IHC was performed by two experienced board certified endocrine pathologists, blinded to clinical, biochemical and radiological outcome information.
Quantitative Polymerase Chain Reaction (qPCR)
qPCR was used to quantify the level of mRNA expression of the genes of interest. FNB samples of adrenal tissue were homogenised in lysis buffer for 20s using FastPrep-24 5G Sample Preparation System (MP-Biomedicals.) Total RNA was extracted using PureLink RNA minikit as per protocol (#12183018A, Invitrogen) and reverse transcribed to cDNA using High Capacity RNA-to-cDNA Kit (#4387406, Applied Biosystem). Quantification of mRNA expression was performed using TaqMan Fast Gene Expression Master Mix (#4444557, Applied Biosystems) and commercially available probes from Life Technology: CYP11B2 (Hs01597732_m1), CYP11B1 (Hs01596404_m1) and 18S rRNA (Hs99999901_s1) as a housekeeping gene. Results were analysed using the 2-ΔΔCT method8.
Where sufficient RNA was available, RNAseq was performed by the Barts and The London Genome Centre, United Kingdom; for analysis of somatic genotype and full transcriptome as previously described9.
Biochemistry Methods
All blood tests were analysed at United Kingdom Accreditation Service-accredited clinical laboratories. Serum aldosterone was measured by either an automated chemiluminescence immunoassay (LIAISON; DiaSorin) or by tandem mass spectrometry (in-house method adapted from10). The direct renin mass was measured at two centres by automated chemiluminescence immunoassay (LIAISON; DiaSorin). Plasma renin activity was measured in the final centre by liquid chromatography tandem mass spectrometry (North West London Pathology). Conversion between activity (nmol/L/h) to renin mass (mU/L) was performed using a conversion factor of 10.989.2
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