Differential Phenotype of Primary Bilateral Macronodular Adrenal Hyperplasia and Other Bilateral Adrenal Lesions With Associated Subclinical Hypercortisolism. Study of 98 Patients.

Purpose: To evaluate the prevalence of primary bilateral macronodular adrenal hyperplasia (PBMAH) in patients with adrenal incidentalomas (AIs) with subclinical hypercortisolism. Also to analyse the differential phenotype of patients with PBMAH compared to other bilateral adrenal lesions which do not meet PBMAH denition. Methods: Retrospective study of patients with AIs diagnosed in our centre between 2013 and 2019 (n=730). Patients with bilateral disease and associated subclinical hypercortisolism (possible ACS or ACS) were included (n=98). Possible ACS and ACS were dened as a cortisol post-1mg-dexamethasone suppression test (DST)>1.8µg/dl but ≤ 5.0µg/dl and >5.0µg/dl. without specic clinical signs of Cushing ´s syndrome, respectively. PBMAH diagnosis was established in patients with subclinical hypercortisolism, hyperplasia and bilateral adrenal nodules >1cm. Results: PBMAH was conrmed in 31.6% of bilateral AIs with subclinical hypercortisolism. Patients with PBMAH presented a higher prevalence of ACS than non-PBMHA (OR 4.1, 95%CI 1.38-12.09, P=0.010), but differences disappeared after adjusting by tumour size and total adenomatous mass (adjusted OR 2.3, 95%CI=0.65-8.27 and 2.3, 95%CI 0.47-11.21, respectively). However, no signicant differences in the cardiometabolic prole of both groups were observed. Tumour size and total adenomatous mass were signicantly higher in PBMAH (30.2±12.16 vs 24.3±8.47, P=0.010 and 53.9±20.8 vs 43.3±14.62, P=0.023). Conclusion: PBMAH is common in patients with incidentally detected bilateral adrenals lesions with associated subclinical hypercortisolism. The higher prevalence of ACS in PBMAH compared to non-PBMAH is associated with a higher tumour size and total adenomatous mass in PBMHA, but no differences in the cardiometabolic prole were observed between both groups.


Introduction
Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of Cushing's syndrome and is frequently diagnosed as bilateral adrenal incidentalomas (AIs) with modest autonomous cortisol secretion (MACE) [1]. It is a highly heterogeneous entity, both regarding the severity of cortisol excess and the morphologic appearance of the adrenals. Moreover, no consensus in its de nition has been reached. Its reported prevalence is usually low [2][3] [4] [5][6] and it represents less than 2.0% of the cases of endogenous Cushing´s syndrome [7].
It is well known that 3-5% of the general population may harbour AIs [8], 15% of them bilateral. The latter may have associated possible autonomous cortisol secretion (ACS) or ACS in up to 40% [9] [10]. Therefore, the actual prevalence of PBMHA is expected to be higher than that reported from series of patients with Cushing´s syndrome. Further, few data exist about the clinical, hormonal, and radiological features of this entity which may differ from those presented by other adrenal lesions associated with MACE that do not meet PBMHA criteria.
The aim of our study was to evaluate the prevalence of PBMAH in a large series of 730 patients with AIs and analyse the clinical, hormonal, and radiological phenotype of these patients. We then compared them with the features of patients with bilateral adrenal hyperplasia and bilateral adrenal nodules with possible ACS or ACS that do not meet the criteria for PBMHA de nition (non-PBMHA).

Patient cohorts
Patients were selected from the ADRENAL INCIDENTALOMA database. Patients with AIs were identi ed through an electronic search in the Biochemical database of our hospital. All 1mg-dexamethasone suppression tests (DST) performed between 2013 and 2019 were identi ed. Only those patients who met the inclusion criteria to enter the ADRENAL INCIDENTALOMA database were included. The inclusion and exclusion criteria were previously published (5). These were patients between 18 and 90 years old, with incidentally discovered unilateral and/or bilateral AIs, of at least 10 mm in largest diameter. Exclusion criteria were: i) known diagnosis of hereditary syndromes associated with adrenal tumours; ii) chronic treatment with glucocorticoids or drugs that affect dexamethasone metabolism; iii) current treatment with oral hormonal contraceptives (treatment should be suspended for at least 6 weeks before performing the functionality study) and iv) AIs identi ed during the extension study of an extra-adrenal primary cancer. Only patients with MACE (possible ACS or ACS) and bilateral AIs with or without hyperplasia were included in this study, so patients with primary aldosteronism, pheochromocytoma and overt Cushing´s syndrome were excluded. Neither patients with missing information in the hormonal or radiological study were included (Fig. 1). The study was approved by the local ethics committee of our hospital (Approval date: 23th September 2019).
Clinical de nitions AI was de ned as the presence of an asymptomatic adrenal lesion greater than 1 cm detected in imaging tests not performed in the context of suspected adrenal disease (such as the extension study of a primary extra-adrenal cancer, or other abdominal diseases) [11]. The de nitions of hypertension, type 2 diabetes, obesity and dyslipidaemia were the same as a previous research shows [12]. Cardiovascular disease was de ned as ischemic heart disease or heart failure, and cerebrovascular disease as transient ischemic attack or acute stroke. Moreover, the presence of active smoking was analysed.
For the MACE de nition, the most sensitive criterion was used to optimize the identi cation of patients with AI with increased cardiometabolic risk, considering that there was possible ACS when the cortisol post-DST was > 1.8µg/dL and equal or lower than 5.0µg/dL in the absence of speci c data of hypercortisolism [11], and ACS was con rmed when cortisol post-DST was > 5.0µg/dL [8]. The diagnosis of PBMHA was based on the presence of possible ACS or ACS due to bilateral adrenal hyperplasia associated with the presence of one or more AIs greater than 1 cm in each adrenal gland [1] [13]. Those patients with possible ACS or ACS and bilateral AIs without hyperplasia or bilateral AIs < 1cm were classi ed as non-PBMHA.

Biochemical and hormonal study
All patients underwent a 1mg-DST (n = 98). Measurement of adrenocorticotropic hormone (ACTH) (n = 85), dehydroepiandrosterone sulphate (DHEA-S) (n = 62), late-night salivary cortisol (n = 76) and 24urinary free cortisol (UFC) (n = 73) was performed based on physician judgment. The DST was repeated at the follow-up visit in 75 patients. Moreover, in 48 patients an intermediate DST between rst and last visit was performed. Other hormones were also determined at the discretion of the treating physician. A biochemical study including fasting plasma glucose, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides, was performed in all patients. Besides, HbA1c was measured in 43 patients.

Study of aberrant receptors
Aberrant receptors study was performed on ve patients. The protocol of our centre was performed on an outpatient basis over 4 consecutive or non-consecutive days. On the rst day, 250 µg of cosyntropin was administered intravenously to serve as a reference for the cortical response capacity. This day, a wandering test was also performed, taking a baseline sample and repeating the extraction after 2 walking hours. The second day, a mixed standard food test was conducted with a meal of 450 Kcal with 46% of carbohydrates, 32% of lipids and 22% of proteins. The third day, an intravenous administration of 100µg of GnRH and an intravenous administration of 200µg of TRH were performed. The fourth day, the patient received an intravenous dose of 1 mg of glucagon, then 4 µg of desmopressin and, nally, an oral dose of 10 mg of metoclopramide. In all tests, baseline, and serial determinations of cortisol, aldosterone and DHEA-S were performed. A change of less than 25% of cortisol level was de ned as no response, a 25-49% change as partial response, and a 50% or greater as a positive response to the test.

Laboratory assays
The following assays were employed: serum and urine cortisol were measured by chemiluminescence assays in Architect i2000 systems Abbott Diagnostics with an intra-assay coe cient of variation (CV) < 10%; the normal range for serum cortisol was 3,7-19,4 µg/dl and for urine cortisol was 140 µg/24h. ACTH was measured by chemiluminescence in Immulite 2000 Siemens system with an intra-assay CV of < 10%, and in Liaison XL Diasorin from 2019 with an intra-assay CV of < 10%. The normal range for ACTH was 9-46 pg/ml in Immulite systems and 4.7-48.8 pg/ml in Liaison XL systems. DHEA-S was measured by chemiluminescence assay in Immulite 2000 Siemens system with an intra-assay CV of < 15%. Salivary cortisol was measured in Cobas 6000 Roche by electrochemiluminescence with an intra-assay CV of < 10% and normal range lower than 5.74 nmol/L.

Radiological study
Abdominal computed tomography (CT) or MRI were performed in all patients at diagnosis (CT and MRI in 53 patients, only CT in 38 and only MRI in 7 patients). The size of the largest adenoma was included in the analyses. Moreover, we calculated the total adenomatous mass as the sum of the largest diameters of both adrenal incidentalomas [12]. Lipidic content was analysed in 85 patients, and the presence of necrosis, haemorrhage or other atypical radiological features were described in all AIs. During follow-up, radiological studies were repeated in 89 patients (CT and MRI in 8 patients, only CT in 52 and only MRI in 27 patients).
In 15 patients, norcholesterol scintigraphy (potassium iodide was administered before the radiopharmaceutical administration) was performed, evaluating grade and laterality of the uptake.

Statistical analysis
All statistical analyses were performed with STATA.15. Shapiro-Wilk test was used to check the normality assumption and Levene's test to evaluate variance homogeneity. Categorical variables are expressed as percentages and absolute values. Quantitative variables are expressed as mean ± standard deviation or median and interquartile range (IQR) depending on the normal distribution of the variable. Odds ratios (with 95% con dence intervals) and mean differences were calculated as association measures using logistic regression model, and β coe cient using linear regression model. For variables following the normal distribution, we used Student's t test to compare differences between two groups. The chi-square test was performed for the comparison of categorical variables between independent groups. In all cases, a two-tailed P value < 0.05 was considered statistically signi cant.

Results
Differential phenotype of patients with PBMHA and non-PBMHA Ninety-eight patients were enrolled in the study, 31 patients with PBMHA and 67 with no-PBMHA. Patients with PBMHA presented a higher prevalence of ACS compared to non-PBMHA (OR 4.1, 95%CI 1.38 to 12.09, P = 0.010). This nding could be explained by the higher tumour size and total adenomatous mass in patients with PBMHA than non-PBMHA, as differences in the prevalence of ACS disappeared after adjusted by tumour size (adjusted OR 2.3, 95%CI = 0.65-8.27) or by total adenomatous mass (OR 2.3, 95%CI 0.47 to 11.21) ( Table 1 and Fig. 2). Moreover, a positive correlation was found between total adenomatous mass (r = 0.45, P < 0.001), although not with tumour size (r = 0.19, P = 0.084). Nevertheless, no signi cant differences were found in the cardiometabolic pro le of both groups. The only observed difference in the hormonal pro le was the presence of lower DHEAS levels in patients with PBMHA (Table 1). Norcholesterol scintigraphy was performed in 9 patients with BMHA and 6 patients with non-PBMHA. In all patients, bilateral uptake was observed, with the exception of one patient with non-PBMHA with no uptake (Fig. 2).

Follow-up study
Two patients with PBMHA underwent unilateral adrenalectomy of the one with the largest adenoma, improving their cardiometabolic comorbidities and cortisol secretion. Two patients with non-PBMHA died (one due to an infection and the other with no identi ed cause). After a median follow-up of 33.7 (range 3.7 to 194.8) months, 7 of 58 patients (12.1%) with possible ACS developed ACS. 10 patients developed dyslipidaemia, 5 type 2 diabetes, 5 cardiovascular disease, 4 obesity, and 2 high blood pressure. No new cases of cerebrovascular events were reported. No differences in the risk of developing comorbidities or tumour growth were observed between PBMHA and non-PBMHA (Table 2).  [14] and showed that only 1 patient (0.1%) had PBMHA. Lomte et al. [5] retrospectively analysed a cohort of 70 patients with bilateral adrenal masses and they found that only 3 patients (4.3%) harboured PBMHA. The prevalence of PBMHA in our cohort is higher than in those two other studies. This may be explained by the fact that in the cohort of Song et al. [14] the diagnosis of PBMHA was established by histology (the number of biopsied masses was low, only 12) and not on the basis of the secretory pro le and radiological features. The differences with Lomte et al.´s study [5] can be attributed to the higher prevalence of familial pheochromocytoma (40%) and tuberculosis (27.1%) which is more prevalent in India than in our country.
Patients with PBMHA presented a prevalence four times greater of ACS than non-PBMHA, but these differences were related to a higher tumour size and total adenomatous mass of patients with PBMHA. The ACS prevalence in AIs is widely variable in the previous reported studies [12] [15][16]. In two observational studies performed in Italian population, the prevalence of ACS varied from 9,2% in a multicentre cohort with more than a thousand patients with AIs [15] to 29% in a small group of 41 patients with AIs and typical adrenal adenoma image [16]. However, no previous study compared the prevalence of ACS in PBMHA vs. other bilateral AIs without PBMHA de nition criteria. Therefore, we believe that larger prospective and multicentre studies should be carried out to con rm our ndings.
Regarding tumour size and ACS risk, there is some discrepancy in previous studies about the prevalence of ACS and the size of AIs. In accordance with our ndings, Vassiliadi et al. [9] found a signi cantly higher size and levels of cortisol after DST in bilateral AIs when compared with unilateral lesions.
Besides, the ACS was diagnosed in more patients with bilateral than in unilateral AIs (41.5% vs 12.2%, respectively), even when various criteria were used to de ne the ACS. In another small retrospective study with 33 bilateral AIs (5 of them PBMAH), those subjects with positive responses to the aberrant receptor study had bigger adenomas, higher cortisol levels after DST, higher night cortisol levels and a tendency to lower levels of ACTH [17]. According to these results, it seems that the signi cant higher size of the bilateral lesions could justify the more frequency of ACS in this group. Perhaps, the larger tumour size in bilateral than in unilateral AIs could be related to a longer duration of the disease in bilateral tumours, which may also explain the higher risk of ACS. On the other hand, Morelli et al [18] performed a prospective study with 175 patients with unilateral AIs and 38 with bilateral AIs (30 of them corresponding to PBMAH), and although the maximum diameter of the adrenal lesions was signi cantly higher in bilateral than unilateral forms, the magnitude of hypercortisolism was similar in both unilateral and bilateral AIs.
Despite the higher prevalence of ACS in patients with PBMHA than in non-PBMHA, no differences in the cardiometabolic risk were observed between them. To our knowledge, no previous study has analysed this issue. Previous AIs incidentalomas series focused in the differential phenotype of unilateral and bilateral AIs have described similar results [19][18] [20]. Actually, the lack of association of bilateral AIs with ACS-related comorbidities was observed in some studies reporting an association between the prevalence of ACS and bilaterality of AIs [19][18]. This nding could be related to the presence of different sensitivity to hypercortisolism, in patients with bilateral AIs [21]. In this sense, Majnik et al [21] found that the prevalence of T2DM in patients with bilateral AIs (40.9%) was signi cantly higher compared with unilateral adrenal tumours (23.2%). This could be related with the higher prevalence of N363S variant in bilateral AIs suggesting a role in the pathogenesis in bilateral AIs. Even though, the studies that focused on the role of single-nucleotide polymorphisms (SNP) of the glucocorticoid (GC) receptor gene analysis in the risk of ACS in AIs found controversial results [21] [22]. Some authors showed an association between GC receptor SNP and ACS risk [23] [24], other authors [22][21] [25] found that the presence of SNP of GC receptor did not in uence the development of Cushing's disease, or adrenal dependent Cushing´s syndrome. So, scanty data are available in patients with AIs to con rm the hypothesis that single polymorphic variant could in uence cortisol secretion and risk of comorbidities, and even less about the differences in GC receptor SNP prevalence in bilateral and unilateral AIs. The presence of abnormal receptors can lead to innovative pharmacological therapies as alternatives to adrenalectomy, including suppression of the ligands or the use of speci c receptor antagonists [27]. However, it should be noted that in none of the 2 patients of our study the medical treatment was effective. Libé et al. [28] found an inhibition of cortisol secretion by octreotide in the three Cushing's syndrome patients who positively responded to the mixed meal, in all the glucagon responsive patients, and in 12 of 13 (92%) patients with ACS. In accordance with this, previous studies demonstrated a correction of hypercortisolism with chronic subcutaneous octreotide administration in patients with fooddependent Cushing's syndrome [31] [32], and endogenous GIP levels inhibition is thought to be the possible underlying mechanism [17]. Moreover, long-term control of hypercortisolism has also been obtained by blockade of ectopic -adrenergic receptor with propranolol [27] [33] and by inhibition of LH secretion with leuprolide acetate [27] [34]. In contrast, Albiger et al. [35] reported, on the follow-up of 16 patients with PBMAH and Cushing's syndrome, one case with food-dependent CS treated with octreotide LAR and two patients with a positive postural test treated with propranolol, with a limited clinical response despite marked improvement in biochemical values. Thus, surgery is the current recommended therapy for PBMAH [36]. However, adrenal steroidogenesis inhibitors remain a valid option for nonsurgical candidates.
One of the major strengths of our study is that it includes a large number of subjects who were consecutively evaluated in our centre between 2013 and 2019, and the long-term follow-up. Moreover, this is the rst study focusing on the differential phenotype of PBMHA compared to other bilateral AIs with associated MACE. The limitations are mainly related to the retrospective nature of the study, and the possible bias induced by individual decisions for adrenalectomy or medical treatment of comorbidities. Moreover, information in the last visit was not available in all patients, so a follow-up bias should be considered.

Conclusions
PBMHA is relatively common in patients with incidentally detected bilateral adrenals lesions with associated subtle hypercortisolism. The higher prevalence of ACS in PBMHA compared to non-PBMHA is related with the higher tumour size and total adenomatous mass in patients with PBMHA, but no differences in the cardiometabolic pro le was observed between both groups.
Con ict of Interest: The authors have no con ict of interest.