EJE
From hyperplasia to carcinoma: a molecular driven adrenal disease
Elisa Dybal,1,2,*[D Myriam Decaussin-Petrucci,2,3 Françoise Descotes,4 Gerald Raverot, 1,2[D Jean-Christophe Lifante,5 Christophe Sajous, 60D Jonathan Lopez,4 and Helene Lasolle *
1Endocrinology Department, Reference Centre for Rare Pituitary Diseases HYPO, “Groupement Hospitalier Est” Hospices Civils de Lyon, 69500 Bron, France
2Lyon 1 University, Claude Bernard University Medical School, 69003 Lyon, France
3Service D’anatomopathologie, Hôpital Lyon Sud, Hospices Civils de Lyon, 69495 Oullins-Pierre-Benite, France
4Service de Biochimie et de Biologie Moléculaire, Hôpital Lyon Sud, Hospices Civils de Lyon, 69495 Oullins-Pierre-Benite, France
5Service de Chirurgie Endocrinienne, Hôpital Lyon Sud, Hospices Civils de Lyon, 69495 Oullins-Pierre-Benite, France
6Service D’oncologie, Hôpital Lyon Sud, Hospices Civils de Lyon, 69495 Oullins-Pierre-Benite, France
*Corresponding authors: Fédération d’Endocrinologie, Hospices Civils de Lyon, 69 Boulevard Pinel, Bron 69500, France. Email: elisa.dybal@chu-lyon.fr (E.D.); Fédération d’Endocrinologie, Hospices Civils de Lyon, 69 Boulevard Pinel, Bron 69500, France. Email: helene.lasolle@chu-lyon.fr (H.L.)
Abstract
Bilateral Macronodular Adrenocortical Disease (BMAD) is characterized by bilateral benign macronodules and, frequently, autonomous cortisol secretion. Germline molecular alterations of tumor suppressor genes are identified in around 30% of cases, the most frequent being ARMC5. Even if adrenocortical nodular disease often occurs with tumor suppressor gene pathogenic variant, the association with adrenal cortical carcinoma (ACC) is rare and no functional studies have proven a link between these two diseases. We reported the case of a woman with an adrenal Cushing’s syndrome developed on BMAD. Over 20 years later, ACC was diagnosed, developed inside a benign nodule of macronodular adrenal gland. Germline genotyping showed no alteration in CDKN1B, KDM1A, PRKACA, PRKAR1A, MEN1, APC, ARMC5, or TP53 genes. Next-generation sequencing has been performed in the ACC and the adjacent macronodular tissue, showing a progressive accumulation of somatic protumoral molecular alterations between the benign nodular part of the adrenal gland and the ACC. Therefore, we hypothesize that BMAD could be an early event of ACC development and may beneficiated from more systematic radiological monitoring.
Keywords: bilateral macronodular adrenocortical disease, adrenal cortical carcinoma, Cushing’s syndrome, mutational burden
Significance
This observation leads us to question the possibility of malignant tumor occurrence in BMAD due to a neoplastic process with accumulation of protumoral molecular alterations, and therefore the possible need for radiological monitoring.
Introduction
Adrenal tumors are mainly diagnosed as incidentalomas, which concern 4% of the population. Around 10% of adrenal incidentalomas are bilateral. The World Health Organization published their latest classification of adrenal cortical tumors in 2022.1 This new nomenclature divides adrenocortical nodular diseases into 3 categories: Sporadic nodular adrenocortical disease, bilateral micronodular adrenocortical disease, and bilateral macronodular adrenocortical disease (BMAD). This last category, formerly known as “Primary Bilateral Macronodular Adrenal Hyperplasia” (PBMAH) then “BMAD”, is increasingly well defined. Patients with BMAD usually show diffuse enlargement of both adrenals with multiple nodules more than 1 cm. Radiological presenta- tion can sometimes be variable, in particular initially showing asymmetric involvement mimicking a unilateral nodule. Overt Cushing’s syndrome is quite rare and biological explorations show preferentially mild cortisol autonomous secretion.2
Histologically, BMAD is characterized by multiple clear cell-rich nodules of over 1 cm lacking features of malignancy.
Knowledge of molecular alterations in adrenal tumorigen- esis has improved in the last decade. Next-generation sequen- cing enabled the identification of germline alterations in almost 30% of BMAD cases,3 often associated with a somatic alteration in the second allele leading to the disease, suggesting a neoplastic-like process. The most frequently reported consti- tutional alterations concern the ARMC5 gene, as initially described in 2013.2 Germline variants of KDM1A, MEN1, FH, and APC have also been reported.4-6
At a molecular level, ARMC5 is now known to be a tumor suppressor gene with a proapoptotic role in malignant adreno- cortical cells.7 Its inactivation increases cellular viability by decreasing apoptosis and increasing proliferation.8 Thus, the development of macronodules could be a combination of increased proliferation and decreased apoptosis. It has also been shown, both in vitro and in vivo, that its inactivation
leads to lower levels of cortisol secretion. Faced with this confusing hypothesis, it was assumed that this inefficiency in producing cortisol may be overcompensated by adrenal hyper- plasia and increased adrenal mass in patients with BMAD.6
Despite a neoplastic process being observed in BMAD, the association with adrenal cortical carcinoma (ACC) is not clas- sically described. Management is based on hormonal secretion and its consequences with no recommendation concerning radiological follow-up.
Adrenal cortical carcinomas are rare aggressive malignant tu- mors with bad prognosis. Adrenal cortical carcinoma is defined by invasive growth, architectural and cytologic features, and high proliferative activity. Different histological scores are used to establish a diagnosis of malignancy.9 Somatic next- generation sequencing and single nucleotide polymorphism (SNP) array enabled the identification of driver alteration impli- cated in the Wnt/beta-catenin pathway, cell cycle regulation, and DNA repair, such as CDKN2A, PRKAR1A, ATM, and RB1.10,11 Copy number variation studies reported amplifica- tion of prooncogenes such as TERT and PIK3R1 and losses of CDKN2A.12 Sporadic in 95% of cases, ACC can arise along germline TP53, MEN1, APC, or PRKAR1A pathogenic var- iants. Recently, germline analysis in an Italian cohort of 150 patients found a ARMC5 variant in 3/150 patients with ACC, one patient having concurrent macronodular disease.13 But this finding is still to be interpreted with caution as the au- thors reported a variant of unknown significance with a minor allele frequency in the general population higher than expected for a rare disease as ACC. Moreover, ARMC5 is known to act as a tumor suppressor gene following a two-hit inactivation model which has not been described with this variant at time. 14
We report here a case of ACC developed inside a nodule of BMAD patient with no germline pathogenic variant (in CDKN1B, KDM1A, PRKACA, PRKAR1A, MEN1, APC, ARMC5, or TP53). This case allows us to focus on the neo- plastic process in nodular adrenal disease.
Case presentation, diagnostic assessment, and treatment
In 2000, a diagnosis of adrenal Cushing syndrome was estab- lished in a 36-year-old woman with severe hypertension and type 2 diabetes. She underwent a left adrenalectomy. The histo- logical analysis revealed a 40 mm adrenal cortical adenoma (Weiss score = 0) associated with micronodular adrenal hyper- plasia. At that time, the size and aspect of the contralateral ad- renal was not described. After surgery, clinical symptoms improved; she kept low-dose hydrocortisone supplementation due to corticotrope deficiency with low adrenocorticotropic hormone (ACTH) level. She was considered to have adreno- cortical adenoma in her medical history and had no further endocrinological follow-up.
Twenty-two years later in 2022, a tomodensitometry was carried out due to abdominal pain and found a new nodular lesion on the right adrenal gland. The spontaneous density was 38 Hounsfield Unities with a relative wash-out of 52%, therefore not fulfilling benignity imaging criteria.15
Radiological monitoring after 6 months showed a rapidly growing tumor measured from 25 to 45 mm (Figure 1A). The performed 18 F-fluorodeoxyglucose (FDG)-Positron Emission Tomography (PET) showed an abnormal metabolic activity of the adrenal lesion leading to the decision of coelioscopic right adrenalectomy (Figure 1B). Interestingly, this hypermetabolic signal was heterogeneous within the lesion.
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DoBl: C Ex:A
4.8 cm
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ROI 5 (PET): 52.23cm3 M=9.21 Av=2
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Biological explorations found a mild autonomous cortisol se- cretion (low 8H AM ACTH level <3 ng/L, loss of cortisol nych- themeral variation with a mean level of 300 nmol/L, elevated cortisol after 1 mg dexamethasone 295 nmol/L [<50], normal 24 h urinary cortisol 41 nmol/24 h [N <180]) with no food- induced secretion; associated with high dehydroepiandrosterone sulfate (DHAS) level (11 300 nmol/L [N 510-5560]), normal tes- tosterone level 0.21 nmol/L (N 0.42-1.49). There was no clinical sign of androgen excess, no clinical Cushing syndrome; nor metabolic complications, and especially no hypertension, no dia- betes mellitus, no dyslipidemia, no osteoporosis, or fractures.
The macroscopic evaluation showed irregular enlargement of the adrenal gland with a 45 mm yellow nodule. Histological analysis revealed a 2.7 cm ACC included in a benign clear cell- rich nodule (Figure 2). The Weiss score was 5 (nuclear grade, diffuse architecture, necrosis, mitotic rate, and abnormal mi- toses), Ki67 up to 40%, and ENSAT stage1, pT1NxR0.
The patient refused mitotane adjuvant treatment. Unfortunately, she presented local recurrence 5 months after the initial surgery and underwent a new surgical removal. The suprarenal and retro-renal pedicle lymph node dissection con- firmed six metastases measuring 4-20 mm with capsular and lymphatic invasion. After failure to attempt mitotane treat- ment, she developed lung distant metastasis a few months later.
We analyzed the ACC tumor by whole genome sequencing (mean coverage 61x, >98% of the genome with coverage
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greater than 30x) and whole transcriptome sequencing (140 M unique reads). We also sequenced a matched blood sam- ple (whole genome sequencing (WGS) with a mean coverage of 113x, >99% of the genome with coverage <30x) to detect germline variants. Tumor content was evaluated in silico around 85%. Our molecular study did not include WGS for the hyper- plasia part as benign tumors are not part of the indications of France Médicine Génomique (https://www.auragen.fr/).
We identified a somatic homozygous deletion of CDKN2A, CDKN2B, and MTAP (1.16 Mb deletion on locus 9p21.3), a somatic pathogenic variant of ARID1A associated with a loss of heterozygosity (NM_006015.6 c.6282C> A p.[Cys2094Ter], variant allele frequency (VAF): 44%, ref:alt 36:28) and a somatic variant of unknown significance of KDM1A also associated with a loss of heterozygosity (ENST00000400181.8 c.913G> T p.[Gly305Cys], VAF:59%, ref:alt 21:31). No pathogenic or likely pathogenic germline vari- ant was retained (including CDKN1B, KDM1A, PRKACA, PRKAR1A, MEN1, APC, ARMC5, or TP53 genes). We also identified an in-frame CSNK2A2 :: BBX fusion of unknown sig- nificance (breakpoints: chr16:581967733 [CSNK2A2 exon2]/ chr3:107710452 [BBX exon3]).
To detect subclonal events, we then performed targeted DNA sequencing to get deeper coverage and compared the ACC (90% tumor content, mean coverage 884x) to the
hyperplasia counterpart (60% tumor content, mean coverage 1051x). The complete list of analyzed genes is in the Supplementary data of this article.
In the ACC fraction, we confirmed the homozygous deletion of CDKN2A. We also identified a gain in TERT (8 copies) and a sub- clonal likely pathogenic variant of CTNNB1 (NM_001904.3 c.14-1G> T p.(?), VAF:16.8%, ref:alt 1161:234). When com- paring to the hyperplasia counterpart, we only identified a het- erozygous loss of CDKN2A but no amplification of TERT or variant in CTNNB1. Of note MTAP and KDM1A were not included in the sequencing panel.
All the genetic alterations have been summarized in Figure 3. Molecular analysis methods are fully detailed in Supplementary data.
Patient signed a written consent for molecular analysis and for this publication. This brief report concerns only one pa- tient and did not involve any examinations outside the usual clinical procedures. As a result, it was not submitted to an in- dependent Ethics Committee. This research complies with the Declaration of Helsinki.
Discussion
Few cases of ACC developed in congenital adrenal hyperplasia patients have been described.16 It has been hypothesized that
Normal Adrenal Gland
Macronodular Adrenocortical Disease
Adrenal Cortical Carcinoma
00
DE
CDKN2A homozygous deletion
CDKN2A heterozygous deletion
MTAP homozygous deletion
Mutation Load
ARID1A exon 20 variation
MTAP unknown status ARID1A exon 20 variation
ARID1A loss of heterozygosity CTNNB1 exon 3 variation TERT amplification
insufficient treatment leading to ACTH hypersecretion could be part of the tumorigenesis pathway. However in BMAD ACTH levels are usually low.17 The occurrence of ACC in BMAD has been reported in 10 cases at time, notably one similar to ours by Libé et al.18,19 but with radiologically distin- guishable parts.
We observed heterozygous somatic alterations in the non- malignant gland in genes regulating the cell cycle and from the SWI (switch mutants)/SNF (sucrose non-fermenting) chromatin remodeling complex, such as heterozygous CDKN2A deletion and ARID1A pathogenic variant. These alterations were also present in the ACC, associated with a second hit in the same gene, such as ARID1A LOH and homozygous CDKN2A dele- tion. Accumulation of other protumoral alterations was ob- served in the ACC such as CTNNB1 pathogenic variant and TERT amplification not found in the nonmalignant part, sug- gesting ACC developed from a precancerous lesion.
From a physiopathological point of view, the CDKN2A gene, also known as the P16 gene, plays a role in cell cycle arrest by encoding multiple tumor suppressor 1 (MTS1). Alterations of CDKN2A lead to abnormal cell cycle regulation and proliferation ability.20
CTNNB1 pathogenic variants are common in ACC and are found in around 20% of cases. CTNNB1 plays a role in the Wnt/beta-catenin signaling pathway, which is often dysregu- lated in ACC. It has been shown experimentally that its consti- tutional activation in mice leads to adrenal cell expansion and the development of malignant characteristics such as uncon- trolled vascularization.21
CTNNB1 pathogenic variants and homozygous deletion of CDKN2A have already been described as molecular altera- tions linked to ACC prognosis.
Alterations affecting the SWI/SNF chromatin remodeling complex such as ARID1A have been described less frequently in ACC. There are currently few descriptions of its alteration in ACC and no experimental evidence of its physiopathologi- cal mechanism.23 However, this gene has emerged as recur- rently mutated in a broad array of tumor types, and it has been suggested that it could act as an epigenetic tumor suppressor.24 It can be associated with characteristics predict- ive of response to immunotherapies, such as microsatellite instability and higher tumor mutational burden. However, this was not observed in our patient.
In this patient, morphology of the controlateral adrenal was not available at the time of the first surgery. However, initial bilateral involvement was suggested, as histological review of the first affected adrenal showed micronodular adrenal hyperplasia with the adrenocortical adenoma and the lifelong persistence of a low ACTH level. This highlights the import- ance of careful radiological analysis of both adrenals for prop- er diagnosis and follow-up.
To our knowledge, this is the first published case of ACC developed inside a benign adrenal macronodule, with somatic molecular analysis available in both benign and malignant adrenal tumors, showing an accumulation of pro-tumorigenic genetic alterations.
In this BMAD context, current guidelines propose to analyze each nodule individually. Unfortunately, we do not have initial radiological examination of our patient also no information con- cerning the radiological aspect of the initial benign nodule and its evolution. However, this observation suggests to be more cau- tious in this BMAD context, and the surveillance of indetermin- ate nodules with no clear benign features could be prolonged longer than proposed in current guidelines.15
This case of BMAD with an internal ACC raises the ques- tion of the precancerous nature of adrenal hyperplasia, in which genetic alterations accumulation may be the basis for the development of ACC. This observation raises the question of radiological monitoring frequency in BMAD with benign or indeterminate characteristics, the importance of appropriate initial diagnosis and genetic analysis.
Supplementary material
Supplementary material is available at European Journal of Endocrinology online.
Funding
None declared.
Authors’ contributions
Elisa Dybal (Data curation [lead], Writing-original draft [lead]), Myriam Decaussin-Petrucci (Investigation [equal], Writing-review & editing [equal]), Françoise Descostes (Formal analysis [equal]), Gerald Raverot (Writing-review & editing [equal]), Jean-Christophe Lifante (Data curation [equal], Writing-review & editing [equal]), Christophe Sajous (Writing-review & editing [equal]), Jonathan Lopez (Data cur- ation [equal], Investigation [equal], Writing-review & editing [equal]), and Hélène Lasolle (Investigation [equal], Supervision [equal], Writing-review & editing [equal])
Conflict of interest: None declared.
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