Primary macronodular adrenal hyperplasia: morphology, immunohistochemistry, and correlation with molecular genetic alterations

Author(s)

Maria Luiza Anhaia de Arruda Botelho

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2022-12-08

Examining board members

Maria Claudia Nogueira Zerbini; Katia Ramos Moreira Leite; Chin Jia Lin; Romulo Loss Mattedi

• Advisor: Maria Claudia Nogueira Zerbini

Abstract

Introduction: First described in 1964, primary macronodular adrenal hyperplasia (PMAH) is a rare cause of adrenocorticotropic hormone (ACTH)-independent hypercortisolism, characterized by the presence of functioning adrenal nodules > 1.0 cm in diameter. The recent discovery that PMAH is associated with pathogenic allelic variants of ARMC5, a putative tumor suppressor gene, indicates that genetic determination of the disease is more common than previously thought. The role that ARMC5 mutations play in determining the morphological and immunohistochemical characteristics of PMAH has not been widely studied. Here, we revisit PMAH in light of this discovery. Objectives: This study aims: to review in detail the macroscopic and microscopic characteristics of resected adrenal glands from patients diagnosed with PMAH, comparing the patients with and without pathogenic allelic variants of the ARMC5 gene; to perform quantitative analyses of the phenomenon of cell proliferation, of the mitotic index, and of the Ki-67 proliferation index, comparing the groups with and without an ARMC5 mutation (M+ and M-, respectively), as well as among those groups and a control group; and to evaluate the expression of the BCL-2, BAX, p53, SF1, ACTH, CYP11B1, and CYP11B2 proteins, as well as that of the protein related to the ARMC5 gene, comparing the M+ and M- groups with each other and with the control group. Material: We studied 22 surgical specimens - 14 M+ group specimens and 8 M- group - obtained from adult patients diagnosed with PMAH between January 2009 and December 2017 in the Divisão de Endocrinologia do Departamento de Clínica Médica do Hospital das Clínicas da Universidade de São Paulo. We also studied a control group of 11 surgical samples obtained from the normal adrenal glands of patients undergoing radical nephrectomy for renal carcinoma. Methods: Macroscopic data were obtained by reviewing the pathology reports and photographic documentation available. Microscopic data were obtained by evaluating all the hematoxylin & eosin-stained slides available for each case, and the analysis included the following parameters: four architectural patterns (alveolar, diffuse, trabecular, and pseudo glandular); extension of adrenocortical cells, through the capsule, to the adjacent adipose tissue; histological subtype according to Hsiao; Fürhman grade; cytomegaly; mitoses; adrenalitis; and other findings. For the immunohistochemical study, a paraffin block was selected, preferably from an adrenal gland that had been completely resected (largest specimen), that best represented the entire case of each patient. The 11 control cases were used to compare the cell proliferation index (quantitative analysis of Ki-67); apoptosis (expression of BCL-2, BAX, and p53); expression of SF1, ACTH, and the protein related to the ARMC5 gene (qualitative analysis); and steroidogenesis (semiquantitative analysis of CYP11B1 and CYP11B2 expression). Results: Morphology - The pseudo glandular architectural pattern behaved as a marker of the presence of a mutation, being observed in all 14 cases in the M+ group and in none of the 8 cases in the M group (p < 0.001). The trabecular pattern was also more common in the M+ group; it was observed in 13 of the 14 cases, compared with only 1 of the 8 cases in the M group (p < 0.001). The alveolar pattern was equally present in both groups. The diffuse pattern was the least common, being observed in only 5 of the 22 cases evaluated. Extracapsular extension was identified in all 14 of the M+ group cases, compared with only 2 of the 8 M group cases (p < 0.001). More than one architectural pattern was observed in all 14 M+ group cases and in 4 of the 8 M group cases (p < 0.001). In both groups, the most common histological subtype was Type 2 of Hsiao (diffuse hyperplasia with no residual normal or surrounding atrophic adrenal cortex). Immunohistochemistry - In all the specimens evaluated, including PMAH and control specimens, expression of ACTH and BCL-2 was identified only in the medullary region and not in adrenocortical cells. In all cases, diffuse positivity for BAX and SF-1 was observed in adrenocortical cells, with no statistical difference among the M+, M-, and control groups. Anti-CYP11B1 and anti-CYP11B2 antibodies proved to be useful markers of the zona fasciculata and zona glomerulosa, respectively. However, no statistically significant associations of those markers were observed with the M+ or M- groups. Expression of CYP11B2 was less common in the PMAH specimens than in the control group. The Ki-67 proliferation index was generally quite low (< 4%) and did not differ significantly between the M+ and M- groups or between either of those groups and the control group. None of the specimens tested positive for the p53 protein. Expression of the protein related to the ARMC5 gene was observed in 6 of the cases of PMAH (4 M+ group cases and 2 M- group cases). Diffuse cytoplasmic positivity for that protein in adrenocortical cells was observed in all 11 control group cases. Expression of the protein was significantly lower in both PMAH groups than in the control group (p < 0.001). Conclusions: The morphological changes observed, especially in the cases of PMAH in which there were ARMC5 gene mutations, lead us to consider the hypothesis that this is a biological situation with a behavior intermediate between that of hyperplasia and that of neoplasia. The fact that expression of the protein related to the ARMC5 gene was significantly lower in the glands of patients with PMAH than in the normal glands, particularly in those of the patients with pathogenic allelic variants, might be related to the loss of apoptosis inhibitory effect of that protein and the consequent increase in gland volume. The disorganized increase in adrenal gland volume in PMAH was not found to be related to increased cell proliferation or changes in the expression of apoptosis regulatory proteins, such as Ki-67, BCL2, BAX, and p53. In both the PMAH groups and the control group, expression of ACTH was observed only in the medullary region. Therefore, the disruption of the corticomedullary architecture in PMAH might be an important destabilizing factor in the functional interaction between the adrenal cortex and medulla in PMAH, particularly concerning the ACTH produced by the latter (AU)

• FAPESP's process: 17/11495-1 - Primary Macronodular Adrenocortical Hyperplasia: morphological, immunohistochemical study and its correlation with genetic-molecular alterations
• Grantee: Maria Luiza Anhaia de Arruda Botelho
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)