The adrenal cortex is divided into three zones: zona glomerulosa, zona fasciculata and zona reticularis where three main pathways of steroidogenesis occur. Adrenocortical carcinoma is a rare malignancy originating from the cortex of the adrenal gland with a poor prognosis due to its aggressive nature and unresponsiveness to conventional chemotherapeutic strategies. Although most ACC cases are sporadic and without a known cause, a minority of cases occur within other syndromes. The most common of these are Li-Fraumeni syndrome (TP53 gene germline and somatic mutation), Lynch syndrome (MSH2, MLH1, MSH6, PMS2, EPCAM genes), multiple endocrine neoplasia type 1 (MEN1 gene), Beckwith–Wiedemann syndrome (11p151 gene, IGF-2 overexpression), familial adenomatous polyposis (FAP gene, β catenin somatic mutations), neurofibromatosis type 1 (NF1 gene) and Carney complex (PRKAR1A gene) ^[1][2][3][5,7,9]. In spite of evident progress, molecular mechanisms of ACC tumorigenesis have not been yet fully understood ^[4][10]. Several molecular alterations and signaling pathways are thought to have a main role in tumor development. Monoclonality indicates that tumor progression is the end result of an intrinsic genetic tumor driver mutation ^[5][11]. Most common mutations implicated in sporadic ACC are insulin-like growth factor 2 (IGF2), β-catenin (CTNNB1 or ZNRF3) and TP53 mutations ^[6][7][8][12,13,14].

The main proposed oncogene in ACC tumorigenesis is insulin-like growth factor 2. The IGF-2 gene is located at 11p15 region that consists of a telomeric domain including the IGF-2 and H19 that might modulate IGF-2 expression and a centromeric domain including cyclin dependent kinase inhibitor (CDKNIC) involved in the G1/S phase of the cell cycle ^[5][11]. IGF-2 gene encodes IGF-2 protein and it is expressed by both fetal and adult adrenal glands and as a part of complex signaling system which plays an important role in normal growth and development, cell survival and proliferation as well as in malignant alteration ^[9][15]. IGF-2 overexpression was proven in more than 85% of ACCs although it is low or absent at the beginning of clonal proliferation ^[10][16]. Different studies have shown that IGF2 mRNA expression was 10–20-fold higher and IGF2 protein expression 8–80-fold greater in ACC compared to normal adrenal glands or adrenocortical adenomas (ACA), speculating that different IGF2 concentrations could be responsible for different biological behaviors of ACC ^{[11][12][13][14][15][16]}[17,18,19,20,21,22]. IGF2 activates tyrosine kinase receptors that in turn lead to mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathway activation. Activated Akt is then able to trigger the subsequent activation of the mammalian target of rapamycin (mTOR) pathway ^[16][22]. These pathways are involved in proliferation, survival, and metastasis of cancer cells ^[16][22].

Another signaling pathway included in ACC tumorigenesis is the canonical Wnt/β-catenin pathway where β-catenin protein plays a central role. The Wnt signaling pathway is normally activated during embryonic development where β-catenin stimulates and maintains proliferation of adrenal cortical cells, but it is also required for cell renewal in the adult adrenal cortex ^[17][23]. It has a structural role in cell–cell adhesion, and it is a transcription cofactor with T-cell factor/lymphoid enhancer factor mediating transcriptional activation of target genes of the Wnt signaling pathway ^[17][23]. Constitutive activation of Wnt/β-catenin is involved in many tumor types and, in experimental studies, it has been shown to act as an adrenal oncogene ^[18][24]. The Wnt/beta-catenin pathway in ACC can be activated by CTNNB1 mutations and by ZNRF3 (zinc and ring finger protein 3) inactivation ^[18][24]. According to the data from the literature almost 50% of ACCs show increased cytoplasmic or nuclear β-catenin accumulation ^[19][25].

Tumor protein 53 (TP53, p53) is a protein product of tumor suppressor gene located on chromosome 17 (17p13.1). P53 plays a role in regulation of the cell cycle, apoptosis, genomic stability and activation of DNA repair proteins. It is the most frequently altered gene in sporadic cancers, with greater than 50% of human tumors harboring somatic mutation ^[5][20][11,26]. According to genomic analyses, germline mutations in TP53 were observed in 50–80% of children with sporadic ACC, while somatic TP53 mutation was observed in 20% to 30% of sporadic ACC patients where it correlates with poor outcome ^[21][27]. In immunohistochemical studies diffuse p53 staining correlates positively with increased Ki-67 expression ^[22][28].

2. Pathological Approach to Adrenocortical Carcinoma

Adrenocortical tumors are a unique group of tumors whose differentiation between adenomas and carcinomas is a great challenge even for pathologists since no single pathohistological marker indicates malignancy ^[23][72]. Pathological assessment, crucial for the diagnosis of ACC after surgical resection relies on morphological features, margin identification and immunohistochemical staining ^[24][73]. Biopsy of a specimen of adrenal tumors is usually contraindicated due to possible complications and the fact of it not being completely informative ^[25][42]. Relative indication remains to exclude/prove secondary etiology of non-functional adrenal tumor in patients with positive anamnesis of extra-adrenal neoplasm ^[25][42]. Weight and size of the resected tumor should be the first to raise the suspicion for malignancy ^[23][72]. In the literature, different cut off values can be found to determine it: >95, >50, >100 g, but also in some cases tumors <50 g had malignant potential ^{[23][24][26][27]}[72,73,74,75]. Most morphological studies confirm the size of the malignant lesion to be greater than 50–65 mm, ranging from 20 to 196 mm ^{[23][24][26][28]}[72,73,74,76]. It is important to keep in mind that tumor size might be underestimated by radiological investigation and not correlate with the real size of tumor lesion ^[28][29][76,77]. Except that above mentioned, further examination should include evaluation of capsule integrity and the presence of hemorrhage, necrosis and invasion ^[28][76]. Beside the classical form, adrenocortical carcinoma can have other rare histological variants like oncocytic, myxoid and sarcomatoid ^[30][78]. ACC arise from the different zones of the adrenal cortex and they most often have the cellular morphology characteristic of different adrenocortical cells ^[24][73]. According to the latest guidelines, immunohistochemical panel staining should be done, including steroidogenesis factor 1 (SF1), adrenocortical-specific marker or alternatively inhibin-alpha, calretinin and melan-A for identification of adrenocortical tumors, chromogranin A for identification of pheochromocytoma and paraganglioma as well as synaptophysin for both ^[25][42]. Adrenocortical cells express SF-1, a transcriptional factor, during fetal and adult life, mostly in the zona glomerulosa and fasciculate ^[31][79]. Experimental studies have confirmed that its high expression positively correlates with high mitotic count, high Ki-67 index, and high European Network for the Study of Adrenal Tumors (ENSAT) stage and negatively with loss of functionality, presence of oncocytic features and decreased survival ^[31][79]. Therefore, steroidogenic factor 1 can be used as diagnostic and prognostic marker in adrenocortical carcinoma ^[31][32][79,80]. Ki-67 is also routinely measured and, although nonspecific for ACC, it has a prognostic role. Ki-67 is a protein expressed in all cell cycle phases except G0 and represents a cell proliferation index. Ki-67 labeling index of more than 5% confirms the diagnosis of ACC ^[2][28][7,76]. Ki67 index >10% correlates with higher risk of recurrence in ACCs and it is associated with worse overall survival in patients with advanced disease or rapid disease recurrence ^[28][33][76,81]. Although practical utility of Ki-67 staining was indisputable and confirmed in many studies, one should keep in mind that it is hard to set a diagnostic threshold because of possible interobserver variations ^[34][82]. According to some authors, a combination of insulin-like growth factor 2 (IGF2) and Ki67 index might be useful for differentiating malignant etiology of adrenal masses ^[35][36][83,84]. Beside abovementioned markers, steroidogenic enzymes, p53, cyclin E and β-catenin expression might be also histologically analyzed ^[2][7]. Several novel markers and some other roles of already known biomarkers were investigated in experimental studies using immunohistochemistry (± other methods) on a different number of patients with benign and malign adrenal tumors. The aim of analyses was to elucidate their utility in the diagnostic approach of discriminating malignant lesions, to investigate possible pathophysiological role and, finally, to analyze their prognostic and targeted therapy efficiency (Table 12). Further studies on larger cohorts are needed for their implementation in routine praxis. ACCs can be graded into low- and high-grade based on their mitotic rates (≤20 mitoses per 50 high-power fields (HPF) or >20 mitoses per 50 HPF ^[74][1]. In clinical practice, several scoring systems have been developed to help distinguish malignant from benign adrenal tumors. The most widely used diagnostic tool is the Weiss score. The Weiss score includes nine histopathological parameters, related to tumor and cellular structure as well as invasion. A score of ≥3 suggests malignancy ^[28][76]. For an oncocytic variant of ACC Lin–Weiss–Bisceglia (LWB) scoring is proposed and Wieneke criteria are more reliable than Weiss scoring for the pediatric population ^[75][76][77][121,122,123]. Another simplified diagnostic algorithm termed the Reticulin algorithm was proposed several years ago, with a sensitivity and specificity of 100% for ACC ^[35][83]. It includes evaluation of disruption of the reticular network (highlighted by histochemical staining) and at least one of following parameters: mitotic rate >5/50 high-power fields, necrosis and vascular invasion ^[35][83]. In 2015, the Helsinki score was developed for more precisely predicting occurrence of metastases in adrenocortical carcinoma ^[78][124]. According to Duregon et al., who performed analysis on 225 ACC patients, it presents the most useful tool with an impact on prognosis, outperforming other prognostic parameters such as clinical stage, mitotic index and Ki-67 proliferation index, also applicable in all histological variants of disease ^[30][78]. The Helsinki score accounts for morphological (mitoses and necrosis) and immunohistochemistry parameters (the absolute value of the Ki-67 proliferation index), meaning 3× mitotic rate greater than 5/50 high-power fields + 5× presence of necrosis + proliferation index in the most proliferative area of the tumor ^[30][78]. With a cut off value of 8.5, this scoring has 100% sensitivity and 99.4% specificity for diagnosing metastatic ACC ^[35][78][83,124]. In summary, the Helsinki and Weiss score are predictors of poor prognosis, while the Helsinki score and Ki-67 index are the best predictors of disease-related death ^[30][78]. It is important to mention that in different studies, some other cut off values of the abovementioned scores were proven, i.e., <13 and ≥19 for the Helsinki score ^[30][78]. Further studies are needed to elucidate this area and its reproducibility.