Keywords

3.1 Introduction

In the last decade, integrated multi-omic platforms revealed that adrenocortical carcinoma (ACC) is characterized by a complex genomic organization (germline and somatic DNA genes, aneuploidy, DNA proliferation and translation, epigenetics), and complex proteomic and metabolomic profiles. This pathological complexity can be seen as the result of dysregulation of the tight controls that regulate the development and the multiple functions of the normal adrenal gland [1]. There are several clinical consequences of this biological complexity:

  • ACC is not a monogenic but rather a polygenic disease;

  • one therapeutic target does not fit all ACC heterogeneity;

  • ACC is an adaptive disease characterized by temporal heterogeneity.

3.2 Germline DNA Mutations and the Hereditary Component of Adrenocortical Carcinoma

The heritable fraction of ACC is estimated at 5–10% and 50–80% of adult and pediatric cases, respectively [2]. ACC is a rare cancer and population-based registries of patients with hereditary ACC are lacking worldwide except in Southern Brazil where there is abundance of specific germline mutations of the TP53 gene causing a higher incidence of ACC [3]. Elsewhere, ACC can arise in the context of cancer-predisposing syndromes: Li-Fraumeni syndrome (TP53), Lynch syndrome (NMR genes), Beckwith-Wiedemann syndrome (CDKN1C, H19, IGF2, KCNQ1OT1), Carney complex (PRKAR1A), multiple endocrine neoplasia type 1 (MEN1) [2]. Other rarer germinal variants potentially predisposing to ACC have been described in succinate dehydrogenase genes (SDHx) [4] and in Armadillo-containing repeat protein 5 gene (ARMC5) [5]. In the majority of adult cases, however, ACC is diagnosed as a sporadic cancer with acquired genomic alterations of the somatic DNA.

The Cancer Genome Atlas (TCGA) project analyzed germline variants (GVs) relevant to adult ACC in a pan-cancer study and in a specific ACC study. From the analysis of the core dataset of 91 ACC cases in the TCGA pan-cancer study [6], a low rate of GVs was found that places adult ACC in the lowest quartile among the 33 cancers screened. In the TCGA-ACC study [7], 9 GVs were found among 177 genes potentially linked to ACC. In a recent Italian study, 21 GVs among 17 genes (including TP53, ARMC5 and DNA Damage Repair [DDR] genes) were found in 150 (9.3%) patients with sporadic ACC that correlated with shorter survival [8].

There are practical implications concerning germline DNA in ACC. First, because ACC is not included in any screening program of hereditary cancer syndromes (like colorectal, breast or thyroid cancers), patients with a hereditary cancer syndrome should undergo surveillance for ACC. Second, patients with a diagnosis of ACC should be tested to identify germline mutations in cancer-predisposing genes in their families [9]. Third, GVs affecting DDR genes could have therapeutic and prognostic relevance.

3.3 Chromosomal Number Alteration (Aneuploidy)

Whole chromosome or chromosome arm imbalance is called aneuploidy, a hallmark of human cancers [10]. Chromosome copy number aberrations (CNA) include whole chromosome or single-arm alterations as well as smaller changes, like loss of heterozygosity (LOH), or larger changes, like whole genome doubling (WGD). Higher tumor aneuploidy negatively correlates with T cell infiltration, T cell clonality, expression of immune-related genes and overall survival [11].

ACC is frequently hypodiploid compared with other cancer types. However, copy number gains and losses can occur in up to 60% of cases (a pattern called “noisy” in the TCGA-ACC study). This pattern of high aneuploidy is often associated with WGD, which is related to alteration of the telomeres’ length regulation machinery leading to cell immortalization and is a clinical marker of poor prognosis [7].

3.4 Somatic DNA Mutations and Tumor Mutation Burden

Several somatic DNA gene mutations and corresponding functional pathways have been found in ACC. Most of our current knowledge of ACC genomics derives from three main multi-omics studies of structural and functional alterations in this disease [7, 12, 13]. While the list of single somatic gene mutations is constantly increasing, there are recurring mutations (frequency >10% indicated in square brackets) of candidate driver genes in ACC that are summarized as follows:

  • genes involved in cell cycle (TP53 [21%], CDKN2A [15%], RB1, CDK4, CCNE1);

  • genes involved in Wnt/β-catenin signaling (CTNNB1 [16%], ZNRF3 [19%]);

  • genes involved in chromatin remodeling (MEN1, DAXX);

  • genes involved in telomere maintenance (TERT [14%], TERF2);

  • genes involved in protein kinase cAMP-dependent regulatory type I alpha (cAMP/PKA signaling) (PRKAR1A [11%]) [14];

  • genes involved in DNA transcription (MED12) and RNA translation (RPL22).

An exception is represented by the alteration of the insulin-like growth factor-2 gene (IGF2) that is a hallmark of ACC (loss of heterozygosity in 90% of cases) [15, 16] but overexpression of the corresponding insulin-like growth factor-2/receptor-1 (IGF2/IGF1R) axis seems not to be a driver pathway in ACC, as demonstrated by failure of a phase III clinical intervention with the anti-IGF2/IGF1R drug linsitinib [17].

Collectively, the two most frequently altered genes and functional pathways in ACC are the TP53/RB1 cell cycle and the Wnt/β-catenin pathways (33–45% and 41% of cases, respectively) [7, 12, 13].

  • DNA damage repair (DDR) genes

    In the TCGA pan-cancer study, >80% of ACC samples displayed at least one DDR gene alteration including genes involved in:

    • mismatch repair (MMR): MLH1–3, MSH2–6, PMS2;

    • homologous recombination (HR): TP53BP1, BRCA1–2, BRIP1, RAD51, TOP3A;

    • damage sensor (DS): ATM, ATR, CHEK2;

    • translesion synthesis (TS): REV3L;

    • base excision repair (BER): POLB.

    Other minor DDR gene alterations involve direct repair (ALKBH3, MGMT), Fanconi anemia (FANCA, FANCD2) and nonhomologous end joining (LIG4, XRCC4, XRCC6) [18].

    Many of these gene alterations are found at the level of both germinal and somatic DNA: in particular, germinal MMR gene alterations observed in familial ACC cases identify familial ACC as a Lynch syndrome (LS)-associated cancer [19].

  • Microsatellite instability (MSI)

    Defects of the MMR system cause microsatellite instability (MSI) that is both prognostic and predictive of response to therapy in many cancer types including colorectal and endometrial cancers. In a pan-cancer re-analysis of TCGA data, Bonneville et al. found a MSI-high (MSI-H) phenotype in 4.3% of ACC cases placing ACC as the fifth neoplasm with the highest MSI-H rate among 39 different cancers [20].

  • Tumor mutation burden (TMB)

    In the TCGA-ACC study, the median somatic mutation density was 0.9 mutation/Mb (range 0.2–14.0 mutations/Mb) [7]. In a pan-cancer analysis, ACC had a median TMB less than 5 mutations/Mb and less than 10% of cases had a TMB >10 mutations/Mb. Therefore, despite all of the above considerations, ACC is placed among tumors with the lowest TMB [21].

From the above considerations it does appear clear that ACC is not driven by one single gene alteration (like the GIST with c-Kit). A mosaic of multiple, concurrent and not exclusive gene alterations can be present, generating a high degree of pleiotropism.

Thus, translation of genomic data into effective target therapies has been hampered because of two main reasons. First, many gene alterations in ACC are master regulators of fundamental processes in eukaryotic cells (e.g., TP53/Rb or Wnt/β-catenin) for which there are no specific drugs, or they could cause unacceptable toxicities. Second, while one biological pathway may be predominant in ACC, there are multiple ways to escape or circumvent that pathway.

3.5 Epigenetic (Post-Translational) Changes

In biology, epigenetics refers to changes of gene expression without changes of the DNA sequence that occur by activation or repression of specific transcription factors or biochemical modifications (acetylation, methylation) of specific target genes. At least seven studies identified DNA methylation as an important mechanism of epigenetic control of gene expression in ACC. Both hypomethylation and hypermethylation of promoter regions can occur at a higher frequency in ACC compared to adrenal adenomas [22, 23]. In the European Network for the Study of Adrenal Tumors (ENSAT) and TCGA-ACC studies, analysis of the hypermethylation at CpG-rich islands defined three clusters of methylation (CpG island methylator phenotype [CIMP]-high, -intermediate, and -low) that showed a significant prognostic value. In particular, the CIMP-high profile identified ACC patients with higher proliferative index and worse prognosis [7, 12]. For further reading, a comprehensive review by Ettaieb et al. on the role of epigenetic alterations in ACC and their potential role as prognostic factors and therapeutic targets has been published [24].

3.6 An Integrative View of Molecular Biology of Adrenocortical Carcinoma

The availability of multi-omics platforms enabled researchers to provide an integrative view of the biology of ACC by combining germinal and somatic DNA, epigenetic changes, chromosomal aberrations, RNA, proteins and other metabolites. In particular, the TCGA-ACC study proposed a molecular classification of ACC with identification of three different clusters of diseases each characterized by homogeneous molecular subtypes in terms of DNA, RNA, proteins, etc. The final clusters were called “cluster of clusters” (COC) numbered from 1 to 3.

Patients in COC1 had better prognosis, lower grade of aneuploidy, lower somatic mutations, lower genome-wide methylation rate (CIMP-low), high expression of IGF2, low expression of steroidogenic machinery and higher immune cell infiltration signature. Patients in COC3 had the worst prognosis, frequent mutations involving the cell cycle and DNA damage repair pathways, overexpression of Wnt/β-catenin pathway, higher degree of aneuploidy and WGD, high CIMP profile, high expression of steroidogenic machinery and the lowest expression of immune cell infiltration signature. Patients in COC2 have features of intermediate prognosis and biology [7, 25].

Despite the fact that a molecular classification of ACC is not validated and, therefore, not ready yet for clinical use, this approach will match clinical and molecular profiles and it will hopefully generate new venues for a more rationale therapeutic strategy.

3.7 Spatial/Temporal Molecular Heterogeneity in Adrenocortical Carcinoma

Clinical decisions are taken on the basis of a biological snapshot of the ACC disease taken at a given time (usually at diagnosis). However, little is known about whether metastases from ACC share the same genomic alterations of their primary tumor and, more importantly, if their genotype confers different sensitivity or resistance to therapy. In a study investigating genomic heterogeneity in 33 metastatic ACCs, investigators demonstrated that ACC is characterized by a significant heterogeneity among different metastatic sites in the same patient and metastases had a 2.8-times higher mutation rate than the primary ACC [26].