CrossMark
REVIEW
Adrenocortical carcinoma: the dawn of a new era of genomic and molecular biology analysis
R. Armignacco1 . G. Cantini1 . L. Canu1 . G. Poli1 . T. Ercolino1 . M. Mannelli1 . M. Luconi1 D
Received: 6 March 2017 / Accepted: 29 September 2017 @ Italian Society of Endocrinology (SIE) 2017
Abstract Over the last decade, the development of novel and high penetrance genomic approaches to analyze bio- logical samples has provided very new insights in the comprehension of the molecular biology and genetics of tumors. The use of these techniques, consisting of exome sequencing, transcriptome, miRNome, chromosome altera- tion, genome, and epigenome analysis, has also been suc- cessfully applied to adrenocortical carcinoma (ACC). In fact, the analysis of large cohorts of patients allowed the stratification of ACC with different patterns of molecular alterations, associated with different outcomes, thus provid- ing a novel molecular classification of the malignancy to be associated with the classical pathological analysis. Improv- ing our knowledge about ACC molecular features will result not only in a better diagnostic and prognostic accuracy, but also in the identification of more specific therapeutic tar- gets for the development of more effective pharmacological anti-cancer approaches. In particular, the specific molecular alteration profiles identified in ACC may represent targetable events by the use of already developed or newly designed drugs enabling a better and more efficacious management of the ACC patient in the context of new frontiers of personal- ized precision medicine.
R. Armignacco, G. Cantini, and L. Canu have equally contributed to this study.
☒ M. Luconi michaela.luconi@unifi.it
1 Endocrinology Unit, Department of Clinical and Experimental Biomedical Sciences “Mario Serio”, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy
Keywords Adrenal cancer . Genomics · Molecular biology
Introduction
In spite of its rarity (1-2 cases per million prevalence), adrenocortical carcinoma (ACC) is an aggressive malig- nancy of the steroidogenic component of the adrenal gland with poor prognosis, in particular when already metastatic at diagnosis. The current poor and heterogeneous prognosis is due to the lack of both reliable clinically diagnostic criteria and specific/effective therapeutic treatments [1, 2]. To date, in fact, the radical surgery (R0) represents the only effective therapeutic option [3]. In advanced carcinoma, therapy with mitotane, an adrenolytic derivative of the insecticide DDT, is recommended, since it associates with a more prolonged disease-free interval after surgery [4].
ACC diagnosis on the tumor mass [5] is based on his- topathological criteria within the Weiss Score system (Table 1), which can also provide a prognostic index [6-8] together with the tumor resection status (R0/R1) [3] and the tumor staging at the diagnosis [9]. This new classification system (Table 2) has been proposed by the European Net- work for the Study of the Adrenal Tumors (ENSAT) [10]: it revises the original TNM classification to identify four tumor stages associated with different disease outcomes. The proliferation index, evaluated by the cell positivity to Ki67 staining, is also considered as an independent prog- nostic factor [11]. Although the staging system offers a good prognostic tool, ACC outcome is often highly variable and heterogeneous, even within the same stage: some cases are less aggressive and can be successfully treated by surgery, while others can progress despite an apparently complete mass removal. These aspects, along with the few available
Table 1 Weiss score
Nuclear atypia Atypical mitotic figures Mitotic rate > 5/50 HPF Cytoplasm: ≤ 25% clear of vacuolated cells Diffuse architecture Necrosis Venous invasion Sinusoid invasion Invasion of tumor capsule
Presence of three or more cri- teria is related to malignancy (specificity 96%, sensitivity 100%)
| Tumor stage | |
| I | T1, N0, M0 |
| II | T2, N0, M0 |
| III | T1-T2, N1, M0; |
| T3-T4, NO-N1, MO | |
| IV | T1-T4, NO-N1, M1 |
T1 tumor size ≤ 5 cm, T2 tumor size > 5 cm, T3 tumor infiltration in surrounding tissue, T4 tumor invasion in adjacent organs or venous tumor thrombus in vena cava or renal vein, N0 no positive lymph nodes, N1 positive lymph nodes, M0 no distant metastasis, M1 pres- ence of distant metastasis
and efficacious therapeutic approaches, contribute to make the management of this disease generally complex and dif- ficult [1, 2, 5].
In addition to the rarity and the clinical heterogeneity, the molecular mechanisms underlying ACC onset and pro- gression still remain to be fully elucidated. Moreover, the relation between ACC and the benign form (adrenocortical adenomas, ACA) is still debated: an independent origin is suggested by the low prevalence of carcinoma against the high prevalence of the benign forms. Conversely, the dis- covery of some mutations and altered pathways common to both forms [12] may support the hypothesis of carcino- mas progressing from adenomas [13]. In addition, in animal models, activation of beta-catenin and insulin-like growth factor 2 (IGF2) sequentially leads to adenoma progressing to carcinoma [14, 15]. In humans, however, if this progression sequence occurs, it may represent an “exceptional random event” [16].
The currently accepted hypothesis is that the intrinsic molecular heterogeneity of the tumor drives the variable clinical features and the disease progression. Thanks to many international scientific collaborations, large cohorts of ACC samples have been analyzed so far by integrated
pan-genomic approaches, highlighting, on one side, the het- erogeneity of the tumor molecular profile, but, on the other, also isolating specific recurrent molecular alterations in these tumors, by assessing the alterations at the level of the transcriptome, miRNome, methylome/epigenome, as well as the aberrations of the chromosome structure (copy number alteration, CNA; loss of heterozygosity, LOH).
In this review, we aim at summarizing the results of the recent studies that have contributed to define a new ACC classification based on a specific panel of molecular mark- ers. Moreover, we address the potential transferability of the novel molecular information derived from this genomic analysis to personalized treatment approaches for ACC. To date, the three largest studies of ACC pan-genomic analysis have been performed on cohorts of adult patients enrolled all over the world [17-19] and have contributed to validate this new molecular classification [20]. Of note, these stud- ies resulted from large international collaborations and were able to assess cohorts of 41, 122, and 91 ACC patients, which are consistent numbers for such a rare tumor.
The future challenge in the field will be to integrate the genomic information with the “classical clinical parameters” used so far for ACC characterization, to develop a novel integrated classification of this malignancy with a greater prognostic value to also open the new era of precision-based medicine for ACC. In addition, techniques, cheaper, and more rapid than the pan-genomic approach, applying for instance targeted molecular analysis of the alterations in spe- cific driver genes are urgently needed to transfer the acquired molecular knowledge to the routine patients’ screening and to personalized medicine.
Gene expression (transcriptome)
The genomics of the adrenocortical tumors has been exten- sively validated on large series of ACCs for differential diagnosis to discriminate between the benign and malignant forms. Of note, such molecular approach led to the identi- fication of new molecular clusters of ACCs with different outcomes. At the level of the transcriptome (gene expression in the entire genome), ACC and ACA show a differential gene expression profile [21-23]: genes involved in several processes, such as cell cycle, chromosomal maintenance, cell survival, inflammation, and immunity, are deregulated in ACC compared with ACA. Among them, IGF2 is clearly the most up-regulated gene in the malignant forms [22, 23]. An early study applying cDNA array-based gene expres- sion profiling of 230 candidate genes correlated with the clinical follow-up to screen a cohort of 57 adrenal cortical tumors identified two independent clusters of genes (includ- ing, respectively, IGF2 and other 7 IGF2-related genes or 14 steroidogenic enzymes) able to significantly discriminate
between two populations of tumors with distinct clinical out- comes [23]. The relevance of the IGF2-specific signature cluster in predicting recurrence was further confirmed by immunohistochemical analysis of ACC [25, 26].
Independent transcriptome analyses identified two main ACC subgroups with different prognosis associated with the overall survival. These expression profiles include hyper-expression of genes involved in cell proliferation and cycle [18, 22, 24, 27-29] (Fig. 1a): the C1A cluster, which includes the most aggressive tumors, and the C1B cluster, which identifies the most indolent forms. This reflects the clearly different tumor biology, likely resulting from various genetic and epigenetic alterations. Unsupervised clustering analysis identified two groups of malignant tumors with very different outcome based on the combined expression of PINK1 with DLG7 or BUB1B. Their combined expres- sion is the best predictor of disease-free and overall survival,
respectively, also remaining significant after adjustment to the Weiss score. PINK1 and BUB1B combined expression has also been recently validated as the best predictor of dis- ease-free in a cohort of pediatric patients [30]. Moreover, some discrepancies found between proliferation index/tumor stage and tumor outcome may be explained on the basis of the molecular expression profile [20]. ACC stratification, based on such specific expression profiles, provides a valid prognostic tool independent from the proliferation index and the tumor stage [30].
DNA methylation (methylome)
Gene expression is strictly regulated by the methylation sta- tus of the CpG islands, DNA sequences enriched in “CG” bases that are located in the promoter sequences of genes.
a
mRNA subgroup
C
C1A
C1B
C1A
C1B
Omics subgroups
mRNA
DNA methylation subgroup
1.0
DNA methylation
C1B Mi1
CIMP-high
miRNA
CIMP-low
Deregulated miRNAs
miR-506-514
Non-CIMP
Overall survival rate
0.8
C1B Mi2
DLK1-MEG3
miRNA cluster
Mi1
ZNRF3
Mi2
0.6
Driver genes
CTNNB1
Mi3
TP53
Drivers and pathways
Altered
0.4
C1A non-CIMP
Wnt/B-catenin
Not altered
Pathways
p53/Rb
Deregulated miRNAs
Chromatin remodeling
0.2
C1A CIMP-low
Down
Up
Log-rank
Mutation rate
Mutation rate (per Mb)
0
P = 3.67 x 10
C1A CIMP-high
0.05
1.4
0
24
48
72
96
120
144
Time (months)
b
d
Cluster of clusters
Stage
1234
1.0
Vital status
Diease progression
ny
CTNNB1 Mutation
0.8
CIMP-intermediate
Event Free Survival
Steroid-pheno-high
Group I (n=33)
miRNA 6
miRNA 4
0.6
miRNA 2
Steroid-pheno-low+prolif
Quiet
Noisy
0.4
Group II (n=19)
Steroid-pheno-high+prolif
CIMP-high
miRNA 3
C1A
0.2
p value, log-rank test:
miRNA 1
Group III (n=24)
I vs II: 1.36×103
C1B
I vs III: 3.55×10-15
Steroid-pheno-low
Il vs III: 4.36×104
CIMP-low
0.0
miRNA 5
Chromosomal
0
50
100
150
Genomic studies have highlighted how a global hypo-meth- ylation induces genomic instability, loss of parental imprint- ing, and reactivation of transposable elements, features that can often be observed in cancer [31]. On the other hand, hyper-methylation of CpG islands is negatively correlated with the expression of tumor suppressor genes [32].
Pan-genomic studies have recently shown that, in terms of DNA methylation, ACCs are globally hypo-methylated compared with ACAs, mainly in intergenic regions [33, 34]. Conversely, about 220 CpG islands in the promoter regions are hyper-methylated in ACC [34, 35], with a possible down- regulation of the tumor suppressor genes involved in cell cycle, apoptosis, and DNA transcription. The methylation levels of CpG islands vary among ACCs, but they have been demonstrated to correlate with some prognostic features. In particular, a hyper-methylated profile, referred as CIMP (CpG Island Methylator Phenotype), associates with a worse outcome of ACC (Fig. 1a-d) [18, 19, 35].
As already reported, an altered DNA methylation status of the IGF2 locus is associated with ACC tumorigenesis. Although IGF2 over-expression alone has not an accurate diagnostic value, it has been recently proposed that the meth- ylation status of three regulating regions of the IGF2 locus may represent a reliable tool for the differential diagnosis of adrenocortical tumors [36]. Moreover, the global CpG island methylation level in ACC specimens calculated by averaging the percentage of methylation of four probes assessed by routine laboratory methods (i.e., Methylation-Specific Multi- plex-Ligation70 dependent Probe Amplification, MS-MLPA) resulted as an independent predictor for recurrence and death in ACC, together with the classic clinical parameters [37].
MicroRNAs (miRNome)
The total microRNA expression profile (miRNome) has also been shown to discriminate ACC from ACA. MicroRNAs (miRNAs) are small non-coding RNAs that play a pivotal role in the post-translational regulation of gene expression, by targeting specific mRNAs for cleavage or translational repression. A deregulated expression of miRNAs has been demonstrated to alter gene expression in several types of cancer (i.e., activating oncogenes or silencing suppres- sor tumor genes), thus providing new putative biomarkers for cancer diagnosis and prognosis. MiRNome analysis revealed that specific miRNA expression can be a useful pathogenetic and diagnostic marker also in ACC [38-40]. Globally, several miRNAs are differentially expressed in ACCs compared to ACAs; the most consistent signature is the over-expression of miR483-5p and miR483-3p and concomitant down-regulation of miR-195 [25, 41-43]. The combination of different altered miRNAs seems to corre- late with malignancy [25, 41, 44, 45]. Although the global impact of miRNA deregulation on ACC pathogenesis and
evolution has to be fully elucidated [38-40], miRNome anal- ysis stratifies patients in three different miRNA clusters of ACC associated with different tumor recurrence and overall survival [18]: Mi1, Mi2, and Mi3 (Fig. 1a). Furthermore, the assessment of the circulating levels of specific miRNAs in ACC patients may provide novel non-invasive biomarkers of malignancy and recurrence [40, 43, 46].
Chromosomal alterations
Numerous quantitative alterations are often present in ACC genome compared to benign adenomas, with extended chro- mosomal gains, losses, and loss of heterozygosity (LOH) (see for rev [47]). Analysis of the three independent ACC cohorts has identified common profiles of quantitative chro- mosomal alterations (Copy Number Alterations, CNAs) [17-19]. In particular, specific amplifications have been observed in the chromosomal regions containing the TERT gene (5p15.33), encoding the inverse transcriptase of telom- erase, and the CDK4 gene (12q14). On the other hand, dele- tions are more commonly present in chromosome 22, in the region of the ZNRF3 gene (22q12.1), and in chromosomes 9 and 13, containing CDKN2A (9p21.3) and RB1 (13q14) genes, respectively [17, 18] (Fig. 1a). The presence of mutations in these genes has also been confirmed by exome sequencing analysis (see below). Interestingly, also the LOH profiles are consistent with the observed CNAs [18, 19].
One of these analyses of ACC [19] shows how LOH can be associated with a whole-genome doubling (WGD), which associates with tumor aggressiveness, thus suggesting that WGD may represent a marker of tumor progression.
Both Assie and colleagues and Zheng et al. identified, in their independent pan-genomic analyses, a concordant stratification of ACC according to the CNA pattern (defined as quiet, noisy, and chromosomal), which was proposed as a further interesting prognostic molecular marker [18, 19] as the noisy pattern was found associated with a significantly shorter event-free survival [19].
Mutations in driver genes
Advances in DNA sequencing and bioinformatic analysis techniques have allowed shedding new light onto the muta- tional landscape of ACC, resulting in identification of spe- cific driver genes. In particular, the development of next- generation sequencing (NGS) technique that allows rapid parallel sequencing of a panel of genes and of the Exome Sequencing, that enables through a specific enrichment to selectively sequence the all encoding DNA, has been applied to identify any genomic alterations in driver genes associ- ated with ACC. In 2014, the European ENSAT-CANCER
consortium reported the first ACC genomic profile based on the Exome sequencing and SNP-array analysis by comparing tumor somatic mutations and germline DNA profile [18]. Few months later, data were confirmed by the two other studies on independent ACC cohorts [17, 19], resulting in validation of a list of recurrent ACC driver genes.
Among these drivers, ZNRF3 is the most common altered gene (maximal percentage of mutations among the three studies: 21%), inactivated by homologous deletion or inactivating mutations. It encodes an E3 ubiquitin ligase that negatively regulates the Wnt/beta-catenin pathway: the presence of inactivating mutations/gene loss specifically triggers this signaling pathway, which is also constitutively activated as the result of activating mutations of the beta- catenin encoding gene, CTNNB1 (16%). These two types of mutations are found to be mutually exclusive in all ACC series [17-19] (Fig. 1a, b).
Other recurrently mutated genes are those related to the cell cycle regulation, such as TP53 (21%), the tumor sup- pressor genes CDKN2A (11%) and RB1 (7%), and onco- genes such as MDM2 (5%) and CDK4 (2%). Even at lower frequency, other mutations are found in genes involved in chromatin remodeling (MEN1, DAXX, and ATRX, all below 7%) and chromatin maintenance (TERT, 7%, and TERF2) [17-19] (Fig. 1a, b). Some somatic mutations in genes involved in PKA activation, such as the PKA regula- tory subunit PRKAR1A (8%), which is typically present as germline inactivating mutations in the Carney’s Syndrome, as well as germline or somatic mutations in some adreno- cortical adenomas have also been identified in ACC [19].
The tumor liquid biopsy: a new non-invasive tool for diagnosis/prognosis and monitoring the disease progression
The tumor gene expression profile can be traced by means of the “liquid biopsy” through assessing the presence in the bloodstream of specific molecular markers derived from the tumor [48-50]. The liquid biopsy is a novel and minimally invasive technique established in other tumors but now applied also to ACC [43, 46, 51-54] which enables by a simple blood draw to obtain and analyze material derived from tumor and released in the bloodstream. According to the method of extraction from the blood, circulating tumor cells (CTCs), miRNAs, exosomes, as well as cell-free DNA can be analyzed [55-57]. Among cell-free DNA (cfDNA) species found in the bloodstream, circulating tumor DNA (ctDNA) is released from the lesion. This marker is repre- sentative of the tumor genomic profile and although often correlated with CTCs gives different information from the DNA that can be extracted from CTCs, since the lat- ter is associated with the progression and the potentially
metastatic profile of the tumor cells detached from the lesion [55]. Thus, the liquid biopsy represents a solid and non- invasive tool to not only characterize the genomic profile of the tumor but to also monitor the tumor evolution and iden- tify drug-sensitivity/resistance-associated markers, finally guiding therapy towards a real-time personalized anti-cancer approach [48-50, 55-57].
Potentially, the liquid biopsy tool may also provide rel- evant information for ACC. Ongoing studies within the ENSAT consortium are exploring the potential relevance of the liquid biopsy for ACC diagnosis and prognosis.
Isolation and characterization of circulating tumor cells (CTCs) in the peripheral blood of cancer patients could provide precious information about the tumor evolution and progression [48, 49]. CTCs originate from subclones of the primary tumor cells, harboring mutations that favor the cell entrance in the bloodstream and organ colonization, and sustain the metastatic process. Our group demonstrated the detection of CTCs in a pilot study performed on blood samples obtained from a small cohort of ACC patients [51]. The number of CTCs found in ACC is also significantly correlated with the absolute levels of another circulating marker associated with aggressive ACC, miR483-5p [52]. A recent study reported mutations in cfDNA in a patient with ACC, showing variability in the fraction of ctDNA in cfDNA between the patients analyzed [54].
The development of techniques of analysis of the DNA obtained from CTCs to be compared with ctDNA in ACC would add pivotal molecular information on tumor progres- sion, similar to what already obtained in monitoring other solid tumors [55].
Transferability of the molecular information derived from the genomic analysis of ACC to develop personalized treatment approaches
So far, chemotherapy with mitotane remains the goal stand- ard for the treatment of advanced/metastatic ACC. The First International Randomized (FIRM-ACT) Trial in metastatic ACC reported that the association of mitotane with the chemotherapeutic agents’ etoposide, doxorubicin, and cis- platin (EDPM) is superior to mitotane plus streptozotocin in extending progression-free survivor [58], but with serious adverse events in more than 50% of patients. Thus, novel markers characterizing differential ACC evolution derived from the analysis of the molecular profile of ACC are urgently required for their potential to represent novel targets to develop more effective and specific therapies for ACC.
The -omics exploration of the ACC genomic landscape reveals a significant heterogeneity in the tumor profiles also highlighting novel molecular markers able to identify sub- groups with different prognosis [18, 19, 23]. These markers
could be useful targets to be translated into new precision medicine therapies [20, 47, 59-63]. Indeed, in 59% of the 29 ACC patients with metastatic or locally advanced adrenocor- tical carcinoma, at least one genomic alteration was found to be potentially associated with the currently available specific therapeutic options [61]. A similar percentage of 69% was found in the 91 ACC patients’ cohort of Zheng et al. study, with 51 potentially actionable alterations in 22 ACCs [19], suggesting that molecular profiling of each ACC may pro- vide useful information to drive precision therapy.
The recent update of the completed and ongoing clinical trials in advanced ACC testing agents active on the spe- cific molecular targets emerged from -omics ACC analysis reports a disappointing scenario [59, 60, 62, 63]. So far,
therapies utilizing tyrosine kinase inhibitors targeting epi- dermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR), as well as multikinase inhibitors (sorafenib and sunitinib) or monoclonal antibod- ies (bevacizumab) to target vascular endothelial growth factor receptor (VEGFR), resulted in minimal effects when used in advanced ACC. Similarly, interfering with the IGF2/mTOR pathway gave disappointing results. However, some of these frustrating results may be affected by the concomitant use of mitotane, which is a potent inducer of drug metabolism, resulting in decreased bioavailability of the drugs [64]. In this scenario, the new molecular targets identified in pan-genomic analysis of the large cohorts of ACCs could be assessed (beta-catenin/Wnt, ZNRF3, TP53,
Adrenocortical carcinoma
DIAGNOSIS
WEISS SCORE
け
steroid secretion
PROLIFERATION
Ki67%
TUMOR STAGE
IV
III
II
I
PROGNOSIS
Poor
Intermediate
Better
CHROMOSOMAL ALTERATIONS
MUTATIONS
ZNRF3, CTNBB1, TP53, RB1, CDKN2A, MEN1, DAXX, ATRX, TERT, TERF2 RPL22, PRKAR1A
GENOMICS
Transcriptome
C1A
C1B
miRNome
Mi3
Mi2
Mi1
Metilome
CIMP-High
CIMP-low / Non-CIMP
IGF2
SIGNALING PATHWAYS
p53, RB
Wnt/beta catenin
Personalized therapy
RB1, PRKAR1A, and gene methylation) as more selective actionable targets for ACC [61]. Molecular profiling of ACCs would also be pivotal to select those ACCs potentially best-responders to the molecular target-therapy, opening up a promising application of personalized medicine for ACC. Some interesting drugs interfering with the Wnt/beta-catenin pathway are currently under study in solid tumors for their potential antitumor effects [65], together with agents able to selectively reactivate impaired TP53 pathway [66]. Assess- ment of their potential efficacy in selected ACCs will repre- sent a challenging future perspective.
Conclusion
The use of “genomics” to study adrenocortical cancer has shed new light on the comprehension of the molecular pro- cesses underlying the pathogenesis of this disease. Notably, a new molecular classification of the malignant tumors has been defined, thanks to the integrated genomic analyses performed on large cohorts of ACC patients. On the basis of these molecular features, it is now possible to stratify ACC patients in three prognostic subgroups with different expected outcomes (Fig. 1c, d).
The genomic approach allows obtaining ACC gene expression profiles, as well as the miRNA expression pro- file, the methylation status, and the subset of chromosomal alterations; moreover, a list of the most recurrently altered driver genes is now available for ACC. By integrating all these profiles, we have now obtained a reliable molecular classification of ACC with a potential prognostic value. The challenge is now represented by understanding how this complex information can be integrated with the clas- sic clinical and pathological parameters, utilized so far, to improve the diagnosis, prognosis, and global management of ACC patients (Fig. 2).
Classifying such a heterogeneous tumor on the basis of specific molecular features opens new perspectives not only for a better diagnosis and prognosis of ACC, but also for the development of novel pharmacological strategies, which can be specifically designed for each single patient, in the light of the new frontier of personalized medicine.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Funding The research leading to these results received funding from the Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 259735 ENS@T-Cancer, and was supported by Associazione Italiana Ricerca sul Cancro (AIRC) Investigator grant to M.L. (Grant # IG2015-17691). All authors are members of the ENS@T (European Network for the Study of Adrenal Tumors).
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
Informed Consent No informed consent.
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