@ 2018 EDIZIONI MINERVA MEDICA Online version at http://www.minervamedica.it REVIEW Rossella LIBÉ ABSTRACT Introduction A drenocortical carcinoma (ACC) originates from the adrenal cortex and as such is typi- cally defined by positive immunostaining for steroidogenic factor 1 (SF1), melanA (Mart1) markers but without staining for cytokeratins and chromogranin A.1-4 Malignancy is ascertained by the presence of local or distant spread. In case of local presentation, the diagnosis of malignancy is based on pathology findings, in particular a Weiss Score >3.3, 4 58 MINERVA ENDOCRINOLOGICA to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher. or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically KEY WORDS: Adrenocortical carcinoma - Mortality - Survival rate - Prognosis.

Minerva Endocrinologica 2019 March;44(1):58-69 DOI: 10.23736/S0391-1977.18.02900-0

UNILATERAL NON-ALDOSTERONE-PRODUCING ADRENOCORTICAL TUMORS

Clinical and molecular prognostic factors in adrenocortical carcinoma

French Network for Adrenal Cancer, Department of Endocrinology, Cochin Hospital, Paris, France *Corresponding author: Rossella Libé, Department of Endocrinology, Cochin Hospital, 75014 Paris, France. E-mail: rossella.libe@aphp.fr

INTRODUCTION: Adrenocortical carcinoma (ACC) is a rare cancer, with an incidence less than 0.7-1.5 per 1 million people per year, with a poor prognosis. The overall survival (OS) depends on the ENSAT stage: in particular in metastatic ACC the OS varies from 10 to 20 months, with a 5-year survival around 10%. ACC has a different behavior, probably due to a different biology. For this reason, a careful prognostic classification is mandatory, in order to stratify the patients and propose a specific management.

EVIDENCE ACQUISITION: Prognostic factors can be divides in three groups: clinical factors (tumor stage, age, hor- mone-related symptoms), pathological factors (Weiss Score, mitotic count, Ki-67, SF-1 and AVA2, P53, beta-catenin immunohistochemistry, resection status), molecular factors (chromosomal aberrations, methylation profile, altered gene expression and miRNA expression, gene mutations).

EVIDENCE SYNTHESIS: The best way to stratify ACC patients and propose the best therapeutic option is to combine clinical, pathological and molecular factors.

CONCLUSIONS: Individualizing patients’ prognosis and tumor biology appears as a necessary step for personalized medicine. In addition to tumor stage and tumor grade, the genomic classification may precise the risk stratification and thus help defining therapeutic strategy.

(Cite this article as: Libé R. Clinical and molecular prognostic factors in adrenocortical carcinoma. Minerva Endocrinol 2019;44:58-69. DOI: 10.23736/S0391-1977.18.02900-0)

The incidence of ACC is less than 0.7-1.5 per 1 million people per year.5-7 At the time of diagnosis, ACC must be precisely characterized according to standardized criteria as defined by ENSAT and ESMO recommendations.4 In particular, an accu- rate hormonal and radiological work-up, including thoracic-abdominal CT scan, 18F-FDG PET, are rec- ommended in order to obtain a careful staging of the disease. In 2009, ENSAT recommended a new stag- ing, the ENSAT stage, which is defined as follow: stage I: < 5 cm; stage II: >5 cm; stage III: locore- gional; stage IV: presence of metastasis (Table I).8

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

TABLE I .- TNM classifications in patients with ACC and proposals for new classifications.
Stage	UICC	ENSAT	mENSAT	mENSAT+GRAS **
I	T1 (<5 cm) NO, MO	T1 (<5 cm) NO, MO	-	T1-2, favorable GRAS
II	T2 (>5 cm) NO, MO	T2 (>5 cm) NO, MO	-	II-A: T1-2, unfavorable GRAS II-B: T1-2, pejorative GRAS
III	T3 N0 or N1	T3-T4 or, any T-N1, MO	T3, or T4, NO, MO	III-A: mENSAT stage III and favorable GRAS III-B: mENSAT stage III and unfavorable GRAS III-C: mENSAT stage III and pejorative GRAS
IV	T3N1 or T4 or M1	M1	Anu T-N1, M1 IV-A: 2 tumor organs* IV-B: 3 tumor organs IV-C: >3 tumor organs	IV-A: mENSAT stage IV-A or B and favorable GRAS IV-B: mENSAT stage IV-A or B and unfavorable GRAS IV-C: mENSAT stage IV-C or mENSAT IV-A or B and pejorative GRAS

M1: presence of distant metastasis; N1: positive lymph nodes; T1: 5 cm; T2: < 5 cm; T3: infiltration of surrounding tissue; T4: invasion of adjacent organs or renal vein/vena cava.

*Tumor organ counts include the primary and lymph nodes if not resected; ** GRAS parameters are considered favorable if grading defined by Ki-67 is <20%, primary RO resection status performed, age <50 years and absence of symptoms at diagnosis. GRAS parameters are classified unfavorable in case of age >50 years, or presence of symptoms at diagnosis. GRAS parameters are classified as pejorative in case of grading as defined by Ki-67 >20% and/or primary R1-2 resection status.

Based on thoracic and abdomen CT scan, ACC are classified ENSAT stage I, II, III, or IV in 5-6%, 33-50%, 10-26%, or 21-35% of cases, respectively.7-10

ACC resection is the only curative treat- ment.11, 12 As stated in recommendations13-15 surgery should respect the rules of oncologic surgery, be performed within expert centers by expert surgeons and through laparotomy.

In patients with stage III/IV disease medical treatment is recommended. The mitotane is a com- pound which combine antitumor and antisecretory effects in order to reduce steroid production by tu- mor cells.16-18 Partial responses were reported in 13% to 33% of cases with response duration of 2 to 190 months. In the absence of placebo-con- trolled randomized trials, the real benefit of mito- tane therapy on OS remains unknown. Recently, in a multicenter cohort study of three German referral centers, the objective response rate was slightly lower than previously reported (20.5%). However, over 20% of patients experienced long- term disease control at >1 year. The authors sug- gest that patients with late diagnosis of advanced disease and low tumor burden might especially benefit from mitotane monotherapy, whereas pa- tients with early advanced disease and high tumor burden are probably better candidates for com- bined therapy of mitotane and cytotoxic drugs. 19

Interventional radiology techniques may of- fer an alternative approach to surgery to control tumor growth but also to improve the secretory status in the palliative setting. They are recom- mended in combination with mitotane therapy in advanced ACC patients with favorable prognos- tic factors.4

In metastatic disease, the first line of che- motherapy is represented by the combination of cisplatin, etoposide and doxorubicin (EDP) plus mitotane, as described in the first phase III trial, the FIRM ACT trial, with a response rate of 23.2% and a median progression-free survival of 5.0 months.20 As second line, a gemcitabine- based chemotherapy was considered with a par- tial response or stable disease achieved in 4.9% and 25.0% of cases, respectively, and a median PFS of 12 weeks.21

The median overall survival (OS) of metastatic ACC patients varies between 10 and 20 months, with a 5-year survival around 10%.22 However, some long survivors in metastatic ACC have been described, suggesting the heterogeneity of the disease.23

These observations suggest that, even at meta- static stage, ACC present a different behavior, probably due to a different biology. For these reasons, a careful prognostic classification is mandatory, in order to stratify the patients and

to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove,

or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

purpose a specific management. This article pro- vides a review of the main clinical and molecular prognostic factors in ACC.

Evidence acquisition

Clinical prognostic factors

Tumor stage

Historically, presence of metastases and tumor resection constituted the keystones of the prog- nostic stratification.5, 24 More recently the UICC and European Network for the Study of Adre- nal Tumors (ENSAT) established the first TNM stage classifications but also the resection status was implemented allowing a refined prognostica- tion.8, 11, 12, 25, 26 Within the TNM staging system, ENSAT classification has been found to more accurately predict the outcome of ACC patients but recent studies suggest that the N status or se- vere vena cava invasion may behave like stage IV ACC patients suggesting that refinements in the stratification of TNM are still needed.11, 27-29 Also, the relevance of stage I or II ENSAT sub- categories is debated.8, 10, 12, 30-33 In ACC patients with advanced stage III-IV, another recent study of the ENSAT network, which analyzed of 444 advanced stage III-IV ACC patients, found that the stage as redefined by a new ENSAT classi- fication (mENSAT) in which the presence of N positive moves from stage III to IV and that takes into account the number of tumor organs, has a major prognostic role.27 Table I summarizes the evolution of TNM classifications in ACC pa- tients and new proposals based of recent studies.

Age

ACC occurs at any age, with two peak incidenc- es: the first one in the first decade and the sec- ond one between 40-50 years. ACC in children displays a better prognosis than in adults.34, 35 Younger patients presented lower tumor stages, whereas adults were more likely to show aggres- sive tumors with shorter prognosis. After com- plete tumor resection, 5-year DFS was above 80% for children under 3 years but only 40% for children over 13 years, similar to that of adults.35 However, pathology and molecular biology sug-

gest that pediatric ACC are different tumors than adults ACC.36 In adult several studies demon- strated that older age is associated with a de- creased OS.9, 11, 27, 30-32, 36 A novel staging system have been proposed: stage I and II ACC were dif- ferentiated by age (≤55 years for stage I versus >55 years for stage II) and not by size, like it is done in the ENSAT-staging system,12 with a significant difference in OS between stage I and stage II.

On the other hand, some studies did not found age as a predictor of recurrence30, 32, 33 or can- cer-specific mortality.26 Probably, this is due to a possible other confusing factor, as comorbid- ity which seems to be associated with a poorer outcome.37

Hormonal hypersecretion (glucocorticoids, an- drogens and mineralocorticoids) is present in about 40-60% of patients with ACC. In particu- lar, cortisol secretion was considered as a nega- tive prognostic factor in different series. In a large single institution French series including 202 patients with different disease stages, cortisol excess was found to be an independent prognos- tic factor for OS.36 Similar results were obtained from a series of 72 Italian patients submitted to chemotherapy with etoposide, doxorubicin, and cisplatin plus mitotane.38 More recently, a large cohort of 524 patients with ACC followed at re- ferral centers for ACC (Europe and USA) was studied and, in ACC patients after resection, the prognostic significance of cortisol excess was highly significant for both RFS and OS after ad- justment for sex, age, tumor stage, and mitotane treatment.32 On the contrary, in other series, cor- tisol secretion failed to demonstrate prognostic value in localized33 or metastatic ACC.39

Pathological prognostic factors

Weiss Score and mitotic count

Weiss Score is the reference method to diagnose ACC. It uses nine items, each being a type of his- topathological alteration; they include 3 cytolog- ical items (high nuclear grade, high mitotic rate, atypical mitoses), 3 structural items (low per- centage of clear cells, necrosis, diffuse architec-

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

Figure 1 .- Kaplan-Meier curves of Ki-67 LI on recurrence-free survival (A) and overall survival (B) in a German cohort of ACC (from Beuchlein et al.).33

100

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48 60 72 84 96 108 120 132 144 156 168 1

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ture), and 3 items related to invasion (invasion of sinusoidal structures, invasion of venous struc- tures, invasion of capsule). A score of 1 is given for each item present and the final score is the sum of items present; a final score >3 indicates malignancy. The mitotic count is the most im- portant item associated with prognostic value. A cut-off of 20 mitotic figures per 50 HPF was pro- posed to separate low from high grade ACC.39-41 Some limitations exist for using mitotic count as a prognostic factor in clinical routine: it is nota- bly time consuming and subject to inter-observer variability, although inter-observer variability may decrease with trained pathologists.42

Ki-67 labeling index

Ki-67 is a proliferation index, frequently used in pathology and, according to recent data gen- erated by ENSAT network, constitute with the resection status in both localized and advanced ACCs the most relevant prognostic parameters. In accordance, Duregon et al. demonstrated that the Ki-67 labeling index (LI) is the most power- ful tool in terms of prognostic stratification.43 In addition to its emerging value as a critical deter- minant of prognosis, Ki-67 LI has been recently integrated in treatment flow charts for ACC pa- tients suffering from tumors either amenable to radical resection or at advanced presentation.27, 33

Accordingly, thresholds of 10%, 20%, and 30% seem to be crucial in therapeutic deci- sions. In particular in localized ACC, Ki-67 was found as the most powerful prognostic factor of DFS after surgery, and an independent prognos-

tic factor of OS33 (Figure 1).33 In recent series, evaluating both mitotic count and Ki-67, the Ki-67 proliferation index proved to be the most powerful tool to predict patient’s survival.33, 43 In advanced diseases (stages III and IV), Ki-67 index was not consistently associated with OS.27 Some limitation of the Ki-67 LI has been dem- onstrated. First, immunohistochemistry proto- cols in terms of time to fixation, type of fixative, are not standardized. Second, different type of Ki-67 antibody are used: up to now the mouse anti-human Ki-67 monoclonal antibody, MIB1 clone, is considered the gold standard. Third, current practices in Ki-67 scoring assessment vary greatly, and interobserver variation sets particular limitations to the clinical utility of Ki- 67 LI, especially around clinically relevant cut- off values, in ACC.44

Recently, it has been introduced the Helsinki Score which proposed diagnostic and prognos- tic system based on the combined evaluation of morphological (mitoses and necrosis) and im- munohistochemical (Ki-67) parameters. For ma- lignant tumors, a score >17 was associated to a poor outcome45 and it have been validated in an independent cohort.46

SF-1 and VAV2

The transcription factor steroidogenic factor-1 (SF-1) has a pivotal role in regulating adrenocor- tical cell proliferation and differentiation.47 In a large cohort of ACC, SF-1 protein staining was detectable in 98% of evaluable ACC samples including 30% with strong SF-1 staining. More-

over, strong SF-1 protein expression significant- ly correlated with poor clinical outcome.2

SF-1 overexpression is associated with adre- nocortical tumorigenesis through regulation of a specific set of SF-1 dosage-dependent target genes.48, 49 One of these genes encodes VAV2, a guanine nucleotide exchange factor for small GTPases of the Rho family. Recently it has been shown that VAV2 overexpression induced by an increased SF-1 dosage in ACC is an essential factor driving tumor cell invasion.50 Sbiera et al. reported that VAV2 expression in the tumor was strongly correlated to both PFS and OS. In particular, patients with strong VAV2 expression had a 2.8-fold higher risk of experiencing a re- currence and 1.6-fold increased risk of dying. Moreover, they showed that combined assess- ment of VAV2 expression and Ki-67 improved patient stratification to low-risk and high-risk groups.51

P53 and beta-catenin immunohistochemistry

Targeted genetic analyses, such as sequencing and single-strand confirmation analyses have identified somatic genetic changes in TP53 or CTNNB1. The associated immunohistochem- istry studies for these two genes allowed to use as surrogates for molecular alterations in the two major signaling pathways of ACC.52, 53 Somatic mutations or loss of heterozygosity of TP53 result in aberrant P53 expression and are linked with aggressive phenotype,52, 54, 55 with higher tumor stage and poorer DFS. Activation of the Wnt/CTNNB1 pathway is associated with CTNNB1 nuclear staining and correlates with high mitotic rate56, 57 and poor survival.53, 54, 56, 57 However, these markers did not show indepen- dent prognostic value in multivariate analyses including tumor grade.57

Resection status “R”

In localized ACC, surgery is the single most important intervention and the complete resec- tion (RO) correlates with a better prognosis.11 In fact, an incomplete microscopic resection (R1), an incomplete macroscopic resection (R2) or unknown resection (Rx) are associated with the worst overall survival of 20% and 15%, respec- tively.11

Figure 2 .- Overall survival according to mENSAT in 444 advanced ACC patients (from Libé et al.).27

1.0

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Combination of clinical and pathological pa- rameters

GRAS

In order to better stratify ACC patients, a combi- nation of mENSAT stage with four other param- eters label GRAS have been recently studies in advanced ACC.27 Indeed, after adjustment for a new mENSAT TNM classifications, GRAS pa- rameters, as defined by Grade (Weiss Score be- low or above 6 or Ki-67 below or above 20%), Resection status of the primary, Age below or above 50 years and, absence or presence of tu- mor- or hormone related Symptoms at diagno- sis were also found prognostic on OS. Interest- ingly, the similar parameters were previously found to play a major role to predict recurrence in localized ACC patients. Based on these re- sults, a risk stratification of the management of ACC stage III-IV patients in the palliative set- ting can be envisioned based on mENSAT new TNM staging and GRAS parameters as shown in Figure 2.27

Molecular classification

The development of the genomics in the last years allowed to study chromosomal aberration, gene expression, genetic and epigenetic altera- tion in ACC in order to better understand the heterogeneity in ACC biology and provide new classification and stratification in clinical out- come.58

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

LIBÉ

Chromosomal aberrations

Comparative genomic hybridization (CGH) can identify structural chromosomal abnormalities at a higher resolution within ACCs. A number of studies have shown that whereas ACAs have few regions of chromosomal losses and gains, ACCs presented complex chromosomal alterations. In ACCs, chromosomal gains were frequently observed in regions 4q, 4p16, 5p15, 5q12-13, 5q32-qter, 9q34, 12q13, 12q24, and 19p, and chromosomal losses were observed at 1p, 2q, 11q 17p, 22p, and 22q.59 A follow-up study ex- amining 35 adrenal tumors and six adrenocorti- cal hyperplasias identified unique events within 12 of 12 ACCs compared with 15 of 23 ACAs. Specific events in ACC were gains at 5q12-13, 5q22-ter, 9q32-qter, 12q13-14, 12q24, and 20q and losses at 1p21-31, 3p, 2q, 3q, 6q, 9p, and 11q14-qter.60 A confirmatory study of 25 adreno- cortical tumor samples, including 14 ACCs and 8 ACAs as well as NCI-H295 and SW13 cell lines revealed similar gains in chromosomes 5 and 12 with additional gains in chromosomes 7 and 16 in ACC.61 Moreover, a study using higher-res- olution CGH arrays revisited this phenomenon through examination of 138 adrenal neoplasms encompassing 86 ACAs and 52 ACCs to assess the diagnostic and prognostic value of chromo- somal abnormalities.62 The study confirmed in- creased alterations in ACCs (44%) compared with ACAs (10%). Although survival prediction using these data could not be established, a sepa- rate CGH study that identified a similar increase in copy number in chromosomes 5, 6q, 7, 8q, 12, 16q, and 20 and allelic losses in 1, 2q, 3, 6p, 7p, 8p, 9, 10, 11, 13q, 14q, 15q, 16, 17, 19q, and 22q determined that some of these alterations (gains in 6q, 7q, and 12q and losses in chromosomes 3, 8 10p, 16q, 17q, and 19q) were associated with decreased overall survival.63 In a recent cancer project of The Cancer Genome Atlas (TCGA), clustering of 89 tumors based on their arm-level alterations produced three groups with striking differences: chromosomal, noisy, and quiet. The “chromosomal” group showed the highest fre- quency of whole chromosome arm gains and losses. The “noisy” group was characterized by a significantly higher number of chromosomal

breaks as well as frequent loss of 1p with 1q in- tact. Tumors in the “quiet group” had few large copy number alterations. Kaplan-Meier analysis demonstrated a significant decrease in survival in the noisy group relative to the chromosomal and quiet subtypes, suggesting that this copy number phenotype is characteristic of aggressive disease. The correlation between the noisy group and its survival association were recovered in an inde- pendent cohort.64 These results are encouraging, but the prognostic values of these 3 groups based on chromosomal alteration needs to be evaluated after adjustment on other prognostic factors, as ENSAT stage and pathological grade.

Methylome

DNA methylation involves the addition of a methyl group to the cytosine pyrimidine ring or adenine purine ring, occurring typically at CpG dinucleotides. In a normal cell, it acts as a regu- latory mechanism for proper gene expression. However, in cancer, frequent dysregulation in this process is observed. A recent study of 51 ACCs and 84 ACAs revealed hypermethylation of promoters in ACCs with correlation to poor survival and identified H19, PLAGL1, GOS2, and NDRG2 as silenced genes.62 This observational study also provided insight into the possible role of methylation in ACC tumorigenesis, particular- ly in the 11p15 locus containing IGF2 and H19. Recently, a targeted prognostic marker measuring methylation of 4 genes (PAX5, PAX6, PYCARD, GSTP1) was developed using a commercial kit by methylation specific multiplex ligation-depen- dent probe amplification (MS-MLPA).65 A strong correlation between the methylation profile of these 4 genes and the DFS and OS was demon- strated, independently of ENSAT stage and Ki-67 proliferation index.65 Recently, a correlation was observed between relative telomere length and the gene expression of TERT. It was concluded that epigenetic alterations of the TERT promoter are frequent and associated with advanced dis- ease and poorer clinical outcome in ACC.66

Gene expression arrays

Global gene expression studies aim to identify biomarkers that could provide diagnostic and

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

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prognostic utility in addition to the classic his- tological analyses and hold the promise of new potential targets for therapy. An initial study identified elevated expression of genes involved in cell proliferation in ACCs, such as IGF2, com- pared with increased expression of steroidogenic genes in ACAs (steroidogenic cluster).67 More recently, 2 large studies have correlated expres- sion profiles in ACC with clinical outcome. Spe- cifically, Giordano et al.68 determined that ACCs with high histological grade exhibited marked overexpression of cell cycle and functional an- euploidy genes, which correlated with decreased overall survival. In another study, cluster analy- sis of ACCs again revealed two distinct groups with different genetic signatures and concomi- tant distinct clinical outcomes. ACCs with poor outcome were enriched for genes involved in cell cycle and proliferation, whereas ACCs in the bet- ter outcome group exhibited overexpression of genes involved in differentiation, metabolism, and intracellular transport. Expression levels of BUB1B and PINK1 alone identified subgroups of ACCs with different overall survival, regard- less of tumor stage: C1A (poor prognosis) and C1B (good prognosis). Similarly, the expression levels of DLG7 and PINK1 identified subgroups of ACCs with distinct disease-free survival, re- gardless of tumor grade69 and these findings were later validated in a separate cohort of adult patients.70 The two gene predictors BUB1B and PINK1 showed an excellent prognostic value- with 5 years OS <20% in the “poor prognosis” subgroup versus >80% in the “good prognosis” subgroup. Recently, the histone methyltransfer- ase EZH2 was found overexpressed in ACC. High EZH2 expression was also associated with proliferation and poor prognosis.71 The major limit of this study is that EZH2 expression was not confronted to other prognostic factors of ACC, such as tumor stage.

MicroRNAs

MicroRNAs (miRNAs) are evolutionarily con- served, small, noncoding, 18- to 25-nucleotide RNAs that are important in posttranscriptional regulation of gene expression. Mature miRNAs in association with the RNA-induced silencing complex are loaded onto the 3’-untranslated

region of the targeted mRNA to inhibit transla- tion or to cause degradation.72 Numerous miR- NAs have been identified and implicated in the regulation of various cellular processes such as proliferation, apoptosis, and differentiation. In addition, dysregulation of miRNAs, such as overexpression or deletion, plays an important role in diseases, including various cancers.73, 74 Mistargeting of the miRNAs, resulting in inhi- bition or activation of various oncogenes, tumor suppressors, and/or other factors important in tumor angiogenesis, epithelial-mesenchymal transition, and metastasis, have been identified.74 Several studies demonstrated a different expres- sion of miRNAs between normal adrenal, ACA and ACCs.75, 76 Interestingly, the mi RNA most frequently found as up-regulated are miR-483- 3p, miR-483-5p, miR-210 and miR-503, while miR-195 and miR-335 were down-regulated.75-80 Unsupervised classifications of ACC based on miR expression showed different subgroups of ACC, which are associated with different out- comes.75, 78 MiRNA clusters were correlated with transcriptome subgroups, with rather miR- NA overexpression in the transcriptome ‘CIA’ cluster. Several studies have proposed RT-qPCR prognostic markers based on miRNA expression, including miR-195 and miR-483-5p,75 miR- 503,78 and miR-210.81 However, the prognostic value of selected miRNA was either not tested after adjustment on other prognostic factors or dropped in multivariate analysis for miR-210.81 Recently, Agosta et al. examined the involve- ment of miR-483-5p and miR-139-5p in adreno- cortical cancer aggressiveness. Using bioinfor- matics predictions and mRNA/miRNA expres- sion profiles, they identified N-myc downstream- regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR- 139-5p, respectively. Moreover, they showed miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with poten- tial implications for prognosis and therapeutic interventions in adrenocortical malignancies.82

Gene mutations

Targeted genetic analyses, such as sequencing and single-strand confirmation analyses have identified somatic genetic changes in TP53,

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

MEN1, IGF2, IGF2R, RB1 and p16/INK4A (CDKN2A).16, 83, 84 Recently, three pangenomic studies have investigated instead exome se- quencing and SNP arrays to identify somatic mutations, homozygous deletions and high-level amplifications in adult’s ACC.64, 85, 86 Recurrent gene alterations have been observed in about 50% of ACC: in particular, the most frequent al- terations were CTNNB1 and TP53 mutations and ZNRF3 and CDKN2A homozygous deletions.

About 20% of ACC harbored gain-of-function mutations in CTNNB1.64, 85, 86 Thus, CTNNB1 mutations correlated with nuclear CTNNB1 staining.55, 56 Moreover, CTNNB1 mutations were associated with higher mitotic count,56 poor prognosis transcriptome cluster54, 85 and poor survival.55, 56 However, the prognostic value of CTNNB1 was not tested after adjustment on tu- mor grade.

Loss-of-function mutations of TP53 occurred in about 20% of adult’s ACC,64, 85 often associ- ated with loss of heterozygosity (LOH) of the 17p13 region, on which TP53 is located.52 TP53 mutations were almost mutually exclusive from CTTNB1 mutations.54, 64, 86 TP53-mutated tu- mors were associated with aberrant P53 stain- ing,52, 55 advanced stage,52 poor prognosis tran- scriptome cluster21, 55 and shorter DFS.52 No sig- nificant association with OS was reported.52, 55, 86

Another 20% presented ZNRF3 deletions64, 85 -a negative regulator of Wnt/CTNNB1 -which results in the activation of the Wnt/ CTNNB1 pathway. A trend toward OS was also noted for ZNFR3 deletions.86

Zheng et al. reported two MSH6 and one MSH2 mutation.64 The MSH6 and MSH2 muta- tions support recent observations that ACC is a Lynch syndrome-associated cancer.87

Combination of molecular studies toward to a mo- lecular classification

The integration of the different molecular stud- ies, as the transcriptome, the whole genome sequencing, the methylome and miRNAome helped to generate a global genomics classifica- tion, in order to better stratify ACC patients with different outcome.64, 85 In the first study, the mo- lecular classification was based on the two tran- scriptome signatures which led to distinguish

two group, one with a “good prognosis” (C1B) and the second with “bad prognosis” (CIA). The first group showed a low mutation rate, very rare mutation in ACC driver genes and no hypermeth- ylation. The second one harbored high mutation rate of alterations in ACC driver genes and, in a subset of ACC, an hypermethylated profile cor- related with a poorer prognosis.85 In the second study, the integrated genomics allowed to de- fine three groups of ACC: the first with a “good prognosis” was characterized by transcriptome C1B profile, “chromosomal” SNP profile and no methylation. The second and the third group are more aggressive ACC. In particular the second presented intermediate level of hypermethylation and “chromosomal” SNP profile and was asso- ciated with a intermediate outcome. The third group showed high levels of hypermethylation, noisy SNP profile and poorer prognosis. Disease progression rates of the three groups were 7%, 56%, and 96%, respectively. Survival analysis showed a dismal median event-free survival of eight months for the third group.64

The next step will be to integrate both molecu- lar classification and clinical prognostic factors in the clinical practice in order to offer the better therapeutic options to ACC patients.

Evidence synthesis

Adrenocortical carcinoma is a rare endocrine cancer with limited therapeutic options and over- all poor outcome. It depends on the initial stage, but at metastatic stage the overall survival is about 10% at 5 years. Recently, long-term sur- vivors in metastatic ACC patients have been de- scribed, including a few cases with synchronous metastases at the time of diagnosis. These obser- vations suggest that even metastatic ACC present a different tumor biology and incite to a careful evaluation at diagnosis of ACC in order to iden- tify some possible prognostic factors. The stage, i.e. ENSAT stage, represent the most robust of clinical prognostic factor. Moreover, the tumor grade, in particular, the mitotic count and the Ki-67 are useful and simple tools, even if they suffer from inter-observer reproducibility. The new genomics techniques provide new insight in ACC biology, that lead to identify new genetic

PROGNOSTIC FACTORS IN ADRENOCORTICAL CARCINOMA

mechanisms associated to the ACC pathogen- esis, but also molecular prognostic factors. The main studies on ACC genomics clearly identify different subgroup of ACC patients. On the basis of this classification, a personalized management can be purposed. For example, for “chromosom- al” patients, who present with a very low rate of relapse, probably no adjuvant treatment is neces- sary, but a simple follow-up after complete re- section. On the other hand, “bad prognosis” ACC could benefit from an adjuvant systemic therapy. The main problem associated to the molecular classification, is related to their feasibility. Actu- ally, gene and miRNA expression can be assayed by targeted measurement using RT-qPCR. How- ever, additional studies are needed to precise the best set of molecular markers for prognosis and to confront their prognostic value to the already validated prognostic markers, especially ENSAT stage and Ki-67 index. Moreover, the reproduc- ibility of the discriminant molecular tools within a single tumor has also to be evaluated. Finally, the molecular markers will have to be simple and cost-effective for transfer to clinical routine. The use of immunostaining coupled with gene expression analysis in fresh frozen tissue could be a good, simple and cost-effective technique, as shown by Ronchi et al. which found an activa- tion of Notch1 signaling in ACC and, in particu- lar, a correlation between JAG1 overexpression and a better clinical outcome.88

The best way in order to stratify ACC pa- tients and purpose the best therapeutic option is to combine clinical, pathological and molecular factors. Recently, Papotti et al.89 showed that genomic signature in adrenocortical carcinoma differs among histological variants (i.e. onco- cytic, mixoid, conventional) and is associated to specific clinical and pathological characteristics. The p53/Rb pathway alterations are the most relevant prognostic molecular markers in adre- nocortical carcinoma and the genomic signature in ACC is highly unstable along tumor progres- sion. Very recently, Lippert et al. identified a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine. In- deed, they demonstrated that molecular profiling of formalin-fixed paraffin-embedded tumor sam-

ples improves prognostication of ACC beyond clinical/histopathological parameters and identi- fies new potential drug targets.90

Conclusions

In conclusion, several advances have been done in prognosis of ACC, mostly due to the develop- ment of genetics studies. Individualizing patients’ prognosis and tumor biology appears as a neces- sary step for personalized medicine. In addition to tumor stage and tumor grade, the genomic classification may precise the risk stratification and thus help defining therapeutic strategies.

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Conflicts of interest .- The author certifies that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Article first published online: September 12, 2018. - Manuscript accepted: September 7, 2018. - Manuscript received: July 31, 2018.

Quartz 4

Explorer

30221891

Clinical and molecular prognostic factors in adrenocortical carcinoma

Evidence acquisition

Clinical prognostic factors

Pathological prognostic factors

Weiss Score and mitotic count

Ki-67 labeling index

SF-1 and VAV2

P53 and beta-catenin immunohistochemistry

GRAS

Molecular classification

Chromosomal aberrations

Methylome

Gene expression arrays

MicroRNAs

Gene mutations

Evidence synthesis

Conclusions

References

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Table of Contents

Backlinks

Quartz 4

Explorer

30221891

Clinical and molecular prognostic factors in adrenocortical carcinoma

Evidence acquisition

Clinical prognostic factors

Hormone-related symptoms

Pathological prognostic factors

Weiss Score and mitotic count

Ki-67 labeling index

SF-1 and VAV2

P53 and beta-catenin immunohistochemistry

GRAS

Molecular classification

Chromosomal aberrations

Methylome

Gene expression arrays

MicroRNAs

Gene mutations

Evidence synthesis

Conclusions

References

Graph View

Table of Contents

Backlinks