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Detailed genomic characterization identifies high heterogeneity and histotype-specific genomic profiles in adrenocortical carcinomas
Simona Vatrano1 . Marco Volante1 . Eleonora Duregon1 . Jessica Giorcelli1 . Stefania Izzo1 . Ida Rapa1 . Arianna Votta1 . Antonina Germano1 . Giorgio Scagliotti1 . Alfredo Berruti2 . Massimo Terzolo3 . And Mauro Papotti4
Received: 21 December 2017 / Revised: 30 January 2018 / Accepted: 31 January 2018 @ United States & Canadian Academy of Pathology 2018
Abstract
Molecular characterization of adrenocortical carcinoma has been recently established, but the correlation between molecular profiles and clinical and pathological characteristics is still poorly defined with no data available about genetic heterogeneity along disease progression. In this scenario, a detailed molecular profile was correlated with clinical and pathological characteristics in adrenocortical carcinoma patients to identify potentially novel biomarkers. Targeted next-generation sequencing and copy number variation analyses for 18 most frequently altered genes in adrenocortical carcinoma were assessed on 62 adult cases (including 10 with matched primary and metastatic/recurrence samples) and results correlated with major clinical and pathological characteristics of tumors. A total of 433 somatic deleterious genetic alterations (328 gene mutations and 105 copy number variations) were identified in 57/62 cases, five resulted wild type for all genes tested. TERT, CDK4, ZNRF3,and RB1 were altered in more than 30% of cases. Among histological variants genotypes were significantly different. Lowest mutation burden was found in the oncocytic type (p=0.006), whereas the highest with a prevalence of RB1 (p = 0.001) and CDK4 (p=0.002) was found in the conventional and myxoid ones, respectively. None of the 10 cases with matched samples showed a stable genotype along tumor progression, although allelic frequencies or percentages of altered nuclei at fluorescence in situ hybridization were in most cases similar among different tumor samples for genes that were stable along tumor progression. Among individual genes, an altered p53/Rb1 pathway was the strongest adverse molecular signature, being associated with high Ki-67 index, high tumor stage, aggressive disease status, and shorter disease-free survival. The genomic signature in adrenocortical carcinoma is changing along tumor progression and is associated with specific clinical and pathological features, including histological variant and prognosis.
Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41379-018-0042-6) contains supplementary material, which is available to authorized users.
☒ Marco Volante marco.volante@unito.it
1 Department of Oncology, University of Turin at San Luigi Hospital, Orbassano, Turin, Italy
2 Oncology Unit, University of Brescia, Brescia, Italy
3 Internal Medicine, Department of Clinical and Biological Sciences, University of Turin at San Luigi Hospital, Orbassano, Turin, Italy
4 Department of Oncology, University of Turin at Molinette Hospital, Turin, Italy
Introduction
Despite better definition of the standard of care, adrenocortical carcinoma is a rare malignancy that has still a dismal prognosis, with a 20-40% 5-year survival. Adrenocortical carcinoma is not a uniform entity but is characterized by remarkable mor- phological (with several histological variants identified), clin- ical (with variable and poorly predictable outcome), and genetic heterogeneity. Pediatric adrenocortical carcinomas are frequently associated to Beckwith-Wiedemann syndrome (alterations of the insulin-like growth factor/IGF2) [1] and to Li-Fraumeni syndrome (TP53 mutations). TP53 germline mutations (p.R337H) are associated to pediatric adrenocortical carcinoma in Southern Brazil [2]. Recently, it has been recognized that in the adult population adrenocortical carci- noma can be associated with the Lynch syndrome [3]. Few driving somatic variants have been identified so far, and most of the available molecular data on adrenocortical carcinomas
are restricted to gene expression, array-CGH, or methylome profiles assessed to compare adrenocortical carcinoma with the benign counterpart of adrenocortical adenoma. Mutations in TP53 gene are detectable as somatic events in about one-third of adult cases [2]. Mutations in CTNNB1 gene, coding for beta- catenin protein, are detectable in a subset of adrenocortical carcinoma (about 40%) but also in a similar percentage of adrenocortical adenomas. The role of these genes in the pathogenesis and progression of adrenocortical carcinoma is still controversial [4]. Recently, highly prevalent mutations of ZNRF3 (Zinc and ring finger protein 3) gene have been reported in adrenocortical carcinoma [5]. However, none of the above mentioned genomic alterations demonstrated to be prognostic or predictive markers of response to treatment. Many other mutations have been reported by whole genome sequencing though at very low prevalence. This confirms the heterogeneity of the molecular background of adrenocortical carcinoma and underlines the need for adequate investigations to define biomarkers that could have a positive impact in the clinical setting.
On the basis of these findings, we performed a detailed genomic characterization on 62 adrenocortical carcinoma cases and correlated mutational profile results with clinical and pathological characteristics. Furthermore, in ten cases of matched primary and recurrent/metastatic tissues the molecular signatures were compared to evaluate molecular heterogeneity during progression.
Material and methods
Tissues samples
Tumor samples from adrenocortical carcinoma patients undergoing surgery with curative intent were collected from the pathology files at the University of Torino, San Luigi Hospital (Orbassano, Turin, Italy). A total of 62 were selected based on enough leftover tissue from primary tumors for molecular analyses. Pathological re-classification included the recognition of major histological variants, namely the oncocytic, myxoid, and sarcomatoid as coded by the current WHO classification [6], applying the pathological criteria described in the available literature [7-11]. To define malignancy, the Weiss score for conventional and myxoid and adrenocortical carcinoma the Lin-Weiss-Bisceglia system for pure oncocytic ones were applied [7, 12]. Furthermore, the Helsinki Score was evaluated, as it has been recently indicated as a relevant prognostic system in adrenocortical carcinoma [13, 14]. Main clinical and pathological data (including age, gender, European Network for the Study of Adrenal Tumors-ENSAT-stage, all para- meters included in the Weiss score-WS, Helsinki score, hormonal secretion, and Ki-67 proliferation index) were also recorded. Clinical data concerning outcome were available for
all but one case. In ten cases, primary tumors were matched with corresponding metachronous recurrent or metastatic (liver or lung) lesions (for a total of 85 tissue samples overall enrolled in the study). All samples were de-identified, and cases anon- ymized by a pathology staff member not involved in the study. The study received ethical approval from the local review board of our Institution (protocol 20840 dated 22nd December 2016).
DNA isolation and mutational analysis
Genomic DNA was obtained from formalin-fixed paraffin- embedded tissues after manual microdissection, for neo- plastic cell enrichment (at least 50% of tumor cells), as previously reported [15]. Targeted next-generation sequen- cing analyses were performed in all samples using the Ion Torrent Personal Genome Machine (PGM, ThermoFisher Scientific), and an AmpliSeq custom panel targeting the entire coding sequence of 15 genes previously reported as adrenocortical carcinoma related: TP53, MSH2, MSH6, MLH1, PMS2, ATRX, DAXX, CTNNB1, MED12, MEN1, CTNNB1, PTCH1, APC, PTEN, ZNRF3. Targeted primers were designed using the AmpliSeq designer software v4.0 (ThermoFisher Scientific), see Supplementary Table S1. Twenty-five nanograms of genomic DNA were used for each multiplex PCR amplification. Library quality was evaluated by Agilent 2100 Bioanalyzer on-chip electro- phoresis (Agilent Technologies) and libraries passing this step went in emulsion PCR performed with the OneTouch2 system (ThermoFisher Scientific). The libraries were loaded on a Ion 318chipv2 and then sequenced on the Ion PGM (ThermoFisher Scientific). Data analysis, includ- ing alignment to the reference genome and variant calling, was done using the Torrent Suite Software v.5.0 (Thermo- Fisher Scientific).
Bioinformatics pipeline and experimental validation
All genome coordinates are human genome build GRCh37/ hg19 and RefSeq sequences used for gene annotations are detailed in Supplemental Table 1. Genetic variants were annotated and filtered using a custom pipeline as previously reported [16]. To minimize the risk of calling alterations derived from sequencing errors or polymerase chain reac- tion (PCR)-based enrichment artifacts, all reads with a quality score <30 were excluded from further analyses. The discrimination of likely germline or likely somatic variants was performed on the basis of the following criteria: (i) known polymorphisms recorded in the public polymorph- isms databases were removed from the analysis [17]; (ii) a variant read frequency range of 5-50% for all candidate alterations different from well-characterized somatic known hotspots [18, 19]. The following databases were used to
(a)
(b)
(c)
annotate the genetic alterations: COSMIC (version 70), Single Nucleotide Polymorphism (dbSNPs, build 137), 1000 Genomes Project, 6500 exomes of the National Heart, Lung and Blood Institute Exome Sequencing Project, and Non-synonymous Single Nucleotide Polymorphisms’ Functional Predictions (dbNSFP, light version 1.3). In silico analyses were carried out to predict both the amino acid changes potentially deleterious to protein function and the clinical implications of genetic alterations not previously identified by means of sorting intolerant from tolerant and polyphen-2 (polymorphism phenotyping) algorithms. In addition, alignments and sequences were visually verified with the Integrative Genomics Viewer (IGV) v2.3 to further confirm the presence of the identified mutations [20]. Path Visio and Caleydo pathway-based analysis tools were used to cluster genetic alterations and copy number varia- tions, respectively, into pathway [21].
DNA copy number analysis
Fluorescence in situ hybridization analysis was performed on 5-um sections as previously reported [15]. Different probe sets were used based on genomic loci to analyze according to the manufacturer’s instructions. Three to five areas on each slide were analyzed to count at least 100-150 tumor cell nuclei. For RB1/CEP13 FISH (Zytolight, Zyto- vision), all cases with absence of one or both RB1 orange signal/s, but intact 13q12 green signals in more than 22% of nuclei, were reported as heterozygous or homozygous deleted, respectively [22]. For CDKN2A/CEP9 FISH (Zytolight, Zytovision), the nuclear probe patterns were normal if two green and two red signals were seen, whereas abnormal if 2Green/1Red as loss of heterozygosity (LOH),
2Green/0Red as homozygous deletion and 1Green/1Red as monosomy of chromosome 9 were seen. The cut-off value applied was 18% of altered nuclear signal for both 9p loss of heterozygosity and homozygous loss of 9p (2Green/ 1Red-2Green/0Red) [23]. For ZNRF3/CEP22 FISH (Empire genomics), given the absence of fluorescence in situ hybridization interpretation criteria of this genomic locus, we followed the international guidelines [24]. Cases with absence of one or both ZNRF3 orange signal/s, but intact CEP22 green signals in more than 25% of nuclei, were reported as heterozygous or homozygous deleted, respectively. Finally, for both CDK4/CEP12 and TERT/ 5q31 FISH (Zytolight, Zytovision), amplification was defined when the ratio of signal counts Locus/Centromere was >2, with a cut-off of 10% of positive nuclei.
Statistical analyses
The x test, Fisher’s exact test, and Mann-Whitney tests were used to analyze the association between mutations and clinical pathological variables or to evaluate concordance between different results, as appropriate. Disease-free sur- vival (DFS) was defined as the time between diagnosis and the first failure (either recurrence/metastasis or death). Univariate analysis was performed with Kaplan-Meier curves and the significance was verified by the log-rank test. Multivariate analysis was performed using a Cox propor- tional hazards model. Overall survival analysis was not performed due to the limited number of events. All analyses were performed using GraphPad software (Graphpad Soft- ware Inc., La Jolla, CA) and SPSSsoftware (IBM corpora- tion, Armonk, USA). A p value lower than 0.05 was considered statistically significant in all analyses.
| Features | |
| Total number | 62 |
| Median age, | 45 (18-82) |
| years (range) | |
| Sex no. (%) | |
| Male | 22 (35%) |
| Female | 40 (65%) |
| ENSAT stage no. (%) | |
| I-II | 31 (58%) |
| III-IV | 22 (42%) |
| Not known | 9 |
| Hormonal | |
| hypersecretion | |
| no. (%) | |
| Cortisol | 22 (37%) |
| producing | |
| 29 (49%) | |
| Nonfunctional | |
| Other | 8 (14%) |
| Not known | 3 |
| Histologic type | |
| no. (%) | |
| Conventional | 38 (62%) |
| Myxoid | 7 (11%) |
| Oncocytic | 17 (27%) |
| Weiss score | |
| distribution no. | |
| (%) | |
| <6 | 13 (21%) |
| ≥6 | 49 (79%) |
| Helsinki score distribution no. | |
| (%) | |
| <19 | 16 (26%) |
| ≥19 | 46 (74%) |
| Ki-67 indexª no. | |
| (%) | |
| ≤22% | 34 (55%) |
| >22% | 28 (45%) |
ENSAT European Network for the Study of Adrenal Tumors
a The selected cut-off corresponds to median value obtained in the present series.
Results
Study cohort
Adrenocortical carcinoma samples included 38 conven- tional, 7 myxoid, and 17 oncocytic variants (Fig. 1). No case of the sarcomatoid variant was recognized. Twenty- two were males and 40 females, with a median age at
diagnosis of 45 years (range 18-82). According to the ENSAT stage, 5 patients presented with stage I, 26 patients with stage II, 15 patients with stage III, and 7 patients with stage IV. After a mean follow up of 34 months, 18 died of disease (conventional = 13, myxoid = 3; oncocytic = 2). Thirty cases were hormone producing, 22 of which being cortisol-hypersecreting (Table 1). No significant differences were found between adrenocortical carcinoma variants, with the exception of age distribution (lower in conventional histotype; p = 0.02) and a more favorable outcome in the oncocytic histotype (p=0.05) (Supplementary Table S2).
Prevalence of somatic alterations by means of targeted next-generation sequencing and fluorescence in situ hybridization
The entire genomic profile (328 gene alterations and 105 somatic copy number variants) clusterized according to adrenocortical carcinoma histotypes is illustrated in Fig. 2a. Taken together, the results of DNA copy number alterations (105 somatic copy number variations) and targeted next- generation sequencing (328 genetic variations) analyses showed a high prevalence of somatic alterations, with 57/62 (92%) cases harboring at least one alteration among studied genes, whereas five cases (8%), which incidentally were all oncocytic adrenocortical carcinomas, did not harbor any somatic aberration. Somatic variants were clustered into four main pathways, among which the ß-catenin pathway harbored the highest number of alterations (40%), followed by p53/Rb (28%), mismatch repair (18%), and chromatin- remodeling (14%) pathways. Mutually exclusive alterations were observed in 13 cases (Fig. 2b).
All 62 samples were informative for targeted next- generation sequencing analysis, with adequate libraries of 690 amplicons each. According to bioinformatic pipeline, after read quality filtering, mapping, and alignment to the reference genome (hg19), the median sequencing depth was 896x and a total of 986 genetic variants (median 14; range: 1-44) were identified, either in exonic (284 synonymous and 338 non-synonymous, respectively, both likely germ- inal and likely somatic alterations) or regulatory regions (364 both likely germinal and likely somatic alterations). The nucleotide change spectrum was characterized by a predominance of cytosine: guanine → thymine:adenine transitions, as previously reported [25] and without sig- nificant difference among individual genes. All genetic variants which do not lead to a protein change (i.e., synonymous and regulative variants) or that were categor- ized as likely germline, based on the presence in public polymorphism databases or of their allelic frequency in the sample, were excluded from further analysis. Thus, we focused on 328 non-synonymous genetic alterations
(median 5; range: 1-22), more likely having a causative role in tumorigenesis (Supplementary Table S3), that included 265 missense and 44 nonsense variations, 6 frameshifts, and 6 in-frame InDels variations. Almost half of them (40%) were already reported in official mutational databases. The mean number of genetic alterations was 8 for conventional adrenocortical carcinomas, 7 for myxoid, and 2 for onco- cytic ones, respectively; 42/62 (68%) cases showed at least one alterations among sequenced genes, whereas 20 cases (32%) were wild type for somatic genetic variants in the targeted genes. One conventional case (ACC14) showed a hypermutated profile with high number (128) of inactivat- ing somatic variants mainly in APC, MED12, and MSH6 genes, as previously reported [25, 26]. This case was
excluded from all prevalence and statistical analyses of mutation burden, because of the large number of somatic alterations with skewing effect.
Among single genes, MED12, CTNNB1, and TP53 were the most commonly mutated (Supplementary Figure S1a). MED12 alterations were mainly missense and truncating mutations; four of these alterations were identified in more than one case, p.T1681I-p.P1824L-p.T2171L (in two patients), p.Q2115del (in three patients). Even though never reported before, they affected C-terminal region of the protein, known to be important for physically interact with ß-catenin. In silico analyses confirmed the pathogenic role of these variants. Furthermore, in six cases truncating mutations were detected with a premature stop codon
a
Mutation Legend
Copy Number Alterations Legend
b
Missense
0-1 copies
Nonsense
110
Total number of somatic alterations among cases
>2 copies (focal amplification)
Frameshift
10%
100
ÎnFrame
40
Splice Site
30
2
25
Different Multiple Variants
30
as
Total number of somatic alterations among genes 15 20 25 30 $
MMR pathway
Wild Type
A
Morphologie variants
Đ
35
TP53
RB1
p53/Rb Pathway
CDK4
2
0
CDKN2A
2
PTEN
CTNNB1
B-catenin Pathway
MED12
ß catenin pathway
5
ChrRem pathway
APC
ZNRF3
11
0
TERT
Chromatin Remodelling Pathway
MEN1
5
1
DAXX
ATRX
19
4
0
MLH1
Mismatch Repair Pathway
MSH2
MSH6
PMS2
PTCH1
p53/Rb1 pathway
Sex
Clinical and Pathological Characteristics
Hormone Hypersecretion
Male
ENSAT stage I-II ENSAT stage III-IV
Weiss Score
Female
5
Helsinki Score
Ki-67 index
Cortisol producing
NED/Alive DODAWD
ENSAT Stage
Non hyperfunctional
STATUS
Other
Weis scorec6 Weis score26
Conventional
Mynoid
Oncecytic
Ki67 indes>22
Helsinki score <19
Ki67 inden≤22
Helsinki score 219
Not Known
| Genes (locus) analyzed | Conventional (n=38) | Myxoid (n=7) | Oncocytic (n= 17) | ||||
|---|---|---|---|---|---|---|---|
| Loss | Gain | Loss | Gain | Loss | Gain | ||
| RB1/13q12 | (13q14.2) | 18 (47%) | / | 2 (28%) | / | 1 (6%) | / |
| CDKN2A/CEN9 | (9p21.3) | 15 (39%) | / | 2 (28/%) | / | 1 (6%) | / |
| ZNRF3/22q13 | (22q12.1) | 13 (34%) | / | 1 (14%) | / | / | / |
| TERT/5q31 | (5p15.33) | / | 16 (42%) | / | 3 (43%) | / | 4 (23%) |
| CDK4/CEN12 (12q14.1) | / | 14 (37%) | / | 6 (86%) | / | 2 (12%) | |
expected to downregulate protein levels. TP53 mutations were different and mostly clustered into DNA-binding or oligomerization domains. All of them have been already reported as somatic pathogenic mutations in several cancers, especially the mutation p.S116F, which was identified in two cases. Immunohistochemical analysis for p53 was performed as a surrogate validation test of the TP53 mutational status, with significant correlation between the two tests (Mann-Whitney test p = 0.005) (Supplementary Figure S1b). Mutations in CTNNB1 gene were mainly localized in exon 3. Among these, three (p.Q28 *- p.E53K-p. S45P) were recurrent and identified in two cases, respec- tively. All other variants are detailed in Supplementary Table S3.
All fluorescence in situ hybridization cases were able to generate an informative genomic profile. Most cases (43/62, 69%) exhibited either gains of oncogenes (CDK4 and TERT) or loss of tumor suppressor genes (CDKN2A, RB1, and ZNRF3) (Supplementary Table S4, Table 2, Fig. 3). TERT and ZNRF3 genes, although showing also somatic variants, more frequently harbored copy number variations, indeed 23/62 (37%) cases harboring amplification of TERT and 19/62 (30%) cases deletions of ZNRF3. Loss of CDKN2A and RB1 loci was detected in 18/62 (%) and 21/62 (%) cases, respectively; whereas amplifications of CDK4 locus were identified in 22/62 (35%) patients. Moreover, 13/62 (21%) cases had polysomy of CDK4 and/or TERT
loci, although these were not considered altered for statis- tical purposes due to their equivocal functional and patho- genic significance.
Clinical and pathological correlation with integrated genomic profile
Correlation analysis between molecular data and clinical pathological variables was performed and summarized in Table 3. Of note, we observed higher prevalence of RB1 (p=0.01), CDK4 (p=0.003), and CDKN2A (p=0.04) alterations in the conventional and myxoid histotypes; TP53 mutations were associated with a higher Ki-67 index, higher Helsinki score, higher tumor stage, and aggressive disease status, either as single gene mutation (all p<0.04) or as mutational burden of the p53/Rb pathway (all p<0.04); tumors with TERT and CTNNB1 alterations were associated with higher Weiss score (p=0.03) and higher Ki-67 index (p=0.04), respectively. Furthermore, PMS2 and PTCH1 genetic alterations were more prevalent in male compared with female patients (both p<0.001), whereas CDK4 alterations were associated with higher Helsinki score (p = 0.03). Interestingly, the overall mutational bur- den, as well as the mutational burden of ß-catenin and p53/Rb pathways, were significantly different among his- tological variants, with the lowest rate in the oncocytic one (p=0.006, p = 0.02 and p = 0.0002, respectively) (Fig. 4).
(a)
(b)
(c)
(d)
(e)
Detailed genomic characterization identifies high heterogeneity and histotype-specific genomic …
| Clinico-pathological features | Mutational profile | ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CTN- | p value | TERT | p value | RB1 | p value | CDK- | p value | CDK4 | p value | TP53 | p value | PMS2 | p value | PTC- | p value | ||||||||
| NB1 | N2A | H1 | |||||||||||||||||||||
| W | M | W | M | W | M | W | M | W | M | W | M | W | M | W M | |||||||||
| Gender (n = 62) | 0.4 | 0.8 | 1 | 0.4 | 1 | 0.2 | 0.006 | 0.003 | |||||||||||||||
| Male | 18 | 4 | 14 | 8 | 15 | 7 | 14 | 8 | 14 | 8 | 14 | 8 | 16 | 6 | 14 8 | ||||||||
| Female | 8 | 12 | 23 | 17 | 26 | 14 | 30 | 10 | 26 | 14 | 32 | 8 | 39 | 1 | 38 2 | ||||||||
| ENSAT stage (n=53) | 0.4 | 0.2 | 0.3 | 0.6 | 0.05 | 0.008 | 0.1 | 0.8 | |||||||||||||||
| I-II | 24 | 7 | 20 | 11 | 22 | 9 | 24 | 7 | 25 | 9 | 27 | 4 | 29 | 2 | 26 5 | ||||||||
| III-IV | 15 | 7 | 11 | 11 | 13 | 9 | 16 | 6 | 9 | 10 | 12 | 10 | 18 | 4 | 18 4 | ||||||||
| Outcomes (n=61) | 0.2 | 0.2 | 0.4 | 0.3 | 0.4 | 0.01 | 0.09 | 0.09 | |||||||||||||||
| Ned/alive | 22 | 5 | 14 | 13 | 19 | 8 | 21 | 6 | 21 | 13 | 24 | 3 | 26 | 1 | 25 2 | ||||||||
| Awd/Dod | 23 | 11 | 23 | 11 | 21 | 13 | 23 | 11 | 8 | 21 | 13 | 28 | 6 | 26 8 | |||||||||
| Histologic type (n= 62) | 0.4 | 0.2 | 0.01 | 0.04 | 0.003 | 0.07 | 0.3 | 0.6 | |||||||||||||||
| Conventional | 28 | 10 | 20 | 18 | 20 | 18 | 23 | 15 | 24 | 14 | 26 | 12 | 43 | 4 | 33 5 | ||||||||
| Myxoid | 4 | 3 | 4 | 3 | 5 | 2 | 5 | 2 | 1 | 6 | 4 | 3 | 5 | 2 | 5 2 | ||||||||
| Oncocytic | 14 | 3 | 13 | 4 | 16 | 1 | 16 | 1 | 15 | 2 | 16 | 1 | 16 | 1 | 14 3 | ||||||||
| Weiss score distribution (n = 62) | 0.5 | 0.03 | 0.5 | 0.5 | 0.3 | 0.3 | 0.6 | 0.6 | |||||||||||||||
| <6 | 9 | 5 | 12 | 2 | 8 | 6 | 9 | 5 | 3 | 2 | 12 | 2 | 11 3 | ||||||||||
| ≥6 | 37 | 11 | 25 | 23 | 33 | 17 | 35 | 13 | 29 | 19 | 34 | 14 | 43 | 5 | 41 7 | ||||||||
| Helsinki score distribution (n = 62) | 1 | 0.2 | 0.7 | 1 | 0.03 | 0.04 | 1 | 0.2 | |||||||||||||||
| <19 | 34 | 12 | 25 | 10 | 31 | 11 | 33 | 14 | 26 | 15 | 31 | 41 | 12 40 | ||||||||||
| ≥19 | 4 | 12 | 4 | 21 | 6 | 15 | 5 | 13 | 2 | 20 | 1 | 15 | 2 | 5 | 4 6 | ||||||||
| Ki-67 indexª (n=62) | 0.04 | 0.7 | 0.7 | 0.4 | 0.001 | 0.4 | 0.3 | ||||||||||||||||
| ≤22 | 29 | 5 | 21 | 13 | 0.7 | 23 | 11 | 25 | 9 | 24 | 10 | 31 | 3 | 32 | 2 | 30 4 | |||||||
| >22 | 17 | 11 | 16 | 12 | 18 | 10 | 19 | 9 | 17 | 11 | 15 | 13 | 23 | 5 | 22 6 | ||||||||
W wild type, M mutated, ENSAT European Network for the Study of Adrenal Tumors, Ned no evidence of disease, Dod death of disease, Awd alive with disease
a Cut-off value correspond to median value obtained in the present series
(a)
p=0.006
p=0.02
p=0.0002
Overall Mutational Burden
40
20
15
30
Mutational Burben of Bcat Pathway
15
Mutational Burben of p53Rb Pathway
10
20
10
5
10
5
0
0
0.
-10
Conventional
Myxoid
Oncocytic
-5
-5
Conventional
Myxoid
Oncocytic
Conventional
Myxoid
Oncocytic
ACC Variants
ACC Variants
ACC Variants
(b)
p=0.03
p=0.04
p=0.01
15-
15.
15
Mutational Burben of p53Rb Pathway
Mutational Burben of p53Rb Pathway
10
Mutational Burben of p53Rb Pathway
10
10
5
5
5
0
0
0-
-5
-5
-5
I/II
III/IV
NED/ALIVE
DOD/AWD
⇐ 22
>22
ENSAT Stage
OUTCOMES
Ki67 Index
Survival analysis
Median DFS was of 22.5 months. At univariate analysis, a conventional/myxoid histotype (p=0.01), a high Weiss score (p = 0.01), a high Helsinki score (p = 0.002), a high mitotic index (p=0.004), and high Ki-67 index (p= 0.0004) were all predictive of poor DFS (Fig. 5); as well as harboring an high mutational burden of ß-catenin (p = 0.04), p53/Rb (p=0.02), and chromatin-remodeling (p= 0.03) pathways, using the median number of somatic alterations in each pathway as the arbitrary cut-off value (Fig. 5, Table 4). In multivariate analysis, Ki-67 and Hel- sinki score remained independent predictors of DFS (hazard ratio (HR) = 2.60, 95% confidence interval (CI): 0.89-7.64, p = 0.04; HR = 2.59, 95% CI: 0.72-9.13, p = 0.01), toge- ther with an high mutational burden of mismatch repair pathway (HR = 0.16, 95% CI: 0.03-0.73, p = 0.01).
Re-assessment of the genomic profiling at the time of disease progression
The genomic profile obtained from matched samples is illustrated in Fig. 6 (Supplementary Table S5). Overall, a very high degree of genetic heterogeneity was observed. None of the patients showed a stable genotype between diagnosis and tumor progression and both acquisition and loss of somatic genomic alterations were observed. The number of molecular events did not allow a statis- tical comparison and in most instances the heterogeneity
was linked to molecular alterations encountered at low allelic frequency or percentage of nuclei with copy number variations. However, arbitrarily considering only cases with a change in the percentage of allelic fre- quency/nuclei with copy number variations above 50%, the events acquired during progression were more fre- quently gene mutations (13/14), acquired rather than lost as compared to the primary tumor (13/14) and involving a relative small set of genes, mostly (4/13) belonging to the mismatch repair pathway. Indeed, allelic frequencies or percentages of altered nuclei at fluorescence in situ hybridization were in most cases similar among different tumor samples for genes that were stable along tumor progression (Fig. 7).
Discussion
The genomic characterization of adrenocortical carcinoma has been recently assessed by high-throughput technologies approaches, leading to the identification of specific mole- cular profiles that allow adrenocortical carcinoma stratifi- cation in molecular subtypes with distinct clinical outcome [5, 25-27]. This is the first study focusing on the most prevalent molecular alterations previously identified in adrenocortical carcinoma with the aim to investigate their correlation with pathological and clinical characteristics and to assess genetic heterogeneity along the process of tumor progression. The use of molecular markers for diagnostic
ACC Variants
Weiss Score
Mitotic Index
Ki67 index
Disease Free Survival
100
Conventional
Disease Free Survival
100
Disease Free Survival
100
-L. Myxoid
- < 6
- ⇐ 18
Disease Free Survival
100
- ⇐ 22
>=6
Oncocytic
-L. >18
-L. >22
50
50
50
50
0
0
0
0
0
50
100
150
200
250
300
0
50
100
150
200
250
300
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Months
Months
Months
Months
Helsinki Score
MB Bcat pathway
MB p53/Rb pathway
MB Chr Rem pathway
Disease Free Survival
100
<19
… >=19
Disease Free Survival
100
Low MB
Disease Free Survival
100
Low MB
100
Low MB
-L. High MB
High MB
Disease Free Survival
High MB
50
50
50
50
0
0
0
0
0
50
100
150
200
250
300
0
50
100
150
200
250
300
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Months
Months
Months
Months
| Parameters | Univariate analysis of disease-free survival | Multivariate analysis of disease-free survival | ||
|---|---|---|---|---|
| HR (CI 95%) | p value | HR (CI 95%) | p value | |
| Sex (M vs. F) | 0.87 (0.43-1.73) | 0.69 | 1.02 (0.33-3.16) | 0.8 |
| Age (above vs. below median) | 1.25 (0.64-2.47) | 0.4 | 1.98 (0.75-5.28) | 0.2 |
| Histotype (conventional /myxoid vs. oncocytic) | 2.49 (1.08-4.46) | 0.011 | 0.49 (0.17-1.44) | 0.2 |
| Mitotic indexa (>18 vs. ≤18) | 2.83 (1.39-5.76) | 0.004 | 2.13 (0.22-2.08) | 0.06 |
| Weiss score (26 vs. < 6) | 2.41 (1.18-5.26) | 0.01 | 1.07 (0.31-3.68) | 0.9 |
| Helsinki score (≥19 vs. < 19) | 3.10 (1.49-6.44) | 0.002 | 2.59 (0.72-9.13) | 0.01 |
| Ki-67 indexª (>22 vs. ≤22) | 3.76 (1.80-7.83) | 0.0004 | 2.60 (0.89-7.64) | 0.04 |
| ENSAT stage (I/II vs. III/IV) | 0.58 (0.26-1.17) | 0.13 | 1.50 (0.52-4.33) | 0.4 |
| Overall mutational burden (above vs. below median) | 0.59 (0.30-1.21) | 0.16 | 0.85 (0.25-2.89) | 0.1 |
| P53/Rb pathway (MB>2 vs. MB ≤2) | 2.90 (1.18-7.13) | 0.02 | 2.18 (0.48-9.78) | 0.3 |
| Chr/Rem. pathway (MB>1 vs. MB ≤1) | 2.79 (1.10-7.11) | 0.03 | 1.6 (0.37-7.05) | 0.5 |
| Bcat pathway (MB>3 vs. MB ≤3) | 3.61 (1.04-12.48) | 0.04 | 1.71 (0.26-11.95) | 0.5 |
| MMR pathway (MB >2 vs. MB ≤2) | 0.96 (0.36-2.51) | 0.9 | 0.19 (0.03-0.73) | 0.01 |
ENSAT European Network for the Study of Adrenal Tumors, HR hazard ratio, CI confidence interval, Chr. Rem chromatin remodeling, Bcat ß- catenin, MMR mismatch repair, M male, F female, vs. versus, MB mutational burden
a The selected cut-off corresponds to median value obtained in the present series
purposes was not an aim of the present study and for this reason benign adrenocortical tumors were not included.
Our analysis, performed on 62 sporadic adult adreno- cortical carcinomas at the genetic and genomic level by targeted next-generation sequencing and fluorescence in situ hybridization techniques, revealed a very high mutational burden in the studied cohort of patients, with 92% of the samples having at least one somatic alteration in the
evaluated genes. Although the genetic assessment in our study was targeted to a group of 18 genes and not to the whole genome, this prevalence of mutations is higher than others previously reported [25-27]. This might be partially explained by specific pre-analytical and analytical work- flows of our targeted next-generation sequencing molecular analysis aimed to identify somatic alterations [16, 28]. The greater depth of sequencing (more than 500x) performed by
Clinical and Pathological Characteristics
Mutation Legend
Copy Number Alterations Legend
Male Female
ENSAT stage I-II
Missense
0-1 copies
ENSAT stage III-IV
Nonsense
☐ >2 copies (focal amplification)
Cortisol producing Non hyperfunctional Other
NED/Alive DOD/AWD
Sex
Frameshift
Hormone Hvpersecretion
Conventional Myxoid Oncocytic
InFrame
Weiss Score
Weis score<6 Weis score26
Helsinki Score
Splice Site
Ki-67 index
Ki67 index>22 Ki67 index_22
Helsinki score <19 Helsinki score ≥19
ENSAT Staae
Different Multiple Variants
STATUS
Not Known
☐ Wild Type
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
PMPM PMIM2 PM PR PRMPM P MP R P RIR2
Morphologic variants
0
1
2
3
4
5
6
7
8
TP53
RB1
p53/Rb Pathway
CDK4
CDKN24
PTEN
Distribution of timeframe alterations among genes analyzed
CTNNB1
ß-catenin Pathway
MED12
APC
ZNRF3
TERT
Alteration acquired
Chromatin Remodelling Pathway
MEN1
Alteration lost
DAXX
ATRX
Alteration maintained
MLH1
Mismatch Repair Pathway
MSH2
MSH6
PMS2
PITCH1
us could explain the differences between the studies and might improve detection of subclonal events. This is parti- cularly relevant when the specimens show a low tumor purity and high heterogeneity as adrenocortical carcinoma, which calls into question the suitability of whole-exome sequencing as the platform of choice with diagnostic and clinical purposes. Moreover, it is worth of consideration that in the vast majority of cases, molecular alterations in the four main pathways covered by our analysis were not mutually exclusive and that we selected these genes based on results obtained by most wide genomic analysis. This highlights how an additive rather than single pathway deregulation mechanisms is the hallmark of adrenocortical carcinoma.
Molecular characterization of adrenocortical carcinoma variants
The mutational burden was different among adrenocortical carcinoma histological variants, showing a higher pre- valence of somatic alterations in conventional and myxoid variants as compared to the oncocytic one (p =0.006). Indeed, a small proportion of patients (8%) did not harbor any somatic alterations in the analyzed genes, and, inter- estingly, they were all oncocytic. These data might partly be associated with the previously described more indolent clinical behavior of oncocytic adrenocortical carcinoma [8, 29]. In fact, also in the present series, this histotype was
corresponding recurrence/metastasis. Left: genes are clustered according to specific signaling pathways; right: histograms of time- frame alterations among genes. Main clinical and pathological features are detailed above
associated with a more favorable prognosis at univariate analysis for DFS, as compared to conventional and myxoid tumor types (HR = 2.49, 95% CI: 1.08-4.46, p = 0.01). However, among the 8% of cases with no detectable molecular alterations, 3 out of 5 had a Weiss score >6 and 3 out of 5 only had a indolent clinical behavior. Thus, at least in the small number of cases available, there were no spe- cific clinical or pathological characteristics in these tumors, apart from being oncocytic. The favorable prognosis of oncocytic adrenocortical carcinoma is furthermore high- lighted in the present study by the identification of a sig- nificant correlation with a lower prevalence of mutations in the Wnt/B-catenin (p=0.02) and p53/Rb (p=0.0002) pathways, both known to be individually associated to a more aggressive adrenocortical carcinoma phenotype. This finding was also confirmed in studies of patients harboring these alterations associated with poor DFS either as indi- vidually mutated genes or as altered pathways (see below). On the other hand, the more frequent inactivation of RB1- CDKN2A and gain of function alterations of CDK4, crucial in G1-S phase cell-cycle progression, in myxoid and con- ventional adrenocortical carcinomas are in line with the more aggressive and proliferative phenotype of these tumors. This is also confirmed by the association of CDK4 (p = 0.03) alterations with higher Helsinki score, which has been recently identified as a valuable system to predict prognosis in morphological variants of adrenocortical
Allelic Frequency/nuclei with Copy Number Variations [%]
110%
MES
Recidive
Primitive
@Mts2
OMts1 Primitive
90%
70%
50%
30%
10%
10%
TNNB1 (545F)
RB1 LOH)
DKNZA (LOH)
CTNNB1 (R376H]
CINNB1 (E692”)
TP53 (F 134L]
TP53 (Y126N)
APC (A53 [V]
MSH2 (E $29K)
MSH6|[E286K]
MSH6 (E744”)
MEN1(W203”)
MENI (ART)
ATRX (Q539*)
PTCH1 (W844*)
DAXX (V661M)
LOH)
TP53 (R280G)
RB1 (LOH)
APC (Q1242’
APCIET132K)
ATRX (5381L)
ATRX (T3630
ATRX (02377f)
CTNNB1 (Q193”)
CTNNB1 (D770N)
DAXX (R328H)
DAXX (P24L)
DAXX (0437”)
MSH6 [E546K]
MSH6 (CH475”]
MED12 (516/UE
MED12 (8621”]
MSH2 (G827H)
PM52(P4705)
TP53 (052”)L RB1 (LOH)
YERLIFA)
TERT FA)
ZNREB LOH]
COKATFAL
APC (625025)
TR53 (A161T
PMS2IM6220
COKNZA ILDHI
COKNZA LOHE
TERTIFAL
DAXX (E477KJL
ATRX (N1993K)
CDK4 (EA)
RBL ILOH)
COKNZA (LOH)
CDKA [FAL
TERT (HA)
ZNRF HILOHI
TPSB Y163C)
CTNNB1 (5718E)
APC (51995’L
PTCH1 [G1396EL
RB1 | COHI
CDKA (FA)
TERT (FA)
CINNB1 (545F)
MED12 [R1862H]
COKNZA (LOH
30%
COK
CDK
ZNRF3
50%
70%
ACC34
ACCS
ACC9
ADC11
ACC29
ACC35
ACC71
90%
110%
110%
Allelic Frequency/nuclei with Copy Number Variations (%)
90%
Il Recidive 2
DRecidive1
WPrimitive
70%
50%
30%
10%
10%
APC(C2020V)
APC (52296
APC001131
DAXX [A3161)
MED12 |H9841)
MED12 (W1354”
MSH6 (15234)
MASH6-8976
PIEN [F233
CININ81 (0601
ZMPF3 (R436H
MEN] [Y326Tfs*47)
MEN1 6239
ABRX 118
ATRX (0-138
MLH [V/520Fh15]
MASH2 |0402
PICH3 JH201
APCIC947VI
APC (GB4851)
APC (G2597R)
AC002727*1
DAXX (55SWF]
MIDE2 [0948’
TP53-(13361)
APC (35139]
APC 0599
CININ61 |8486C)
CINN81 (38735_1756
PAXX (E 484k)
PICH1 [4701
PICHE (WS63-
21991|[]14F)
MED12 (R266℃)
411032 (91455”
MED12 [F16811
MED12 |7382 41)
1753-0011580
1P53P1 6
30%
50%
70%
ACC72
40C32
00%
110%
carcinoma, outperforming the currently established prog- nostic parameters [13, 14]. Drugs targeting the cell-cycle pathway, as the CDK4 inhibitor (PD0332991), currently tested in phase I clinical trials (www.clinicaltrials.gov), may represent a new therapeutic strategy for patients with advanced adrenocortical carcinomas.
Correlation of molecular profile with other clinical and pathological features
Among the different molecular alterations associated with specific clinical and pathological parameters, TP53 and CTNNB1 alone were the most altered (>20%), with somatic variations already reported and cancer associated in somatic genomic databases. These genes are well known to have a role in adrenocortical carcinoma pathogenesis [26, 27], as early (CTNNB1) or late (TP53) events in the oncogenesis. Among TP53 variants identified in our cohort, none of our patients harbored the germline mutation p.R337H, whereas we detected either several TP53 mutations with a known dominant negative function or less common mutations associated with absence of protein expression. This finding could explain the good concordance between p53 immu- noreactivity and the TP53 genetic status. TP53 mutations were significantly associated with high Ki-67 index, high Helsinki score, high tumor stage, and aggressive disease status (all p<0.04). These results confirm that TP53 inac- tivation should be a late event in adult adrenocortical car- cinoma oncogenesis [30], and highly prevalent in sporadic adrenocortical carcinoma with a more advanced stage and decreased DFS [31-33]. Our data expand these observations to the concurrent analysis of several genes acting in the p53/ Rb1 pathway, reinforcing the association of a high muta- tional rate in p53/Rb pathway genes with poor DFS.
variation identified in 10 adrenocortical carcinoma samples with matched primary and recurrent/metastatic. ACC adrenocortical carci- noma, Mts metastasis
Similarly, CTNNB1 somatic alterations are associated with poor outcome in adrenocortical carcinoma [34]. In line with these results, in our study a high mutational rate of Wnt/B-catenin pathway genes showed a poor DFS. The detected CTNNB1 gene alterations were mainly localized in exon 3 and known to be pathogenic activating mutations with leading to stabilization of ß-catenin. CTNNB1 altera- tions are also associated with a higher Ki-67 index (p= 0.04). However, only approximately one quarter of adre- nocortical carcinoma cases with ß-catenin nuclear deloca- lization is explained by activating mutations of CTNNB1 gene [4, 34]. Interestingly, in our study, molecular altera- tions of TERT and MED12 were among the most frequent ones, as previously reported [25]. As they are involved in Wnt/ß-catenin pathway interacting physically with ß- catenin [35], their alterations could influence the ß-catenin protein activity in adrenocortical carcinoma.
We also identified an association with poor survival for the chromatin-remodeling pathway (HR = 0.19, 95% CI: 0.03-0.73, p=0.01), that (together with Wnt/ß-catenin and p53/Rb pathways) was previously found altered in a subset of unfavorable outcome patients [25], but not by other groups [26, 27]. The somatic alterations identified in DAXX, ATRX, and MEN1 genes were mainly loss of function and/ or affecting important functional domains of the corre- sponding proteins.
Genomic heterogeneity along tumor progression
The comparison of the genomic profiles from matched primary vs. recurrent/metastatic samples showed a high degree of genetic heterogeneity. None of the patients ana- lyzed (n = 10) showed a stable genotype between diagnosis and the time of tumor progression. In fact, in all cases either
gain or loss of somatic genomic alterations during disease progression were observed and no significant association was observed between genes and cases although-with all the limitations due to small sample size-an enrichment of mismatch repair pathway alterations was observed. On the other hand, for those genes whose alterations were main- tained in the different tumor samples of individual patients, a relative stability of percentage of allelic frequency or tumor cell nuclei with copy number variation was observed. Even if molecular heterogeneity has been already described in other tumors, such as breast, lung, and colon cancer [36, 37], it has never been reported in adrenocortical carcinoma, so far. Absence of recurrent gains or losses of molecular alterations in matched samples, makes difficult to dis- criminate between those that are crucial for survival and proliferation during the metastatic process from passenger ones. These data, provide additional support to a branched evolution model during oncogenesis in adrenocortical car- cinoma, as recently reported in other malignancies [38, 39]. Moreover, the high degree of heterogeneity would represent a relevant pitfall for adrenocortical carcinoma molecular testing in the clinical practice and should be considered in future studies aimed to identify prognostic or predictive markers.
In conclusion, in this study we demonstrated that: (a) the genomic signature in adrenocortical carcinoma differs among histological variants and is associated to specific clinical and pathological characteristics; (b) p53/Rb path- way alterations are the most relevant prognostic molecular markers in adrenocortical carcinoma; (c) the genomic sig- nature in adrenocortical carcinoma is highly unstable along tumor progression, thus emphasizing the potential clinical problem of molecular biomarker testing in advanced adre- nocortical carcinoma patients.
Acknowledgements This work was supported by grants from Asso- ciazione Italiana per la Ricerca sul Cancro (AIRC, Milan; No. IG/ 14820/2013 to MP).
Compliance with ethical standards
Conflict of Interest The authors declare that they have no conflict of interest.
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