Inactivation of the APC Gene Is Constant in Adrenocortical Tumors from Patients with Familial Adenomatous Polyposis but Not Frequent in Sporadic Adrenocortical Cancers
Sébastien Gaujoux1,2,3, Stéphane Pinson13, Anne-Paule Gimenez-Roqueplo6,9,10,11, Laurence Amar7, Bruno Ragazzon1,2, Pierre Launay1,2, Tchao Meatchi8, Rossella Libé1,2,4,11, Xavier Bertagna1,2,4,9,11, Anne Audebourg5, Jessica Zucman-Rossi9,12, Frédérique Tissier1,2,5,9,11, and Jérôme Bertherat1,2,4,9,11
Abstract
Purpose: In adrenocortical tumors (ACT), Wnt/B-catenin pathway activation can be explained by B-catenin somatic mutations only in a subset of tumors. ACT is observed in patients with familial ade- nomatous polyposis (FAP) with germline APC mutations, as well as in patients with Beckwith-Wiede- mann syndrome with Wilms’ tumors reported to have WTX somatic mutations. Both APC and WTX are involved in Wnt/B-catenin pathway regulation and may play a role in ACT tumorigenesis. The aim of this study was to report if APC and WTX may be associated with FAP-associated and sporadic ACT.
Experimental Design: ACTs from patients with FAP and sporadic adrenocortical carcinomas (ACC) with abnormal ß-catenin localization on immunohistochemistry but no somatic B-catenin mutations were studied. APC was analyzed by denaturing high-performance liquid chromatography followed by direct sequencing and by multiplex ligation-dependent probe amplification when allelic loss was sus- pected. WTX was studied by direct sequencing.
Results: Four ACTs were observed in three patients with FAP and were ACC, adrenocortical adenoma, and bilateral macronodular adrenocortical hyperplasia, all with abnormal ß-catenin localization. Biallelic inactivation of APC was strongly suggested by the simultaneous existence of somatic and germline altera- tions in all ACTs. In the 20 sporadic ACCs, a silent heterozygous somatic mutation as well as a rare het- erozygous polymorphism in APC was found. No WTX mutations were observed.
Conclusions: ACT should be considered a FAP tumor. Biallelic APC inactivation mediates activation of the Wnt/B-catenin pathway in the ACT’s of patients with FAP. In contrast, APC and WTX genetic altera- tions do not play a significant role in sporadic ACC. Clin Cancer Res; 16(21); 5133-41. º2010 AACR.
Several studies support the importance of activation of the Wnt/B-catenin pathway in adrenocortical tumorigene- sis, as previously shown also in a variety of cancers (1-3). Gene profiling studies have reported overexpression of tar-
Authors’ Affiliations: 1Institut Cochin, Université Paris Descartes-Faculté de médecine, CNRS (UMR 8104); 2INSERM, U1016; 3Department of Digestive and Endocrine Surgery, 4Department of Endocrinology, Center for Rare Adrenal Diseases, and 5Department of Pathology, Hôpital Cochin, AP-HP; Departments of 6Genetics, 7Hypertension, and 8Pathology, Hôpital Européen Georges Pompidou, Assistance Publique-Hopitaux de Paris; 9Université Paris Descartes-Faculté de médecine; 10INSERM, UMR-970, Paris Cardiovascular Research Center at HEGP; 11Rare Adrenal Cancer Reference Center-COMETE, Institut National du Cancer (INCa); 12INSERM, UMR-674, Génomique fonctionnelle des tumeurs solides, Paris, France; and 13HCL (Hospice Civil de Lyon), Service de Génétique Clinique et Moléculaire, Hôpital Edouard Herriot, Lyon, France
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Jérôme Bertherat, Service des Maladies Endo- criniennes et Métaboliques, Hôpital Cochin, 27 rue du Faubourg Saint- Jacques, 75014 Paris, France. Phone: 33-1-58-41-18-95; Fax: 33-1-46- 33-80-60; E-mail: jerome.bertherat@cch.aphp.fr.
doi: 10.1158/1078-0432.CCR-10-1497
@2010 American Association for Cancer Research.
get genes in the Wnt/B-catenin pathway in adrenocortical carcinoma (ACC; ref. 4) as well as in adrenocorticotropin- independent macronodular adrenocortical hyperplasia (AIMAH; ref. 5). It seems that activation of this pathway may play an important role in adrenocortical tumorigene- sis through activating mutations of the ß-catenin gene (CTNNB1) in adrenocortical adenoma (ACA), ACC, and primary pigmented micronodular adrenal dysplasia (PPNAD; refs. 1-3). This is demonstrated by the frequent nuclear and cytoplasmic ß-catenin accumulation on immu- nohistochemistry (1-3). Currently, ß-catenin-activating mu- tations are the most frequent genetic defect observed in sporadic ACA and ACC (1, 3). However, somatic ß-catenin- activating mutations are present in only a quarter to a third of ACCs (1). This strongly suggests the possible existence of alternative genetic defects or crosstalks with other sig- naling pathways that could activate the Wnt/B-catenin signaling pathway.
Familial adenomatous polyposis (FAP; OMIM #175100) is an autosomal dominant inherited disorder characterized by an early onset of hundreds to thousands of adenomatous polyps throughout the colon and rectum
AR American Association for Cancer Research
Translational Relevance
Activation of the Wnt/B-catenin pathway frequently occurs in adrenocortical tumors (ACT). This is ex- plained by ß-catenin somatic mutations but only in a subset of tumors, suggesting the possible involve- ment of other genetic defects. Our results support that ACT tumorigenesis, at least in patients with familial adenomatous polyposis (FAP), can be mediated by biallelic APC inactivation leading to activation of the Wnt/B-catenin pathway. Thus, ACT should be consid- ered a FAP tumor, and screening for ACT should be in- cluded in the workup of patients with FAP.
and a predisposition to various extracolonic manifesta- tions, including endocrine tumors such as thyroid tumors (6) and adrenocortical tumors (ACT; refs. 7, 8). The genet- ic defect in FAP is an inactivating germline mutation of the tumor suppressor gene adenomatous polyposis coli (APC; ref. 9). This results in a truncated nonfunctional protein, leading to constitutive activation of the Wnt/B-catenin pathway (10). Adrenal masses have been associated with FAP (7, 8). Nevertheless, although ACTs in FAP have been reported from a clinical perspective (11-17), there is a paucity of research into the role of APC mutations in the ACT from FAP (11, 13, 16, 18).
Similarly, ACT is associated with Wilms’ tumor in Beckwith-Wiedemann syndrome. This overgrowth disorder is caused by overexpression of insulin-like growth factor II, which has also been detected in sporadic ACC (19) and Wilms’ tumor (20, 21). As in the case of FAP, Wnt/B-catenin signaling is constitutively activated in Wilms’ tumor, in part through CTNNB1 mutations (22). Recently, the WTX
(Wilms’ tumor gene on the X chromosome) gene has been reported to be inactivated by deletion or mutation in 7% to 29% of Wilms’ tumors (23-25). This gene is a tumor suppressor gene and, like APC, operates through Wnt/B- catenin signaling, downregulating its activity by forming a complex with ß-catenin (26).
Given these findings, this study was undertaken to in- vestigate the hypothesis that Wnt/B-catenin signaling path- way activation may be involved in ACTs through APC or WTX mutations in patients with FAP and sporadic ACC.
Materials and Methods
Patients and tissue collection
Patients with FAP and ACT. Three FAP patients from two different families, who presented with four ACTs, includ- ing one bilateral AIMAH, one ACA, and one ACC, were included in this study.
Patients with sporadic ACC. Twenty sporadic ACC samples presenting Wnt/B-catenin pathway activation (i.e., nuclear and/or cytoplasmic ß-catenin protein accu- mulation by immunohistochemistry) but no somatic B-catenin-activating mutation were studied.
For all samples, clinical, hormonal, and radiological in- vestigations were done as previously reported (27, 28). Tu- mor and tissue fragments obtained during surgery were immediately dissected by a pathologist and then frozen and stored in liquid nitrogen until use.
The study was carried out under the approval of the In- stitutional Review Board (Comité Consultatif de Protec- tion des Personnes dans la Recherche Biomédicale) of Cochin Hospital, Paris, France. Informed consent was ob- tained for clinical information, tumor analysis, and gen- etic diagnosis as part of the COMETE network research activities.
| Table 1. Characteristics of the three patients with ACTs in the context of FAP | |||||||
|---|---|---|---|---|---|---|---|
| Patient | Age (y)/sex | Side | Secretion | Pathologic diagnosis | Gross aspect (weight in g and diameter in cm) | Weiss score | ß-Catenin immunohistochemistry |
| 1 (mother) | 59/F | Right | Glucocorticoid | AIMAH | 6 g, 3 cm (3 nodules) | NA | Diffuse |
| Left | AIMAH | 18 g, 7 cm (including several nodules, with one of 25 mm) | cytoplasmic accumulation | ||||
| 2 (son) | 29/M | Right | Mineralocorticoid | ACA | 32 g, 5 cm | 0 | Cytoplasmic |
| and glucocorticoid | and nuclear accumulation | ||||||
| 3 | 68/F | Right | None | ACC | 230 g, 12 cm | 6 | Cytoplasmic and nuclear accumulation |
| (Continued on the following page) | |||||||
Pathology examination and immunohistochemistry
For each patient, diagnosis was confirmed by pathologic examination, and a Weiss score established when appro- priate (29, 30). Immunohistochemical staining for B- catenin was done as previously described (1, 2) and assessed in a blinded fashion with respect to the mutational status and clinical data. B-Catenin immunohistochemistry was evaluated for the presence of cytoplasmic and nuclear staining. Cytoplasmic and/or nuclear staining on immuno- histochemistry was recorded as intracellular accumulation.
Nucleic acid extraction and mutation analysis of CTNNB1, the mutation cluster region, APC, and WTX
DNA was extracted from peripheral blood leukocytes us- ing the Wizard Genomic DNA Purification Kit (Promega Corp.), and tumor DNA was purified by cesium chloride gradient ultracentrifugation (31). Because mutations of the main proteins in the Wnt/B-catenin pathway are re- ported to be exclusive (32), specimens were first se- quenced for mutations in CTNNB1; if absent, this was followed by sequencing for mutations in APC, and then WTX. For mutation analysis, PCR was used to amplify the exon 3 and flanking 5’ intron sequences of CTNNB1 in the tumors (1); the mutation cluster region of APC in the tumors and leukocytes of patients with FAP; and WTX in the tumors (see Supplementary Data). Both strands of the amplified products were directly sequenced on an au- tomated sequencer (ABI 3700, Perkin-Elmer). Nucleotides were numbered in accordance with the reference sequences of CTNNB1 (GenBank accession no. NM_001904), APC (GenBank accession no. NM_001127510), and WTX (GenBank accession no. NM_152424).
Denaturing high-performance liquid chromatography analysis
When two mutations were not found in the mutation cluster region of APC, denaturing high-performance liquid
chromatography (DHPLC), optimized for rapid screening of the 15 exons and splice junctions of the APC gene, was used to screen for mutations. Touchdown PCR was used to reduce nonspecific primer annealing by gradually lowering the annealing temperature as PCR cycling pro- gressed. Amplifications were done using the Optimase polymerase (Transgenomic). Following PCR, reaction pro- ducts were denatured by heating at 94℃ for 5 minutes, followed by cooling at 25℃ over a 25-minute period to enhance heteroduplex formation. Five microliters of PCR products were analyzed directly by DHPLC using the WAVE DNA fragment analysis system (Transge- nomic) with 1 to 3 injections per amplicon, for a total of 71 injections for a complete analysis. Abnormal pro- files detected by DHPLC were subsequently sequenced using the BigDye Terminator reaction kit on the ABI Prism 3100 genetic analyzer system (Applied Biosys- tems). Primer sequences and annealing temperatures for PCR and analysis parameters for DHPLC are listed in Supplementary Data.
Multiplex ligation-dependent probe amplification
When large deletions or loss of heterozygosity was sus- pected, APC was screened using the multiplex ligation- dependent probe amplification (MLPA) using the SALSA P043 kit (MRC-Holland), which had 23 probe sets for the APC region. In short, 120 ng of genomic DNA were used as starting material, and after hybridization at 60℃ for 16 hours followed by ligation and amplification, the PCR products were separated on a 16-channel ABI Prism 3100 genetic analyzer (Applera) using capillary electrophoresis. Fragment analysis, normalization of data by regression line of control probe, and quantitative and statistical analysis were done using three methods, GeneMapper (Applera), GeneMarker (Softgenetics), and Excel (Microsoft), using normalization by the regression line of the control probe. All three methods yielded similar results.
| Patient | CTNNB1 somatic mutation | APC germline mutation | APC somatic mutation (additional tumoral mutation) | WTX somatic mutation | Status |
|---|---|---|---|---|---|
| 1 (mother) | None | c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 | c.1863_1866del4 (TTAC; heterozygous state) | None | Alive |
| None | (heterozygous state) | c.4667_4668insA/p.Thr_1556fsX1558 (heterozygous state) | None | Alive | |
| 2 (son) | None | c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (heterozygous state) | c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (homozygous state*) | None | Alive |
| 3 | None | c.4689_4690 ins 4 (CCTA)/p.Leu1563fsX1567 (heterozygous state) | c.3234 T>A/p.Tyr1078X (heterozygous state) | None | Dead |
| Abbreviation: NA, nonapplicable. | |||||
| *No APC gene deletion detected by MLPA. | |||||
Results
ACTs in patients with FAP
Three patients from two different families with FAP were identified and presented with four various ACTs including one ACA, one ACC, and a bilateral AIMAH (Table 1). On B- catenin immunohistochemistry, all tumors showed diffuse cytoplasmic and/or nuclear accumulation, revealing activa- tion of the Wnt/B-catenin pathway (Fig. 1). There were no
somatic CTNNB1 mutations, but all of the tumors showed evidence of germline and somatic APC mutations accord- ing to Knudson’s two-hit model for a tumor suppressor gene (Fig. 2).
Patients 1 and 2 were mother and son belonging to the same family with FAP that had been characterized by a heterozygous germline mutation (c.4549_4550ins6/ p.Glu1517fsX1519) in both the blood cells and tumors. Patient 1 had AIMAH, and in this patient, a heterozygous
Aa
Ab
Ba
Bb
Ca
Cb
Da
Db
Ea
Eb
Fig. 2. Electropherogram of APC mutations in an ACT from a patient with FAP (* indicating mutations in the forward sequence). A, germline DNA from patients 1 and 2 showing heterozygous germline mutations (c.4549_4550ins6/ p.Glu1517fsX1519). B, tumor DNA from the main nodule of the right adrenal gland of patient 1 showing an additional somatic heterozygous deletion (c.1863_1866del4/ p.Thr621fsX628). C, tumor DNA from the main nodule of the left adrenal gland of patient 1 showing an additional somatic heterozygous insertion (c.4667_4668insA/ p.Thr_1556fsX1558) leading to a frameshift mutation. D, tumor DNA from patient 2 showing the same mutation as germline DNA (c.4549_4550ins6/ p.Glu1517fsX1519) in a homozygous state. E, germline DNA from patient 3 showing a heterozygous germline mutation (c.4689_4690ins4/ p.Leu1563FsX1567). F, tumor DNA from patient 3 showing an additional heterozygous somatic mutation (c.3234T>A/p.Tyr1078X) found in ACC.
| Germline | Somatic |
|---|---|
| A ATT TAT A CRER NAN | B * CTCTT a CTTACC GG MAMMAANDAM |
| C ATTCCAATGAGAAAA | |
| D ATTTATACGTGTATA | |
| E OGACCTATTAMATO * | F GTTTAAACTGA |
deletion (c.1863_1866del4/p.Thr621fsX628) was detected in the main nodule of the right adrenal gland, and a heterozygous insertion (c.4667_4668insA/p.Thr_1556fsX1558) was detected in the main nodule of the left gland (Fig. 3A). Unfortunately, nodules smaller than 2 mm were not available for sequencing.
In patient 2, who had ACA, tumor analysis was surprising for a homozygous germline APC mutation (c.4549_4550ins6/ p.Glu1517fsX1519). An analysis of known intragenic polymorphisms of APC detected a change of status between the blood cells, which were heterozygous for the mutation, and the tumor, which was homozygous. Partial or complete APC gene deletion was ruled out by MLPA, which detected 2 copies of the gene. This homozygous status in the tumor could be attributed to large genomic rearrangements with loss of the normal allele and duplication of the mutated allele involving at least the entire APC gene (Fig. 3B).
With regard to patient 3, who was not related to the oth- er patients and had ACC, a heterozygous germline muta- tion (c.4689_4690ins4/p.Leu1563FsX1567) was detected in both the blood cells and tumor. An additional hetero- zygous somatic mutation (c.3234T>A/p.Tyr1078X) was al- so found in the tumor.
Overall, five known silent single-nucleotide polymorph- isms (http://www.umd.be/APC/ and http://www.ensembl.org) were observed in the four tumors as well as the corres- ponding germline DNA.
ACTs in patients with sporadic ACC
Twenty cases of sporadic ACCs were identified and characterized to have Wnt/B-catenin pathway activation (i.e., abnormal ß-catenin localization by immunohisto- chemistry but no somatic ß-catenin activating muta- tions); the data are presented in Table 2. During sequencing for APC, a silent heterozygous variant (c.3340C>A/p.Arg1114Arg) that had not been described previously was discovered. In another tumor, there was a rare heterozygous polymorphism (c.3949G>C/p. Glu1317Gln) that might represent a colonic adenoma- carcinoma predisposition allele (33-36). These two var- iants were found in the DNA of both the leukocytes and tumors. In addition, four tumors were homozygous for all single-nucleotide polymorphisms analyzed, and no large deletions were found on MLPA, excluding a hemizygous status. Additionally, 11 known heterozy- gous single-nucleotide polymorphisms without any amino acid modification were observed in 16 tumors. The frequency of these polymorphisms was similar to that in the general population according to National Center for Biotechnology Information databases (http://www.umd.be/APC/ and http://www.ensembl. org). Of the 20 sporadic ACC analyzed, none had any somatic WTX mutations, and 5 known polymorph- isms of WTX (http://www.ensembl.org) were observed in all these tumors.
Discussion
This study shows that APC inactivation is one of the mechanisms of Wnt/B-catenin pathway activation in adre-
nocortical tumorigenesis. The important role of the Wnt/ B-catenin pathway in ACT is supported by gene profiling studies showing overexpression of target genes in ACC (4) as well as AIMAH (5) and PPNAD (37). Activation of this
A
| Localisation | Mother | Son | ||||||
|---|---|---|---|---|---|---|---|---|
| Right tumor | Left tumor | Blood sample | Blood sample | Tumor | ||||
| Detected mutations | Exon 14 | c.4689_4690ins4 | Mutation detected 2nd hit mutation | Non detected | Non detected | Non detected | Non detected | |
| Exon 15 | c.4549_4550ins6 | First hit | mutation according the Knudson's Two-Hit Mutation detected in blood and in tumors | Model | Homozygous or hemizygous for the detected mutation ? 2nd hit mutation | |||
| c.4667_4668insA | Non detected | Mutation detected 2nd hit mutation | Non detected | Non detected | Non detected | |||
| Known Polymorphisms | Exon 11 | c.1458T>C | Heterozygous polymorphism T/C | Heterozygous polymorphism T/C | C/C Or C/- | |||
| Exon 13 | c.1635A>G | Heterozygous polymorphism A/G | Heterozygous polymorphism A/G | A/A Or A/- | Homozygous or hemizygous ? | |||
| Exon 15 | c.5465T>A | Heterozygous polymorphism T/A | Heterozygous polymorphism T/A | A/A Or A/- | ||||
| c.5880A>G | Heterozygous polymorphism A/G | Heterozygous polymorphism A/G | A/A Or A/- | |||||
B
| The Hypotheses for the second hit in the son's tumor | ||||
|---|---|---|---|---|
| Blood | ||||
| SALSA MLPA KIT P043-B1 APC | ||||
| Tumor | Hypothesis A | A large deletion of the normal allele occurs in the tumor cells It could explain that the constitutional mutation has been detected as a hemizygous mutation. | Deletion | |
| Hypothesis B | After the deletion of the normal allele, the mutated allele was duplicated in the tumor cells. Then, cells of the tumor has two alleles for the APC gene locus which carry both the same constitutional mutation | Normal | ||
Fig. 3. Details of germline and somatic mutations including polymorphisms in patients 1 and 2. A, mutations and polymorphisms in patients 1 and 2. B, hypothesis for the second-hit mutation in patient 2 (the son). Because no deletions were found on MLPA, a duplication of the mutated allele in the tumor cells was likely.
pathway can be explained by somatic CTNNB1 mutations of ß-catenin in a subset of ACA, ACC, and PPNAD (1-3, 38). From immunohistochemical and molecular studies, it is likely that activation of the Wnt/B-catenin pathway is a major event in ACC (1, 39). As it is observed more fre- quently than somatic CTNNB1 mutations (1, 2), activa- tion of the pathway could be explained by other genetic defects such as APC or WTX mutations.
Our results showed that all ACTs in patients with FAP presented with a secondary somatic alteration of the APC gene, in addition to the germline mutation. All so- matic mutations led to a stop codon, resulting in a trun- cated APC protein. This is consistent with the finding that the residual allele of the APC gene is inactivated in tumors of patients with FAP (40) and suggests that ACT can be mediated by biallelic APC inactivation.
The majority of APC mutations are nucleotide substitu- tions and frameshift mutations that result in truncated pro- teins. Interestingly, uncommon APC mutations were observed in patients with FAP in our study. According to the APC mutation database (http://www.umd.be/APC/), none of the mutations in our study have been described pre- viously. Moreover, one of them presented with a mutation [c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519] in the homozygous state with a diploid APC copy number on MLPA. This suggests the loss of the normal allele with a duplication of the mutated APC locus. As previously shown in FAP, loss of heterozygosity tends to occur by a mechanism that does not result in a change of the APC gene dosage, with a diploid APC copy number, probably because of chromo- some rearrangement tied to mitotic recombination (41). Overall, this is consistent with the previously reported high incidence of chromosomal gains, frequently on chromo- some 5 where the APC locus is located (42). Interestingly, a similar mechanism of loss of the wild-type allele has been described in a functional ACT from a patient with FAP, which was associated with a nonsense germline mutation at codon 1577 (17). Overall, the simultaneous existence of somatic and germline alterations was found in all ACTs from patients with FAP that were analyzed in our series.
APC promoter methylation has been shown to silence transcription and has therefore been proposed as an alternative mechanism of inactivation of APC (43). This mechanism may be involved in sporadic ACCs, which do not carry the APC or CTNNB1 mutation. Nevertheless, this mechanism can be ruled out as the dominant mechanism. Indeed, no decrease in APC mRNA expression was observed in our previously published gene expression profiling mi- croarray in which APC mRNA expression was compared in ACC with the CTNNB1 mutation, ACC without the CTNNB1 mutation, ACA, and normal adrenal gland (44).
The higher incidence of adrenal lesions in patients with FAP patients in retrospective studies, such as those by Smith and colleagues (7) from St. Mark’s Hospitals and by Mar- chesa and colleagues (8) from the Cleveland Clinic Founda- tion, could have been attributed to the extensive and frequent imaging done in these expert, high-volume cen- ters. Our study is the largest series of patients with
| n (%)/mean (range) | |
|---|---|
| Age at diagnosis (y) | 44 (18-68) |
| Sex ratio (male/female) | 5/15 |
| Side (right/left) | 10/10 |
| Presentation at diagnosis | |
| Endocrine features | 16 |
| Local or regional manifestations | 2 |
| Incidentaloma | 4 |
| Secretion | |
| Cortisol + androgen | 6 |
| Cortisol | 4 |
| Nonsecreting | 4 |
| Cortisol + mineralocorticoid | 2 |
| Cortisol + mineralocorticoid + androgen | 1 |
| Cortisol + precursor | 1 |
| Androgen | 1 |
| Cortisol + precursor + androgen | 1 |
| Tumor size (cm) | 10.5 (4-24) |
| Tumor weight (g) | 314 (40-2,160) |
| MacFarlane stage | |
| 2 | 10 |
| 3 | 2 |
| 4 | 8 |
| Weiss score | 6 (3-9) |
| Follow-up (mo) | 44 (1-119) |
| Status | |
| Alive without recurrence | 8 |
| Alive with recurrence | 0 |
| Dead | 12 |
genetic analysis and gives strong molecular evidence for a causal association between ACT and FAP. Indeed, we be- lieve that ACT tumorigenesis, at least in patients with FAP, can be mediated by biallelic APC inactivation and thus complete loss of function of the APC gene according to Knudson’s two-hit model (45). The abnormal cytoplasmic and nuclear ß-catenin localization on immunohistochem- istry confirms the activation of the pathway in the ACTs with APC mutations in our study. Further experimental studies with APC knockdowns in mice will help toward a better understanding of APC-mediated ACT tumorigenesis.
Given the present genetic findings and the previous find- ings that ACT was more prevalent in patients with FAP by 7.4% to 13% than the general population (7, 8), ACT should be considered a FAP tumor. Therefore, screening for ACT could be offered to patients with FAP. Although no evidence-based recommendations on screening (i.e., methods for screening, the frequency and intervals, and the age at which it should start) for ACT are available, our suggestion would be to consider an initial screening by ab- dominal magnetic resonance imaging before adolescence,
which would simultaneously screen for other extraintest- inal manifestations such as desmoid tumors, pancreatic cancer, and hepatomas. Referral to an endocrinologist would be another option for patients with a significant family history of extraintestinal manifestations and would facilitate physical examination for any thyroid nodules in the surveillance for thyroid carcinoma. Special attention to the adrenal glands should be given to these patients at each interval imaging. Owing to improved survival as a result of better prevention of colorectal cancer, the incidence of extraintestinal manifestations, including ACT, will further in- crease. Education of medical personnel to these rare manifes- tations would be the best way to avoid adverse consequences.
One limitation of our study is the small number of pa- tients with FAP and ACT, as the combination is uncom- mon. Consequently, our findings cannot be generalized to the ACT’s in all patients with FAP. Yamakita and col- leagues (46) suggested that second-hit mutations are not always found in ACT from FAP. Because most of the previous genetic data on ACTs in patients with FAP are not available, we cannot rule out some unrelated sporadic adrenal disease. It is also possible that some APC muta- tions in FAP create a mutant protein displaying a tissue- specific dominant negative effect (47, 48) by reducing the function of the APC tumor suppressor wild-type allele in the adrenal cortex. Such a germline mutation conferring a particular genetic susceptibility to tumor development has been hypothesized in thyroid carcino- mas associated with FAP (6), which usually occur in the absence of biallelic inactivation of the APC gene. Never- theless, to date, no specific familial, gender, or genotype association that increases the risk of ACT has been described. An association of desmoid tumors and ACTs has been suggested in FAP (8), but the evidence for such an association remains weak (7).
The results of our genetic study of APC in sporadic ACC contrasted with that in FAP-associated ACT. All the spo- radic ACC tumors studied presented with activation of the Wnt/B-catenin pathway on immunohistochemistry. In this way, they were similar to the ACT’s in patients with FAP. However, only two changes in the APC gene were found in 20 cases of sporadic ACC. These two genetic al- terations were a silent variant and a rare polymorphism, and therefore, their functional or pathologic value remains questionable and probably minor. Furthermore, these changes presented in both the tumor and germline DNA and were not associated with secondary alterations of the APC gene at the somatic level. APC has already been
implicated in other tumors with Wnt/B-catenin pathway activation, such as sporadic hepatocellular carcinoma (49). Our results do not exclude that biallelic APC inactiva- tion could still occur in some rare sporadic ACC with Wnt/ B-catenin pathway activation. Our study also indicates that the somatic mutations of WTX that are observed frequently in Wilms’ tumors do not play a significant role in sporadic ACC in adults. This suggests that along with somatic B- catenin mutations, other genetic or epigenetic defects could explain the Wnt/B-catenin pathway activation in sporadic ACC in adults. One explanation would be activation of the Wnt/B-catenin pathway by the overexpression of its extra- cellular ligands. Crosstalks with other signaling pathways such as the insulin-like growth factor pathway, which fre- quently overexpresses insulin-like growth factor II in these tumors (49), might be another explanation.
In conclusion, our results support that ACT tumorigen- esis, at least in patients with FAP, can be mediated by bial- lelic APC inactivation leading to activation of the Wnt/ B-catenin pathway. ACT should be considered a FAP tu- mor and might be screened during the workup of patients with FAP. The role of the APC or WTX gene in sporadic ACC remains to be seen, but these genes do not seem to play a significant role.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Gabrielle Couchy and Karine Perlemoine for excellent technical assistance; Professors Anne Berger-Petit, Bertrand Dousset, and Franck Zinzindohoue, who performed the operations on the patients; Professor Pierre Laurent-Puig for genetic diagnosis of the FAP families; Franck Letourneur (Plate-forme séquencage et génomique, of Cochin Institute) for help in the sequencing; Christine Perret, Marie-Cecile Vacher-Lavenue, and Catherine Cavard for helpful discussions; and Dana L. Haviland and Au Joyce for their valuable help in the preparation of the manuscript.
Grant Support
Contrat d’Initiation à la Recherche Clinique (Grant CIRC 05045, AP-HP) and the Plan Hospitalier de Recherche Clinique (AOM06179), Recherche Translationnelle DHOS/INCA 2009 grant RTD09024, to the COMETE Net- work. S. Gaujoux was the recipient of a research fellowship from the Fond d’Etude et de Recherche du Corps Medical (FERCM)-AP-HP, Paris, France.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 06/02/2010; revised 08/18/2010; accepted 08/20/2010; published OnlineFirst 10/26/2010.
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AAGR American Association for Cancer Research
Clinical Cancer Research
Inactivation of the APC Gene Is Constant in Adrenocortical Tumors from Patients with Familial Adenomatous Polyposis but Not Frequent in Sporadic Adrenocortical Cancers
Sébastien Gaujoux, Stéphane Pinson, Anne-Paule Gimenez-Roqueplo, et al. Clin Cancer Res 2010;16:5133-5141. Published OnlineFirst October 26, 2010.
Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-1497
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