Purpose: In adrenocortical tumors (ACT), Wnt/β-catenin pathway activation can be explained by β-catenin somatic mutations only in a subset of tumors. ACT is observed in patients with familial adenomatous polyposis (FAP) with germline APC mutations, as well as in patients with Beckwith-Wiedemann syndrome with Wilms' tumors reported to have WTX somatic mutations. Both APC and WTX are involved in Wnt/β-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 β-catenin localization on immunohistochemistry but no somatic β-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 suspected. 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 β-catenin localization. Biallelic inactivation of APC was strongly suggested by the simultaneous existence of somatic and germline alterations in all ACTs. In the 20 sporadic ACCs, a silent heterozygous somatic mutation as well as a rare heterozygous 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/β-catenin pathway in the ACTs of patients with FAP. In contrast, APC and WTX genetic alterations do not play a significant role in sporadic ACC. Clin Cancer Res; 16(21); 5133–41. ©2010 AACR.

Translational Relevance

Activation of the Wnt/β-catenin pathway frequently occurs in adrenocortical tumors (ACT). This is explained by β-catenin somatic mutations but only in a subset of tumors, suggesting the possible involvement 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/β-catenin pathway. Thus, ACT should be considered a FAP tumor, and screening for ACT should be included in the workup of patients with FAP.

Several studies support the importance of activation of the Wnt/β-catenin pathway in adrenocortical tumorigenesis, as previously shown also in a variety of cancers (13). Gene profiling studies have reported overexpression of target genes in the Wnt/β-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 tumorigenesis through activating mutations of the β-catenin gene (CTNNB1) in adrenocortical adenoma (ACA), ACC, and primary pigmented micronodular adrenal dysplasia (PPNAD; refs. 13). This is demonstrated by the frequent nuclear and cytoplasmic β-catenin accumulation on immunohistochemistry (13). Currently, β-catenin–activating mutations are the most frequent genetic defect observed in sporadic ACA and ACC (1, 3). However, somatic β-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 signaling pathways that could activate the Wnt/β-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 and a predisposition to various extracolonic manifestations, including endocrine tumors such as thyroid tumors (6) and adrenocortical tumors (ACT; refs. 7, 8). The genetic 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/β-catenin pathway (10). Adrenal masses have been associated with FAP (7, 8). Nevertheless, although ACTs in FAP have been reported from a clinical perspective (1117), 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/β-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 (2325). This gene is a tumor suppressor gene and, like APC, operates through Wnt/β-catenin signaling, downregulating its activity by forming a complex with β-catenin (26).

Given these findings, this study was undertaken to investigate the hypothesis that Wnt/β-catenin signaling pathway activation may be involved in ACTs through APC or WTX mutations in patients with FAP and sporadic ACC.

Patients and tissue collection

Patients with FAP and ACT.

Three FAP patients from two different families, who presented with four ACTs, including one bilateral AIMAH, one ACA, and one ACC, were included in this study.

Patients with sporadic ACC.

Twenty sporadic ACC samples presenting Wnt/β-catenin pathway activation (i.e., nuclear and/or cytoplasmic β-catenin protein accumulation by immunohistochemistry) but no somatic β-catenin–activating mutation were studied.

For all samples, clinical, hormonal, and radiological investigations were done as previously reported (27, 28). Tumor 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 Institutional Review Board (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale) of Cochin Hospital, Paris, France. Informed consent was obtained for clinical information, tumor analysis, and genetic diagnosis as part of the COMETE network research activities.

Pathology examination and immunohistochemistry

For each patient, diagnosis was confirmed by pathologic examination, and a Weiss score established when appropriate (29, 30). Immunohistochemical staining for β-catenin was done as previously described (1, 2) and assessed in a blinded fashion with respect to the mutational status and clinical data. β-Catenin immunohistochemistry was evaluated for the presence of cytoplasmic and nuclear staining. Cytoplasmic and/or nuclear staining on immunohistochemistry 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 using 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/β-catenin pathway are reported to be exclusive (32), specimens were first sequenced 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 automated 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 progressed. Amplifications were done using the Optimase polymerase (Transgenomic). Following PCR, reaction products were denatured by heating at 94°C for 5 minutes, followed by cooling at 25°C 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 (Transgenomic) with 1 to 3 injections per amplicon, for a total of 71 injections for a complete analysis. Abnormal profiles detected by DHPLC were subsequently sequenced using the BigDye Terminator reaction kit on the ABI Prism 3100 genetic analyzer system (Applied Biosystems). 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 suspected, 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°C 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.

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 β-catenin immunohistochemistry, all tumors showed diffuse cytoplasmic and/or nuclear accumulation, revealing activation of the Wnt/β-catenin pathway (Fig. 1). There were no somatic CTNNB1 mutations, but all of the tumors showed evidence of germline and somatic APC mutations according to Knudson's two-hit model for a tumor suppressor gene (Fig. 2).

Table 1.

Characteristics of the three patients with ACTs in the context of FAP

PatientAge (y)/sexSideSecretionPathologic diagnosisGross aspect (weight in g and diameter in cm)Weiss scoreβ-Catenin immunohistochemistryCTNNB1 somatic mutationAPC germline mutationAPC somatic mutation (additional tumoral mutation)WTX somatic mutationStatus
1 (mother) 59/F Right Glucocorticoid AIMAH 6 g, 3 cm (3 nodules) NA Diffuse cytoplasmic accumulation None c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (heterozygous state) c.1863_1866del4 (TTAC; heterozygous state) None Alive 
Left AIMAH 18 g, 7 cm (including several nodules, with one of 25 mm) None c.4667_4668insA/p.Thr_1556fsX1558 (heterozygous state) None Alive 
2 (son) 29/M Right Mineralocorticoid and glucocorticoid ACA 32 g, 5 cm Cytoplasmic and nuclear accumulation None c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (heterozygous state) c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (homozygous state*None Alive 
68/F Right None ACC 230 g, 12 cm Cytoplasmic and nuclear accumulation None c.4689_4690 ins 4 (CCTA)/p.Leu1563fsX1567 (heterozygous state) c.3234 T>A/p.Tyr1078X (heterozygous state) None Dead 
PatientAge (y)/sexSideSecretionPathologic diagnosisGross aspect (weight in g and diameter in cm)Weiss scoreβ-Catenin immunohistochemistryCTNNB1 somatic mutationAPC germline mutationAPC somatic mutation (additional tumoral mutation)WTX somatic mutationStatus
1 (mother) 59/F Right Glucocorticoid AIMAH 6 g, 3 cm (3 nodules) NA Diffuse cytoplasmic accumulation None c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (heterozygous state) c.1863_1866del4 (TTAC; heterozygous state) None Alive 
Left AIMAH 18 g, 7 cm (including several nodules, with one of 25 mm) None c.4667_4668insA/p.Thr_1556fsX1558 (heterozygous state) None Alive 
2 (son) 29/M Right Mineralocorticoid and glucocorticoid ACA 32 g, 5 cm Cytoplasmic and nuclear accumulation None c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (heterozygous state) c.4549_4550ins6 (GTGTGA)/p.Glu1517fsX1519 (homozygous state*None Alive 
68/F Right None ACC 230 g, 12 cm Cytoplasmic and nuclear accumulation 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.

Fig. 1.

H&E-safran staining at ×200 magnification (a) and β-catenin immunohistochemical staining at ×200 magnification (b) in ACTs from patients with FAP (A-C) and sporadic ACC as controls (D and E). A, adrenocorticotropin-independent macronodular adrenocortical hyperplasia in patient 1 with FAP, showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. B, ACA from patient 2 with FAP showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. C, ACC from patient 3 with FAP showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. D, sporadic ACC showing no β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. E, sporadic ACC showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry.

Fig. 1.

H&E-safran staining at ×200 magnification (a) and β-catenin immunohistochemical staining at ×200 magnification (b) in ACTs from patients with FAP (A-C) and sporadic ACC as controls (D and E). A, adrenocorticotropin-independent macronodular adrenocortical hyperplasia in patient 1 with FAP, showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. B, ACA from patient 2 with FAP showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. C, ACC from patient 3 with FAP showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. D, sporadic ACC showing no β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry. E, sporadic ACC showing β-catenin cytoplasmic and nuclear accumulation on immunohistochemistry.

Close modal
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.

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.

Close modal

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 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.

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.

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.

Close modal

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 other patients and had ACC, a heterozygous germline mutation (c.4689_4690ins4/p.Leu1563FsX1567) was detected in both the blood cells and tumor. An additional heterozygous somatic mutation (c.3234T>A/p.Tyr1078X) was also found in the tumor.

Overall, five known silent single-nucleotide polymorphisms (http://www.umd.be/APC/ and http://www.ensembl.org) were observed in the four tumors as well as the corresponding germline DNA.

ACTs in patients with sporadic ACC

Twenty cases of sporadic ACCs were identified and characterized to have Wnt/β-catenin pathway activation (i.e., abnormal β-catenin localization by immunohistochemistry but no somatic β-catenin activating mutations); 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 (3336). These two variants 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 heterozygous 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 polymorphisms of WTX (http://www.ensembl.org) were observed in all these tumors.

Table 2.

Characteristics of the 20 patients with sporadic ACCs

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 
    Incidentaloma 
Secretion  
    Cortisol + androgen 
    Cortisol 
    Nonsecreting 
    Cortisol + mineralocorticoid 
    Cortisol + mineralocorticoid + androgen 
    Cortisol + precursor 
    Androgen 
    Cortisol + precursor + androgen 
Tumor size (cm) 10.5 (4-24) 
Tumor weight (g) 314 (40-2,160) 
MacFarlane stage 
    2 10 
    3 
    4 
Weiss score 6 (3-9) 
Follow-up (mo) 44 (1-119) 
Status 
    Alive without recurrence 
    Alive with recurrence 
    Dead 12 
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 
    Incidentaloma 
Secretion  
    Cortisol + androgen 
    Cortisol 
    Nonsecreting 
    Cortisol + mineralocorticoid 
    Cortisol + mineralocorticoid + androgen 
    Cortisol + precursor 
    Androgen 
    Cortisol + precursor + androgen 
Tumor size (cm) 10.5 (4-24) 
Tumor weight (g) 314 (40-2,160) 
MacFarlane stage 
    2 10 
    3 
    4 
Weiss score 6 (3-9) 
Follow-up (mo) 44 (1-119) 
Status 
    Alive without recurrence 
    Alive with recurrence 
    Dead 12 

This study shows that APC inactivation is one of the mechanisms of Wnt/β-catenin pathway activation in adrenocortical tumorigenesis. The important role of the Wnt/β-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 pathway can be explained by somatic CTNNB1 mutations of β-catenin in a subset of ACA, ACC, and PPNAD (13, 38). From immunohistochemical and molecular studies, it is likely that activation of the Wnt/β-catenin pathway is a major event in ACC (1, 39). As it is observed more frequently than somatic CTNNB1 mutations (1, 2), activation 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 somatic mutations led to a stop codon, resulting in a truncated 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 substitutions and frameshift mutations that result in truncated proteins. 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 previously. 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 chromosome rearrangement tied to mitotic recombination (41). Overall, this is consistent with the previously reported high incidence of chromosomal gains, frequently on chromosome 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 microarray 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 Marchesa and colleagues (8) from the Cleveland Clinic Foundation, could have been attributed to the extensive and frequent imaging done in these expert, high-volume centers. Our study is the largest series of patients with genetic analysis and gives strong molecular evidence for a causal association between ACT and FAP. Indeed, we believe 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 β-catenin localization on immunohistochemistry 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 findings 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 abdominal magnetic resonance imaging before adolescence, which would simultaneously screen for other extraintestinal 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 increase. Education of medical personnel to these rare manifestations would be the best way to avoid adverse consequences.

One limitation of our study is the small number of patients with FAP and ACT, as the combination is uncommon. Consequently, our findings cannot be generalized to the ACTs in all patients with FAP. Yamakita and colleagues (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 mutations 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 carcinomas associated with FAP (6), which usually occur in the absence of biallelic inactivation of the APC gene. Nevertheless, 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 sporadic ACC tumors studied presented with activation of the Wnt/β-catenin pathway on immunohistochemistry. In this way, they were similar to the ACTs in patients with FAP. However, only two changes in the APC gene were found in 20 cases of sporadic ACC. These two genetic alterations 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/β-catenin pathway activation, such as sporadic hepatocellular carcinoma (49). Our results do not exclude that biallelic APC inactivation could still occur in some rare sporadic ACC with Wnt/β-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 β-catenin mutations, other genetic or epigenetic defects could explain the Wnt/β-catenin pathway activation in sporadic ACC in adults. One explanation would be activation of the Wnt/β-catenin pathway by the overexpression of its extracellular ligands. Crosstalks with other signaling pathways such as the insulin-like growth factor pathway, which frequently overexpresses insulin-like growth factor II in these tumors (49), might be another explanation.

In conclusion, our results support that ACT tumorigenesis, at least in patients with FAP, can be mediated by biallelic APC inactivation leading to activation of the Wnt/β-catenin pathway. ACT should be considered a FAP tumor 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.

No potential conflicts of interest were disclosed.

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 Network. 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.

1
Tissier
F
,
Cavard
C
,
Groussin
L
, et al
. 
Mutations of β-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors
.
Cancer Res
2005
;
65
:
7622
7
.
2
Gaujoux
S
,
Tissier
F
,
Groussin
L
, et al
. 
Wnt/β-catenin and 3′,5′-cyclic adenosine 5′-monophosphate/protein kinase A signaling pathways alterations and somatic β-catenin gene mutations in the progression of adrenocortical tumors
.
J Clin Endocrinol Metab
2008
;
93
:
4135
40
.
3
Tadjine
M
,
Lampron
A
,
Ouadi
L
,
Bourdeau
I
. 
Frequent mutations of β-catenin gene in sporadic secreting adrenocortical adenomas
.
Clin Endocrinol (Oxf)
2008
;
68
:
264
70
.
4
Giordano
TJ
,
Thomas
DG
,
Kuick
R
, et al
. 
Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis
.
Am J Pathol
2003
;
162
:
521
31
.
5
Bourdeau
I
,
Antonini
SR
,
Lacroix
A
, et al
. 
Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators
.
Oncogene
2004
;
23
:
1575
85
.
6
Cetta
F
,
Curia
MC
,
Montalto
G
, et al
. 
Thyroid carcinoma usually occurs in patients with familial adenomatous polyposis in the absence of biallelic inactivation of the adenomatous polyposis coli gene
.
J Clin Endocrinol Metab
2001
;
86
:
427
32
.
7
Smith
TG
,
Clark
SK
,
Katz
DE
,
Reznek
RH
,
Phillips
RK
. 
Adrenal masses are associated with familial adenomatous polyposis
.
Dis Colon Rectum
2000
;
43
:
1739
42
.
8
Marchesa
P
,
Fazio
VW
,
Church
JM
,
McGannon
E
. 
Adrenal masses in patients with familial adenomatous polyposis
.
Dis Colon Rectum
1997
;
40
:
1023
8
.
9
Kinzler
KW
,
Nilbert
MC
,
Su
LK
, et al
. 
Identification of FAP locus genes from chromosome 5q21
.
Science
1991
;
253
:
661
5
.
10
Munemitsu
S
,
Albert
I
,
Souza
B
,
Rubinfeld
B
,
Polakis
P
. 
Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein
.
Proc Natl Acad Sci U S A
1995
;
92
:
3046
50
.
11
Blaker
H
,
Sutter
C
,
Kadmon
M
, et al
. 
Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli
.
Genes Chromosomes Cancer
2004
;
41
:
93
8
.
12
Beuschlein
F
,
Reincke
M
,
Koniger
M
,
D'Orazio
D
,
Dobbie
Z
,
Rump
LC
. 
Cortisol producing adrenal adenoma-a new manifestation of Gardner's syndrome
.
Endocr Res
2000
;
26
:
783
90
.
13
Wakatsuki
S
,
Sasano
H
,
Matsui
T
,
Nagashima
K
,
Toyota
T
,
Horii
A
. 
Adrenocortical tumor in a patient with familial adenomatous polyposis: a case associated with a complete inactivating mutation of the APC gene and unusual histological features
.
Hum Pathol
1998
;
29
:
302
6
.
14
Traill
Z
,
Tuson
J
,
Woodham
C
. 
Adrenal carcinoma in a patient with Gardner's syndrome: imaging findings
.
AJR Am J Roentgenol
1995
;
165
:
1460
1
.
15
Painter
TA
,
Jagelman
DG
. 
Adrenal adenomas and adrenal carcinomas in association with hereditary adenomatosis of the colon and rectum
.
Cancer
1985
;
55
:
2001
4
.
16
Seki
M
,
Tanaka
K
,
Kikuchi-Yanoshita
R
, et al
. 
Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis
.
Hum Genet
1992
;
89
:
298
300
.
17
Hosogi
H
,
Nagayama
S
,
Kanamoto
N
, et al
. 
Biallelic APC inactivation was responsible for functional adrenocortical adenoma in familial adenomatous polyposis with novel germline mutation of the APC gene: report of a case
.
Jpn J Clin Oncol
2009
;
39
:
837
46
.
18
Hsiao
HP
,
Kirschner
LS
,
Bourdeau
I
, et al
. 
Clinical and genetic heterogeneity, overlap with other tumor syndromes, and atypical glucocorticoid hormone secretion in adrenocorticotropin-independent macronodular adrenal hyperplasia compared with other adrenocortical tumors
.
J Clin Endocrinol Metab
2009
;
94
:
2930
7
.
19
Gicquel
C
,
Bertagna
X
,
Schneid
H
, et al
. 
Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors
.
J Clin Endocrinol Metab
1994
;
78
:
1444
53
.
20
Scott
J
,
Cowell
J
,
Robertson
ME
, et al
. 
Insulin-like growth factor-II gene expression in Wilms' tumour and embryonic tissues
.
Nature
1985
;
317
:
260
2
.
21
Reeve
AE
,
Eccles
MR
,
Wilkins
RJ
,
Bell
GI
,
Millow
LJ
. 
Expression of insulin-like growth factor-II transcripts in Wilms' tumour
.
Nature
1985
;
317
:
258
60
.
22
Maiti
S
,
Alam
R
,
Amos
CI
,
Huff
V
. 
Frequent association of β-catenin and WT1 mutations in Wilms tumors
.
Cancer Res
2000
;
60
:
6288
92
.
23
Perotti
D
,
Gamba
B
,
Sardella
M
, et al
. 
Functional inactivation of the WTX gene is not a frequent event in Wilms' tumors
.
Oncogene
2008
;
27
:
4625
32
.
24
Rivera
MN
,
Kim
WJ
,
Wells
J
, et al
. 
An X chromosome gene, WTX, is commonly inactivated in Wilms tumor
.
Science
2007
;
315
:
642
5
.
25
Ruteshouser
EC
,
Robinson
SM
,
Huff
V
. 
Wilms tumor genetics: mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors
.
Genes Chromosomes Cancer
2008
;
47
:
461
70
.
26
Major
MB
,
Camp
ND
,
Berndt
JD
, et al
. 
Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling
.
Science
2007
;
316
:
1043
6
.
27
Groussin
L
,
Horvath
A
,
Jullian
E
, et al
. 
A PRKAR1A mutation associated with primary pigmented nodular adrenocortical disease in 12 kindreds
.
J Clin Endocrinol Metab
2006
;
91
:
1943
9
.
28
Bertherat
J
,
Groussin
L
,
Sandrini
F
, et al
. 
Molecular and functional analysis of PRKAR1A and its locus (17q22–24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity
.
Cancer Res
2003
;
63
:
5308
19
.
29
Weiss
LM
. 
Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors
.
Am J Surg Pathol
1984
;
8
:
163
9
.
30
Weiss
LM
,
Medeiros
LJ
,
Vickery
AL
 Jr
. 
Pathologic features of prognostic significance in adrenocortical carcinoma
.
Am J Surg Pathol
1989
;
13
:
202
6
.
31
Groussin
L
,
Kirschner
LS
,
Vincent-Dejean
C
, et al
. 
Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD
.
Am J Hum Genet
2002
;
71
:
1433
42
.
32
Polakis
P
. 
Wnt signaling and cancer
.
Genes Dev
2000
;
14
:
1837
51
.
33
Lamlum
H
,
Al Tassan
N
,
Jaeger
E
, et al
. 
Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q
.
Hum Mol Genet
2000
;
9
:
2215
21
.
34
Frayling
IM
,
Beck
NE
,
Ilyas
M
, et al
. 
The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history
.
Proc Natl Acad Sci U S A
1998
;
95
:
10722
7
.
35
Popat
S
,
Stone
J
,
Coleman
G
, et al
. 
Prevalence of the APC E1317Q variant in colorectal cancer patients
.
Cancer Lett
2000
;
149
:
203
6
.
36
Hall
MJ
,
Liberman
E
,
Dulkart
O
, et al
. 
Risk of colorectal neoplasia associated with the adenomatous polyposis coli E1317Q variant
.
Ann Oncol
2009
;
20
:
1517
21
.
37
Horvath
A
,
Mathyakina
L
,
Vong
Q
, et al
. 
Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation
.
J Clin Endocrinol Metab
2006
;
91
:
584
96
.
38
Tadjine
M
,
Lampron
A
,
Ouadi
L
,
Horvath
A
,
Stratakis
CA
,
Bourdeau
I
. 
Detection of somatic β-catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD)
.
Clin Endocrinol (Oxf)
2008
;
69
:
367
73
.
39
Giordano
TJ
,
Kuick
R
,
Else
T
, et al
. 
Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling
.
Clin Cancer Res
2009
;
15
:
668
76
.
40
Morin
PJ
,
Sparks
AB
,
Korinek
V
, et al
. 
Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC
.
Science
1997
;
275
:
1787
90
.
41
Crabtree
M
,
Sieber
OM
,
Lipton
L
, et al
. 
Refining the relation between “first hits” and “second hits” at the APC locus: the “loose fit” model and evidence for differences in somatic mutation spectra among patients
.
Oncogene
2003
;
22
:
4257
65
.
42
Dohna
M
,
Reincke
M
,
Mincheva
A
,
Allolio
B
,
Solinas-Toldo
S
,
Lichter
P
. 
Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications
.
Genes Chromosomes Cancer
2000
;
28
:
145
52
.
43
Esteller
M
,
Sparks
A
,
Toyota
M
, et al
. 
Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer
.
Cancer Res
2000
;
60
:
4366
71
.
44
de Reynies
A
,
Assie
G
,
Rickman
DS
, et al
. 
Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival
.
J Clin Oncol
2009
;
27
:
1108
15
.
45
Knudson
AG
 Jr
. 
Mutation and cancer: statistical study of retinoblastoma
.
Proc Natl Acad Sci U S A
1971
;
68
:
820
3
.
46
Yamakita
N
,
Murai
T
,
Ito
Y
, et al
. 
Adrenocorticotropin-independent macronodular adrenocortical hyperplasia associated with multiple colon adenomas/carcinomas which showed a point mutation in the APC gene
.
Intern Med
1997
;
36
:
536
42
.
47
Dihlmann
S
,
Gebert
J
,
Siermann
A
,
Herfarth
C
,
von Knebel Doeberitz
M
. 
Dominant negative effect of the APC1309 mutation: a possible explanation for genotype-phenotype correlations in familial adenomatous polyposis
.
Cancer Res
1999
;
59
:
1857
60
.
48
Tighe
A
,
Johnson
VL
,
Taylor
SS
. 
Truncating APC mutations have dominant effects on proliferation, spindle checkpoint control, survival and chromosome stability
.
J Cell Sci
2004
;
117
:
6339
53
.
49
Katoh
H
,
Shibata
T
,
Kokubu
A
, et al
. 
Genetic inactivation of the APC gene contributes to the malignant progression of sporadic hepatocellular carcinoma: a case report
.
Genes Chromosomes Cancer
2006
;
45
:
1050
7
.