Six pediatric patients with ACT, all with wild type TP53, were selected from the International Pediatric Adrenocortical Tumor Registry (IPACTR) at St. Jude Children’s Research Hospital (St. Jude). Written informed consent was obtained from parents or legal guardians for inclusion in the St Jude Children’s Research Hospital (St. Jude) International Pediatric Adrenocortical Tumor Registry (IPACTR; http://clinicaltrials.gov/show/NCT00700414). One patient (#1) was diagnosed with BWS at the time of cancer diagnosis. A second patient (#2) developed lateralized overgrowth and the ACT was found when surveilling for abdominal tumors. No features of BWS were seen during cancer diagnosis for the remaining four cases (#3,4,5,6) but patients were subsequently found to harbor constitutional chromosome 11p15 alterations ( Table 1 ).

Due to the complexity of the chromosome 11p15 imprinting regions and their interactions, the interpretation of copy number variations (16) and epigenetic changes (6, 16) in that region requires a series of molecular assays. In this series of 6 patients, whole genome or whole exome sequencing was examined for three (#1,5,6) patients and single nucleotide polymorphism array was offered to patient #3.

Targeted techniques were combined to determine 11p15 status in germline DNA for all 6 patients. Genotyping of a panel of five microsatellite markers (D11S1363, D11S922, D11S4046, HUMTH01 and D11S988) covering positions 1,061,991 to 4,539,851 at chromosome 11p15 (GRCh37/hg19) was performed by using a fluorescently labeled forward and conventional reverse primers as previously described (1). An informative result allows us to discriminate maternal and paternal alleles for each marker. A pattern of homozygosity is suggestive of paternal uniparental disomy (UPD). In addition, genomic DNA was analyzed by using a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA; ME030-C3 BWS/RSS, MRC-Holland), commercially designed specifically for the 11p15 region and currently the most rapid and robust technique to assess methylation. DNA was processed in parallel with and without digestion with the methylation sensitive HhaI enzyme to detect both chromosome copy number alteration and methylation deregulation. Data analysis was performed using the Coffalyser software (MRC Holland) which provides two outputs, one related to chromosome 11p15 copy number changes and the other methylation status of imprinting control 1 (ICR1; regulating H19 and IGF2; position 1,976,280 to 1,982,450) and ICR2 (regulating CDKN1C, KCNQ1 and KCNQ1OT1; position 2,677,130 to 2,678,030) by the ratio of digested to undigested DNA. DNA from control individuals shows reduction of 50% of the MS-MLPA signal, corresponding to the presence of methylated alleles (ICR1 is unmethylated in maternal allele and ICR2 unmethylated in the paternal allele) and the contribution of both parental chromosomes ( Figure 2A ). Patients with UPD exhibited hypermethylation at ICR1 and hypomethylation at ICR2 consistent with the absence of maternal chromosome and duplication of paternal chromosome ( Figure 2B ). These epigenetic and structural changes at chromosome 11p15 leads to biallelic expression of IGF2 and inactivation of H19 and CDKN1C.

Enrollment on the IPACTR requires central review for the diagnosis of pediatric adrenocortical tumors in individuals up to 21 years of age at the time of initial diagnosis. The histological diagnosis was based on a combination of morphologic (hematoxylin and eosin) and histochemical criteria including chromogranin, cytokeratin, Cam 5.2, inhibin ( Figure 3 ), Melan-A and synaptophysin. In addition, Ki-67 and beta-catenin were analyzed using standard assays (1). The p53 expression by immunohistochemistry was performed on deparaffinized tissue sections using the avidin-biotin complex method. The slides were incubated with monoclonal antibodies directed against p53 protein (1:50, DO-7; Dako Carpinteria, CA) ( Table 1 ). There was no attempt to reclassify the tumors using specific histological criteria.

The patient was an 8.5-year-old female with presumptive diagnosis of BWS who presented to the local physician in 2003 with new onset of herculean habitus, clitoromegaly, pubic hair, acne, and apocrine odor. Abdominal computed tomography imaging revealed synchronous bilateral adrenal masses. There was no evidence of disease elsewhere. The patient underwent laparotomy with excision of the left adrenal gland and tumor enucleation of the right gland. The left tumor weighed 21.5 g and measured 5.5 × 1.5 × 0.7 cm, and right tumor measured 1.3 × 1.2 × 1.0 cm. Sections of both tumors showed similar cellular constituents. Tumors were composed of a uniform population of cells with ample acidophilic cytoplasm and moderate anisonucleosis that recapitulated the zona reticularis, consistent with the diagnosis of adrenocortical adenoma. The p53 staining was negative and Ki-67 labeling index (LI) was less than 2%. Inhibin-α was positive in a subset of cells. No chemotherapy was indicated. The patient remained asymptomatic until 2008, and then developed bilateral small breast nodules. The right breast mass measured 3.5 × 2.5 × 2.0 cm and the left inferior breast mass measured 3.0 × 2.0 × 2.0 cm and both were surgically removed. Histology of the first mass had features of tubular adenoma and that of the left mass of juvenile fibroadenoma. The patient remained clinically stable until the age of 18 years when she was taken off the protocol.

Molecular findings: Blood and tumor DNA of this patient was examined by whole exome sequencing (WES) (17). Results showed that the patient had a wild type sequence for TP53 in both germline and tumor DNA as well as wild type sequence for CTNNB1 and ATRX in the tumor (1, 17). Additional findings included the somatic p.E150K variant in the imprinted MKRN3 and the p.D155fs in the cytochrome P450 Family 17 Subfamily A Member 1, CYP17A1. Of note, the patient harbored a germline p.E257K variant in EGFR (17) and the p.M595T variant in the regulator of epigenetic gene silencing SIRT1. Both variants were present in the tumor in the heterozygous state (17). The variant in EGFR was classified as likely benign in CLINVAR (variant ID 1123013) and the SIRT1 variant was not reported. Therefore, both variants have been reported in the Genome Aggregation Database (gnomAD) originated from unrelated individual sequences as part of population genetic studies supporting lack of biological significance for both variants in the context of adrenocortical tumors. WES analysis of blood-derived DNA did not detect 11p15 UPD. However, a pattern of homozygosity at chromosome 11p15 was verified by microsatellite markers analyses and confirmed by MS-MLPA consistent with paternal uniparental disomy (UPD) in the germline sample ( Figure 2 ).

Patient was a 2.4-year-old male with a past medical history consistent with BWS (hemihypertrophy) who had an abdominal mass in the left adrenal gland during surveillance for abdominal tumors. In addition, a hepatic mass was noted. The patient underwent laparotomy with resection of the left adrenal gland and partial hepatectomy. The adrenal tumor weighed 56 g and measured 5.9 × 5.9 × 3.6 cm. The liver lesion weighed 73 g and measured 6.5 × 4.5 × 4.0 cm. Tumors in adrenal gland and liver were histologically identical. The immune profile of both tumors was positive for pan keratin, vimentin, synaptophysin, melan-A, and was negative for inhibin-α, chromogranin, and alpha-feto protein, anti-endomysial antibody (EMA), and calretinin. Liver and adrenal masses were also negative for p53, and Ki-67 LI was <5% in both tumors. There was vascular invasion in the adrenal tumor. Peri-aortic lymph nodes were negative. Tumor margins were free of tumor. The patient was not treated with chemotherapy and remains free of disease 11 years after the diagnosis of adrenocortical carcinoma (ACC).

Molecular findings: Molecular studies were done for the germline sample and the patient was wild type for TP53. Microsatellite analysis covering the position 1,061,991 to 4,539,851 at chromosome 11p15 (GRCh37/hg19) revealed a heterozygous pattern for all studied markers but MS-MLPA revealed a pattern consistent with mosaic paternal UPD.

The patient was an 8.4-year-old girl diagnosed with stage II ACC at age 7 months. In 2013, she went to the local pediatric endocrinologist as she had Cushing syndrome. Her medical history revealed that she was a full-term infant with a birth weight of 6lb 12 oz (~3kg). She did not have umbilical hernia, omphalocele, macroglossia, nevus flammeus or lateralized overgrowth. An ultrasound imaging study revealed an abdominal mass in the adrenal gland region. The mass, which measured 8.2 × 7.5 × 4.5 cm was completely resected and the diagnosis of ACC confirmed. Tumor was positive for vimentin and melan-A and inhibin-α was moderate in occasional tumor cells. The Ki-67 LI was 30%. The patient was not treated with chemotherapy and has remained disease-free.

Molecular findings: Blood and tumor samples were available for molecular studies. No TP53 variants were observed in the germline or tumor (1). The tumor harbored the p.S45P missense variant in CTNNB1 and was wild type for ATRX. Whole genome SNP microarray (Reveal; Integrated Genetics) analysis of germline DNA revealed normal chromosome copy number but 50% mosaicism for chromosome 11p covering a 45.6 Mb region. A pattern consistent with mosaic paternal UPD was confirmed by MS-MLPA.

Patient was a 28-year-old healthy female diagnosed with non-functional metastatic ACT at age 15 months. She did not have an antecedent of endocrine manifestations, and the tumor was discovered during the one-year routine visit to the pediatrician. She did not have a history of growth and developmental abnormalities. CT imaging of the abdomen and chest revealed a left supra-renal mass measuring 5 cm in the largest diameter and bilateral pulmonary nodules (Stage IV). She underwent resection of the primary tumor that measured 7.0 × 6.0 × 4.0 cm and weighed 130 g. The histopathological examination confirmed the diagnosis of ACC. She was transferred to St. Jude and treated with cisplatin, etoposide and mitotane. However, after four courses of chemotherapy, several pulmonary lesions remained. She underwent resection of the bilateral pulmonary metastatic nodules, and most of them showed viable metastatic tumor by histology. Chemotherapy was stopped and the patient was monitored for tumor progression. The pulmonary lesions regressed and remained very small for over 10 years. She continues to be free of disease and in good health to date, 26.5 years after the diagnosis.

Molecular findings: No TP53 variants were observed in blood and tumor samples (1). In addition, the primary tumor was negative for CTNNB1 and ATRX. Furthermore, microsatellite analysis of germline DNA revealed a pattern of homozygosity consistent with paternal UPD that was confirmed by MS-MLPA.

The patient was a 21-year-old female with past history of stage 3 ACT diagnosed at 4.9 years. Her development was normal except for an incidental note per the family of longer and larger right upper and lower extremities throughout her life that had not been specifically investigated as far as the patient and her family were aware, but was associated with chronic left-leaning posture and asymmetric shoe size. She had been in her usual state of good health until the age 4 years when she complained of abdominal pain. She also had weight loss, increased headaches, decreased activity and energy, increased tiredness, and sweating. Given the persistence of symptoms, she was examined by a physician who palpated an abdominal mass and noted hypertension and slight androgen elevations. Magnetic resonance imaging showed a large abdominal mass in the right adrenal gland, with direct extension into the inferior vena cava and nearly completely filling of the right atrium. She underwent a partial sternotomy, and extraction of the caval portion of the adrenal tumor and resection of the right retroperitoneal tumor. The tumor weight was 550 g and measured 13.5 × 10.5 × 6.0 cm. The pathology confirmed ACC (low Ki-67 LI, rare mitotic figures and negative for inhibin-α). There were large areas of necrosis, and vascular invasion with tumor penetration through the capsule. Immunohistochemistry analysis was negative for p53 and positive for beta-catenin. She was treated with the combination of cisplatin, doxorubicin, etoposide, and mitotane. Features of right-sided lateral overgrowth, including right-sided tongue enlargement as well as increased right vs. left arm circumferences (51 cm vs 50 cm), calf circumferences (39 cm vs 37 cm), and a slight leg length discrepancy (88 cm vs 85 cm) were first noted during exams performed at St. Jude as part of a multidisciplinary rare endocrine tumor clinic when she was 18 years old, and led to additional testing subsequently confirming chromosome 11p15 UPD. She has been alive and tumor free for 17 years.

Molecular findings: Blood DNA of this patient was analyzed by whole exome sequencing (WES). No reportable structural, single nucleotide variants, and indel or copy number changes were identified in APC, CDKN1C, MEN1, PRKAR1A, and TP53. Chromosome paternal 11p15 UPD was confirmed by MS-MLPA.

The patient was a 23-year-old female who had hypertension during a routine medical visit in 2009 at age 11 years. Examination for causes of hypertension revealed a large, and well-defined mass in the left upper quadrant above the left kidney and deep to the spleen. The mass was approximately 8.7 × 9.7 × 9.7 cm and was relatively round. The kidneys and liver were normal in appearance. There was no regional adenopathy. Laboratory investigation showed remarkably elevated levels of deoxycorticosterone, aldosterone, and dehydroepiandrosterone. The mass was removed one month later, and weighed 388 g and measured 11 × 10 × 6 cm. There was a 3 × 3 cm defect on the surface suggestive of tumor rupture. The tumor was composed of nests of moderate-sized cells with round nuclei, prominent nucleoli, and moderate to abundant foamy to amphophilic non-foamy cytoplasm. There was only one mitosis per ten 400× fields and there was no atypical mitosis. However, the tumor demonstrated prominent venous invasion, necrosis, and extracapsular extension. Calcifications were noted. Immunohistochemical stains were positive for melan-A, vimentin, and cytokeratin (focal). Epithelial membrane antigen, chromogranin, and inhibin-α were negative. Ki-67 LI was <5%. Occasional areas of the tumor showed moderate nuclear staining for p53. The patient was referred to St. Jude for treatment. At that time, her adrenal hormones had returned to normal levels. She underwent comprehensive imaging studies, including FDG-PET scans. There was no evidence of residual disease. The recommendation was observation. She remains disease free 12 years from diagnosis.

Molecular findings: Blood and tumor DNA of this patient was analyzed by whole genome sequencing (WGS) (17). The patient had a wild type sequence for TP53 in both germline and tumor DNA (1, 17). ACT was wild type for ATRX and acquired the p.G34E variant in CTNNB1 and p.R201H in GNAS (17). In addition, the patient harbored the germline p.R307* variant in the PDE11A (18). WES analysis of blood-derived DNA was not sufficiently sensitive to detect chromosome 11p15 UPD, but MS-MLPA revealed a mosaic pattern of paternal 11p15 UPD.