As we continue to learn more about adrenocortical tumors (ACT) through our research, we will inform you of advances. We periodically update aggregate data so you have the most recent statistics from the IPACTR.
There are two main registries for pediatric ACT information in the United States:
- IPACTR, International Pediatric Adrenocortical Tumor Registry (St. Jude Children’s Research Hospital). Learn more about IPACTR.
- Children’s Oncology Group (COG) rare tumor research registry
The International Pediatric Adrenocortical Tumor Registry (IPACTR) was built in two distinct periods. During the first period, clinical and demographic information from pediatric patients with adrenocortical tumors (ACT) was collected. The results, obtained since 1990, are published and available.
St. Jude physicians and researchers recently amended the IPACTR protocol. The second period of this study allows us to bank biological samples according to established procedures and country-specific guidelines. We developed a translational program that includes analyzing TP53 mutations and polymorphisms, interrogating pathways involved in tumor initiation and progression, performing gene expression analysis, and establishing cell lines and xenograft models for drug testing.
Detailed clinical and molecular findings are being shared with the scientific community in several publications.
Between January 1990 and December 2001, 254 patients younger than 20 years of age with newly diagnosed or previously treated ACT were registered in the IPACTR database. A histologic diagnosis of ACT was required, although central review was not mandatory. Follow-up information was periodically requested from the referring physician. Treatment was chosen by the primary physician. Here a brief descriptive analysis of these patients:
Demographics and Clinical Findings
- Most of the study patients (79.5%) were from southern Brazil; 13% were from the USA, and 7.5% were from nine other countries.
- There were 156 girls and 98 boys. The overall female-male ratio was 1.6:1, but it varied widely across age groups. Girls predominated in the age groups 0 to 3 years (ratio, 1.7:1) and older than 13 years (ratio, 6.2:1). In the 4- to 12-year-old group, neither sex predominated.
- The median age was 3.2 years (range, 0 to 19 years) with 60% of them younger than 4 years of age and only 14% older than 13 years of age. Of interest, one child had a diagnosis of ACT during the neonatal period and another 18 patients had a history compatible with onset of overproduction of adrenal hormones during the first few months of life.
- Approximately 90% of the children had clinical evidence of an endocrine syndrome. Virilization, alone or in combination with signs of overproduction of other adrenal hormones, was the most common clinical presentation (84.3%). Tumors that did not cause clinical manifestations of hormone excess (nonfunctional tumors) were observed in 10.2% of patients. Isolated Cushing’s syndrome (median age, 12.6 years) and nonfunctional tumors (median age, 5.7 years) tended to occur in the older children without sex differences.
- The median interval between the first symptoms and diagnosis was 5 months, with a range from 0 years to 90 months.
Pathologic Findings and Disease Stage
- ACT cases were classified as carcinomas (n=228) or adenomas (n=26).
- It was considered stage I and II as limited disease. These cases accounted for about two-thirds of the total. Of these, 112 patients (44.1%) had stage I disease, and 80 patients (31.5%) had stage II disease. Among patients with advanced disease (stages III and IV), 25 patients (9.8%) had stage III and 37 patients (14.6%) had stage IV disease.
- Among those with stage IV disease, the most common metastasis sites included lung and liver. Bone and brain metastasis occurred rarely.
- In cases in which tumor spillage during surgery was reported, 18 of 86 patients (21%) had stage I or II disease, whereas intravenous tumor thrombus was found in 9 of 64 such patients (14%).
In addition to surgery, 7 of 116 patients with stage I disease received adjuvant treatment (5 patients received mitotane, 1 patient received local radiotherapy, and 1 patient received combination chemotherapy). Of 80 patients with stage II disease, 16 received adjuvant treatment (13 patients received mitotane; 2 patients received combination chemotherapy; and 1 patient received local radiation). All patients with stage III or IV disease received intensive chemotherapy.
Follow-up and Outcomes
At a median follow-up of 2 years and 5 months, with a range from 5 days to 22 years:
- 157 patients (61.8%) remained alive.
- 97 patients (38.2%) had died. In 92 cases, death was associated with disease progression (including 22 patients who had active disease at the time of the last follow-up visit). Five patients died from causes unrelated to tumor progression.
- The 5-year event-free survival was 54.2%, and the overall survival estimate was 54.7%.
We examined several clinical features for association with outcome (age, sex, clinical syndrome, interval between first symptoms and diagnosis, blood pressure, disease stage, tumor spillage during surgery, intravenous tumor thrombus and tumor weight). Please note that histologic criteria were not consistently used to classify pediatric ACT as benign (adenoma) or malignant (carcinoma); therefore, we did not evaluate these data as prognostic factors.
- Among 26 patients reported to have adrenocortical adenoma, only 1 experienced relapse.
- In patients with localized disease we found the following factors were associated with a greater probability of EFS: age between 0 and 3 years, virilization alone, normal blood pressure, disease stage I, absence of spillage during surgery and tumor weight ≤200 grams.
- Disease stage I, virilization alone, and age 0 to 3 years were independently associate with a greater probability of EFS and overall results did not change with the exclusion of adenomas. We were unable to do a factor analysis for patients with stages III and IV disease because of the small number and dismal outcome of such patients.
The IPACTR protocol was amended in 2002 in order to allow us to obtain relevant biological material (tumor tissue and blood samples) from study participants with ACT, their biological parents and relatives. These materials helped identify determination of the TP53 germline status with genetic counseling implications, and research in molecular and biological mechanism implicated in adrenocortical tumorigenesis.
We have enrolled 123 pediatric ACT patients and 3 relatives who developed cancer since the protocol amendment. Patients are from United States, South America (Brazil, Chile and Argentina), Central America (Honduras, Guatemala and Mexico) and Europe (Spain, Greece, Poland and UK). We have also established a collaborative study of pediatric ACT with the Children’s Oncology Group, and tumor samples from 56 cases were studied at molecular level at St. Jude Children’s Research Hospital.
Case descriptions, the development of a xenograft model and the establishment of the detailed genomic landscape of pediatric ACT are results of our research during the second period of IPACTR.
After the amendment to the IPACTR protocol, we have enrolled 123 pediatric patients with ACT. These patients have been diagnosed in several countries outside the United States, including Brazil, Argentina, Chile, Honduras, Guatemala, Mexico, Spain, Greece, Poland, Saudi Arabia and England. For many of these patients, we have collected biological material, including tumor samples (frozen or paraffin blocks), blood from patients and parents and other relatives with cancer.
Because the strong association between ACT and germline TP53 mutations, St. Jude clinicians recommend TP53 sequencing analysis for children enrolled on IPACTR. The result is clinically relevant for the patient regarding treatment and follow-up guidelines. Before blood is obtained for this study, patients and families received information about the implications of a positive test (presence of a germline TP53 mutation) and whether they would like to know the results of this testing. St. Jude investigators also obtained blood and tumors samples for research purposes. These latter are not used for clinical decisions.
The study of a large series of cases from different countries suggests that the frequency of the germline TP53 mutations is about 50% to 60%. Few cases were demonstrated to be “de novo” TP53 mutations (n=8 cases), but the number could be underestimated because not all patients with germline TP53 have parents tested. Among ACT cases without germline TP53 mutations, study of tumor DNA showed that about 10% acquired TP53 mutations. For genetic counseling purposes, parents and family members of patients with TP53 mutations restricted to the tumor tissue do not need to be tested because there are no implications for inheritance.
Mutations in TP53 from our IPACTR cases include changes in one base pair (missense or nonsense mutation), as well as complex mutations (insertion, deletion and duplication). The range of mutations in our IPACTR cohort is illustrated below. All were previously reported in IARC database.
In our more recent publication, DNA of blood and tumor of 37 pediatric patients with ACT were analyzed by whole genome and whole exome sequencing. Germline TP53 mutations were present in 25 of the 37 patients (68%) in this cohort, 12 of which were the Brazilian founder R337H mutation.
Mutations were located in the DNA-binding domain as well others domains of TP53 supporting the concept that a subtle germline TP53 mutation may contribute to adrenocortical tumorigenesis by promoting chromosomal instability. In fact, those cases with detected TP53 mutations were those with higher chromosomal copy number alteration and high background mutation rates. However, chromosomal copy number gains was almost universal, and occurring in almost all cases, independent of TP53 status. In the figure, the light blue color represent chromosomal copy number gains.
Circos is a representative way to visualize data in a circular layout. It has changed the way the scientific community visualizes genomic alterations (changes in a genome over time or differences between two or more genomes). One timely application of this approach is creating effective figures showing how cancer genomes differ from healthy ones. Cancer samples can exhibit chaotic genomes. By definition, cancer results from an accumulation of genetic alterations that lead a cell population from initiation through promotion and then progression. These genetic alterations include subtle changes, such as small gains and losses and nucleotide substitutions, and more conspicuous alterations, such as changes in chromosomal copy number, translocations and high-level amplifications.
Our study involving the genetic landscape of pediatric ACT reveal cases with “quiet” genomes, as well cases with more complex or chaotic genomes. By visual inspection, it’s clear which ones accumulate more genomic alterations.
Another important finding in our genomic analysis of pediatric adrenocortical tumorigenesis refers to somatic mutations in a gene named ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked).
Several studies suggest that ATRX regulate the expression of other genes through a process known as chromatin remodeling. Chromatin is the complex of DNA and protein that packages DNA into chromosomes. Chromatin remodeling is one way gene expression is regulated during development. When DNA is tightly packed, gene expression is lower than when DNA is loosely packed. Mutations in ATRX have been shown to cause diverse changes in the pattern of DNA methylation, which may provide a link between chromatin remodeling, DNA methylation and gene expression in developmental processes.
In our recent study, it was observed few cases with disruptive ATRX mutation including nonsense or structural variations leading to complete or partial exons deletions. Of interest, all cases with ATRX mutation were associated with the presence of a TP53 mutation. Additionally, the association of TP53 and ATRX mutations was strongly associated with tumor weight, late-stage disease and poor prognosis. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. The association of TP53 and ATRX showing a massive and complex genome is illustrated in these circos plots. The association of both mutations cause more BMR, SNVs and SVs. Cases with TP53 mutation as exemplified by R337H could be very quiet as WT ones (with few other mutations or chromosomal alterations) are more complex as represented in the circos plots by a case with a DNA binding domain disruptive TP53 mutation.
The third more representative recurrent mutation observed in our recent project occurred at CTNNB1. Constitutive activation of Wnt signaling leading to accumulation and translocation of β-catenin to the nucleus is well known to be an initiator of transformation and tumorigenesis in multiple organ types. We have extended our study and identified mutations in β-catenin (CTNNB1) in our IPACTR cases. The image represents all CTNNB1 mutations observed, all of them located at exon 3. Overall, CTNNB1 mutations were observed in the context of WT TP53 sequence. However, few cases with somatic CTNNB1 had also acquired a TP53 mutation.
Chromosomal copy number alteration (CNA) is a hallmark of pediatric ACT. A mechanism of CNA observed in our cases was copy-neutral loss of heterozygosity or cn-LOH. Cn-LOH represents one example of a genomic abnormality in which no net change in copy number occurs, yet the abnormality can contribute to tumorigenesis. Copy-neutral LOH can occur due to duplication of one chromosome segment along with loss of the corresponding homologous region, so that the cell retains two copies derived from one parental source and no copies derived from the other parental source. The acquired homozygosity can contribute to tumorigenesis by activating potential oncogenes, unmasking mutated tumor suppressor genes or contributing to pathogenicity as a result of altered gene expression due to imprinting. This is the case for cn-LOH for chromosomes 11 and 17 in our cohort.
Most mammalian autosomal genes are expressed from both the maternally inherited and paternally inherited copies of the chromosomes. However, some genes are expressed in a parent-of-origin–manner. This phenomenon, known as genomic imprinting, is regulated by epigenetic mechanisms. Epigenetics refers to transmissible changes in gene expression that are not accompanied by a change in primary nucleotide sequence. There is considerable evidence of dysregulation of imprinted gene expression in human disorders of growth and development. This can occur via primary epigenetic alterations or genetic alterations that change the contribution of parental alleles, such as duplications, deletions and UPD. UPD refers to the presence of two chromosomal regions from one parent and none from the other.
The regulation of imprinted genes on chromosome 11p15.5 is functionally divided into two domains. Domain 1 contains the imprinted genes insulin-like growth factor 2 (IGF2) and H19, and a differentially methylated region, “DMR1” postulated to be an imprinting center. The maternally expressed H19 gene encodes an apparently untranslated transcript, and the IGF2 gene encodes a paternally expressed fetal growth factor. Up-regulation of IGF2 is thought to be important in the pathogenesis of Beckwith-Wiedemann syndrome (BWS) and a variety of tumors. Increased expression of IGF2 may be caused by paternal chromosome duplications of chromosome 11p15, paternal uniparental disomy (two copies of the paternal chromosome region), or alterations to differential methylation.
Domain 2 contains the CDKN1C (p57KIP2), a maternally expressed gene that encodes a cyclin-dependent kinase inhibitor and negatively regulates cell proliferation. In tumors, CDKN1C shows aberrant methylation associated with cell cycle dysregulation; however, this gene is rarely mutated in tumors. Interestingly, mutations in CDKN1C do cause BWS and are often associated with autosomal dominant inheritance of the syndrome. The maternally expressed KCNQ1 gene product forms part of a potassium channel. Six known translocation sites spanning the length of this gene are strongly associated with BWS. Intron 10 of the KCNQ1 gene contains another DMR called KvDMR1 or ‘DMR2’. The paternal allele is non-methylated, permitting the paternal expression of a long transcript called KCNQ1OT1, also known as LIT1. This transcript originates near DMR2 and is transcribed in an antisense direction to the KCNQ1 gene in which it originates.
Whole-genome sequencing (WGS) analysis in our pediatric ACT cases reveals cn-LOH for chromosome 11. More detailed analysis reveals a selective loss of maternal chromosome 11p15 (represented in purple). With exclusive presence of paternal chromosome, IGF2 is overexpressed and genes with maternal expression were lost. Of interest, few cases was showed to have UPD for chromosome 11 in germline tissue point out the importance to study all the abnormalities involving chromosome 11 in pediatric ACT.
Since cn-LOH for 11 and 17 were a common event in pediatric ACT, we decided to infer the temporal order of events. Our analysis demonstrates that cn-LOH of chromosomes 11p and 17p occurs early in adrenocortical tumorigenesis and precedes the accumulation of mutations in these regions. We propose that germline TP53 mutations may contribute to adrenocortical tumorigenesis by promoting chromosomal instability.
In this setting, aneuploid adrenocortical cells that experience chromosome 11p LOH and deregulation of imprinted genes on 11p15 may be selected and expanded via constitutive overexpression of IGF2, which encodes a potent mitogen and fetal growth-promoting protein. This mechanism is consistent with chromosome 11p abnormalities and IGF2 overexpression in all cases of ACT with germline TP53 mutations. Clones that undergo chromosome 17 LOH and lose wild-type TP53, become more unstable, accumulate additional mutations and are selected for further expansion. In support of this hypothesis, our temporal studies place cn-LOH of chromosomes 11p and 17 during early tumorigenesis, before the acquisition of widespread genomic alterations.
Our genomic analysis opens opportunities to improve the current tumor size-histology-disease stage prognostic scheme for pediatric ACT. Notably, tumors with both germline TP53 and somatic ATRX mutations (Group 1) were significantly associated with high tumor weight, advanced disease (COG stage III/IV) and poor event-free survival. Moreover, five of six patients in Group 1 had adverse events (relapse or death) consistent with the genomic findings indicative of an aggressive phenotype. Cases with germline TP53 mutations and wild-type ATRX (Group 2) are clinically and molecularly heterogeneous.
Although all cases in this group carried TP53 mutations (eight of the nine cases harbor the founder R337H), they had fewer genomic abnormalities, smaller tumors and generally much better clinical outcome than cases in Group 1. Finally, ACT cases with wild-type TP53 exhibit relatively simple genomes and, despite their large size in some cases, patients generally have a good outcome. Because of the small number of these cases, additional genomic studies will be needed to clarify the role of molecular changes in this subset of patients.