Identification of Clinical and Biologic Correlates Associated With Outcome in Children With Adrenocortical Tumors Without Germline TP53 Mutations: A St Jude Adrenocortical Tumor Registry and Children's Oncology Group Study

Abstract

Purpose The clinical features, pathogenesis, and outcomes in children with adrenocortical tumors (ACTs) without germline TP53 mutations have not been systematically studied. Herein, we describe these correlates and analyze their association with outcome. Patients and Methods Genomic DNA was analyzed for TP53, CTNNB1, CDKN1C, ATRX, and chromosome 11p15 abnormalities. β-catenin expression and Ki-67 labeling index (LI) were evaluated by immunostaining. Primary end points were progression-free (PFS) and overall survival. Results Median age of 42 girls and 18 boys was 3.3 years (range, 0.25 to 21.7 years). Complete resection (stages I and II) was achieved in 32 patients, and 28 patients had stage III or IV disease. Constitutional abnormalities of chromosome 11p15 occurred in nine of 40 patients, with six patients not showing phenotype of Beckwith-Wiedemann syndrome. Three-year PFS and overall survival for all patients were 71.4% and 80.5%, respectively. In single-predictor Cox regression analysis, age, disease stage, tumor weight, somatic TP53 mutations, and Ki-67 LI were associated with prognosis. Ki-67 LI and age remained significantly associated with PFS after adjusting for stage and tumor weight. Three-year PFS for 27 patients with Ki-67 LI ≥ 15% was 48.5% compared with 96.2% for 29 patients with Ki-67 LI < 15% (log-rank P = .002), and the rate of relapse increased by 24% with each 1-year increase in age at diagnosis (hazard ratio, 1.24; P = .0057). Conclusion Clinicopathologic features and outcomes of children with ACTs without germline TP53 mutations overlapped those reported for children with germline TP53 mutations. Our findings highlight the central role of genetic or epigenetic alterations on chromosome 11p15 in pediatric ACTs. Ki-67 LI is a strong prognostic indicator and should be investigated to improve the histologic classification of pediatric ACTs.

Fig 1.

Somatic TP53 mutations in patients with adrenocortical tumors (ACTs) without TP53 mutations. (A) Schematic diagram of acquired TP53 mutations. (B) Kaplan-Meier estimates for progression-free survival (B) in pediatric patients with ACTs with or without acquired TP53 mutations and (C) by TP53 status and disease stage. After discriminating for disease stage and including stage IV disease in the Cox regression model, presence of somatic TP53 mutations was no longer associated with outcome (P = .21). hom, homozygous.

Fig 2.

Somatic CTNNB1 mutations in patients with adrenocortical tumors (ACTs) without TP53 mutations. (A) Schematic diagram of activating CTNNB1 mutations. (B) Immunohistochemical analysis of β-catenin and yes-associated protein 1 (YAP1) in pediatric ACTs. YAP serves as a surrogate marker for β-catenin expression. Moderate nuclear immunostaining of (i) β-catenin and (ii) YAP in a tumor with the S45P mutation in CTNNB1. Strong nuclear immunoreactivity for (iii) β-catenin and (iv) YAP in a pediatric patient with unknown CTNNB1 molecular status. These results are indicative of Wnt signaling pathway activation. Size bars represent 50 μm. (C) Kaplan-Meier estimates for progression-free survival by CTNNB1 status (P = .80).

Fig 3.

(A) Hematoxylin and eosin staining and Ki-67 labeling index (LI) in pediatric adrenocortical tumors without TP53 mutations: (i, ii) tumor with a high (≥ 15%) Ki-67 labeling index (LI; red arrows indicate mitotic figures), (iii, iv) tumor with a low (< 5%) LI. Size bars represent 50 μm. (B) Kaplan-Meier estimates for progression-free survival (PFS) according to Ki-67 LI showing that Ki-67 LI < 15% was significantly associated with longer PFS (P = .002). (C) Kaplan-Meier estimates for PFS according to Ki-67 LI and disease stage. Ki-67 LI remained significantly associated with outcome after adjusting for disease stage (P = .0048).

Fig A1.

Chromosome 11p15 abnormalities as visualized by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and microsatellite analysis in DNA from pediatric patients with adrenocortical tumors (ACTs) without TP53 mutations. (A) MS-MLPA analysis of WT031 blood DNA showing (upper panel) normal copy number of chromosome 11p15 and (lower panel) gain of methylation at imprinting control region 1 (IC1) with loss of methylation at imprinting control region 2 (IC2) indicating uniparental disomy. (B) WT002 blood DNA showing partial gain of methylation at IC1 and partial loss of methylation at IC2. (C) MS-MLPA analysis of WT023 blood DNA showing loss of methylation at IC2. (D) Representative microsatellite marker analysis of parents (blood) and patient (blood and tumor) DNA. The father (F) is homozygous (185/185) and the mother (M) heterozygous (192/194) for the D11S4046 marker. Patient blood DNA shows inheritance of alleles 185 from father and 194 from mother; (bottom panel) allele 194 (maternal origin) is selected against in the tumor. (E) Western blot analysis performed with 50 µg of protein with goat polyclonal antihuman antibody directed against insulin-like growth factor 2 (IGF2; 1:500 dilution; Sigma-Aldrich, St Louis, MO) as previously reported. β-actin (1:2,000; Sigma-Aldrich) was used as the loading control. This analysis included patients with 11p15 abnormalities on germline (n = 6) and with germline-mutated TP53, as indicated. Levels of IGF2 were higher in samples than in control (two normal adrenocortical tissues obtained during nephrectomy for Wilms tumor) and were not affected by genotype (wild-type or mutated TP53) or histology (carcinoma, adenoma, or undetermined [Und]). ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; CNV, copy number variation; al, allele; ht, height.

Fig A2.

Association of PTTG1, BUB1B, AURKB, HLA-DPA1, and MKI67 mRNA expression with progression-free survival (PFS). mRNA expression was obtained from Gene Expression Omnibus databases GSE76019 and GSE76021. Plots represent mRNA expression according to each probe set for those selected genes. Higher expression of AURKB, BUB1B, PTTG1, and MKI67 was significantly associated with worse PFS.

Fig A3.

Correlations of mRNA expression of selected cell-cycle genes within the (A) Children’s Oncology Group (GSE76019) and International Pediatric Adrenocortical Tumor Registry cohorts (GSE76021). The panels provide the Spearman correlation and P value for each pair of cell-cycle gene probe sets in both cohorts. Each entry in the upper triangle gives the Spearman correlation of the expression of the pair of genes indicated by the row and column labels. Each entry in the lower triangle gives the P value for the association of the pair of genes indicated by the row and column labels. NA, not applicable.

Fig A4.

Representative plots of mRNA expression of MKI67, AURKB, BUB1B, and PTTG1 according to TP53 status from adrenocortical tumor (ACT) samples in available public databases (Gene Expression Omnibus databases GSE76019 and GSE7602). Expression levels of PTTG1, BUB1B, and AURKB were significantly higher in ACTs with TP53 mutations than in those without. Expression of IGF2 and MKI67 was not significantly different,according to TP53 status. Expression levels are represented on the positive quantile transformation (PQT) scale.