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Pintotable1.xlsxTable S1a. Cohort characteristicsTable S1b. Frequencies and features of TP53 variants at codon 125 in public databasesTable S1c. Annotation and interpretation of TP53 variants at codon 125 and surrounding basesTable S1d. Splice site predictionTable S1e. Primers sequences for mutagenesis constructs and cDNA sequencing Table S1f. STR profiling of Saos-2 and H1299 cell lines

S1. Proband pedigrees. S2. TP53 status as determined by Multiplex Ligation Probe Amplification for Saos-2 and H1299 cell lines. S3. TP53 altering splice variants in the (a) IARC database (b) PCGP database. (c) Total germline TP53 variants observed in IPACTR and COG studies Circles represent number of cases. S4. Chromatogram representing the constructs for the TP53 variants included in this study. S5. p53 Immunostaining of pediatric tumors associated with germline mutations at codon 125 and surrounding sequences.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up.Significance:A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

PDF file - 285K, Identification of TP53-G245C mutation in the germline, primary ACC and xenograft tumor

Gene set enrichment analysis of control and IR-treated WT and p53-R334H thymocytes.

Supplementary Figures 1-4. Supplementary Figure S1: Full Pedigrees from TP53 c.1000G>C;p.G334R clinically ascertained families. Fam ID allows reference to Table 1; Supplementary Figure S2: Constructs encoding p53 wild-type (TP53-WT), p53-R175H (TP53-R175H), and p53-G334R (c.1000G>A and c.1000G>C) were transiently transfected into p53 deficient Saos-2 cells; Supplementary Figure S3: p53 levels after treatment with 10uM cisplatin (CDDP); Supplementary Figure S4: Heatmap showing fold activation of a canonical p53 signature at 24 hours post nutlin with wildtype versus mutant lymphoblastoid cell lines.

Mouse Cohorts and Associated Pathology

Purpose: Pediatric adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. Conventional chemotherapeutic agents have shown limited utility and are largely ineffective in treating children with advanced ACC. The lack of cell lines and animal models of pediatric ACC has hampered the development of new therapies. Here we report the establishment of the first pediatric ACC xenograft model and the characterization of its sensitivity to selected chemotherapeutic agents.Experimental Design: A tumor from an 11-year-old boy with previously untreated ACC was established as a subcutaneous xenograft in immunocompromised CB17 scid−/− mice. The patient harbored a germline TP53 G245C mutation, and the primary tumor showed loss of heterozygosity with retention of the mutated TP53 allele. Histopathology, DNA fingerprinting, gene expression profiling, and biochemical analyses of the xenograft were conducted and compared with the primary tumor and normal adrenal cortex. The second endpoint was to assess the preliminary antitumor activity of selected chemotherapeutic agents.Results: The xenograft maintained the histopathologic and molecular features of the primary tumor. Screening the xenograft for drug responsiveness showed that cisplatin had a potent antitumor effect. However, etoposide, doxorubicin, and a panel of other common cancer drugs had little or no antitumor activity, with the exception of topotecan, which was found to significantly inhibit tumor growth. Consistent with these preclinical findings, topotecan as a single agent in a child with relapsed ACC resulted in disease stabilization.Conclusion: Our study established a novel TP53-associated pediatric ACC xenograft and identified topotecan as a potentially effective agent for treating children with this disease. Clin Cancer Res; 19(7); 1740–7. ©2013 AACR.

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li–Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS.Significance:TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish–predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

PDF file - 73K, Expression of p53 and IGF2 in normal adrenal cortex and xenograft tumor

Supplemental Materials and Methods and Supplementary Figure Legends

This study comprehensively evaluated the landscape of genetic and epigenetic events that predispose to synchronous bilateral Wilms tumor (BWT). We performed whole exome or whole genome sequencing, total-strand RNA-seq, and DNA methylation analysis using germline and/or tumor samples from 68 patients with BWT from St. Jude Children’s Research Hospital and the Children’s Oncology Group. We found that 25/61 (41%) of patients evaluated harbored pathogenic or likely pathogenic germline variants, with WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%) and the BRCA-related genes (5%) BRCA1, BRCA2, and PALB2 being most common. Germline WT1 variants were strongly associated with somatic paternal uniparental disomy encompassing the 11p15.5 and 11p13/WT1 loci and subsequent acquired pathogenic CTNNB1 variants. Somatic coding variants or genome-wide copy number alterations were almost never shared between paired synchronous BWT, suggesting that the acquisition of independent somatic variants leads to tumor formation in the context of germline or early embryonic, post-zygotic initiating events. In contrast, 11p15.5 status (loss of heterozygosity, loss or retention of imprinting) was shared among paired synchronous BWT in all but one case. The predominant molecular events for BWT predisposition include pathogenic germline variants or post-zygotic epigenetic hypermethylation at the 11p15.5 H19/ICR1 locus (loss of imprinting). This study demonstrates that post-zygotic somatic mosaicism for 11p15.5 hypermethylation/loss of imprinting is the single most common initiating molecular event predisposing to BWT. Evidence of somatic mosaicism for 11p15.5 loss of imprinting was detected in leukocytes of a cohort of BWT patients and long-term survivors, but not in unilateral Wilms tumor patients and long-term survivors or controls, further supporting the hypothesis that post-zygotic 11p15.5 alterations occurred in the mesoderm of patients who go on to develop BWT. Due to the preponderance of BWT patients with demonstrable germline or early embryonic tumor predisposition, BWT exhibits a unique biology when compared to unilateral Wilms tumor and therefore warrants continued refinement of its own treatment-relevant biomarkers which in turn may inform directed treatment strategies in the future.

PURPOSE Adrenocortical carcinoma (ACC) is a rare aggressive pediatric malignancy with distinct biology. Its treatment follows the principles developed for adults; pediatric-specific studies are scarce. PATIENTS AND METHODS Prospective single-arm risk-stratified interventional study. Study objectives were (1) to describe the outcome of patients with stage I ACC treated with adrenalectomy alone; (2) to describe the outcome of stage II patients (completely resected > 200 cc or > 100 g) treated with adrenalectomy and retroperitoneal lymph node dissection; and (3) to describe the outcome of patients with stage III or IV treated with mitotane and chemotherapy. RESULTS Between September 2006 and May 2013, 78 patients (77 eligible, 51 females) were enrolled. The 5-year event-free survival estimates for stages I (24 patients), II (15 patients), III (24 patients), and IV (14 patients) were 86.2%, 53.3%, 81%, and 7.1%, respectively. The corresponding 5-year overall survival estimates were 95.2%, 78.8%, 94.7%, and 15.6%, respectively. On univariate analysis, age, stage, presence of virilization, Cushing syndrome, or hypertension, germline TP53 status, and presence of a somatic ATRX mutation were associated with outcome. On multivariable analysis, only stage and age were significantly associated with outcome. The probabilities of mitotane and chemotherapy feasibility events were 10.5% and 31.6%, respectively. CONCLUSION Outcome for children with stage I ACC is excellent with surgery. Outcome for patients with stage II disease is inferior despite retroperitoneal lymph node dissection. Patients with stage III ACC have an excellent outcome combining surgery and chemotherapy. Patients with stage IV ACC are older and have a poor outcome; new treatments should be explored for this high-risk group. The combination of mitotane and chemotherapy as prescribed in ARAR0332 resulted in significant toxicity; one third of patients with advanced disease could not complete the scheduled treatment.

Outcomes for pediatric patients with unresectable, metastatic, or relapsed adrenocortical carcinoma (ACC) are dismal despite aggressive management with multimodality therapies. CAR T cell therapy (CAR T) is a promising approach to improve outcomes for patients with this disease. However, progress is hindered by a lack of targetable surface antigens and limited availability of relevant preclinical models. B7-H3 is a promising immunotherapeutic target but little is known about its expression in pediatric ACC. Thus, we sought to i) evaluate B7-H3 expression in primary pediatric ACC samples and ii) assess B7-H3-CAR T activity in two novel pediatric patient-derived xenograft (PDX) models. First, we assessed B7-H3 expression in 44 pediatric adrenocortical tumors (ACTs) using a CLIA-approved immunohistochemistry (IHC) assay. ACTs had a range of B7-H3 positivity, including 24 (55%) with expression above the threshold for eligibility on our active B7-H3-CAR T clinical trial (H-score ≥ 100; NCT04897321). Univariate analyses demonstrated B7-H3 expression was correlated with the presence of germline TP53 mutations and poor prognostic factors including the inability to achieve complete resection, Cushing phenotype, and the absence of TP53 by IHC. Given a dearth of in vivo models, we next established two clinically relevant pediatric ACC PDX models. SJ-ACC4 was derived from a metastatic tumor and had somatic mutations in TP53 (p.C135Lfs*14) and CTNNB1 (p.S45F). SJACT030812_X1 (www.stjude.org/cstn) was derived from a primary tumor and had a germline TP53 mutation (p.R110Pfs*39). Both had partial ATRX deletions (exons 11-30; 3-28), uniparental disomy at chromosome 11p15, and were B7-H3-positive. Notably, SJ-ACC4 was resistant to a battery of chemotherapeutic agents. To evaluate B7-H3-CAR T anti-ACC activity in vivo, SJ-ACC4 tumor fragments were implanted subcutaneously in NOD SCID gamma (NSG) mice and treated with B7-H3-CAR or control T cells. Strikingly, B7-H3-CAR T eradicated 100% of SJ-ACC4 tumors and mice survived long term compared to controls that met endpoints within 35 days (n=8 per group, p< 0.0001). Finally, we asked if B7-H3-CAR T had antitumor activity in an orthotopic pediatric ACC PDX model. SJACT030812_X1 cells were injected into the para-adrenal region of NSG mice under ultrasound guidance and monitored by serial volumetric ultrasonography. B7-H3-CAR T again had significant antitumor activity, and mice survived long-term compared to controls that met endpoints within 42 days (n=5 per group, p< 0.01). In conclusion, B7-H3 is expressed in a majority of pediatric ACTs and B7-H3-CAR T have potent anti-ACC activity. These findings provide a strong rationale for clinically evaluating B7-H3-CAR T for pediatric patients with chemotherapy-resistant ACC. Additionally, the newly established pediatric ACC PDX models provide a valuable resource for investigating different aspects of this rare and aggressive disease. Citation Format: Rebecca Epperly, Emilia M. Pinto, Teresa Santiago, Heather Sheppard, Michael R. Clay, Christopher Morton, Phuong Nguyen, Yinmei Zhou, Mary Woolard, Carla O'Reilly, Julie Justice, Walter Akers, Melissa Johnson, Asa Karlstrom, Cheng Cheng, Gerard Zambetti, Andrew Davidoff, Raul Ribeiro, Christopher DeRenzo. B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1197.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. Significance: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Two major concerns associated with cancer development in Paraná state, South Brazil, are environmental pollution and the germline

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li–Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS. Significance: TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish–predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

The p53 tumor suppressor transcriptionally regulates a myriad of genes involved in cell cycle control, DNA repair, cell survival, and cell metabolism and represents one of the most well‐studied inhibitors of tumorigenesis. Since the discovery of TP53 in 1979, somatic mutations have been shown to be extremely common; more than 50% of human cancers carry loss‐of‐function mutations in TP53. Inherited or germline TP53 mutations are rare and are involved in complex hereditary cancer predisposition disorders, and affected family members can develop diverse tumor types and multiple primary cancers at young ages. In Brazil, a fascinating history of p53 and cancer predisposition began in the year 2000 with identification of the TP53 p.R337H mutation in close association with the development of adrenocortical tumors. In these past 20 years, much has been learned about the genetics and biochemistry of this mutation, which is widespread in Brazil because of a founder effect. This review highlights the contributions of TP53 p.R337H research over the last 20 years, the findings of which have sparked passionate debate among researchers worldwide, to understanding cancer predisposition in Brazilian individuals and families., This review highlights the impact of TP53 p.R337H research in cancer predisposition studies in the Brazilian population. In addition, these studies serve as a model for carriers of hypomorphic TP53 alleles.

Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.

Context Adrenocortical carcinoma (ACC) is a rare endocrine malignancy that affects patients across the age spectrum. Although the overall survival in patients with ACC is poor, there is significant heterogeneity in terms of outcomes, presentation, and underlying genetic drivers. Evidence Acquisition This review is based on the evidence collected from primary research studies, expert reviews, and published guidelines. The studies were identified through PubMed search with key words “adrenocortical carcinoma,” “prognosis,” “pathology,” and “genetics.” The PubMed search was complemented by authors’ expertise, research, and clinical experience in the field of ACC. Evidence Synthesis Identification of biomarkers has been critical to gain better insight into tumor behavior and to guide therapeutic approach to patients. Tumor stage, resection status, and Ki67 are pathological tumor characteristics that have been identified as prognosticators in patients with ACC. Cortisol excess also correlates with worse prognosis. Clinical and histopathological characteristics help stratify patient outcomes, yet still up to 25% of patients have a different outcome than predicted. To bridge this gap, comprehensive genomic profiling studies have characterized additional profiles that correlate with clinical outcomes. In addition, studies of clinically applicable molecular markers are under way to further stratify outcomes in patients with ACC tumors. Conclusions Clinical predictors in combination with pathological markers play a critical role in the approach to patients with ACC. Recent advances in genetic prognosticators will help extend the stratification of these tumors and contribute to a personalized therapeutic approach to patients with ACC.

Germline TP53 splicing variants are uncommon, and their clinical relevance is unknown. However, splice-altering variants at exon 4–intron 4 junctions are relatively enriched in pediatric adrenocortical tumors (ACT). Nevertheless, family histories of cancer compatible with classic Li-Fraumeni syndrome are rarely seen in these patients. We used conventional and in silico assays to determine protein stability, splicing, and transcriptional activity of 10 TP53 variants at exon 4–intron 4 junctions and analyzed their clinical correlates. We reviewed public databases that report the impact of TP53 variants in human cancer and examined individual reports, focusing on family history of cancer. TP53 exon 4–intron 4 junction germline variants were identified in 9 of 75 pediatric ACTs enrolled in the International Pediatric Adrenocortical Tumor Registry and Children's Oncology Group ARAR0332 study. An additional eight independent TP53 variants involving exon 4 splicing were identified in the Pediatric Cancer Genome Project (n = 5,213). These variants resulted in improper expression due to ineffective splicing, protein instability, altered subcellular localization, and loss of function. Clinical case review of carriers of TP53 exon 4–intron 4 junction variants revealed a high incidence of pediatric ACTs and atypical tumor types not consistent with classic Li-Fraumeni syndrome. Germline variants involving TP53 exon 4–intron 4 junctions are frequent in ACT and rare in other pediatric tumors. The collective impact of these germline TP53 variants on the fidelity of splicing, protein structure, and function must be considered in evaluating cancer susceptibility. Implications: Taken together, the data indicate that splice variants at TP53 codon 125 and surrounding bases differentially impacted p53 gene expression and function.

Li-Fraumeni syndrome (LFS), caused by the germline mutations in the TP53 gene, leads to significant lifetime risk to cancer in the central nervous system. Recognition of LFS, and elucidating its underlying cause has had a remarkable effect on our knowledge of the biology of brain tumors and represents a significant opportunity for cancer surveillance and screening. In this review, we discuss the historical context of the LFS with an emphasis on the clinicopathologic implications in clincal diagnosis, germline testing, and clinical management of brain tumor patients.

Childhood adrenocortical tumors (ACTs) are rare, representing ∼0.2% of all pediatric malignancies and having an incidence of 0.2-0.3 new cases per million per year in the United States, but incidences are remarkably higher in Southern Brazil. At diagnosis, most children show signs and symptoms of virilization, Cushing syndrome, or both. Less than 10% of patients with ACT exhibit no endocrine syndrome at presentation, although some show abnormal concentrations of adrenal cortex hormones. Pediatric ACT is commonly associated with constitutional genetic and/or epigenetic alterations, represented by germline TP53 mutations or chromosome 11p abnormalities. Complete tumor resection is required to achieve cure. The role of chemotherapy is not established, although definitive responses to several anticancer drugs are documented. For patients undergoing complete tumor resection, favorable prognostic factors include young age, small tumor size, virilization, and adenoma histology. Prospective studies are necessary to further elucidate the pathogenesis of ACT and improve patient outcomes.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

The XAF1-E134* variant increases the cancer risk for carriers of the TP53-R337H allele., Cancer risk is highly variable in carriers of the common TP53-R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma (P = 0.003) and subsequent malignancies (P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1-E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1-E134* and TP53-R337H mutations leads to a more aggressive cancer phenotype than TP53-R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers.

Multifocal synchronous or metachronous atypical teratoid rhabdoid tumors (ATRTs) and non-central nervous system malignant rhabdoid tumors (extra-CNS MRTs) are rare cancers. We reviewed the clinical and radiologic characteristics of affected patients seen at our institution. Genotyping and analysis of copy number abnormalities (CNAs) in SMARCB1 were performed in germline and tumor samples. Tumor samples underwent genome-wide DNA methylation and CNA analysis. The median age at diagnosis of 21 patients was 0.6 years. Two-thirds of ATRTs and extra-CNS MRTs were diagnosed synchronously. Although kidney tumors predominated, including two patients with bilateral involvement, at least 30% of cases lacked renal involvement. Histopathologic review confirmed MRTs in all cases and INI1 expression loss in all tumors tested. Fourteen (78%) of 18 patients tested had heterozygous germline SMARCB1 abnormalities. At least one allelic SMARCB1 abnormality was confirmed in 81 and 88% of ATRTs and extra-CNS MRTs, respectively. Unsupervised hierarchical clustering analysis of DNA methylation in 27 tumors and comparison with a reference group of 150 ATRTs classified the CNS tumors (n = 14) as sonic hedgehog (64%), tyrosinase (21%), and MYC (14%). The MYC subgroup accounted for 85% of 13 extra-CNS MRTs. Of 16 paired ATRTs and extra-CNS MRTs, the tumors in seven of eight patients showed a different pattern of genome-wide DNA methylation and/or CNAs suggestive of non-clonal origin. CNS and extra-CNS tumors had an identical SMARCB1 amplification (n = 1) or very similar DNA methylation pattern (n = 1) suggestive of clonal origin. All patients died of tumor progression. The clinical and molecular characteristics of multifocal ATRTs and extra-CNS MRTs are heterogeneous with most patients harboring a cancer predisposition. Although independent tumor origin was confirmed in most cases, metastatic spread was also documented. The recognition of their distinct molecular characteristics is critical in selecting new biologic therapies against these deadly cancers.

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.

PURPOSE Pediatric adrenocortical carcinomas (ACCs) are aggressive; the overall survival of patients with ACCs is 40%-50%. Appropriate staging and histologic classification are crucial because children with incomplete resections, metastases, or relapsed disease have a dismal prognosis. The clinical course of pediatric adrenocortical tumors (ACTs) is difficult to predict using the current classification schemas, which rely on subjective microscopic and gross macroscopic variables. Recent advances in adult ACT studies have revealed distinct DNA methylation patterns with prognostic significance that have not been systematically interrogated in the pediatric population. PATIENTS AND METHODS We performed DNA methylation analyses on 48 newly diagnosed ACTs from the International Pediatric Adrenocortical Tumor Registry and 12 pediatric adrenal controls to evaluate for distinct methylation groups. Pediatric methylation data were also compared systematically with the adult ACC cohort from The Cancer Genome Atlas (TCGA). RESULTS Two pediatric ACT methylation groups were identified and showed differences in selected clinicopathologic and outcome characteristics. The A1 group was enriched for CTNNB1 variants and unfavorable outcome. The A2 group was enriched for TP53 germline variants, younger age at onset, and favorable outcome. Pediatric ACT methylation groups were maintained when International Pediatric Adrenocortical Tumor Registry cohort data were combined with TCGA cohort data. The CpG-island hypermethylator phenotype characterizing the TCGA cohort was not identified in the pediatric patients. When methylome findings were combined with independent histopathologic review using the Wieneke criteria, a high-risk population was identified with uniform fatal outcome. CONCLUSION Our results indicate DNA methylation analysis can enhance current diagnostic algorithms. A combination of methylation and histologic classification produced the strongest prediction model and may prove useful in future risk-adapted therapeutic trials.

Background Genome-wide association studies (GWASs) have enriched the fields of genomics and drug development. Adrenocortical carcinoma (ACC) is a rare cancer with a bimodal age distribution and inadequate treatment options. Paediatric ACC is frequently associated with TP53 mutations, with particularly high incidence in Southern Brazil due to the TP53 p.R337H (R337H) germline mutation. The heterogeneous risk among carriers suggests other genetic modifiers could exist. Methods We analysed clinical, genotype and gene expression data derived from paediatric ACC, R337H carriers, and adult ACC patients. We restricted our analyses to single nucleotide polymorphisms (SNPs) previously identified in GWASs to associate with disease or human traits. Results A SNP, rs971074, in the alcohol dehydrogenase 7 gene significantly and reproducibly associated with allelic differences in ACC age-of-onset in both cohorts. Patients homozygous for the minor allele were diagnosed up to 16 years earlier. This SNP resides in a gene involved in the retinoic acid (RA) pathway and patients with differing levels of RA pathway gene expression in their tumours associate with differential ACC progression. Conclusions These results identify a novel genetic component to ACC development that resides in the retinoic acid pathway, thereby informing strategies to develop management, preventive and therapeutic treatments for ACC.

Individuals harboring a germline TP53 mutation have a high lifetime risk of a broad spectrum of cancers, but the age of onset, tumor types and penetrance vary among carriers. The TP53-R337H variant is common in South and Southeastern Brazil due to founder effect and detected in 0.3% of the general population. TP53-R337H predisposes children to adrenocortical tumors (ACTs) and adults to various other Li-Fraumeni syndrome (LFS)-associated core cancers. Assessment of the family history of cancer in TP53-R337H carriers has revealed families fulfilling the classic criteria for LFS as well as those showing an apparently sporadic pattern of cancer presentation, suggesting that genetic and/or environmental factors modulate cancer penetrance in TP53-R337H carriers. Given the incomplete penetrance and heterogeneous familial cancer pattern in TP53-R337H carriers, we hypothesized that additional allelic variants in cis could act as modifiers of TP53-R337H activity and contribute to cancer risk. We used whole genome sequencing for fine mapping analysis of 2-Mb region flanking TP53 locus in ACT samples. Selected common and rare variants were genotyped in 204 unrelated TP53-R337H cancer patients and a control group of 67,359 newborns. Segregation analysis was performed by analyzing 682 family members. Cancer risk was ascertained, and in-vitro functional analysis was performed. We identified a common shared haplotype containing the E134* variant in the XAF1 gene in a subset (42%) of ACT patients harboring the TP53-R337H mutation. This rare variant (minor allele frequency worldwide= 0.004) was identified in 70% of TP53-R337H carrying chromosomes. Co-segregation of both variants was observed in 79% of cancer patients, however, it was significantly enriched in individuals with sarcoma (odds ratio, 5.6; 95% CI, 1.3 to 50.8; P=0.010) and multiple primary malignancies (odds ratio, 3.1; 95% CI, 1.0 to 12.9; P=0.033). Breast cancer patients harboring the extended haplotype developed multiple primary tumors three times more frequently than patients with the TP53-R337H-only haplotype. In vitro studies demonstrated that wild-type XAF1 enhances wild-type and p53-R337H transactivation whereas XAF1-E134* markedly attenuated this activity, thereby establishing a functional interaction between p53 and XAF1. These findings demonstrate that a tolerated and common polymorphism (XAF1-E134*) cooperates with the low-penetrance TP53-R337H variant to modulate cancer phenotype and highlights the potential biological contribution of additional variants that act in cis to influence the cancer phenotype conferred by low-penetrance TP53 alleles. Understanding the nature of the germline TP53 mutation and the contribution of other modifiers that influence its function, such as XAF1-E134*, will have implications for genetic counseling, surveillance and clinical management of these patients. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Pediatric adrenocortical tumors (ACTs) are heterogeneous and have a poor prognosis. Overall survival is around 60%. In pediatric ACT that is managed only with surgery, features associated with adverse outcome include age greater than 3 years, non-functioning tumors, and metastatic (Stage IV) or residual tumor after surgery (Stage III). These factors individually do not account for most treatment failures, however. As current histopathologic classification systems are hindered by poor reproducibility and lack of predictive value, new prognostic markers are critically needed. In adults, methylation profiling can distinguish benign from malignant ACT, but similar data on pediatric ACT have not been reported. We performed DNA methylation profiling (MethylationEPIC BeadChip Array) on 52 pediatric ACTs and 13 normal pediatric adrenocortical tissue samples. The median age of 38 girls and 14 boys was 2.8 years (range, 0.3-17). ACTs were histologically classified as carcinoma (n=29), adenoma (n=10), and indeterminate (n=13). Overall survival was used as a primary endpoint. Unsupervised analysis of the 20,000 most variably methylated probes segregated cases into two distinct subgroups (designated A1 and A2). Differentially methylated genes (q-value 4 years (HR = 16.8, p = 0.008) and A1 methylation group (HR 7.7, p=0.0011) were each significantly correlated with overall survival. A1 subgroup remained significantly associated with overall survival in a series of models that adjusted for features including: clinical diagnosis, histopathologic characteristics, Ki67 index, and advanced stage. Methylation profiling provides prognostic significance that is independent of most current predictive variables, and in the future, may serve as a powerful biomarker in the prognostication of pediatric ACT.

Purpose: Histologic markers that differentiate benign and malignant pediatric adrenocortical tumors are lacking. Previous studies have implicated an association of MHC class II expression with adrenocortical tumor prognosis. Here, we determined the expression of MHC class II as well as the cell of origin of these immunologic markers in pediatric adrenocortical tumor. The impact of MHC class II gene expression on outcome was determined in a cohort of uniformly treated children with adrenocortical carcinomas. Experimental Design: We analyzed the expression of MHC class II and a selected cluster of differentiation genes in 63 pediatric adrenocortical tumors by Affymetrix Human U133 Plus 2.0 or HT HG-U133+PM gene chip analyses. Cells expressing MHC class II were identified by morphologic and immunohistochemical assays. Results: MHC class II expression was significantly greater in adrenocortical adenomas than in carcinomas (P = 4.8 ×10−6) and was associated with a higher progression-free survival (PFS) estimate (P = 0.003). Specifically, HLA-DPA1 expression was most significantly associated with PFS after adjustment for tumor weight and stage. HLA-DPA1 was predominantly expressed by hematopoietic infiltrating cells and undetectable in tumor cells in 23 of 26 cases (88%). Conclusions: MHC class II expression, which is produced by tumor-infiltrating immune cells, is an indicator of disease aggressiveness in pediatric adrenocortical tumor. Our results suggest that immune responses modulate adrenocortical tumorigenesis and may allow the refinement of risk stratification and treatment for this disease. Clin Cancer Res; 22(24); 6247–55. ©2016 AACR.

Phosphodiesterases (PDEs) form a superfamily of enzymes that catalyze the hydrolysis of cyclic nucleotides adenosine 3&prime, 5&prime, cyclic monophosphate (cAMP) and guanosine 3&prime, cyclic monophosphate (cGMP) to their inactive 5&prime, monophosphates. cAMP plays a critical role as a second messenger in endocrine tissues, and activation of cAMP signaling has been reported in endocrine tumors. Germline variants in PDEs have been associated with benign cortisol-secreting adrenocortical adenomas and testicular germ cell cancer but not adrenocortical carcinoma. We performed whole genome sequencing (WGS) and whole exome sequencing (WES) of paired blood and tumor samples from 37 pediatric adrenocortical tumors (ACTs). Germline inactivating variants in PDEs were observed in 9 of 37 (24%) patients. Tumor DNA analysis revealed loss of heterozygosity, with maintenance of the mutated allele in all cases. Our results suggest that germline variants in PDEs and other regulators of the cAMP-signaling pathway may contribute to pediatric adrenocortical tumorigenesis, perhaps by cooperating with germline hypomorphic mutant TP53 alleles and uniparental disomy of chromosome 11p15 (Beckwith&ndash, Wiedemann syndrome).

Multifocal synchronous/metachronous ATRTs and extra-CNS MRTs are rare. We reviewed clinicoradiologic characteristics of affected patients. Genotyping and copy number abnormality (CNA) analysis in SMARCB1 were performed in germline and tumor samples. Tumor samples underwent DNA methylation and CNA analysis. Median age at diagnosis (n=21 patients) was 0.6 years. Two-thirds of patients had synchronous tumors. Although kidney tumors predominated, at least 30% of cases lacked renal involvement. Histopathologic review confirmed MRTs in all cases and INI1 expression loss in all tumors tested. Fourteen (78%) of 18 patients tested had heterozygous germline SMARCB1 abnormalities. Mono and biallelic SMARCB1 abnormalities were confirmed in at least 50% and 80% of tumors, respectively. Unsupervised hierarchical clustering analysis of DNA methylation in 27 tumors and comparison to 150 reference ATRTs classified 14 multifocal ATRTs as SHH (64%), tyrosinase (21%), and MYC (14%). The MYC subgroup accounted for 85% of 13 extra-CNS MRTs. Of 16 paired ATRTs and extra-CNS MRTs, 7 of 8 patients had tumors with different patterns of DNA methylation and/or CNAs suggestive of non-clonal origin. CNS and extra-CNS tumors had an identical SMARCB1 amplification (n=1) or similar DNA methylation pattern (n=1) suggestive of clonal origin. All patients died of tumor progression. The clinical and molecular characteristics of multifocal ATRTs and extra-CNS MRTs are heterogeneous with most patients harboring a cancer predisposition. Although independent tumor origin was confirmed in most cases, metastatic spread was also documented. The recognition of their distinct molecular characteristics is critical in selecting new biologic therapies against these deadly cancers.

The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes., The progress in pre-clinical drug discovery for Wilms tumor (WT) is limited by a lack of disease models. Here, the authors develop 45 heterotopic WT patient-derived xenografts including several anaplastic models that recapitulate the biological heterogeneity of WT, and propose this as a resource for evaluating future therapeutics for WT.

Laron syndrome (LS) is a genetic disorder caused by mutations in the growth hormone receptor (GHR) gene. The most frequent GHR mutation is E180splice (rs121909360), which was initially found in an inbred population of Spanish descent in Ecuador and subsequently in Israel, Brazil, Chile, and the United States. The aim of the present study is to determine if the E180splice mutation arose from a common origin. We studied 22 patients with LS from Ecuador, Israel (of Moroccan origin), Brazil, Chile, and the United States (of Mexican origin) who were homozygous for the E180splice mutation and compared them to control individuals for markers surrounding the GHR, intragenic polymorphisms, and Y-chromosome STR. An identical haplotype was found in all but one of the subjects carrying the E180splice mutation: D5S665: 150/150; D5S2082: 192/192; D5S2087: 246/246; rs6179 G/G; and rs6180 C/C. One patient differed from the others only at D5S2082 (168/192). This haplotype is rare (~1%) in control individuals and confirmed that the E180splice-associated haplotype was not derived from independent origins but represented recombination from a common ancestor. The analysis of paternal lineage markers showed that 50% belong to haplogroup R1b (found in Portugal and Spain) and 40% to haplogroups J and E (typical in the Middle East and in Eastern European Jews). The germline E180Splice mutation appears to have originated from a single common ancestor. The presence of Y-chromosome markers associated with Sephardic populations in persons harboring the E180splice mutation provides genetic evidence in support of the historical tracking of the exodus of this specific population.

Li–Fraumeni syndrome (LFS) is a highly penetrant cancer predisposition syndrome caused by heterozygous germline mutations in the TP53 gene. Although more than 200 missense and null TP53 mutations are well established as disease-causing, little is known about the pathogenicity and cancer risks associated with small in-frame deletions. This leads to challenges in variant classification and subsequent difficulty making a molecular diagnosis. We report the genetic testing process for a pediatric patient diagnosed with an undifferentiated high-grade brain tumor following his mother's diagnosis of early-onset bilateral breast cancer. Sequential testing revealed that both harbored a heterozygous three-nucleotide deletion in exon 7 of TP53 (c.764_766delTCA; I255del), which was classified as a variant of uncertain significance. Because the maternal family history was void of any other LFS spectrum tumors, additional information was needed to effectively classify the variant. Targeted TP53 testing of the patient's maternal grandparents confirmed that neither carried the variant; this new de novo data upgraded the variant classification to likely pathogenic. To assess the impact of this mutation on the encoded p53 protein, additional in vitro analyses were performed. Structural modeling predicted that the deletion of isoleucine at codon 255 would disrupt the architecture of the DNA-binding domain, suggesting that it might negatively impact p53 function. Consistent with this notion, the I255del mutant protein exhibited significantly impaired transcriptional activity and greatly reduced growth suppressive properties, similar to more well-characterized LFS-associated p53 mutants. This report illustrates the importance of seeking additional evidence to assign proper pathogenicity classification, which enables optimal genetic counseling and medical management of individuals with LFS and their at-risk relatives.

Purpose: Pediatric adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. Conventional chemotherapeutic agents have shown limited utility and are largely ineffective in treating children with advanced ACC. The lack of cell lines and animal models of pediatric ACC has hampered the development of new therapies. Here we report the establishment of the first pediatric ACC xenograft model and the characterization of its sensitivity to selected chemotherapeutic agents. Experimental Design: A tumor from an 11-year-old boy with previously untreated ACC was established as a subcutaneous xenograft in immunocompromised CB17 scid−/− mice. The patient harbored a germline TP53 G245C mutation, and the primary tumor showed loss of heterozygosity with retention of the mutated TP53 allele. Histopathology, DNA fingerprinting, gene expression profiling, and biochemical analyses of the xenograft were conducted and compared with the primary tumor and normal adrenal cortex. The second endpoint was to assess the preliminary antitumor activity of selected chemotherapeutic agents. Results: The xenograft maintained the histopathologic and molecular features of the primary tumor. Screening the xenograft for drug responsiveness showed that cisplatin had a potent antitumor effect. However, etoposide, doxorubicin, and a panel of other common cancer drugs had little or no antitumor activity, with the exception of topotecan, which was found to significantly inhibit tumor growth. Consistent with these preclinical findings, topotecan as a single agent in a child with relapsed ACC resulted in disease stabilization. Conclusion: Our study established a novel TP53-associated pediatric ACC xenograft and identified topotecan as a potentially effective agent for treating children with this disease. Clin Cancer Res; 19(7); 1740–7. ©2013 AACR.

Prolonged exposure to ambient particles is associated with premature mortality due to cardio-respiratory diseases and lung cancer. The size and composition of these particles determine their toxicity, which is aggravated by their long-term retention in the lungs.To compare the elemental profile of particles retained along the bronchial tree and lymph nodes by combining laser capture microdissection (LCM) and elemental composition analysis through energy dispersive x-ray (EDX) and scanning electron microscopy (SEM).Twenty-four right lung middle lobes from autopsied cases were obtained from two cities with different pollution backgrounds. Lung samples were collected from three distinct sites within the lung at the time of autopsy: peribronchial tissue, peripheral parenchyma and hilar lymph nodes. Areas of potentially increased particle deposition were microdissected using LCM and analyzed for elemental composition through EDX "allied" with SEM.Elemental analyses of the particles retained along the bronchial tree showed two groups of distribution: peribronchiolar or lymph node deposition. The elemental profile of peribronchial areas were significantly different between the two cities and were better discriminators of past air pollution exposure.Our data suggest that particle uptake varies along the bronchial tree and human lung tissue retains particles indicative of regional air pollution background.

Childhood adrenocortical tumor (ACT), a very rare malignancy, has an annual worldwide incidence of about 0.3 per million children younger than 15 years. The association between inherited germline mutations of the TP53 gene and an increased predisposition to ACT was described in the context of the Li-Fraumeni syndrome. In fact, about two-thirds of children with ACT have a TP53 mutation. However, less than 10% of pediatric ACT cases occur in Li-Fraumeni syndrome, suggesting that inherited low-penetrance TP53 mutations play an important role in pediatric adrenal cortex tumorigenesis. We identified a novel inherited germline TP53 mutation affecting the acceptor splice site at intron 10 in a child with an ACT and no family history of cancer. The lack of family history of cancer and previous information about the carcinogenic potential of the mutation led us to further characterize it. Bioinformatics analysis showed that the non-natural and highly hydrophobic C-terminal segment of the frame-shifted mutant p53 protein may disrupt its tumor suppressor function by causing misfolding and aggregation. Our findings highlight the clinical and genetic counseling dilemmas that arise when an inherited TP53 mutation is found in a child with ACT without relatives with Li-Fraumeni-component tumors.

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17-hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A22%, B 3-7%, C20%. Twenty-one children with neonatal symptoms and/or 17OHP levels80 ng/ml carried A genotypes, except two virilized girls (17OHP50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false-positives and help distinguish between clinical forms of CAH.

Nelson's syndrome (NS) is characterized by the appearance and/or progression of ACTH-secreting pituitary macroadenomas in patients who had previously undergone bilateral adrenalectomy for the treatment of Cushing's disease. Such corticotroph macroadenomas respond poorly to currently available therapeutic options which include surgery, radiotherapy and chemotherapy. P53 protein accumulation may be detected by immunohistochemistry in pituitary corticotroph adenomas and it has been suggested that it might be causally related to tumor development. Wild type P53 protein plays an important role in the cellular response to ionizing radiation and other DNA damaging agents and is mutated in many human tumors. In this study we report an adult male patient with NS who underwent both transsphenoidal and transcranial pituitary surgeries, conventional and stereotaxic radiotherapy and brachytherapy. Despite of the efforts to control tumor mass and growth, this macroadenoma displayed relentless growth and aggressive behavior. DNA extracted from the first two surgical samples, as well as DNA from peripheral blood leukocytes disclosed normal p53 sequence. DNA extracted from tumor samples obtained at surgeries performed after pituitary irradiation carried a somatic heterozygous mutation, consisting of a deletion of four cytosines between nucleotides 12,144-12,149 in exon 4 of the p53 gene. This frameshift mutation creates a stop codon in exon 4 excluding the expression of a functional protein from the defective allele. These data demonstrate a possible association between the P53 protein loss of function induced by radiotherapy and the aggressive course of the disease in this patient.

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

Rare activating mutations of the human (h)FSHR have been reported in some women with spontaneous ovarian hyperstimulation in pregnancy, where follicular growth is inappropriately stimulated by elevated concentrations of human chorionic gonadotropin acting through the hFSHR. It is not known whether ovarian hyperstimulation in peripubertal girls with untreated primary hypothyroidism is caused by hFSHR mutations and/or influenced by hFSHR allelic variants, rendering the hFSHR more sensitive to circulating TSH.The aim of the study was to determine whether mutations of the hFSHR and/or hFSHR allelic variants are associated with greater sensitivity of the hFSHR to TSH.The hFSHR gene was sequenced from eight pediatric patients displaying gonadal hyperstimulation due to primary hypothyroidism. HEK293 cells expressing different hFSHR allelic combinations were studied for their responsiveness to recombinant (r)hTSH.The study was conducted at university research centers.Eight unrelated patients (seven girls and one boy) who exhibited primary hypothyroidism and gonadal hyperstimulation were included in the study.There were no interventions.DNA sequencing of the hFSHR gene was the main outcome measure. Basal, rhFSHR- and rhTSH receptor-stimulated cAMP levels were assayed in HEK293 cells transfected with the hTSH receptor or different hFSHR allelic combinations. Cell surface receptor numbers were also determined.No hFSHR mutations were identified in the patient population, but we did identify two known polymorphisms. In vitro experiments demonstrated a dose-dependent and specific rhTSH-dependent increase in cAMP production in HEK293 cells expressing the wild-type hFSHR, regardless of hFSHR isoform.Pediatric gonadal hyperstimulation associated with severe primary hypothyroidism is likely due to the actions of the elevated concentrations of TSH on the wild-type hFSHR, and this response is not dependent upon the hFSHR isoform.

Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis. Pediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here the authors analyse the genomes, exomes and transcriptomes of 37 such tumours and identify genetic alterations whose nature, timing and potential interactions are key events with prognostic significance in pediatric adrenocortical tumorigenesis.

The human p53 tumor suppressor gene is located at the short arm of chromosome 17. A germinative mutation (Arg337His) in the tetramerization domain of p53 has been frequently identified in Brazilian children with sporadic adrenocortical tumors. Loss of heterozygosity at this region was demonstrated in the majority of the cases. In the present study, we performed deletion mapping of chromosome 17 in 30 adrenocortical tumors from 29 Brazilian patients (15 children and 14 adults). One boy had bilateral adrenocortical tumor. Sixteen patients had the germinative Arg337His mutation. Loss of heterozygosity analysis using six polymorphic microsatellite markers disclosed loss of the entire chromosome 17 in 18 tumors (10 adenomas and eight carcinomas) from 17 patients. The Arg337His mutation was present in 13 of them. Chromosomal instability involving chromosomes 2, 9, and 11 was also found in 47, 47, and 70% of the 17 patients who exhibited chromosome 17 losses, respectively. The concomitant loss of chromosomes 2, 9, 11, and 17 was evidenced exclusively in malignant tumors. Therefore, chromosomal instability involving three or more chromosomes may contribute to define the malignant adrenocortical lesions. In conclusion, we demonstrated a high frequency of biallelic inactivation of p53 derived from two distinct events, the germinative Arg337His mutation and the acquired loss of the entire chromosome 17. In addition, the isolated loss of the entire chromosome 17 did not correlate with aggressive tumor behavior in these patients with adrenocortical tumors.