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Children diagnosed with pediatric adrenocortical tumors (pACT) have variable outcomes, and, to date, the disease lacks robust prognostic biomarkers. The prognostic potential of tumor methylation has been demonstrated in several cancers. We aimed to evaluate the pACT methylation profile and its association with disease presentation and survival. In this cross-sectional study, we accessed the DNA methylation (MethylationEPIC Array, Illumina) of 57 primary pACT from Southeastern Brazil and the respective patients' clinicopathological features. We also applied our analysis in an independent 48 pACT methylation dataset. Unsupervised learning whole-methylome analysis showed two groups with distinct methylation signatures: pACT-1 and pACT-2. Compared to pACT-2, pACT-1 tumors were enriched with higher methylation in CpG islands, mainly in gene promoter regions. The topmost hypermethylated gene in these samples was shown to be underexpressed. Patients in the pACT-1 group were older at diagnosis and were more likely to have carcinomas and nonlocalized/advanced and recurrent/metastatic disease. Univariate and bivariate regressions showed that pACT-1 methylation signature confers superior hazard ratio of disease progression and death than known prognostic features. The methylation groups had similar frequencies of germline mutations in the TP53 gene, including the regionally frequent p.R337H. Our analysis replication validated our findings and reproduced those recently described in pACT. We demonstrated the existence of different tumor methylation signatures associated with pACT presentation and clinical evolution, even in the context of germline TP53 mutations. Our data support tumor methylation profiling as a robust and independent prognostic biomarker for pACT and suggest a list of candidate genes for further validation.

Background: Pediatric head and neck cancer (PHNC) is rare and its nonspecific clinical manifestations may often lead to delayed diagnosis. We aimed to describe the signs, symptoms, and clinicopathological characteristics of PHNC., Material and Methods: Medical records were retrospectively reviewed for all PHNC cases diagnosed from 1986 to 2016 affecting patients aged 19-years and younger from a tertiary referral center in Brazil. Demographic variables, anatomical site of primary tumors, histopathological diagnoses, signs and symptoms, and patterns of misdiagnosis were collected and interpreted by statistical and descriptive analysis., Results: A total of 253 PHNC cases were included. The mean age was 9.3 years and male patients were more frequently affected (60.9%). Burkitt lymphoma (23.7%), nasopharyngeal carcinoma (15.8%), and rhabdomyosarcoma (15.4%) were the most common cancer types. The nasopharynx (28.9%), cervical/lymph node region (25.3%), and craniofacial bones (8.3%) were the predominant anatomical sites. Tumor/swelling (68.4%), was the clinical finding often presented. The univariable analysis showed association between tumor histology and clinical variables such as sex (p=0.022), age (p<0.0001), anatomical location (p<0.0001) tumor/swelling (p=0.034), pain (p=0.031), systemic/general manifestations (p=0.004), nasal/breathing alterations (p=0.012), orbital/ocular alterations (p<0.0001). Misdiagnosis such as tonsillitis, otitis, and abscess were frequent., Conclusions: Although the clinical findings of PHNC are often unspecific, this study provided signs and symptoms with significant correlations between tumor histology. The suspicion of malignancy should be considered when the main signs and symptoms reported here appear and persist, in order to conduct a timely diagnosis.

Introduction: Medulloblastoma (MB) is an embryonal tumour that originates from genetic deregulation of cerebellar developmental pathways and is classified into 4 molecular subgroups: SHH, WNT, group 3, and group 4. Hydroxymethylation levels progressively increases during cerebellum development suggesting a possibility of deregulation in MB pathogenesis. The aim of this study was to investigate global hydroxymethylation levels and changes in TET and IDH gene expression in MB samples compared to control cerebellum samples., Methods: The methods utilized were qRT-PCR for gene expression, dot-blot and immunohistochemistry for global hydroxymethylation levels and sequencing for the investigation of IDH mutations., Results: Our results show that global hydroxymethylation level was decreased in MB, and low 5hmC level was associated with the presence of metastasis. TET1 expression levels were decreased in the WNT subgroup, while TET3 expression levels were decreased in the SHH subgroup. Reduced TET3 expression levels were associated with the presence of events such as relapse and death. Higher expression of IDH1 was observed in MB group 3 samples, whereas no mutations were detected in exon 4 of IDH1 and IDH2., Conclusion: These findings suggest that reduction of global hydroxymethylation levels, an epigenetic event, may be important for MB development and/or maintenance, representing a possible target in this tumour and indicating a possible interaction of TET and IDH genes with the developmental pathways specifically activated in the MB subgroups. These genes could be specific targets and markers for each subgroup.

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1μM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth., (© 2016 Society for Endocrinology.)

Background: Pediatric adrenocortical tumors (ACT) are rare malignancies and treatment has a small impact on survival in advanced disease and the discovery of potential target genes could be important in new therapeutic approaches., Methods: The mRNA expression levels of spindle checkpoint genes AURKA, AURKB, BUB, and BUBR1 were analyzed in 60 children with ACT by quantitative real time PCR. The anticancer effect of ZM447439, an experimental AURK inhibitor, was analyzed in a primary childhood ACT culture carrying the TP53 p.R337H mutation., Results: A significant association was observed between malignancy as defined by Weiss score ≥3 and higher AURKA (2.0-fold, P = 0.01), AURKB (7.0-fold, P = 0.007), and BUBR1 (5.8-fold, P = 0.007) gene expression, and between unfavorable event (death or relapse) and higher expression of AURKA (6.0-fold, P = 0.034) and AURKB (17-fold, P = 0.013). Overexpression of AURKA and AURKB was associated with lower event-free survival in uni- (P < 0.001 and P = 0.006, respectively) and multivariate (P = 0.002 and P = 0.03, respectively) analysis. Significant lower Event free survival (EFS) was also observed in patients with moderate/strong immunostaining to AURKA (P = 0.012) and AURKB (P = 0.045). ZM447439 was able to induce inhibition of proliferation and colony formation in a primary childhood ACT culture carrying the TP53 p.R337H mutation., Conclusion: Our results suggest that AURKA and AURKB overexpression in pediatric ACT may be related to more aggressive disease and the inhibition of these proteins could be an interesting approach for the treatment of these tumors., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Impaired apoptosis has been implicated in the development of childhood adrenocortical tumors (ACT), although the expression of apoptosis-related gene expression in such tumors has not been reported., Methods: The mRNA expression levels of the genes CASP3, CASP8, CASP9, FAS, TNF, NFKB, and BCL2 were analyzed by quantitative real-time PCR in consecutive tumor samples obtained at diagnosis from 60 children with a diagnosis of ACT and in 11 non-neoplastic adrenal samples. BCL2 and TNF protein expression was analyzed by immunohistochemistry., Results: A significant association was observed between tumor size ≥100 g and lower expression levels of the BCL2 (P=0.03) and TNF (P=0.05) genes; between stage IV and lower expression levels of CASP3 (P=0.008), CASP9 (P=0.02), BCL2 (P=0.002), TNF (P=0.05), and NFKB (P=0.03); Weiss score ≥3 and lower expression of TNF (P=0.01); unfavorable event and higher expression values of CASP9 (P=0.01) and lower values of TNF (P=0.02); and death and lower expression of BCL2 (P=0.04). Underexpression of TNF was associated with lower event-free survival in uni- and multivariate analyses (P<0.01). Similar results were observed when patients with Weiss score <3 were excluded., Conclusion: This study supports the participation of apoptosis-related genes in the biology and prognosis of childhood ACT and suggests the complex role of these genes in the pathogenesis of this tumor.

Background: The inherited, low-penetrance arginine-to-histidine substitution at codon 337 (R337H) of the tumor protein 53 gene (TP53) is clustered in southeast Brazil (estimated frequency, 0.3%). Although its tumorigenic effect initially appeared to be tissue-specific, recent evidence suggests its association with a broader range of tumors. Therefore, the authors of this report investigated the spectrum of pediatric malignancies associated with the TP53 R337H mutation at a single referral institution in southeast Brazil., Methods: Genomic DNA samples from 493 children with malignancies were screened for the R337H mutation. Available tumor samples from carriers were investigated for loss of heterozygosity (LOH) and nuclear p53 accumulation. Clinical data were obtained from medical records., Results: Sixty-five of 70 patients (93%) with adrenocortical tumors (ACTs), 9 of 13 patients (69%) with choroid plexus carcinoma (CPC), and 3 of 41 patients (7.3%) with osteosarcoma carried the mutation. The proportion of CPC to choroid plexus papilloma (CPP) was much higher than that reported elsewhere. Osteosarcoma in carriers had a significantly poorer outcome (P = .02). The mutation was not identified in patients who had acute lymphoblastic leukemia (ALL) (n = 187), recurrent ALL (n = 49), acute myeloid leukemia (n = 44), lymphoma (n = 30), non-CPC central nervous system tumors (n = 26), Ewing sarcoma (n = 25), or rhabdomyosarcoma (n = 8). Among the tumors that were available for analysis, LOH with retention of the mutant allele was confirmed in 21 of 21 ACTs, in 2 of 2 CPCs, and in 2 of 3 osteosarcomas that were positive for R337H. CPCs and osteosarcomas that were positive for R337H had marked nuclear accumulation of p53., Conclusions: The current findings demonstrated compellingly that the TP53 R337H mutation is associated not only with ACT but also with CPC and, to a lesser extent, with osteosarcoma, both of which are core-component tumors of the Li-Fraumeni syndrome., (2010 American Cancer Society.)

Purpose: Renal scintigraphy has been used for many years in the evaluation of renal transplants and can help in the diagnosis of graft complications, leading to prompt clinical management and preventing further deterioration of renal function. The purpose of this study was to evaluate the overall accuracy of renal scintigraphy with (99m)Tc-DTPA in the diagnosis of acute renal graft complications., Materials and Methods: Seventy-six scintigraphic studies performed in 55 patients (ages ranging from 6 to 65 years), were reviewed. Scintigraphy results were compared to biopsies performed within 5 days of imaging. (99m)Tc-DTPA study was performed within a mean time of 19 days after kidney transplants. Dynamic images were performed in the anterior position of the abdomen and pelvis every 2 seconds for 80 seconds (flow phase) and every 15 seconds for 30 minutes (functional phase), after an intravenous injection of 370 MBq (10 mCi) of (99m)Tc-DTPA., Results: The scintigraphic results were concordant with the biopsies in 86% of the cases studied. The sensitivities of renal scintigraphy for detection of acute tubular necrosis (ATN), acute rejection (AR) and cortical necrosis (CN) were 98%, 87% and 100%, respectively. Specificities and accuracies for detection of ATN, AR and CN were 89%, 86% and 100%, and 95%, 87% and 100%, respectively., Conclusion: Renal scintigraphy with (99m)Tc-DTPA showed a good overall accuracy in the detection of acute renal graft complications. It can be used as a reliable tool in the routine evaluation of these patients.

Background: Dysgenetic male pseudohermaphroditism (DMP) is a sexual differentiation disorder characterized by bilateral dysgenetic testes, persistent müllerian structures, and cryptorchidism in individuals with a 46,XY karyotype. However, the histologic criteria for the diagnosis of DMP are poorly established., Objective: To determine gonadal histology in children with DMP., Patients and Methods: Between 1996 and 1998, 13 patients with DMP were evaluated on our service. The clinical diagnosis of DMP was based on a 46,XY karyotype, sex ambiguity, high levels of follicle-stimulating hormone and low levels of antimüllerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of müllerian structures. Molecular sequencing the HMGbox region of the SRY gene did not reveal any mutations. Biopsies were performed for 22 of 26 gonads (patient age at the time of biopsy, 16 months to 10 years). Conventional microscopy was used to evaluate mean tubular diameter, tubular fertility index, and number of Sertoli cells per tubular profile., Results: All 26 gonads were located outside of the labioscrotal folds. Their histologic features varied from only a reduction in tubular size to features of a streak gonad. Five of the 22 gonads grossly resembled a streak gonad. The mean tubular diameter was severely reduced (>30% reduction relative to the normal tubular diameter for the patient's age) in 4 gonads, markedly reduced (10%-30%) in 11 gonads, slightly reduced (<10%) in one gonad, and normal in one gonad. The tubular fertililty index, expressed as the percentage of tubular profiles containing germ cells, was severely reduced (<30% of normal values) in 9 gonads, markedly reduced (50%-30%) in 2 gonads, and normal in 6 gonads. The number of Sertoli cells per tubular profile was elevated in 16 gonads and normal in one gonad. Thin tubules surrounded by fibrous tissue were occasionally observed., Conclusion: The histologic findings confirmed the clinical diagnosis of DMP in every patient in the present series. However, gonadal histology was variable, and careful morphometric evaluation may be necessary to establish the diagnosis.

Osteolytic lesions rarely occur in acute myeloid leukemia (AML). We reported an atypical form of the disease, with marrow fibrosis and osteolytic lesions, in a 17-year-old patient, whose main symptom was lumbar pain. Diagnosis of AML was established by bone marrow and lymph node histological analysis. Computed tomography (CT) scan and 99mTc-MDP bone scintyscan revealed osteolytic lesions. After remission-induction, bone marrow aspirate and biopsy showed no evidence of leukemic infiltration, nevertheless bone abnormalities persisted on 99mTc-MDP bone scintyscan, suggesting residual disease. Suspect bone areas were irradiated with symptomatic improvement and 99mTc-MDP bone scintyscan showed the appearance of more condensed bone compared with the pre-radiotherapy pattern. Twelve months later he was readmitted to the hospital due to relapse of AML and died of sepsis within a few weeks. This report illustrates the usefulness of histological studies to establish diagnosis of AML in atypical cases, as well as the importance of CT scan and bone scintigraphy scan for the identification of osteolytic lesions. It also provides additional data as evidence that although osteolytic lesions indicate an adverse prognosis in AML, local irradiation results symptomatic relief.

The hemophagocytic syndrome (HS) is characterized by a clinical picture of fever, hepatosplenomegaly, lymphadenopathy and peripheral pancytopenia. The morphologic hallmark of this syndrome is the phagocytosis of hematopoietic elements by morphologically normal macrophages. HS is considered rare and may be a primary disease or associated to viral, infection, neoplasias or autoimmune diseases. Treatment is controversial and its evolution is often fatal. Anatomo-pathological evaluation shows the phenomenon of hemophagocytosis in several organs, especially the hematopoietic tissues. We describe a case of HS, discuss its possible causes, its clinical and pathologic features, its pathophysiology and therapeutic possibilities.

Mielodysplastic syndromes (MDS) are clonal disorders of the hemopoietic stem cell. About one third of the cases terminate in an acute leukemia, usually acute myeloblastic leukemia. However, few cases of transformation into acute lymphoblastic leukemia (ALL) have been described. We present a case of refractory anemia that transformed into ALL two months after diagnosis and was successfully treated with conventional chemotherapy. Two years later a hyperfibrotic form of MDS was detected in the patient, that soon after terminated in acute megakaryoblastic leukemia. The course of MDS in the present case provides evidence that MDS can involve a pluripotent stem cell, presenting clonal evolution, documented by successive changes in its clinical and hematological features.

Counts of nucleolar regions (NORs) demonstrated by a silver staining technique in paraffin sections, have been used to distinguish benign from malignant lesions. AgNORs were studied in 24 biopsies from oral cavity (5 cases of normal oral mucosa, 5 of pseudocarcinomatous hyperplasia and 14 of squamous cell carcinoma, subdivided according to degree of differentiation: 5 grade 1, 5 grade 2 and 4 grade 3) to find whether they were helpful in distinguishing pseudocarcinomatous hyperplasia due to chronic parasitic infections from squamous cell carcinoma. Two methods of counting AgNORs were used: (A) a simpler one which counts nucleolar clusters (AgNU) and satellite AgNORs and the other (B) counting all individual AgNORs, including those within AgNUs. In both methods the lowest mean values were observed for grade 3 carcinoma, while the highest belonged to grade 2 carcinoma. The simpler method (A) was the most useful because AgNU counts showed significant difference when pseudocarcinomatous hyperplasia was compared with grade 1 and 2 carcinomas, which are the most difficult to discriminate from it. However the overlapping of values render the technique of limited use in individual cases.

The authors report one case of granulocytic sarcoma infiltrating the larynx and cervical lymph nodes in a 50-year-old smoking patient. At the time of diagnosis there was no clinical and laboratory evidence of acute myeloid leukemia or chronic myeloproliferative disease. Four months after diagnosis, bone marrow morphology was consistent with chronic myeloid leukemia, accelerated phase. Cytogenetic abnormalities (Ph 1 chromosome, t(1; 12) (p36; p13), and trisomy of chromosome 20) were also found in hemopoetic cells. Granulocytic sarcoma preceding installation of chronic myeloid leukemia, as described here, seems to be a rare clinical event.