Iberê C. Soares

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and β-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal β-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal β-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized β-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized β-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.

Steroidogenic Factor 1 Overexpression and Gene Amplification Are More Frequent in Adrenocortical Tumors from Children than from Adults

BACKGROUND Steroidogenic factor 1 (SF-1) is a key determinant of endocrine development and function of adrenal cortex. SF-1 overexpression and gene amplification were previously demonstrated in a small group of pediatric adrenocortical tumors. OBJECTIVE Our objective was to determine the frequency of SF-1 protein expression and gene amplification in a large cohort of pediatric and adult adrenocortical tumors. PATIENTS SF-1 protein expression was assessed in a cohort of 103 adrenocortical tumors from 36 children and 67 adults, whereas gene amplification was studied in 38 adrenocortical tumors (17 from children). METHODS Tissue microarray, multiplex ligation-dependent probe amplification, and quantitative real-time PCR were used. RESULTS A strong nuclear SF-1 expression was detected by tissue microarray in 56% (20 of 36) and 19% (13 of 67) of the pediatric and adult adrenocortical tumors, respectively (P = 0.0004). Increased SF-1 copy number was identified in 47% (eight of 17) and 10% (two of 21) of the pediatric and adult adrenocortical tumors, respectively (P = 0.02). All adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five (29%) pediatric adrenocortical tumors without SF-1 amplification. The frequency of SF-1 overexpression and gene amplification was similar in adrenocortical adenomas and carcinomas. CONCLUSION We demonstrated a higher frequency of SF-1 overexpression and gene amplification in pediatric than in adult adrenocortical tumors, suggesting an important role of SF-1 in pediatric adrenocortical tumorigenesis.

Acromegaly: correlation between expression of somatostatin receptor subtypes and response to octreotide-lar treatment

About one-third of acromegalics are resistant to the clinically available somatostatin analogs (SA). The resistance is related to density reduction or different expression of somatostatin receptor subtypes (SSTR). This study analyzes SSTR’s expression in somatotrophinomas, comparing to SA response, hormonal levels, and tumor volume. We analyzed 39 somatotrophinomas; 49% were treated with SA. The most expressed SSTR was SSTR5, SSTR3, SSTR2, SSTR1, and SSTR4, respectively. SSTR1 and SSTR2 had higher expression in patients that had normalized GH and IGF-I. SSTR3 was more expressed in patients with tumor reduction. There was a positive correlation between the percentage of tumor reduction and SSTR1, SSTR2 and SSTR3 expression. Also, a positive correlation between SSTR2 mRNA expression and the immunohistochemical reactivity of SSTR2 was found. Our study confirmed the association between the SA response to GH and IGF-I and the SSTR2. Additionally, this finding was also demonstrated in relation to SSTR1.

Isolated familial somatotropinoma: 11Q13-LOH and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

OBJECTIVE Non-pituitary tumors have been reported in a subset of patients harboring germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene. However, no detailed investigations of non-pituitary tumors of AIP-mutated patients have been reported so far. PATIENTS We examined a MEN1- and p53-negative mother-daughter pair with acromegaly due to somatotropinoma. Subsequently, the mother developed a large virilizing adrenocortical carcinoma and a grade II B-cell non-Hodgkin’s lymphoma. DESIGN Mutational analysis was performed by automated sequencing. Loss-of-heterozygosity (LOH) analysis was carried out by sequencing and microsatellite analysis. AIP expression was assessed through quantitative PCR (qPCR) and immunohistochemistry. RESULTS The functional inactivating mutation c.241C>T (R81X), which blocks the AIP protein from interacting with phosphodiesterase 4A (PDE4A), was identified in the heterozygous state in the leukocyte DNA of both patients. Analyzing the tumoral DNA revealed that the AIP wild-type allele was lost in the daughter’s somatotropinoma and the mother’s adrenocortical carcinoma. Both tumors displayed low AIP protein expression levels. Low AIP gene expression was confirmed by qPCR in the adrenocortical carcinoma. No evidence of LOH was observed in the DNA sample from the mother’s B-cell lymphoma, and this tumor displayed normal AIP immunostaining. CONCLUSIONS Our study presents the first molecular analysis of non-pituitary tumors in AIP-mutated patients. The finding of AIP inactivation in the adrenocortical tumor suggests that further investigation of the potential role of this recently identified tumor suppressor gene in non-pituitary tumors, mainly in those tumors in which the cAMP and the 11q13 locus are implicated, is likely to be worthwhile.

Coordinated expression of ER, PR and HER2 define different prognostic subtypes among poorly differentiated breast carcinomas

Aims:  Histological grade is one of the most important prognostic factors in breast carcinomas, but poorly differentiated neoplasms still have quite heterogeneous biological behaviour, since they can be genetically classified as basal‐like, HER2+ or even luminal. The aim was to analyse the frequency of oestrogen receptor (ER), progesterone receptor (PR) and HER2 expression profiles among breast carcinomas with <10% tubular formation, and their correlation with classic prognostic factors.

Expression of LIN28 and its regulatory microRNAs in adult adrenocortical cancer

LIN28 control cells reprogramming and pluripotency mainly through miRNA regulation and has been overexpressed in many advanced cancers. In this study, we evaluated the prognostic role of LIN28 and its regulatory miRNAs in a large cohort of adrenocortical tumours (ACTs).

PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas with or without β-catenin mutations

INTRODUCTION: Activating mutations in exon 3 of the β-catenin gene are involved in the pathogenesis of adamantinomatous craniopharyngiomas. Recently, the interaction between β-catenin and PROP1 has been shown to be responsible for pituitary cell lineage determination. We hypothesized that dysregulated PROP1 expression could also be involved in the pathogenesis of craniopharyngiomas. OBJECTIVES: To determine whether dysregulated gene expression was responsible for tumor pathogenesis in adamantinomatous craniopharyngiomas, the β-catenin gene was screened for mutations, and the expression of the β-catenin gene and PROP1 was evaluated. METHODS: The β-catenin gene was amplified and sequenced from 14 samples of adamantinomatous craniopharyngiomas. PROP1 and β-catenin gene expression was assessed by real-time RT-PCR from 12 samples, and β-catenin immunohistochemistry was performed on 11 samples. RESULTS: Mutations in the β-catenin gene were identified in 64% of the adamantinomatous craniopharyngiomas samples. Evidence of β-catenin gene overexpression was found in 71% of the tumors with β-catenin mutations and in 40% of the tumors without mutations, and β-catenin immunohistochemistry revealed a nuclear staining pattern for each of the analyzed samples. PROP1 expression was undetectable in all of the tumor samples. CONCLUSION: We found evidence of β-catenin gene overexpression in the majority of adamantinomatous craniopharyngiomas, and we also detected a nuclear β-catenin staining pattern regardless of the presence of a β-catenin gene mutation. These results suggest that WNT signaling activation plays an important role in the pathogenesis of adamantinomatous craniopharyngiomas. Additionally, this study was the first to evaluate PROP1 expression in adamantinomatous craniopharyngiomas, and the absence of PROP1 expression indicates that this gene is not involved in the pathogenesis of this tumor, at least in this cohort.

Rebmab200, a Humanized Monoclonal Antibody Targeting the Sodium Phosphate Transporter NaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: A Novel Reagent for Targeted Antibody Therapy of Cancer

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibodys full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

The role of fibroblast growth factor receptor 4 overexpression and gene amplification as prognostic markers in pediatric and adult adrenocortical tumors

In the last decade, relevant progresses in the molecular basis of adrenocortical tumors (ACTs) were achieved and abnormalities involving growth pathway deregulation were frequently associated with malignancy. Remarkably, upregulation of insulin-like growth factor 2 (IGF2) and its receptor (IGF1R) has been demonstrated in a significant proportion of ACTs, and the presence of these abnormalities has both prognostic and therapeutic implications (Almeida et al. 2008). In fact, clinical trials involving pharmacological blockade of IGF1R are currently under process. Besides the IGF system, other growth signaling pathways have been suggested to be important for ACC progression and are possible therapeutic targets. Among these, fibroblast growth factor receptor 4 (FGFR4) overexpression has been observed in both adult and pediatric ACT by genome-wide expression studies in the same extent as the IGF system (de Fraipont et al. 2005, Laurell et al. 2009). However, these data have not been validated in an independent cohort and the molecular mechanisms responsible forFGFR4 upregulation have not been assessed. Therefore, we studied the expression levels of FGFR4 and gene amplification in a cohort of ACT patients from our institution. This study was approved by the Ethics Committee of Hospital das Clinicas, Sao Paulo, Brazil, and an informedwritten consentwas obtained from all patients and/or parents. Our cohort consisted of 57 patients – 32 adults (ageR18 years, 18–66 years) and 25 pediatric (age!18 years, 0.9–17 years) with the diagnosis of ACT, with a mean follow-up period of 77.5G53 months. FGFR4 transcript levels were assessed through quantitative real-time RT-PCR in all 57 samples using TaqMan gene expression assays (Applied Biosystem, Foster City, CA,USA). The endogenous control gene used was ACTB for each sample and the reactions were carried out in triplicate. The relative expression levels of FGFR4 were calculated using the 2t method as described previously (Livak & Schmittgen 2001). A commercial pool of 61 human adrenal glands of autopsy was the reference sample (Clontech). The criteria used for underand overexpression was a twofold change in comparison

Decreased immunoexpression of survivin could be a potential marker in human non-alcoholic fatty liver disease progression?

Background/aim: Regulation of apoptosis in non‐alcoholic fatty liver disease (NAFLD) has been a theme of growing debate. Although no other study assessed the role of survivin in NAFLD, its expression has been reported in hepatic carcinogenesis because of other aetiological factors with relevant discrepancies. The aim of this study was to assess the pattern of survivin immunoexpression by tissue microarray along the whole spectrum of NAFLD, including non‐alcoholic steatohepatitis (NASH)‐related hepatocelular carcinoma (HCC).