Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Wong Pak Cheung Ronald. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 121-142). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / Abbreviation --- p.VI / List of figures --- p.IX / List of tables --- p.XI / Content --- p.XII / Content / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The p53 tumor suppressor gene --- p.2 / Chapter 1.1.1 --- Structure and function --- p.2 / Chapter 1.1.2 --- Regulation of p53 stability and activity --- p.3 / Chapter 1.1.3 --- p53 and cell cycle arrest --- p.4 / Chapter 1.1.4 --- p53 and apoptosis --- p.4 / Chapter 1.2 --- Mutation in p53 gene --- p.9 / Chapter 1.2.1 --- Loss of function through dominant negative effect --- p.9 / Chapter 1.2.2 --- Gain-of-function through transactivation by mutant p53 --- p.10 / Chapter 1.2.3 --- Mutation in p53 and resistance to cancer therapy --- p.10 / Chapter 1.3 --- Objective of the study --- p.14 / Chapter Chapter 2: --- Mutant p53 induced interferon resistance and its regulation of the Jak/Stat pathway --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- IFN classification and biological activities --- p.16 / Chapter 2.1.2 --- IFN signaling --- p.17 / Chapter 2.1.3 --- IFN direct antitumor effect: cell cycle arrest and apoptosis --- p.18 / Chapter 2.1.4 --- IFN in immunotherapy --- p.20 / Chapter 2.1.5 --- Resistance to IFN therapy --- p.21 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Cell lines --- p.24 / Chapter 2.2.2 --- Drugs and antibodies --- p.24 / Chapter 2.2.3 --- Cell Proliferation assay- MTT assay --- p.24 / Chapter 2.2.4 --- Cell cycle analysis --- p.25 / Chapter 2.2.5 --- DNA fragmentation assay --- p.25 / Chapter 2.2.6 --- Western blot analysis --- p.26 / Chapter 2.2.7 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Mutant p53-V143A and p53-R273H induced IFN resistance: the role of IFN induced apoptosis and cell cycle arrest --- p.27 / Chapter 2.3.2 --- IFN induction of apoptosis: a p53-independent and caspase-dependent pathway --- p.28 / Chapter 2.3.3 --- Mutant p53 regulation of Jak/Stat pathway --- p.36 / Chapter 2.3.4 --- Janus kinases (Jaks) and IFN-alpha sensitivity in Saos-2 cells --- p.41 / Chapter 2.3.5 --- Janus kinases (Jaks) and IFN-gamma sensitivity --- p.49 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Mutant p53-V143 and p53-R273H induced IFN resistance in Saos-2 cells --- p.56 / Chapter 2.4.2 --- Role of Jaks in IFN sensitivity in Saos-2 cells --- p.57 / Chapter 2.4.3 --- IFN signaling in Saos-2 cells --- p.57 / Chapter 2.4.4 --- Jak2 and IFN induced apoptosis --- p.58 / Chapter Chapter 3: --- Mutant p53 induced drug resistance --- p.60 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.1.1 --- The multidrug resistance (MDR) --- p.61 / Chapter 3.1.2 --- Anticancer drugs used in the study: action mechanisms and resistance --- p.67 / Chapter 3.1.3 --- Jak/Stat pathway and MDR --- p.68 / Chapter 3 .2 --- Materials and Methods --- p.72 / Chapter 3.2.1 --- Cell lines --- p.72 / Chapter 3.2.2 --- Drugs and antibodies --- p.72 / Chapter 3.2.3 --- Caspase 3 activity assay --- p.72 / Chapter 3.2.4 --- Cell Proliferation assay- MTT assay --- p.73 / Chapter 3.2.5 --- Cell cycle analysis --- p.73 / Chapter 3.2.6 --- DNA fragmentation assay --- p.73 / Chapter 3.2.7 --- Reverse transcription polymerase chain reaction --- p.73 / Chapter 3.2.8 --- Western blot analysis --- p.74 / Chapter 3.2.9 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- Mutant p53 and drug sensitivity --- p.75 / Chapter 3.3.2 --- Mutant p53 and drug induced apoptosis and cell cycle arrest --- p.75 / Chapter 3.3.3 --- Classical drug resistance factors in mutant p53 induced drug resistance --- p.87 / Chapter 3.3.4 --- The role of Jaks in drug sensitivity of Saos-2 cells --- p.89 / Chapter 3.3.5 --- The role of Jaks in drug induced DNA fragmentationin Saos-2 cells --- p.89 / Chapter 3.3.6 --- Jak signaling and caspase activation in MTX induced apoptosis in Saos-2 cells --- p.100 / Chapter 3.3 --- Discussion --- p.108 / Chapter 3.3.1 --- Mutant p53-V143A and p53-R273H induced drug resistance in Saos-2 cells --- p.108 / Chapter 3.3.2 --- Role of Jaks in drug sensitivity in Saos-2 cells --- p.109 / Chapter 3.3.3 --- Jak/Stat signaling in Saos-2 cells --- p.109 / Chapter 3.3.4 --- Jak2 and MTX induced apoptosis --- p.110 / Chapter Chapter 4: --- General discussion --- p.112 / Chapter 4.1 --- Mutant p53 induced immunotherapy and chemotherapy resistance --- p.113 / Chapter 4.2 --- Gain of new function of mutant p53-V143A and p53-R273H in regulating Jak/Stat pathway leading to resistance to IFN and chemotherapeutic drugs --- p.114 / Chapter 4.3 --- The role of Jaks in MTX sensitivity --- p.114 / Chapter 4.4 --- Future work --- p.115 / Chapter 4.5 --- Perspective --- p.120 / References --- p.121

by Tsang Wing Pui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 195-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Poon Hong Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-161). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT (ENGLISH/CHINESE) --- p.ii / ABBREVIATIONS --- p.vi / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.viii / TABLE OF CONTENTS --- p.x / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.4 / Chapter I. --- Models for studying photoreceptor cell death --- p.4 / Chapter II. --- Photic retinopathy --- p.5 / Chapter II.1. --- Proposed Mechanisms of Photic Retinopathy --- p.5 / Chapter II.2. --- Factors Affecting Photic Retinopathy --- p.6 / Chapter II.3. --- Pathologic Processes of Photic Retinopathy --- p.7 / Chapter III. --- P53 --- p.9 / Chapter III.1. --- Historical Perspective --- p.9 / Chapter III.2. --- Structure of p53 --- p.10 / Chapter III.2.1. --- The p53 Gene & mRNA --- p.10 / Chapter III.2.2. --- The p53 Protein --- p.10 / Chapter III.3. --- Modifications of p53 Protein --- p.13 / Chapter III.4. --- Functions of p53 --- p.14 / Chapter III.4.1. --- p53 & Tumors --- p.14 / Chapter III.4.2. --- p53 & DNA Damage --- p.15 / Chapter III.4.3. --- p53 & Apoptosis --- p.16 / Chapter III.4.3.1. --- p53-dependent Apoptosis --- p.16 / Chapter III.4.3.2. --- p53-independent Apoptosis --- p.20 / Chapter IV. --- clusterin --- p.22 / Chapter IV. 1. --- Historical Perspective --- p.22 / Chapter IV.2. --- Structure of Clusterin --- p.22 / Chapter IV.2.1. --- Clusterin Gene & mRNA --- p.22 / Chapter IV.2.2. --- Clusterin Protein --- p.22 / Chapter IV.3. --- Functions of Clusterin --- p.22 / Chapter IV.3.1. --- Clusterin & Apoptosis --- p.23 / OBJECTIVES --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter V. --- Sample collections --- p.26 / Chapter V.l. --- Induction of Photic Injury in Rat --- p.26 / Chapter V.2. --- Tissue Processing --- p.26 / Chapter V.2.1. --- Paraffin-embedded Tissues --- p.26 / Chapter V.2.2. --- Epoxy-embedded Tissues --- p.27 / Chapter V.3. --- Cell Culture --- p.28 / Chapter VI. --- Light-microscopic study --- p.29 / Chapter VI. 1. --- Histopathology --- p.29 / Chapter VI.1.1. --- Toluidine Blue --- p.29 / Chapter VI. 1.2. --- Morphometry of the ONL Thickness --- p.29 / Chapter VI.2. --- In situ TUNEL --- p.29 / Chapter VI.2.1. --- Morphometry of TUNEL --- p.29 / Chapter VI.3. --- Immunohistochemistry --- p.30 / Chapter VI.3.1. --- Single-labeling --- p.30 / Chapter VI.3.1.1. --- Immunolabeling of p53 Protein --- p.31 / Chapter VI.3.1.2. --- Immunolabeling of p21 Protein --- p.31 / Chapter VI.3.1.3. --- Immunolabeling of bax Protein --- p.31 / Chapter VI.3.1.4. --- Immunolabeling of c-fos Protein --- p.32 / Chapter VI.3.1.5 --- Immunolabeling of Clusterin Protein --- p.32 / Chapter VI.3.2. --- Double-labeling --- p.32 / Chapter VI.3.2.1. --- TUNEL & Fluorescent-labeling of p53 --- p.32 / Chapter VI.3.2.2. --- TUNEL & Fluorescent-labeling of p21 --- p.33 / Chapter VI.3.3. --- Grading of Immunoreactivities --- p.33 / Chapter VI.3.4. --- Morphometry of c-fos Immuno-positive Nuclei --- p.33 / Chapter VI.4. --- In situ RT-PCR --- p.34 / Chapter VI.4.1. --- Isolation of Retinal DNA --- p.34 / Chapter VI.4.2. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter VI.4.3. --- In situ Reverse Transcriptase 226}0ؤ PCR (RT-PCR) --- p.35 / RESULTS --- p.37 / Chapter VII. --- LIGHT-MICROSCOPY --- p.37 / Chapter VII.1. --- Histopathology & Morphometry --- p.37 / Chapter VII. 1.1. --- Histopathology --- p.37 / Chapter VII.1.2. --- Morphometry of the ONL Thickness --- p.49 / Chapter VII.2. --- DNA Nicks Detection --- p.42 / Chapter VII.2.1. --- In situ TUNEL --- p.42 / Chapter VII.2.2. --- Morphometry of TUNEL --- p.42 / Chapter VII.3. --- p53 --- p.51 / Chapter VII.3.1. --- Immunohistochemistry of p53 --- p.51 / Chapter VII.3.2. --- Grading of p53 Immunoreactivities --- p.51 / Chapter VII.3.3. --- p53 in situ RT-PCR --- p.51 / Chapter VII.3.4. --- Double-labeling ofp53 with in situ TUNEL --- p.59 / Chapter VII.4. --- p21 --- p.74 / Chapter VII.4.1. --- Immunohistochemistry of p21 --- p.74 / Chapter VII.4.2. --- Grading of p21 Immunoreactivities --- p.74 / Chapter VII.4.3. --- Double-labeling of p21 with in situ TUNEL --- p.74 / Chapter VII.5. --- bax --- p.84 / Chapter VII.5.1. --- Immunohistochemistry of bax --- p.84 / Chapter VII.5.2. --- Grading of bax Immunoreactivities --- p.84 / Chapter VII.5.3. --- bax in situ RT-PCR --- p.84 / Chapter VII.6. --- c-fos --- p.96 / Chapter VII.6.1. --- Immunohistochemistry of c-fos --- p.96 / Chapter VII.6.2. --- Morphometry of c-fos immuno-positive nuclei & TUNEL --- p.96 / Chapter VII.7. --- Clusterin / Chapter VII.7.1. --- Immunohistochemistry of Clusterin --- p.108 / Chapter VII.7.2. --- Grading of Clusterin Immunoreactivities --- p.108 / discussion --- p.116 / Chapter VIII. 1. --- Summary --- p.116 / Chapter VIII.1.1. --- Histopathology & Morphometry --- p.118 / Chapter VIII.1.2. --- In situ TUNEL --- p.119 / Chapter VIII.1.3. --- p53 & c-fos --- p.119 / Chapter VIII.1.4. --- p27 --- p.124 / Chapter VIII.1.5. --- bax --- p.124 / Chapter VIII. 1.6. --- Clusterin --- p.124 / Chapter VIII.2. --- Hypothesis --- p.125 / Chapter VIII.3. --- Conclusion --- p.127 / bibliography --- p.129

by Chan Ka Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 94-112). / Abstracts in English and Chinese. / Acknowledgements --- p.i / abstract(english/chinese) --- p.iii / Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / Chapter I --- Introduction --- p.1 / Chapter I.1 --- Gastric Lymphoma --- p.1 / Chapter I.1.1 --- Background --- p.1 / Chapter I.1.2 --- Mucosa-Associated Lymphoid Tissue --- p.2 / Chapter I.1.3 --- Classification of Primary Gastric Lymphomas --- p.3 / Chapter I.1.3.1 --- Mucosa-Associated Lymphooid Tissue Type Lymphomas --- p.3 / Chapter I.1.3.2 --- High Grade Primary Gastric Lymphomas --- p.5 / Chapter I.1.3.3 --- Other Gastric Lymphomas --- p.7 / Chapter I.2 --- Helicobater Pylori --- p.8 / Chapter I.3 --- Epstein-Barr Virus --- p.9 / Chapter I.3.1 --- Epidemiology --- p.9 / Chapter I.3.2 --- Virus and Genome Structure --- p.9 / Chapter I.3.3 --- Latent Infection --- p.11 / Chapter I.3.4 --- Latent Membrane Protein-1 --- p.12 / Chapter I.3.5 --- "EBV-Encoded, Small Non-polydenylated RNAs (EBERs)" --- p.13 / Chapter I.3.6 --- Disease Associated with EBV --- p.13 / Chapter I.3.7 --- EBV and PGL --- p.14 / Chapter I.4 --- Genetic Alterations --- p.15 / Chapter I.4.1 --- Background --- p.15 / Chapter I.4.2 --- Tumor Suppressor Genes (TSGs) --- p.16 / Chapter I.4.2.1 --- Origin and Structure of p53 Gene and Protein --- p.16 / Chapter I.4.2.2 --- Functions of p53 Gene --- p.18 / Chapter I.4.2.3 --- Inactivation Mechanisms of p53 --- p.21 / Chapter I.4.2.4 --- p53 Mutations and Protein Expression in NHLs --- p.24 / Chapter I.4.3 --- Oncogene --- p.25 / Chapter I.4.3.1 --- Bcl-2 --- p.25 / Chapter I.4.3.2 --- Other Oncogenes --- p.27 / Chapter II --- OBJECTIVES OF STUD Y --- p.30 / Chapter III --- ma terials and methods --- p.31 / Chapter III.1 --- Materials --- p.31 / Chapter III.2 --- Detection of EB V Latent Gene Product by In-situ Hybridization --- p.33 / Chapter III.2.1 --- Pretreatment of Paraffin-embedded Tissues and Apparatus --- p.33 / Chapter III.2.2 --- In-situ Hybridization of EBERs --- p.34 / Chapter III.3 --- Detection of p53 and bcl-2 and LMP-1 Protein Expression by Immunohisiochemistry --- p.35 / Chapter III.4 --- Microdissection of Formalin-fixed Paraffin-embedded Tissues --- p.37 / Chapter III.5 --- Extraction of Genomic DNA from Formalin-fixed Paraffin-embedded Tissues --- p.38 / Chapter III.5.1 --- Phenol / Chloroform Extraction --- p.38 / Chapter III.5.2 --- Commercially Available DNA Extraction Kit --- p.40 / Chapter III.6 --- Mutational Analysis p53 --- p.41 / Chapter III.6.1 --- Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) Analysis --- p.41 / Chapter III.6.1.1 --- PCR primers --- p.41 / Chapter III.6.1.2 --- PCR Amplification ofp53 gene --- p.42 / Chapter III.6.1.3 --- Non-denaturing Polyacrylamide Gel Electrophoresis --- p.42 / Chapter III.6.2 --- DNA Sequencing Analysis --- p.44 / Chapter III.6.2.1 --- Purification of DNA from Shifts on Non-denaturing Gels --- p.44 / Chapter III.6.2.2 --- 5' end Labeling of Primer --- p.45 / Chapter III.6.2.3 --- Cycle Sequencing --- p.45 / Chapter III.6.2.4 --- Denaturing Gel Electrophoresis --- p.46 / Chapter III.7 --- Loss of Heterozygosity (LOH) Analysis on Chromosome 17p --- p.47 / Chapter III.7.1 --- Microsatellite Markers --- p.49 / Chapter III.7.2 --- PCR Amplification of DNA Fragments Containing Polymorphic Microsatellites --- p.49 / Chapter III.7.3 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.50 / Chapter III.7.4 --- Determination of Allelic Abnormalities --- p.51 / Chapter III. 8 --- Statistical Analysis --- p.52 / Chapter IV --- results --- p.53 / Chapter IV.1 --- Association with Helicobactor Pylori (HP) --- p.53 / Chapter IV.2 --- Detection of EBERs by ISH --- p.53 / Chapter IV.3 --- Immunohistochemical Analysis --- p.54 / Chapter IV.3.1 --- Protein Expression of EBV LMP-1 --- p.54 / Chapter IV.3.2 --- Protein Expression of p53 --- p.54 / Chapter IV.3.3 --- Protein Expression of bcl-2 --- p.55 / Chapter IV.3.4 --- Correlation between p53 and bcl-2 Protein Expression --- p.55 / Chapter IV.4 --- Mutational Analysis of p53 --- p.56 / Chapter IV.5 --- LOH Analysis on Chromosome 17p --- p.57 / Chapter V --- DISCUSSION --- p.58 / Chapter V.1 --- Helicobactor Pylori Association --- p.58 / Chapter V.2 --- Association with EE V --- p.60 / Chapter V.3 --- Protein Expression of p53 and bcl-2 --- p.61 / Chapter V.3.1 --- p53 --- p.61 / Chapter V.3.2 --- Bcl-2 --- p.62 / Chapter V.3.3 --- Correlation between p53 and bcl-2 Expression --- p.63 / Chapter V.4 --- p53 Gene Molecular Analysis --- p.65 / Chapter V.5 --- Distribution of Mutations and Molecular Fingerprinting --- p.67 / Chapter V.6 --- Possible Role of p53 Mutation in EBV+ Gastric Lymphomas --- p.69 / ILLUSTRATIONS --- p.71 / references --- p.94

Introdução: A incidência de tumores adrenocorticais em crianças é particularmente elevada nas regiões sudeste e sul do Brasil, correlacionandose com a ocorrência da mutação germinativa p.R337H do supressor tumoral p53, entretanto, o carcinoma adrenocortical é uma neoplasia endócrina maligna rara em todo o mundo com uma incidência aproximada de 0,5 - 2 casos por milhão por ano. Esta condição é uma doença heterogênea, apresentando frequentemente comportamento clínico agressivo e letal. A cascata de sinalização Wnt é uma via importante de transdução de sinal em cânceres humanos e tem sido implicada na tumorigênese adrenocortical. A atividade desta via de sinalização é dependente da quantidade de beta-catenina citoplasmática e nuclear. Mutações ativadoras no gene da beta-catenina (CTNNB1) foram relatadas em diversas neoplasias humanas. Estudos demonstraram que mutações no gene CTNNB1 são os defeitos genéticos mais frequentemente encontrados em adenomas e em carcinomas adrenocorticais. O estudo destas mutações demonstrou que as alterações no gene CTNNB1 localizam-se principalmente exon 3, que codifica a porção amino terminal da beta- catenina. Objetivos: determinar a ocorrência e a frequência das mutações somáticas no exon 3 do gene CTNNB1. Adicionalmente, determinar a imunorreatividade de beta-catenina e de p53 em tumores adrenocorticais benignos e malignos de crianças e adultos. Correlacionar os resultados da análise de mutações gênicas e os dados de imunorreatividade com as características hormonais, a mutação p.R337H do p53, o diagnóstico histológico e a evolução dos tumores adrenocorticais de crianças e adultos. Métodos: Neste estudo, a análise de imunohistoquímica para beta-catenina e p53 foi realizada em 103 tumores adrenocorticais benignos e malignos (40 crianças e 63 adultos), estando as amostras histológicas alocadas em micromatriz tecidual (TMA). A pesquisa de mutações no exon 3 do gene CTNNB1 foi determinada por seqüenciamento automático em 64 tumores adrenocorticais. Resultados: a imunorreatividade para beta-catenina em citoplasma e/ou núcleo foi evidenciada de maneira similar nos tumores adrenocorticais benignos e malignos de crianças e de adultos (15% e 23,8%, respectivamente). O percentual das células neoplásicas imunorreativas para beta-catenina em citoplasma e/ou núcleo não foi significativamente diferente entre os tumores clinicamente benignos e malignos pediátricos (15,6% vs. 12,5%, respectivamente; p=0,93) e entre adenomas e carcinomas adrenocorticais de adultos (28,5% vs. 17,8%, respectivamente; p=0,38). A síndrome endócrina causada pelo perfil de secreção hormonal foi similar entre os tumores adrenocorticais com presença ou ausência de acúmulo citoplasmático e/ou nuclear de beta-catenina em crianças e adultos. A associação entre acúmulo anormal de beta-catenina e diminuição de sobrevida foi avaliada nos pacientes adultos portadores de carcinomas adrenocorticais isoladamente (n=25), sendo observada na curva de Kaplan- Meier uma tendência de significância (log-rank p=0,07). A análise do gene CTNNB1 revelou mutações somáticas em heterozigose em 10 tumores adrenocorticais (4 crianças e 6 adultos). As mutações encontradas no gene CTNNB1 foram, sobretudo do tipo missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Outras mutações encontradas compreenderam: a inserção de um único nucleotídeo (p.E9GfsX14), dando origem a uma desvio de leitura do exon 3; além da deleção dos três nucleotídeos do códon 45 (p.Ser45del). Todos os tumores com mutações somáticas no gene CTNNB1 mostraram acúmulo anormal para ?-catenina, com exceção de um caso. A presença de alterações no gene CTNNB1 não se associou ao tamanho do tumor (Teste de Mann-Whitney: p=0,75), desfecho desfavorável tanto no grupo pediátrico (log-rank p=0,29) como no grupo de pacientes adultos (log-rank p=0,77). Todos os pacientes portadores da mutação germinativa do gene TP53 apresentaram imunorreatividade nuclear de p53 nas células tumorais. Não foi encontrada correlação entre a presença de acúmulo anormal de beta-catenina e imunorreatividade nuclear de p53, considerando os grupos de crianças e de adultos portadores de tumores adrenocorticais. Adicionalmente, não foi observada correlação entre mutações no gene CTNNB1, bem como acúmulo anormal de beta-catenina, com a imunorreatividade nuclear de p53 no grupo de tumores adrenocorticais de pacientes adultos, porém, interessantemente, avaliando isoladamente o grupo de tumores adrenocorticais pediátricos, foi observada relação entre a presença de mutações no gene CTNNB1 e a presença de acúmulo nuclear de p53 (X2: p=0,009). Conclusões: Estes dados confirmam a participação da via Wnt na tumorigênese adrenocortical de crianças e de adultos, que apresenta uma prevalência de ativação semelhante entre crianças e adultos. O acúmulo citoplasmático e/ou nuclear de beta-catenina provavelmente é um marcador biológico de mau prognóstico do carcinoma de adrenocortical de adultos. Adicionalmente, observamos evidências de uma correlação positiva entre mutações no gene CTNNB1 e acúmulo nuclear de p53 em tumores adrenocorticais pediátricos, confirmando uma possível relação destas duas vias na tumorigênese do córtex da glândula suprarrenal / Introduction: The incidence of adrenocortical tumors in children is particularly high in the southeastern and southern regions of Brazil, correlating with the occurrence of p.R337H p53 tumor suppressor germline mutation. However, adrenocortical carcinoma is a worldwide rare endocrine malignancy with an approximate incidence of 0.5 to 2 cases per million per year. This condition is a heterogeneous disease and is often lethal. The Wnt signaling pathway is an important signal transduction pathway in human cancers and has been implicated in adrenocortical tumorigenesis. The activity of this signaling pathway is dependent on the amount of nuclear and cytoplasmic beta-catenin. Activating mutations of ?-catenin (CTNNB1) gene have been reported in several human malignancies. Studies have shown that CTNNB1 mutations are the most common genetic defect found in adrenocortical adenomas and carcinomas. The study of these mutations demonstrated that the changes in CTNNB1 gene are mainly located in exon 3, which encodes the amino terminal portion of the beta- catenin. Objectives: to determine the occurrence and frequency of CTNNB1 somatic mutations and the abnormal beta-catenin and p53 accumulation in benign and malignant adrenocortical tumors in both children and adults. We also evaluated the correlation of the gene mutations analysis and immunohistochemistry data with the hormonal characteristics, the p.R337H germline mutation, the histological diagnosis and the prognosis of adrenocortical tumors in children and adults. Methods: In this study, immunohistochemistry for beta-catenin and p53 was performed in 103 benign and malignant (40 children and 63 adults) adrenocortical tumors. The histological samples were allocated in a tissue microarray (TMA). The study of the CTNNB1 gene was performed by direct sequencing of 64 adrenocortical tumors. Results: The beta-catenin abnormal accumulation was similar in benign and malignant adrenocortical tumors of children and adults (15 % and 23.8 %, respectively). The percentage of cells with beta-catenin abnormal accumulation was not significantly different between benign and malignant pediatric adrenocortical tumors (15.6% vs. 12.5 %, respectively; P=0.93) and between adrenocortical adenomas and carcinomas in adults (28.5% vs 17.8 %, respectively; p=0.38). The endocrine syndrome caused by hormonal tumor secretion was similar in patients with and without beta-catenin abnormal accumulation both in pediatric and adult patients. The association between beta-catenin abnormal accumulation and decreased survival was evaluated in adult patients with adrenocortical carcinomas (n=25) and a trend toward significance was observed (log-rank p=0,07). The analysis of the CTNNB1 gene revealed heterozygous somatic mutations in 10 adrenocortical tumors (6 adults and 4 children). The mutations found in CTNNB1 gene were mainly missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Other mutations found included: a single nucleotide insertion (p.E9GfsX14) and a deletion within codon 45 of exon 3 of CTNNB1 gene, (p.Ser45del). All tumors with somatic mutations in the CTNNB1 gene showed abnormal beta -catenin accumulation, except for one case. The mutations in CTNNB1 gene was not associated with tumor size (Mann - Whitney: p=0.75), unfavorable outcome in both pediatric (log -rank p=0.29) and adult group of patients (log-rank p=0.77). All patients with TP53 germline mutation showed p53 nuclear accumulation in the tumor cells. No correlation was found between the presence of beta-catenin abnormal accumulation and p53 nuclear accumulation in adrenocortical tumor cells of children and adults. In addition, no correlation was observed between CTNNB1 mutations, as well as beta-catenin abnormal accumulation, with p53 nuclear accumulation in adults adrenocortical tumors. Interestingly, the evaluation of pediatric adrenocortical tumors revealed a relationship between the occurrence of CTNNB1 mutations and the presence of p53 nuclear accumulation (X2: p=0.009). Conclusions: These data confirm the involvement of the Wnt pathway in adrenocortical tumorigenesis of children and adults, which has a prevalence similar activation between children and adults. We observed that abnormal beta-catenin accumulation in adults adrenocortical carcinoma is probably associated with a dismal prognosis. Additionally, we found evidence of a positive relationship between CTNNB1 mutations and p53 nuclear accumulation in pediatric adrenocortical tumors, confirming a possible connection of these two pathways in the pediatric adrenocortical tumorigenesis

Introdução: A incidência de tumores adrenocorticais em crianças é particularmente elevada nas regiões sudeste e sul do Brasil, correlacionandose com a ocorrência da mutação germinativa p.R337H do supressor tumoral p53, entretanto, o carcinoma adrenocortical é uma neoplasia endócrina maligna rara em todo o mundo com uma incidência aproximada de 0,5 - 2 casos por milhão por ano. Esta condição é uma doença heterogênea, apresentando frequentemente comportamento clínico agressivo e letal. A cascata de sinalização Wnt é uma via importante de transdução de sinal em cânceres humanos e tem sido implicada na tumorigênese adrenocortical. A atividade desta via de sinalização é dependente da quantidade de beta-catenina citoplasmática e nuclear. Mutações ativadoras no gene da beta-catenina (CTNNB1) foram relatadas em diversas neoplasias humanas. Estudos demonstraram que mutações no gene CTNNB1 são os defeitos genéticos mais frequentemente encontrados em adenomas e em carcinomas adrenocorticais. O estudo destas mutações demonstrou que as alterações no gene CTNNB1 localizam-se principalmente exon 3, que codifica a porção amino terminal da beta- catenina. Objetivos: determinar a ocorrência e a frequência das mutações somáticas no exon 3 do gene CTNNB1. Adicionalmente, determinar a imunorreatividade de beta-catenina e de p53 em tumores adrenocorticais benignos e malignos de crianças e adultos. Correlacionar os resultados da análise de mutações gênicas e os dados de imunorreatividade com as características hormonais, a mutação p.R337H do p53, o diagnóstico histológico e a evolução dos tumores adrenocorticais de crianças e adultos. Métodos: Neste estudo, a análise de imunohistoquímica para beta-catenina e p53 foi realizada em 103 tumores adrenocorticais benignos e malignos (40 crianças e 63 adultos), estando as amostras histológicas alocadas em micromatriz tecidual (TMA). A pesquisa de mutações no exon 3 do gene CTNNB1 foi determinada por seqüenciamento automático em 64 tumores adrenocorticais. Resultados: a imunorreatividade para beta-catenina em citoplasma e/ou núcleo foi evidenciada de maneira similar nos tumores adrenocorticais benignos e malignos de crianças e de adultos (15% e 23,8%, respectivamente). O percentual das células neoplásicas imunorreativas para beta-catenina em citoplasma e/ou núcleo não foi significativamente diferente entre os tumores clinicamente benignos e malignos pediátricos (15,6% vs. 12,5%, respectivamente; p=0,93) e entre adenomas e carcinomas adrenocorticais de adultos (28,5% vs. 17,8%, respectivamente; p=0,38). A síndrome endócrina causada pelo perfil de secreção hormonal foi similar entre os tumores adrenocorticais com presença ou ausência de acúmulo citoplasmático e/ou nuclear de beta-catenina em crianças e adultos. A associação entre acúmulo anormal de beta-catenina e diminuição de sobrevida foi avaliada nos pacientes adultos portadores de carcinomas adrenocorticais isoladamente (n=25), sendo observada na curva de Kaplan- Meier uma tendência de significância (log-rank p=0,07). A análise do gene CTNNB1 revelou mutações somáticas em heterozigose em 10 tumores adrenocorticais (4 crianças e 6 adultos). As mutações encontradas no gene CTNNB1 foram, sobretudo do tipo missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Outras mutações encontradas compreenderam: a inserção de um único nucleotídeo (p.E9GfsX14), dando origem a uma desvio de leitura do exon 3; além da deleção dos três nucleotídeos do códon 45 (p.Ser45del). Todos os tumores com mutações somáticas no gene CTNNB1 mostraram acúmulo anormal para ?-catenina, com exceção de um caso. A presença de alterações no gene CTNNB1 não se associou ao tamanho do tumor (Teste de Mann-Whitney: p=0,75), desfecho desfavorável tanto no grupo pediátrico (log-rank p=0,29) como no grupo de pacientes adultos (log-rank p=0,77). Todos os pacientes portadores da mutação germinativa do gene TP53 apresentaram imunorreatividade nuclear de p53 nas células tumorais. Não foi encontrada correlação entre a presença de acúmulo anormal de beta-catenina e imunorreatividade nuclear de p53, considerando os grupos de crianças e de adultos portadores de tumores adrenocorticais. Adicionalmente, não foi observada correlação entre mutações no gene CTNNB1, bem como acúmulo anormal de beta-catenina, com a imunorreatividade nuclear de p53 no grupo de tumores adrenocorticais de pacientes adultos, porém, interessantemente, avaliando isoladamente o grupo de tumores adrenocorticais pediátricos, foi observada relação entre a presença de mutações no gene CTNNB1 e a presença de acúmulo nuclear de p53 (X2: p=0,009). Conclusões: Estes dados confirmam a participação da via Wnt na tumorigênese adrenocortical de crianças e de adultos, que apresenta uma prevalência de ativação semelhante entre crianças e adultos. O acúmulo citoplasmático e/ou nuclear de beta-catenina provavelmente é um marcador biológico de mau prognóstico do carcinoma de adrenocortical de adultos. Adicionalmente, observamos evidências de uma correlação positiva entre mutações no gene CTNNB1 e acúmulo nuclear de p53 em tumores adrenocorticais pediátricos, confirmando uma possível relação destas duas vias na tumorigênese do córtex da glândula suprarrenal / Introduction: The incidence of adrenocortical tumors in children is particularly high in the southeastern and southern regions of Brazil, correlating with the occurrence of p.R337H p53 tumor suppressor germline mutation. However, adrenocortical carcinoma is a worldwide rare endocrine malignancy with an approximate incidence of 0.5 to 2 cases per million per year. This condition is a heterogeneous disease and is often lethal. The Wnt signaling pathway is an important signal transduction pathway in human cancers and has been implicated in adrenocortical tumorigenesis. The activity of this signaling pathway is dependent on the amount of nuclear and cytoplasmic beta-catenin. Activating mutations of ?-catenin (CTNNB1) gene have been reported in several human malignancies. Studies have shown that CTNNB1 mutations are the most common genetic defect found in adrenocortical adenomas and carcinomas. The study of these mutations demonstrated that the changes in CTNNB1 gene are mainly located in exon 3, which encodes the amino terminal portion of the beta- catenin. Objectives: to determine the occurrence and frequency of CTNNB1 somatic mutations and the abnormal beta-catenin and p53 accumulation in benign and malignant adrenocortical tumors in both children and adults. We also evaluated the correlation of the gene mutations analysis and immunohistochemistry data with the hormonal characteristics, the p.R337H germline mutation, the histological diagnosis and the prognosis of adrenocortical tumors in children and adults. Methods: In this study, immunohistochemistry for beta-catenin and p53 was performed in 103 benign and malignant (40 children and 63 adults) adrenocortical tumors. The histological samples were allocated in a tissue microarray (TMA). The study of the CTNNB1 gene was performed by direct sequencing of 64 adrenocortical tumors. Results: The beta-catenin abnormal accumulation was similar in benign and malignant adrenocortical tumors of children and adults (15 % and 23.8 %, respectively). The percentage of cells with beta-catenin abnormal accumulation was not significantly different between benign and malignant pediatric adrenocortical tumors (15.6% vs. 12.5 %, respectively; P=0.93) and between adrenocortical adenomas and carcinomas in adults (28.5% vs 17.8 %, respectively; p=0.38). The endocrine syndrome caused by hormonal tumor secretion was similar in patients with and without beta-catenin abnormal accumulation both in pediatric and adult patients. The association between beta-catenin abnormal accumulation and decreased survival was evaluated in adult patients with adrenocortical carcinomas (n=25) and a trend toward significance was observed (log-rank p=0,07). The analysis of the CTNNB1 gene revealed heterozygous somatic mutations in 10 adrenocortical tumors (6 adults and 4 children). The mutations found in CTNNB1 gene were mainly missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Other mutations found included: a single nucleotide insertion (p.E9GfsX14) and a deletion within codon 45 of exon 3 of CTNNB1 gene, (p.Ser45del). All tumors with somatic mutations in the CTNNB1 gene showed abnormal beta -catenin accumulation, except for one case. The mutations in CTNNB1 gene was not associated with tumor size (Mann - Whitney: p=0.75), unfavorable outcome in both pediatric (log -rank p=0.29) and adult group of patients (log-rank p=0.77). All patients with TP53 germline mutation showed p53 nuclear accumulation in the tumor cells. No correlation was found between the presence of beta-catenin abnormal accumulation and p53 nuclear accumulation in adrenocortical tumor cells of children and adults. In addition, no correlation was observed between CTNNB1 mutations, as well as beta-catenin abnormal accumulation, with p53 nuclear accumulation in adults adrenocortical tumors. Interestingly, the evaluation of pediatric adrenocortical tumors revealed a relationship between the occurrence of CTNNB1 mutations and the presence of p53 nuclear accumulation (X2: p=0.009). Conclusions: These data confirm the involvement of the Wnt pathway in adrenocortical tumorigenesis of children and adults, which has a prevalence similar activation between children and adults. We observed that abnormal beta-catenin accumulation in adults adrenocortical carcinoma is probably associated with a dismal prognosis. Additionally, we found evidence of a positive relationship between CTNNB1 mutations and p53 nuclear accumulation in pediatric adrenocortical tumors, confirming a possible connection of these two pathways in the pediatric adrenocortical tumorigenesis