FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF P53 AND RB

拟议研究的目的是继续表征成人软组织肉瘤（STS）中细胞周期调节因子的突变和异常表达模式，因为它们对肿瘤发生和肿瘤进展的影响。我们的工作假设是TP53和RB基因的突变和表达改变对STS患者的肿瘤生长和侵袭行为产生选择性优势。它们的关键作用需要其他因子如细胞周期蛋白、细胞周期蛋白依赖性激酶（Cdk）和细胞周期蛋白依赖性激酶抑制剂（Cki）的严格多级调控。对这些调节器的研究是我们以前工作的合理延伸。我们项目的主要目标是将基础和临床研究成果转化为临床研究。具体目标概述如下：目标#1。TP53和RB在成人软组织肉瘤中的预后意义：分子和功能分析。这个目标分为两个研究。在研究A中，我们计划：1)前瞻性验证TP53和RB的预后价值；2)确定不同TP53突变的功能意义；3)确定pRB表达改变的分子基础以及与化疗反应相关的E2F水平。在研究B中，我们将确定特定STS亚型在p53和pRB方面是否具有特定的分子“特征”。目标# 2。p53通路的分子表征：对细胞周期阻滞和凋亡的影响。我们将确定：1)p21表达模式改变的临床相关性；2)缺乏mdm2扩增而过表达mdm2的STS比例；3) mdm2过表达而p19/ARF不表达的STS病例比例；4)关节p53和mdm2改变的潜在临床影响。目标# 3。rb通路的分子表征：对S相进入和增殖的影响。我们将确定：1)pRB过表达的频率、临床意义和性质；2) INK4A在STS中的突变频率，以及这些突变是否涉及p16、p19/ARF，或两者兼而有之；3) cyclin D1和Cdk4过表达的病例所占比例，以及基因扩增所占比例；4) CDK4和MDM2共扩增频率；5)检测E2F蛋白（主要是E2F1、E2F4和E2F5）表达模式改变的频率和潜在的临床相关性。

The objective of the proposed studies is the continued characterization of mutations and aberrant patterns of expression of cell cycle regulators as they rate to tumorigenesis and tumor progression in adult soft tissue sarcomas (STS). Our working hypothesis has been that mutations and altered expression of the TP53 and RB genes produce a selective advantage for tumor growth and aggressive behavior in STS patients. Their critical role requires stringent multi-level regulation by other factors such as cyclins, cyclin-dependent kinases (Cdk), and cyclin-dependent kinase inhibitors (Cki). Studies of these regulators are a logical extension of our previous work. The main goal of our project is to translate basic and clinical research findings into clinical studies. The Specific Aims are outlined as follows: Aim #1. Prognostic Significance of TP53 and RB in Adult Soft Tissue Sarcomas: Molecular and Functional Analyses. This aim is divided into two studies. In Study A we plan to: 1) prospectively validate the prognostic value of TP53 and RB; 2) determine the functional significance of different TP53 mutations; 3) determine the molecular basis of altered pRB expression and relating E2F levels with response to chemotherapy. In Study B we will determine if particular STS subtypes have specific molecular "signatures" with respect to p53 and pRB. Aim #2. Molecular Characterization of the p53-Pathway: Impact on Cell Cycle Arrest and Apoptosis. We will determine: 1) the clinical relevance of altered patterns of p21 expression; 2) the proportion of STS that over- express mdm2 by lack MDM2 amplification; 3) the proportion of STS cases that over-express mdm2 but do not express p19/ARF; and 4) the potential clinical impact of joint p53 and mdm2 alterations. Aim #3. Molecular Characterization of the RB-Pathway: Impact on S- Phase Entry and Proliferation. We will determine: 1) the frequency, clinical significance, and nature of pRB over-expression; 2) the mutation frequency of INK4A in STS, and if these mutations involve p16, p19/ARF, or both; 3) the proportion of cases that over-express cyclin D1 and Cdk4, and what proportion is due to gene amplifications; 4) the frequency of co-amplification of CDK4 and MDM2; and 5 ) the frequency and potential clinical relevance of detecting altered expression patterns of E2F proteins (mainly E2F1, E2F4, and E2F5).