FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF P53 AND RB

本研究的目的是继续表征细胞周期调节因子在成人软组织肉瘤(STS)中的突变和异常表达模式，因为它们与肿瘤的发生和发展有关。我们的工作假设是，TP53和Rb基因的突变和表达变化对STS患者的肿瘤生长和侵袭性行为产生了选择性优势。它们的关键作用需要其他因素的严格多水平调控，如细胞周期蛋白、细胞周期蛋白依赖性蛋白激酶(CDK)和细胞周期蛋白依赖性蛋白激酶抑制因子(CKI)。对这些监管机构的研究是我们之前工作的合乎逻辑的延伸。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：目的#1.成人软组织肉瘤中TP53和Rb的预后意义：分子和功能分析。这一目标分为两个研究。在研究A中，我们计划：1)前瞻性地验证TP53和Rb的预后价值；2)确定不同的TP53突变的功能意义；3)确定pRb表达改变的分子基础以及E2F水平与化疗反应的关系。在研究B中，我们将确定特定的STS亚型是否具有与p53和pRb相关的特定分子“签名”。目的#2.P53通路的分子特征：对细胞周期停滞和细胞凋亡的影响。我们将确定：1)p21表达模式改变的临床相关性；2)由于缺乏MDM2扩增而过度表达MDM2的STS的比例；3)过度表达MDM2但不表达p19/ARF的STS病例的比例；以及4)联合p53和MDM2改变的潜在临床影响。目的#3.RB通路的分子特征：对S期进入和增殖的影响。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，或两者都涉及；3)细胞周期蛋白D1和CDK4过表达的病例比例，以及与基因扩增有关的比例；4)CDK4和MDM2共扩增的频率；以及5)检测E2F蛋白(主要是E2F1、E2F4和E2F5)表达模式变化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).