BRCA1 和 RB1 基因体细胞突变在乳腺癌预后中的作用

Part 1: Background：Triple-negative breast cancer (TNBC) is a subtype of aggressive breast cancer with generally poor outcome. Lacking molecular targets, chemotherapy is the main adjuvant treatment for TNBC patients. Recent studies demonstrated that breast cancers with BRCA1 mutation would benefit from poly ADP-ribose polymerase (PARP) inhibitors. TNBC harbor a high prevalence of BRCA1 germline mutation, but there was no research focus on BRCA1 somatic mutation of TNBC. Material and methods：222 tumor tissues, including 112 core needle biopsy samples and 110 tumor tissues procured following surgery, from TNBC patients without BRCA1/2 germline pathogenic mutation were involved in this study. Tumor RNA was extracted from core-needle biopsy samples, then reverse-transcribed to cDNA; Tumor DNA was purified from tumor tissues procured following surgery. PCR- direct sequencing analysis was used to examine the whole coding sequences of the BRCA1 gene in tumor DNA/cDNA. Patients who carried a BRCA1 mutation in their tumor tissues were further investigated to determine whether the mutation was present in the corresponding blood DNA. PolyPhen, SIFT and Pmut were used for functional prediction for those BRCA1 missense mutations with unknown significance. SPSS17.0 was used to analyze the association between BRCA1 somatic status and clinicopathological characteristics. Result: 10 (4.5%) somatic mutations were identified by sequencing of the entire coding regions of BRCA1 gene. Among them, 8 (3.6%) mutations were pathogenic, including four frame-shift (c.1664delA, c.3671_3672insTTCC, c.4674_4675+17del, c.191_212del22) mutations, one nonsense (C.1162A>T) mutations, one splice site mutation (c.134+2T>C), and two missense mutations (c.286G>A, c.5511G>C) which were proved to affect BRCA1 protein function. Breast cancer patients with BRCA1 somatic pathogenic mutation had a tendency to be early-onset (37.5% vs 13.1%, P=0.085) and lymph node metastasis (50% vs 30.3%, P=0.258). Conclusion: Eight (3.6%) pathogenic somatic mutations of BRCA1 were detected in this study, and carriers may had a more aggressive phenotype. Hence, more TNBC patients may benefit from PARP inhibitor. Part 2: Background: RB1 is the first tumor suppressor gene to be cloned which has been implicated in many cellular processes, such as regulation of the cell cycle, DNA repair, DNA replication, differentiation, and apoptosis. The RB-pathway status can provide critical information related to therapeutic interventions of breast cancer. Recent studies have demonstrated that RB-deficiency led to increased sensitivity to cytotoxic therapies and was associated with resistance to hormonal therapy. Multiple mechanisms can contribute to RB inactivation, such as RB1 mutation, deletion, epigenetic silencing or Rb protein phosphorylation/inactivation by CDK4/CyclinD1. Most studies about RB in breast cancers are focused on RB expression or loss of heterozygosity. However, the prevalence and prognostic value of RB1 mutation in breast cancers is unclear. Purpose: We aimed to investigate the prevalence of RB1 mutation in a relatively large cohort of breast cancer patients and further investigate the association between RB1 somatic mutations and survival in breast cancer patients. Material and methods: This study included 1187 primary breast cancer (Stage I-III) patients who were treated at Breast Center, Peking University Cancer Hospital from July 2002 to July 2012. Total RNA was purified from tumor tissue samples, then reverse-transcribed to cDNA. The whole coding sequences of the RB1 gene in tumor cDNA was screened by using polymerase chain reaction -direct sequencing (sanger sequencing) analysis. Patients who carried RB1 mutations in breast tumors were further investigated to determine whether the mutation was present in the corresponding blood or normal tissue. SPSS17.0 was used to analyze the association between RB1 somatic status and clinicopathological characteristics, and to compare the difference between the RB1 somatic mutation carriers and non-carriers about disease-free survival (DFS), distant-disease-free survival (DFS) and Overall Survival (OS). Results: 36 mutations (34 carriers) were identified in this cohort of 1187 breast cancers, including 9 germline mutations, 24 somatic mutations (22 carriers, 1.9%), 3 unclassified. RB1 somatic mutation was correlated with triple-negative breast cancer and high histopathologic grade, but had no association with tumor size, age and lymph node status. RB1 somatic status was not associated with Overall Survival (P=0.388), but had a significantly worse DFS and DDFS (5-year DFS: 65.2% vs 83.7%, P=0.043; 5-year DDFS: 65.2% vs 86.1%, P=0.012). In multivariate analysis, RB1 somatic status was an independent prognostic factor for DFS and DDFS （DFS, adjusted HR=2.67, 95% CI, 1.15-6.19, P=0.022; DDFS, adjusted HR=3.27, 95% CI, 1.40-7.63, P=0.006） Conclusion: The prevalence of RB1 somatic mutation in breast cancers is 1.9%. RB1 somatic mutation is correlated with triple-negative breast cancer and high histopathologic grade. RB1 somatic mutation carriers have worse survival than those with wide-type.