表观遗传学修饰在妇科肿瘤诊断中的应用价值研究
|
摘要
表观遗传学修饰(Epigenetics)是指在不改变DNA序列的前提下,细胞内其他可遗传物质发生改变而引起的基因表达或细胞表型变化,这种改变在胚胎发育和细胞增殖过程中能稳定遗传且具有可逆潜能。表观遗传学修饰主要包括两大类,一类是基因选择性转录表达的调控,主要包括DNA甲基化、染色质重塑、组蛋白修饰、基因组印记、基因沉默、核仁显性及休眠转座子激活等,另一类为基因转录后的调控,主要包括基因组中非编码RNA.MicroRNA.反义RNA.内含子及核糖开关等。与经典遗传学以研究基因序列影响生物学功能为核心相比,表观遗传学主要研究这些“表观遗传现象”建立和维持的机制,作为经典遗传学的补充,表观遗传学让我们更全面的理解基因调节的机制。近年来研究已证实表观遗传学修饰在干细胞分化,早期胚胎发育及多种疾病(肿瘤、自身免疫疾病及代谢性疾病等)的发生发展过程中具有重要作用；特别是在肿瘤研究领域,表观遗传学修饰与原癌基因激活、抑癌基因失活、DNA损伤修复缺陷及肿瘤干细胞分化等密切相关。
针对肿瘤表观遗传学机制研究的最终目的是为了应用于临床诊断及预防治疗。DNA甲基化和MicroRNA在诊断方面的研究已经趋于成熟,Fujiwara等研究表明5个基因血清游离DNA甲基化谱在诊断早期肺癌方面特异性和阳性预测值分别为85%和75%,同样众多研究表明MicroRNA表达谱可应用于乳腺癌的病理分型(如雌孕激素受体状态、肿瘤分期、肿瘤转移及Her2状态等)。本研究主要研究DNA甲基化及MicrORNA表达谱在早期上皮性卵巢癌及子宫平滑肌肿瘤诊断分类中的应用。
第一部分：多重巢式甲基化特异性PCR在早期上皮性卵巢癌诊断中的应用研究
背景和目的
卵巢癌发病率位居女性恶性生殖系统肿瘤第三位,病死率居第一位,占所有因癌症死亡女性患者的3%,其中90%以上为上皮性卵巢癌。卵巢癌发病隐匿,临床确诊时约85%患者疾病己为晚期(FIGOⅡ-Ⅳ期),5年生存率仅为30-44%,而早期卵巢癌(FIGOⅠ期)患者5年生存率高达93%。因此,除积极寻找新的有效的治疗方式外,探讨卵巢癌发生发展的生物学机制,研发新型卵巢癌早期诊断技术迫在眉睫。
目前尚无简便有效的、可常规应用的卵巢癌早期诊断方法。CA125是目前普遍使用的卵巢癌标志物,但仅有50%的Ⅰ期卵巢癌患者CA125水平升高,且其他疾病(如子宫内膜异位症、卵巢纤维瘤、盆腔炎性疾病及某些其他恶性肿瘤等)亦可引起CA125水平升高,故其敏感性和特异性较差,卵巢癌阳性预测值仅有100%～35%。总体而言,对于血清CA125检测、经阴道超声探查等传统的诊断方法单一或联合应用,目前均无证据显示可降低人群的卵巢癌发病率和(或)病死率,其主要原因在于这些方法的假阴性或假阳性率均过高,敏感性和特异性达不到临床需要。
DNA甲基化,即在DNA甲基转移酶的催化下将甲基转移到胞嘧啶的第5位碳原子上生成5-甲基胞嘧啶,是重要的表观遗传学机制。DNA启动子区异常高甲基化是抑癌基因失活的重要机制,在某些情况下可能是唯一的机制,在癌症发生发展中发挥重要作用。作为癌症发生的早期事件,DNA异常甲基化检测可以在患者出现临床表现或者影像学证据之前做到分子诊断,为癌症早期诊断提供新的途径。现己证实肿瘤患者中血清游离DNA含量明显升高,其来源主要有2种机制：1、增殖旺盛的肿瘤细胞持续释放DNA进入血液循环；2、肿瘤细胞的坏死、凋亡或直接入血裂解使血清游离DNA含量增高。血清游离DNA具有与原发肿瘤组织相一致的分子遗传学改变(如启动子区高甲基化、基因突变、微卫星不稳定和杂合性缺失等),可间接反映肿瘤的发生发展情况。使用血清游离DNA甲基化检测在其他癌症如肺癌、头颈部肿瘤、前列腺癌等己被证实可行,因此卵巢癌患者血清游离DNA甲基化检测,是一种可行的具有临床实用价值的卵巢癌早期诊断技术。针对血清游离DNA微量,甲基化特异性PCR敏感性不足的特点,课题组将多重PCR、巢式PCR与甲基化特异性PCR相结合,开发出全新的多重巢式甲基化特异性PCR (Multiplex-MSP)检测方法,已满足血清微量肿瘤游离DNA样本多基因甲基化检测需要。
材料和方法
1.上皮性卵巢癌特异性甲基化基因的筛选。
2.临床卵巢肿瘤及正常对照病人血清及组织样本的收集。
3.引物的设计合成及多重巢式甲基化特异性PCR反应体系的优化。
4.多重巢式甲基化特异性PCR检测血清及组织样本甲基化水平。
5.卵巢癌血清与组织甲基化谱对照研究。
6.回顾性分析评估多重巢式甲基化特异性PCR检测在上皮性卵巢癌早期诊断中的意义。
7.双盲实验进一步验证多重巢式甲基化特异性PCR检测在上皮性卵巢癌早期诊断中的作用。
结果
1.通过Pubmed检索筛选出7个与卵巢癌发生发展密切相关的高频甲基化基因APC, RASSF1A, CDH1, RUNX3, TFP12, SFRP5和OPCML。
2.将多重PCR、巢式PCR与甲基化特异性PCR相结合,开发出全新的多重巢式甲基化特异性PCR检测方法。
3.应用多重巢式甲基化特异性PCR对20例卵巢癌血清及配对组织进行检测,证实血清游离DNA甲基化谱完全包含于肿瘤组织DNA甲基化谱。
4.应用多重巢式甲基化特异性PCR回顾140例卵巢肿瘤及正常女性血清标本,结果证实该方法的特异性为90.57%,敏感性为89.66%；特别是在
背景和目的
临床上,常见的子宫平滑肌肿瘤主要包括良性子宫平滑肌瘤和恶性子宫平滑肌肉瘤,它们的分类主要基于以下三个标准：细胞异型性、有丝分裂相、肿瘤细胞坏死,典型的子宫平滑肌瘤形态学上主要表现为无细胞异型性,小于10MF/10HPF的有丝分裂相,可能会表现出玻璃样坏死(如缺血性),但缺乏典型的凝固性肿瘤细胞坏死；相反,恶性子宫平滑肌肉瘤至少具备以下3个特征中的2个,明显的细胞异型性,大于10MF/10HPF的有丝分裂相及典型的凝固性肿瘤细胞坏死。
在典型的子宫平滑肌瘤和子宫平滑肌肉瘤之间,存在几大类中间类型的子宫平滑肌肿瘤,它们表现一些但不是所有的恶性肿瘤的特征,主要包括以下几大类：不典型性、恶性潜能未定型、有丝分裂活跃型、富于细胞性等,它们的诊断标准主要依据1994年发表的Bell准则及2003年WHO肿瘤分类标准。除典型的良性子宫平滑肌瘤外,其他类型的子宫平滑肌肿瘤发病率很低,诊断也存在一定的分歧,对它们的研究大部分集中于临床及病理阶段,其分子生物学研究较少。
传统观点认为,1、子宫平滑肌肉瘤为突然发生(de novo),没有癌前病变；2、不典型性子宫平滑肌瘤是普通子宫平滑肌瘤的一个变种,属于良性病变。但随着目前对各类型子宫平滑肌肿瘤的分子生物学研究进展,这两个观点受到了挑战。临床上对不典型性子宫平滑肌瘤没有明确的诊疗指南,但根据文献报道,这类肿瘤虽然具有高治愈率和低复发率的特点,但也有病例(3/18)死于这类疾病,但与子宫肉瘤相比有更长的潜伏期(>6年vs2.1年),在临床实践中,我们也看到极个别的不典型性子宫平滑肌瘤在短时间内进展成为子宫肉瘤的情况；另外不典型性子宫平滑肌瘤部分存在染色体1p缺失,与典型的子宫肌瘤相比与平滑肌肉瘤更为相似。在前期研究中,课题组对167例不同类型的子宫平滑肌肿瘤从组织形态学、基因突变、染色体变异及免疫组化角度进行了整体分析。研究显示,MED12基因突变在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中的突变率均低于15%(P>0.05),普通子宫平滑肌瘤突变率为75%；P53基因突变在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中的突变率分别为12%和24%(P>0.05),而普通子宫平滑肌瘤未发现突变；PTEN缺失在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中也具有相似的缺失频率(P>0.05)。课题组进一步挑选与子宫平滑肌肉瘤发生发展相关的关键蛋白,进行免疫组化染色,聚类分析显示不典型性子宫平滑肌瘤与子宫平滑肌肉瘤具有相似的蛋白表达谱。基于以上研究基础,课题组假设不典型性子宫肌瘤可能代表有能力演变为子宫平滑肌肉瘤的中间形式,可能是其癌前病变。
本研究着重从MicroRAN表达谱、DNA甲基化角度对子宫平滑肌肿瘤进行研究,以寻求不典型性子宫肌瘤可能是子宫平滑肌肉瘤的癌前病变这一假说的更多证据。
材料和方法
1.收集美国Northwestern University, Northwestern memorial hospital及山东大学齐鲁医院1993～2013年185例不同类型的子宫平滑肌肿瘤(包括38例子宫平滑肌肉瘤、42例不典型性子宫平滑肌瘤、18例恶性潜能未定的子宫平滑肌瘤、22例富于细胞性子宫平滑肌瘤、7例有丝分裂活跃型子宫平滑肌瘤及58例普通子宫平滑肌瘤),所有病例均有完整的临床、病理及随访资料。
2.提取组织形态学最典型的子宫平滑肌肉瘤,不典型性子宫平滑肌瘤,恶性潜能未定的子宫平滑肌瘤,富于细胞性子宫平滑肌瘤,普通子宫平滑肌瘤及正常子宫肌层各8例RNA,利用FirePlexTM平台行miRNA表达谱分析。
3.提取18例普通子宫平滑肌瘤及配对子宫肌层新鲜组织DNA,利用Infinium Human Methylation27K BeadChip行全基因组甲基化谱分析。
4.提取30例子宫平滑肌肉瘤,25例不典型性子宫平滑肌瘤,12例普通子宫平滑肌瘤及6例正常子宫肌层DNA,利用Sequenom MassArray甲基化检测平台对候选基因(KLF11、DLEC1及RUNX3)进行启动子CpG岛甲基化分析。
结果
1.非监督聚类分析及马氏距离分析均显示不典型性子宫平滑肌瘤与子宫平滑肌肉瘤有相似的MicroRAN表达谱。
2. Infinium Human Methylation27K BeadChip筛选出子宫平滑肌瘤相对正常子宫肌层显著高甲基化的基因KLF11及DLEC1(P<0.001).
3.不典型性子宫平滑肌瘤中KLF11、DLEC1及RUNX3甲基化谱与子宫平滑肌肉瘤相近(P>0.05),而与子宫平滑肌瘤差异显著(P<0.05)。
4.结合课题组前期研究结果,对全部类型的子宫平滑肌肿瘤进行主成成分3D距离分析,发现不典型性子宫平滑肌瘤与子宫平滑肌肉瘤距离最为接近。
结论
1.相对于普通子宫平滑肌瘤,不典型性子宫平滑肌瘤与子宫平滑肌肉瘤有相似的MicroRAN表达谱及甲基化谱,从表观遗传学角度提示不典型子宫平滑肌瘤可能为子宫平滑肌肉瘤的癌前病变。
2.临床上,不典型性平滑肌瘤的处理原则应不同于普通子宫平滑肌瘤,须严密随访。
Epigenetics is the study of heritable changes in gene activity that are not caused by changes in the DNA sequence, these changes can be stable heredity in development and cell proliferation and have potential to be reversed. There are two parts of epigenetics, one is a selective transcriptional regulation of gene expression, including DNA methylation, chromatin remodeling, histone modification, genomic imprinting, gene silence, nucleolar dominance and dormant transposon activation. The other part is post-transcriptional regulation, including no-encoding RNA, MicroRNA, antisense RNA, intron and riboswitches. Comparing to classical genetics which studies the effects of the biological function of the gene sequences, the main task of epigenetic is the establishment and maintain mechanisms of these "epigenetic phenomenon". As the supplement to classical genetics, epigenetics make us have more comprehensive understanding of the mechanisms of gene regulation. Recent studies have confirmed that epigenetic modifications played an important role in stem cell differentiation and early embryonic development and a variety of diseases (cancer, autoimmune diseases and metabolic diseases, etc.), especially in the field of cancer research, such as oncogene activation, tumor suppressor gene inactivation, DNA damage repair defects and cancer stem cell differentiation.
The ultimate goal for the study of epigenetic mechanism is to be applied to clinical diagnosis and preventive treatment. The studies of DNA methylation and MicroRNA have been well established. Fujiwara and other studies have shown that the specificity and positive predictive values of cell-free serum DNA methylation in five genes were85%and75%in the early diagnosis of lung cancer. Many studies also indicated that MicroRNA expression profiling can be used for classification of breast cancer (such as estrogen and progesterone receptor status, tumor stage, metastasis and Her2status, etc.). The main task of this research is to explore the potential application of DNA methylation and MicroRNA spectrum in early epithelial ovarian cancer diagnosis and uterine smooth muscle tumor classification.
Part Ⅰ:A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA
Background and Purpose
Epithelial ovarian cancer (EOC) is the third leading type of cancer among female gynecological system carcinomas, with the highest mortality rate. Due to the lack of early diagnosis methods and no obvious symptoms,85%of patients are first diagnosed at advanced stages (FIGO Ⅱ-Ⅳ), and the survival rates for advanced-stage patients are confined to30%-44%. However, if diagnosed at the localized stage (FIGO I), the5-year survival rate is93%. In addition to exploring the tumorigenesis of ovarian cancer and generating novel effective chemotherapies, the establishment of effective early diagnosis methods is paramount to improving the survival of women with ovarian cancer.
There is no effective method for ovarian cancer screening. Clinically, CA125is a routinely used serum marker; however, it is elevated in less than50%of stage I ovarian cancers. Moreover, the specificity of CA125is poor, and a large number of benign and malignant conditions (such as endometriosis, pregnancy, pelvic inflammatory disease, lung cancer and colon cancer) may result in falsely elevated CA-125values. The positive predictive value for detection is only10%-35%. Therefore, the traditional methods for early diagnosis (such as serum CA125, trans-vaginal ultrasound probe and magnetic resonance imaging), which exhibit high false-negative rates, as well as lower sensitivity and specificity for clinical needs, have not demonstrated the capacity to reduce the population morbidity and/or mortality.
DNA methylation is an important epigenetic phenomenon that affects gene expression without changing the DNA sequence. Aberrant hypermethylation occurring in promoter CpG islands is a significant mechanism of tumor suppressor gene (TSG) silencing and, in certain cases, may be the only mechanism. Recent studies have shown that aberrant DNA methylation, which usually occurs before patients develop clinical manifestations and radiographic evidence, provides a new molecular approach for the early diagnosis of cancer. A large number of genes have been identified as hypermethylated and have been associated with molecular, clinical and pathological features of ovarian carcinomas. The levels of cell-free serum DNA are abnormally high in both early and advanced-stage tumors. Two primary mechanisms proposed for this phenomenon include the following:1) cells in cancer tissues in situ undergo apoptosis and/or necrosis, and2) cells detach and extravasate into the bloodstream and undergo lysis. Tumor-specific aberrant methylation of cell-free serum DNA has been detected in patients with various tumor types, such as prostate, lung, breast and colon cancer tumors, and altered methylation has also been confirmed to be an independent prognostic marker of progression-free and overall survival. Despite its potential clinical applications, the methylation detection of cell-free serum DNA has several limitations, such as the extremely small amounts of available cell-free serum DNA, missing bisulfite-conversions and the low sensitivity conferred by a single marker. In the present study, we developed a novel Multiplex-MSP assay to detect the methylation status of cell-free serum DNA. We confirmed that this novel assay had higher efficiency in the detection of EOC, especially at early stages, compared to the conventional tumor marker CA125.
Materials and Methods
1. Screening for epithelial ovarian cancer-specific methylated genes.
2. Collecting ovarian tumors and normal control serum and tissue samples
3. Designing and optimization of Multiplex-MSP assay
4. Detecting methylation statues of serum and tissue samples by Multiplex-MSP assay
5. Comparison of the methylation status in tissue and serum of EOC
6. Retrospective study of serum samples to determine the sensitivity, specificity and accuracy of the multiplex MSP assay for the detection of ovarian cancer
7. Ovarian cancer screening using multiplex MSP assay in patients with pelvic masses
Result
1. A PubMed search of the English literature published between January1,2000, and January1,2012, was performed with the keywords "methylation" and "epithelial ovarian cancer" and finally seven tumor suppressor genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5and OPCML) with hypermethylation rate were selected to construct the Multiplex-MSP assay.
2. Successfully combining multiplex PCR, nested PCR and methylation-specific PCR together to construct the multiplex-MSP assay
3. Compared the methylation status in tissue and serum of EOC and found the serum methylation profiles were included in the tissue methylation profiles in both early and advanced-stage EOC.
4. An evaluation of the ability to differentiate EOC from benign pelvic masses showed that the sensitivity, specificity and accuracy of the Multiplex-MSP assay were90.57%,89.66%and91.26%, respectively, all of which were higher than the respective values for CA125. When dividing patients in advanced-stage EOC, no significant superiority of the multiplex-MSP assay compared to CA125was found (P=0.5817). However, the specificity, sensitivity and accuracy of the Multiplex-MSP assay were significantly higher than CA125in early-stage EOC (P=0.0036)
5. In screening test, the specificity, sensitivity and accuracy of the multiplex-MSP assay compared to CA125were83.33%vs.50.00%,82.76%vs.72.41%and91.27%vs.89.70%, respectively.
Conclusion
1. The status of cell-free serum DNA methylation can accurately reflect the DNA methylation status of tumor tissue and can be used as a marker of tumor earluy diganosis.
2. The multiplex-MSP assay serves as a new powerful tool to identify the methylation status of cell-free serum DNA.
3. The multiplex-MSP assay is confirmed to be sensitive and specific enough to detect ovarian cancers at an early stage, thereby holding great promise for the possible early detection of other cancers.
Background and Purpose
Uterine smooth muscle tumors (USMTs) range from conventional leiomyomas (ULM) to highly aggressive and fully malignant leiomyosarcomas (LMS). Conventional leiomyomas are characterized by bland cytology and mitotic inactivity (usually<10MF/10HPF). Leiomyomas may often demonstrate hyaline (ischemic type) necrosis but lack classic coagulative tumor cell necrosis. In contrast, uterine leiomyosarcomas are morphologically defined by the presence of at least2of the following3features:(1) cytologic atypia,(2) increased mitotic rate (usually>10MF/10HPF), and (3) coagulative tumor cell necrosis.
Between conventional leiomyomas and leiomyosarcoma, there are several categories of uterine smooth muscle tumors of intermediate types. They show some but not all of the characteristics of malignant tumors, including the following categories:uncertain malignant potential (STUMP), atypical leiomyoma (ALM), mitotically-active leiomyoma (MALM) and cellular leiomyoma (CLM). The diagnostic criteria are based on WHO and Stanford (Bell) scheme. In addition to the typical uterine fibroids, other types of uterine smooth muscle tumors have very low incidence. The studies were mainly on the clinic and pathology, less on molecular analysis.
There are two conventional views about USMTs:1. LMS arise de novo, rather than from any precursor lesions;2. ALM is a benign variant of ULM. But with advances in molecular research in USMTs, these two views have been challenged. The current World Health Organization (Stanford) schema uses the term atypical leiomyoma; older synonymous names include pleomorphic, symplastic, and bizarre leiomyoma. There is no clinical guideline treatment. According to the literature, ALM often had a high cure rate and a low recurrence rate. But rare exceptions to this may exist, a small case series reported that3of18women with cellular or atypical variants died of their disease, of note, there was a longer latency between diagnosis and death in these patients compared with those with LMS (>6years versus median of2.1years). In clinical practice, we have seen very few ALMs progress to LMSs in a short time. Chromosome lp deletion can also be found in some ALM, which is more similar to LMS than ULM.
In the previous study, we collected167cases of the six different USMT variants, then reviewed and confirmed the diagnoses based on the WHO and Stanford criteria. We conducted molecular and genetic analyses of these cases using a group of gene markers that are most relevant to USMT. These included the P53, MED12and PTEN genes. The gene signatures and the genetic fingerprints of each tumor variants were further analyzed. MED12mutations were high at75%in ULM cases (30/40). MED12mutations were significantly low in ALM (10%,4/42) and LMS (10%,4/38)(P>0.05). P53mutation rate in ALM and LMS were12%and24%, respectively, while no mutations were found in ULM. PTEN loss detection also showed ALMs and LMSs had similar frequency (P>0.05). A total of17oncogenic markers were selected for next study. They were all relevant to LMS in the functional pathways involving in tumor growth, differentiation, and tumor stem cells, including Bcl-2, CD24, C-Kit, EGFR, ER, Fascin, HMGA2, Ki-67, PR, p16, p21, p53, pAKT, PTEN, Rb, PS6, and RBL2. Overall, the immunoprofile of these17markers clearly separated LMS from other variants. The IHC signature for LMS requires ER, PR, Bcl-2, P16, P21, P53, and Ki-67. When we removed ER and PR, more than70%of ALM aggregated with LMS. Interestingly, ALM shared several oncogenic markers with LMS, including P16, P53, Bcl-2, and Fascin. Unsupervised cluster analysis showed ALM and LMS had similar protein expression profiles. Based on the above basis, the research group assumed ALM may be the precursor lesion of LMS.
This research focuses the MicroRAN profiling and DNA methylation analysis on USMTs, to seek more evidence to support the hypothesis ALM may be the precursor lesion of LMS.
Materials and Methods
1. We reviewed the pathology database from1993to2003in two institutions (Northwestern Memorial Hospital, Northwest University and Qilu Hospital, Shandong University) to retrieve160patients with a diagnosis of usual type leiomyomas (ULM), cellular leiomyomas (CLM), atypical leiomyomas (ALM), Uterine smooth muscle tumors of uncertain malignant potential (STUMP) and leiomyosarcomas (LMS). Among160patients,4CLMs,15ALMs and5STUMPs were from Qilu Hospital. Each case was reviewed by at least two pathologists to confirm the diagnosis based on Stanford scheme and WHO criteria (2003).
2. Extracted RNA from8tumor samples from each of5different tumor types (LMS, STUMP, ALM, CLM and ULM). RNA was examined on custom designed miRNA chip of FirePlexTM.
3. Extracted DNA from18ULM and paired myometrium. Genomic methylation analysis was performed by Infinium Human Methylation27K BeadChip.
4. Used Sequenom Methylation MassArray to detect the selected genes{KLF11、 DLEC1and RUNX3) methylation status in30LMS,25ALM,12ULM,6MM.
Result
1. Unsupervised clustering analysis showed ALM and LMS had similar miRNA expression profiling.
2. Two significantly hypermethylated genes (KLF11and DLEC1) were found by Infinium Human Methylation27K BeadChip in ULM (P<0.001).
3. ALM showed similar methylation pattern in selected genes (KLF11、DLEC1and RUNX3) with LMS.
4. Principal component analysis which showed the3D distance of all type uterine smooth muscle tumors found that ALM was much closer to LMS than other types.
Conclusion
1. Rather than ULM, ALM had similar miRNA and methylation profiles with LMS. This suggested that ALM may be precursor lesion of LMS.
2. Because of ALM shared similar epigenetic and other molecule characteristics with LMS, the clinical treatment of ALM patients should be different from ULM, should be closely followed up.
针对肿瘤表观遗传学机制研究的最终目的是为了应用于临床诊断及预防治疗。DNA甲基化和MicroRNA在诊断方面的研究已经趋于成熟,Fujiwara等研究表明5个基因血清游离DNA甲基化谱在诊断早期肺癌方面特异性和阳性预测值分别为85%和75%,同样众多研究表明MicroRNA表达谱可应用于乳腺癌的病理分型(如雌孕激素受体状态、肿瘤分期、肿瘤转移及Her2状态等)。本研究主要研究DNA甲基化及MicrORNA表达谱在早期上皮性卵巢癌及子宫平滑肌肿瘤诊断分类中的应用。
第一部分：多重巢式甲基化特异性PCR在早期上皮性卵巢癌诊断中的应用研究
背景和目的
卵巢癌发病率位居女性恶性生殖系统肿瘤第三位,病死率居第一位,占所有因癌症死亡女性患者的3%,其中90%以上为上皮性卵巢癌。卵巢癌发病隐匿,临床确诊时约85%患者疾病己为晚期(FIGOⅡ-Ⅳ期),5年生存率仅为30-44%,而早期卵巢癌(FIGOⅠ期)患者5年生存率高达93%。因此,除积极寻找新的有效的治疗方式外,探讨卵巢癌发生发展的生物学机制,研发新型卵巢癌早期诊断技术迫在眉睫。
目前尚无简便有效的、可常规应用的卵巢癌早期诊断方法。CA125是目前普遍使用的卵巢癌标志物,但仅有50%的Ⅰ期卵巢癌患者CA125水平升高,且其他疾病(如子宫内膜异位症、卵巢纤维瘤、盆腔炎性疾病及某些其他恶性肿瘤等)亦可引起CA125水平升高,故其敏感性和特异性较差,卵巢癌阳性预测值仅有100%～35%。总体而言,对于血清CA125检测、经阴道超声探查等传统的诊断方法单一或联合应用,目前均无证据显示可降低人群的卵巢癌发病率和(或)病死率,其主要原因在于这些方法的假阴性或假阳性率均过高,敏感性和特异性达不到临床需要。
DNA甲基化,即在DNA甲基转移酶的催化下将甲基转移到胞嘧啶的第5位碳原子上生成5-甲基胞嘧啶,是重要的表观遗传学机制。DNA启动子区异常高甲基化是抑癌基因失活的重要机制,在某些情况下可能是唯一的机制,在癌症发生发展中发挥重要作用。作为癌症发生的早期事件,DNA异常甲基化检测可以在患者出现临床表现或者影像学证据之前做到分子诊断,为癌症早期诊断提供新的途径。现己证实肿瘤患者中血清游离DNA含量明显升高,其来源主要有2种机制：1、增殖旺盛的肿瘤细胞持续释放DNA进入血液循环；2、肿瘤细胞的坏死、凋亡或直接入血裂解使血清游离DNA含量增高。血清游离DNA具有与原发肿瘤组织相一致的分子遗传学改变(如启动子区高甲基化、基因突变、微卫星不稳定和杂合性缺失等),可间接反映肿瘤的发生发展情况。使用血清游离DNA甲基化检测在其他癌症如肺癌、头颈部肿瘤、前列腺癌等己被证实可行,因此卵巢癌患者血清游离DNA甲基化检测,是一种可行的具有临床实用价值的卵巢癌早期诊断技术。针对血清游离DNA微量,甲基化特异性PCR敏感性不足的特点,课题组将多重PCR、巢式PCR与甲基化特异性PCR相结合,开发出全新的多重巢式甲基化特异性PCR (Multiplex-MSP)检测方法,已满足血清微量肿瘤游离DNA样本多基因甲基化检测需要。
材料和方法
1.上皮性卵巢癌特异性甲基化基因的筛选。
2.临床卵巢肿瘤及正常对照病人血清及组织样本的收集。
3.引物的设计合成及多重巢式甲基化特异性PCR反应体系的优化。
4.多重巢式甲基化特异性PCR检测血清及组织样本甲基化水平。
5.卵巢癌血清与组织甲基化谱对照研究。
6.回顾性分析评估多重巢式甲基化特异性PCR检测在上皮性卵巢癌早期诊断中的意义。
7.双盲实验进一步验证多重巢式甲基化特异性PCR检测在上皮性卵巢癌早期诊断中的作用。
结果
1.通过Pubmed检索筛选出7个与卵巢癌发生发展密切相关的高频甲基化基因APC, RASSF1A, CDH1, RUNX3, TFP12, SFRP5和OPCML。
2.将多重PCR、巢式PCR与甲基化特异性PCR相结合,开发出全新的多重巢式甲基化特异性PCR检测方法。
3.应用多重巢式甲基化特异性PCR对20例卵巢癌血清及配对组织进行检测,证实血清游离DNA甲基化谱完全包含于肿瘤组织DNA甲基化谱。
4.应用多重巢式甲基化特异性PCR回顾140例卵巢肿瘤及正常女性血清标本,结果证实该方法的特异性为90.57%,敏感性为89.66%；特别是在
背景和目的
临床上,常见的子宫平滑肌肿瘤主要包括良性子宫平滑肌瘤和恶性子宫平滑肌肉瘤,它们的分类主要基于以下三个标准：细胞异型性、有丝分裂相、肿瘤细胞坏死,典型的子宫平滑肌瘤形态学上主要表现为无细胞异型性,小于10MF/10HPF的有丝分裂相,可能会表现出玻璃样坏死(如缺血性),但缺乏典型的凝固性肿瘤细胞坏死；相反,恶性子宫平滑肌肉瘤至少具备以下3个特征中的2个,明显的细胞异型性,大于10MF/10HPF的有丝分裂相及典型的凝固性肿瘤细胞坏死。
在典型的子宫平滑肌瘤和子宫平滑肌肉瘤之间,存在几大类中间类型的子宫平滑肌肿瘤,它们表现一些但不是所有的恶性肿瘤的特征,主要包括以下几大类：不典型性、恶性潜能未定型、有丝分裂活跃型、富于细胞性等,它们的诊断标准主要依据1994年发表的Bell准则及2003年WHO肿瘤分类标准。除典型的良性子宫平滑肌瘤外,其他类型的子宫平滑肌肿瘤发病率很低,诊断也存在一定的分歧,对它们的研究大部分集中于临床及病理阶段,其分子生物学研究较少。
传统观点认为,1、子宫平滑肌肉瘤为突然发生(de novo),没有癌前病变；2、不典型性子宫平滑肌瘤是普通子宫平滑肌瘤的一个变种,属于良性病变。但随着目前对各类型子宫平滑肌肿瘤的分子生物学研究进展,这两个观点受到了挑战。临床上对不典型性子宫平滑肌瘤没有明确的诊疗指南,但根据文献报道,这类肿瘤虽然具有高治愈率和低复发率的特点,但也有病例(3/18)死于这类疾病,但与子宫肉瘤相比有更长的潜伏期(>6年vs2.1年),在临床实践中,我们也看到极个别的不典型性子宫平滑肌瘤在短时间内进展成为子宫肉瘤的情况；另外不典型性子宫平滑肌瘤部分存在染色体1p缺失,与典型的子宫肌瘤相比与平滑肌肉瘤更为相似。在前期研究中,课题组对167例不同类型的子宫平滑肌肿瘤从组织形态学、基因突变、染色体变异及免疫组化角度进行了整体分析。研究显示,MED12基因突变在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中的突变率均低于15%(P>0.05),普通子宫平滑肌瘤突变率为75%；P53基因突变在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中的突变率分别为12%和24%(P>0.05),而普通子宫平滑肌瘤未发现突变；PTEN缺失在不典型性子宫平滑肌瘤与子宫平滑肌肉瘤中也具有相似的缺失频率(P>0.05)。课题组进一步挑选与子宫平滑肌肉瘤发生发展相关的关键蛋白,进行免疫组化染色,聚类分析显示不典型性子宫平滑肌瘤与子宫平滑肌肉瘤具有相似的蛋白表达谱。基于以上研究基础,课题组假设不典型性子宫肌瘤可能代表有能力演变为子宫平滑肌肉瘤的中间形式,可能是其癌前病变。
本研究着重从MicroRAN表达谱、DNA甲基化角度对子宫平滑肌肿瘤进行研究,以寻求不典型性子宫肌瘤可能是子宫平滑肌肉瘤的癌前病变这一假说的更多证据。
材料和方法
1.收集美国Northwestern University, Northwestern memorial hospital及山东大学齐鲁医院1993～2013年185例不同类型的子宫平滑肌肿瘤(包括38例子宫平滑肌肉瘤、42例不典型性子宫平滑肌瘤、18例恶性潜能未定的子宫平滑肌瘤、22例富于细胞性子宫平滑肌瘤、7例有丝分裂活跃型子宫平滑肌瘤及58例普通子宫平滑肌瘤),所有病例均有完整的临床、病理及随访资料。
2.提取组织形态学最典型的子宫平滑肌肉瘤,不典型性子宫平滑肌瘤,恶性潜能未定的子宫平滑肌瘤,富于细胞性子宫平滑肌瘤,普通子宫平滑肌瘤及正常子宫肌层各8例RNA,利用FirePlexTM平台行miRNA表达谱分析。
3.提取18例普通子宫平滑肌瘤及配对子宫肌层新鲜组织DNA,利用Infinium Human Methylation27K BeadChip行全基因组甲基化谱分析。
4.提取30例子宫平滑肌肉瘤,25例不典型性子宫平滑肌瘤,12例普通子宫平滑肌瘤及6例正常子宫肌层DNA,利用Sequenom MassArray甲基化检测平台对候选基因(KLF11、DLEC1及RUNX3)进行启动子CpG岛甲基化分析。
结果
1.非监督聚类分析及马氏距离分析均显示不典型性子宫平滑肌瘤与子宫平滑肌肉瘤有相似的MicroRAN表达谱。
2. Infinium Human Methylation27K BeadChip筛选出子宫平滑肌瘤相对正常子宫肌层显著高甲基化的基因KLF11及DLEC1(P<0.001).
3.不典型性子宫平滑肌瘤中KLF11、DLEC1及RUNX3甲基化谱与子宫平滑肌肉瘤相近(P>0.05),而与子宫平滑肌瘤差异显著(P<0.05)。
4.结合课题组前期研究结果,对全部类型的子宫平滑肌肿瘤进行主成成分3D距离分析,发现不典型性子宫平滑肌瘤与子宫平滑肌肉瘤距离最为接近。
结论
1.相对于普通子宫平滑肌瘤,不典型性子宫平滑肌瘤与子宫平滑肌肉瘤有相似的MicroRAN表达谱及甲基化谱,从表观遗传学角度提示不典型子宫平滑肌瘤可能为子宫平滑肌肉瘤的癌前病变。
2.临床上,不典型性平滑肌瘤的处理原则应不同于普通子宫平滑肌瘤,须严密随访。
Epigenetics is the study of heritable changes in gene activity that are not caused by changes in the DNA sequence, these changes can be stable heredity in development and cell proliferation and have potential to be reversed. There are two parts of epigenetics, one is a selective transcriptional regulation of gene expression, including DNA methylation, chromatin remodeling, histone modification, genomic imprinting, gene silence, nucleolar dominance and dormant transposon activation. The other part is post-transcriptional regulation, including no-encoding RNA, MicroRNA, antisense RNA, intron and riboswitches. Comparing to classical genetics which studies the effects of the biological function of the gene sequences, the main task of epigenetic is the establishment and maintain mechanisms of these "epigenetic phenomenon". As the supplement to classical genetics, epigenetics make us have more comprehensive understanding of the mechanisms of gene regulation. Recent studies have confirmed that epigenetic modifications played an important role in stem cell differentiation and early embryonic development and a variety of diseases (cancer, autoimmune diseases and metabolic diseases, etc.), especially in the field of cancer research, such as oncogene activation, tumor suppressor gene inactivation, DNA damage repair defects and cancer stem cell differentiation.
The ultimate goal for the study of epigenetic mechanism is to be applied to clinical diagnosis and preventive treatment. The studies of DNA methylation and MicroRNA have been well established. Fujiwara and other studies have shown that the specificity and positive predictive values of cell-free serum DNA methylation in five genes were85%and75%in the early diagnosis of lung cancer. Many studies also indicated that MicroRNA expression profiling can be used for classification of breast cancer (such as estrogen and progesterone receptor status, tumor stage, metastasis and Her2status, etc.). The main task of this research is to explore the potential application of DNA methylation and MicroRNA spectrum in early epithelial ovarian cancer diagnosis and uterine smooth muscle tumor classification.
Part Ⅰ:A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA
Background and Purpose
Epithelial ovarian cancer (EOC) is the third leading type of cancer among female gynecological system carcinomas, with the highest mortality rate. Due to the lack of early diagnosis methods and no obvious symptoms,85%of patients are first diagnosed at advanced stages (FIGO Ⅱ-Ⅳ), and the survival rates for advanced-stage patients are confined to30%-44%. However, if diagnosed at the localized stage (FIGO I), the5-year survival rate is93%. In addition to exploring the tumorigenesis of ovarian cancer and generating novel effective chemotherapies, the establishment of effective early diagnosis methods is paramount to improving the survival of women with ovarian cancer.
There is no effective method for ovarian cancer screening. Clinically, CA125is a routinely used serum marker; however, it is elevated in less than50%of stage I ovarian cancers. Moreover, the specificity of CA125is poor, and a large number of benign and malignant conditions (such as endometriosis, pregnancy, pelvic inflammatory disease, lung cancer and colon cancer) may result in falsely elevated CA-125values. The positive predictive value for detection is only10%-35%. Therefore, the traditional methods for early diagnosis (such as serum CA125, trans-vaginal ultrasound probe and magnetic resonance imaging), which exhibit high false-negative rates, as well as lower sensitivity and specificity for clinical needs, have not demonstrated the capacity to reduce the population morbidity and/or mortality.
DNA methylation is an important epigenetic phenomenon that affects gene expression without changing the DNA sequence. Aberrant hypermethylation occurring in promoter CpG islands is a significant mechanism of tumor suppressor gene (TSG) silencing and, in certain cases, may be the only mechanism. Recent studies have shown that aberrant DNA methylation, which usually occurs before patients develop clinical manifestations and radiographic evidence, provides a new molecular approach for the early diagnosis of cancer. A large number of genes have been identified as hypermethylated and have been associated with molecular, clinical and pathological features of ovarian carcinomas. The levels of cell-free serum DNA are abnormally high in both early and advanced-stage tumors. Two primary mechanisms proposed for this phenomenon include the following:1) cells in cancer tissues in situ undergo apoptosis and/or necrosis, and2) cells detach and extravasate into the bloodstream and undergo lysis. Tumor-specific aberrant methylation of cell-free serum DNA has been detected in patients with various tumor types, such as prostate, lung, breast and colon cancer tumors, and altered methylation has also been confirmed to be an independent prognostic marker of progression-free and overall survival. Despite its potential clinical applications, the methylation detection of cell-free serum DNA has several limitations, such as the extremely small amounts of available cell-free serum DNA, missing bisulfite-conversions and the low sensitivity conferred by a single marker. In the present study, we developed a novel Multiplex-MSP assay to detect the methylation status of cell-free serum DNA. We confirmed that this novel assay had higher efficiency in the detection of EOC, especially at early stages, compared to the conventional tumor marker CA125.
Materials and Methods
1. Screening for epithelial ovarian cancer-specific methylated genes.
2. Collecting ovarian tumors and normal control serum and tissue samples
3. Designing and optimization of Multiplex-MSP assay
4. Detecting methylation statues of serum and tissue samples by Multiplex-MSP assay
5. Comparison of the methylation status in tissue and serum of EOC
6. Retrospective study of serum samples to determine the sensitivity, specificity and accuracy of the multiplex MSP assay for the detection of ovarian cancer
7. Ovarian cancer screening using multiplex MSP assay in patients with pelvic masses
Result
1. A PubMed search of the English literature published between January1,2000, and January1,2012, was performed with the keywords "methylation" and "epithelial ovarian cancer" and finally seven tumor suppressor genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5and OPCML) with hypermethylation rate were selected to construct the Multiplex-MSP assay.
2. Successfully combining multiplex PCR, nested PCR and methylation-specific PCR together to construct the multiplex-MSP assay
3. Compared the methylation status in tissue and serum of EOC and found the serum methylation profiles were included in the tissue methylation profiles in both early and advanced-stage EOC.
4. An evaluation of the ability to differentiate EOC from benign pelvic masses showed that the sensitivity, specificity and accuracy of the Multiplex-MSP assay were90.57%,89.66%and91.26%, respectively, all of which were higher than the respective values for CA125. When dividing patients in advanced-stage EOC, no significant superiority of the multiplex-MSP assay compared to CA125was found (P=0.5817). However, the specificity, sensitivity and accuracy of the Multiplex-MSP assay were significantly higher than CA125in early-stage EOC (P=0.0036)
5. In screening test, the specificity, sensitivity and accuracy of the multiplex-MSP assay compared to CA125were83.33%vs.50.00%,82.76%vs.72.41%and91.27%vs.89.70%, respectively.
Conclusion
1. The status of cell-free serum DNA methylation can accurately reflect the DNA methylation status of tumor tissue and can be used as a marker of tumor earluy diganosis.
2. The multiplex-MSP assay serves as a new powerful tool to identify the methylation status of cell-free serum DNA.
3. The multiplex-MSP assay is confirmed to be sensitive and specific enough to detect ovarian cancers at an early stage, thereby holding great promise for the possible early detection of other cancers.
Background and Purpose
Uterine smooth muscle tumors (USMTs) range from conventional leiomyomas (ULM) to highly aggressive and fully malignant leiomyosarcomas (LMS). Conventional leiomyomas are characterized by bland cytology and mitotic inactivity (usually<10MF/10HPF). Leiomyomas may often demonstrate hyaline (ischemic type) necrosis but lack classic coagulative tumor cell necrosis. In contrast, uterine leiomyosarcomas are morphologically defined by the presence of at least2of the following3features:(1) cytologic atypia,(2) increased mitotic rate (usually>10MF/10HPF), and (3) coagulative tumor cell necrosis.
Between conventional leiomyomas and leiomyosarcoma, there are several categories of uterine smooth muscle tumors of intermediate types. They show some but not all of the characteristics of malignant tumors, including the following categories:uncertain malignant potential (STUMP), atypical leiomyoma (ALM), mitotically-active leiomyoma (MALM) and cellular leiomyoma (CLM). The diagnostic criteria are based on WHO and Stanford (Bell) scheme. In addition to the typical uterine fibroids, other types of uterine smooth muscle tumors have very low incidence. The studies were mainly on the clinic and pathology, less on molecular analysis.
There are two conventional views about USMTs:1. LMS arise de novo, rather than from any precursor lesions;2. ALM is a benign variant of ULM. But with advances in molecular research in USMTs, these two views have been challenged. The current World Health Organization (Stanford) schema uses the term atypical leiomyoma; older synonymous names include pleomorphic, symplastic, and bizarre leiomyoma. There is no clinical guideline treatment. According to the literature, ALM often had a high cure rate and a low recurrence rate. But rare exceptions to this may exist, a small case series reported that3of18women with cellular or atypical variants died of their disease, of note, there was a longer latency between diagnosis and death in these patients compared with those with LMS (>6years versus median of2.1years). In clinical practice, we have seen very few ALMs progress to LMSs in a short time. Chromosome lp deletion can also be found in some ALM, which is more similar to LMS than ULM.
In the previous study, we collected167cases of the six different USMT variants, then reviewed and confirmed the diagnoses based on the WHO and Stanford criteria. We conducted molecular and genetic analyses of these cases using a group of gene markers that are most relevant to USMT. These included the P53, MED12and PTEN genes. The gene signatures and the genetic fingerprints of each tumor variants were further analyzed. MED12mutations were high at75%in ULM cases (30/40). MED12mutations were significantly low in ALM (10%,4/42) and LMS (10%,4/38)(P>0.05). P53mutation rate in ALM and LMS were12%and24%, respectively, while no mutations were found in ULM. PTEN loss detection also showed ALMs and LMSs had similar frequency (P>0.05). A total of17oncogenic markers were selected for next study. They were all relevant to LMS in the functional pathways involving in tumor growth, differentiation, and tumor stem cells, including Bcl-2, CD24, C-Kit, EGFR, ER, Fascin, HMGA2, Ki-67, PR, p16, p21, p53, pAKT, PTEN, Rb, PS6, and RBL2. Overall, the immunoprofile of these17markers clearly separated LMS from other variants. The IHC signature for LMS requires ER, PR, Bcl-2, P16, P21, P53, and Ki-67. When we removed ER and PR, more than70%of ALM aggregated with LMS. Interestingly, ALM shared several oncogenic markers with LMS, including P16, P53, Bcl-2, and Fascin. Unsupervised cluster analysis showed ALM and LMS had similar protein expression profiles. Based on the above basis, the research group assumed ALM may be the precursor lesion of LMS.
This research focuses the MicroRAN profiling and DNA methylation analysis on USMTs, to seek more evidence to support the hypothesis ALM may be the precursor lesion of LMS.
Materials and Methods
1. We reviewed the pathology database from1993to2003in two institutions (Northwestern Memorial Hospital, Northwest University and Qilu Hospital, Shandong University) to retrieve160patients with a diagnosis of usual type leiomyomas (ULM), cellular leiomyomas (CLM), atypical leiomyomas (ALM), Uterine smooth muscle tumors of uncertain malignant potential (STUMP) and leiomyosarcomas (LMS). Among160patients,4CLMs,15ALMs and5STUMPs were from Qilu Hospital. Each case was reviewed by at least two pathologists to confirm the diagnosis based on Stanford scheme and WHO criteria (2003).
2. Extracted RNA from8tumor samples from each of5different tumor types (LMS, STUMP, ALM, CLM and ULM). RNA was examined on custom designed miRNA chip of FirePlexTM.
3. Extracted DNA from18ULM and paired myometrium. Genomic methylation analysis was performed by Infinium Human Methylation27K BeadChip.
4. Used Sequenom Methylation MassArray to detect the selected genes{KLF11、 DLEC1and RUNX3) methylation status in30LMS,25ALM,12ULM,6MM.
Result
1. Unsupervised clustering analysis showed ALM and LMS had similar miRNA expression profiling.
2. Two significantly hypermethylated genes (KLF11and DLEC1) were found by Infinium Human Methylation27K BeadChip in ULM (P<0.001).
3. ALM showed similar methylation pattern in selected genes (KLF11、DLEC1and RUNX3) with LMS.
4. Principal component analysis which showed the3D distance of all type uterine smooth muscle tumors found that ALM was much closer to LMS than other types.
Conclusion
1. Rather than ULM, ALM had similar miRNA and methylation profiles with LMS. This suggested that ALM may be precursor lesion of LMS.
2. Because of ALM shared similar epigenetic and other molecule characteristics with LMS, the clinical treatment of ALM patients should be different from ULM, should be closely followed up.
引文
[1]Choudhuri S. From Waddington's epigenetic landscape to small noncoding RNA: some important milestones in the history of epigenetics research. Toxicology mechanisms and methods.2011;21:252-74.
[2]Wu C, Morris JR. Genes, genetics, and epigenetics:a correspondence. Science (New York, NY).2001;293:1103-5.
[3]Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature.2004;429:457-63.
[4]Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature. 2007;445:214-8.
[5]Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nature reviews Molecular cell biology.2005;6:376-85.
[6]Fujiwara K, Fujimoto N, Tabata M, Nishii K, Matsuo K, Hotta K, et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clinical cancer research:an official journal of the American Association for Cancer Research.2005;11:1219-25.
[7]Cuk K, Zucknick M, Heil J, Madhavan D, Schott S, Turchinovich A, et al. Circulating microRNAs in plasma as early detection markers for breast cancer. International journal of cancer Journal international du cancer.2013;132:1602-12.
[8]Teodoridis JM, Hall J, Marsh S, Kannall HD, Smyth C, Curto J, et al. CpG island methylation of DNA damage response genes in advanced ovarian cancer. Cancer research.2005;65:8961-7.
[9]Akahira J, Sugihashi Y, Ito K, Niikura H, Okamura K, Yaegashi N. Promoter methylation status and expression of TMS1 gene in human epithelial ovarian cancer. Cancer science.2004;95:40-3.
[10]Terasawa K, Sagae S, Toyota M, Tsukada K, Ogi K, Satoh A, et al. Epigenetic inactivation of TMS1/ASC in ovarian cancer. Clinical cancer research:an official journal of the American Association for Cancer Research.2004; 10:2000-6.
[11]Kaneuchi M, Sasaki M, Tanaka Y, Shiina H, Yamada H, Yamamoto R, et al. WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma. Cancer.2005; 104:1924-30.
[12]Kaneuchi M, Sasaki M, Tanaka Y, Shiina H, Verma M, Ebina Y, et al. Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer. Biochemical and biophysical research communications. 2004;316:1156-62.
[13]Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer research. 2005;65:7065-70.
[14]Mattie MD, Benz CC, Bowers J, Sensinger K, Wong L, Scott GK, et al. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Molecular cancer. 2006;5:24.
[15]Siegel R, Naishadham D, Jemal A. Cancer statistics,2012. CA Cancer J Clin. 2012;62:10-29.
[16]Woolas RP, Xu FJ, Jacobs IJ, Yu YH, Daly L, Berchuck A, et al. Elevation of multiple serum markers in patients with stage I ovarian cancer. J Natl Cancer Inst. 1993;85:1748-51.
[17]Powell CB, Kenley E, Chen LM, Crawford B, McLennan J, Zaloudek C, et al. Risk-reducing salpingo-oophorectomy in BRCA mutation carriers:role of serial sectioning in the detection of occult malignancy. Journal of clinical oncology:official journal of the American Society of Clinical Oncology.2005;23:127-32.
[18]Lee Y, Miron A, Drapkin R, Nucci MR, Medeiros F, Saleemuddin A, et al. A candidate precursor to serous carcinoma that originates in the distal fallopian tube. The Journal of pathology.2007;211:26-35.
[19]American cancer society, Cancer Facts and Figures.2011.
[20]Shih Ie M, Kurman RJ. Ovarian tumorigenesis:a proposed model based on morphological and molecular genetic analysis. The American journal of pathology. 2004;164:1511-8.
[21]Smith Sehdev AE, Sehdev PS, Kurman RJ. Noninvasive and invasive micropapillary (low-grade) serous carcinoma of the ovary:a clinicopathologic analysis of 135 cases. The American journal of surgical pathology.2003;27:725-36.
[22]Singer G, Kurman RJ, Chang HW, Cho SK, Shih Ie M. Diverse tumorigenic pathways in ovarian serous carcinoma. The American journal of pathology. 2002;160:1223-8.
[23]Singer G, Oldt R,3rd, Cohen Y, Wang BG, Sidransky D, Kurman RJ, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst.2003;95:484-6.
[24]Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nature genetics.1998;19:83-6.
[25]Francis-Thickpenny KM, Richardson DM, van Ee CC, Love DR, Winship IM, Baguley BC, et al. Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma. British journal of cancer.2001;85:687-91.
[26]Moreno-Bueno G, Gamallo C, Perez-Gallego L, de Mora JC, Suarez A, Palacios J. beta-Catenin expression pattern, beta-catenin gene mutations, and microsatellite instability in endometrioid ovarian carcinomas and synchronous endometrial carcinomas. Diagnostic molecular pathology:the American journal of surgical pathology, part B.2001;10:116-22.
[27]Obata K, Morland SJ, Watson RH, Hitchcock A, Chenevix-Trench G, Thomas EJ, et al. Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors. Cancer research.1998;58:2095-7.
[28]Chan WY, Cheung KK, Schorge JO, Huang LW, Welch WR, Bell DA, et al. Bcl-2 and p53 protein expression, apoptosis, and p53 mutation in human epithelial ovarian cancers. The American journal of pathology.2000; 156:409-17.
[29]Kohler MF, Marks JR, Wiseman RW, Jacobs IJ, Davidoff AM, Clarke-Pearson DL, et al. Spectrum of mutation and frequency of allelic deletion of the p53 gene in ovarian cancer. J Natl Cancer Inst.1993;85:1513-9.
[30]Kupryjanczyk J, Thor AD, Beauchamp R, Merritt V, Edgerton SM, Bell DA, et al. p53 gene mutations and protein accumulation in human ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:4961-5.
[31]Wen WH, Reles A, Runnebaum IB, Sullivan-Halley J, Bernstein L, Jones LA, et al. p53 mutations and expression in ovarian cancers:correlation with overall survival. International journal of gynecological pathology:official journal of the International Society of Gynecological Pathologists.1999;18:29-41.
[32]Clarke-Pearson DL. Clinical practice. Screening for ovarian cancer. The New England journal of medicine.2009;361:170-7.
[33]Buys SS, Partridge E, Black A, Johnson CC, Lamerato L, Isaacs C, et al. Effect of screening on ovarian cancer mortality:the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA:the journal of the American Medical Association.2011;305:2295-303.
[34]Menon U, Gentry-Maharaj A, Hallett R, Ryan A, Burnell M, Sharma A, et al. Sensitivity and specificity of multimodal and ultrasound screening for ovarian cancer, and stage distribution of detected cancers:results of the prevalence screen of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). The lancet oncology. 2009; 10:327-40.
[35]Menon U, Jacobs IJ. Recent developments in ovarian cancer screening. Current opinion in obstetrics & gynecology.2000;12:39-42.
[36]Perlin E, Moquin RB. Serum DNA levels in patients with malignant disease. American journal of clinical pathology.1972;58:601-2.
[37]Ibanez de Caceres I, Battagli C, Esteller M, Herman JG, Dulaimi E, Edelson MI, et al. Tumor cell-specific BRCA1 and RASSFlA hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer research.2004;64:6476-81.
[38]Nelissen EC, van Montfoort AP, Dumoulin JC, Evers JL. Epigenetics and the placenta. Human reproduction update.2011;17:397-417.
[39]Nakao M. Epigenetics:interaction of DNA methylation and chromatin. Gene. 2001;278:25-31.
[40]Singal R, Ginder GD. DNA methylation. Blood.1999;93:4059-70.
[41]Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:11995-9.
[42]Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science (New York, NY).2001;293:1068-70.
[43]Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer research.1977;37:646-50.
[44]Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer research.1999;59:67-70.
[45]Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients:quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer research.2001;61:1659-65.
[46]The Guide to Clinical Preventive Services 2010-2011:Recommendations of the U.S. Preventive Services Task Force. Rockville MD2010.
[47]Philipp AB, Stieber P, Nagel D, Neumann J, Spelsberg F, Jung A, et al. Prognostic role of methylated free circulating DNA in colorectal cancer. International journal of cancer Journal international du cancer.2012;131:2308-19.
[48]Yagyu S, Gotoh T, Iehara T, Miyachi M, Katsumi Y, Tsubai-Shimizu S, et al. Circulating methylated-DCR2 gene in serum as an indicator of prognosis and therapeutic efficacy in patients with MYCN nonamplified neuroblastoma. Clinical cancer research:an official journal of the American Association for Cancer Research. 2008;14:7011-9.
[49]Liggett TE, Melnikov A, Yi Q, Replogle C, Hu W, Rotmensch J, et al. Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors. Gynecologic oncology.2011; 120:113-20.
[50]Song JZ, Stirzaker C, Harrison J, Melki JR, Clark SJ. Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells. Oncogene. 2002;21:1048-61.
[51]Barton CA, Hacker NF, Clark SJ, O'Brien PM. DNA methylation changes in ovarian cancer:implications for early diagnosis, prognosis and treatment. Gynecologic oncology.2008;109:129-39.
[52]Sellar GC, Watt KP, Rabiasz GJ, Stronach EA, Li L, Miller EP, et al. OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer. Nature genetics.2003;34:337-43.
[53]Czekierdowski A, Czekierdowska S, Szymanski M, Wielgos M, Kaminski P, Kotarski J. Opioid-binding protein/cell adhesion molecule-like (OPCML) gene and promoter methylation status in women with ovarian cancer. Neuro endocrinology letters.2006;27:609-13.
[54]Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis:practical aspects and biological significance. Mutation research.2007;635:105-17.
[55]Shapiro B, Chakrabarty M, Cohn EM, Leon SA. Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease. Cancer. 1983;51:2116-20.
[56]Fraga MF, Esteller M. DNA methylation:a profile of methods and applications. BioTechniques.2002;33:632,4,6-49.
[57]Yamamoto N, Nakayama T, Kajita M, Miyake T, Iwamoto T, Kim SJ, et al. Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARbeta2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay. Breast cancer research and treatment. 2012;132:165-73.
[58]Hibi K, Taguchi M, Nakayama H, Takase T, Kasai Y, Ito K, et al. Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma. Clinical cancer research:an official journal of the American Association for Cancer Research.2001;7:3135-8.
[59]Usadel H, Brabender J, Danenberg KD, Jeronimo C, Harden S, Engles J, et al. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer research. 2002;62:371-5.
[60]Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women:ultrasound evidence. American journal of obstetrics and gynecology.2003; 188:100-7.
[61]Leibsohn S, d'Ablaing G, Mishell DR, Jr., Schlaerth JB. Leiomyosarcoma in a series of hysterectomies performed for presumed uterine leiomyomas. American journal of obstetrics and gynecology.1990; 162:968-74; discussion 74-6.
[62]Giuntoli RL,2nd, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL, et al. Retrospective review of 208 patients with leiomyosarcoma of the uterus: prognostic indicators, surgical management, and adjuvant therapy. Gynecologic oncology.2003;89:460-9.
[63]Bell SW, Kempson RL, Hendrickson MR. Problematic uterine smooth muscle neoplasms. A clinicopathologic study of 213 cases. The American journal of surgical pathology.1994;18:535-58.
[64]IARC. WHO Classification of Tumors.2003.
[65]Lee WY, Tzeng CC, Chou CY. Uterine leiomyosarcomas coexistent with cellular and atypical leiomyomata in a young woman during the treatment with luteinizing hormone-releasing hormone agonist. Gynecologic oncology.1994;52:74-9.
[66]Evans HL, Chawla SP, Simpson C, Finn KP. Smooth muscle neoplasms of the uterus other than ordinary leiomyoma. A study of 46 cases, with emphasis on diagnostic criteria and prognostic factors. Cancer.1988;62:2239-47.
[67]Sung CO, Ahn G, Song SY, Choi YL, Bae DS. Atypical leiomyomas of the uterus with long-term follow-up after myomectomy with immunohistochemical analysis for p16INK4A, p53, Ki-67, estrogen receptors, and progesterone receptors. International journal of gynecological pathology:official journal of the International Society of Gynecological Pathologists.2009;28:529-34.
[68]Ly A, Mills AM, McKenney JK, Balzer BL, Kempson RL, Hendrickson MR, et al. Atypical leiomyomas of the uterus:a clinicopathologic study of 51 cases. The American journal of surgical pathology.2013;37:643-9.
[69]Giuntoli RL,2nd, Gostout BS, DiMarco CS, Metzinger DS, Keeney GL. Diagnostic criteria for uterine smooth muscle tumors:leiomyoma variants associated with malignant behavior. The Journal of reproductive medicine.2007;52:1001-10.
[70]Christacos NC, Quade BJ, Dal Cin P, Morton CC. Uterine leiomyomata with deletions of Ip represent a distinct cytogenetic subgroup associated with unusual histologic features. Genes, chromosomes & cancer.2006;45:304-12.
[71]Mittal KR, Chen F, Wei JJ, Rijhvani K, Kurvathi R, Streck D, et al. Molecular and immunohistochemical evidence for the origin of uterine leiomyosarcomas from associated leiomyoma and symplastic leiomyoma-like areas. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc. 2009;22:1303-11.
[72]Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al. MED 12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science (New York, NY).2011;334:252-5.
[73]Zhai YL, Nikaido T, Toki T, Shiozawa A, Orii A, Fujii S. Prognostic significance of bcl-2 expression in leiomyosarcoma of the uterus. British journal of cancer. 1999;80:1658-64.
[74]Miyajima K, Tamiya S, Oda Y, Adachi T, Konomoto T, Toyoshiba H, et al. Relative quantitation of p53 and MDM2 gene expression in leiomyosarcoma; real-time semi-quantitative reverse transcription-polymerase chain reaction. Cancer letters.2001;164:177-88.
[75]Ito M, Barys L, O'Reilly T, Young S, Gorbatcheva B, Monahan J, et al. Comprehensive mapping of p53 pathway alterations reveals an apparent role for both SNP309 and MDM2 amplification in sarcomagenesis. Clinical cancer research:an official journal of the American Association for Cancer Research.2011; 17:416-26.
[76]de Vos S, Wilczynski SP, Fleischhacker M, Koeffler P. p53 alterations in uterine leiomyosarcomas versus leiomyomas. Gynecologic oncology.1994;54:205-8.
[77]Hall KL, Teneriello MG, Taylor RR, Lemon S, Ebina M, Linnoila RI, et al. Analysis of Ki-ras, p53, and MDM2 genes in uterine leiomyomas and leiomyosarcomas. Gynecologic oncology.1997;65:330-5.
[78]Zhai YL, Nikaido T, Orii A, Horiuchi A, Toki T, Fujii S. Frequent occurrence of loss of heterozygosity among tumor suppressor genes in uterine leiomyosarcoma. Gynecologic oncology.1999;75:453-9.
[79]Jeffers MD, Farquharson MA, Richmond JA, McNicol AM. p53 immunoreactivity and mutation of the p53 gene in smooth muscle tumours of the uterine corpus. The Journal of pathology.1995; 177:65-70.
[80]Hernando E, Charytonowicz E, Dudas ME, Menendez S, Matushansky I, Mills J, et al. The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nature medicine.2007; 13:748-53.
[81]Yamada C, Ozaki T, Ando K, Suenaga Y, Inoue K, Ito Y, et al. RUNX3 modulates DNA damage-mediated phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The Journal of biological chemistry.2010;285:16693-703.
[82]Shio S, Kodama Y, Ida H, Shiokawa M, Kitamura K, Hatano E, et al. Loss of RUNX3 expression by histone deacetylation is associated with biliary tract carcinogenesis. Cancer science.2011;102:776-83.
[83]Lin FC, Liu YP, Lai CH, Shan YS, Cheng HC, Hsu PI, et al. RUNX3-mediated transcriptional inhibition of Akt suppresses tumorigenesis of human gastric cancer cells. Oncogene.2012;31:4302-16.
[84]Gao J, Chen Y, Wu KC, Liu J, Zhao YQ, Pan YL, et al. RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells. Experimental cell research.2010;316:149-57.
[85]Voon DC, Wang H, Koo JK, Nguyen TA, Hor YT, Chu YS, et al. Runx3 protects gastric epithelial cells against epithelial-mesenchymal transition-induced cellular plasticity and tumorigenicity. Stem cells (Dayton, Ohio).2012;30:2088-99.
[86]Danielson LS, Menendez S, Attolini CS, Guijarro MV, Bisogna M, Wei J, et al. A differentiation-based microRNA signature identifies leiomyosarcoma as a mesenchymal stem cell-related malignancy. The American journal of pathology. 2010;177:908-17.
[87]Wang T, Zhang X, Obijuru L, Laser J, Aris V, Lee P, et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas. Genes, chromosomes & cancer.2007;46:336-47.
[88]Ip PP, Cheung AN, Clement PB. Uterine smooth muscle tumors of uncertain malignant potential (STUMP):a clinicopathologic analysis of 16 cases. The American journal of surgical pathology.2009;33:992-1005.
[89]Guntupalli SR, Ramirez PT, Anderson ML, Milam MR, Bodurka DC, Malpica A. Uterine smooth muscle tumor of uncertain malignant potential:a retrospective analysis. Gynecologic oncology.2009;113:324-6.
[90]Ng JS, Han A, Chew SH, Low J. A clinicopathologic study of uterine smooth muscle tumours of uncertain malignant potential (STUMP). Annals of the Academy of Medicine, Singapore.2010;39:625-8.
[91]Makinen N, Heinonen HR, Moore S, Tomlinson IP, van der Spuy ZM, Aaltonen LA. MED 12 exon 2 mutations are common in uterine leiomyomas from South African patients. Oncotarget.2011;2:966-9.
[92]McGuire MM, Yatsenko A, Hoffner L, Jones M, Surti U, Rajkovic A. Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED 12 mutations in majority of uterine leiomyomas. PloS one. 2012;7:e33251.
[93]Perot G, Croce S, Ribeiro A, Lagarde P, Velasco V, Neuville A, et al. MED 12 Alterations in Both Human Benign and Malignant Uterine Soft Tissue Tumors. PloS one.2012;7:e40015.
[94]Je EM, Kim MR, Min KO, Yoo NJ, Lee SH. Mutational analysis of MED12 exon 2 in uterine leiomyoma and other common tumors. International journal of cancer Journal international du cancer.2012;131:E1044-7.
[95]Markowski DN, Bartnitzke S, Loning T, Drieschner N, Helmke BM, Bullerdiek J. MED 12 mutations in uterine fibroids--their relationship to cytogenetic subgroups. International journal of cancer Journal international du cancer.2012; 131:1528-36.
[96]Kampjarvi K, Makinen N, Kilpivaara O, Arola J, Heinonen HR, Bohm J, et al. Somatic MED 12 mutations in uterine leiomyosarcoma and colorectal cancer. British journal of cancer.2012; 107:1761-5.
[97]Ravegnini G, Marino-Enriquez A, Slater J, Eilers G, Wang Y, Zhu M, et al. MED 12 mutations in leiomyosarcoma and extrauterine leiomyoma. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc.2013;26:743-9.
[98]Schwetye KE, Pfeifer JD, Duncavage EJ. MED12 exon 2 mutations in uterine and extrauterine smooth muscle tumors. Human pathology.2013.
[99]de Graaff MA, Cleton-Jansen AM, Szuhai K, Bovee JV. Mediator complex subunit 12 exon 2 mutation analysis in different subtypes of smooth muscle tumors confirms genetic heterogeneity. Human pathology.2013;44:1597-604.
[100]Matsubara A, Sekine S, Yoshida M, Yoshida A, Taniguchi H, Kushima R, et al. Prevalence of MED 12 mutations in uterine and extrauterine smooth muscle tumours. Histopathology.2013;62:657-61.
[101]Makinen N, Vahteristo P, Kampjarvi K, Arola J, Butzow R, Aaltonen LA. MED 12 exon 2 mutations in histopathological uterine leiomyoma variants. European journal of human genetics:EJHG.2013;21:1300-3.
[102]Bertsch E, Qiang W, Zhang Q, Espona-Fiedler M, Druschitz S, Liu Y, et al. MED 12 and HMGA2 mutations:two independent genetic events in uterine leiomyoma and leiomyosarcoma. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc.2014.
[103]Quade BJ, Pinto AP, Howard DR, Peters WA,3rd, Crum CP. Frequent loss of heterozygosity for chromosome 10 in uterine leiomyosarcoma in contrast to leiomyoma. The American journal of pathology.1999; 154:945-50.
[104]El-Rifai W, Sarlomo-Rikala M, Knuutila S, Miettinen M. DNA copy number changes in development and progression in leiomyosarcomas of soft tissues. The American journal of pathology.1998;153:985-90.
[105]Levy B, Mukherjee T, Hirschhorn K. Molecular cytogenetic analysis of uterine leiomyoma and leiomyosarcoma by comparative genomic hybridization. Cancer genetics and cytogenetics.2000; 121:1-8.
[106]Hu J, Khanna V, Jones M, Surti U. Genomic alterations in uterine leiomyosarcomas:potential markers for clinical diagnosis and prognosis. Genes, chromosomes & cancer.2001;31:117-24.
[107]Hu J, Rao UN, Jasani S, Khanna V, Yaw K, Surti U. Loss of DNA copy number of lOq is associated with aggressive behavior of leiomyosarcomas:a comparative genomic hybridization study. Cancer genetics and cytogenetics.2005;161:20-7.
[108]Raish M, Khurshid M, Ansari MA, Chaturvedi PK, Bae SM, Kim JH, et al. Analysis of molecular cytogenetic alterations in uterine leiomyosarcoma by array-based comparative genomic hybridization. Journal of cancer research and clinical oncology.2012;138:1173-86.
[109]Ellenrieder V. TGFbeta regulated gene expression by Smads and Sp1/KLF-like transcription factors in cancer. Anticancer research.2008;28:1531-9.
[110]Yin P, Lin Z, Reierstad S, Wu J, Ishikawa H, Marsh EE, et al. Transcription factor KLF11 integrates progesterone receptor signaling and proliferation in uterine leiomyoma cells. Cancer research.2010;70:1722-30.
[111]Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, Miller L, Urrutia R. An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation. The EMBO journal.2003;22:4748-58.
[112]Wang H, Mahadevappa M, Yamamoto K, Wen Y, Chen B, Warrington JA, et al. Distinctive proliferative phase differences in gene expression in human myometrium and leiomyomata. Fertility and sterility.2003;80:266-76.
[113]Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E, Nakamura Y. Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3. Cancer research.1999;59:1966-72.
[114]Zhang JS, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. A conserved alpha-helical motif mediates the interaction of Spl-like transcriptional repressors with the corepressor mSin3A. Molecular and cellular biology. 2001;21:5041-9.
[115]Kwong J, Lee JY, Wong KK, Zhou X, Wong DT, Lo KW, et al. Candidate tumor-suppressor gene DLEC1 is frequently downregulated by promoter hypermethylation and histone hypoacetylation in human epithelial ovarian cancer. Neoplasia (New York, NY).2006;8:268-78.
[116]Rathi A, Virmani AK, Schorge JO, Elias KJ, Maruyama R, Minna JD, et al. Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women. Clinical cancer research:an official journal of the American Association for Cancer Research.2002;8:3324-31.
[117]Makarla PB, Saboorian MH, Ashfaq R, Toyooka KO, Toyooka S, Minna JD, et al. Promoter hypermethylation profile of ovarian epithelial neoplasms. Clinical cancer research:an official journal of the American Association for Cancer Research. 2005;11:5365-9.
[118]Wu Q, Lothe RA, Ahlquist T, Silins I, Trope CG, Micci F, et al. DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets. Molecular cancer. 2007;6:45.
[119]Tam KF, Liu VW, Liu SS, Tsang PC, Cheung AN, Yip AM, et al. Methylation profile in benign, borderline and malignant ovarian tumors. Journal of cancer research and clinical oncology.2007; 133:331-41.
[120]Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J, et al. Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene.2001;20:1509-18.
[121]Bondurant AE, Huang Z, Whitaker RS, Simel LR, Berchuck A, Murphy SK. Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer. Gynecologic oncology. 2011;123:581-7.
[122]Cul'bova M, Lasabova Z, Stanclova A, Tilandyova P, Zubor P, Fiolka R, et al. [Methylation of selected tumor-supressor genes in benign and malignant ovarian tumors]. Ceska gynekologie/Ceska lekarska spolecnost J Ev Purkyne. 2011;76:274-9.
[123]Yoon JH, Dammann R, Pfeifer GP. Hypermethylation of the CpG island of the RASSF1A gene in ovarian and renal cell carcinomas. International journal of cancer Journal international du cancer.2001;94:212-7.
[124]Zhang S, Wei L, Zhang A, Zhang L, Yu H. RUNX3 gene methylation in epithelial ovarian cancer tissues and ovarian cancer cell lines. Omics:a journal of integrative biology.2009; 13:307-11.
[125]Yuecheng Y, Hongmei L, Xiaoyan X. Clinical evaluation of E-cadherin expression and its regulation mechanism in epithelial ovarian cancer. Clinical & experimental metastasis.2006;23:65-74.
[126]Hibi K, Goto T, Kitamura YH, Yokomizo K, Sakuraba K, Shirahata A, et al. Methylation of TFPI2 gene is frequently detected in advanced well-differentiated colorectal cancer. Anticancer research.2010;30:1205-7.
[127]Sato N, Parker AR, Fukushima N, Miyagi Y, Iacobuzio-Donahue CA, Eshleman JR, et al. Epigenetic inactivation of TFPI-2 as a common mechanism associated with growth and invasion of pancreatic ductal adenocarcinoma. Oncogene.2005;24:850-8.
[128]Ho CM, Lai HC, Huang SH, Chien TY, Lin MC, Chang SF. Promoter methylation of sFRP5 in patients with ovarian clear cell adenocarcinoma. European journal of clinical investigation.2010;40:310-8.
[129]Su HY, Lai HC, Lin YW, Liu CY, Chen CK, Chou YC, et al. Epigenetic silencing of SFRP5 is related to malignant phenotype and chemoresistance of ovarian cancer through Wnt signaling pathway. International journal of cancer Journal international du cancer.2010;127:555-67.
[130]Kuhn E, Yemelyanova A, Wang T-L, Kurman R, Shih I-M. Abstract 5536:TP53 and MED12 mutations in uterine smooth muscle tumors. Cancer research. 2012;72:5536.
[2]Wu C, Morris JR. Genes, genetics, and epigenetics:a correspondence. Science (New York, NY).2001;293:1103-5.
[3]Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature.2004;429:457-63.
[4]Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature. 2007;445:214-8.
[5]Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nature reviews Molecular cell biology.2005;6:376-85.
[6]Fujiwara K, Fujimoto N, Tabata M, Nishii K, Matsuo K, Hotta K, et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clinical cancer research:an official journal of the American Association for Cancer Research.2005;11:1219-25.
[7]Cuk K, Zucknick M, Heil J, Madhavan D, Schott S, Turchinovich A, et al. Circulating microRNAs in plasma as early detection markers for breast cancer. International journal of cancer Journal international du cancer.2013;132:1602-12.
[8]Teodoridis JM, Hall J, Marsh S, Kannall HD, Smyth C, Curto J, et al. CpG island methylation of DNA damage response genes in advanced ovarian cancer. Cancer research.2005;65:8961-7.
[9]Akahira J, Sugihashi Y, Ito K, Niikura H, Okamura K, Yaegashi N. Promoter methylation status and expression of TMS1 gene in human epithelial ovarian cancer. Cancer science.2004;95:40-3.
[10]Terasawa K, Sagae S, Toyota M, Tsukada K, Ogi K, Satoh A, et al. Epigenetic inactivation of TMS1/ASC in ovarian cancer. Clinical cancer research:an official journal of the American Association for Cancer Research.2004; 10:2000-6.
[11]Kaneuchi M, Sasaki M, Tanaka Y, Shiina H, Yamada H, Yamamoto R, et al. WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma. Cancer.2005; 104:1924-30.
[12]Kaneuchi M, Sasaki M, Tanaka Y, Shiina H, Verma M, Ebina Y, et al. Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer. Biochemical and biophysical research communications. 2004;316:1156-62.
[13]Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer research. 2005;65:7065-70.
[14]Mattie MD, Benz CC, Bowers J, Sensinger K, Wong L, Scott GK, et al. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Molecular cancer. 2006;5:24.
[15]Siegel R, Naishadham D, Jemal A. Cancer statistics,2012. CA Cancer J Clin. 2012;62:10-29.
[16]Woolas RP, Xu FJ, Jacobs IJ, Yu YH, Daly L, Berchuck A, et al. Elevation of multiple serum markers in patients with stage I ovarian cancer. J Natl Cancer Inst. 1993;85:1748-51.
[17]Powell CB, Kenley E, Chen LM, Crawford B, McLennan J, Zaloudek C, et al. Risk-reducing salpingo-oophorectomy in BRCA mutation carriers:role of serial sectioning in the detection of occult malignancy. Journal of clinical oncology:official journal of the American Society of Clinical Oncology.2005;23:127-32.
[18]Lee Y, Miron A, Drapkin R, Nucci MR, Medeiros F, Saleemuddin A, et al. A candidate precursor to serous carcinoma that originates in the distal fallopian tube. The Journal of pathology.2007;211:26-35.
[19]American cancer society, Cancer Facts and Figures.2011.
[20]Shih Ie M, Kurman RJ. Ovarian tumorigenesis:a proposed model based on morphological and molecular genetic analysis. The American journal of pathology. 2004;164:1511-8.
[21]Smith Sehdev AE, Sehdev PS, Kurman RJ. Noninvasive and invasive micropapillary (low-grade) serous carcinoma of the ovary:a clinicopathologic analysis of 135 cases. The American journal of surgical pathology.2003;27:725-36.
[22]Singer G, Kurman RJ, Chang HW, Cho SK, Shih Ie M. Diverse tumorigenic pathways in ovarian serous carcinoma. The American journal of pathology. 2002;160:1223-8.
[23]Singer G, Oldt R,3rd, Cohen Y, Wang BG, Sidransky D, Kurman RJ, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst.2003;95:484-6.
[24]Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nature genetics.1998;19:83-6.
[25]Francis-Thickpenny KM, Richardson DM, van Ee CC, Love DR, Winship IM, Baguley BC, et al. Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma. British journal of cancer.2001;85:687-91.
[26]Moreno-Bueno G, Gamallo C, Perez-Gallego L, de Mora JC, Suarez A, Palacios J. beta-Catenin expression pattern, beta-catenin gene mutations, and microsatellite instability in endometrioid ovarian carcinomas and synchronous endometrial carcinomas. Diagnostic molecular pathology:the American journal of surgical pathology, part B.2001;10:116-22.
[27]Obata K, Morland SJ, Watson RH, Hitchcock A, Chenevix-Trench G, Thomas EJ, et al. Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors. Cancer research.1998;58:2095-7.
[28]Chan WY, Cheung KK, Schorge JO, Huang LW, Welch WR, Bell DA, et al. Bcl-2 and p53 protein expression, apoptosis, and p53 mutation in human epithelial ovarian cancers. The American journal of pathology.2000; 156:409-17.
[29]Kohler MF, Marks JR, Wiseman RW, Jacobs IJ, Davidoff AM, Clarke-Pearson DL, et al. Spectrum of mutation and frequency of allelic deletion of the p53 gene in ovarian cancer. J Natl Cancer Inst.1993;85:1513-9.
[30]Kupryjanczyk J, Thor AD, Beauchamp R, Merritt V, Edgerton SM, Bell DA, et al. p53 gene mutations and protein accumulation in human ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:4961-5.
[31]Wen WH, Reles A, Runnebaum IB, Sullivan-Halley J, Bernstein L, Jones LA, et al. p53 mutations and expression in ovarian cancers:correlation with overall survival. International journal of gynecological pathology:official journal of the International Society of Gynecological Pathologists.1999;18:29-41.
[32]Clarke-Pearson DL. Clinical practice. Screening for ovarian cancer. The New England journal of medicine.2009;361:170-7.
[33]Buys SS, Partridge E, Black A, Johnson CC, Lamerato L, Isaacs C, et al. Effect of screening on ovarian cancer mortality:the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA:the journal of the American Medical Association.2011;305:2295-303.
[34]Menon U, Gentry-Maharaj A, Hallett R, Ryan A, Burnell M, Sharma A, et al. Sensitivity and specificity of multimodal and ultrasound screening for ovarian cancer, and stage distribution of detected cancers:results of the prevalence screen of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). The lancet oncology. 2009; 10:327-40.
[35]Menon U, Jacobs IJ. Recent developments in ovarian cancer screening. Current opinion in obstetrics & gynecology.2000;12:39-42.
[36]Perlin E, Moquin RB. Serum DNA levels in patients with malignant disease. American journal of clinical pathology.1972;58:601-2.
[37]Ibanez de Caceres I, Battagli C, Esteller M, Herman JG, Dulaimi E, Edelson MI, et al. Tumor cell-specific BRCA1 and RASSFlA hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer research.2004;64:6476-81.
[38]Nelissen EC, van Montfoort AP, Dumoulin JC, Evers JL. Epigenetics and the placenta. Human reproduction update.2011;17:397-417.
[39]Nakao M. Epigenetics:interaction of DNA methylation and chromatin. Gene. 2001;278:25-31.
[40]Singal R, Ginder GD. DNA methylation. Blood.1999;93:4059-70.
[41]Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:11995-9.
[42]Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science (New York, NY).2001;293:1068-70.
[43]Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer research.1977;37:646-50.
[44]Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer research.1999;59:67-70.
[45]Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients:quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer research.2001;61:1659-65.
[46]The Guide to Clinical Preventive Services 2010-2011:Recommendations of the U.S. Preventive Services Task Force. Rockville MD2010.
[47]Philipp AB, Stieber P, Nagel D, Neumann J, Spelsberg F, Jung A, et al. Prognostic role of methylated free circulating DNA in colorectal cancer. International journal of cancer Journal international du cancer.2012;131:2308-19.
[48]Yagyu S, Gotoh T, Iehara T, Miyachi M, Katsumi Y, Tsubai-Shimizu S, et al. Circulating methylated-DCR2 gene in serum as an indicator of prognosis and therapeutic efficacy in patients with MYCN nonamplified neuroblastoma. Clinical cancer research:an official journal of the American Association for Cancer Research. 2008;14:7011-9.
[49]Liggett TE, Melnikov A, Yi Q, Replogle C, Hu W, Rotmensch J, et al. Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors. Gynecologic oncology.2011; 120:113-20.
[50]Song JZ, Stirzaker C, Harrison J, Melki JR, Clark SJ. Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells. Oncogene. 2002;21:1048-61.
[51]Barton CA, Hacker NF, Clark SJ, O'Brien PM. DNA methylation changes in ovarian cancer:implications for early diagnosis, prognosis and treatment. Gynecologic oncology.2008;109:129-39.
[52]Sellar GC, Watt KP, Rabiasz GJ, Stronach EA, Li L, Miller EP, et al. OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer. Nature genetics.2003;34:337-43.
[53]Czekierdowski A, Czekierdowska S, Szymanski M, Wielgos M, Kaminski P, Kotarski J. Opioid-binding protein/cell adhesion molecule-like (OPCML) gene and promoter methylation status in women with ovarian cancer. Neuro endocrinology letters.2006;27:609-13.
[54]Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis:practical aspects and biological significance. Mutation research.2007;635:105-17.
[55]Shapiro B, Chakrabarty M, Cohn EM, Leon SA. Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease. Cancer. 1983;51:2116-20.
[56]Fraga MF, Esteller M. DNA methylation:a profile of methods and applications. BioTechniques.2002;33:632,4,6-49.
[57]Yamamoto N, Nakayama T, Kajita M, Miyake T, Iwamoto T, Kim SJ, et al. Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARbeta2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay. Breast cancer research and treatment. 2012;132:165-73.
[58]Hibi K, Taguchi M, Nakayama H, Takase T, Kasai Y, Ito K, et al. Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma. Clinical cancer research:an official journal of the American Association for Cancer Research.2001;7:3135-8.
[59]Usadel H, Brabender J, Danenberg KD, Jeronimo C, Harden S, Engles J, et al. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer research. 2002;62:371-5.
[60]Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women:ultrasound evidence. American journal of obstetrics and gynecology.2003; 188:100-7.
[61]Leibsohn S, d'Ablaing G, Mishell DR, Jr., Schlaerth JB. Leiomyosarcoma in a series of hysterectomies performed for presumed uterine leiomyomas. American journal of obstetrics and gynecology.1990; 162:968-74; discussion 74-6.
[62]Giuntoli RL,2nd, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL, et al. Retrospective review of 208 patients with leiomyosarcoma of the uterus: prognostic indicators, surgical management, and adjuvant therapy. Gynecologic oncology.2003;89:460-9.
[63]Bell SW, Kempson RL, Hendrickson MR. Problematic uterine smooth muscle neoplasms. A clinicopathologic study of 213 cases. The American journal of surgical pathology.1994;18:535-58.
[64]IARC. WHO Classification of Tumors.2003.
[65]Lee WY, Tzeng CC, Chou CY. Uterine leiomyosarcomas coexistent with cellular and atypical leiomyomata in a young woman during the treatment with luteinizing hormone-releasing hormone agonist. Gynecologic oncology.1994;52:74-9.
[66]Evans HL, Chawla SP, Simpson C, Finn KP. Smooth muscle neoplasms of the uterus other than ordinary leiomyoma. A study of 46 cases, with emphasis on diagnostic criteria and prognostic factors. Cancer.1988;62:2239-47.
[67]Sung CO, Ahn G, Song SY, Choi YL, Bae DS. Atypical leiomyomas of the uterus with long-term follow-up after myomectomy with immunohistochemical analysis for p16INK4A, p53, Ki-67, estrogen receptors, and progesterone receptors. International journal of gynecological pathology:official journal of the International Society of Gynecological Pathologists.2009;28:529-34.
[68]Ly A, Mills AM, McKenney JK, Balzer BL, Kempson RL, Hendrickson MR, et al. Atypical leiomyomas of the uterus:a clinicopathologic study of 51 cases. The American journal of surgical pathology.2013;37:643-9.
[69]Giuntoli RL,2nd, Gostout BS, DiMarco CS, Metzinger DS, Keeney GL. Diagnostic criteria for uterine smooth muscle tumors:leiomyoma variants associated with malignant behavior. The Journal of reproductive medicine.2007;52:1001-10.
[70]Christacos NC, Quade BJ, Dal Cin P, Morton CC. Uterine leiomyomata with deletions of Ip represent a distinct cytogenetic subgroup associated with unusual histologic features. Genes, chromosomes & cancer.2006;45:304-12.
[71]Mittal KR, Chen F, Wei JJ, Rijhvani K, Kurvathi R, Streck D, et al. Molecular and immunohistochemical evidence for the origin of uterine leiomyosarcomas from associated leiomyoma and symplastic leiomyoma-like areas. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc. 2009;22:1303-11.
[72]Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al. MED 12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science (New York, NY).2011;334:252-5.
[73]Zhai YL, Nikaido T, Toki T, Shiozawa A, Orii A, Fujii S. Prognostic significance of bcl-2 expression in leiomyosarcoma of the uterus. British journal of cancer. 1999;80:1658-64.
[74]Miyajima K, Tamiya S, Oda Y, Adachi T, Konomoto T, Toyoshiba H, et al. Relative quantitation of p53 and MDM2 gene expression in leiomyosarcoma; real-time semi-quantitative reverse transcription-polymerase chain reaction. Cancer letters.2001;164:177-88.
[75]Ito M, Barys L, O'Reilly T, Young S, Gorbatcheva B, Monahan J, et al. Comprehensive mapping of p53 pathway alterations reveals an apparent role for both SNP309 and MDM2 amplification in sarcomagenesis. Clinical cancer research:an official journal of the American Association for Cancer Research.2011; 17:416-26.
[76]de Vos S, Wilczynski SP, Fleischhacker M, Koeffler P. p53 alterations in uterine leiomyosarcomas versus leiomyomas. Gynecologic oncology.1994;54:205-8.
[77]Hall KL, Teneriello MG, Taylor RR, Lemon S, Ebina M, Linnoila RI, et al. Analysis of Ki-ras, p53, and MDM2 genes in uterine leiomyomas and leiomyosarcomas. Gynecologic oncology.1997;65:330-5.
[78]Zhai YL, Nikaido T, Orii A, Horiuchi A, Toki T, Fujii S. Frequent occurrence of loss of heterozygosity among tumor suppressor genes in uterine leiomyosarcoma. Gynecologic oncology.1999;75:453-9.
[79]Jeffers MD, Farquharson MA, Richmond JA, McNicol AM. p53 immunoreactivity and mutation of the p53 gene in smooth muscle tumours of the uterine corpus. The Journal of pathology.1995; 177:65-70.
[80]Hernando E, Charytonowicz E, Dudas ME, Menendez S, Matushansky I, Mills J, et al. The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nature medicine.2007; 13:748-53.
[81]Yamada C, Ozaki T, Ando K, Suenaga Y, Inoue K, Ito Y, et al. RUNX3 modulates DNA damage-mediated phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The Journal of biological chemistry.2010;285:16693-703.
[82]Shio S, Kodama Y, Ida H, Shiokawa M, Kitamura K, Hatano E, et al. Loss of RUNX3 expression by histone deacetylation is associated with biliary tract carcinogenesis. Cancer science.2011;102:776-83.
[83]Lin FC, Liu YP, Lai CH, Shan YS, Cheng HC, Hsu PI, et al. RUNX3-mediated transcriptional inhibition of Akt suppresses tumorigenesis of human gastric cancer cells. Oncogene.2012;31:4302-16.
[84]Gao J, Chen Y, Wu KC, Liu J, Zhao YQ, Pan YL, et al. RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells. Experimental cell research.2010;316:149-57.
[85]Voon DC, Wang H, Koo JK, Nguyen TA, Hor YT, Chu YS, et al. Runx3 protects gastric epithelial cells against epithelial-mesenchymal transition-induced cellular plasticity and tumorigenicity. Stem cells (Dayton, Ohio).2012;30:2088-99.
[86]Danielson LS, Menendez S, Attolini CS, Guijarro MV, Bisogna M, Wei J, et al. A differentiation-based microRNA signature identifies leiomyosarcoma as a mesenchymal stem cell-related malignancy. The American journal of pathology. 2010;177:908-17.
[87]Wang T, Zhang X, Obijuru L, Laser J, Aris V, Lee P, et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas. Genes, chromosomes & cancer.2007;46:336-47.
[88]Ip PP, Cheung AN, Clement PB. Uterine smooth muscle tumors of uncertain malignant potential (STUMP):a clinicopathologic analysis of 16 cases. The American journal of surgical pathology.2009;33:992-1005.
[89]Guntupalli SR, Ramirez PT, Anderson ML, Milam MR, Bodurka DC, Malpica A. Uterine smooth muscle tumor of uncertain malignant potential:a retrospective analysis. Gynecologic oncology.2009;113:324-6.
[90]Ng JS, Han A, Chew SH, Low J. A clinicopathologic study of uterine smooth muscle tumours of uncertain malignant potential (STUMP). Annals of the Academy of Medicine, Singapore.2010;39:625-8.
[91]Makinen N, Heinonen HR, Moore S, Tomlinson IP, van der Spuy ZM, Aaltonen LA. MED 12 exon 2 mutations are common in uterine leiomyomas from South African patients. Oncotarget.2011;2:966-9.
[92]McGuire MM, Yatsenko A, Hoffner L, Jones M, Surti U, Rajkovic A. Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED 12 mutations in majority of uterine leiomyomas. PloS one. 2012;7:e33251.
[93]Perot G, Croce S, Ribeiro A, Lagarde P, Velasco V, Neuville A, et al. MED 12 Alterations in Both Human Benign and Malignant Uterine Soft Tissue Tumors. PloS one.2012;7:e40015.
[94]Je EM, Kim MR, Min KO, Yoo NJ, Lee SH. Mutational analysis of MED12 exon 2 in uterine leiomyoma and other common tumors. International journal of cancer Journal international du cancer.2012;131:E1044-7.
[95]Markowski DN, Bartnitzke S, Loning T, Drieschner N, Helmke BM, Bullerdiek J. MED 12 mutations in uterine fibroids--their relationship to cytogenetic subgroups. International journal of cancer Journal international du cancer.2012; 131:1528-36.
[96]Kampjarvi K, Makinen N, Kilpivaara O, Arola J, Heinonen HR, Bohm J, et al. Somatic MED 12 mutations in uterine leiomyosarcoma and colorectal cancer. British journal of cancer.2012; 107:1761-5.
[97]Ravegnini G, Marino-Enriquez A, Slater J, Eilers G, Wang Y, Zhu M, et al. MED 12 mutations in leiomyosarcoma and extrauterine leiomyoma. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc.2013;26:743-9.
[98]Schwetye KE, Pfeifer JD, Duncavage EJ. MED12 exon 2 mutations in uterine and extrauterine smooth muscle tumors. Human pathology.2013.
[99]de Graaff MA, Cleton-Jansen AM, Szuhai K, Bovee JV. Mediator complex subunit 12 exon 2 mutation analysis in different subtypes of smooth muscle tumors confirms genetic heterogeneity. Human pathology.2013;44:1597-604.
[100]Matsubara A, Sekine S, Yoshida M, Yoshida A, Taniguchi H, Kushima R, et al. Prevalence of MED 12 mutations in uterine and extrauterine smooth muscle tumours. Histopathology.2013;62:657-61.
[101]Makinen N, Vahteristo P, Kampjarvi K, Arola J, Butzow R, Aaltonen LA. MED 12 exon 2 mutations in histopathological uterine leiomyoma variants. European journal of human genetics:EJHG.2013;21:1300-3.
[102]Bertsch E, Qiang W, Zhang Q, Espona-Fiedler M, Druschitz S, Liu Y, et al. MED 12 and HMGA2 mutations:two independent genetic events in uterine leiomyoma and leiomyosarcoma. Modern pathology:an official journal of the United States and Canadian Academy of Pathology, Inc.2014.
[103]Quade BJ, Pinto AP, Howard DR, Peters WA,3rd, Crum CP. Frequent loss of heterozygosity for chromosome 10 in uterine leiomyosarcoma in contrast to leiomyoma. The American journal of pathology.1999; 154:945-50.
[104]El-Rifai W, Sarlomo-Rikala M, Knuutila S, Miettinen M. DNA copy number changes in development and progression in leiomyosarcomas of soft tissues. The American journal of pathology.1998;153:985-90.
[105]Levy B, Mukherjee T, Hirschhorn K. Molecular cytogenetic analysis of uterine leiomyoma and leiomyosarcoma by comparative genomic hybridization. Cancer genetics and cytogenetics.2000; 121:1-8.
[106]Hu J, Khanna V, Jones M, Surti U. Genomic alterations in uterine leiomyosarcomas:potential markers for clinical diagnosis and prognosis. Genes, chromosomes & cancer.2001;31:117-24.
[107]Hu J, Rao UN, Jasani S, Khanna V, Yaw K, Surti U. Loss of DNA copy number of lOq is associated with aggressive behavior of leiomyosarcomas:a comparative genomic hybridization study. Cancer genetics and cytogenetics.2005;161:20-7.
[108]Raish M, Khurshid M, Ansari MA, Chaturvedi PK, Bae SM, Kim JH, et al. Analysis of molecular cytogenetic alterations in uterine leiomyosarcoma by array-based comparative genomic hybridization. Journal of cancer research and clinical oncology.2012;138:1173-86.
[109]Ellenrieder V. TGFbeta regulated gene expression by Smads and Sp1/KLF-like transcription factors in cancer. Anticancer research.2008;28:1531-9.
[110]Yin P, Lin Z, Reierstad S, Wu J, Ishikawa H, Marsh EE, et al. Transcription factor KLF11 integrates progesterone receptor signaling and proliferation in uterine leiomyoma cells. Cancer research.2010;70:1722-30.
[111]Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, Miller L, Urrutia R. An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation. The EMBO journal.2003;22:4748-58.
[112]Wang H, Mahadevappa M, Yamamoto K, Wen Y, Chen B, Warrington JA, et al. Distinctive proliferative phase differences in gene expression in human myometrium and leiomyomata. Fertility and sterility.2003;80:266-76.
[113]Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E, Nakamura Y. Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3. Cancer research.1999;59:1966-72.
[114]Zhang JS, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. A conserved alpha-helical motif mediates the interaction of Spl-like transcriptional repressors with the corepressor mSin3A. Molecular and cellular biology. 2001;21:5041-9.
[115]Kwong J, Lee JY, Wong KK, Zhou X, Wong DT, Lo KW, et al. Candidate tumor-suppressor gene DLEC1 is frequently downregulated by promoter hypermethylation and histone hypoacetylation in human epithelial ovarian cancer. Neoplasia (New York, NY).2006;8:268-78.
[116]Rathi A, Virmani AK, Schorge JO, Elias KJ, Maruyama R, Minna JD, et al. Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women. Clinical cancer research:an official journal of the American Association for Cancer Research.2002;8:3324-31.
[117]Makarla PB, Saboorian MH, Ashfaq R, Toyooka KO, Toyooka S, Minna JD, et al. Promoter hypermethylation profile of ovarian epithelial neoplasms. Clinical cancer research:an official journal of the American Association for Cancer Research. 2005;11:5365-9.
[118]Wu Q, Lothe RA, Ahlquist T, Silins I, Trope CG, Micci F, et al. DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets. Molecular cancer. 2007;6:45.
[119]Tam KF, Liu VW, Liu SS, Tsang PC, Cheung AN, Yip AM, et al. Methylation profile in benign, borderline and malignant ovarian tumors. Journal of cancer research and clinical oncology.2007; 133:331-41.
[120]Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J, et al. Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene.2001;20:1509-18.
[121]Bondurant AE, Huang Z, Whitaker RS, Simel LR, Berchuck A, Murphy SK. Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer. Gynecologic oncology. 2011;123:581-7.
[122]Cul'bova M, Lasabova Z, Stanclova A, Tilandyova P, Zubor P, Fiolka R, et al. [Methylation of selected tumor-supressor genes in benign and malignant ovarian tumors]. Ceska gynekologie/Ceska lekarska spolecnost J Ev Purkyne. 2011;76:274-9.
[123]Yoon JH, Dammann R, Pfeifer GP. Hypermethylation of the CpG island of the RASSF1A gene in ovarian and renal cell carcinomas. International journal of cancer Journal international du cancer.2001;94:212-7.
[124]Zhang S, Wei L, Zhang A, Zhang L, Yu H. RUNX3 gene methylation in epithelial ovarian cancer tissues and ovarian cancer cell lines. Omics:a journal of integrative biology.2009; 13:307-11.
[125]Yuecheng Y, Hongmei L, Xiaoyan X. Clinical evaluation of E-cadherin expression and its regulation mechanism in epithelial ovarian cancer. Clinical & experimental metastasis.2006;23:65-74.
[126]Hibi K, Goto T, Kitamura YH, Yokomizo K, Sakuraba K, Shirahata A, et al. Methylation of TFPI2 gene is frequently detected in advanced well-differentiated colorectal cancer. Anticancer research.2010;30:1205-7.
[127]Sato N, Parker AR, Fukushima N, Miyagi Y, Iacobuzio-Donahue CA, Eshleman JR, et al. Epigenetic inactivation of TFPI-2 as a common mechanism associated with growth and invasion of pancreatic ductal adenocarcinoma. Oncogene.2005;24:850-8.
[128]Ho CM, Lai HC, Huang SH, Chien TY, Lin MC, Chang SF. Promoter methylation of sFRP5 in patients with ovarian clear cell adenocarcinoma. European journal of clinical investigation.2010;40:310-8.
[129]Su HY, Lai HC, Lin YW, Liu CY, Chen CK, Chou YC, et al. Epigenetic silencing of SFRP5 is related to malignant phenotype and chemoresistance of ovarian cancer through Wnt signaling pathway. International journal of cancer Journal international du cancer.2010;127:555-67.
[130]Kuhn E, Yemelyanova A, Wang T-L, Kurman R, Shih I-M. Abstract 5536:TP53 and MED12 mutations in uterine smooth muscle tumors. Cancer research. 2012;72:5536.