乳腺增生病p53基因第5外显子突变及其蛋白表达

目的：探讨p53基因在乳腺癌发生早期的作用。方法：用免疫组化方法检测36例乳腺单纯性增生、31例不典型增生、14例原位癌和16例浸润癌中p53蛋白的表达，用PCR－SSCP检测了上述组织中p53基因第5外显子突变。结果：p53蛋白在单纯性增生、不典型增生、导管内癌、浸润癌中的表达率分别为0、22.6%(7/31)、42.8%(6/14)、50％（8／16），PCR－SSCP在各组中均未检测到该基因第5外显子突变。结论：乳腺癌发生早期阶段有p53基因的参与，但与第5外显子突变无明显关系。     

端粒酶基因在乳腺导管不典型增生中的表达

目的：探讨端粒酶基因与乳腺导管不典型增生恶性转化的关系及其作用。方法：应用原位杂交方法检测端粒酶基因hTR和hTRT在50例乳腺导管增生组织（其中单纯性增生6例,轻度不典型增生9例,中度不典型增生12例、重度不典型增生23例）及26例乳腺癌中的表达。结果：在乳腺导管单纯性增生组织hTR、hTRT、mRNA呈弱表达（1／6）和阴性。在乳腺导管轻、中度不典型增生组织hTR、hTRT、mRNA呈弱表达（2／9、1／9;4／12、3／12）,在乳腺导管重度不典型增生组织中hTR、hTRT、mRNA表达增强（14／23,60．9％,12／23,52．1％）,在乳腺癌组织中端粒酶基因hTR、hTRT、mRNA呈较强阳性表达（23／26,88．5％;21／25,80．8％）。乳腺导管重度不典型增生组织中的端粒酶基因表达与在轻、中度不典型增生、浸润性导管癌组织中的表达比较差异有显著性意义（P＜0．05）,但与导管内癌表达差异无显著性意义（P＞0．05）。结论：端粒酶基因hTR、hTRT的表达与乳腺导管不典型增生细胞的恶性转化密切相关,端粒酶的重新激活可能在乳腺癌的组织发生中起关键性作用。     

散发性乳腺癌及乳腺不典型导管增生组织BRCA1基因启动子区甲基化分析

目的 了解散发性乳腺癌及癌旁增生组织、乳腺不典型导管增生组织BRCA1基因启动子区甲基化状态,探讨其与乳腺癌发生的关系.方法 采用甲基化特异性PCR(MSP)结合巢式PCR技术,研究23例散发性乳腺癌及其癌旁增生组织、6例乳腺不典型导管增生组织及5例健康成人女性外周血淋巴细胞中BRCA1基因启动子区甲基化状态.结果 5例健康成人女性外周血淋巴细胞均表现BRCA1基因启动子区甲基化阴性;23例原发性乳腺癌组织中,BRCA1基因启动子区CpG岛甲基化率为65.22%(15/23);癌旁增生组织检出CpG岛甲基化者11例,甲基化率为47.83%(11/23),且均为癌组织阳性患者;6例乳腺不典型导管增生组织中,BRCA1基因启动子区CpG岛甲基化阳性者2例,甲基化率为33.33%(2/6);统计学检验结果表明,乳腺癌、癌旁增生组织之间,BRCA1基因启动子区甲基化阳性率无显著差异.结论 BRCA1基因启动子区CpG 岛甲基化是散发性乳腺癌发生过程中的早期事件,可能在乳腺癌发生中和乳腺增生病癌变过程中起重要生物学作用.     

ras癌基因产物P21在乳腺良,恶性病变中的表达

女性乳腺癌４０例，良性肿瘤３７例，乳腺增生病５２例用ＡＢＣ法做了免疫组化Ｐ２１蛋白水平检测，其阳性率分别是７２．５％，３２．４％，２３．１％。阳性表达主要在分化差的浸润性导管癌细胞；乳腺增生病中５例导管上皮细胞不典型增生有３例阳性表达，提示Ｐ２１蛋白检测可为估价乳腺组织癌变和病变预后提供分子生物学依据。     

乳腺癌发生过程中p53、BRCA1、BRCA2、PTEN、Rb蛋白异常表达

目的探讨p53、BRCA1、BRCA2、PTEN、Rb基因蛋白异常表达在乳腺癌发生中的作用。方法选取同时存在浸润癌、导管内癌、不典型增生和单纯性增生的乳腺癌档案蜡块,应用免疫组化S-P法检测p53、BRCA1、BRCA2、PTEN、Rb基因蛋白在各例的异常表达。结果(1)在24．3％(17／70)的乳腺癌及其癌旁不典型增生中检测到突变型p53蛋白表达,分别在2．9％(2／69)、6．3％(4／63)、5．1％(3／59)、5．4％(3／56)的乳腺癌及其癌旁不典型增生中分别检测到BRCA1、BRCA2、PTEN、Rb表达缺失。(2)在44．6％的病例同时检测到2种基因蛋白异常表达,在5．4％的病例同时检测到3种基因蛋白异常表达,在3．6％的病例同时检测到4种基因蛋白异常表达。结论(1)p53、BRCA1、BRCA2、PTEN、Rb蛋白异常表达可出现于乳腺癌发生的早期阶段,可能在乳腺癌发生过程中起作用。(2)多种抑癌基因的失活共同参与了乳腺癌的发生。     

乳腺癌和乳腺增生组织中TIMP-3基因突变谱研究

目的获得乳腺癌发生中TIMP-3基因的突变谱,探讨该基因突变与乳腺癌发生的关系。方法以50例乳腺癌及相应癌旁增生组织和正常乳腺组织、50例乳腺良性增生病变及15例正常人血淋巴细胞为研究对象,采用PCR-SSCP技术和DNA直接测序技术检测TIMP-3基因全部5个外显子的突变情况。结果在40/50例(80%)乳腺癌、36/50例(72%)相应的癌旁增生、40/50例(80%)乳腺良性增生病变,37/50(74%)例正常乳腺组织及13/15(86.67%)例正常人血液淋巴细胞中检测到TIMP-3基因外显子3突变,外显子1、2、4、5均未检测到突变,1～5号外显子的相应剪接点也未检测到基因序列改变。DNA测序证实上述外显子3的突变全部集中在249和261两个位点处,均为同义突变(249TC和261CT,对编码的氨基酸无影响),表明这两处位点的改变可能均为单核苷酸多态性。结论突变不是TIMP-3在乳腺癌中作用失活的主要原因,TIMP-3可能是以基因突变以外的其它方式在乳腺癌的发生发展过程中起作用。     

RUNX3基因表达对判断人乳腺癌预后的价值

目的探讨乳腺癌组织中RUNX3基因的表达及其与乳腺癌生物学特征和预后的关系。方法采用免疫组化SP法检测RUNX3蛋白在88例乳腺癌、40例乳腺纤维腺瘤和40例乳腺增生病组织中的表达。结果（1）RUNX3在乳腺癌中的阳性表达率为35．23％,明显低于在乳腺纤维腺瘤（85％）及乳腺增生病（87．5％）组织中的表达率,差异具有统计学意义（P〈0．05）。（2）RUNX3蛋白表达与乳腺癌有无浸润、临床分期、淋巴结转移、ER、PR表达相关,而与病人的年龄、肿瘤类型、病理分级无关。（3）RUNX3表达阳性者的：生存率高于表达阴性者的生存率（P〈0．05）。RUNX3阳性表达者,术后生存时间长。结论（1）乳腺癌组织中RUNX3蛋白表达降低,证明RUNX3基因可能作为一个抑癌基因参与乳腺癌的发生。（2）随着乳腺癌的临床进展,RUNX3表达下降。（3）RUNX3在乳腺癌中的表达对评价患者的预后有一定价值。     

乳腺癌中PTEN表达及其临床病理意义

目的　探讨PTEN蛋白表达在乳腺癌发生发展中的临床病理意义。方法　用免疫组织化学S P法检测 2 3例正常乳腺组织、2 8例乳腺增生组织 (其中普通型增生 2 0例 ,非典型增生 8例 )、8例导管原位癌、98例浸润性乳腺癌组织PTEN蛋白的表达 ,并对 98例浸润癌组织进行微血管密度计数。结果　正常乳腺组织的PTEN多呈弱阳性表达 (6 5 4 % ) ,非典型增生上皮和导管原位癌的PTEN蛋白水平高于正常乳腺腺上皮 (P <0 0 5 )。浸润癌组织中PTEN的失表达率为 7 1% ,高表达率为4 9 0 % ,与正常乳腺组织比较差异有显著性 (P <0 0 1) ,与增生的乳腺组织和导管原位癌组间差异无显著性。浸润癌组织学分级愈高 ,PTEN高表达率愈低 (P <0 0 5 )。随临床分期升高 ,PTEN高表达率亦有降低趋势。乳腺癌中PTEN表达与微血管密度计数、腋淋巴结转移均呈负相关。结论　PTEN表达的改变是乳腺癌发生的早期事件 ,PTEN的表达下调可作为乳腺癌分化和转移的一个潜在标志     

Ras、MAPK、Cyclin D1与皮肤瘢痕癌的相关性研究

目的探讨Ras/Raf/MAPK信号通路和通路下游靶基因Cyclin D1与皮肤瘢痕癌的相关性。方法 (1)用激光扫描共聚焦显微技术对病理性瘢痕和瘢痕癌进行K-ras、H-ras、N-ras免疫荧光双标记;(2)提取DNA,检测病理性瘢痕和瘢痕癌组织中K-ras、H-ras、N-ras第12、13位密码子的突变;(3)采用免疫组化SP法检测正常皮肤、病理性瘢痕和瘢痕癌组织中MAPK、Cyc-lin D1蛋白的表达;(4)采用原位杂交技术检测3组组织中MAPK mRNA、Cyclin D1 mRNA的表达。结果 (1)免疫荧光双标记K-ras、H-ras、N-ras在病理性瘢痕上皮中呈较弱荧光为弱阳性,在瘢痕癌组织中呈较强荧光为强阳性;(2)在病理性瘢痕和瘢痕癌中未发现K-ras、H-ras、N-ras第12、13位密码子突变;(3)MAPK和Cyclin D1的蛋白及mRNA在正常皮肤表皮均呈阴性或弱阳性,在皮肤病理性瘢痕上皮中呈弱阳性,在瘢痕癌组织中呈强阳性。瘢痕癌组表达水平(阳性面积)、表达强度(平均光密度)与正常皮肤、病理性瘢痕组比较,差异均有统计学意义(P<0.01),正常皮肤组与病理性瘢痕组比较,差异无统计学意义(P>0.05)。结论 (1)Ras、MAPK、Cyclin D1基因的高表达与瘢痕癌的发生密切相关,各种基因共同发挥了协同作用;(2)K-ras、H-ras、N-ras第12、13位密码子突变与瘢痕癌的发生无相关性。     

H-ras、c-erbB-2、p53蛋白在乳腺增生病中的表达及意义

我们通过检测Ｈｒａｓ、ｃｅｒｂＢ２、ｐ５３蛋白在乳腺增生病和乳腺癌中的表达，探讨不同增生程度的乳腺增生病与乳腺癌的关系。一、材料与方法１．标本：取自成都军区昆明总医院病理科１９９０～１９９６年的存档蜡块，包括导管上皮轻度增生、导管上皮中重度增生、导管上皮不典型增生和导管癌各６０例，共２４０例。标本均经４％甲醛固定，逐级乙醇脱水和常规石蜡包埋。２．乳腺增生病的分级标准：参照Ｐａｇｅ分类［１，２］、李维华和纪小龙［３］提出的标准将导管增生分为轻度增生、中重度增生和不典型增生３级。轻度增生指导管…     

聚合酶链反应检测胃癌组织K—ras基因突变60例分析

目的：了解胃癌Ｋ－ｒａｓ基因突变情况及意义。方法；病理确诊的胃癌组织６０例，蛋白酶Ｋ法裂解，ＰＣＲ法扩增Ｋ－ｒａｓ基因密码子１２。结果：胃癌组织Ｋ－ｒａｓ基因突变率８．３３％，但胃上１／３部位的胃癌组织中，Ｋ－ｒａｓ基因突变率（２１．０５％）比胃其余部位显著高（Ｐ＜０．０５）。结论：Ｋ－ｒａｓ基因突变与否对胃癌的诊治、预后未见明显相关，突变率与病灶所处部位相关可能反映癌变的机理不同。     

K-ras基因突变与结直肠癌生物学行为的关系

目的: 观察结直肠癌组织中k-ras基因突变情况,探讨k-ras基因突变与结直肠癌生物学行为的关系。方法: 采用实时荧光定量PCR法检测123例结直肠癌组织中k-ras基因1号外显子12、13密码子突变情况,结合其临床病理资料分析。结果: 123例结直肠癌组织中k-ras基因突变者53例(40.8%),其中12密码子突变42例(34.1%),13密码子突变者11例(8.9%)。基因突变率与肿瘤大小、肿瘤侵润深度、分化程度无明显相关性,与淋巴结转移、肝脏转移及TNM分期有相关性（P<0.05）。淋巴结转移多者k-ras基因突变率高,有肝脏转移者基因突变率高,TNM分期越晚基因突变率越高。结论: K-ras基因突变可能在结直肠癌的发生、发展中起重要作用,而且与淋巴结转移和肝脏转移有密切相关,可作为判断结直肠癌恶性程度的一个分子生物学指标。     

乳腺癌发展过程中的bcl-2甲基化及其与蛋白表达的关系

目的建立bcl-2基因甲基化特异的PCR（MSP）检测方法,检测乳腺癌发展过程中的bcl-2甲基化．并探讨bcl-2甲基化与蛋白表达的关系。方法设计bcl-2基因MSP引物,采用MSP方法检测30例正常、25例不典型增生（癌前病变）、40例腋窝淋巴结阴性（癌症初期）和45例腋窝淋巴结阳性（癌症后期）乳腺组织的bcl-2基因5’端启动子CpG岛甲基化状态。另外采用免疫组织化学S-P法检测bcl-2的蛋白表达。结果正常、不典型增生、淋巴结阴性和淋巴结阳性乳腺组织的bcl-2甲基化率分别为10．0％、32．0％、40．0％和53．3％。乳腺癌发展过程中的bcl-2甲基化率渐增（P〈0．01）,bcl-2蛋白表达率渐减（P〈0．01）。不典型增生较正常乳腺组织的bcl-2甲基化水平显著提高（P〈0．05）。在乳腺癌发展过程中的4个阶段,bcl-2甲基化与蛋白表达两者之间均呈显著负相关性（P〈0．01）。结论该研究建立了bcl-2基因MSP检测方法,bcl-2基因MSP引物的设计是合理的。bcl-2启动子甲基化可能成为乳腺癌前病变的分子指标之一。在乳腺癌发展过程中,bcl-25’端调控区CpG岛甲基化可能是下调bcl-2表达的因素。     

Cyclin D1 expression in ductal carcinoma in situ, atypical ductal hyperplasia and usual ductal hyperplasia: an immunohistochemical study

The cell cycle regulatory gene, Cyclin D1, plays a critical role in the growth and progression of several types of human cancer, including breast cancer. Immunohistochemical study of Cyclin D1 expression has been extensively reported in invasive ductal carcinoma (IDC). In contrast, there have been few reports concerning Cyclin D1 expression in ductal carcinoma in situ (DCIS) and their positive rates are variable. The differences in the reported frequency may be largely due to the differences in antibodies used, immunohistochemical methods and the positive cut-off point. However, we speculated that the strictness of diagnosis of DCIS might be somewhat responsible for these differences in frequency. Therefore, we selected cases of DCIS by carefully eliminating cases of predominantly intraductal carcinoma (PIC). Moreover, to clarify whether Cyclin D1 expression is involved in multistep carcinogenesis or the progression of human breast cancer, we immunohistochemically investigated Cyclin D1 expression in 57 DCIS, 10 atypical ductal hyperplasia (ADH), 70 usual ductal hyperplasia (UDH), 44 PIC and 92 IDC. Cyclin D1 expression was detected in 41 DCIS cases (72%), 22 PIC cases (50%) and 40 IDC cases (43%). No expression of Cyclin D1 was observed in either ADH or UDH. There were no significant correlations between Cyclin D1 expression and histological grade or estrogen receptor expression in DCIS. These results suggest that Cyclin D1 expression may play an important role in the early stages of carcinogenesis, and that immunohistochemical detection of Cyclin D1 expression may be helpful in differentiating low-grade DCIS from ADH.     

Role of ras mutation in the progression of thyroid carcinoma of follicular epithelial origin

The histological differentiation of thyroid carcinoma is known to correlate with prognosis. Ras oncogene mutations, which have been identified in various human cancers, have been suspected playing an important role in carcinogenesis and tumor progression. The purpose of this study was to clarify the mechanism of thyroid tumor progression, focusing on ras oncogenes. We examined ras mutations using nested polymerase chain reaction (PCR) and direct sequencing methods. The ras oncogene product was also examined immunohistochemically. Our results indicated that the incidence of ras mutations correlated with the histological differentiation of thyroid cancer. Three poorly differentiated carcinomas showed a higher rate of ras mutations than did 17 well-differentiated counterparts. Hot spots were not identified except for a relative accumulation of the N-ras gene at codon 61. There was a correlation between the immunoreactivity of the ras oncogene product and ras mutation, although the immunoreactivity of ras-p21 did not correlate with the histological differentiation. Mutation of the ras gene seemed to be one of the important events in the progression from well-differentiated carcinoma to poorly differentiated thyroid carcinoma.     

KRAS gene mutations in lung cancer: Particulars established and issues unresolved

Lung cancer, like other cancers, is considered to develop through the accumulation of genetic alterations. Mutation of the KRAS gene is one of the most important events in carcinogenesis of the lung. The KRAS gene, belonging to the RAS gene family, encodes a membrane‐bound 21‐kd guanosine triphosphate (GTP)‐binding protein. Single point mutations in this protein result in continuous activation to transmit excessive signals, promoting a variety of biological events. In lung cancers, the mutations concentrate at codon 12 and mostly affect adenocarcinomas (ADCs). They also affect atypical adenomatous hyperplasia, the precursor of ADCs. Therefore, mutation of the KRAS gene is suggested to confer a growth advantage to airway epithelial cells enabling them to expand clonally early in the development of ADCs. The mutation is also a reliable marker of an unfavorable response to certain molecular‐targeting therapies. Furthermore, patients with ADCs affected by mutations have been reported to exhibit a significantly higher risk of postoperative disease recurrence. Thus, the significance of KRAS gene mutations has been investigated extensively. However, not all the details emerged. In this review, particulars that have been established are introduced, and important issues remaining to be resolved are discussed, with special reference to carcinogenesis of the lung.     

High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype

Zardawi S J, Zardawi I, McNeil C M, Millar E K A, McLeod D, Morey A L, Crea P, Murphy N C, Pinese M, Lopez‐Knowles E, Oakes S R, Ormandy C J, Qiu M R, Hamilton A, Spillane A, Soon Lee C, Sutherland R L, Musgrove E A & O’Toole S A
(2010) Histopathology 56, 286–296 High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype Aims: Activation of Notch signalling results in hyperplasia and tumorigenesis in murine mammary epithelium. However, there is little information regarding the expression of Notch1 in premalignant lesions and early breast cancer. We investigated expression of Notch1 in breast cancer development and its association with molecular subtypes. Methods and results: Immunohistochemical expression of Notch1 was determined in a murine model of mammary carcinogenesis and in breast tissue from two cohorts of breast cancer patients, the first (n = 222) comprising a histological progression series and the second an outcome series of 228 patients with operable invasive ductal carcinoma. Enhanced expression of Notch1 protein was an early event in both murine and human breast cancer development with progressive increases in expression with the development of hyperplasia and malignancy. High Notch1 was not prognostic in the outcome cohort. There was, however, a highly significant association of high Notch1 protein with the HER‐2 molecular subtype of breast cancer (P = 0.008). Conclusions: These data demonstrate that aberrant Notch regulation is an early event in mammary carcinogenesis and is associated with the HER‐2 molecular subtype of breast cancer, and suggest the Notch signalling pathway may be a potential therapeutic target worthy of further investigation.