霍奇金淋巴瘤IκBα基因第5外显子突变初步分析

目的分析单个霍奇金淋巴瘤细胞IκBα基因第5外显子的碱基序列,探讨IκBα基因突变与霍奇金淋巴瘤的关系。方法用免疫组织化学EnVision法检测7例霍奇金淋巴瘤CD30抗原,采用激光显微切割技术捕获CD30阳性表达的单个霍奇金淋巴瘤细胞和其周围反应性增生的淋巴细胞,以这些标本的基因组DNA为模板,用半巢式聚合酶链反应(PCR)方法扩增IκBα基因的第5外显子,PCR产物经纯化后直接测序。结果分析测序图发现3例霍奇金淋巴瘤细胞IκBα第5外显子出现点突变,均形成TGA终止密码子。结论霍奇金淋巴瘤中IκBα基因第5外显子出现点突变,可能是霍奇金淋巴瘤恶性转化过程中除EB病毒感染外另一重要分子机制。     

肝细胞癌病人中p53基因突变状况探讨

目的 了解肿瘤抑制基因p53在肝细胞癌中的突变情况,探讨乙型肝炎病毒（HBV）感染与p53基因突变之间的关系。方法 提取50例有乙型肝炎病毒感染史肝癌患者手术样本中的DNA,用聚合酶链式反应（PCR）扩增5－9外显子,作单链构象多态性（SSCP）分析。结果 p53基因突变率超过26％,突变主要分布于5－8外显子,5、6、7、8外显子分别有3、3、4、3例,另仍4个可疑突变。结论 p53基因突变可能是肝细胞癌的病因之一,而乙型肝炎病毒感染在中国肝癌患者p53基因突变中可能起到比较重要的作用。     

p53基因第8外显子在喉癌中的缺失和点突变

目的 探讨抑癌基因p53第8外显子突变在喉鳞癌分子发病机理中所起的作用。方法 应用聚合酶链反应－单链构象多态性（PCR－SSCP）银染技术和DNA直接测序法,检测喉鳞癌新鲜组织中抑癌基因p53基因第8外显子的突变情况。结果 60例喉鳞癌组织中,15例发生迁移率异常的SSCP区带。在SSCP阳性样本中随机抽出4例进行测序分析,发现两个标本在288位密码子缺失一个A,两个标本在292位密码子上缺失一个A。285、287密码子上共发生3次G→T的颠换,286密码子第3位碱基发生A→T的颠换。结论 喉鳞癌p53基因点突变与碱基缺失常常同时并存。p53基因第8外显子突变在喉癌的发生中可能起着重要作用。     

p53基因在内分泌肿瘤形成中的作用:用PCR单链构象多态性检测突变

作者使用PCR-SSCP技术检测了134例原发性内分泌肿瘤和6株人内分泌肿瘤衍生细胞株中p53基因的突变。通过PCR扩增和SSCP技术检测p53基因,发现p53基因突变区域为外显子5到10,它包含了人肿瘤中大多数p53基因的突变区域。在所检的三株细胞株中没有移动的带与对照组类似,表明     

冠心病新致病基因MEF2A第一、第八外显子突变的检测

目的研究冠心病新致病基因MEF2A在中国人群突变情况。方法利用聚合酶链反应-单链构象多态性(polym erase chain reaction-single strand conform ation polymorph ism,PCR-SSCP)和DNA测序技术对156例冠心病(CAD)患者第1外显子及第8外显子进行基因突变检测。结果MEF2A基因第1外显子区域6例患者SSCP泳动异常,第8外显子区域7例患者SSCP泳动异常,但DNA直接测序未发现MEF2A基因第1、8外显子区域基因突变。结论冠心病患者在MEF2A基因第1、8外显子未发现新的突变,PCR-SSCP结果与DNA测序结果并非平行关系,SSCP同样存在假阳性。     

免疫组织化学-激光显微切割-聚合酶链反应技术在胃癌石蜡切片中检测p53突变的应用

目的 探索在石蜡切片中应用免疫组织化学-激光显微切割-聚合酶链反应(PCR)技术,检测进展期胃癌p53蛋白表达阴性或阳性的p53基因外显子5～8的突变情况。方法 对41例进展期胃癌标本进行p53蛋白的免疫组织化学标记,再用激光显微切割(LMD)技术切取p53表达一致的癌组织及远离癌灶的正常胃黏膜腺体。经消化后直接进行p53基因外显子5～8的PCR扩增、单链构象多态性分析及直接测序。结果 41例进展期胃癌标本均扩增出特异的目的条带。其中有11例p53蛋白表达阳性,15例p53基因检测到突变。蛋白表达阳性者中有8例突变(8／11);表达阴性者中有7例突变(7／30,23．3％)。p53蛋白表达和p53基因突变具显著相关(P=0．004)。结论 免疫组织化学-LMD-PCR技术应用于石蜡切片可获得满意的结果,此技术可将细胞特定基因的结构状态和表达水平很好地结合起来检测分析。     

霍奇金淋巴瘤中ΙκΒα基因突变的研究

目的分析霍奇金淋巴瘤细胞中IκBα基因突变的特点、可能造成的蛋白结构异常,并探讨其分子病理学意义。方法应用激光显微切割技术分离16例霍奇金淋巴瘤切片中的HRS细胞,并对IκBα基因进行半巢式PCR扩增及DNA直接测序,检测HRS细胞IκBα基因6个外显子突变。结果16例标本中6例发现IκBα基因突变,突变率为37.5%,突变分别位于1、4、5和6外显子上。其中2例第五外显子出现点突变,形成TGA终止密码子;1例出现第四外显子单碱基缺失突变,形成框移,可造成IκBα蛋白羧基端缺失。霍奇金淋巴瘤背景中反应性淋巴细胞没有发现IκBα突变。结论霍奇金淋巴瘤IκBα基因突变率为37.5%,高于文献报道(10%～25%)。突变可能造成IκBα蛋白的结构异常,通常造成羧基端截短改变。背景中反应性增生的淋巴细胞未发现IκBα基因突变,表明IκBα的基因突变是HRS细胞的特征,并可能在霍奇金淋巴瘤发病机制中起重要作用。     

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。     

p53基因突变与食管贲门多源癌的相关性

目的 探讨食管贲门双源癌组织p53基因突变的特征及其与p53蛋白表达的关系。方法 采用显微切割、PCR、DNA测序和免疫组化ABC法，分析4例食管贲门多源癌p53基因突变和蛋白表达状况。结果 4例食管贲门双源癌中，1例食管鳞癌和贲门腺癌同时发生p53基因突变，并均发生第7外显子231、232密码子的点突变和225、232～233、234密码子的缺失/插入改变。食管鳞癌p53基因突变率为50％(2／4)，贲门腺癌为75％(3／4)。所有检测到的突变均发生在第7和8外显子。食管癌和贲门癌分别有1例p53蛋白阴性表达的患者检测到p53基因突变，食管癌中1例p53蛋白阳性表达患者未检测到基因突变。结论 同一个体食管鳞癌和贲门腺癌组织同时发生相似位点的p53基因突变，提示二者具有相似的发病因素和分子机制，为进一步揭示林州地区相似的食管／贲门癌区域分布特征提供了重要的理论依据和线索。     

大肠癌K-ras和p53基因序列分析

目的 研究大肠癌变与K－ras和p53基因突变的关系。方法 采用PCR－DNA双链四色荧光单道测序方法对31例大肠癌组织、13例大肠息肉伴不典型增生、11例大肠癌术后并发的腹水标本进行K－ras基因外显子1、2和p53基因外显子5、6、7、8测序分析。结果 31例大肠癌组织中发现K－ras基因突变7例，p53基因突变11例，阳性率分别为22.58%和35．48％;13例大肠息肉伴不典型增生组织中发现p53基因突变2例；11例术后腹水标本中发现K-ras基因突变1例和p53基因突变2例，且突变与患者原包埋组织结果一致。但未发现这两个基因同时突变的病例。结论 大肠组织癌变与这两个基因突变密切相关，K－ras和p53基因突变可能为大肠癌变的早期事件；DNA测序方法是研究癌基因突变的最精确可靠的方法。     

p53 protein expression in non-Hodgkin's lymphomas is infrequently related to p53 gene mutations

Accumulation of the p53 protein has been found In several types of lymphomas. However, p53 gene mutations have been infrequently demonstrated in some specific types of lymphomas. In the present study, a correlation between p53 immunoreactivity and p53 gene mutations in a large panel of non-Hodgkin's lymphoma (NHL) cases is attempted. A panel of 202 cases of NHL was evaluated by immuno-hlstochemical staining for p53 protein. All cases that were immunohistochemically positive for p53 protein were analyzed by the polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) method to identify mutations within the pS3 gene. In order to confirm the mutation, sequencing of PCR-amplified p53 gene segments was performed. Overexpression of p53 protein was found in 59 of the 202 cases of NHL, but only four of these 59 cases showed a shift on SSCP analysis, and point mutations were detected in three of them by the subsequent sequencing. p53 Immunoreactivity was generally greater in high-grade lymphoma. The results of this study suggest that Immunohistochemlcal reactivity for p53 protein is not a reliable Indicator of the presence of their structural alterations of p53 gene exons 4–9 in NHL.     

Mutation of the <Emphasis Type="Italic">p53</Emphasis> tumour suppressor gene and overexpression of its protein in 62 Japanese non-Hodgkin’s lymphomas

To clarify whether p53 mutation could be involved in the pathogenesis of various subtypes of lymphoma, we investigated 62 Japanese cases of non-Hodgkin’s lymphomas (NHLs) for p53 gene mutations and their relationship with the expression of p53 protein. Mutations in exons 5–9 of the p53 gene were screened for using the non-isotopic RNase cleavage assay (NIRCA) and confirmed by direct sequencing, followed by immunohistochemical analysis for p53 protein. Missense and/or nonsense mutations of p53 were detected in 3 (10.7%) of 28 diffuse large B-cell lymphomas (DLBLs) and 2 (15.4%) of 13 T-cell NHLs (15.4%). A single missense mutation at codon 157 (Val to Phe) in exon 5 and at codon 273 (Arg to Pro) in exon 8 was found respectively in 2 DLBLs and in one peripheral T-cell lymphoma (unspecified). In these 3 cases harbouring a missense mutation, overexpression of p53 protein was observed in more than 80% of tumour cells. Double transversion mutations comprising of a missense mutation at codon 167 (Gln to His) in exon 5 and a nonsense mutation at codon 183 (Ser to stop codon) in exon 5 were detected in one DLBL that had apparently transformed from follicular lymphoma and in one advanced adult T-cell lymphoma (ATL). In these two cases harbouring p53 nonsense mutation, no cells positive for p53 protein immunostaining were detected, as well as lymphomas without p53 mutation.     

中国人Burkitt淋巴瘤中ID3、TCF3及MYC基因突变分析

目的观察中国人Burkitt淋巴瘤中ID3、TCF3和MYC基因突变情况,并探讨其意义。方法提取32例Burkitt淋巴瘤组织DNA,经PCR扩增部分DNA片段,并采用Sanger测序法对PCR产物进行DNA序列测定。结果 ID3、TCF3的突变率分别为35.5%(11/31)、18.8%(6/32)。MYC的突变率为50%,MYC exon1的突变率为3.3%(1/30),MYC exon2的突变率为50%(15/30),MYC exon3的突变率为7.7%(2/26)。结论通过Sanger测序证实在中国人Burkitt淋巴瘤组织中存在ID3、TCF3、MYC基因的重现性突变。3例中发现新的TCF3基因突变位点c.2202GC p.L569V。2例发现新的MYC基因突变位点c.1070AG p.G182D。     

Gene mutations in lymphoproliferative disorders of T and NK/T cell phenotypes developing in renal transplant patients

Post-transplantation lymphoproliferative disorder (PT-LPD) is characterized by lymphoid proliferation after organ or bone marrow transplantation. In Western countries, most cases of PT-LPD are B-cell-derived and Epstein-Barr virus-associated, in which alterations of c-myc, p53, and N-ras genes might play a role in disease progression. In Japan, PT-LPD of T- and NK/T-cell types are not uncommon in renal transplant patients. Mutations of p53 (exons 4 through 8), K-ras (exons 1 and 2), c-kit (exons 11 and 17), and beta-catenin genes (exon 3) in 12 cases of these diseases were analyzed by PCR single strand conformation polymorphism and then by direct sequencing. p53 gene mutations were detected in 5 of 5 cases of peripheral T-cell lymphoma, 3 (60%) of 5 cases of adult T-cell leukemia/lymphoma, and 1 of 2 cases of NK/T cell lymphoma. Twenty-five percent of T and NK/T cell lymphomas showed K-ras mutations. Mutations of c-kit and beta-catenin genes were found in 33% of cases. Among a total of 42 substitution mutations, 40 were transitions with involvement of CpG sites in 20 to 30% of cases. Most cases had at least one mutation that changed an amino acid, which might have provided the selection pressure for expansion. These findings suggested that p53 gene mutations might play a central role in development of peripheral T-cell lymphoma including adult T-cell leukemia/lymphoma in renal transplant patients.     

Missense mutation with/without nonsense mutation of the p53 gene is associated with large cell morphology in human malignant lymphoma

Mutations in p53 gene exons 5-9 were studied in 44 non-Hodgkin's lymphomas (NHL) consisting of 35 B-NHL and 9 T-NHL. Missense mutations were found in two diffuse large B-cell lymphomas (DLBL) and one peripheral T-cell lymphoma (unspecified). Double transversion missense and nonsense mutations were detected in one DLBL and one adult T-cell leukemia/lymphoma. Silent mutations were found in two DLBL. Detailed histomorphological study showed that cases harboring p53 missense mutation with/without nonsense mutation tended to have larger nuclei with much more prominent nucleoli. Cytomorphometric analysis was therefore conducted by measuring the gross area of 100 lymphoma cell nuclei in 44 cases and the results were compared between lymphomas harboring p53 missense mutation with/without nonsense mutation and lymphomas harboring p53 silent mutation or lacking mutation. It was found that the lymphomas harboring p53 missense mutation with/without nonsense mutation had a highly significantly larger nuclear gross area than lymphomas with silent p53 mutation or lacking mutation (two-sample t-test, P < 0.00001; Exact Wilcoxon rank-sum test, P < 0.00001). This result suggests that p53 mutation might induce enlargement of neoplastic cell nuclei by some molecular mechanism.     

Midgut Carcinoids and Solid Carcinomas of the Intestine: Differences in Endocrine Markers and p53 Mutations

Fifteen midgut carcinoid tumors and 5 solid carcinomas of the intestine with carcinoid-like morphological features were evaluated histochemically and immunohistochemically with respect to various endocrine markers and expression of mutant p53 protein. Direct sequencing of the p53 gene after PCR amplification was carried out on microdissected cells from all tumors. All investigated carcinoid tumors showed chromogranin and argentaffin reaction, but lacked nuclear immunostaining with p53 antibodies. In 14 immunohistochemically negative midgut carcinoid tumors, no mutations were identified. One carcinoid tumor devoid of p53 staining was, however, found to contain mutation in exon 6 of the p53 gene. In contrast, the solid carcinomas were essentially chromogranin negative but all displayed clearly positive p53 staining in a variable number of cell nuclei. Sequence analysis of exons 5–8 of the p53 gene in the 5 carcinomas showed mutations in exons 6 and 7 in 2 tumors and in exon 8 in 2 other tumors, while no mutation was detected in the fifth tumor. The carcinoid tumors and the solid carcinomas of the small intestine are thought to derive histogenetically from endocrine cells and enterocytes, respectively. The present results substantiate that divergent mechanisms operate in the development of the two tumor types.     

大肠癌组织p14ARF与p53基因变异研究

目的：观察大肠癌组织p14ARF基因遗 传和表观遗传变化及p53基因突变状况，探讨两者改变的关系及p14ARF-p53通路功能 破坏在大肠癌发生中的作用。方法：应用PCR、直接测序、甲基化特异PC R和RT-PCR分别检测56例原发性大肠癌及相应癌旁正常组织p14ARF基因纯合性缺失、 突变、5′CpG岛甲基化、mRNA表达及p53基因突变状况。结果：①大肠 癌组织p14ARF总变异率为27% (15/56)，其中1例纯合性缺失，14例5′CpG岛甲基化,未见突变发生。②15例p14ARF基因变异的大肠癌组织其相应mRNA表达阴性（13例）或 低水平表达（2例），41例未发生基因变异的大肠癌组织和所有癌旁正常组织p14ARF mRNA均显示明显表达。③大肠癌组织p53突变率为48% (27/56)。④56例大肠癌组织中，12例 仅发生p14ARF变异，24例仅显示p53突变，3例同时有p14ARF 5′CpG岛甲基化 和p53突变，17例p14ARF和p53均无变异，p14ARF-p53通路总变异率为70%（39/ 56），p14ARF 5′CpG岛高甲基化与野生型p53状态有关（P＜0.05）。⑤低分化 腺癌组p14ARF变异率（44%，7/16）明显高于高中分化腺癌组（20%，8/40）（P＜ 0.05），但两组间p53突变率无显著差异（P＞0.05）。结论:①大肠癌p14ARF基因5′ CpG岛高甲基化是其表达失活的主要机制；②大肠癌p14 ARF基因高甲基化与p53基因突变可能是相互独立的事件，两者的失活可能各自出现于不同 的大肠癌亚组中；③p14ARF高甲基化或p53突变引起的p14ARF-p53通路功能 破坏在大肠癌的发生中具有重要作用。     

BCL10 in malignant lymphomas--an evaluation using fluorescence in situ hybridization.

BCL10 is a tumour suppressor gene originally cloned from a t(1;14)(p22;q32) breakpoint in a case of mucosa-associated lymphoid tissue (MALT) lymphoma. Translocations involving this gene, though uncommon, are sometimes encountered in MALT lymphomas. This gene is thought to play an important role in the development of malignant lymphomas. Fluorescence in situ hybridization (FISH) was therefore undertaken on 22 cases of malignant lymphoma of varying histology to establish the incidence of rearrangements involving the BCL10 gene. Initially, one case with a novel t(1;2)(p22;p12) translocation involving the BCL10 gene was identified, in a marginal zone lymphoma of the MALT type, and was reported elsewhere. Seven other cases were subsequently identified with abnormalities in the 1p region, including a translocation with a breakpoint in the 1p22 region in a case of lymphoblastic lymphoma. However, none of these involved the BCL10 gene. Mutation analysis of BCL10 was then performed on 57 cases of malignant lymphoma, including 17 MALT lymphomas, by single-strand conformational polymorphism (SSCP) analysis of tumour DNA. Tissue was obtained for mutation analysis for 12 of the 22 cases analysed by FISH. Selected cases with SSCP band shifts were further studied by direct sequencing. Polymorphisms were identified in eight cases, but no mutations of pathogenic significance were identified. Further RT-PCR and mutation analysis was performed on cDNAs from 12 cases (four MALT, seven diffuse large B-cell lymphoma, one Hodgkin's disease) in which DNA analysis had already been completed. This included the MALT lymphoma with the t(1;2)(p22;p12) rearrangement. Again, no mutations were identified in the coding sequence. This study confirms that rearrangements of the BCL10 gene are uncommon in lymphoma (1/22) and may be limited tothe MALT subtype of non-Hodgkin's lymphomas. It was also found that breakpoints or rearrangements in the 1p22 region do not necessarily involve the BCL10 gene. Moreover, the absence of mutations at both the DNA (0/60) and the mRNA (0/12) level indicates that this gene is not frequently inactivated by mutation, in those tumours in which it is not involved in translocations. Our findings suggest that the BCL10 gene is unlikely to have a frequent or key role in general lymphomagenesis.     

Mutations of the p53 gene in nasal NK/T-cell lymphoma

Mutations of the p53 tumor suppressor gene are reported in various kinds of malignancies including lymphomas. However, p53 gene mutations in nasal NK/T-cell lymphoma have not been reported because most parts of tumors are necrotic and a small amount of living tumor tissues is available for the molecular study. Expression and mutations of the p53 gene were examined in the paraffin-embedded specimens of the nasal lesions from 42 Chinese (Beijing and Chengdu) and Japanese (Okinawa and Osaka) patients with nasal NK/T-cell lymphoma by the immunohistochemistry and single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified products followed by direct sequencing. Thirty single-nucleotide substitution mutations were observed in 20 of 42 cases (47.6%). Among the 30 mutations, 18 were missense (mainly G:C to A:T transitions), 9 were silent, and 1 was a nonsense mutation. The remaining 2 mutations involved intron 5 and exon 5 terminal points. Abnormal expression of the p53 protein was also observed in 19 of 42 (45.2%) cases. The incidence was significantly (4-fold) higher in the cases of Osaka than those in other areas, although the incidence of p53 mutations in the cases of Osaka was one-half to one-third of those in the other three areas. The results may suggest some racial, environmental, or lifestyle differences in the cause of nasal tumorigenesis.     

Molecular Genetic Alterations in Radiation-Induced Astrocytomas

Astrocytic tumors occasionally arise in the central nervous system following radiotherapy. It is not clear if these gliomas represent a unique molecular genetic subset. We identified nine cases in which an astrocytoma arose within ports of previous radiation therapy, with total doses ranging from 2400 to 5500 cGy. Irradiated primary lesions included craniopharyngioma, pituitary adenoma, Hodgkin's lymphoma, ependymoma, pineal neoplasm, rhabdomyosarcoma, and three cases of lymphoblastic malignancies. Patients ranged from 9 to 60 years of age and developed secondary tumors 5 to 23 years after radiotherapy. The 9 postradiation neoplasms presented as either anaplastic astrocytoma (3 cases) or glioblastoma multiforme (6 cases). Two of the latter contained malignant mesenchymal components. We performed DNA sequence analysis, differential polymerase chain reaction (PCR), and quantitative PCR on DNA from formalin-fixed, paraffin-embedded tumors to evaluate possible alterations of p53, PTEN, K-ras, EGFR, MTAP, and p16 (MTS1/CDKN2) genes. By quantitative PCR, we found EGFR gene amplification in 2 of 8 tumors. One of these demonstrated strong immunoreactivity for EGFR. Quantitative PCR showed chromosome 9p deletions including p16 tumor suppressor gene (2 of 7 tumors) and MTAP gene (3 of 7). Five of 9 tumors demonstrated diffuse nuclear immunoreactivity for p53 protein. Sequencing of the p53 gene in these 9 cases revealed a mutation in only one of these cases, a G-to-A substitution in codon 285 (exon 8). Somewhat unexpectedly, no mutations were identified in PTEN, a commonly altered tumor suppressor gene in de novo glioblastoma multiformes. Unlike some radiation-induced tumors, no activating point mutations of the K-ras proto-oncogene or base pair deletions of tumor suppressor genes were noted. These radiation-induced tumors are distinctive in their high histological grade at clinical presentation. The spectrum of molecular genetic alterations appears to be similar to that described in spontaneous high grade astrocytomas, especially those of the de novo type.