Replication failure,genome instability,and increased cancer susceptibility in mice with a point mutation in the DNA ligase I gene

DNA ligase I has a key role in DNA replication in the joining together of short replication intermediates.We used gene targeting to introduce a point mutation into the mouse DNA ligase I gene that was present in a human cancer patient with immunodeficiency and a cellular accumulation of DNA replication intermediates. Mutant mice grew more slowly and showed hematopoietic defects at critical stages at which the demands for DNA replication were highest. In the spleen and thymus of mutant mice, the accumulation of a sub-G1, but nonapoptotic, population was observed that we believe may represent cells with single-strand DNA breaks. In mutant bone marrow, occasional DNA replication failure was observed. The level of genome instability was significantly elevated in the spleens of DNA ligase I mutant mice and, because we have found no evidence for any DNA repair defect associated with DNA ligase I deficiency, we believe that this may result directly from the accumulation of replication intermediates. Mutant mice showed an increased incidence of spontaneous cancers with a diverse range of epithelial tumors, particularly cutaneous adnexal tumors that are rare in mice. The origin of the tumors from generalized genome instability, rather than the inactivation of one key control gene, should make DNA ligase I mutant mice a useful model to investigate the relationship between genome instability and cancer in humans.     

Whole genome analysis for liver metastasis gene signatures in colorectal cancer

Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. To understand this process, we investigated whether the gene expression profiling of matched colorectal carcinomas and liver metastases could reveal key molecular events involved in tumor progression and metastasis. We performed experiments using a cDNA microarray containing 17,104 genes with the following tissue samples: paired tissues of 25 normal colorectal mucosa, 27 primary colorectal tumors, 13 normal liver and 27 liver metastasis, and 20 primary colorectal tumors without liver metastasis. To remove the effect of normal cell contamination, we selected 4,583 organ-specific genes with a false discovery rate (FDR) of 0.0067% by comparing normal colon and liver tissues using significant analysis of microarray, and these genes were excluded from further analysis. We then identified and validated 46 liver metastasis-specific genes with an accuracy of 83.3% by comparing the expression of paired primary colorectal tumors and liver metastases using prediction analysis of microarray. The 46 selected genes contained several known oncogenes and 2 ESTs. To confirm that the results correlated with the microarray expression patterns, we performed RT-PCR with WNT5A and carbonic anhydrase II. Additionally, we observed that 21 of the 46 genes were differentially expressed (FDR=2.27%) in primary tumors with synchronous liver metastasis compared with primary tumors without liver metastasis. We scanned the human genome using a cDNA microarray and identified 46 genes that may play an important role in the progression of liver metastasis in CRC.     

Direct serum and tissue assay for EGFR mutation in non-small cell lung cancer by high-resolution melting analysis

Biological therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have noted promising outcomes for patients with non-small cell lung carcinoma (NSCLC), especially those with mutated EGFR. Tissue EGFR gene mutation testing can predict the benefit of taking a first-line EGFR-TKI, thus, allowing the physician to prescribe the most suitable therapy. Unfortunately, most lung cancer patients, especially NSCLC patients present with advanced disease that is surgically unresectable. The goal of this study was to develop high-resolution melting (HRM) assays to detect EGFR mutations in exons 18 to 21, compare their sensitivity and concordance to direct sequencing, and evaluate the feasibility and reliability of serum as a tissue alternate for routine EGFR mutation screening. EGFR mutations of 126 Formalin-Fixed Paraffin-Embedded (FFPE), 47 fresh frozen tissues and from 47 matched pre-operation serum specimens of NSCLC patients were screened by the HRM assays. EGFR mutations by HRM were confirmed through sequencing. We found 78 EGFR mutations in 70 FFPE tissues, 25 EGFR mutations in 24 fresh frozen tissues, with a mutation rate of 55.56% (70/126) and 51.06% (24/47), respectively. Most mutations were correctly identified by sequencing. EGFR mutations were detected in 22 serum samples from 24 tissue EGFR mutation-positive patients. The concordance rate between serum and tissue in EGFR mutation screening was 91.67%. We conclude that the HRM assay can provide convincing and valuable results both for serum and tissues samples, thus, it is suitable for routine serum EGFR mutation screening for NSCLC patients, especially those surgically unresectable.     

子宫内膜癌中EGFR蛋白的表达及EGFR基因突变

目的:研究表皮生长因子受体(epidermal growth factor receptor,EGFR)在子宫内膜癌组织中的表达及其基因突变情况。方法:取江西省肿瘤医院及南昌大学第二附属医院2007年1月至2011年4月期间的子宫内膜石蜡包埋组织104例,其中子宫内膜癌组织56例、非典型增生子宫内膜组织18例、正常子宫内膜组织30例,利用免疫组化检测上述各组织中EGFR蛋白的表达,PCR法扩增子宫内膜癌或正常子宫内膜组织中EGFR基因外显子19、21序列并测序检测其突变情况。结果:子宫内膜癌组织中EGFR的阳性表达率高于正常子宫内膜[73.2%(41/56)vs 30.0%(9/30),P<0.01]及非典型增生子宫内膜[73.2%(41/56)vs 44.4%(8/18),P<0.05]。进一步分析表明:G3级子宫内膜癌中EGFR的阳性表达率明显高于G1级[81.8%(9/11)vs 66.7%(12/18),P<0.01]及G2级[81.8%(9/11)vs 74.1%(20/27),P<0.05],在肌层浸润>1/2组中的阳性表达率也明显高于肌层浸润≤1/2组[86.8%(33/38)vs 44.4%(8/18),P<0.01]。但EGFR的表达与子宫内膜癌的FIGO分期、有无淋巴结转移无明显相关性。发现1例子宫内膜癌组织中,EGFR基因外显子19有G2281A突变,而外显子21无突变。结论:EGFR的阳性表达率与子宫内膜癌的组织学分级及肌层浸润深度有关,子宫内膜癌组中存在EGFR外显子19的G2281A点突变。     

KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis

Background:

The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.

Methods:

After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.

Results:

HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan–Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.

Conclusions:

HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients.     

一先天性头皮单纯少毛症家系CDSN基因突变分析

目的:对一个先天性头皮单纯少毛症家系编码角化桥粒素的CDSN基因进行突变分析,旨在寻找致病原因。方法:收集家系中4例患者及正常家系成员的详细临床资料和外周血样本,提取基因组DNA,采用PCR技术扩增CDSN基因的2个外显子,用直接双向测序、BLAST比对进行突变分析。结果:在CDSN基因中发现了7个变异序列,其中4个已被报道为多态,3个为新发现的突变。所有序列变异均存在于患者的正常亲属中,与疾病表型无共分离现象。结论:排除了CDSN外显子突变导致该家系先天性头皮单纯少毛症的可能性。     

The mutation analysis for CDSN gene in a Chinese family with hypotrichosis simplex of the scalp

目的:对一个先天性头皮单纯少毛症家系编码角化桥粒素的CDSN基因进行突变分析,旨在寻找致病原因.方法:收集家系中4例患者及正常家系成员的详细临床资料和外周血样本,提取基因组DNA,采用PCR技术扩增CDSN基因的2个外显子,用直接双向测序、BLAST比对进行突变分析.结果:在CDSN基因中发现了7个变异序列,其中4个已被报道为多态,3个为新发现的突变.所有序列变异均存在于患者的正常亲属中,与疾病表型无共分离现象.结论:排除了CDSN外显子突变导致该家系先天性头皮单纯少毛症的可能性.     

子宫内膜癌p16基因突变及蛋白表达异常的分子学探讨

目的　研究 p16基因的突变及蛋白表达异常在人子宫内膜癌发生中的作用。方法　应用 PCR- SSCP、DNA测序及免疫组化对 4 1例子宫内膜癌中 p16基因第二外显子进行检测 ,研究 p16基因的蛋白表达情况。结果　4 1例子宫内膜癌中 12例存在 p16基因第二外显子 5 2 2 bp的纯合缺失 ,10例存在点突变 (二个突变位点相同 ) ,突变位点之一为第 12 6密码子碱基 GTC→ AAT(错义突变 ) ,另一突变点为 12 7密码子 GCA→ GCG(同义突变 )。缺失及突变共占 5 3.7%。 12例第二外显子缺失标本全部呈现 p16蛋白表达阴性 ,10例点突变标本有 9例出现 p16蛋白表达积分下降呈弱阳性 ,p16蛋白表达异常占 5 6 .0 %。结论　 p16基因的突变及蛋白表达异常与子宫内膜癌的发生有明显相关性     

肝细胞肝癌中T细胞转录因子4基因表达及突变

目的 :研究人T细胞转录因子 4(hTcf 4 )基因在肝细胞肝癌中表达及突变和肝癌发生发展的关系。方法 :用逆转录 -聚合酶链反应 (RT PCR)检测了 32例肝癌组织及癌旁组织中hTcf 4基因表达 ,并用聚合酶链反应 -单链构像多态性分析 (PCR SSCP)方法检测了肝癌组织中hTcf 4外显子 1、4、9、15的突变情况。结果 :hTcf 4mRNA在癌组织及癌旁组织中表达率分别为 90 .6 %和 71.9%。且癌组织中表达水平明显高于癌旁组织 [(0 .71±0 .13)vs (0 .2 9± 0 .0 5 ) ,P <0 .0 0 1]。同时 ,此基因在包膜不完整及有转移的癌组织表达增高更为显著。 32例肝癌中仅检测到 2例hTcf 4基因外显子 15突变 ,突变率为 6 .2 5 %。结论 :hTcf 4基因在肝细胞肝癌 ,尤其伴转移肝癌组织中表达增高 ,并与肝癌侵袭转移有关 ,而该基因突变与肝癌关系不密切。     

Gene expression analysis and immunohistochemistry in evaluation of cancer of unknown primary: time for a patient-centered approach

Molecular medicine is rapidly changing the diagnosis and management of cancer of unknown primary. The science, business, and economics of the genomic revolution have moved at such a pace that coordinating practical application of all available tools, such as gene expression analysis and immunohistochemistry, often seems to clash. In fact, very little work has been done to actively coordinate use of these techniques, each of which can be very resource-intensive. The Institute of Medicine proposed the STEEEP principles, a basic set of guidelines that maintain that the best patient care is safe, timely, effective, efficient, equitable, and patient-centered. Application of these principles will help lead to a better understanding of the most appropriate use of modern diagnostic modalities.     

Annual screening strategies in BRCA1 and BRCA2 gene mutation carriers: a comparative effectiveness analysis

BACKGROUND:

Although breast cancer screening with mammography and magnetic resonance imaging (MRI) is recommended for breast cancer‐susceptibility gene (BRCA) mutation carriers, there is no current consensus on the optimal screening regimen.

METHODS:

The authors used a computer simulation model to compare 6 annual screening strategies (film mammography [FM], digital mammography [DM], FM and magnetic resonance imaging [MRI] or DM and MRI contemporaneously, and alternating FM/MRI or DM/MRI at 6‐month intervals) beginning at ages 25 years, 30 years, 35 years, and 40 years, and 2 strategies of annual MRI with delayed alternating DM/FM versus clinical surveillance alone. Strategies were evaluated without and with mammography‐induced breast cancer risk using 2 models of excess relative risk. Input parameters were obtained from the medical literature, publicly available databases, and calibration.

RESULTS:

Without radiation risk effects, alternating DM/MRI starting at age 25 years provided the highest life expectancy (BRCA1, 72.52 years, BRCA2, 77.63 years). When radiation risk was included, a small proportion of diagnosed cancers was attributable to radiation exposure (BRCA1, <2%; BRCA2, <4%). With radiation risk, alternating DM/MRI at age 25 years or annual MRI at age 25 years/delayed alternating DM at age 30 years was the most effective, depending on the radiation risk model used. Alternating DM/MRI starting at age 25 years also produced the highest number of false‐positive screens per woman (BRCA1, 4.5 BRCA2, 8.1).

CONCLUSIONS:

Annual MRI at age 25 years/delayed alternating DM at age 30 years is probably the most effective screening strategy in BRCA mutation carriers. Screening benefits, associated risks, and personal acceptance of false‐positive results should be considered in choosing the optimal screening strategy for individual women. Cancer 2012. © 2011 American Cancer Society.     

核糖核酸酶抑制因子基因在人乳腺癌中的表达及突变分析

目的　通过核糖核酸酶抑制因子 (ribonucleaseinhibitor,RI)基因在人乳腺癌中的表达及其是否突变的分析 ,进一步探讨RI与肿瘤细胞生长的关系。方法　采用Westernblotting方法检测人乳腺癌组织中RI的表达量 ;采用SSCP的方法分析肿瘤细胞RI基因中是否有突变。结果　人乳腺癌组织中RI的表达明显低于同体癌旁正常组织 ,但用MboI酶切及SSCP分析未发现人乳腺癌组织中RI基因有突变。结论　乳腺癌的生长可能与RI表达的下降有关 ,从而可能使癌组织周边新血管的形成未受到有效抑制的结果。     

子宫内膜癌组织中PTEN基因突变及蛋白表达的检测

背景与目的:肿瘤抑制基因———第10染色体同源丢失性磷酸酶-张力蛋白基因(phosphataseandtensinhomologdeletedonchromosometen,PTEN)被称为子宫内膜的看家基因。但有关PTEN基因在子宫内膜癌发生发展中的确切作用尚不清楚。本研究旨在检测子宫内膜癌组织中PTEN基因的突变及蛋白表达情况。方法:应用聚合酶链反应-单链构象多态性分析(polymerasechainreaction-singlestranconformationpolymorphism,PCR-SSCP)和DNA序列分析法,检测52例子宫内膜癌组织和10例正常子宫内膜组织中PTEN基因第5和第8外显子的突变;免疫组织化学法检测PTEN蛋白的表达,并结合临床病理特征进行分析。结果:子宫内膜癌组织的PTEN基因突变率和蛋白缺失率分别为25%和60%,高于正常子宫内膜组织(0%),差异有显著性(P<0.05)。病理学分级为G1、G2及肌层浸润深度<1/2的组织的PTEN基因突变率高于病理学分级为G3及肌层浸润深度≥1/2者,而病理学分级为G1、G2组织的PTEN蛋白表达缺失率低于病理学分级为G3者,差异有显著性(P<0.05)。PTEN基因突变率和蛋白表达缺失率在子宫内膜样腺癌和其它组织类型之间比较,差异有显著性(P<0.05),而在不同手术分期之间差异无显著性(P>0.05)。结论:PTEN基因突变和蛋白阳性表达常发生在病理学分级较低的子宫内膜癌病例     

Analysis of c-KIT expression and KIT gene mutation in human mucosal melanomas

Recent data suggested an increased frequency of KIT aberrations in mucosal melanomas, whereas c-KIT in most types of cutaneous melanomas does not appear to be of pathogenetic importance. However, studies investigating the status of the KIT gene in larger, well-characterised groups of patients with mucosal melanomas are lacking. We analysed 44 archival specimens of 39 well-characterised patients with mucosal melanomas of different locations. c-KIT protein expression was determined by immunhistochemistry, KIT gene mutations were analysed by PCR amplification and DNA sequencing of exons 9, 11, 13, 17 and 18. c-KIT protein expression could be shown in 40 out of 44 (91%) tumours in at least 10% of tumour cells. DNA sequence analysis of the KIT was successfully performed in 37 patients. In 6 out of 37 patients (16%) KIT mutations were found, five in exon 11 and one in exon 18. The presence of mutations in exon 11 correlated with a significant stronger immunohistochemical expression of c-KIT protein (P=0.015). Among the six patients with mutations, in two patients the primary tumour was located in the head/neck region, in three patients in the genitourinary tract and in one patient in the anal/rectal area. In conclusion, KIT mutations can be found in a subset of patients with mucosal melanomas irrespective of the location of the primary tumour. Our data encourage therapeutic attempts with tyrosine kinase inhibitors blocking c-KIT in these patients.