Detection of k-ras gene point mutation in fine needle aspiration and pancreatic juice by sequence special primer method and its clinical significance

INTRODUCTIONThe point mutation rate of k-ras gene at codon 12 inpancreatic adenocarcinoma is reported to be as highas 90%,and with no mutations in normalpancreas tissues or other pancreatic disorders.Wehave detected the presence of k-ras gene

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.

Early detection of pancreatic cancer

The diagnosis of pancreatic cancer associates an appalling significance.Detection of preinvasive stage of pancreatic cancer will ameliorate the survival of this deadly disease.Premalignant lesions such as Intraductal Papillary Mucinous Neoplasms or Mucinous Cystic Neoplasms of the pancreas are detectable on imaging exams and this permits their management prior their invasive development.Pancreatic intraepithelial neoplasms(PanIN)are the most frequent precursors of pancreatic adenocarcinoma(PDAC),and its particular type PanIN high-grade represents the malignant non-invasive form of PDAC.Unfortunately,PanINs are not detectable on radiologic exams.Nevertheless,they can associate indirect imaging signs which would rise the diagnostic suspicion.When this suspicion is established,the patient will be enrolled in a follow-up strategy that includes performing of blood test and serial imaging test such as computed tomography or magnetic resonance imaging,which will cost in the best-case scenario a burden of healthcare systems,and potential mortality in the worst-case scenario when the patient underwent resection surgery,worthless when there is no moderate or high grade dysplasia in the final histopathology.This issue will be avoid having at its disposal a diagnostic technique capable of detecting high-grade PanIN lesions,such is the cytology of pancreatic juice obtained by nasopancreatic intubation.Herein,we review the possibility of detection of early malignant lesions before they become invasive PADC.

KAI1 gene is differently expressed in papillary and pancreatic cancer:influence on metastasis

AIM To compare KAI1 in cancer of papilla ofVater and pancreas to evaluate whether there aredifferences in biologic behavior which mightaccount for prognosis.METHODS We compared the expression in 24papillay and 29 pancreatic cancers usingNorthern blot analysis,immunochemical assayand in situ hybridization,and investigatedwhether early diagnosis or molecular differencespredict the outcome in these tumor entities.RESULTS By Northern blot analysis there is nostatistical difference of KAI1 levels in normaland cancerous papilla.No association betweenKAI1 mRNA expression and tumor stage or tumordifferentiation was found in the tumors.Byimmunohistochemical assay,KAI1 staining incytoplasm of papillary cancer cells was similarto that of normal papillary cells.By in situhybridization,the results of KAI1 mRNAexpression in normal and cancerous papilla weresimilar to those with immunohistochemicalassay.The normal and cancerous pancreastissues were also analyzed by the methods usedin papillary samples.CONCLUSION Although the biologic roles of KAI1 have not been clarified, our results suggest that KAI1 may restrict the progression of malignant papillary cancer, but its expression might not have any effect on the characteristics of papillary tumor, whereas by the analysis of KAl1 gene, its reduced expression is closely related to the progression and metastases of pancreatic cancer.

Epigenetic changes of pituitary tumor-derived transforming gene 1 in pancreatic cancer

BACKGROUND: Pancreatic cancer is a devastating disease with abnormal genetic changes. The pituitary tumor-derived transforming gene (PTTG) is considered to be implicated in the tumorigenesis of cancers when the gene is epigenetically transformed. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the PTTG1 gene in pancreatic cancer. METHODS: We chose 4 cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues. After using restriction isoschizomer endonucleases (Msp I /Hpa II) to digest the DNA sequence (5'-CCGG-3'), we performed PCR reaction to amplify the product. And RT-PCR was applied to determine the gene expression. RESULTS: The mRNA expression of the PTTG1 gene was higher in pancreatic tumor than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the PTTG1 gene were almost identical in normal, tumor pancreatic tissues and the 4 PDAC cell lines. Some (5'-CCGG-3') areas in the upstream region of PTTG1 were methylated, while some others were unmethylated. CONCLUSIONS: The oncogene PTTG1 was overexpressed in pancreatic tumor tissues and verified by RT-PCR detection. The methylation status of DNA in promoter areas was involved in the gene expression with the help of other factors in pancreatic cancer.

Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (Msp I/Hpa II) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as verified by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

Studies on the Relationship between Multidrug Resistance 1 Gene and Pancreatic Cancer

Objective: To study the relationship between the expressions of multidrug-resistance, 1 (mdr1) gene-coded mRNA and its product P-glycoprotein (P-gp) and biological characteristics of tumor cells in patients with previously untreated primary pancreatic cancer (PC) for guiding signi?cance to the clinical treatment. Methods: Expression of mdr1 mRNA and P-gp on para?n embedded sections was detected by in situ polymerase chain reaction (ISPCR) and immunohistochemistry correspondingly from 150 cases of normal and abnormal pancreatic tissues including 97, 32 and 21 cases of pancreatic cancer, pancreatitis and normal pancreatic tissues respectively. Results: Distributions of positive staining in mdr1 mRNA and P-gp were mainly found on the apical plasma membranes and in cytoplasms of endothelial duct cells in tumor and normal tissues. The positive staining rates of expression of the mdr1 mRNA and P-gp detected in all pancreatic tumors were signi?cantly higher than that in pancreatitis and normal tissues correspondingly (P <0.05). Moreover, higher expressions of mdr1 mRNA and P-gp in tumor cells were correlated with some biological characteristics of PC, such as the degree of di?erentiation, aggressiveness and TNM stage of tumors (P <0.05). However, there was no correlation between the rate of expression of mdr1 mRNA and P-gp and some clinical ?ndings including age, sex, location and tumor size. Conclusion: The expression of mdr1 gene was associated with “natural” multi-drug resistance in PC. There was an important guiding signi?cance between the detection of expression of mdr1 gene and prediction of the sensitivity to chemotherapy of PC. Meanwhile, it probably could be used as one of profoundly parameters to assess the degrees of di?erentiation and prognosis in PC.

Detecting K-ras and p53 gene mutation from stool and pancreatic juice for diagnosis of early pancreatic cancer

OBJECTIVE: To explore new methods for the early diagnosis of pancreatic cancer through detection of K-ras and p53 mutations in pancreatic juice and stool. METHODS: 201 patients in PUMC Hospital from 1994 - 2000 and 60 control individuals were enrolled in this study. K-ras point mutation was detected by PCR-RFLP while p53 mutation was detected by PCR-SSCP. RESULTS: K-ras mutation was found in pancreatic juice in 87.8% (36/41) of pancreatic cancer patients and 23.5% (4/17) of benign pancreatic disease patients. In 261 stool specimens, amplification found mutations successfully in 235 patients (90%). K-ras mutation was found in stool in 88% (66/75) of pancreatic cancer patients, 51.1% (24/47) of benign pancreatic disease patients and 19.6% (9/46) of normal individuals. p53 mutation was found in pancreatic juice in 47.4% (18/38) of pancreatic cancer patients and 12.5% (2/16) of benign pancreatic disease patients. p53 mutation was found in stool in 37.1% (23/62) and 19.1% (4/21) of chronic pancreatitis patients. CONCLUSION: K-ras mutation in pancreatic juice has higher diagnosis sensitivity and specificity, and therefore may be used as a supplement in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can aid in the screening of pancreatic cancer.

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Evaluation of diagnostic cytology via endoscopic naso-pancreatic drainage for pancreatic tumor

AIM: To evaluate the usefulness of cytology of the pancreatic juice obtained via the endoscopic naso-pancreatic drainage tube(ENPD-C).METHODS: ENPD was performed in cases where a diagnosis could not be made other than by using en-doscopic retrograde cholangiopancreatography and in cases of pancreatic neoplasms or cystic tumors, includ-ing intraductal papillary mucinous neoplasm(IPMN) suspected to have malignant potential. 35 patients(21 males and 14 females) underwent ENPD between January 2007 and June 2013. The pancreatic duct was imaged and the procedure continued in one of ENPD-C or ENPD-C plus brush cytology(ENPD-BC). We checked the cytology result and the final diagnosis.RESULTS: The mean patient age was 69 years(range, 48-86 years). ENPD-C was performed in 24 cases andENPD-C plus brush cytology(ENPD-BC) in 11 cases. The ENPD tube was inserted for an average of 3.5 d. The final diagnosis was confirmed on the basis of the resected specimen in 18 cases and of follow-up findings at least 6 mo after ENPD in the 18 inoperable cases. Malignancy was diagnosed in 21 cases and 14 patients were diagnosed as having a benign condition. The ratios of class Ⅴ/Ⅳ:Ⅲ:Ⅱ/Ⅰ?findings were 7:7:7 in malignant cases and 0:3:11 in benign cases. The sensitivity and specificity for all patients were 33.3% and 100%, re-spectively. The cytology-positive rate was 37.5%(6/16) for pancreatic cancer. For IPMN cases, the sensitivity and specificity were 33% and 100%, respectively.CONCLUSION: Sensitivity may be further increased by adding brush cytology. Although we can diagnosis cancer in cases of a positive result, the accuracy of ENPD-C remains unsatisfactory.

Preventing pancreatic fistula after distal pancreatectomy: An invagination method

Following an increase in the use of the GIA stapler for treating a pancreatic stump, more techniques to prevent postoperative pancreatic juice leakage have been required. We describe one successful case using our new technique of invaginating the cut end of the pancreas into the stomach to prevent a pancrea-tic fistula(PF) from occurring. A 50-year-old woman with pancreatic cancer in the tail of the pancreas underwent distal pancreatectomy, causing a grade A PF. We resected the distal pancreas without additional reinforcement to invaginate the stump into the gastric posterior wall with single layer anastomosis using a 3-0 absorbable suture. The drain tubes were removed on the third postoperative day. Although a grade A PF was noted, the patient was discharged on foot on the eleventh postoperative day. Our technique may be a suitable method for patients with a pancreatic body and tail tumor.

Pancreatic neuroendocrine tumors G3 and pancreatic neuroendocrine carcinomas: Differences in basic biology and treatment

In 2017 the World Health Organization revised the criteria for classification of pancreatic neuroendocrine neoplasms(p NENs) after a consensus conference at the International Agency for Research on Cancer. The major change in the new classification was to subclassify the original G3 group into well-differentiated pancreatic neuroendocrine tumors G3(p NETs G3) and poorly differentiated pancreatic neuroendocrine carcinomas(p NECs), which have been gradually proven to be completely different in biological behavior and clinical manifestations in recent years. In 2019 this major change subsequently extended to NENs involving the entire digestive tract. The updated version of the p NENs grading system marks a growing awareness of these heterogeneous tumors. This review discusses the clinicopathological, genetic and therapeutic features of poorly differentiated p NECs and compare them to those of well-differentiated p NETs G3. For p NETs G3 and p NECs(due to their lower incidence), there are still many problems to be investigated. Previous studies under the new grading classification also need to be reinterpreted. This review summarizes the relevant literature from the perspective of the differences between p NETs G3 and p NECs in order to deepen understanding of these diseases and discuss future research directions.

Peripancreatic paraganglioma:Lesson from a round table

We described the case of a peripancreatic paraganglioma(PGL)misdiagnosed as pancreatic lesion.Surgical exploration revealed an unremarkable pancreas and a large well-defined cystic mass originating at the mesocolon root.Radical enucleation of the mass was performed,preserving the pancreatic tail.Histologically,a diagnosis of PGL was rendered.Interestingly,two previously unreported mutations,one affecting the KDR gene in exon 7 and another on the JAK3 gene in exon 4 were detected.Both mutations are known to be pathogenetic.Imaging and cytologic findings were blindly reviewed by an expert panel of clinicians,radiologists,and pathologists to identify possible causes of the misdiagnosis.The major issue was lack of evidence of a cleavage plane from the pancreas at imaging,which prompted radiologists to establish an intraparenchymal origin.The blinded revision shifted the diagnosis towards an extrapancreatic lesion,as the pancreatic parenchyma showed no structural alterations and no dislocation of the Wirsung duct.Ex post,the identified biases were the emergency setting of the radiologic examination and the very thin mesocolon sheet,which hindered clear definition of the lesion borders.Original endoscopic ultrasonography diagnosis was confirmed,emphasizing the intrinsic limit of this technique in detecting large masses.Finally,pathologic review favored a diagnosis of PGL due to the morphological features and immonohistochemical profile.Eighteen months after tumor excision,the patient is asymptomatic with no disease relapse evident by either radiology or laboratory tests.Our report strongly highlights the difficulties in rendering an accurate preoperative diagnosis of PGL.

Inhibitary effects of antisense oligonucleotide specific to K-ras point mutation on the target gene expression in human pancreatic carcinoma cells

The prognosis of pancreatic carcinoma is disappointing due to the difficulty of early and accurate diagnosis,low operative resection rate, insensitivity to radiation therapy and chemotherapy. The incidence of pancreatic carcinoma has increased considerably in the past few years, the progress of diagnosis and treatment, however, has not been significantly improved. The overall 5-year survive rate is still as low as 5%-10%, and the improved 5 years survive rate is about 20%-40% after successive Whipple-operation. The early diagnosis is critical to the successful surgical treatment. It depends on the establishment of the new way for that.

胰液K-ras12密码子点突变检测对胰腺癌诊断的临床价值

目的 :探讨胰液中 K- ras 12密码子点突变对胰腺癌诊断的临床应用价值。 方法 :采用内镜下置鼻胰管方法收集 2 0例胰腺疾病的胰液标本 ,聚合酶链反应 -限制性片段长度多态性分析 (PCR- RFL P)检测胰液 K- ras基因第 12密码子点突变 ,与肿瘤标记物 CA 19- 9及 CEA检测结果比较。结果 :12例胰腺癌患者胰液标本中 K- ras突变率为 5 8.3% (7/ 12 ) ;8例慢性胰腺炎患者标本 K- ras突变率为 12 .5 % (1/ 8) ,胰腺癌胰液 K- ras基因突变检测的敏感性为 5 8.3% ,特异性为 87.5 % ,阳性预示值为 87.5 % ,阴性预示值为 5 8.3%。同一组病例以血清 CA19- 9>37U / m l、CEA >4 .5μg/ ml为阳性界值。胰腺癌血清CA19- 9、CEA阳性率分别为 5 8.3% (7/ 12 )、4 1.7% (5 / 12 )。结论 :K- ras基因突变与胰腺癌相关性好 ,胰液中 K- ras 12密码子突变率高 ,特异性强 ,为胰腺癌早期诊断的初步筛选提供了手段。

应用肽核酸钳制PCR技术检测胰腺癌K-ras基因突变

目的:应用肽核酸钳制PCR技术检测胰腺癌K-ras基因突变,探讨其诊断价值。方法:结合特异性肽核酸(针对野生型序列)与实时定量PCR技术建立一步K-ras基因检测法,与传统限制性片段长度多态性PCR法检测结果进行比较,评价新方法的诊断价值。结果:肽核酸钳制PCR法可同时对胰腺癌标本中K-ras基因的第12、13密码子突变情况进行检测,操作简便;可在105倍的野生型K-ras基因背景下检测出突变,灵敏度为0.001%,可检测突变基因最低限为102拷贝;该方法可同时进行较大规模的样品检测,单次检测时间仅需1h。而传统方法可检测的突变基因最低限为103拷贝,检测灵敏度为0.01%;需花费约2d完成同样数量的样本检测。结论:肽核酸钳制实时定量PCR法较限制性片段长度多态性PCR法有明显优势,适用于胰腺癌大规模临床样本的K-ras基因突变检测,可作为胰腺癌筛查的有效手段。

检测胰液中K-ras基因突变对胰腺癌诊断的作用

目的:检测经逆行胰胆管造影(ERCP)过程中所获胰液中的K-ras基因突变。探讨其在胰腺癌诊断中的作用和意义。方法:收集ERCP过程中注射促胰泌素后的胰液,应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)的方法检测胰腺良、恶性疾病胰液上清和细胞中的K-ras基因突变。结果:胰腺癌病例的胰液上清液中K-ras基因突变检出率为60.0%(18/30),细胞中的检出率为76.7%(23/30)。胰腺良性疾病胰液细胞中有15.4%(2/13)的K-ras基因突变,上清液和其他非胰腺疾病对照组胰液中无K-ras基因突变检出。胰腺癌患者的胰液中检测到K-ras基因突变明显高于胰腺良性疾病患者和对照组(P<0.001)。K-ras基因突变检出率与肿瘤的部位及大小无明显关系。结论:胰液K-ras基因突变检出对于胰腺癌的诊断具有一定作用。

血浆K-ras突变和肿瘤标志物诊断胰腺癌的临床价值

目的 :进一步研究血浆K ras基因突变和肿瘤标志物诊断胰腺癌的价值。方法 :胰腺癌病人 90例 ,非胰腺癌组 6 0例。入院时抽取患者外周静脉血 ,分离血浆并提取DNA。采用突变富集PCR RFLP法分析K ras基因密码子 12突变。同时测定血清CA19 9、CA2 4 2、CEA。结果 :胰腺癌组患者血浆K ras基因突变阳性率为 73% (6 6 / 90 ) ,显著高于非胰腺癌组 15 % (9/ 6 0 ) (χ2 =4 9.0 ,P <0 .0 0 1)。血浆K ras和血清CA19 9、CA2 4 2诊断胰腺癌均有较高的敏感性。与单个指标相比 ,平行法联合检测可以提高敏感性 ,系列法联合检测可提高特异性。结论 :测定血浆K ras突变对诊断胰腺癌有重要的临床意义 ,联合测定血清肿瘤标志物可提高诊断价值。

人胰腺癌组织K-ras,p53基因表达及其意义

目的：探讨Ｋ－ｒａｓ，ｐ５３基因表达与人胰腺癌临床生物学行为之间的关系。方法：采用免疫组织化学与斑点杂交方法，检测４８例人胰腺癌组织Ｋ－ｒａｓ，ｐ５３基因表达。结果：胰腺癌组织Ｐ２１ｒａｓ蛋白表达较正常胰腺组织增强（Ｐ＜０．０５），Ⅰ～Ⅱ期较Ⅲ～Ⅳ期表达增强（Ｐ＜０．０５），组织学分级Ｇ１，Ｇ２级较Ｇ３级表达增强（Ｐ＜０．０１），Ｐ２１ｒａｓ蛋白表达阳性患者生存期较表达阴性者延长（Ｐ＜０．０１）；胰腺癌组织Ｐ２１ｒａｓ蛋白与Ｋ－ｒａｓｍＲＮＡ表达呈正相关（ｒ＝０．８９，Ｐ＜０．０１）；胰腺癌组织Ｐ５３蛋白表达较正常胰腺及慢性胰腺炎组织增强（Ｐ＜０．０５），Ⅲ～Ⅳ期胰腺癌较Ⅰ～Ⅱ期Ｐ５３蛋白表达增强（Ｐ＜０．０５），随肿瘤分化程度降低Ｐ５３蛋白表达而增强（Ｐ＜０．０１），Ｐ５３蛋白与ｐ５３ｍＲＮＡ表达呈负相关（ｒ＝－０．４４，Ｐ＜０．０１）。结论：Ｐ２１ｒａｓ与Ｐ５３蛋白表达对估价胰腺癌患者的预后具重要价值；

血浆K-ras突变联合CA19-9检测对胰腺癌的诊断价值

目的 :探讨血浆K ras基因突变联合CA19 9检测对胰腺癌的诊断价值。方法 :对连续 5 8例疑为胰腺肿瘤患者 ,入院时抽取外周静脉血 ,分离血浆并提取DNA。采用突变富集PCR RFLP法分析K ras基因密码子 12突变 ,用放射免疫法测定血清CA19 9。所有病例的血标本分析均在术前完成 ,并将检测结果与手术探查、病理诊断进行比较。结果 :4 1例胰腺癌患者中 ,血浆K ras基因突变者 2 9例 (占 70 .7% ) ,血清CA19 9升高者 30例 (占 73.2 % )。联合K ras与CA19 9检测胰腺癌的敏感性为 90 .2 % (37/ 4 1)。在其他 17例非胰腺癌患者中 ,有 3例血浆K ras突变 ,8例血清CA19 9有升高。结论 :血浆K ras突变可能是诊断胰腺癌的一个有价值的指标 ,若同时测定血清CA19 9。