胰腺癌患者胰液中K-ras基因突变的检测及其意义

目的 通过对经逆行胰胆管造影（ＥＲＣＰ）所获胰液的Ｋ－ｒａｓ基因突变检测,以探索胰腺癌诊断的新方法。方法 应用聚合酶链反应（ＰＣＲ）－限制性片段长度多态性的方法检测胰腺良、恶性疾病组织物、胰液上清和细胞的Ｋ－ｒａｓ突变。结果 胰腺癌和胰腺良性疾病标本分别有７１％（１５／２１）,而胰腺良性疾病无一例有Ｋ－ｒａｓ的突变。用胰液细胞有４％ＰＣＲ未能获得成功,胰腺癌胰液细胞Ｋ－ｒａｓ的突变率为６５％（１１     

p53 and K-ras mutations in pancreatic juice samples from patients with chronic pancreatitis

BACKGROUND: Mutations in p53 and ras genes are frequent in pancreatic carcinoma. Several ras mutations are consistently detected in the pancreatic juice from patients with chronic pancreatitis. The p53 gene mutations have been detected occasionally in chronic pancreatitis tissue. It was the aim of this study to evaluate the presence and clinical significance of p53 and ras mutations in clinical pancreatic juice samples from patients with chronic pancreatitis. METHODS: Pancreatic juice was obtained from 66 patients with chronic pancreatitis and no evidence of pancreatic carcinoma (51 men, 15 women; age 17-86 years [mean 49.6 +/- 12.9]). Patients were followed prospectively for 26 +/- 3 (4-54) months. Detection of p53 gene mutations was by temperature gradient gel electrophoresis (TGGE) and single strand conformation polymorphism (SSCP) for exons 5-8. Analysis of ras mutations was performed by SSCP/polymerase chain reaction, restriction fragment length polymorphism/polymerase chain reaction. All mutations were confirmed by sequencing. RESULTS: Five of 66 (7.5%) pancreatic juice samples contained p53 mutations, and ras mutations were detected in 6 cases (9%). Cytology was negative in all cases. No pancreatic carcinoma developed during follow-up and neither cancer cells nor preneoplastic lesions could be detected histologically in resected specimens. Although no correlation between p53 mutations and duration of pancreatitis or drinking habits was found, K-ras mutations correlated with both heavy smoking and severity of the disease. CONCLUSION: p53 and ras mutations can be detected in a minority of pancreatic juice samples from patients with chronic pancreatitis in the absence of malignancy.     

A meta-analysis of the diagnostic value of detecting K-ras mutation in pancreatic juice as a molecular marker for pancreatic cancer

BackgroundK-ras codon 12 mutation is one of the earliest genetic changes in the development of pancreatic cancer (PC) and accurate detection of K-ras mutations is gaining increasing attention in the field of molecular diagnosis.MethodsOriginal research articles which evaluated the diagnostic accuracy of K-ras mutation detection in PC were selected. Data were presented as forest plots and summary receiver operating characteristic curve analysis was used to summarize the overall test performance.ResultsWe assessed 16 studies from 15 published articles. The pooled sensitivity and specificity were 59% (95%CI: 54%–64%) and 87% (95%CI: 84%–89%), respectively. The pooled positive likelihood ratio and negative likelihood ratio were 4.13 (95%CI: 2.73–6.25) and 0.42 (95%CI: 0.32–0.56), respectively, and the pooled diagnostic odds ratio was 13.66 (95% CI: 7.25–25.74).ConclusionsOur results indicate that the analysis of K-ras mutations in pancreatic juice has a considerable diagnostic value in PC. Further studies with rigorous design, large sample size, and multi-regional co-operation are needed.     

Significance of K-ras mutation and CEA level in pancreatic juice in the diagnosis of pancreatic cancer

The early diagnosis of pancreatic carcinoma is essential for increasing patient survival rates. In this study, 52 patients with suspected pancreatic diseases were examined to investigate the value of K‐ras codon 12 point mutation, levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19‐9), and cytology of pancreatic juice in the diagnosis of pancreatic carcinoma. Pancreatic juice was taken without secretin stimulation. K‐ras mutation was detected by enriched polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). K‐ras mutation in pancreatic juice was more frequent in carcinoma than in benign diseases (P = 0.0448). The positive predictive value of K‐ras mutation for the diagnosis of neoplastic disease was 83%. The CEA level in pancreatic juice in carcinoma was significantly greater than that in benign disease (P < 0.0001). When the cutoff level of CEA was set at 50 ng/ml, its accuracy for the diagnosis of carcinoma was 85%. A multivariate analysis showed that K‐ras mutation and CEA level in pancreatic juice, as well as serum CA19‐9 level and age of the patient were independent variables for the diagnosis of carcinoma, and the accuracy of diagnosis by this analysis was increased to 90%. In conclusion, both K‐ras mutation and CEA level in pancreatic juice may be valuable for the diagnosis of carcinoma. Better discrimination was possible with a multivariate analysis.     

Clinical usefulness of K-ras gene mutation detection and cytology in pancreatic juice in the diagnosis and screening of pancreatic cancer.

BACKGROUND: The significance of K-ras codon 12 mutation in pancreatic juice is still unclear. Although considerable controversy surrounds this question, the diagnostic utility of K-ras in patients with clinical suspicion of pancreatic cancer (PC) and in PC-risk patients remains unknown. OBJECTIVE: To study prospectively the utility of the K-ras gene mutation and cytology in the diagnosis and screening of PC, and to assess its contribution to clinical decision making. METHODS: Pancreatic juice samples obtained from 90 patients were evaluated prospectively. Group I (n = 40) comprised patients with clinical suspicion of PC; group II (n = 50) comprised 49 patients with chronic pancreatitis and one patient proceeding from a PC family screening. The K-ras mutation was detected by means of artificial restriction fragment length polymorphisms (RFLP) in DNA after polymerase chain reaction (PCR) amplification. RESULTS: In group I, of those patients with a definitive diagnosis of PC, malignant cells were found in 27% and K-ras mutation in 44%. In five cases, molecular analysis contributed to diagnosis (4/11 with negative cytology and 1/2 with insufficient cytological material). K-ras mutation revealed an early tumour in one patient, and was the only sample available for diagnosis in another. In group II, the K-ras gene mutation was detected in 8/49 patients (16%) with chronic pancreatitis, one of whom developed PC (2%). CONCLUSIONS: K-ras mutation analysis of pancreatic juice may complement cytological evaluation in the diagnosis of PC, in spite of its limited contribution to clinical decision making. The presence of K-ras mutation in chronic pancreatitis classifies a subgroup of PC-risk patients who should be evaluated carefully by long-term follow-up.     

Early detection of pancreatic cancer following the diagnosis of chronic pancreatitis.

AIM: To assess whether patients with misdiagnoses of chronic pancreatitis (CP), followed at an early stage by a diagnosis of pancreatic cancer (PCr), present different epidemiological characteristics from patients suffering either from CP alone or from CP with late degeneration to PCr. METHODS: We arbitrarily subdivided our patient series into three groups: (1) 12 CP who developed PCr within 4 years after onset of symptoms; (2) 12 CP developing PCr after the 4th year, and (3) 701 CP with no subsequent development of PCr. The variables studied were age, sex, drinking and smoking habits, tumor localization, and presence of intraductal calcifications and diabetes mellitus at the time of diagnosis of CP. RESULTS: There were no significant differences between CP and 'late' PCr in any of the study variables considered. As compared with the CP group, the 'early' PCr cases were older (58.7 vs. 40.7 years; p < 0.0001), with a lower proportion of males (58 vs. 88%; p < 0.01), smaller proportions of both smokers (42 vs. 88%; p < 0.0001) and subjects drinking more than 40 g of alcohol/day (42 vs. 86%; p < 0. 0001), and a greater incidence of non-insulin-dependent diabetics at the time of diagnosis of CP (25 vs. 3.7%; p < 0.012). As compared with the 'late' PCr group, the malignancies in the 'early' PCr cases were more often located in the head of the pancreas (100 vs. 50%; p < 0.01). Multivariate logistic regression analysis selected age over 50 (odds ratio OR 13.5, 95% confidence interval CI 2.79-65.5; p < 0. 001), smoking habits (OR 0.14, 95% CI 0.04-0.49; p < 0.002), and non-insulin-dependent diabetes (OR 5.91, 95% CI 1.20-29.1; p < 0. 028) as variables identifying subjects with 'early' PCr. CONCLUSIONS: A high suspicion of a pancreatic tumor is necessary when CP is diagnosed in a patient with atypical epidemiological characteristics for this condition, possibly female, aged over 50, who is not a smoker or drinker, and suffers from non-insulin-dependent diabetes.     

Significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas

The significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas is still unclear. The aim of this study was to evaluate the significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas. All of the 78 reports written from 1988 to 1996 on K-ras point mutation of carcinoma, mucin-producing tumors, and hyperplastic epithelia of the pancreas in both surgical or autopsy specimens and pancreatic juice are reviewed. As results, in surgical or autopsy specimens, K-ras mutation was found in 81% of ordinary duct cell carcinoma and in 53% of mucin-producing tumor of the pancreas; this mutation was also found in hyperplastic epithelia in chronic pancreatitis (7%) and in autopsy cases without pancreatic diseases. In pancreatic juice, K-ras mutation was found in 72% of ordinary pancreatic carcinoma and in 53% of mucin-producing tumor, respectively. In conclusion, most previous reports have indicated that K-ras mutation in pancreatic juice is useful for a diagnosis of pancreatic carcinoma. However, since K-ras gene mutation was also detected in non-tumorous lesions, the diagnosis of pancreatic carcinomas is not necessarily correct if it is based solely on the detection of K-ras mutation in pancreatic juice. Future studies should focus on analyzing the amino acid sequence of K-ras mutation or the combination of this mutation with other parameters such as tumor markers in pancreatic juice, to enhance its specificity and accuracy.     

Secretory immunoglobulin A in pancreatic juice and pancreatic tissue of patients with chronic pancreatitis

Background—The predominace of secretory IgA(S-IgA) in intestinal secretions compared with blood is wellestablished, but concentrations of this protein in pancreatic juice andits origin, especially in chronic pancreatitis, are unknown.
Aims—To investigate the role of S-IgA in chronic pancreatitis.
Patients—Twenty one patients with chronicpancreatitis (group I), three patients with proven malignancies (groupII), and 12 patients without pancreatic disease (group III).
Methods—Pure human pancreatic juice was collectedendoscopically in four fractions after consecutive stimulation withsecretin and cholecystokinin (CCK). Samples were analysed for S-IgA,protein, trypsinogen, and proteolytic activity.
Results—The S-IgA level was significant increasedin fraction 1 of pancreatic juice of group I (1210 (1411) ng/ml)compared with controls (33 (70) ng/ml). Protein concentrations andtrypsinogen content were lower in group I than in the other groups.Proteolytic activity could be observed in 53% of all 133 pancreaticjuice samples, but in 87% of fraction 1. In pancreatic tissue of three patients with chronic pancreatitis both IgA and secretory component were detected by immunohistology. Expression of the secretory componentby human pancreatic epithelial cells was increased in patients withchronic pancreatitis compared with normal controls. The concentrationof S-IgA in pancreatic juice did not correlate with the serum S-IgAlevel. In contrast, serum levels of S-IgA were decreased in patientswith chronic pancreatitis.
Conclusion—There are high levels of S-IgA inhuman pancreatic juice following chronic inflammation and a protectiverole is suggested for this immunoglobulin.

Keywords:chronic pancreatitis; pancreatic juice; proteaseactivity; protease inhibitors; secretory IgA; immunohistochemistry

     

Telomerase activity in pancreatic juice differentiates pancreatic cancer from chronic pancreatitis: A meta-analysis

Background/objectiveTo evaluate the usefulness of genetic markers in pancreatic juice (PJ), and the combination of these markers with telomerase activity in the differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis.MethodsWe conducted a meta-analysis for the diagnostic utility of the four major altered genes in PDAC (KRAS, CDKN2A/p16, TP53, and SMAD4/DPC4), telomerase activity, and a combination assay using PJ samples. A literature search was conducted in MEDLINE, Cochrane Library, and Web of Science. Data were pooled and presented as diagnostic sensitivity and specificity with 95% confidence intervals (CIs).ResultsThirty-nine studies fulfilled the inclusion criteria. Pooled estimates of KRAS analysis were as follows: sensitivity was 0.67 (95% CI, 0.63–0.71) and specificity, 0.82 (95% CI, 0.79–0.85). For telomerase activity analysis, sensitivity was 0.82 (95% CI, 0.76–0.87) and specificity, 0.96 (95% CI, 0.90–0.99). The other three tumor suppressors demonstrated low sensitivity. The data did not suggest any publication bias. A combined analysis of KRAS and telomerase activity showed a higher diagnostic sensitivity (0.94; 95% CI, 0.83–0.99) than KRAS alone. A combined analysis of telomerase activity and cytology revealed more reliable diagnostic accuracy than telomerase activity alone, with high sensitivity (0.88; 95% CI, 0.74–0.96) and specificity (1.00; 95% CI, 0.91–1.00).ConclusionsThe most reliable marker in PJ samples for diagnosis of PDAC was telomerase activity. Telomerase activity can play a central role in diagnostic analysis using PJ samples, and can increase diagnostic accuracy when combined with KRAS mutations or cytological examination.     

Telomerase activity in pure pancreatic juice for the diagnosis of pancreatic cancer may be complementary to K-ras mutation

BACKGROUND: The usefulness of K-ras mutation in pancreatic juice for the diagnosis of pancreatic cancer is questionable. Telomerase is positive in pancreatic cancer but rarely in benign pancreatic diseases. We conducted this study to determine the usefulness of K-ras mutation and telomerase activity in pancreatic juice for the diagnosis of pancreatic cancer. METHODS: Pancreatic juice collected during endoscopic retrograde cholangiopancreatography was examined in 31 patients: 12 with pancreatic cancer, 11 with chronic pancreatitis, and 8 control patients. The K-ras gene was detected by using the restriction fragment length polymorphism method. Telomerase activity was detected by using the telomeric repeat amplification protocol. RESULTS: K-ras mutation was positive in 75% (9 of 12) of pancreatic cancers and in 27% (3 of 11) of cases of chronic pancreatitis but in none of the control patients. Telomerase activity was detected in 92% (11 of 12) of pancreatic cancers and in 18% (2 of 11) of cases of chronic pancreatitis. The diagnostic value in pancreatic cancer was comparable between K-ras mutation and telomerase when evaluated separately. However, by combining these 2 methods, the specificity rose to 100%. CONCLUSIONS: For the diagnosis of pancreatic cancer, telomerase activity in pancreatic juice may possibly be complementary to K-ras mutation because it may decrease the rate of false-positive diagnosis.     

Pure pancreatic juice studies in normal subjects and patients with chronic pancreatitis

Pure pancreatic juice was obtained from within the pancreatic duct in 54 patients after endoscopic cannulation of the papilla of Vater. In all 20 normal subjects there was a brisk response to intravenous injections of GIH secretin in small dosage (1 and 4 CU). Peak bicarbonate concentrations occurred after a 4 CU stimulus, whereas volumes, and bicarbonate and protein outputs were greatest after 70 CU. Total protein and amylase concentrations were highest in the first specimens collected from each patient, and fell rapidly after stimulation. Plateau levels for all indices were achieved 10-20 minutes after starting infusions of secretin and pancreozymin. When normal patients and those with chronic pancreatitis were compared, there was considerable overlap in all indices (volume, bicarbonate and total protein concentrations) after bolus injections of secretin. Most patients with chronic pancreatitis achieved a peak bicarbonate concentration in excess of 100 mmol/l. The median concentrations were not significantly different from normal after any dose of secretin when pooled 10 minute samples were analysed. However there were significant differences in peak bicarbonate concentrations (after 1 and 4 CU, but not after 70 CU), when one minute samples were compared. There were also statistically significant differences in the median 10 minute responses for volume after 1 and 70 CU, for bicarbonate output after 1, 4, and 70 CU, and for protein output after 70 CU. The results of juice studies in patients believed to have early chronic pancreatitis did not differ significantly from those in normal subjects or those with chronic pancreatitis. Endoscopic duct cannulation cannot guarantee complete recovery of pancreatic secretions, and measurements of volume and output may be inaccurate. When standard biochemical indices are used, the diagnostic role of pure juice studies is limited; further research may reveal more specific disease markers.     

顺序特异性引物法快速检测胰腺癌K—ras基因点突变

目的：研究简捷、特异、敏感的检测胰腺癌K－ras基因点突变的方法及其在胰腺疾病中定性诊断的价值。方法：采用针对K－ras基因点突变方式（CGT、GAT、GTT）设计的顺序特异性引物（SSP），先后对胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液进行多聚酶链反应（PCR），扩增产物借助常规电泳和染色检测有无K－ras基因突变及突变方式。结果：胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液中K－ras基因点突变率分别为74．2％、95．1％、91．4％及94．1％，而所有被检测的慢性胰腺炎、胰岛素瘤、壶腹癌、胆管癌、十二指肠乳头癌及外伤胰腺的组织标本和胰液标本均无K－ras基因突变发生。结论：该检测法简便、快速、特异、敏感，具有临床实用性，可以作为鉴别胰腺肿块良恶性和诊断胰腺癌的一种方法。     

Diagnosis of pancreatic cancer by detecting telomerase activity in pancreatic juice: comparison with K-ras mutations

OBJECTIVE: Telomerase activity is reported to be specific and very frequent in human malignancy. K-ras mutations are also very frequently detected in pancreatic cancer, but their specificity for pancreatic cancer is controversial. We examined the telomerase activity and K-ras mutations in pancreatic juice from patients with pancreatic disease. METHODS: Pancreatic juice was obtained endoscopically at endoscopic retrograde pancreatography from 10 patients with pancreatic cancer, three with chronic pancreatitis, and three with a normal pancreas. The telomerase activity in pancreatic juice was assayed by telomeric repeat amplification protocol. K-ras mutations in exon 1 codon 12 were examined by the two-step polymerase chain reaction combined with restriction enzyme digestion, followed by single-strand conformation polymorphism analysis and direct sequencing. RESULTS: Telomerase activity of >5.0 was detected in eight of 10 (80%) subjects with pancreatic cancer, but in none with chronic pancreatitis or normal pancreas. K-ras mutations were detected not only in eight of 10 (80%) subjects with pancreatic cancer but also in two of three with chronic pancreatitis and in one of three with a normal pancreas. CONCLUSIONS: It was shown that the detection of telomerase activity in pancreatic juice is a more useful diagnostic tool for pancreatic cancer than that of K-ras mutations.     

顺序特异性引物法快速检测胰腺癌K-ras基因点突变

目的研究简捷、特异、敏感的检测胰腺癌K ras基因点突变的方法及其在胰腺疾病中定性诊断的价值.方法采用针对K ras基因点突变方式(CGT、GAT、GTT)设计的顺序特异性引物(SSP),先后对胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液进行多聚酶链反应(PCR),扩增产物借助常规电泳和染色检测有无K-ras基因突变及突变方式.结果胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液中K-ras基因点突变率分别为74.2%、95.1%、91.4%及94.1%,而所有被检测的慢性胰腺炎、胰岛素瘤、壶腹癌、胆管癌、十二指肠乳头癌及外伤胰腺的组织标本和胰液标本均无K ras基因突变发生.结论该检测法简便、快速、特异、敏感,具有临床实用性,可以作为鉴别胰腺肿块良恶性和诊断胰腺癌的一种方法.     

Study on chronic pancreatitis and pancreatic cancer using MRS and pancreatic juice samples

AIM:To investigate the markers of pancreatic diseases and provide basic data and experimental methods for the diagnosis of pancreatic diseases.METHODS:There were 15 patients in the present study,among whom 10 had pancreatic cancer and 5,chronic pancreatitis.In all patients,pancreatic cancer or chronic pancreatitis was located on the head of the p-a-ncrea-s.Pa-thology da-ta-of a-ll p-a-tients wa-s confirmed by biopsy and surgery.Among the 10 patients with pancreatic cancer,3 people had a medical history of l...     

Pure pancreatic juice from patients with chronic pancreatitis has an impaired antibacterial activity

Summary Conclusion These data show that pure pancreatic juice of AICP patients has a markedly defective antibacterial activity. This finding might be of potential clinical interest in the understanding of the pathophysiology of the disease. Background The aim of the present study was to test the antibacterial activity of pure pancreatic juice in patients with chronic pancreatitis. Methods The study group consisted of ten patients with ethanol-induced chronic pancreatitis (AICP) and seven control patients free of pancreatic disease. All subjects had recently undergone a secretin-pancreozymin pancreatic function test. After an overnight fast, through a side-viewing endoscope, selective pancreatic duct cannulation was performed. After secretin stimulation, pure pancreatic juice was obtained. Three fractions of different molecular weights were separated. Samples were incubated with 1-mL suspension of 10⁵ Escherichia coli ATCC 25922, and log₁₀ of colony-forming units were counted. Experiments were repeated by grading pancreatic juice concentration, pH of the medium, and inoculum size. Results No significant change of pH of pure pancreatic juice appeared between AICP and controls. Starting from 6-h observation, pure pancreatic juice of AICP patients showed a significant bacterial colonization vs controls (p<0.01). A direct correlation appeared between bacterial colonization and either pH and dilution of pancreatic juice (p<0.001). Antibacterial activity was independent of inoculum size, enzymatic activation or inhibition, and heat treatment. The fraction with 1000–10,000 molecular weight was the one endowed with antibacterial activity.