Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in bile aspirates

Biliary brush cytology is the standard method of sampling a biliary stricture but has a low sensitivity for the detection of malignancy. We have previously shown that minichromosome maintenance (MCM) replication proteins (Mcm2-7) are markers of dysplasia and have utilised these novel biomarkers of growth for the diagnosis of cervical and bladder cancer. We aimed to determine if MCM proteins are dysregulated in malignant pancreaticobiliary disease and if levels in bile are a sensitive marker of malignancy. In 30 tissue specimens from patients with malignant/benign biliary strictures, we studied Mcm2 and -5 expression by immunohistochemistry. Bile samples were also collected prospectively at endoscopic retrograde cholangiopancreatography from 102 consecutive patients with biliary strictures of established (n=42) or indeterminate aetiology (n=60). Patients with indeterminate strictures also underwent brush cytology as part of standard practice. Bile sediment Mcm5 levels were analysed using an automated immunofluorometric assay. In benign biliary strictures, Mcm2 and -5 protein expression was confined to the basal epithelial proliferative compartment - in contrast to malignant strictures where expression was seen in all tissue layers. The percentage of nuclei positive for Mcm2 was higher in malignant tissue (median 76.5%, range 42-92%) than in benign tissue (median 5%, range 0-33%) (P<0.0005), with similar results for Mcm5. Minichromosome maintenance protein 5 levels in bile were significantly more sensitive than brush cytology (66 vs 20%; P=0.004) for the detection of malignancy in patients with an indeterminate stricture, with a comparable positive predictive value (97 vs 100%; P=ns). In this study, we demonstrate that Mcm5 in bile detected by a simple automated test is a more sensitive indicator of pancreaticobiliary malignancy than routine brush cytology.     

Diagnosis of Malignant Biliary Strictures: Conventional or Negative Pressure Brush Cytology?

Background/Objective: The aim of this study was to perform a comparative evaluation of the yields of conventional brush cytology and brush cytology with negative pressure in the diagnosis of malignant biliary strictures. Methods: A total of 132 consecutive patients undergoing endoscopic were identified. Of these, 88.0 had brush cytology after ERCP and 44 were Brush cytology with negative pressure. Retrograde cholangiopancreatography (ERCP) including brush cytology and brush cytology with negative pressure in patients with biliary strictures between 2012-2015. Endoscopic retrograde cholangiography was performed with a standard videoduodenoscope Olympus TFJ 160-R (Olympus, Hamburg, Germany) and brush cytology with a Cook medical Double Lumen Biliary BrushTM (Cytology). Means and standard frequencies were used to calculate variables. Results: Positive results for malignancy were obtained in 22 of 88 patients (25%) by brush cytology and 31 of 44 patients (70.4 %) by brush cytology with negative pressure. Conclusions: Sensitivity of cytology sampling could be maximized by negative pressure during ERCP.     

内镜下球囊扩张术在胆管癌细胞刷检中的应用价值

目的:探索内镜下球囊扩张术联合胆道细胞刷检应用于胆管癌的诊断价值.方法:单中心前瞻性研究2018年1月至2020年1月华中科技大学同济医学院附属武汉中心医院经影像学(CT或MRI)提示胆管恶性狭窄可能或原因不明胆管狭窄并同意逆行胰胆管造影(endoscopic retrograde cholangiopancreato...     

Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis.

Extrahepatic biliary stenosis can be caused by benign and malignant disorders. In most cases, a tissue diagnosis is needed for optimal management of patients, but the sensitivity of biliary cytology for the diagnosis of a malignancy is relatively low. The additional diagnostic value of K-ras mutational analysis of endobiliary brush cytology was assessed. Endobiliary brush cytology specimens obtained during endoscopic retrograde cholangiopancreaticography were prospectively collected from 312 consecutive patients with extrahepatic biliary stenosis. The results of conventional light microscopic cytology and K-ras codon 12 mutational analysis were compared and evaluated in view of the final diagnosis made by histological examination of the stenotic lesion and/or patient follow-up. The sensitivities of cytology and mutational analysis to detect malignancy were 36 and 42%, respectively. When both tests were combined, the sensitivity increased to 62%. The specificity of cytology was 98%, and the specificity of the mutational analysis and of both tests combined was 89%. Positive predictive values for cytology, mutational analysis, and both tests combined were 98, 92, and 94%, whereas the corresponding negative predictive values were 34, 34, and 44%, respectively. The sensitivity of K-ras mutational analysis was 63% for pancreatic carcinomas compared to 27% for bile duct, gallbladder, and ampullary carcinomas. K-ras mutational analysis can be considered supplementary to conventional light microscopy of endobiliary brush cytology to diagnose patients with malignant extrahepatic biliary stenosis, particularly in the case of pancreatic cancer. The presence of a K-ras codon 12 mutation in endobiliary brush cytology per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or equivocal.     

Biliary brush cytology in the assessment of biliary strictures at a tertiary center in Iran

Background: Confirmation of cholangiocarcinoma and other malignant bile duct stenosis is challenging. The aim of the current study was to assess the accuracy of brush cytology for diagnosis of malignant biliary strictures. Methods: 105 patients with hepatic biliary strictures undergoing ERCP were included in this study. Prospectively collected data included symptoms, results of biochemical testing and imaging procedures, as well as details of ERCP. Exclusion criteria were: 1) strictures that would not permit passage of guidewire and brush accession; and 2) post-operative strictures. Brushings of the bile duct strictures were performed. All patients were followed for at least 6 months. The final diagnosis was confirmed following surgery, histopathological diagnosis of the lesion, radiological infiltration of adjacent organs or metastases, or after at least a 6-month follow-up. Results: 88 brush samples from 88 patients were of appropriate quality. The overall diagnostic sensitivity and specificity for malignant nature of biliary strictures were 40.7% and 100%, respectively. The sensitivity was 66.6 % for ampullary carcinomas, 36.3% for pancreatic cancer and 32.5% for cholangiocarcinomas. Conclusions: Despite the low sensitivity, due to the relative ease and safety, brush cytology should remain the first choice for diagnosis of causes of biliary strictures.     

Evaluation of MSX2 mRNA in brush cytology specimens distinguished pancreatic carcinoma from chronic pancreatitis

It is difficult to distinguish pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis (CP) when stricture is present in the pancreatic duct. Endoscopic brushing cytology is a convenient method for investigating strictures in the pancreatic duct, however, the diagnostic sensitivity of this method for PDAC is reported to be low (40-70%). Recently, we revealed that MSX2 is frequently expressed in PDAC cells but not in normal cultured pancreatic duct or stellate cells. Thus, we analyzed MSX2 expression levels in brushing samples to examine whether this would differentiate PDAC from CP. Cytologic brushing specimens were obtained from pancreatic duct strictures during endoscopic retrograde cholangiopancreaticography in 82 patients. The brushing fluid was subjected to cytological diagnosis and RNA extraction. The expression level of MSX2 was evaluated by one-step real-time RT-PCR. MSX2 expression levels were significantly higher in PDAC than in CP (P = 0.0000007), and the expression level was associated with positive cytology (P = 0.013). The sensitivity, specificity, and diagnostic accuracy for PDAC of cytology and MSX2 expression in ductal strictures were: 47.4%, 100%, and 63.4%, and 73.7%, 84.0%, and 79.3%, respectively. The sensitivity and accuracy of MSX2 expression levels for diagnosis were much higher than those of cytology. This suggests that the evaluation of MSX2 levels in endoscopic retrograde cholangiopancreaticography brushing samples would be useful for distinguishing PDAC from CP.     

激素受体阳性早期乳腺癌治疗现状与挑战

在早期发现、有效治疗的情况下,早期乳腺癌是可治愈的,长期无病生存应是早期患者所追求的治疗目标.既往认为乳腺癌各亚型中激素受体(hormone receptor,HR)阳性早期乳腺癌预后最好,然而随着人类表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)阳性乳...     

Problems in the diagnosis of small cell carcinoma of the lungs by fiberoptic bronchoscopy

Fiberoptic bronchoscopy (FOB) with the aid of endoscopic biopsies and brush cytology is recognized as a valuable approach in the diagnosis of lung cancer. However, histologic classification of lung cancer based on tiny specimens obtained from FOB can be difficult. Correct identification of small cell carcinoma of the lung is especially important because its recognition usually precludes surgery. In a review of 770 patients who underwent FOB biopsies at The Mount Sinai Hospital, New York, in individuals with proven lung cancer 150 instances of small cell carcinoma were encountered. In four of these instances subsequent surgery, such as scalene node biopsy, mediastinoscopy, or thoracotomy, was performed because clinically and radiologically the tumors did not behave as small cell carcinomas. Pathologic examination of larger tissue samples from these neoplasms provided the following final diagnoses: bronchial carcinoid, adenocarcinoma, squamous cell carcinoma, and small cell carcinomas-combined type. Analysis of the FOB biopsies and brush cytology usually permit diagnosis of small cell carcinoma of the lung. However, in instances where the biologic behavior of a tumor casts doubt on the diagnosis of small cell carcinoma, further studies should be performed, including radionuclide scans, and bone marrow and other biopsies before denying the patient a chance of surgical cure.     

Differential diagnosis of chronic pancreatitis and pancreatic cancer in brush cytology specimens

Discrimination between chronic pancreatitis and pancreatic carcinoma can be complicated, particularly in brush cytology specimens. Previous studies have shown that the oxygen insensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase activity based on neotetrazolium reduction can be used for discriminating malignant cells from nonmalignant cells. In the present study, we investigated the value of the assay for differential diagnosis between the two pancreatic diseases. Oxygen insensitivity in ductal epithelial cells in normal human pancreas, chronic pancreatitis, and pancreatic carcinoma was determined by quantitative image analysis in sections of biopsies and in brush cytology preparations. In sections, the reaction in the absence of oxygen was a proper reflection of glucose-6-phosphate dehydrogenase activity, whereas in the presence of oxygen only malignant cells showed a significant reaction. Of 39 brush cytology specimens, diagnosis of all 11 cases of pancreatitis and 28 cases of cancer with the oxygen insensitivity test were in agreement with independent measures of chronic pancreatitis and cancer. The oxygen insensitivity test is a simple and valuable tool in addition to conventional pathology for differential diagnosis between pancreatitis and pancreatic cancer, both in biopsies and in brush cytology specimens.     

Analysis of RNA from brush cytology detects changes in B2M, CYP1B1 and KRT17 levels with OSCC in tobacco users

RNA expression analysis of oral keratinocytes can be used to detect early oral cancer, but a limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner, they have not been validated for quantitative analysis of RNA expression. Earlier we showed RNA from brush cytology of hamster Oral Squamous Cell Carcinoma (OSCC) demonstrated differential expression of B2M and CYP1B1 using real time RT-PCR in a dibenz[a,I]pyrene, tobacco carcinogen, induced model of this disease. Here we show reproducibility of this approach to measuring gene expression in humans. Cytology brush samples from 12 tobacco and betel related OSCC and 17 nonmalignant oral lesions revealed B2M mRNA was enriched in tumor samples while CYP1B1 mRNA was reduced, similar to what was seen in the model system. Additionally, we showed that KRT17 mRNA, a gene linked to OSCC in another brush cytology study, was also enriched in OSCC versus nonmalignant lesions, again supporting the promise of using RNA from brush oral cytology to reproducibly monitor oral gene expression.     

Detection of telomerase activity in exfoliated cancer cells obtained from bile

Telomerase is detected by the telomeric repeat amplification protocol (TRAP) assay in more than 85% of primary cancers. In the present study, we determined telomerase activity using exfoliated bile cells obtained from biliary tract neoplasia specimens. The aim of this study was to provide additional information regarding minimally invasive approaches to the detection of biliary tract cancer in combination with routine cytologic examination. We analyzed for telomerase activity bile juice from patients with gallbladder carcinoma, cholangiocarcinoma, cholecystitis and cholangitis. Semiquantitative determination of telomerase activity was performed using both a fluorescence-based TRAP assay on cell extracts and at the cellular level by an in situ TRAP assay. The fluorescence-based TRAP assay detected bile telomerase activity in samples from 4 of 10 patients with biliary tract cancer. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 6 of 10 patients with biliary tract cancer. However, only one of these samples showed class V cytology. A combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase positive cells may improve the diagnosis of biliary tract cancers with the combination of routine cytologic examination.     

痰和纤维支气管镜刷片细胞学检查在肺癌诊断中的意义

目的探讨痰和纤支镜刷片细胞学检查在肺癌诊断中的意义。方法收集532例同时进行痰和纤支镜检查的肺癌病例,分析痰和纤支镜刷片细胞学检查的敏感性和分型的准确性,与纤支镜咬检组织学对比,评价细胞学在肺癌诊断中的意义。结果痰细胞学的敏感性为33．8％,纤支镜刷片细胞学的敏感性为58．7％,纤支镜咬检组织学的敏感性为39．1％。痰细胞学分型诊断与组织学的符合率鳞癌为95．7％,腺癌为87．2％,小细胞癌为100．0％,痰细胞学区分小细胞癌和非小细胞癌的准确性为100．0％。纤支镜刷片细胞学分型诊断与组织学的符合率鳞癌为93．3％,腺癌为85．4％,小细胞癌为95．5％,纤支镜刷片细胞学区分小细胞和非小细胞癌的准确性为98．0％。结论纤支镜刷片细胞学具有较高的敏感性,在肺癌的诊断中有重要的应用价值。痰细胞学敏感性较低,但可作为纤支镜检查阴性和不能耐受纤支镜检查的肺癌患者的补充检查手段。两者的细胞学分型具有较高的可信度。     

相关蛋白标记物辅助液基薄层细胞检测纤维支气管镜刷片细胞学肺癌分型诊断的探讨

目的 探讨相关蛋白标记物辅助液基薄层细胞检测(TCT)纤维支气管镜刷片细胞学肺癌分型诊断的价值.方法 收集纤维支气管镜刷片细胞学临床诊断剩余标本206例,术后肺肿瘤穿刺标本45例,应用TCT制片技术和免疫细胞化学(ICC)法检测相关蛋白CK10/13、CK7、CK18、CD56和突触素(SYN)在各型肺癌中的表达.结果 CK10/13诊断鳞癌的敏感性和特异性分别为94.7%和72.0%;CK7诊断腺癌的敏感性和特异性分别为98.6%和61.5%;CK18诊断腺癌的敏感性和特异性分别为98.6%和37.5%;CD56诊断小细胞肺癌的敏感性和特异性分别为86.3%和82.9%;SYN诊断小细胞肺癌的敏感性和特异性分别为81.6%和93.5%.CK10/13、CK7和CK18在不同分化鳞癌和腺癌中的表达差异均无统计学意义(均P＞0.05).细胞形态学与ICC联合诊断细胞学未分类癌44例,其中鳞癌、腺癌和小细胞肺癌的分型诊断率分别为90.0%、96.3%和100.O%.结论 相关蛋白ICC检测与细胞学相结合,可提高肺癌分型诊断的准确性,可能成为TCT纤维支气管镜刷片细胞学肺癌分型诊断的一种辅助手段.     

Correlation of tissue and blood plasminogen activation system in breast cancer

The plasminogen activation system plays a crucial role during cancer invasion and metastasis. In the solid tumor, urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1) and uPA receptor (uPAR) are considered as prognostic factors. In this study, we have investigated whether secretion of the uPA, PAI-1 and uPAR from the primary breast cancer tissue can be detected in the blood of the patients using the ELISA assay. We have found that the plasminogen activation system (uPA, PAI-1, uPAR) of tumor tissue is activated from the early stage of breast cancer. However, only a number of metastatic lymph nodes was a prognostic factor in multivariate analysis for relapse. The blood level of the plasminogen activation system correlated with that of tissue in an order of uPAR (r²=0.61; P=0.001), uPA (r²=0.35; P=0.001) and PAI-1 (r²=0.11; P=0.001). We conclude that the total uPAR level of cancer tissue can be substituted by that which is detected in the blood for further clinical applications.     

Evaluation of various cytological examinations by bronchoscopy in the diagnosis of peripheral lung cancer

To improve the efficacy of fibreoptic bronchoscopy in the diagnosis of peripheral lung cancer, we evaluated the effectiveness of various techniques for obtaining samples for cytological examination. Between January 1984 and December 2000, flexible fibreoptic bronchoscopy under fluoroscopic guidance was performed in 1372 patients with lung cancer having no visible endoscopic findings. Histological examination of specimens obtained by forceps biopsy and cytological examinations on imprints of biopsy specimens, brushing, selective bronchial lavage, curettage, transbronchial needle aspiration, rinse fluids of the forceps, brush, curette, and aspiration needle, and all fluids aspirated during the bronchoscopic examinations were evaluated for diagnostic power. Using these techniques, the overall diagnostic rate with bronchoscopy was 93.4%. The sensitivity of the histological examination was 76.9%; additional imprint cytology increased the sensitivity to 84.8% (P<0.0001), while additional cytology on the rinse fluid of the forceps increased the sensitivity to 83.7% (P<0.0001). The addition of both imprint cytology and cytology on the rinse fluid of the forceps increased the diagnostic rate to 86.2% (P<0.0001). Our results indicate that cytological examinations of the imprints of biopsy samples and the rinse fluids of the forceps and the brush improve the efficacy of fibreoptic bronchoscopy in the diagnosis of peripheral lung cancer.     

Toxicity of a 24-hour infusion of gemcitabine in biliary tract and pancreatic cancer: a pilot study

The antitumor activity of gemcitabine is not dose-response related but schedule-dependent. Based on the results of a published phase I study in patients with non-small-cell lung cancer we started a pilot study of a 24-hr infusion of gemcitabine in patients with adenocarcinoma of the pancreas and biliary tract cancer. Twenty-five patients were enrolled and received a 24-hr infusion of gemcitabine once weekly on three consecutive out of 4 weeks. Dose levels of gemcitabine ranged from 100 to 150 mg/m². One of 13 chemotherapy-naive patients had a partial response. Dose-limiting toxicity (DLT) was thrombocytopenia in pretreated patients and neutropenia in chemotherapy-naive patients. Other toxicities were oral mucositis, fever, flu-like symptoms, and asthenia. Maximum tolerated dose (MTD), especially in pretreated patients, was 100 mg/m².     

基于纤支镜刷片细胞学的肺癌细胞核形态的多参数定量研究

目的探讨基于纤维支气管镜刷片细胞涂片中,多参数的细胞核形态定量分析对肺癌的鉴别价值。方法采用回顾性的分析方法,利用HMIAS-2000医学图文分析测量系统,对组织学确诊的纤维支气管镜刷片的细胞核进行形态定量研究(腺癌48例、鳞癌28例、小细胞癌22例,阴性对照组40例)。结果在22个形态定量参数中,19项参数有显著统计学差异(P<0.01),2项有统计学差异(P<0.05),1项无统计学差异(P>0.05)。结论多参数的形态计量揭示了纤支镜刷片细胞学的肺癌与肺良性病变细胞核形态学计量特征,对辅助鉴别诊断良恶性具有一定的意义。     

Sensitive Detection of p53 Gene Mutations in Esophageal Endoscopic Biopsy Specimens by Cell Sorting Combined with Polymerase Chain Reaction Single-strand Conformation Polymorphism Analysis

For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis. Mutations in exons 5–8 of the p53 gene were investigated by FCR-SSCF analysis using 10³ sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR-SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR-SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.     

Multicolor in vivo targeted imaging to guide real-time surgery of HER2-positive micrometastases in a two-tumor coincident model of ovarian cancer

One of the primary goals of oncological molecular imaging is to accurately identify and characterize malignant tissues in vivo . Currently, molecular imaging relies on targeting a single molecule that while overexpressed in malignancy, is often also expressed at lower levels in normal tissue, resulting in reduced tumor to background ratios. One approach to increasing the specificity of molecular imaging in cancer is to use multiple probes each with distinct fluorescence to target several surface antigens simultaneously, in order to identify tissue expression profiles, rather than relying on the expression of a single target. This next step forward in molecular imaging will rely on characterization of tissue based on fluorescence and therefore will require the ability to simultaneously identify several optical probes each attached to different targeting ligands. We created a novel 'coincident' ovarian cancer mouse model by coinjecting each animal with two distinct cell lines, HER2⁺/red fluorescent protein (RFP)⁻ SKOV3 and HER2⁻/RFP⁺ SHIN3-RFP, in order to establish a model of disease in which animals simultaneously bore tumors with two distinct phenotypes (HER2⁺/RFP⁻, HER2⁻/RFP⁺), which could be utilized for multicolor imaging. The HER2 receptor of the SKOV3 cell line was targeted with a trastuzumab–rhodamine green conjugate to create green tumor implants, whereas the RFP plasmid of the SHIN3 cells created red tumor implants. We demonstrate that real-time in vivo multicolor imaging is feasible and that fluorescence characteristics can then serve to guide the surgical removal of disease. ( Cancer Sci 2009; 100: 1099–1104)