Role of biliary brush cytology in primary sclerosing cholangitis

OBJECTIVE: To assess the role of brush cytology in the routine evaluation of patients with primary sclerosing cholangitis (PSC). STUDY DESIGN: From January 1995 to June 2000, 64 brush cytology specimens were obtained from 21 patients who had at least one cytologic sample obtained during endoscopic retrograde cholangiography. All patients had a diagnosis of primary sclerosing cholangitis. Cases were classified as benign, atypical or malignant according to major cytologic criteria (nuclear contour and chromatin irregularities) and minor cytologic criteria (polarity, cellularity, nuclear enlargement, mitosis, increased nuclear/cytoplasmic ratio) used by us to diagnose biliary brush cytology. Follow-up was available in all cases. RESULTS: Diagnoses were benign (13), atypical (5) and malignant (3) on cytology. Follow-up of the 13 benign cases showed bile duct stones (2), gallbladder adenocarcinoma at cholecystectomy (1), ascending cholangitis (1) and clinically/cytologically by benign follow-up (9). Five of 13 benign cases had subsequent liver transplantation for liver failure, with explants showing changes of primary sclerosing cholangitis. Of the 3 malignant cases, 1 had carcinoma in situ on biopsy, with the explanted liver showing high grade dysplasia; the second patient had cholangiocarcinoma on explant; and the third had hepatocellular carcinoma on liver five needle aspiration. The 5 patients with atypical cytology were reclassified on review as reactive (3) and atypical not otherwise specified (2). Follow-up showed benign disease in 3 of 3 atypical cases reclassified as reactive; 2 of 2 reclassified as atypical not otherwise specified showed low grade dysplasia in the explant. CONCLUSION: The overall incidence of malignancy was low (3 of 21) in patients with PSC. Bile duct brushing is a sensitive method of detecting neoplasia in the setting of PSC when well-defined cytologic criteria are applied.     

Immunocytochemical detection of cyclin, a proliferation-associated protein, in cytologic preparations.

Cyclin is a nuclear protein associated with DNA-polymerase delta, whose expression correlates with cell proliferation in vitro. To assess the value of cyclin staining in diagnostic cytology, an anticyclin monoclonal antibody was used to survey cyclin expression in cytologic preparations obtained as "bench top" aspirates from surgically resected specimens. The tissues assayed included carcinomas and normal and benign proliferative tissues of renal, mammary, prostatic and colonic origin. Staining was performed via the avidin-biotin-complex immunoperoxidase method. The staining of tumor cells was nuclear, with sparing of the nucleoli; the results were variable in different areas of a given tumor and varied significantly between tumors of the same histopathologic type. Benign proliferative tissues also showed staining. Nonproliferative tissues, such as renal tubules adjacent to a renal cell adenocarcinoma, were largely, but not entirely, nonstaining. The percentage of cyclin-positive nuclei was sometimes much higher than the typical percentages of tumor cells found in S phase. This observation was confirmed in two cases in which cyclin staining was much greater than the percentage of S-phase cells detected by flow cytometry. This suggests either stabilization of the protein beyond S phase in cells and/or dysregulation of cyclin expression in malignant cells. The viability of unfixed surgically resected tissue may also have affected the detection of cyclin, a problem that should not exist with clinically aspirated tissue fixed immediately after aspiration. These preliminary observations suggest that the selective use of cyclin staining may facilitate cytologic diagnoses. Furthermore, the wide range of cyclin expression within tumors of one histologic type suggests that cyclin expression may serve as a new parameter for investigating tumor behavior and prognosis.     

Detection of IGF2BP3, HOXB7, and NEK2 mRNA Expression in Brush Cytology Specimens as a New Diagnostic Tool in Patients with Biliary Strictures

Introduction

It is a challenging task to distinguish between benign and malignant lesions in patients with biliary strictures. Here we analyze whether determination of target gene mRNA levels in intraductal brush cytology specimens may be used to improve the diagnosis of bile duct carcinoma.

Materials and Methods

Brush cytology specimens from 119 patients with biliary strictures (malignant: n = 72; benign: n = 47) were analyzed in a retrospective cohort study. mRNA of IGF-II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), Forkhead box M1 (FOXM1), kinesin family member 2C (KIF2C) and serine/threonine kinase NEK2 was determined by semi-quantitative RT-PCR using the ΔCt method.

Results

IGF2BP3 (p<0.0001), HOXB7 (p<0.0001), and NEK2 (p<0.0001) mRNA expression levels were significantly increased in patients with cholangiocarcinoma or pancreatic cancer. Median ΔCt values differed by 3.5 cycles (IGF2BP3), 2.8 cycles (HOXB7) and 1.3 cycles (NEK2) corresponding to 11-fold, 7-fold and 2.5-fold increased mRNA levels in malignant versus benign samples. Sensitivity to detect biliary cancer was 76.4% for IGF2BP3 (80.9% specificity); 72.2% for HOXB7 (78.7% specificity) and 65.3% for NEK2 (72.3% specificity), whereas routine cytology reached only 43.1% sensitivity (85.4% specificity). Diagnostic precision was further improved, when all three molecular markers were assessed in combination (77.8% sensitivity, 87.2% specificity) and achieved 87.5% sensitivity and 87.2% specificity when molecular markers were combined with routine cytology.

Conclusions

Our data suggest that measuring IGF2BP3, HOXB7 and NEK2 mRNA levels by RT-PCR in addition to cytology has the potential to improve detection of malignant biliary disorders from brush cytology specimens.     

Exfoliative colonic cytology. A simplified method of collection and initial results

Exfoliative colonic cytology for the diagnosis of colorectal cancer has been largely abandoned due to (1) the widespread use of colonoscopy, (2) the cumbersome methods of cell collection and (3) the occasional difficulty of interpreting the cytologic findings in the presence of inflammatory bowel disease or adenomas. This paper describes a newly formulated bowel preparation for routine colonoscopy, based on imbibing 2 L to 4 L of a balanced electrolyte solution, in which the recovered precolonoscopic effluent (using a convenient disposable collecting kit) yielded cells for cytologic evaluation from 70% of a group of 80 patients at high risk for large bowel neoplasia. Cytology demonstrated neoplastic cells in most cases of endoscopically proven cancer. These results suggest that colonic exfoliative cytology may be useful as a supplemental test to routine colonoscopy. This could be enhanced by further methodologic modifications to the collecting and cytologic methods; large long-term studies are needed to evaluate the potential usefulness of colonic exfoliative cytology.     

Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma

The high affinity neurotensin receptor (NTSR1) mediates most of the biologic effects of neurotensin (NT), a 13-amino acid peptide that stimulates growth in certain cell types. NT is expressed in fetal but not differentiated colonic epithelium and is re-expressed in colonic adenocarcinoma. The cognate receptor, NTSR1, is also not expressed or is present at a low level in adult colonic epithelial cells but is expressed in most colon cancer cell lines. These observations suggest that altered NT-NTSR1 signaling may be associated with malignant transformation in the colon. To further understand the possible role of NTSR1 expression in colonic tumorigenesis and progression, we examined NTSR1 mRNA by in situ hybridization in normal colonic mucosa, adenomas, and colonic adenocarcinomas. NTSR1 mRNA expression was undetectable or weak in superficial differentiated epithelial cells of normal colonic epithelium, but adenomas and adenocarcinomas showed moderate to strong expression (p<0.05). Adenocarcinomas showed a higher level of expression compared to adenomas (p<0.05). Furthermore, adenocarcinomas that infiltrated into and beyond the muscularis propria showed a higher intensity of NTSR1 expression compared with tumors that were localized to the mucosa or submucosa. In some cases, infiltrating margins and foci of lymphovascular invasion showed a higher intensity of expression than the main mass of the tumor. These results suggest that increased NTSR1 expression may be an early event during colonic tumorigenesis and also contribute to tumor progression and aggressive behavior in colonic adenocarcinomas. NTSR1 may thus be a potential target for preventive or therapeutic strategies in colon cancer.     

False "false-positive" results in diagnostic cytology

With the introduction of transbronchial brushings and fine needle aspiration biopsy, which enable us to obtain samples directly from lesions, the diagnostic potential of cytology for the detection of malignancy, including early cancer, has been greatly enhanced. From 1976 to 1982, five positive cytology reports were initially considered to be "false positives" on the basis of negative gross findings, benign operative biopsies or negative histologic findings in the resected surgical specimens. However, these proved to be false "false positives," based upon the clinical follow-up or further examination of the surgical specimens. Presentation is made of three of these cases with positive cytologic findings and initially negative histologic diagnoses, with an analysis of the causes of the latter. From our experience, four types of cancerous lesions seem prone to being missed during gross examination, namely: any small cancer with a consistency similar to that of the parenchyma of the organ in which the tumor is located, superficially invasive carcinoma, scar cancer and a radiologically occult lung cancer in the presence of a coexisting radiologically demonstrable lesion. With more clinical application of these cytologic methods, false "false positives" are expected to occur more often.     

特异性人端粒酶催化亚单位基因小干扰RNA对HeLa细胞生长的抑制作用

通过设计并化学合成人端粒酶催化亚单位(hTERT)特异性siRNA,观察其对hTERT表达水平及肿瘤细胞生长的影响。将hTERT-siRNA以脂质体法转染入HeLa细胞,应用RT-PCR、实时定量TRAP、Western印迹、软琼脂克隆形成实验、荷瘤裸鼠肿瘤内注射等方法检测细胞内hTERTmRNA、蛋白质表达水平及对肿瘤细胞生长的影响。RT-PCR、实时定量TRAP和Western印迹的结果显示hTERT-siRNA明显降低了HeLa细胞内hTERT的mRNA及蛋白质表达水平并伴随有端粒酶活性的下降。克隆形成实验表明hTERT-siRNA组的体外肿瘤形成能力受到抑制。荷瘤裸鼠肿瘤内注射hTERT-siRNA使肿瘤平均体积显著小于对照组。TUNEL凋亡检测表明hTERT-siRNA转染组的凋亡率明显高于对照组。研究表明hTERT特异性siRNA可以明显抑制HeLa细胞内hTERT的表达水平,对其生长有明显抑制作用,是一种有前途的肿瘤治疗新方法。     

A gastrointestinal-specific monoclonal antibody that may be of clinical value in cytologic material

A new monoclonal antibody (MAb), 29-10, produced by immunization of mice with cells from the SW 1116 colorectal carcinoma cell line, detected an antigen present in cytologic touch imprints of surgically resected normal and neoplastic gastrointestinal (GI) tissue, including specimens from the stomach and the colon. These imprints were fixed in 95% ethanol and stained with the avidin-biotin immunoperoxidase technique. In tested cases, 22 (100%) of 22 imprints from GI adenocarcinomas and from normal GI tissue, as well as 13 (56.6%) of 23 imprints from colonic polyps, stained positively while no staining was demonstrable in imprints from other tissues. In histologic sections, only 4 (23%) of 17 colonic adenocarcinomas and 3 (11.5%) of 26 polyps stained positively. The staining ability of MAb 29-10 was compared to that of MAb 19-9, another colorectal antibody, and was found to be markedly superior for binding of the antigen in cytologic preparations. This tissue-specific antibody may be useful in identifying malignant cells of metastatic carcinoma as to their GI tract origin.     

6-姜烯酚对溃疡性结肠炎小鼠结肠上皮细胞Notch信号通路的影响

目的:观察6-姜烯酚对溃疡性结肠炎小鼠结肠上皮细胞Notch信号通路的调控作用。方法:清洁级昆明小鼠40只随机分为正常组(10只)和造模组(30只),造模组采用2%葡聚糖硫酸钠(DSS)自由饮用诱导溃疡性结肠炎模型,造模15 d后分为模型组,6-姜烯酚组,阳性对照组,每组10只。正常组、模型组灌胃生理盐水,6-姜烯酚组采用6-姜烯酚100 mg/(kg·d)灌胃,阳性对照组采用柳氮磺吡啶100 mg/(kg·d)灌胃,给药20 d后处死,观察小鼠结肠病理组织学改变,免疫荧光双标法检测结肠上皮细胞Hes-1、Math-1蛋白的表达,RT-PCR法检测结肠上皮组织Notch-1、Hes-1、Math-1 mRNA的表达,Western blot法检测结肠上皮组织Notch-1、Hes-1、Math-1蛋白的表达。结果:与正常组相比,模型组小鼠结肠上皮组织Notch-1、Hes-1蛋白和mRNA相对表达量显著升高( P＜0.01), Math-1蛋白和mRNA相对表达量显著降低( P＜0.01);与模型组比较,6-姜烯酚组、柳氮磺吡啶组小鼠结肠上皮组织Notch-1、Hes-1蛋白和mRNA相对表达量显著降低( P＜0.01), Math-1蛋白和mRNA相对表达量显著升高( P＜0.01) 。结论:6-姜烯酚能够抑制Notch通路的过度活化,调节结肠上皮吸收细胞系和分泌细胞系之间分化的平衡,修复受损结肠粘膜组织。     

EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

Cyclin E expression and early cervical neoplasia in ThinPrep specimens. A feasibility study

OBJECTIVE: To investigate cyclin E expression as a possible marker for early cervical neoplasia using ThinPrep gynecologic specimens from premenopausal women. STUDY DESIGN: Archived ThinPrep liquid-based cervical/endocervical specimens (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) diagnosed as human papillomavirus infection (HPV) (20), atypical squamous cells of undetermined significance (ASCUS) (48) and within normal limits (WNL)/benign cellular changes (BCC) (21) were resampled in duplicate, fixed in 95% ethanol, subjected to immunocytochemical staining with the cyclin E antibody (clone 13A3, Novocastra Laboratories Ltd., Newcastle upon Tyne, U.K.) and HPV antibody (clone K1H8, Dako Corporation, Carpinteria, California, U.S.A.) and the expression scored by two pathologists and correlated with the cytologic diagnosis. A case was scored as positive if it contained > 10 abnormal squamous cells with nuclear immunocytochemical staining. RESULTS: The cylin E antibody assay was positive in 20 (100%) cases cytologically diagnosed as HPV. These cases were also anti-HPV antibody positive. Four cases (19%) cytologically diagnosed as WNL/BCC were cyclin E positive. Of these, two were anti-HPV antibody positive. Thirty-four (73%) cases cytologically diagnosed as ASCUS were positive for the cyclin E assay and for anti-HPV antibody staining. CONCLUSION: Cyclin E expression correlates strongly with morphologic features of HPV in ThinPrep specimens and may serve as a surrogate marker for HPV infection and early cervical preneoplastic lesions.     

Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 mRNA were detected in LPMC. CRF receptor type 2β mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.     

Bronchial brushing during fiberoptic bronchoscopy for the cytodiagnosis of lung cancer: comparison with sputum and bronchial washings.

In a study of 50 cases of bronchogenic carcinoma in which brushings and washings during fiberoptic bronchoscopy, as well as sputum cytopathologic examinations were performed in the same patients, accuracy rates were respectively: 76 per cent, 76 per cent and 56 per cent. The main cytologic differences setting brush apart from wash and sputum specimens referred to the arrangement of tumor cells as well as the distribution of chromatin within their nuclei. These differences appeared related to cell degeneration which was minimal in brush materials and maximum in sputum specimens. Only six cases were assigned a different cell type of bronchogenic carcinoma when brush cytopathologic diagnoses were compared with results obtained by biopsy or lobectomy specimens. Our findings are consistent with the view that the brush technique is very accurate for the cytodiagnosis of lung cancer and becomes also rather specific once cytologic characteristics of the fresher samples obtained become familiar to the cytopathologist. Non-observance of the special characteristics of these better preserved cellular samples is the major pitfall as to diagnosing, cell typing and judging degree of differentiation of bronchogenic carcinoma in brush cytology specimens.