细胞DNA倍体分析和EBERs原位检测在鼻咽癌早期诊断中的应用

目的　探讨鼻咽脱落细胞进行DNA倍体和EB病毒编码RNA (EBERs)检测在鼻咽癌诊断中的应用。方法　对 38例经细胞学诊断为鼻咽癌和 8例为正常的鼻咽细胞涂片分别进行图像分析测定细胞DNA含量和EB病毒EBERs原位杂交检测。结果　与病理细胞学诊断相比 ,DNA异倍体分析和EBERs检测诊断癌的敏感性分别为 5 0 %和 92 %,其特异性均为 10 0 %;其阴性预测值分别为 30 %和 72 %。结论　与EBERs检测相比 ,DNA异倍体分析的诊断敏感性和阴性预测值较低 ,差异具有显著性 (分别为P <0 0 0 1和P <0 0 5 )。证明在鼻咽细胞学涂片应用EB病毒原位杂交检测诊断鼻咽癌优于应用DNA异倍体分析诊断。与细胞DNA图像分析相比 ,EB病毒原位杂交检测具有客观、实验条件简单等优点 ,在鼻咽癌可疑病人的早期诊断和鉴别诊断上具有重要的实用价值。     

用碘标记核酸检查鼻咽癌上皮细胞中的EB病毒基因

用同位素碘化钠直接标记核酸。以细胞内原位杂交实验检查鼻咽癌上皮细胞中的EB病毒基因。共检查病理确诊的10例鼻咽癌活检标本的细胞涂片,其中9例直接证实有EB病毒核酸。本文建立并改进了细胞内原位核酸杂交技术,首先使用重组的单链DNA作探针,并将碘标记的核酸用于原位核酸杂交实验。     

EBER原位杂交结合CK免疫组织化学双染在诊断鼻咽癌中的应用价值

目的探讨全自动免疫组织化学仪行EB病毒早期RNA(Epstein-Barr virus early RNA,EBER)与细胞角蛋白(cytokeratin,CK)双染在鼻咽癌诊断中的应用价值。方法收集我院2018年1月至2019年12月鼻咽活检组织60例,根据诊断结果进行分组,A组为鼻咽非角化型未分化性癌30例,B组为上皮性病变30例。在VENTANA全自动免疫组织化学仪平台上分别对这两组病变进行CK免疫组织化学染色、EBER原位杂交、EBER原位杂交结合CK免疫组织化学(EBERCK)双染标记,其中双染再分为是否复染苏木素,比较染色情况。结果免疫组织化学CK单染A、B组均可见阳性细胞;原位杂交EBER单染A组鼻咽癌细胞阳性,B组表达呈阴性;EBER-CK双染A组鼻咽癌细胞呈双阳性,B组病变细胞呈CK阳性、EBER呈阴性,未见双阳性表达;EBER-CK双染复染苏木素后的表达与未复染苏木素的表达相同,但对比差,不易观察。结论全自动免疫组织化学仪行EBER-CK双染有助于鼻咽癌组织学诊断,不复染苏木素效果更佳,值得推广。     

EB病毒诱导的胸腺恶性T细胞淋巴瘤细胞体外长期培养研究

建立含有EB病毒的T细胞淋巴瘤细胞系,为探讨EB病毒的致瘤机理,研究EB病毒在T细胞淋巴瘤发生过程中的作用提供手段.在TPA协同EB病毒诱导胸腺恶性T细胞淋巴瘤动物模型的基础上,联合应用IL-2,将诱导的肿瘤组织进行体外细胞培养,成功地分离获得一株在体外长期存活的淋巴细胞TET.T细胞亚群分类实验证实TET细胞为CD4阳性的T淋巴细胞,PCR和原位杂交可检测到EB病毒的EBERs、LMP1和BARF1,并有LMP1蛋白的表达.TET细胞的获得,有望在体外建立转化细胞系,为体外研究EB病毒的致瘤机理及防治提供理想的实验材料.     

携带绿色荧光报告基因的EBV-LMP1真核表达载体构建与鼻咽癌细胞中的表达

潜伏膜蛋白1(LMP1)是由EB病毒编码的致瘤蛋白,众多研究表明LMP1蛋白可通过NF-κB、p38 MAPK、c-JNK等多条重要信号通路引起鼻咽癌细胞的生物学行为改变。我们从EB病毒阳性的B95-8狨猴淋巴瘤细胞中克隆EB病毒LMP1 c DNA,构建携带绿色荧光基因的真核表达质粒p IRES2-Zs-Green1-LMP1,通过脂质体转染的方法将质粒导入鼻咽癌细胞株CNE1、CNE2中,利用质粒所携带的绿色荧光蛋白表达粗略计算转染的效率,通过免疫细胞化学(ICC)、RT-PCR、Western-Blot检测该质粒的表达。本实验室所构建的p IRES2-Zs-Green1-LMP1表达质粒能在鼻咽癌细胞内表达LMP1蛋白,为后续的实验研究奠定基础。     

三种常用宫颈癌检查方法比较

目的比较常规细胞学、高危型HPV-DNA病毒测定及DNA倍体分析方法在诊断宫颈癌及癌前病变(cervical intraepithelial neoplasia, CIN)的优缺点。方法 374名妇女做宫颈活检前进行了宫颈细胞常规细胞学诊断、高危型HPV-DNA病毒测定和DNA倍体分析。用宫颈刷刷取宫颈细胞,每个样本制成二张液基薄层玻片,一张经巴氏染色用于常规细胞学诊断(TBS),另一张经Feulgen染色做DNA倍体分析,剩余样本做HC2-HPV-DNA检测。宫颈活检样本进行组织病理诊断。结果 374例宫颈活检样本中发现167例宫颈炎,92例CIN1,63例CIN2,37例CIN3和15例宫颈浸润癌。在259例宫颈炎和CIN1病例中,高危型HPV检测201例为阴性,58例为阳性;DNA倍体分析发现有异倍体细胞的病例为40例,219例为阴性;TBS诊断ASCUS及以上级别的病例30例,229例为正常。在115例CIN2、CIN3和宫颈浸润癌中,高危型HPV病毒测定阳性为98例,阴性为17例;DNA倍体分析阳性为93例,阴性为22例;TBS诊断ASCUS及以上级别的有79例,正常为36例。诊断CIN2及以上级别的敏感性、特异性、阳性预测值和阴性预测值,高危型HPV测定分别为85.2%、77.6%、62.8%和92.2%;DNA倍体分析分别为80%、84.6%、69.9%和90.9%;TBS诊断为68.7%、88.4%、72.5%和86.4%。结论在诊断宫颈高级别癌前病变(CIN2和CIN3)和宫颈癌上,HPV-DNA病毒方法敏感性高,特异性低;常规细胞学方法敏感性低,特异性高;DNA倍体分析方法正好介于二种方法之间。三种方法均有优缺点,具体运用应根据实际情况而定。     

细胞核内特征值的改变可引起DNA倍体的变化

目的通过测定不同DNA倍体细胞,研究细胞核内特征值的改变。方法用宫颈刷刷出宫颈细胞,经固定后,用涂片离心机制成二张玻片,一张行巴氏染色作TBS诊断,另一张行Feulgen染色做DNA定量测定。通过对宫颈细胞核图像内像素的统计,计算出细胞核内多种特征值,比较不同DNA倍体细胞内特征值的不同。结果 161873例妇女行宫颈细胞学检查,常规细胞学检查发现2454例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)和523例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL);而DNA倍体分析发现3412例有3个以上>5c细胞。84%以上的LSIL和HSIL病例均可见倍体异常细胞。与2c细胞相比,4c、5c、7c及9c细胞核面积及核半径明显增大;7c、9c细胞核内平均光学密度和紧实度均值也有明显改变,而光密度方差和灰度熵无变化。结论宫颈细胞DNA倍体改变往往伴有细胞形态和DNA核内分布等特征值的改变。     

褪色HE染色切片ALK基因融合检测可行性探索

目的 探索利用HE染色切片褪色样本进行ALK基因融合检测的可行性。方法 选取3例ALK基因融合阳性的福尔马林固定石蜡包埋（formalin fixed and paraffin embedded,FFPE）组织样本的HE染色切片,分别经盐酸乙醇褪色法和高锰酸钾草酸氧化漂白法褪色后,提取RNA,采用荧光定量RT-PCR(qRT-PCR)技术,检测ALK基因融合。结果 从经盐酸乙醇褪色法或高锰酸钾草酸氧化漂白法褪色的HE染色切片中提取的RNA的纯度与从对照切片中提取者无显著差异,但其浓度较从对照切片中提取者显著降低。在模板量相同的条件下,经盐酸乙醇褪色的HE染色切片与对照组切片均检测到明确的ALK基因融合阳性,而经高锰酸钾草酸褪色的HE染色切片无任何曲线升起。从经盐酸乙醇褪色的HE染色切片中提取的RNA的完整性与从对照切片中提取的RNA的完整性无差异,但显著高于从经高锰酸钾草酸褪色的HE染色切片中提取者。为保证研究的准确性,本研究采用免疫组织化学与荧光原位杂交法对选取的ALK基因融合阳性样本进行验证,结果发现所选样本均为明确阳性。结论 HE染色切片经盐酸乙醇褪色后提取的RNA可以用于ALK...     

EB病毒介导人鼻咽上皮细胞永生化早期阶段的生物学观察

EB病毒转化人鼻咽上皮细胞,建立体外多阶段细胞模型有利于从细胞和分子水平对肿瘤发病机制作深入研究.我们利用与鼻咽癌密切相关的EB病毒和TPA的协同作用,观察原代人胚鼻咽上皮细胞逃避老化期后其生物学特性的变化.结果表明,EB病毒感染的人胚鼻咽上皮细胞在原代培养后期老化相关半乳糖苷酶(SA-β-Gal)表达降低,形态学上发生改变,出现转化灶样集落,群体倍增时间降低,体外培养寿命延长,表明EB病毒促使部分原代人胚鼻咽上皮细胞逃避老化期、进入永生化早期阶段.这些研究资料为进一步阐明上皮细胞永生化分子机制及建立人鼻咽上皮细胞永生化模型提供实验依据.关键词EB病毒人鼻咽上皮细胞老化期永生化     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16^INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16^INK4a、p53、p21^WAF1／CIP1和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16^INK4a表达而阻断p16^INK4a／Rb途径,上调转录因子E2F1,而对p53、p21^WAF1／CIP表达无明显的影响。结果初步揭示,EB病毒介导的p16^INK4a／Rb／E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Roles of DNA cytometry and detection of EBERs in predicting a diagnosis of nasopharyngeal carcinoma.

OBJECTIVE: To analyze the suitability of DNA cytometry and detection of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) on nasopharyngeal brushings for predicting a diagnosis of nasopharyngeal carcinoma (NPC). STUDY DESIGN: Cytologic preparations in 66 cases suspicious for NPC were evaluated for NPC diagnosis in comparison with the histologic diagnosis. Based on cytologic examination, 38 cases containing cytologically proven cancer and 8 cases interpreted as cytologically negative for cancer with adequate cellularity in the smear specimens were analyzed for DNA ploidy with an image analyzer and for EBER expression by in situ hybridization (ISH). RESULTS: Based on the cytologic diagnosis, DNA aneuploidy analysis, DNA nondiploidy analysis and EBER detection demonstrated a sensitivity of 50%, 84% and 92%, respectively, with the same specificity, 100%, for predicting a diagnosis of cancer. Their negative predictive values were 30%, 57% and 73%, respectively. There was a significant difference between DNA aneuploidy analysis and EBER analysis in sensitivity (P < .001) and in negative predictive value (P < .05) but not between DNA nondiploidy analysis and EBER analysis even though EBER analysis showed a slightly higher value in both parameters (P > .1 and P > .5, respectively). CONCLUSION: ISH for EBERs in cytologic smears showed a role superior to that of DNA aneuploidy analysis in the diagnosis of NPC. Considering its advantages of simple experimental conditions and lower cost as compared with DNA measurement, EBER detection can play a practical and important diagnostic role in patients suspected of having primary NPC.     

HER-2/neu evaluation by fluorescence in situ hybridization on destained cytologic smears from primary and metastatic breast cancer

OBJECTIVE: To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain. STUDY DESIGN: Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears. RESULTS: HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears. CONCLUSION: HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.     

Detection of Human Polyomavirus Dna In Papanicolaou Stained Smears of Urinary Sediment By In Situ Hybridization

Papanicolaou stained smears of urinary sediment containing inclusion bearing urothelial cells suggestive of human polyomavirus infection were destained and reprocessed for in situ hybridization using a biotinylated probe for human polyomavirus DNA. Seven slides were processed in this way. A hybridization signal for viral DNA was noted in each case, even in smears that had previously been stored for 11 years. This simple and rapid non-radioactive detection system is a valuable supplement to routine urinary cytology for the definitive diagnosis of this virus infection.     

A quantitative evaluation of cytophotometric DNA analysis in retrospective studies using archival tumor specimens.

Methods developed for the cytophotometric analysis of archival tumor specimens used in retrospective studies were evaluated quantitatively. May-Grünwald-Giemsa-stained cytologic slides up to 20 years old could be restained by the Feulgen reaction with excellent results if they were destained in methanol and refixed in formaldehyde prior to Feulgen staining. Storage time had only a minor influence on Feulgen stainability. However, a considerable variation in the intensity of the Feulgen stain was observed between different slides stained simultaneously; this variation was not related to storage time. As a consequence of this variation, the use of internal staining controls, such as granulocytes, is an absolute necessity in the quantitative comparison of different slides. By expressing DNA data from the tumor cells in relative values (c values) related to the internal staining control (with a defined mean value of 2c), Feulgen ploidy level determinations could be made as accurately from measurements on old, destained slides as on cells obtained from fresh tumor material. The ploidy level could also be accurately determined in most cases of prostatic carcinoma from measurements on histopathologic sections.     

Evaluation of applying feulgen stain for DNA analysis on destained hematoxylin-eosin-stained cytologic smears

OBJECTIVE: To evaluate the feasibility and reliability of DNA analysis performed on the original hematoxylin-eosin (HE)-stained cytologic specimens by destaining the slides and restaining with the Feulgen method. STUDY DESIGN: Image cytometric analysis of DNA ploidy status was performed in a comparative study on 20 cytologic preparations from 10 nasopharyngeal carcinomas. Ten smears were stained directly by the Feulgen method, and the others were Feulgen stained after HE destaining. RESULTS: There was 90% overall concordance in DNA determination and a good correlation (r = .97, P < .001) between the DNA indices determined by the 2 methods. Discordance was probably due to tumor heterogeneity. CONCLUSION: This study demonstrated that image cytometric DNA analysis on previously routinely HE-stained cytologic preparations is feasible and reliable. This method permits retrospective studies on archival cytologic material from patients with long term follow-up data.     

Predictive value of DNA cytometry in CIN 1 and 2. Image analysis of 193 cases

OBJECTIVE: To investigate DNA image cytometry for predicting the prognosis of cervical intraepithelial neoplasia (CIN). STUDY DESIGN: Smears from 151 women affected by CIN 1 or 2 on cytology with minimal follow-up of three years were included. Sixty-seven showed progression, with histologically confirmed carcinoma in situ or invasive cancer. Eighty-four cases showed regression of the disease, which was cytologically, histologically and colposcopically confirmed. Papanicolaou-stained smears were destained, and the Feulgen reaction was performed with consecutive image DNA cytometry of suspicious cells using an image analysis system (Cires, Zeiss, Germany). The DNA index of the greatest stemline and the number of single aneuploid cells, using 9c exceeding events, were computed. RESULTS: In the group with progression, an aneuploid DNA stemline was found in 25 smears (26.9%). In 64 cases (66.7%) more than one aneuploid event was detected. The total number of aneuploid cases in this group was 76 (81%). In the group without progression, the number of aneuploid stemlines was 2 (2%). Single aneuploid cells could be found in five cases (5%). The overall number of aneuploid cases in that group was five. The sensitivity was 74.3%, positive predictive value 85.2% and negative predictive value 77%. CONCLUSION: Aneuploidy is a marker for prospective malignancy in cervical Papanicolaou smears. DNA image cytometry, as an additional method, can be used to predict outcome in patients with CIN 1 and 2 of the cervix. DNA cytometry is not a screening method but can add further information for a treatment decision in doubtful cases.     

Cytophotometric studies in suspicious cervical smears.

Papanicolaou stained smears of various cervical lesions diagnosed as "suspicious" by routine cytology were reviewed with regard to different cell types leading to this diagnosis. The smears were then submitted to Feulgen hydrolysis and redyed by Acriflavin-SO2 for fluorescence-cytophotometry. In nine of 14 cases measured, the DNA content of all types of "suspicious" cells was increased with DNA modes at euploid levels of 2 n, 4 n and 8 n indicating that the "suspicious" cells in those cases are polyploid. However, in five cases aneuploid DNA-distribution patterns were found similar to those observed in carcinoma in situ or severe dysplasia. Since polyploidization may be considered as a cellular response to higher functional requirement (i.e. inflammation or regeneration) a "suspicious" cervical smear with a polyploid DNA-distribution pattern may reverse to normal cervical epithelium after normal conditions are restored. However, a "suspicious" smear with an aneuploid DNA-distribution pattern should be considered more seriously as being related to a precancerous lesion requiring immediate surgical treatment.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.