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。     

应用H.E染色的鼻咽癌细胞涂片进行DNA含量测定和EB病毒基因原位杂交的研究

分析鼻咽癌常规 H.E染色细胞涂片分别作 Feulgen染色检测 DNA含量和原位杂交检测 EB病毒编码 RNAs(EBERs)的效果 ;将常规 HE染色的 9例正常鼻咽细胞涂片和 38例鼻咽癌细胞涂片用 1%酸性酒精褪色 ,然后作 Feulgen染色 ,应用图象分析仪测定涂片细胞 DNA含量 ,并将 38例鼻咽癌细胞病例的阳性涂片进行 EBERs原位杂交检测 ;结果显示正常鼻咽细胞均为 DNA二倍体核型 ,38例癌阳性涂片中呈 DNA二倍体者 6例 ,DNA非二倍体者 32例 (84.2 % ) ,癌细胞核 EBERs阳性者 35例 (92 .1% ) ;结果表明 HE染色的鼻咽癌细胞学涂片经褪色后作 Feulgen染色和原位杂交检测 ,能较满意进行 DNA和 EBERs分析 ,表明常规 H.E染色和褪色处理均不影响 Feulgen染色和 EB病毒原位杂交的质量效果。本检测方法可靠、可行 ,适用于常规染色细胞学样本的回顾性研究和随访研究 ,并可用于鼻咽癌可疑病人的诊断和鉴别诊断     

Epstein–Barr virus noncoding RNAs from the extracellular vesicles of nasopharyngeal carcinoma (NPC) cells promote angiogenesis via TLR3/RIG-I-mediated VCAM-1 expression

Viral noncoding RNAs (Epstein–Barr virus-encoded RNAs, EBERs) are believed to play a critical role in the progression of lymphoma and nasopharyngeal carcinoma (NPC). However, the accurate mechanisms accounting for their oncogenic function have not been elucidated, especially in terms of interaction between tumor cells and mesenchymal cells. Here, we report that, in addition to NPC cells, EBERs are also found in endothelial cells in Epstein–Barr virus (EBV)-infected NPC parenchymal tissues, which implicates NPC-derived extracellular vesicles (EVs) in transmitting EBERs to endothelial cells. In support of this hypothesis, we first ascertained if EBERs could be transferred to endothelial cells via EVs isolated from NPC culture supernatant. Then, we clarified that EVs-derived EBERs could promote angiogenesis through stimulation of VCAM-1 expression. Finally, we explored the involvement of EBER recognition by TLR3 and RIG-I in NPC angiogenesis. Our observations collectively illustrate the significance and mechanism of EVs-derived EBERs in angiogenesis and underlie the interaction mechanisms between EBV-infected NPC cells and the tumor microenvironment.     

Flow cytometric analysis and cytopathology of body cavity fluids

A total of 75 samples of body cavity fluids from 71 patients were analyzed by both flow cytometry (FCM), to detect cells with an abnormal DNA content (aneuploidy), and by conventional cytopathology. Samples included 27 pleural fluids, 35 peritoneal fluids, 11 peritoneal washings and 2 pericardial fluids. For cytologic examination, the samples were prepared using standard techniques. Samples for FCM analysis were centrifuged and exposed to a hypotonic solution containing detergent and propidium iodide, a DNA intercalating fluorescent stain. Aneuploidy as well as cytologic malignancy were found in 17 samples. Forty-seven samples had normal DNA histograms by FCM and were also cytologically negative. Four samples suspicious by cytology but normal by FCM were from patients with renal-cell carcinoma (two samples from the same patient), endometrial adenocarcinoma without metastasis and chronic lymphocytic leukemia. Three samples abnormal by FCM but negative by cytology were from patients with ovarian cystadenoma, cirrhosis and uterine leiomyoma. FCM showed aneuploidy in four cytologically negative samples from patients with histologically proven malignancy (lymphoma, colonic adenocarcinoma, cervical squamous cell carcinoma, and endometrial adenosquamous carcinoma). Based on these results, FCM analysis combined with conventional cytopathology yielded 100% sensitivity, 100% predictive value of a negative result and 94% specificity. This rapid and quantitative FCM analysis of body cavity fluids can be a very useful adjunct to conventional diagnostic cytopathology.     

EB病毒变异型EBER2对鼻咽癌细胞增殖和凋亡的影响

EB病毒(EBV)编码小RNA(EBERs,包括EBER1和EBER2)的致癌作用已在多种细胞系中得到证实,我们前期研究发现在EBER2基因发生6处突变的EB-8m变异型可能与鼻咽癌(NPC)的发生相关。本研究探讨变异型EBER2对NPC细胞增殖和凋亡的影响,以进一步明确EBER2基因变异在NPC发生中的作用。分别以B95-8原型和EB-8m变异型EBER2稳定转染EBV阴性NPC细胞系,MTT法和平板克隆检测细胞增殖,流式细胞仪检测细胞凋亡。与转染原型EBER2细胞及转染载体的对照细胞比较,转染变异型EBER2细胞的MTT吸光度值和平板克隆形成率增高,细胞凋亡率降低(P均小于0.05),原型和对照之间细胞增殖无差异,但原型的细胞凋亡率亦低于对照(P0.05)。上述结果表明,EB-8m变异型EBER2可通过提高NPC细胞增殖及抗凋亡能力而增强其致癌作用。     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Qualitative and quantitative analysis of peritoneal fluids from women with gynecologic diseases. Comparison of cytology and flow cytometry for the detection of malignancy in lavage and ascitic fluids

A prospective study was undertaken to compare flow cytometric (FCM) analysis to conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids (peritoneal lavages and ascitic fluids) from women with gynecologic diseases. The 94 peritoneal fluids analyzed came from 63 cancer patients (with epithelial ovarian carcinomas) and 31 control patients (with benign gynecologic diseases). The FCM DNA histograms were generated using propidium iodide as a DNA fluorochrome. Samples for cytologic analysis were stained with the standard May-Grünwald-Giemsa or Papanicolaou stains. Of the 94 samples, 90 were evaluable cytologically while 70 were suitable for FCM analysis. The sensitivities were 55% for FCM DNA analysis and 80% for cytologic analysis. FCM DNA analysis had a 30% false-positive rate; cytologic analysis produced no false-positive results. These results indicate that there is no advantage in employing FCM analysis instead of conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids from gynecologic cases.     

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.     

Exfoliative cytology of a lymphoepithelioma-like carcinoma in a cervical smear. A case report

BACKGROUND: Lymphoepithelioma-like carcinoma of the cervix (LELC) is cytologically identical to its counterparts at other sites, such as the nasopharynx. LELC can be suspected on a cervical cytologic smear. The differential diagnosis includes nonkeratinizing squamous cell carcinoma with prominent stromal inflammation, carcinoma with intense stromal eosinophilia, glassy cell carcinoma, malignant lymphoma (especially lymphoepitheloid-Lennerts lymphoma) and metastatic Schmincke-Regaud tumor. CASE: A 55-year-old female presented with an ulcerated endophytic tumor in the cervix. Exfoliative cytology showed uniform, large tumor cells, often associated with inflammatory cells, with round or oval nuclei and one or more prominent nucleoli. The cytoplasm was finely granular to flocculent, and the nuclei were uniformly vesicular. The chromatin was peripherally marginated. The cell borders were indistinct. There was no evidence of dyskeratotic or keratinized cells, koilocytes or glandlike formations. These findings were highly suspicious for LELC and were confirmed by biopsy. Flow cytometry showed DNA aneuploidy, with a DNA index of 1.08. In situ hybridization was negative for human papillomavirus 16 and 18. CONCLUSION: LELC of the uterine cervix has cytologic features that are sufficiently characteristic for a specific cytologic diagnosis. The diagnosis, nevertheless, has to be proven by histology.     

DNA analysis of malignant effusions. Comparison with cytologic diagnosis and carcinoembryonic antigen content.

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.     

Flow cytometric DNA analysis combined with fine needle aspiration biopsy in the diagnosis of palpable metastases

To determine the diagnostic value of flow cytometric (FCM) DNA analysis in combination with fine needle aspiration (FNA) biopsy, the nuclear DNA content was analyzed in FNA specimens from 155 superficial metastases and 60 benign lymph nodes. The results of FCM DNA analysis were considered to be positive for malignancy if DNA aneuploidy was found or if more than 12% S+G2M cells were present in the aspirate in diploid cases. The diagnostic accuracy of FCM DNA analysis alone in detecting malignancy was 92%, with a sensitivity of 91% and a specificity of 95%. FCM suggested the correct diagnosis in 5 of the 7 cytologically false-negative cases and in 9 of the 12 cytologically indeterminate or suspicious cases.     

Genital human papillomavirus testing by in situ hybridization in liquid atypical cytologic materials and follow-up biopsies

OBJECTIVE: To describe cases of HPV testing by DNA in situ hybridization performed on atypical cervicovaginal samples collected by a liquidsed method that were negative for HPV DNA on cytology but revealed cervical intraepithelial neoplasia on follow-up biopsies. STUDY DESIGN: Three hundred ninety-five consecutive SurePath atypical squamous cells of undetermined significance (ASC-US) cytologic samples from asymptomatic, reproductive-age women were tested for human papillomaviruses (HPVs) by the in situ hybridization (ISH) method (Ventana Inform HPV Test, Tucson, Arizona, U.S.A). One hundred (25%) cases underwent follow-up colposcopic biopsy within 3 months of cytology. All the tests (cytology, ISH, histology) were independently evaluated without knowledge of the other tests. RESULTS: One hundred twenty-two (33%) cytologic samples were positive for HPVs. Of a total of 100 (HPV positive and negative) follow-up biopsies, 55 were positive for cervical intraepithelial neoplasia (CIN). Fourteen cases of biopsy-proven CIN tested negative for all HPV types in the prior cytologic samples. Retesting of the 14 CIN tissues by ISH was negative in 10, positive for HPV in 2 and inconclusive in 2. CONCLUSION: There is a small but significant (14%) false negative rate with HPV testing by the Ventana ISH method. Clinically suspicious cases should be followed even if an HPV test is negative.     

Image analysis of DNA content and nuclear morphometry for predicting radiosensitivity of nasopharyngeal carcinoma

OBJECTIVE: To investigate the relationship among nuclear DNA content, nuclear morphology, clinical response, and radiosensitivity in nasopharyngeal carcinoma (NPC) and the suitability of image cytometric analysis of DNA content and nuclear morphology for predicting radiosensitivity of NPC prior to radiotherapy. STUDY DESIGN: Nuclear DNA content and morphology features were detected by image cytometric analysis in 51 biopsy specimens of NPC prior to radiotherapy. The radiotherapeutic effect experienced by the NPC patients was classified as CR (complete response [i.e., complete tumor disappearance]) and PR (partial response [i.e., residual tumor]) according to pathologic analysis of tumor specimens after completion of the scheduled treatment. RESULTS: The mean DNA index; the percentage of cells with the DNA pattern of 2C, 5C, aneuploidy respectively; the mean nuclear area; the mean nuclear perimeter and the mean nuclear diameter in the CR group were significantly higher than they were in the PR group. CONCLUSION: DNA content and nuclear morphometry by image cytometric analysis were significantly correlated with patient outcome and radiosensitivity of NPC. Other measurements of more biomarkers for predicting the radiosensitivity of NPC await further study.     

Early diagnosis of mesothelioma in serous effusions using AgNOR analysis

OBJECTIVE: To compare the results of conventional cytology, DNA image cytometry, immunocytochemistry and argyrophilic nucleolar organizer region (AgNOR) analysis for the diagnosis of malignant cells in serous effusions. STUDY DESIGN: One hundred twenty effusions, 40 with carcinoses, 40 with malignant mesotheliomas and 40 without tumor cells on follow-up were studied by conventional cytology and three adjunctive methods. RESULTS: Unequivocal tumor cells were detected in 92.5% of effusions due to carcinoses and in 45% due to mesotheliomas. Applying immunocytochemistry with BerEP-4 positivity and DNA image cytometry with aneuploidy as a marker revealed 100% of carcinoses and 71.7% of mesotheliomas. Applying the experimentally found thresholds of 2.5 AgNORs as "satellites" and 4.5 AgNORs as "satellites and clusters" together as mean values per nucleus resulted in a 95% correct rate of mesothelioma and 100% rate of carcinoma cell identification without false positive diagnoses. CONCLUSION: AgNOR analysis may be a useful adjunct to other methods in the routine diagnosis of malignant serous effusions. It seems to be the most sensitive method in early cytologic diagnosis of mesotheliomas in effusions. Seventy-three percent of malignant mesotheliomas were diagnosed cytologically at first on effusions. Forty-seven percent of patients with malignant mesotheliomas were identified at the early tumor stage T1 N0 M0.     

Colonic cytology. A retrospective study with histopathologic correlation

Three hundred sixty cytologic specimens obtained by colonoscopic brushing from 336 patients were compared with biopsy specimens simultaneously obtained for histologic examination. Of the cytologic specimens, 160 (44%) were positive for malignant cells, 37 (10%) contained suspicious cells, 54 (15%) had atypical glandular cells, 107 were cytologically negative, and 2 were considered unsatisfactory. Eight-four percent of the patients with cytologically positive smears and 54% of those with suspicious smears had malignant neoplasms in the simultaneously obtained tissue biopsies. Of the patients with follow-up, all with cytologically positive findings and nine with suspicious findings on the initial cytologic examination and simultaneous negative tissue biopsies, were subsequently found to have carcinoma of the colon. Cytology proved to have a sensitivity of 0.73 and a specificity of 1.00 while tissue biopsy showed a sensitivity of 0.81 and a specificity of 1.00. By combining the two methods, the sensitivity increased to 0.92. It is concluded that cytologic examination of colonic brushings is a highly accurate and reliable technique for the detection of malignant neoplasms of the colon and can preempt the use of biopsy forceps.     

Earliest detection of oral cancer using non-invasive brush biopsy including DNA-image-cytometry: report on four cases.

OBJECTIVE: We describe four patients presenting early oral cancers, detected cytologically on non-invasive brush biopsies including DNA-image cytometry as an adjunctive method before histology on scalpel biopsies confirmed the evidence of malignancy. METHODS: Brush biopsies were performed and smears thereof investigated cytologically. After Feulgen restaining, DNA-measurements were performed using a DNA-Image-Cytometer. CASE REPORTS: Oral squamous cell carcinomas were diagnosed cytologically in macroscopically suspicious lesions and malignancy confirmed by DNA-cytometry. The initially performed scalpel biopsies did neither supply evidence of oral cancer nor of severe dysplasia. After at least one to 15 months the occurrence of cancer was finally proven histologically on a second scalpel biopsy each (three microinvasive and one in situ carcinoma). CONCLUSION: Non-invasive brush biopsies are a suitable instrument for early cytologic detection of cancer of the mouth. DNA-image-cytometry, as an adjunctive method, can be used to confirm the cytologic diagnosis or suspicion of cancer in patients with doubtful lesions (dysplasias). DNA-aneuploidy is a marker for (prospective) malignancy in smears of the oral cavity, which may detect malignancy months prior to histology. In future this method could be used as a mass screening tool in dentists practice.     

Nipple discharge cytology in mass screening for breast cancer

Since 1977 mass screening for breast cancer has been conducted in Miyagi Prefecture, Japan; inspection, palpation and cytologic examination of any nipple discharge are part of the initial screening procedures. Among 149,681 subjects examined, 404 cancer cases and 63 papilloma cases were detected. The nipple discharges from 20,537 women were examined cytologically; of the 61 cancer cases, the smears were positive in 18 cases, suspicious in 7, negative with atypical findings in 12 and negative in 24. Ten of the cancer cases were detected exclusively by the cytologic examination of a nipple discharge. In eight of these ten cancer cases, there was no other initial evidence of the primary tumor. The cytologic diagnosis of discharges without blood from 28 cancer cases was positive or suspicious in 10 cases and negative in 18. Thirty-seven of the papilloma cases were initially detected only by the cytologic examination of a nipple discharge; neither physical examination nor mammography showed any abnormal findings.     

DNA-cytometric diagnosis of prospective malignancy in borderline lesions of the uterine cervix

Interactive DNA cytometry was used for the diagnosis of prospective malignancy in 48 smears with borderline lesions (mild and moderate dysplasias) of the uterine cervix. In addition, 183 smears with benign squamous epithelia, 38 with carcinoma in situ and 7 with invasive squamous carcinoma were also measured. Nuclear Feulgen-DNA measurements were performed using various methods, and the resulting data were analyzed by an algorithm for a DNA-cytophotometric diagnosis of malignancy. The results were compared with the data on follow-up and subsequent histologic studies in these cases. There was no false-positive diagnosis in the 183 benign smears and only 1 false-negative diagnosis in the 76 histologically proven squamous-cell carcinomas, which yields a specificity of 100% and a sensitivity of 98.6%. The sensitivity for the detection of subsequent histologically proven malignancy in cases with cytologically mild or moderate dysplasia amounted to 97%. In 13 borderline cases, there was a mean interval of 21 months between the taking of the cytologic smear on which the DNA diagnosis of malignancy was made and the date on which the histologic confirmation of malignancy was made. In 17% of the cytologically dysplastic cases, the DNA diagnosis of malignancy was not verified by subsequent histologic investigation. These results indicate that interactive DNA cytometry is able to detect prospective malignancy in smears from borderline lesions of the uterine cervix with a high sensitivity.