人乳头瘤病毒16与胃癌相关性的研究

目的探讨人乳头瘤病毒１６（ＨＰＶ１６）与胃癌发生的关系。方法用聚合酶链式反应（ＰＣＲ）扩增出ＨＰＶ１６早期区Ｅ６的１２０ｂｐＤＮＡ片段，扩增产物与５＇末端３２Ｐ标记的特异性寡核苷酸探针进行斑点杂交及放射性自显影，用此方法对３４６份新鲜的及福尔马林固定石蜡包埋的胃组织标本中ＨＰＶ１６ＤＮＡ进行检测。结果胃腺癌、癌旁粘膜、胃局部淋巴结及正常胃粘膜组织中ＨＰＶ１６的检出率分别为：２２．７２％（３０／１３２）、５．８８％（４／６８）、０％（０／４２）、２．８８％（３／１０４），胃癌组ＨＰＶ１６的检出率高于其他各组，统计学上差异均有显著性（Ｐ＜０．０１）。结论ＨＰＶ１６可感染胃粘膜上皮细胞，可能是胃癌的致癌因素之一。     

Detection of c-Ki-ras oncogene mutation in gastric adenomas with formalin-fixed, paraffin-embedded biopsy materials

Point mutations at codon 12 of the c-Ki-ras gene were investigated in 103 biopsy tissues from 38 patients with gastric adenoma. DNA from minute specimens of formalin-fixed paraffin-embedded tissue was amplified by nested polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) method was used to detect mutations of the c-Ki-ras gene. Mutations were detected in 4 of the 38 gastric adenomas (10.5%), but were not detected in intestinal metaplasias and gastric adenocarcinomas. Of the 4 mutation-positive gastric adenomas, the GGT sequence in codon 12 changed to GAT in 2 cases, to GTT in 1 case and to TGT in 1 case. We classified gastric adenomas into three degrees of atypia; mutations were detected only in biopsy tissues with moderate and severe atypia but were not detected in biopsy tissues with mild atypia. When different biopsy tissues from a mutation-positive gastric adenoma were examined, the mutation was not always detected in every tissue sample examined. This suggests that mutations are not homogeneously distributed, even in the same adenoma. It is possible to follow up the mutation of biopsy materials of gastric adenoma by this method.     

Detection of human papillomavirus DNA in esophageal carcinoma in Greece

AIM:To detect human papillomavirus(HPV)in theesophageal mucosa and the possible relationship with esophageal cancer in Greece.METHODS:Forty-nine patients underwent esophagogastroduodenoscopy(EGD)and esophageal biopsy at a university hospital that acts as a referral center for Northern Greece.Nineteen of these patients(14 male and 5 female)had esophageal squamous cell carcinoma(ESCC)and 30(15 male and 15 female)did not have any reported esophageal malignancy.Histopathological assessment was followed by polymerase chain reaction analysis of all the samples.Patient demographic data(age,sex,and place of birth)and information regarding smoking habits,alcohol consumption or sexual habits were collected.A method of statistical interference,verification of hypotheses based on homogeneity and independentχ2 test,was used.RESULTS:From the 49 patients that underwent EGD and biopsy,19 had ESCC and 30 had normal esophageal mucosa,with a mean age of 65.2 years.Regarding the prevalence of oncogenic risk factors for esophageal carcinoma,an interesting conclusion was that 78%of the patients used tobacco and almost one-third had multiple sexual partners,whereas only 20%of the patients consumed alcohol,which was not statistically significant,when compared to the control group.In the ESCC group,the only two positive samples were among the male patients(2/14 male patients with ESCC,14.5%).No HPV was identified in the control group.The predominant HPV types identified were 11 and 31,which have a low malignancy potential.The presence of HPV DNA in the ESCC group was not statistically significant,95%confidence interval(χ2=3.292,P=0.07).CONCLUSION:This is the first relevant study in Greece,and despite the lack of statistical significance,the issue of HPV infection and ESCC does merit further investigation.     

Highly multiplexed single-cell analysis of formalin-fixed,paraffin-embedded cancer tissue

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.     

Immunohistochemical measurement of endothelial cell apoptosis and proliferation in formalin-fixed, paraffin-embedded human cancer tissue

There is a need for simple, reproducible methodology for measurement of endothelial cell (EC) apoptosis and proliferation in formalin-fixed, paraffin-embedded (FFPE) human tissue obtained in clinical trials of potential anti-angiogenic agents. Therefore, we developed colorimetric, dual-label immunohistochemical techniques for use on FFPE tissue, based on the use of single-stranded (ss) DNA and Ki-67 as markers of EC apoptosis and proliferation, respectively. Digital image analysis was used to obtain the total tumour microvessel density (MVD), from which the EC apoptosis index (AI) and proliferation index (PI) were derived manually as the number of positive ECs per vessel. Immunohistochemical measurement of EC apoptosis and proliferation was validated on human colorectal cancer liver metastases from a randomized, placebo-controlled trial of the selective cyclooxygenase-2 inhibitor rofecoxib. Proliferating and apoptotic ECs clustered in discrete areas of the tumour vasculature. ECAI (median [inter-quartile range, IQR] 0.0018 [0.0003–0.0064]) and ECPI (median [IQR] 0.0043 [0.002–0.014]) values were low and highly variable in tumours from both placebo- and rofecoxib-treated patients. Our novel dual-label immunohistochemical methods will be generally applicable in FFPE human cancer tissue and should prove invaluable for measuring the anti-angiogenic activity of experimental therapies in clinical trials. The low absolute level of and variability in EC apoptosis and proliferation detectable in human colorectal cancer liver metastases indicates that similar intervention studies using these end-points will need to ensure adequate size in order to be able to detect anti-angiogenic activity by immunohistochemical methods.     

人食管鳞状细胞癌标本中乳头状瘤病毒DNA的检测

目的食管鳞状细胞癌有明显的地域性差异,它的发生与遗传、环境、饮食和某些微生物的感染有密切的关系,本研究通过检测食管鳞状细胞癌和相应正常食管组织中HPV-11型和HPV-16型的DNA,阐明HPV与食管鳞状细胞癌的关系.方法用PCR的方法检测22位食管鳞状细胞癌患者手术切除的癌组织和相应癌旁正常食管粘膜(共44例标本)中的HPV-11型和HPV-16型DNA的存在情况,并对其中一例HPV阳性肿瘤标本的HPV序列进行测定. 结果在本组所涉及的22位患者共计44例标本中,对于HPV-11型,12例癌组织为阳性;有7例癌旁组织为阳性;癌组织和癌旁组织均为阳性的有6例.对于HPV-16型,6例癌组织为阳性;6例癌旁组织为阳性;癌组织和癌旁组织均为阳性的有3例.序列分析结果表明所测定的序列与GeneBank登录的NC001525.1(HPV-11)、M14119.1(HPV-11)和AF217526.1(HPV-11)的同源性极高,均为99%.结论通过实验结果分析,我们认为食管鳞状细胞癌与HPV-11型和HPV-16型主要衣壳蛋白L1基因密切相关,HPV可能在一定的地域环境中与食管鳞状细胞癌的发生具有相关性.马群风,男,1961-01-04生,贵州省贵阳市人,汉族.1983年遵义医学院医疗系本科毕业,1993年第四军医大学普胸外科专业硕士毕业.主要从事普胸外科疾病的临床和研究工作.发表论文4篇项目负责人马群风,710038,陕西省西安市,中国人民解放军第四军医大学唐都医院胸外科.     

Detection of human papillomavirus DNA in cytologic specimens derived from esophageal precancer lesions and cancer

A series of 80 esophageal cytologic specimens derived from the same number of patients with previously diagnosed squamous cell dysplasia of the esophagus were examined for the presence of human papillomavirus (HPV) infection by filter in situ hybridization (FISH), using a mixed DNA probe containing HPV types 11, 16, and 18. All the patients came from an area at high risk for esophageal cancer in China. A total of 53 cases (66.3%) were demonstrated as HPV-DNA-positive. HPV DNA was detected in 22.2% (2 of 9) of the patients without cytologic atypia, in 50% (3 of 6) with mild dysplasia, in 80.6% (25 of 31) with moderate dysplasia, in 67.9% (19 of 28) with severe dysplasia, and in 66.7% (4 of 6) with an invasive squamous cell carcinoma. The present results confirm the recent findings on HPV involvement in esophageal squamous cell lesions. They support the hypothesis that HPV is a possible etiologic agent in esophageal carcinogenesis, most probably acting synergistically with physical, chemical, and/or nutritional factors that have previously been related with this malignancy in the high-risk areas of China.     

Seroprevalence of human papillomavirus type 16 in children

We evaluated the prevalence of antibodies to human papillomavirus (HPV) type 16 in a representative sample of children 6-11 years of age in the United States. Serum samples and questionnaire data were collected between 1991 and 1994, for the National Health and Nutrition Examination Survey III. HPV-16-specific immunoglobulin G antibodies were detected by an HPV-16 L1 virus-like particle-based enzyme-linked immunosorbant assay. Overall, 2.4% of 1316 children 6-11 years of age were seropositive. Seroprevalence was higher in boys than in girls (3.5% vs. 1.2%; P=.08) and in children >7 years of age than in children < or =7 years of age (3.3% vs. 0.4%; P<.05). None of the variables tested for, including race/ethnicity, socioeconomic status, and urban or rural residence, were significantly associated with HPV-16 seropositivity. To explain HPV-16 seropositivity in this population, further study is required.     

食管癌组织中人乳头状瘤病毒感染状况分析

为探讨人乳头状瘤病毒（HPV）感染在食管癌发病中的作用，采用荧光定量聚合酶链反应对河北省高发区食管癌、癌前病变、正常食管组织和低发区食管癌组织中人乳头状瘤病毒（HPV16、18）进行检测。结果显示，高发区食管癌组织HPV16、18DNA阳性率为38.7%，癌前病变为25％，正常食管组织为2.5%，低发区食管癌组织为20％。高发区食管癌和癌前病变与正常食管组织HPV16、18感染率相比差异具有显著性（P＜0.01）；低发区食管癌组织HPV16、18感染率与高发区正常食管组织相比差异具有显著性（P＜0.01）；低发区食管癌组织HPV16、18感染率低于高发区食管癌组织，但差异无显著性（P＞0.05）。提示河北省食管癌高低发地区食管癌组织、高发区食管癌前病变组织中均存在较高的人乳头状瘤病毒感染率，为食管癌发病的重要因素之一。     

Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer.

A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity.     

Detection of cervical antibodies to human papillomavirus type 16 (HPV-16) capsid antigens in relation to detection of HPV-16 DNA and cervical lesions

A more sensitive luminescence immunoassay (LIA) for human papillomavirus type 16 (HPV-16) was developed and used to measure HPV-16 antibodies in cervical samples from 292 college-aged women who were examined at 4-month intervals. Of the 609 collected samples, IgG, IgA, and secretory piece-associated antibodies to HPV-16 were detected in 12%, 6%, and 8%, respectively, of samples tested. Cervical IgG antibodies were most strongly associated with HPV-16 DNA detected within the previous 12 months (odds ratio, 3.3; 95% confidence interval, 1.4-7.8). Secretory IgA (cervical IgA- and secretory piece-positive) was most strongly associated with detection of a squamous intraepithelial lesions 4-8 months earlier (odds ratio, 6.4; 95% confidence interval, 1.9-21.8). As with serum HPV-16 antibodies, there appears to be a several-month delay between cervical HPV infection and detection of cervical antibodies.     

Detection of alpha-1-antitrypsin PiZ individuals by SSCP and DNA sequencing in formalin-fixed and paraffin-embedded tissue: a comparison with immunohistochemical analysis

BACKGROUND/AIM: The role of alpha-1-antitrypsin (AAT) deficiency in the development of cirrhosis and carcinoma of the liver can be investigated from the analysis of archival biopsy specimens. Immunohistochemistry can visualize the storage of defective protease inhibitor (Pi) variant Z, but does not allow differentation between homozygous and heterozygous patients. The aim of the study was to establish a method for the detection of the PiZ mutation on the gene level. METHODS: Liver biopsy and autopsy samples in which AAT deficiency was detected immunohistochemically by a monoclonal PiZ-antibody were analyzed by single-strand conformational polymorphism (SSCP) to reliably determine hetero- and homozygote carrier state in the absence of blood samples and to confirm the histological diagnosis. The accuracy of SSCP was verified by direct DNA sequencing. RESULTS: Tissue slices (>0.8 cm2) from 29 consecutive cases with immunohistochemically detected PiZ depositions and from ten PiZ-negative control cases were provided for extraction and amplification of DNA. In comparison to wild-type sequence of AAT exon V, all 29 cases showed band shifts on SSCP analysis, with a heterozygous pattern in 28 patients and a homozygous pattern in one patient. DNA sequence analysis revealed the same single-base mutation at position 342 of AAT exon V. CONCLUSIONS: SSCP analysis proved a sensitive and specific technique for the detection of the PiZ mutation at the gene level, which allowed unequivocal differentiation between heterozygous and homozygous PiZ status from paraffin-embedded archival tissue specimens. Besides a use in diagnostic pathology, this technique could be valuable for prenatal diagnosis and population screening purposes.     

Immunolocalisation of PIVKA-II in paraffin-embedded specimens of hepatocellular carcinoma

Abstract: Aims/Background: Although serum PIVKA-II has been widely used as a tumor marker for hepatocellular carcinoma (HCC), little information is available concerning tissue PIVKA-II, and detection of tissue PIVKA-II in paraffin-embedded tissue specimens of HCC is also considered difficult. Methods: Paraffin-embedded HCC tissues obtained at autopsy from 22 patients were subjected to immunohistochemical staining using the antibody MU-3 (Eisai Co., Ltd.) after microwave antigen retrieval. Results: PIVKA-II was mainly detected in the cytoplasm of HCC cells. The intensity of tissue PIVKA-II staining was not correlated with serum PIVKA-II levels or with the histological differentiation of HCC. However PIVKA-II staining tended to be more intense in tissue from patients with portal tumor thrombus, distant metastases, or a longer duration of HCC. Conclusion: This method of immunohistochemical staining is easy and simple to use and may be helpful for detecting tissue PIVKA-II in paraffin-embedded HCC specimens.     

Detection and typing of human papillomavirus in anal epidermoid carcinomas: Sequence variation in the E7 gene of human papillomavirus type 16

INTRODUCTION: Human papillomavirus, particularly Type 16, plays a central role in the development of anogenital squamous-cell carcinomas. A common sequence variation of human papillomavirus Type 16 in cervical cancer cell lines and in cervical cancer tissues from Korean patients was recently reported. The present study was performed to determine the integration type of human papillomavirus DNA in anal epidermoid carcinoma and to identify the common sequence variations in the human papillomavirus Type 16 E7 gene that had been previously reported. METHODS: Twenty-one formalin-fixed, paraffin-embedded specimens collected from 29 patients with anal epidermoid carcinomas treated at the Seoul National University Hospital over a ten-year period (1989–1998) were investigated. Genomic DNA from the 21 specimens was extracted and analyzed using the polymerase chain reaction with a general primer and a type-specific primer for human papillomavirus Types 16 and 18. Direct sequencing was performed. As a control, 13 normal anal epithelia available from these patients were microdissected. As another control, 21 hemorrhoidal squamous epithelia obtained from a demographically adjusted group were also analyzed. RESULTS: Human papillomavirus Type 16 DNA was present in all 21 anal epidermoid carcinomas. All controls were negative for human papillomavirus DNA. Sequence analysis revealed that 57 percent (12/21) specimens showed two types of sequence variation in the E7 gene. One variant with a single nucleotide change at position 647 (amino acid 29AATAGT, asparagine to serine) was found in 38 percent (8/21) of the samples. This variant has been detected in cervical cancers from Korean patients: 19 (39 percent) of 49 cervical cancer tissues and 6 (50 percent) of 12 cervical cancer cell lines. Another single nucleotide change at position 645 (amino acid 28, TTATTC, leucine to phenylalanine) was found in 19 percent (4/21) of the samples. These two variants exhibit a change of amino acid affecting the critical sites for Rb binding. CONCLUSION: Human papillomavirus Type 16 was found to be present in all 21 anal epidermoid carcinomas. Furthermore, in the Korean population, the most common sequence variant found in cervical cancer is also the major one in anal epidermoid carcinoma.     

结直肠癌组织中人乳头瘤病毒DNA的研究

目的研究１６、１８型人乳头瘤病毒（ＨＰＶ）是否与结直肠癌的发生有关．方法结直肠粘膜活检组织１２３例，其中结直肠癌３５例，乳头状腺瘤１７例，炎性息肉１１例，结肠炎３０例，以及正常结肠粘膜３０例，用对各型ＨＰＶ高度保守的通用引物和１６、１８型特异性引物作聚合酶链反应（ＰＣＲ）检测ＨＰＶＤＮＡ．结果ＨＰＶＤＮＡ总检出率１４６％，正常粘膜，结肠炎和炎性息肉组为３３％（２／７１），乳头状腺瘤为１７６％（３／１７），结直肠腺癌为３７１％（１３／３５）．在正常粘膜，结肠炎和炎性息肉组未发现１６、１８型ＨＰＶＤＮＡ，在乳头状腺瘤组有３例为１８型ＨＰＶＤＮＡ阳性，结直肠腺癌组１３例ＨＰＶ阳性病例中有３例为１６型，９例为１８型感染；在正常组织、癌旁组织和癌组织中ＨＰＶＤＮＡ检出率依次增高．ＨＰＶ在直肠、左半结肠，右半结肠感染率依次为２８６％，１４％，２６％．结论结肠癌的发生与ＨＰＶ１６、１８型有关，腺癌以１８型感染为主．ＨＰＶＤＮＡ检出率在右半结肠，左半结肠和直肠依次增高．     

Detection of human papillomavirus DNA in squamous cell carcinoma of the esophagus by auto-nested PCR

The aim of the present study was to investigate the presence of human papillomavirus (HPV) in surgical specimens of esophageal squamous cell carcinoma. One hundred and sixty-five paraffin-embedded specimens of esophageal carcinoma were analyzed through high-sensitivity auto-nested polymerase chain reaction (PCR) using the consensus GP5+/GP6+ primer. Twenty-six specimens of esophageal mucosa without malignant disease were also studied as a control group. Two different specific primer sets targeting the E6 region of the HPVs 16 and 18 were used for typing. Direct DNA sequence analysis was conducted to confirm positive PCR results. HPV DNA was detected in 26 esophageal carcinomas (15.75%), but in none of the benign esophageal specimens (P < 0.05). Out of the 26 positive cases, 24 were HPV-16 and one was HPV-18. One tumor contained both HPV-16 and -18 DNA. Positive PCR results were confirmed by the amplified viral sequences. Our findings suggest that the presence of either HPV-16 or -18 might be related to development of the malignant phenotype in the esophagus.     

Detection of tuberculosis genes associated with drug-resistance in paraffin-embedded tissue specimens using next generation sequencing technology

<正>Objective To evaluate the use of multiplex PCR amplicon sequencing (mPCR-NGS) technology in detecting gene mutations related to drug resistance of Mycobacterium tuberculosis (MTB) in formalin-fixed paraffin-embedded tissue specimens,and to explore its clinical value in