Detection of human papillomavirus (HPV) DNA in archival specimens of benign prostatic hyperplasia and prostatic cancer using a highly sensitive nested PCR method

Human papillomavirus is thought to be an etiological factor for urological tumors such as penile cancer. However, there is much conflicting data surrounding prostatic cancer. We recently established a highly sensitive nested PCR method with consensus human papillomavirus (HPV) primers for the detection of many high-risk HPV types. HPV DNA from the long-control region (LCR) to E7 open reading frame was amplified with first primer pairs and subsequently amplified with second internal E6–E7 primers. Our nested PCR method could detect HPV16, 18, 31, 33, 35, 52, 58 and some undetermined HPV DNAs. Using this method, we investigated the existence of HPV DNA in formalin-fixed paraffin-embedded tissue of the prostate. We found HPV DNA in three of 71 specimens of benign prostatic hyperplasia (BPH) and in none of 38 prostatic carcinomas. These three samples were infected with HPV 16. These results suggest that HPV is not a causal factor for prostatic cancer and BPH. Received: 7 May 1997 / Accepted: 19 September 1997     

人乳头状瘤病毒与前列腺癌相关性研究

用高敏感聚合酶链反应（ＰＣＲ）技术检测６３例前列腺组织标本（２８例前列腺癌，３０例良性前列腺增生症和５例正常前列腺组织）中人乳头状瘤病毒（ＨＰＶ）阳性率。结果显示：前列腺癌中ＨＰＶ（１６）感染率为４６．４％（１３／２８），ＨＰＶ（１８）感染率为１０．７％（３／２８）。良性前列腺增生症患者ＨＰＶ（１６）阳性率为２０％（６／３０），ＨＰＶ（１８）阳性率为６．１７％（２／３０）。５例正常前列腺组织无ＨＰＶ感染。提示ＨＰＶ感染与前列腺癌有相关性，ＨＰＶ感染可能在前列腺癌发展中起着重要作用。     

Aberrant expression of cystatin C in prostate cancer is associated with neuroendocrine differentiation

OBJECTIVE: To investigate the expression of cystatin C and the relationship with neuroendocrine differentiation and proliferation in benign and malignant prostatic tissues, as cystatin C, the most important inhibitor of human lysosomal cysteine proteases, is considered to be a major regulator of pathological protein degradation in inflammatory and neoplastic diseases. MATERIALS AND METHODS: Immunoreactivity for cystatin C, prostate-specific antigen, Ki-67 and the neuroendocrine marker chromogranin A was examined in whole-mount radical prostatectomy specimens and using tissue microarrays. Cystatin C in tissue homogenates was analysed by Western blotting and enzyme-linked immunosorbent assay (ELISA). The expression and relative levels of cystatin C mRNA were assessed by in situ hybridization and quantitative real-time polymerase chain reaction (QRT-PCR). RESULTS: The intensity of cystatin C immunostaining in Gleason grade 2 and 3 prostate cancer was significantly higher than in benign prostatic tissues, but decreased significantly with increasing Gleason grades. There was strong expression of cystatin C in neuroendocrine-like cells, which increased significantly with increasing Gleason grades. The Ki-67 immunoreactivity also increased significantly during de-differentiation. In situ hybridization showed staining patterns in concordance with the immunohistochemical results. ELISA showed high concentrations of cystatin C in benign and malignant tissue extracts and QRT-PCR further corroborated that the cystatin C gene is highly expressed in both benign and malignant prostatic tissues. CONCLUSIONS: There was a significant decrease in the immunohistochemical expression of cystatin C in non-neuroendocrine prostate cancer cells, concomitant with increasing Gleason grades. That there were more strongly cystatin C-positive neuroendocrine-like cells in prostate cancer than in benign prostatic tissue suggests a connection between cystatin C and neuroendocrine differentiation in prostate cancer progression.     

端粒酶与前列腺癌及其生物学行为的关系

目的 探讨端粒酶（ＴＥ）与前列腺癌（ＰＣａ）及其生物学行为的关系。方法 应用端粒重复片段扩增法（ＴＲＡＰ）法,检测３９例ＰＣａ组织,１５例前列腺增生（ＢＰＨ）组织及１０例正常前列腺（ＮＰ）组织中的ＴＥ活性,并比较ＴＥ活性水平与ＰＣａ病理分化程度,临床分期及转移情况的关系。     

前列腺癌组织中HMGA2的表达及其意义

目的探讨高迁移率族蛋白HMGA2在前列腺癌中的表达及其意义。方法采用免疫组化法及RT—PCR法检测40例前列腺癌组织及良性前列腺增生组织中HMGA2的表达情况。结果RT-PCR法检测示良性前列腺增生组织中HMGA2少量表达;恶性组织中,随着病理分级增加,HMGA2mRNA相对表达量逐渐增高,免疫组化法显示前列腺癌组织中,有75．0％（30／40）HMGA2呈阳性表达。良性前列腺增生组织与前列腺癌组织两组间差异显著（t=3．32,P〈0．001）。结论HMGA2在前列腺癌中呈过度表达,并与前列腺癌病理分级有关,可能成为前列腺癌诊断与良恶性鉴别诊断的指标之一。     

Papillomavirus found in anorectal squamous carcinoma, not in colon adenocarcinoma.

We used polymerase chain reaction DNA amplification methods for the detection and typing of genital human papillomaviruses in paraffin-embedded tissue sections of five patients with anorectal squamous cell carcinoma and 22 patients with colonic adenocarcinoma. The cases were further tested by in situ hybridization with biotin-labeled probes specific for human papillomavirus types 6/11, 16/18, and 31/33/35. By polymerase chain reaction, human papillomavirus DNA was demonstrated in all of the cases of anorectal squamous cell carcinoma and in none of the cases of colonic adenocarcinoma for which analyzable DNA was available. Tumor cell nuclei stained for human papillomavirus DNA by in situ hybridization in four of the five cases of squamous cell carcinoma and in none of the cases of colonic adenocarcinoma. We conclude that human papillomavirus types usually associated with malignant transformation are uniformly present in anorectal squamous cell carcinoma but are absent from adenocarcinoma of the colon.     

前列腺衰老逃逸现象的实验研究

目的探讨良性前列腺增生(BPH)衰老逃逸现象的机理。方法采用端粒重复片段扩增法(TRAP)分别检测13例正常前列腺组织、35例BPH组织及33例增生结节和包膜的端粒酶活性表达情况,比较端粒酶活性水平与BPH衰老逃逸的关系。结果正常前列腺组织中端粒酶阳性2例(15%),BPH组织中端粒酶阳性17例(49%),增生结节端粒酶阳性14例(42%),包膜阳性1例(3%);BPH组织端粒酶阳性表达率高于正常前列腺组织(P<0.05),增生结节阳性率明显高于包膜组织(P<0.01)。结论BPH组织中端粒酶活性升高,且分布不均匀。提示前列腺衰老逃逸可能与端粒酶活性有关。     

DNA flow cytometric study of the hyperplastic and neoplastic canine prostate.

Flow cytometric DNA measurements were carried out on 45 formalin-fixed, paraffin-embedded canine prostate tissues. The tissues were categorized as normal, hyperplastic, or neoplastic on the basis of light microscopic examination, and DNA ploidy was compared with histologic classification. Ten normal prostate samples showed diploid DNA histograms, but with proliferation greater than that found in the normal human prostate. Thirteen specimens of benign prostate hyperplasia showed diploid or near-diploid DNA histograms. Of 22 prostatic carcinomas, 12 were diploid and 10 were aneuploid, with the majority of the aneuploidy being near-triploid. The frequency of DNA aneuploidy recognized in canine prostatic carcinoma is similar to findings in human prostatic carcinoma if all their grades of malignancy are included.     

Histological distribution of polymerase chain reaction--amplified human papillomavirus 6 and 11 DNA in penile lesions.

The purpose of this study was to ascertain the histological pattern of distribution of human papillomavirus (HPV) 6 and 11 DNA in penile lesions by in situ hybridization after amplification by the polymerase chain reaction (PCR). HPV DNA was routinely detected by in situ hybridization with or without PCR amplification in granular layer cells that showed perinuclear halos and nuclear atypia. Cells that lack these histological features rarely exhibited HPV DNA with conventional in situ hybridization. However, after PCR amplification, in situ analysis showed that many of the cells that lacked halos and atypia contained HPV DNA. The hybridization signal often localized to crevices in the epithelium where there was relative hyperkeratosis and a thickened granular layer. HPV DNA was not noted in the basal cells and was rarely identified in other parts of the lesion. It is concluded that penile tissues may contain HPV DNA when lacking the diagnostic features of a condyloma/low-grade intraepithelial lesions and that such tissues usually demonstrate specific histological changes characterized by a focally thickened granular layer often associated with epithelial crevices.     

Association of human papillomavirus and colon neoplasms

Human papillomavirus has been shown to be associated with squamous carcinomas. We evaluated benign and malignant colon tissues for the presence of human papillomavirus infection to determine if a similar relationship exists between human papillomavirus and colon neoplasms. Colon tissues were screened using an immunohistochemical technique to detect human papillomavirus antigen. In situ DNA hybridization was then performed on those tissues that yielded positive results by immunohistochemistry. Groups were compared using chi 2 analysis. Human papillomavirus antigen was present in 23% of normal colon specimens, 60% of benign tumors, and 97% of carcinomas. Human papilloma viral genome was demonstrated in 27% of benign tumors and in nearly 43% of all carcinomas tested. These data indicate that human papillomavirus infects the columnar mucosa of the colon, and that an association exists between human papillomavirus and colon neoplasia.     

Detection of human papillomavirus DNA in penile lesions histologically negative for condylomata. Analysis by in situ hybridization and the polymerase chain reaction

The purpose of this study was to analyze penile lesions that lacked the histological features of condylomata for human papillomavirus (HPV) DNA. Most of these lesions were detected in men whose partners had an HPV-related lesion. Sequences homologous to HPV DNA were detected in 12 of 84 (14%) lesions using in situ hybridization analysis; nine of these lesions contained HPV 6 or 11. HPV DNA was found in 25 of 26 (96%) penile condylomata, all but two of which contained HPV 6 or 11. Using the highly sensitive polymerase chain reaction (PCR), we also detected HPV DNA in four of 20 (20%) of the penile lesions that lacked the histological features of condylomata negative for HPV by in situ analysis. Most of the lesions that were identifiable only after the application of an acetic acid solution were HPV negative when tested with either technique. We conclude that HPV DNA can be found in penile biopsy specimens that do not demonstrate the unequivocal histological features of condylomata but that the detection rate is much lower than for condylomata even when analyzing tissues in men at high risk for HPV-related lesions by PCR. This study underscores the need for caution when interpreting such tissues and the usefulness of in situ analysis for detecting assumed viral proliferation in noncondylomatous lesions.     

Detection of human papillomavirus (HPV) DNA in human prostatic tissues by polymerase chain reaction (PCR)

Human papillomavirus (HPV) infections are strongly linked to the pathogenesis of uterine cervical neoplasms, and have been implicated in other cancers of the female genital tract. In contrast, the association of HPV with the cancers of the male urogenital tract is less evident, except in anal and penile cancers. However, recent studies reporting the prevalence of HPV infections in human prostate cancers (60–100% HPV 16 positive vs. no infection of HPV) have raised controversies regarding the prevalence of HPV in benign and neoplastic human prostate. We investigated the prevalence of HPV infections in prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinomas in 23 surgically resected prostates. Polymerase chain reaction (PCR) was used to amplify HPV 6b/11, 16 and 18 specific DNA sequences, using type specific HPV primers selected from the transforming gene E6-E7. The areas of PIN and cancer in 6 p.m H&E stained tissue sections were identified, and respective areas of PIN and cancer were isolated from the adjacent serial sections and used for DNA amplification and HPV detection (Fig. 1). Our results demonstrated the presence of HPV 16 in three carcinomas (13%), using type specific primers in PCR amplified samples. We were not able to demonstrate the presence of other HPV types (HPV 6b/11 or HPV 18) in any of the samples using specific primers. Two of these prostates showed relatively strong positive signals by dot blot analysis, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. One more sample showed weak positivity, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. Subsequently, we have confirmed these results by Southern hybridization of the samples transferred to nylon membrane after agarose gel electrophoresis and detected by HPV 16 type specific oligonucleotide probe, using chemiluminescent assay. We, therefore, conclude that HPV infections of the prostate in general are not as common as has been previously claimed by other investigators. © 1993 Wiley-Liss, Inc.     

前列腺干细胞抗原在人前列腺癌组织中的表达及意义

目的　探讨前列腺干细胞抗原 (PSCA)在国人前列腺癌 (PCa)组织中表达的临床意义。　方法　采用免疫组织化学 (IHC)和核酸原位杂交 (ISH)方法检测 4 0例PCa、2 0例良性前列腺增生(BPH)和 2 0例前列腺上皮内瘤 (PIN)组织标本PSCA蛋白和mRNA表达 ,半定量法计算PSCA阳性表达细胞百分数和阳性表达强度 ,比较各组织间表达水平的差异及其与PCa分级、临床分期之间的关系。　结果　PCa、BPH、PIN组织PSCA中度阳性到强阳性表达分别为 85 % (34/ 4 0 )、2 0 % (4/ 2 0 )和35 % (7/ 2 0 ) ;PCa组织PSCA表达水平与BPH和PIN比较差异有统计学意义 (P <0 .0 5 ) ,BPH与PIN比较差异无统计学意义 (P >0 .0 5 ) ;PCa组织PSCA表达水平随Gleason评分及临床分期增加而升高。　结论　人PCa组织有PSCA蛋白质和mRNA的过表达 ,且与PCa病理分级、临床分期呈正相关 ,可能对PCa的诊断及判断预后有潜在价值。     

Detection of chromosomal aberrations in prostate cancer by fluorescence in situ hybridization (FISH)

PURPOSE: Fluorescence in situ hybridization (FISH) is a powerful tool for quantitative analysis of chromosomes and genes and can be applied in a variety of specimens, including cell cultures, isolated nuclei from fresh and fixed tissues, and histological tissue sections. For detection of numerical chromosome aberrations, we examined prostatic cancer samples at our department. In addition, we also observed primary and secondary aberrations taking part in the initiation and progression of tumours. MATERIALS AND METHODS: FISH using chromosome-specific alpha-satellite DNA probes for chromosomes 7, 8, 9, 10, 17, X and Y was performed on 19 prostatic cancer and 19 benign prostatic hyperplasia (BPH) samples obtained from transurethral resection (TUR) and archival paraffin-embedded blocks. RESULTS: Numerical aberrations were observed in 41% of the tumours studied. A range of aberrant copy numbers of chromosome 9 (68%), 7 (63%), 8 (58%), 17 (37%), Y (32%) and 10 (26%) was observed. We did not observe significant aberrations in BPH samples. In prostate cancer patients, chromosomes 7 (47%), 8 (58%) and 9 (63%) were monosomic by FISH. Monosomy 8 and 9 were significant differences (p>0.05) between prostate cancer and BPH patients. CONCLUSIONS: FISH analysis could be observed an one of strongest methods of analysis in detecting numerical aberrations of individual chromosomes with application to paraffin-block samples, metaphase and, interphase nuclei. To our knowledge, this analysis is firstly studied in Turkish patients. Therefore, results of this analysis may be important for Turkish patients.     

High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma

PURPOSE: Recent epidemiological data indicate that a history of increased exposure to sexually transmitted diseases is associated with an increased risk of prostate cancer. Human cytomegalovirus (HCMV) is a member of the herpesvirus family, is sexually transmitted in adults and can persistently infect prostatic epithelium in non-immunocompromised hosts. Based on increased awareness of the oncogenic potential of this virus, we decided to reexplore the issue of whether HCMV might be involved in prostate cancer pathogenesis. MATERIALS AND METHODS: Paraffin embedded biopsy specimens from 22 randomly selected patients with prostatic intraepithelial neoplasia (PIN) lesions and prostatic carcinoma were analyzed by immunohistochemistry, in situ hybridization, polymerase chain reaction and DNA sequencing to detect HCMV nucleic acids and determine whether HCMV gene products were specifically associated with neoplastic cells. RESULTS: We detected HCMV proteins and/or nucleic acids in all 22 of the 22 preneoplastic and neoplastic prostate lesions evaluated. HCMV proteins were specifically and often highly expressed in basal cell hyperplasia and PIN lesions, and to a lesser degree in carcinoma cells. RESULTS: To our knowledge these data demonstrate for the first time the specific localization of HCMV nucleic acids and proteins in a high percent of PIN and prostate carcinoma lesions, and raise the possibility that HCMV might contribute to the natural history of prostatic cancer.     

Down-regulated expression of prostasin in high-grade or hormone-refractory human prostate cancers

BACKGROUND: We previously conducted a search for genes which are differentially expressed in hormone-refractory prostate cancers using cDNA-representational difference analysis (RDA). The prostasin gene was isolated as one showing down-regulation in hormone-refractory cancers. In the present study, linkage to the stage in prostate neoplasia was examined. METHODS: Prostasin expressions in 54 prostate cancer cases were examined by mRNA in situ hybridization and immunohistochemistry as well as by northern blot analysis. RESULTS: Expression levels of prostasin in hormone-refractory cancers were approximately one-sixth of those in organ-confined cancers by northern blotting. Glandular components in benign prostatic hyperplasia and high-grade prostatic intraepithelial neoplasias tended to exhibit mild to moderate and relatively strong intensities, respectively. Expression levels of both prostasin mRNA and protein were inversely correlated with histological differentiation but not associated with clinical stage of human prostate cancer. Almost all cases of metastatic and hormone-refractory cancers demonstrated down-regulation of prostasin expression. CONCLUSIONS: These results suggest that prostasin cannot be regarded as a prognostic indicator for human prostate cancer although it may be a useful marker for tumor differentiation.