Human papillomavirus infection in actinic keratosis and bowen's disease: comparative study with expression of cell-cycle regulatory proteins p21(Waf1/Cip1), p53, PCNA, Ki-67, and Bcl-2 in positive and negative lesions

We examined the presence of human papillomavirus (HPV) DNA in tissues of premalignant skin lesions, i.e., actinic keratosis (n = 13) and Bowen's disease (n = 62), taken from 69 Japanese immunocompetent and renal transplant recipient patients. Detection and typing of HPV DNA were performed using polymerase chain reaction (PCR) and sequence analysis or restriction fragment length polymorphism (RFLP) analysis, respectively. The positivity rates of HPV DNA in tissues of actinic keratosis and Bowen's disease were 77% and 65%, respectively. Twenty-seven HPV types were detected in 50 (67%) premalignant skin lesions, in which Z95963 (accession no. in the EMBL Databank), Z95968, AJ010823, and AJ000151 have been described as partial sequences of unknown HPV types. Furthermore, 2 unknown types, HPVX1 and HPVX2, were found in specimens of actinic keratosis. Sequence analysis showed that HPVX1 is related to HPV-37 (86.1% sequence homology) and that HPVX2 is related to HPV-38 (79.7%). These results indicate that various mucosal and epidermodysplasia verruciformis-related HPV types are associated with the pathogenesis of actinic keratosis and Bowen's disease. In addition, 24 specimens of HPV-positive or HPV-negative premalignant skin lesions were examined immunohistochemically for proliferating cells to determine biological differences between HPV-positive and HPV-negative lesions. Immunohistochemistry for p21(Waf1/Cip1), p53, proliferating cell nuclear antigen (PCNA), Ki-67, and Bcl-2 revealed that there was no significant difference in the cell proliferation activity between HPV-positive and HPV-negative lesions, suggesting that HPV infection alone does not induce cell proliferation in those lesions.     

p53 gene mutations and MDM2 amplification are uncommon in primary carcinomas of the uterine cervix.

The p53 gene is the most frequently altered gene known thus far in a wide variety of human cancers. Inactivation of p53, either through mutation or through interaction with the human papillomavirus (HPV) E6 oncoprotein, is a characteristic feature of all cervical carcinoma cell lines that have been studied. These findings suggest that p53 inactivation is required for cervical carcinoma development and that HPV infection and p53 mutation may be mutually exclusive. We have studied the p53 gene in 35 primary cervical carcinomas. DNA sequence and single strand conformational polymorphism analyses were used to evaluate p53 in 27 squamous carcinomas (25 HPV-positive) and eight adenocarcinomas (four HPV-positive). A missense mutation of p53 was observed in one HPV 16-positive squamous carcinoma, demonstrating that p53 mutations can occur in combination with HPV infection. The HPV-negative tumors all lacked p53 gene mutations. The absence of p53 mutations in HPV-negative cases prompted an assessment of tumors for MDM2 gene amplification. The MDM2 gene encodes a p53 binding protein and has been found to be amplified in some human tumors lacking p53 mutations. MDM2 amplification was not identified in any of the tumors we examined, including four HPV-negative cases. Our findings show that HPV infection and p53 gene mutation are not mutually exclusive and suggest that many HPV-negative carcinomas may arise via a pathway independent of p53 inactivation.     

Human papillomavirus genotyping by oligonucleotide microarray and p16 expression in uterine cervical intraepithelial neoplasm and in invasive carcinoma in Korean women

For evaluating the diagnostic significance of p16(INK4A) over-expression in the uterine cervical intraepithelial neoplasm and in invasive carcinoma, human papillomavirus (HPV) was detected and genotyped by oligonucleotide microarray in archival tissues of 117 cervical specimens, including 47 invasive squamous cell carcinomas (SCC), 30 cases of cervical intraepithelial neoplasia (CIN), 20 adenocarcinomas, and 20 cases of non-neoplastic cervix. The expression of p16(INK4A) protein was immunohistochemically studied in these cases and in five HPV-positive and one HPV-negative cervical cancer cell lines. HPV was detected in 50% of CIN, 61.7% of SCC, and 45.5% of adenocarcinomas. p16(INK4A) expression was seen in all 20 cases of adenocarcinoma, 78.7% (37/47) of SCC, and 96.7% (29/30) of CIN, but not in any cases of the non-neoplastic cervix. There was no difference in p16(INK4A) expression between the HPV-positive and HPV-negative cervical lesions. All HPV-positive and -negative cervical cancer cell lines expressed p16(INK4A) protein. In conclusion, the presence of p16(INK4A) expression in cervical squamous and glandular epithelium indicates the existence of dysplasia or malignancy in the uterine cervix, regardless of HPV infection.     

HPV genotype prevalence in cytologically abnormal cervical samples from women living in south Italy

Human papillomavirus (HPV) infection is the commonest sexually transmitted infection, and high-risk HPV types are associated with cervical carcinogenesis. This study investigated: the HPV type-specific prevalence in 970 women with an abnormal cytological diagnosis; and the association of HPV infection and cervical disease in a subset of 626 women with a histological diagnosis. HPV-DNA was researched by nested PCR/sequencing and the INNOLiPA HPV Genotyping assay. The data were analysed by the chi-square test (p相似文献   

Expression of p21/WAF-1, status of apoptosis and p53 mutation in esophageal squamous cell carcinoma with HPV infection

Human papilloma virus (HPV) is regarded as a causative carcinogenic agent in anogenital squamous cell carcinoma (SCC), but there is controversy about its etiologic role in esophageal SCC (ESCC). In this study, we attempted to clarify whether HPV infection plays a crucial role in the development of ESCC by analysis of multiple factors. These included: detection of HPV DNA; evaluation of immunohistochemical assays for HPV-related cell cycle regulators and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method; and genetic analysis of the p53 gene. Twenty of the 48 ESCC examined (42%) were found to be positive for the HPV genome by polymerase chain reaction. They comprised 16 cases with the HPV16 subtype, three with the HPV18 subtype, and one with both HPV16 and 18. Immunohistochemical analysis revealed that the expression of p21/WAF-1 was significantly decreased in HPV-positive cases (chi2 = 9.2614; P = 0.0023). Furthermore, the 10 apoptosis-negative (< or =10%) cases of HPV-positive SCC were almost exclusively p21/WAF-1-negative (chi2 = 12.1406; P = 0.0005), indicating the significance of the relationship between HPV infection and the phenotype that is expected from HPV-induced inhibition of p53. Although 14 cases possessed missense and deletion mutations of the p53 gene (of which four mutations were found in HPV-positive ESCC), no accumulation of the mutation was defined in the phenotype, suggesting that distinct mutation processes might be involved in HPV-negative and -positive ESCC. The data provide significant support for the hypothesis that HPV infection may play a crucial role in the oncogenesis of some ESCC.     

Expression of p16, Rb, and p53 proteins in squamous cell carcinomas of the anorectal region harboring human papillomavirus DNA.

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of squamous cell carcinoma of the anorectal region. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In cervical and head and neck cancers, HPV infection is also associated with an overexpression of p16, a cyclin-dependent kinase inhibitor. The expression of these cell cycle regulators in squamous cell carcinomas of the anorectal region has not been well studied. In the current study, 29 cases of squamous cell carcinoma of the anorectal region were immunohistochemically examined for the expression of p16, Rb, and p53 proteins. Tumor cell DNA was also extracted from paraffin blocks and subjected to broad-spectrum HPV DNA testing and typing. The results show that the tumor cells exhibited a strong and diffuse nuclear stain (with some cytoplasmic positivity) for p16 in all 29 cases (100%). The adjacent nonneoplastic squamous epithelium or colonic mucosa, in contrast, was completely negative. Loss of Rb nuclear staining in tumor cells was observed in 20 cases (69%). The p53 protein was essentially undetectable, with only 6 cases containing <10% positive cells. HPV DNA was detected in every case (100%), with 25 cases (86%) harboring Type 16. In addition, almost identical results were obtained in 12 HPV-positive squamous cell carcinomas of the upper aerodigestive tract. This was in marked contrast to those of HPV-negative tumors, where positive p16 staining and loss of Rb expression were seen in only 2/21 (10%) and 1/21 (5%) cases, respectively. These observations indicate that overexpression of p16 and loss of Rb nuclear staining are commonly associated with high-risk HPV infection, which may serve as useful surrogate biomarkers for identifying squamous cell carcinomas harboring HPV DNA.     

Human papillomavirus DNA sequences and p53 over-expression in laryngeal squamous cell carcinomas in Northeast China

One-hundred-two patients with laryngeal squamous cell carcinomas in Northeast China were examined for human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) coupled with Southern blot hybridization, and for p53 over-expression by immunohistochemical staining. HPV DNAs were found in 60 cases (58.8%). HPV-16, -18, -6, -11, and -33 DNAs were detected in 30 cases, 22 cases, 25 cases, two cases, and one case, respectively. In addition, coinfection either with HPV-6 and -16 or with HPV-6 and -18 was detected in 20 cases (33.3% of HPV DNA-positive cases). p53 over-expression was observed in 60 patients (58.8%). p53 was over-expressed significanty in the poorly-differentiated SCC and in patients with metastasis to lymph nodes (P < 0.05, respectively). Both HPV DNA and p53-expression were positive in 35 patients, and negative in 17 patients. Either HPV DNA or p53-expression were positive in 50 patients (25 cases each). Although p53 was detected in 35 (58.3%) of HPV-positive patients, there was no significant correlation between HPV infection and p53 over-expression in laryngeal squamous cell carcinomas of Northeast China. J. Med. Virol. 54:186–191, 1998. © 1998 Wiley-Liss, Inc.     

p53、p21~(WAF1)蛋白在非小细胞肺癌中的表达及其临床意义

目的　探讨原发性非小细胞肺癌中p5 3、p2 1WAF1蛋白表达与临床病理及预后的关系。方法　应用免疫组织化学 (SP法 )方法。共检测非小细胞肺癌 147例 ,其中腺癌 6 6例 ,鳞癌 6 3例 ,腺鳞癌 14例 ,大细胞癌 4例。结果　p5 3蛋白总阳性率为 6 1.2 % (90 / 147) ,腺癌为 5 7.6 % (38/ 6 6 ) ,鳞癌阳性率为 6 3.5 % (4 0 / 6 3) ,腺鳞癌为 71.4% (10 / 14) ,大细胞癌 2例阳性。p2 1WAF1蛋白总阳性率为40 1% (5 9/ 147) ,腺癌为 42 .4% (2 8/ 6 6 ) ,鳞癌为 41.3% (2 6 / 6 3) ,腺鳞癌 2 8.6 % (4 / 14) ,大细胞癌 1例阳性。肺腺癌p5 3蛋白阳性表达与其预后相关 ,6 6例腺癌中 ,生存率低于 3年组和高于 3年组的p5 3蛋白阳性率分别为 75 % (2 1/ 2 8)和 44 .7% (17/ 38) ,差异有显著性意义 (P <0 .0 2 5 )。p2 1WAF1阳性表达与肺癌预后有关 ,p2 1WAF1阳性表达者 3年生存率 (6 4.4% )高于阴性表达者 (4 6 .6 % ) (P <0 .0 5 )。p5 3阳性而p2 1WAF1阴性的非小细胞肺癌患者的预后比p5 3阴性而p2 1WAF1阳性者差 (P <0 .0 1)。结论　检测p5 3蛋白表达可作为判断肺腺癌预后的指标之一 ;检测p2 1WAF1蛋白表达有利于对非小细胞肺癌预后的判断 ;联合检测p5 3、p2 1WAF1蛋白对判断非小细胞肺癌的预后有重要的意义 ,似可作     

p^53,p^21^WAF1蛋白在非小细胞肺癌中的表达及其临床意义

目的 探讨原发性非小细胞肺癌中的ｐ＾５３、ｐ＾２１＾ＷＡＦ１蛋白表达与临床病理及预后的关系。方法 应用免疫组织化学（ＳＰ法）方法。共检测非小细胞肺癌１４７例,其中腺癌６６例,鳞癌６３例,腺鳞癌１４例,大细胞癌４例。结果 ｐ＾５３蛋白总阳性率为６１．２％（９０／１４７）,腺癌为５７．６％（３８／６６）,鳞癌阳性率为６３．５％（４０／６３）,腺鳞癌为７１．４％（１０／１４）,大细胞癌２例阳性,ｐ＾２１     

Detection of Chlamydia trachomatis and herpes simplex virus type 1 or 2 in cervical samples in human papilloma virus (HPV)-positive and HPV-negative women

This study investigated whether the prevalence of human papilloma virus (HPV) in association with Chlamydia trachomatis, herpes simplex virus (HSV)-1 and/or HSV-2 was greater in high-grade than in low-grade or control cervical biopsy specimens. HPV-positive (n = 86) and HPV-negative (n = 213) women were screened for HPV, HSV and C. trachomatis by PCR. The most common HPV genotypes were HPV-16, HPV-6 and HPV-33; mixed HPV infection (n = 12) was also seen. A higher prevalence of C. trachomatis, HSV-1 and HSV-2 was found in HPV-positive samples. High-risk HPV genotypes and combined HPV + C. trachomatis or HPV + HSV-1, but not HSV-2, infections were associated with a greater risk of developing cervical carcinoma.     

Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents.

Adolescents have high rates of human papillomavirus (HPV) infection, and persistent high-risk HPV infection can lead to the development of cervical cancer. The cyclin-dependent kinase inhibitor, p16(INK4a) is overexpressed in cervical intraepithelial neoplasia (CIN), probably due to a persistent and integrated HPV infection. This study investigated p16(INK4a) expression, grades of CIN, and high-risk HPV infection in adolescent cervical biopsies. Biopsies were immunohistochemically stained for p16(INK4a). The presence of wide-spectrum, low-risk, or high-risk HPV was determined by amplifying DNA extracted from the cervical biopsies. Biopsies were classified as cervicitis, 15 cases; CIN 1, 48 cases; CIN 2, 46 cases, and CIN 3, 52 cases. The distribution of p16(INK4a) staining was graded as patchy, diffuse basal, and diffuse full thickness. Pearson's chi(2) tests analyzed the relationships between p16(INK4a) staining, HPV infection, and CIN. Biopsies of cervicitis were negative for HPV and for p16(INK4a) expression. High-risk HPV 16, 18, and 31 increased from 18% in CIN 1 to 66% in CIN 2/3 (P<0.001). In CIN 1, p16(INK4a) was positive in 44% of biopsies with 35% showing patchy, 7% diffuse basal, and one case (2%) showing diffuse full thickness staining. In CIN 2/3, p16(INK4a) was positive in 97% of biopsies with 23% showing patchy, 21% diffuse basal, and 53% diffuse full thickness staining. The difference in the proportions of biopsies showing patchy p16(INK4a) staining in CIN 1 and diffuse full thickness staining in CIN 2/3 was significant (P<0.001). In CIN 1, 61% of high-risk HPV-positive biopsies were p16(INK4a) negative, while all high-risk HPV-positive CIN 2/3 biopsies were p16(INK4a) positive. Diffuse, full thickness p16(INK4a) expression discriminated low-grade from high-grade CIN and appears to be a marker of persistent high-risk HPV infection.     

Prediction of conserved microRNAs from skin and mucosal human papillomaviruses

Eight human papillomavirus (HPV) types including four cutaneous HPV types (HPV-5, HPV-8, HPV-20 and HPV-38) and four mucosal HPV types (HPV-6, HPV-11, HPV-16 and HPV-18) were selected for this miRNA study. Pre-miRNAs were predicted using a computer programme, and the conserved mature miRNAs were compared to currently known miRNAs. Predicted HPV miRNAs related to miR-466, -467 and -669 were common and specific to the mucosal HPV types. Northern blot hybridization confirmed a predicted miRNA in HPV-positive cervical cancer cell lines encoded by mucosal HPVs. HPV-38 was predicted to express an miRNA conserved to human let-7a and the expression of let-7a, in HPV-38-positive non-melanoma skin cancer (NMSC) biopsies was 10-fold higher than those with HPV-positive (for other types except HPV-38) and HPV-negative NMSCs, suggesting that let-7a expression might be related to HPV-38 infection. Potential gene targets of the predicted miRNA that may aid HPV in infection and pathogenesis were also analysed.     

HPV DNA and p53 alterations in oropharyngeal carcinomas

We have examined a series of 37 oropharyngeal squamous cell carcinomas for the presence of HPV \611, 16, and 18 DNA by polymerase chain reaction (PCR)/Southern blotting and for p53 alterations by immunohistochemistry and mutation screening with temperature gradient gel electrophoresis (TGGE). HPV sequences were found in a total of 26 of 37 cancers (70.3%), most frequently HPV 16 (\2037) followed by HPV 18 (\1137). Double infections with HPV 16 and 18 were present in 5 tumours. p53 accumulation was detectable immunohistochemically in 21 of 37 carcinomas (56.8%). There were remarkable differences in the distribution of immunoreactive tumour cells in relation to the tumour grade. A mutation screening for p53 by TGGE, directed to the amplified exons 5–8, revealed p53 mutations in 14 of 37 carcinomas (37.8%). Mutations in two different exons were present in 3 tumours, 11 tumours being hit once. Exon 7 was mutated in 6 carcinomas, exons 5 and 8 in 4 cases, and exon 6 in 3 cases. When grouping the tumours with p53 mutation according to their HPV state, HPV-positive cases showed slightly more mutations (\1126) than HPV-negative cases (\311). Only 5 of 37 carcinomas (13.5%) contained neither HPV DNA nor p53 alterations. Our results indicate that high-risk HPV and p53 mutations frequently coexist in oropharyngeal carcinomas, in contrast to genital tumours, notably carcinomas of the cervix uteri. This may reflect different pathways in carcinogenesis in squamous cell epithelium from different sites.     

Effect of high-risk human papillomavirus oncoproteins on p53R2 gene expression after DNA damage

The p53R2 protein is a p53-inducible small subunit of ribonucleotide reductase. It plays a crucial role in p53-dependent cellular response to DNA damage and oxidative stress by providing deoxyribonucleotides (dNTPs) to the DNA repair machinery and by scavenging reactive oxygen species (ROS). To investigate the effects of high-risk human papillomavirus (HPV) oncoproteins on p53R2 expression after DNA damage, we analyzed the p53R2 protein levels in human cells ectopically expressing the HPV-16 E6 and E7 genes, and in the HPV-positive cancer cell lines SiHa, CaSki and HeLa, exposed to adriamycin or to H(2)O(2). We found that in normal cells, p53R2 expression is efficiently induced by both H(2)O(2) and adriamycin, supporting the role of p53R2 in cellular response to oxidative stress. Ectopic expression of E6 impaired p53 and p53R2 induction after DNA damage in human fibroblasts. Moreover, SiHa, CaSki and HeLa cells were unresponsive to H(2)O(2) exposure, and adriamycin induced p53R2 levels only in SiHa cells. Our results imply that high-risk HPV infection may suppress the p53R2-dependent dNTPs supply to the DNA repair system and the ROS scavenging activity; they also suggest that an altered p53R2 response to genotoxins and to oxidative stress may contribute to HPV-induced genetic instability and carcinogenesis.     

Prevalence of human papillomavirus DNA in various histological subtypes of cervical adenocarcinoma: a population-based study.

The role of human papilloma virus (HPV) infection in the development of cervical carcinoma is well established, however, the prevalence of HPV DNA in cervical adenocarcinoma varies from study to study. It appears to be caused by a number of factors, one of which is that cervical adenocarcinomas comprise a heterogeneous group of multiple subtypes. To clarify the impact of HPV infection on the development of cervical adenocarcinoma with diverse histological subtypes, we performed a population-based study in Korean women from 15 different institutes for the status of HPV infection in adenocarcinoma of uterine cervix. A total of 432 cervical adenocarcinomas from 1997 to 2001 were reviewed and classified according to the modified WHO classification. For 135 cases, HPV typing was performed with HPV DNA chip (82 cases) and PCR HPV typing (53 cases), using formalin-fixed, paraffin-embedded archival tissue. The overall prevalence of HPV infection in cervical adenocarcinoma was 90%. The infection of HPV 16 and/or HPV 18 accounted for 78% of HPV-positive adenocarcinomas. Multiple HPV types were found in 13% of the cases. The HPV DNA was rarely detected in minimal deviation adenocarcinoma. Interestingly, HPV 16 was a predominant type in endometrioid and villoglandular types, whereas HPV 16 and HPV 18 were detected with equal prevalence in other subtypes. In conclusion, HPV infection, mostly HPV 16 and HPV 18, is highly associated with most of the cervical adenocarcinomas, whereas endometrioid and villoglandular type have a different pattern of HPV infection status. Minimal deviation adenocarcinoma does not seem to be related with HPV infection.     

厦门地区女性人乳头瘤病毒感染状况调查

目的 对厦门地区妇女宫颈人类乳头瘤病毒（human papilloma virus,HPV）亚型进行筛查,以探讨其分布规律.方法 采用凯普医用核酸分子快速杂交仪,对7683名2013年1月-2013年12月到厦门市174医院妇科门诊或病房就诊的女性进行生殖道21种HPV感染基因亚型筛查.结果 7683例样本中,HPV感染者1421例,阳性率18.50％.感染人群主要集中在30～39岁.其中高危型HPV阳性率为16.01％,低危型HPV阳性率为2.49％.单一基因型别者1148例,阳性率为14.94％;双重感染者220例阳性率为2.86％;三重以上感染者53例,阳性率为0.70％;HPV感染阳性率居前6位的基因型分别为HPV-52（3.64％）、HPV-16 （3.33％）、HPV-58（2.98％）、HPV-53（2.16％）、HPV-CP8304（1.91％）和HPV-18（1.09％）.结论 厦门地区妇女HPV感染率高,且以高危型单一基因亚型感染为主;感染基因型别主要以HPV-52、16、58、53、CP8304和18为主,具有一定的地域差异性;52及58型感染率高,对于疫苗的研制和开发有参考意义.     

Monoclonal expansion with integration of high-risk type human papillomaviruses is an initial step for cervical carcinogenesis: association of clonal status and human papillomavirus infection with clinical outcome in cervical intraepithelial neoplasia

To define the natural history of cervical intraepithelial neoplasia (CIN) as related to clonal status, we evaluated 20 cases of CIN1 and 18 cases of CIN2 that had been clinically followed for 7 to 48 months at Osaka University Hospital. These included 10 cases that progressed, 15 cases that persisted, and 13 cases that regressed. We analyzed the clonal status of each case by analysis of the pattern of X-chromosomal inactivation. Human papillomavirus (HPV) infection was detected by PCR-RFLP analysis. CINs that are monoclonal or infected by high-risk HPVs are more likely to progress or persist than cases that are polyclonal or negative for high-risk HPVs (p = 0.009 for monoclonal vs polyclonal, p = 0.024 for high-risk HPV positive vs negative p = 0.024). Eighteen (90%) of 20 monoclonal, high-risk HPV-associated CINs progressed or persisted, whereas 9 (60%) of 15 polyclonal or high-risk HPV-negative CINs regressed. Therefore, the combination of clonality status and high-risk type HPV infection was significantly correlated with clinical outcome (p = 0.003). The physical status of the HPV genome was evaluated in 17 cases of HPV-16 positive CINs by real-time PCR. Of those, the HPV viral genome was present in both episomal and integrated forms in 14 CINs (84%), and 12 of these cases (86%) were monoclonal in composition. By contrast, all three CINs in which the HPV genome was present in episomal form were polyclonal. In one CIN1 that was polyclonal, HPV-16 was originally present in episomal form but after 24 months, the patient developed a monoclonal CIN3 in which the HPV-16 genome was present in mixed form. These results may imply that HPV viral integration into the host genomic DNA is associated with progression from polyclonal to monoclonal status in CIN. These events may play a fundamental role in the progression from low-grade to higher grade dysplasia of the cervical mucosa.     

EXPRESSION OF CYCLIN-DEPENDENT KINASE INHIBITOR p21WAF1/CIP1 IN NON-NEOPLASTIC MUCOSA AND NEOPLASIA OF THE STOMACH: RELATIONSHIP WITH p53 STATUS AND PROLIFERATIVE ACTIVITY

The expression of the p53-inducible cyclin-dependent kinase inhibitor p21^WAF1/CIP1 in non-neoplastic mucosa, adenoma, and adenocarcinoma of the stomach was examined immunohistochemically and its relationship with p53 expression and proliferative activity was analysed. In normal gastric mucosa as well as in intestinal metaplasia the epithelial cells at the surface which showed no proliferative activity expressed p21whereas the cells in the deep area of the glands expressing Ki-67 did not. In the neoplastic lesions, the expression of p21^WAF1/CIP1 was detected in 78 per cent (112/144) of the adenomas and 76 per cent (262/343) of the adenocarcinomas. The incidence of p21^WAF1/CIP1 expression did not differ among histological types of gastric carcinoma. The strong expression of p21^WAF1/CIP1 was more frequently observed in carcinomas invading into submucosa or in cases of stages 2, 3, and 4 than in carcinomas limited to the mucosa or in stage 1 cases. The incidence of strongly positive cases was higher in carcinomas with lymph node metastasis than in those without metastasis. There was no apparent correlation between the expression of p21^WAF1/CIP1 and the abnormal accumulation of p53 or with proliferative activity measured by Ki-67 expression. These findings overall suggest that p21^WAF1/CIP1 might be associated with the senescence of non-neoplastic gastric epithelial cells; that a p53-independent pathway might be substantially involved in the induction of p21^WAF1/CIP1 in gastric neoplasia; and that the proliferative activity of gastric cancer might not be solely dependent on control of the cell cycle by p21^WAF1/CIP1.