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.     

Malignant transformation of mature cystic teratoma to squamous cell carcinoma involves altered expression of p53‐ and p16/Rb‐dependent cell cycle regulator proteins

Ovarian mature cystic teratomas (MCT) uncommonly undergo malignant transformation to squamous cell carcinoma (SCC). While alterations in the p53 tumor suppressor gene and protein have been shown, few studies have analyzed other molecular changes leading to this malignant conversion. The purpose of the present study was to investigate 21 samples of SCC arising in MCT for altered expression in known p53‐ and p16/Rb‐dependent cell cycle regulatory proteins, and the association between their expression and cellular proliferation and histological features. Overexpression of the p53 protein was observed in 14 SCC (67%), while four (19%) had point mutations in the p53 gene. Reduced expression of the p16 protein was observed in 18 SCC (86%), while p16 gene alterations (hypermethylation (29%) and point mutation (33%)) were found in 11 (52%). Furthermore, a statistically significant correlation was observed between p53 and Rb overexpression (P = 0.0010), and the overexpression of both p53 and Rb was respectively significantly correlated with increased cellular proliferation. The results indicate that alterations in both the p53 and p16‐Rb pathways are associated with SCC arising in MCT.     

Uterine cervical squamous cell carcinoma without p16 (CDKN2A) expression: Heterogeneous causes of an unusual immunophenotype

Immunohistochemically p16 (CDKN2A)‐negative uterine cervical squamous cell carcinoma (SCC) is uncommon, and there are few reports about its pathological features. This study explored the causes of p16 negativity in such cases. We analyzed diagnostic tissue samples of five cases of p16‐negative cervical SCC among 107 patients who underwent hysterectomy at Kyoto University Hospital between January 2010 and December 2015. The samples were subjected to immunohistochemical staining, in situ hybridization and a genetic analysis. Two of five cases were positive for human papilloma virus (HPV) by genotyping. One was positive for HPV56 with promoter hypermethylation of CDKN2A and co‐existing Epstein–Barr virus infection. Another was positive for HPV6 categorized as low‐risk HPV with condylomatous morphology. Among the remaining three cases, one had amplification of the L1 gene of HPV with promoter hypermethylation of CDKN2A and TP53 mutation, and one of the other two HPV‐negative cases had a homozygous CDKN2A deletion, while the other was positive for p53 and CK7. p16‐negativity of cervical SCC is often associated with an unusual virus infection status and CDKN2A gene abnormality.     

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2-induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV-negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low-grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non-neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.     

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.     

The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma

Stankiewicz E, Prowse D M, Ktori E, Cuzick J, Ambroisine L, Zhang X, Kudahetti S, Watkin N, Corbishley C & Berney D M
(2011) Histopathology 58 , 433–439
The retinoblastoma protein/p16 ^INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma Aims: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell‐cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell‐cycle proteins: p53, p21, p16^INK4A and retinoblastoma (RB) protein. Methods and results: One hundred and forty‐eight PSCC samples were examined immunohistochemically for RB, p16^INK4A, p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty‐nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P < 0.0001) and p16^INK4A (P < 0.0001) and p21 (P = 0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. Conclusions: HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual‐type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours.     

Telomerase activity, MIB-1, PCNA, HPV 16 and p53 as diagnostic markers for cervical intraepithelial neoplasia

The present investigation evaluated the relationship between dysplasia of the uterine cervix and telomerase activity, expression of p53, MIB-1 and PCNA. Telomerase activity was measured on cervical cytobrush material from 126 women suspected of having dysplasia and 61 controls using the telomeric repeat amplification protocol. Immunohistochemistry was used to detect the tumor suppressor protein p53 and cell proliferation, the latter by MIB-1 and PCNA expression. Infection with human papillomavirus 16 was detected by PCR amplification and Southern blot hybridization of DNA extracted from the same brush material. Positive telomerase activity was found in 5 of 43 (11.6%) normal samples, 12 of 57 (21.1%) samples with inflammation or koilocytosis, 7 of 17 (41.2%) CIN 1 (cervical intraepithelial neoplasia, grade 1), 8 of 20 (40.0%) CIN 2, and 25 of 42 (59.5%) CIN 3/ CIS. Telomerase activity was significantly related to the level of dysplasia (p<0.001) and proliferation measured by MIB-1 (p=0.019), but not to the level of PCNA (p=0.445), HPV 16 status (p=0.098) or staining for p53 (p=0.271). Dysplasia was also related to PCNA, MIB1, p53, and presence of HPV 16. A sequential increase in the examined parameters, paralleling the progression of abnormality, was observed. PCNA and telomerase showed an increase in CIN 1, MIB-1 and HPV16 in CIN 2, and finally p53 in CIN 3/CIS.     

Esophageal small cell carcinomas: clinicopathologic parameters, p53 overexpression, proliferation marker, and their impact on pathogenesis

OBJECTIVE: To evaluate the clinicopathologic features and the roles of p53 and MIB-1 in esophageal small cell carcinoma. METHOD: Twenty patients (14 men and 6 women) with esophageal small cell carcinoma treated in our hospital from 1982 through 1996 were studied. The clinicopathologic features, treatment received, and survival data of these patients were documented. Representative tissue was collected from each tumor, and immunohistochemical preparations for p53 protein and MIB-1 were made. RESULTS: Small cell carcinoma accounted for 1.3% of all esophageal malignant tumors. The median age of patients at presentation was 60 years. On gross examination, the tumors were large ulcerative lesions (median length, 7.5 cm). In 17 patients in whom p53 immunohistochemical study was performed, p53 protein was detected in 65% (9 of 17). All stage IV tumors were negative for p53 expression. The median tumor cell MIB-1 score was high at 855 (range, 810-964) positive cells per 1000. Overall median survival was 3.4 months. In patients who underwent chemotherapy, there was significant response. CONCLUSIONS: Esophageal small cell carcinoma is an aggressive tumor. Overexpression of p53 is associated with early stages of carcinogenesis. The high proliferative index, as defined by the MIB-1 immunohistochemical method, may be related to aggressive behavior and high sensitivity to chemotherapy and radiotherapy.     

Expression of p53, pRB,and p16 in lung tumours: a validation study on tissue microarrays

Tissue microarrays have been created from 326 lung tumours, including 173 squamous cell carcinomas (SCCs) and 132 adenocarcinomas (ADs). In order to evaluate the usefulness of this microarray series, the expression of p53, p16, and Rb proteins was compared by immunohistochemistry on both the tissue microarrays and the corresponding whole sections for all 326 tumours. The presence of replicate punches improved both the yield and the concordance of data relative to the whole section results, so that the consensus score from the replicates agreed with the whole section result in more than 90% of informative tumours. The large number of tumours in this series also allowed significant differences in protein expression patterns to be detected between SCC and AD, the major subtypes of non-small cell lung carcinoma (NSCLC). SCC had higher levels of p53 staining (67% vs 52% in AD) and substantially increased p16 loss (SCC 75%, AD 53%) combined with greater retention of pRB expression (SCC 86% vs 67% in AD). The strong inverse correlation between p16 and pRB seen in SCC was essentially absent in AD. This study represents the largest single immunohistochemical survey of protein expression for p53, p16, and RB in NSCLCs.     

Detection of HPV-DNA, p53 alterations, and methylation in penile squamous cell carcinoma in Japanese men

Penile carcinoma is a rare disease, accordingly there are few studies on molecular changes, and these results also vary greatly. A total of 26 penile squamous cell carcinomas in Japanese men were studied with respect to HPV, p53 alterations, and methylation of gene promoter region. HPV-DNA was detected in three of 26 patients (11.5%). Overexpression of p53 was observed in 13 of 26 patients (50%), and p53 gene mutations were detected in four of 26 patients (15.4%). The frequency of methylation was as follows: DAPK , 26.9% (7/26); FHIT , 88.4% (23/26); MGMT , 19.2% (5/26); p14 , 3.8% (1/26); p16 , 23.1% (6/26); RAR -β, 23.1% (6/26); RASSF1A , 11.5% (3/26); and RUNX3 , 42.3% (11/26). As for correlation between HPV and p53 alterations, and methylation status, mutations of the p53 gene were detected only in HPV-negative patients, and methylation was more frequently found in HPV-negative than in HPV-positive patients. The present results suggest that the majority of penile squamous cell carcinomas in Japanese men are unrelated to HPV, and gene alterations accumulate more frequently in HPV-unrelated penile carcinomas.     

Correlation between human papilloma virus infection in cervical lesions and expression of p53, p21 proteins and Ki-67

BACKGROUND: Human papilloma virus (HPV) is the most important factor in the oncogenic mechanism of cervical tumor. Furthermore, in a separate multi-stage process, abnormality in cell cycle kinetics has been demonstrated. In order to elucidate the oncogenic mechanism, we examined the relationship between cervical carcinoma and HPV infection, and also investigated the expression of p53 and p21 proteins as well as the cell proliferation capability by detecting Ki-67, and analyzed the correlations of these factors. MATERIALS AND METHODS: We studied the biopsy specimens from 107 patients of chronic cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma (SCC). HPV DNA was detected by the hybrid capture method. Immunostaining by LSAB procedures were performed using antibodies to p53 protein, p21 and MIB-1. The PCR-denaturing gradient gel electrophoresis (DGGE) method was used to search for mutation in exons 5, 6, 7 and 8 of p53. RESULTS: Of 107 cases studied, high-oncogenic HPV was detected in 80 cases (74.8%) with a particularly high prevalence in SCC. No correlation was observed between HPV infection and expression of p53, p21 or Ki-67. The degree of positivity of Ki-67 expression tended to be higher with disease progression. Cases strongly positive (2+) for p53 and p21 proteins were weakly positive for Ki-67, and cases positive (1+) or negative for p53 and p21 were strongly positive for Ki-67. CONCLUSION: In oncogenesis of cervical carcinoma, p53 protein, p21 protein and HPV may act separately as independent factors in some cases, and there is a strong possibility that other factors are involved.     

Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases

P63 is a member of the p53 family, which plays a role in the differentiation of urothelium and is supposed to play a role in urothelial carcinogenesis. P53 and MIB-1 are recognised in many studies as predictive markers of progression, but few studies in the literature have examined p63. The aims of our study were to explore the expression of p63 in bladder carcinomas and to compare this expression to p53 and MIB-1, as well as to stage and grade. Tissue microarrays were performed on 158 urothelial carcinomas (56 pTa, 45 pT1 and 57≥pT2). Immunohistochemical studies were performed with p63, p53 and MIB-1 antibodies. In our study we observed that p63 immunostaining is present in all cell layers in papillary urothelial neoplasm of low malignant potential (PUNLMP), but partially lost in non-invasive papillary urothelial carcinoma low grade (NILGC) and in pT1/≥pT2 bladder cancers. P53 and MIB-1 displayed lower expression in PUNLMP/NILGC vs non-invasive papillary urothelial carcinoma high grade (NIHGC)/pT1, but there was no correlation between the expression of p63, p53 and MIB-1. Our study demonstrates that p63 expression distinguishes between PUNLMP/NILGC and NIHGC/pT1 (p=4.10⁵). A statistical difference disserving pTa and pT1/≥pT2 with a statistical significance (p<10⁻⁶) could also be observed. P63 should be considered as an additional biomarker that might help pathologists to classify their patients.     

Expression and prognostic implications of cell cycle regulatory molecules, p16, p21, p27, p14 and p53 in germinal centre and non-germinal centre B-like diffuse large B-cell lymphomas

AIMS: To evaluate the different expression patterns and the prognostic significance of cell cycle regulatory molecules in diffuse large B-cell lymphomas (DLBCLs) of germinal centre (GC) and non-GC phenotypes. METHODS AND RESULTS: Tissue microarray slides composed of 126 extranodal and 88 nodal DLBCLs were immunostained for p16, p21, p27, p14 and p53. DLBCLs were classified into GC and non-GC phenotype according to the immunohistochemical expression of bcl-6, CD10, and MUM1. Aberrant expression of p53 was more frequent in the GC phenotype in nodal cases (P = 0.038), and the loss of p16, p21 and p14 expression was significantly more common in the non-GC phenotype (P = 0.004, P = 0.001, P < 0.001). Concurrent disruptions of the p16-Rb and p14-p53 pathways as represented by the immunoprofile of p16/p14/p53 (-/-/+) were associated with a poor prognosis in the GC phenotype [mean survival 31 months in the p16/p14/p53 (-/-/+) group versus 62 months in the other groups, P =0.0485]. CONCLUSIONS: The expression and prognostic implications of cell cycle regulatory molecules differ between GC and non-GC phenotypes in DLBCLs. The immunoprofile of p16/p14/p53 (-/-/+) within the GC phenotype of DLBCLs can be defined as a poor prognostic subgroup.     

Frequent alteration of p16(INK4a)/p14(ARF) and p53 pathways in the round cell component of myxoid/round cell liposarcoma: p53 gene alterations and reduced p14(ARF) expression both correlate with poor prognosis

In myxoid/round cell liposarcoma (MLS/RCLS), the presence of a round cell (RC) component has been reported to correlate with a worse prognosis for the patients. However, little is known about the molecular genetic differences between conventional myxoid (MX) components and RC components in this tumour. The aim of this study was to investigate the possible implications of molecular alterations of G1 to S-phase check-point genes, especially in the RC component. We evaluated the immunohistochemical expression of p53, MDM2, p14 and p16 protein and assessed proliferative activities using MIB-1 in 29 RC components and 81 MX components from 90 cases. Mutation of the p53 gene, amplification of the MDM2 gene, homozygous deletion, methylation status and mutation of the p16(INK4a)/p14(ARF) genes were also investigated, using concordant paraffin-embedded and frozen material. The data were analysed together with clinicopathological factors to assess their prognostic implications in MLS/RCLS. Immunohistochemically, the over-expression of p53 protein (p = 0.01366) and the reduced expression of p14 (p < 0.0001) and p16 (p < 0.0001) proteins were significantly more frequently observed in RC components than in MX components. Reduced expression of p14 protein correlated significantly with hypermethylation of the p14(ARF) gene promoter (p = 0.0176) and over-expression of p53 protein (p = 0.00837). By univariate analysis, reduced expression of p14 and p53 missense mutation were found to reduce the rate of survival significantly (p < 0.05). Multivariate analysis, including clinicopathological factors, revealed that tumour site (p = 0.0251), the presence of an RC component (p = 0.0113), high MIB-1 labelling index (p = 0.0005) and p53 missense mutation (p = 0.0036) were adverse prognostic factors. In MLS/RCLS, reduction of p14 protein expression and p53 mutation were related to poor prognosis. Accordingly, the p14(ARF)/p53 pathway may contribute to the presence of an RC component and malignant progression in this tumour.     

Immunohistochemical expression of Mdm2 and p53 in penile verrucous carcinoma

Verrucous carcinoma (VC) of the penis is an uncommon squamous tumor that pursues a biologically indolent course. Unlike conventional squamous cell carcinoma (SCC) of the penis, pathogenic roles for human papillomavirus (HPV) infection and p53 mutation have not been reported in VC. We compared the immunohistochemical expression of Mdm2 and p53 in 7 cases of VC and 7 cases of SCC. The Mdm2 gene product preferentially labeled the perinuclear membrane in the granular layer of VC tumor cells, whereas SCC cases showed weak, focal, cytoplasmic staining for Mdm2. The mean labeling index for Mdm2 was higher in VC compared to SCC [79.3 (SE +/- 7.2) in VC vs 18.3 (SE +/- 2.4) in SCC, p < 0.001]. In SCC cases, the normal surrounding skin showed mild granular-layer staining and dysplastic foci that failed to stain with Mdm2 antibody. Weak p53 immunolabeling was seen within nuclei of scattered tumor cells in the cases of VC, whereas the SCC cases showed strong nuclear staining of p53 throughout the tumors. The mean labeling index for p53 was lower in VC compared to SCC [24.8 (SE +/- 3.9) in VC vs 64.7 (SE +/- 9.0) in SCC, p < 0.01]. In SCC cases, the normal surrounding skin showed moderate staining for p53, preferentially confined to the basal layer. Dysplastic foci in the cases of SCC showed increased p53 labeling. In summary, immunohistochemical analysis showed significantly different levels of expression of Mdm2 and p53 in penile VC vs SCC. Overexpression of the Mdm2 gene product may be important in the pathogenesis of VC. Since Mdm2 is a negative regulator of p53, overexpression of Mdm2 may explain why p53 is down-regulated and, therefore, permissive to oncogenic transformation.     

喉癌发生中人乳头瘤病毒感染与Langerhans细胞、p53表达的关系

目的：探讨喉癌的发生机理。方法：应用免疫组化ＬＳＡＢ法对３０例喉鳞状细胞癌人乳头瘤病毒（ＨＰＶ）感染、Ｌａｎｇｅｒｈａｎｓ细胞（ＬＣ）及ｐ５３蛋白表达进行了研究。结果：２６．７％的病例可以检测到ＨＰＶ抗原成分。ＨＰＶ感染的癌旁粘膜内ＬＣ数量明显少于无感染者，且形态也发生改变。ｐ５３蛋白表达阳性率在ＨＰＶ感染组（３７．５％）明显低于ＨＰＶ检测阴性组（８３．３３％）。结论：提示ＨＰＶ、ＬＣ、ｐ５３在喉癌发生发展过程中起一定作用，且相互影响，ＨＰＶ感染引起ＬＣ数量减少，局部免疫功能降低，ＨＰＶ感染还可能通过表达的肿瘤蛋白或其他机制使抑癌基因ｐ５３失活，进而导致肿瘤的发生。     

High frequency of coexpression of maspin with p63 and p53 in squamous cell carcinoma but not in adenocarcinoma of the lung

Maspin, a member of the serpin family of protease inhibitors, has been shown to inhibit tumor growth and suppress metastasis in several malignancies, including lung cancer. Previous studies have reported that p63 and p53 control maspin expression by transactivating the promoter. The present study analyzed immunohistochemical studies to determine the expression and coexpression patterns of maspin, p63 and p53 in non-small cell lung carcinoma, specifically squamous cell carcinoma and adenocarcinoma. The results showed that 83/86 cases (96.5%) of squamous cell carcinoma and 82/161 cases (50.9%) of adenocarcinoma included in this study were positive for maspin. There were 79/86 cases (91.9%) of squamous cell carcinoma and 16/161 cases (9.9%) of adenocarcinoma with positive expression for p63. In addition, 77/86 cases (89.5%) of squamous cell carcinoma and 99/161 cases (61.5%) of adenocarcinoma were positive for p53. Maspin, p63 and p53 expression were each significantly higher in squamous cell carcinoma than adenocarcinoma. Squamous cell carcinomas more highly coexpress maspin and p63, as well as maspin and p53, when compared with adenocarcinomas. The high frequency of coexpression of maspin and p63, as well as maspin and p53, in squamous cell carcinoma, suggests that p63 and p53 may be involved in the pathway to control maspin expression. Therapeutic targeting on maspin, p63 and p53 molecules might be beneficial in the management of patients with squamous cell carcinomas of the lung in the future.