p16INK4a is a Prognostic Marker in Resected Ductal Pancreatic Cancer: An Analysis of p16INK4a, p53, MDM2, an Rb

OBJECTIVE: To identify the prognostic relevance of the G1/S cell cycle regulator genes p16INK4a, p53, MDM2, and Rb in patients with resected ductal pancreatic cancer (PC). SUMMARY BACKGROUND DATA: The tumor suppressor genes p16INK4a, p53, and Rb are altered in PC in 27% to 95%, 40% to 70%, and 5%, respectively. The role of MDM2 is not clearly defined in PC. The prognostic value of these cell cycle regulators has not been clarified. METHODS: Sixty-two patients with PC with complete follow-up who underwent potentially curative resections were included in the study. An extreme group analysis was performed including the 20 patients with the shortest survival and the 20 patients with the longest survival. Protein expression of p16, p53, MDM2, and Rb was investigated, and mutation analysis of p16INK4a and p53 was performed. p16INK4a promoter hypermethylation was examined by methylation-specific polymerase chain reaction. RESULTS: Significantly more tumors in the shortest-surviving patients had p16INK4a alterations compared with tumors of the longest-surviving patients. In contrast, the frequency of p53 alterations was not significantly higher in the shortest-surviving versus the longest-surviving groups. Stabilization of MDM2 and loss of Rb expression were identified in a minority of tumors, independent of survival length. CONCLUSIONS: The presence of p16INK4a alterations in resected tumors of patients with PC is connected with a worse prognosis, indicating patients that might benefit from adjuvant therapy regimens. p53 alterations, MDM2 overexpression, and loss of Rb expression could not be identified as prognostic markers from this study, but a larger study with greater statistical power might show a different result with regard to p53.     

Molecular and Immunohistochemical Cognizance of HPV16 in Oral Leukoplakia,Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma

Prior studies have established the carcinogenic role of HPV16 and also demonstrated its unique biological behavior in cervical and oropharyngeal squamous cell carcinoma (OPSCC) but its role in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is not well explored. Therefore, in the present study, we assessed HPV16 prevalence using PCR and Anti-HPV16 antibodies for the first time and correlated its biological behavior using p16^INK4a and Ki67 proliferation index (PI) in OL, OSCC, and OPSCC. This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by immunohistochemistry with primary antibodies p16^INK4a, Anti-HPV16, and Ki67. The expressions were evaluated and statistical analysis included Chi-square and Spearman’s test. Cumulatively 37% (OL—7%, OSCC—14% & OPSCC—16%) of cases showed positive PCR expression. PCR positivity was observed to be significantly higher (p 0.00) in OPSCC (9/12) than OSCC (9/26) and OL (5/25) cases. Overall immunohistochemical expression of p16^INK4a, Anti-HPV16, and Ki67 were significantly (p 0.02) higher in HPV16 (PCR) positive cases. HPV16 + OSCC cases showed higher grades of p16^INK4a and Ki67 expression. We have demonstrated a prevalence of HPV16 in OL, OSCC, and OPSCC through PCR, which may be concluded as a gold standard for the detection of HPV16 DNA.     

The expression of key cell cycle markers and presence of human papillomavirus in squamous cell carcinoma of the tonsil

BACKGROUND: Chemical carcinogens induce squamous cell carcinoma (SCC) of the head and neck by targeting the p53 and the retinoblastoma (pRb) pathways. Human papillomavirus (HPV) might have an etiologic role in these cancers at particular sites. Few studies have compared cell cycle protein expression in HPV-positive and HPV-negative tumors in this region. METHODS: Fifty tonsil SCCs were analyzed for HPV by PCR and for expression of cell cycle proteins (p53, pRb, p16(INK4A), p21(CIP1/WAF1), p27(KIP1), and cyclinD1) by immunohistochemistry. RESULTS: HPV was present in 42%; almost all were type 16. There were statistical associations between HPV positivity and reduced expression of pRb and cyclinD1, overexpression of p16, and younger patient age. Tumor with down-regulated p27 tended to have down-regulated pRb and p21. CONCLUSIONS: HPV-positive tonsil SCCs have distinct molecular pathways. Their association with younger patient age suggests that they are biologically distinct from HPV-negative tumors.     

Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of primary small cell carcinoma of the uterine cervix, a rare but highly aggressive malignancy. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In squamous cell carcinoma and adenocarcinoma of the cervix, HPV infection is also associated with overexpression of p16, a cyclin-dependent kinase inhibitor. In this study, 22 cases of primary small cell carcinoma of the uterine cervix were subjected to broad-spectrum HPV DNA amplification and typing, and immunohistochemically examined for the expression of p16, Rb, and p53 proteins. The results show that HPV DNA was detected in every case (100%), with 18 cases (82%) harboring type 18. The tumor cells exhibited strong nuclear staining for p16 in 20 cases (91%). This was associated with a complete loss of Rb nuclear staining in tumor cells in 16 cases (73%). The p53 protein was essentially undetectable in all cases. In contrast, HPV DNA was not detected in 9 colorectal and 8 urinary bladder small cell carcinomas included in this study for comparison. While similar p16 and Rb expression patterns were observed in these HPV-negative tumors, a different expression pattern for p53 was noted where strong nuclear staining was seen in 8 cases (47%; P = 0.0004 compared with cervical tumors). These observations indicate that different mechanisms are involved in the pathogenesis of small cell carcinomas of the uterine cervix and support the notion that nuclear p16 overexpression serves as an indication of Rb defunctioning in tumor cells, which may or may not result from high-risk HPV infection.     

Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16^INK4A and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16^INK4A expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16^INK4A and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher’s exact test and Pearson X² test with a p < 0.05 were used. Cyclin D1 and p16^INK4A are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16^INK4A expression. Our findings suggest that p16^INK4A overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.     

Overexpression of p16INK4 is a reliable marker of human papillomavirus-induced oral high-grade squamous dysplasia.

OBJECTIVE: Human papillomavirus (HPV) infection has been implicated in the development of high-grade squamous dysplasia and carcinoma of the oral cavity in the absence of other known risk factors such as smoking. HPV-induced oral dysplasia or carcinoma may be a unique tumor entity in terms of biologic behavior and treatment decisions. In detecting such cases, most reported studies have used techniques that are less sensitive than DNA amplification. Recent reports have suggested that overexpression of the p16INK4 protein is a surrogate marker of HPV-induced high-grade dysplasia or carcinoma. However, the correlation between expression of p16INK4 and the presence of HPV DNA as determined by polymerase chain reaction (PCR) amplification has not been previously reported. The purpose of this research was to determine if immunohistochemistry for p16 would serve as a marker of HPV-associated high-grade oral squamous dysplasia. STUDY DESIGN: Archival formalin-fixed, paraffin-embedded tissue sections from 41 cases of high-grade oral squamous dysplasia were randomly selected. Expression of p16INK4 protein was assessed by immunohistochemical analysis (16P04 Neomarkers, Fremont, CA). Strong and diffuse nuclear staining restricted to the dysplastic region in the epithelium was scored as positive for protein expression, whereas focal or weak nuclear or cytoplasmic staining was scored as negative. The presence of HPV was determined by microdissection, DNA extraction, and PCR DNA amplification using elongated primers that align with corresponding sequences of the L1 region of 23 mucosotropic HPV genotypes. The HPV type was determined by direct sequencing of the PCR product. Normal squamous epithelium was used as an internal negative control, and cases of severe cervical high-grade squamous dysplasia were used as a positive control for immunohistochemical staining and PCR. RESULTS: The results of immunohistochemical analysis for overexpression of p16INK4 were positive in 6 of the 41 tissue sections. The results of PCR DNA amplification were also positive for these 6 sections. HPV-16 was identified in 5 of the positive cases; in the other case, the viral strain could not be determined. CONCLUSIONS: Immunohistochemical detection of p16INK4 is a technically simple and potentially reliable assay for diagnosing cases of HPV-induced oral high-grade squamous dysplasia. Detecting such lesions may influence future therapeutic decisions.     

HPV Status of Oropharyngeal Cancer by Combination HPV DNA/p16 Testing: Biological Relevance of Discordant Results

Background and Purpose

Human papillomavirus (HPV) causes up to 70 % of oropharyngeal cancers (OSCC). HPV positive OSCC has a more favorable outcome, thus HPV status is being used to guide treatment and predict outcome. Combination HPV DNA/p16^ink4 (p16) testing is commonly used for HPV status, but there are no standardized methods, scoring or interpretative criteria. The significance of discordant (HPV DNA positive/p16 negative and HPV DNA negative/p16 positive) cancers is controversial. In this study, 647 OSCCs from 10 Australian centers were tested for HPV DNA/p16 expression. Our aims are to determine p16 distribution by HPV DNA status to inform decisions on p16 scoring and to assess clinical significance of discordant cancers.

Methods

HPV DNA was identified using a multiplex tandem HPV E6 polymerase chain reaction (PCR) assay and p16 expression by semiquantitative immunohistochemistry.

Results

p16 distribution was essentially bimodal (42 % of cancers had ≥70 % positive staining, 52 % <5 % positive, 6 % between 5 and 70 %). Cancers with 5 to <50 % staining had similar characteristics to the p16 negative group, and cancers with 50 to <70 % staining were consistent with the ≥70 % group. Using a p16 cut-point of 50 %, there were 25 % HPV DNA positive/p16 negative cancers and 1 % HPV DNA negative/p16 positive cancers. HPV DNA positive/p16 negative cancers had outcomes similar to HPV DNA negative/p16 negative cancers.

Conclusions

50 % is a reasonable cut-point for p16; HPV DNA positive/p16 negative OSCCs may be treated as HPV negative for clinical purposes; HPV DNA/p16 testing may add no prognostic information over p16 alone.

     

HPV Status of Oropharyngeal Cancer by Combination HPV DNA/p16 Testing: Biological Relevance of Discordant Results

Background and Purpose

Human papillomavirus (HPV) causes up to 70 % of oropharyngeal cancers (OSCC). HPV positive OSCC has a more favorable outcome, thus HPV status is being used to guide treatment and predict outcome. Combination HPV DNA/p16^ink4 (p16) testing is commonly used for HPV status, but there are no standardized methods, scoring or interpretative criteria. The significance of discordant (HPV DNA positive/p16 negative and HPV DNA negative/p16 positive) cancers is controversial. In this study, 647 OSCCs from 10 Australian centers were tested for HPV DNA/p16 expression. Our aims are to determine p16 distribution by HPV DNA status to inform decisions on p16 scoring and to assess clinical significance of discordant cancers.

Methods

HPV DNA was identified using a multiplex tandem HPV E6 polymerase chain reaction (PCR) assay and p16 expression by semiquantitative immunohistochemistry.

Results

p16 distribution was essentially bimodal (42 % of cancers had ≥70 % positive staining, 52 % <5 % positive, 6 % between 5 and 70 %). Cancers with 5 to <50 % staining had similar characteristics to the p16 negative group, and cancers with 50 to <70 % staining were consistent with the ≥70 % group. Using a p16 cut-point of 50 %, there were 25 % HPV DNA positive/p16 negative cancers and 1 % HPV DNA negative/p16 positive cancers. HPV DNA positive/p16 negative cancers had outcomes similar to HPV DNA negative/p16 negative cancers.

Conclusions

50 % is a reasonable cut-point for p16; HPV DNA positive/p16 negative OSCCs may be treated as HPV negative for clinical purposes; HPV DNA/p16 testing may add no prognostic information over p16 alone.

     

Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16^INK4a immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16^INK4a staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16^INK4a negative and two HPV negative samples were p16^INK4a positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16^INK4a positive, PCR and CISH HPV positive/p16 ^INK4a negative, or PCR HPV positive/p16 ^INK4a and CISH negative, also requires further investigation.     

The prevalence and clinicopathologic correlate of p16INK4a, retinoblastoma and p53 immunoreactivity in locally advanced urinary bladder cancer

The purpose of the study was to investigate the prognostic value and clinicopathological correlate of tumor p53, p16 and Rb protein expression in patients with locally advanced urinary bladder cancer. Sixty-five patients (44 men and 21 women; 40 to 84 yrs old) with locally advanced urinary bladder cancer (21 pT2, 27pT3, 17pT4) undergoing radical cystectomy and bilateral pelvic lymph node dissection were followed up for 2 to 116 months (mean +/- SD: 30.02 +/- 6.46 months). Immunohistochemical staining for p53, Rb and p16 proteins were performed on surgically obtained, formalin fixed and paraffin embedded tissue sections. Thirty of the tumors (46.2%) were p53+, 52 of the tumors (80%) were p16- and 41 (63%) were Rb-. Only 5 of the tumors (7.7%) had normal expression of all three proteins. The tumor expression status of p53 could not be correlated with p16 (P = 1.000) or Rb (P = 1.000). Only a marginal inverse relationship was found between the expression of p16 and Rb (P = 0.056). Higher grade tumors had significantly lower percentage of p16 abnormality (P = 0.05), while higher grade (not higher stage) tumors had higher percentage of Rb abnormality (P = 0.0245). Univariate analysis showed that tumor expression of Rb or p16, alone or combined, had no predictive value on progression-free and disease-specific survival. It did, however, show a significant correlation between progression-free survival and tumor p53 and LN status (P = 0.032 and P = 0.0304) and a significant correlation between tumor stage disease-specific survival (P = 0.042). Multivariate analysis showed tumor stage and nodal status to be two significant independent indicators for progression-free survival (P = 0.0038 and P = 0.0049) and disease-specific survival (P = 0.0066 and P = 0.0484). It was also noteworthy that, after receiving postoperative adjuvant systemic M-VEC chemotherapy, patients with node-positive p53-normal tumors had significantly better progression-free and disease-specific survivals than those with node-positive p53-abnormal tumors (P = 0.036 and P = 0.0479, respectively). This study has found tumor expression of p53, p16 and Rb proteins in locally advanced bladder cancer to be frequently abnormal. Although multivariate analysis showed tumor stage and nodal status to be the only two statistically significant parameters, p53 may also serve as an additional prognostic predictor of the outcome of postoperative adjuvant systemic chemotherapy in patients with regional lymph node tumor involvement. Such patients with p53-normal tumors experienced significantly better progression-free and disease-specific survivals than those with p53-abnormal tumors.     

Prevalence of human papillomavirus in oral epithelial dysplasia: Systematic review and meta‐analysis

The purpose of this systematic review and meta‐analysis was to estimate the overall and type‐specific prevalence of human papillomavirus (HPV) DNA in oral epithelial dysplasia and assess p16^INK4a overexpression in relation to HPV‐status. A systematic literature search identified 31 eligible studies (832 cases) evaluating the presence of HPV DNA in oral epithelial dysplasia cases by PCR. Of these, six studies evaluated p16^INK4a overexpression in relation to HPV‐status. The overall pooled prevalence of HPV DNA in oral epithelial dysplasia was 27.2% (95% CI: 17.6‐38.1). We observed substantial interstudy heterogeneity, which could not be explained by differences in continent, tissue type, or severity of epithelial dysplasia. HPV16 was the predominant genotype detected. Moreover, 62.2% of HPV positive and 17.8% of HPV negative oral epithelial dysplasia samples stained intensively positive for p16^INK4a. This meta‐analysis found that 27% of oral epithelial dysplasia harbor HPV DNA. Whether this represents a transient infection or has a carcinogenic role is unknown.     

CDKN2／p16^INK4a基因克隆,探针制备及其在肺癌基因诊断中的 …

OBJECTIVES: To clone CDKN2/p16(INK4a) gene, prepare its probe, and to study the change of CDKN2/p16(INK4a) gene in lung cancers. METHODS: Total RNA of normal lung tissue was extracted, CDKN2/p16(INK4a) gene cDNA synthesized, and CDKN2/p16(INK4a) gene recombinant vector, constructed. Southern blot was used to study CDKN2/p16(INK4a) gene in 46 cases of lung cancers, 3 cases of normal lung tissues, 6 cases of lung tissues near cancer, and 3 cases of lymph nodes with lung cancer metastasis. RESULTS: Cloned CDKN2/p16(INK4a) cDNA was proved by enzyme digestion and sequencing. Southern blot showed 4.3 kb band in normal lung tissues and lung tissues near cancers, and deletion of CDKN2/p16(INK4a) gene in cancer tissues and lymph nodes with lung cancer metastasis, with a deletion rate of 17.4% (8/46). CONCLUSION: CDKN2/p16(INK4a) gene may play a role to some extent in progression of lung cancers.     

RB1, p16, and Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma

While P16 immunohistochemistry (IHC) is a well-established surrogate marker of Human Papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC), Retinoblastoma 1 (RB1) loss may lead to p16 overexpression in the absence of HPV. We determined the proportion of p16-positive/HPV-negative OSCC with RB1 loss and other alterations in RB1/p16 pathway, and tested RB1 IHC as a prognostic biomarker for OSCC, along with the 8th edition of AJCC staging manual. P16 and RB1 IHC and HPV DNA in situ hybridization (ISH) were performed on 257 OSCC. High risk HPV RNA ISH, RB1 fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) were done on p16-positive/HPV DNA ISH-negative OSCC. Disease free survival (DFS) was used as an endpoint. In the entire cohort and in p16-positive (n = 184) and p16-negative (n = 73) subgroups, AJCC 8th edition staging correlated with DFS (p < 0.01). RB1 IHC showed RB1 loss in p16-positive OSCC only (79/184, 43%). RB1 loss by IHC is associated with a better DFS, without providing additional prognostic information for patients with p16-positive OSCC. HPV RNA ISH was positive in 12 of 14 HPV DNA ISH-negative cases. RB1 IHC showed loss in 10 of 15 HPV DNA ISH-negative cases and in 1 of 2 HPV RNA ISH-negative cases. Overall, only one case of p16-positive/HPV RNA ISH-negative OSCC showed RB1 loss by IHC (1/184, 0.5%). Of the 10 p16-positive and HPV DNA ISH-negative cases with RB1 loss by IHC, 2 had RB1 hemizygous deletion and 3 showed Chromosome 13 monosomy by FISH. No RB1 mutations were detected by NGS. Other molecular alterations in p16-positive/HPV DNA ISH-negative cases included TP53 and TERT mutations and DDX3X loss. HPV-independent RB1 inactivation rarely results in false positive p16 IHC. RB1 inactivation by high risk HPV E7 oncoprotein may co-exist with RB1 deletion. RB1 loss is a favorable prognosticator and occurs exclusively in p16-positive OSCC. The 8th edition of the AJCC staging manual satisfactorily predicts DFS of OSCC patients.     

Human papillomavirus and p53 mutational status as prognostic factors in head and neck carcinoma

BACKGROUND: Mutations of the p53 tumor-suppressor gene are common in squamous cell carcinoma of the head and neck (SCCHN) and may portend a worse prognosis. Human papillomavirus (HPV) represents another potential prognostic factor for SCCHN. The oncogenic potential of HPV may be due to the ability of its E6 oncoprotein to promote degradation of wild-type p53 protein. We wish to determine whether there is a lower incidence of p53 mutations in HPV-positive versus HPV-negative tumors, and if HPV and/or p53 status has an impact on survival. METHODS: Thirty-two SCCHN specimens were analyzed for mutations of the p53 gene using single-strand conformational polymorphism (SSCP) analysis followed by DNA sequencing. The HPV status of all specimens was evaluated by use of polymerase chain reaction with HPV consensus primers and Southern blot hybridization. Pertinent clinical information was obtained from chart review. RESULTS: Nonsilent p53 mutations were present in 2 of 15 (13%) of HPV-positive tumors compared with 6 of 17 (35%) of HPV-negative tumors (p =.229; Fisher's exact test, odds ratio.28). A survival advantage was found between HPV-positive compared with HPV-negative specimens (p =.0264) and between p53 wild type compared with p53 mutant specimens (p =.01) by univariate log rank analysis. When stratified according to both HPV and p53 status, a statistically significant survival difference was observed largely because of a 100% survival for the HPV-positive/p53 wild-type group (p =.003). CONCLUSIONS: This preliminary study supports the notion that the presence of HPV confers a survival advantage among HNSCC patients, particularly when p53 is wild type.     

肝硬化组织中p16INK4a和Rb的基因甲基化状态和蛋白表达

目的：探讨肝硬化组织中p16INK4a和视网膜母细胞瘤易感基因（Rb基因）启动子区域的甲基化状态与其蛋白的表达的关系。方法：分别采用PCR和免疫组织化学方法测定65例肝硬化患者和20例正常人肝组织中p16INK4a和Rb启动子区域甲基化状况和蛋白的表达。结果：20例正常肝组织（对照组肝组织）中均未检测到p16INK4a和Rb甲基化异常,肝硬化组肝组织中p16INK4a和Rb基因甲基化率分别为40.0%（26/65）和36.9%（24/65）;p16INK4a和Rb蛋白表达的积分光密度（IOD）在正常肝组织为225.7±27.4和254.7±34.8,在肝硬化组肝组织中为32.4±7.5和45.2±6.4,差异均有统计学意义（均P<0.05）。结论：肝硬化组织中存在p16INK4a和Rb异常甲基化和p16INK4a和Rb蛋白的表达降低,由此导致的细胞周期失控可能参与了肝硬化的发生发展过程。     

Human Papillomavirus and Overexpression of P16INK4a in Nonmelanoma Skin Cancer

BACKGROUND: P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV)-infected cervical (pre)cancer lesions. OBJECTIVE: To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens. METHODS: Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only beta-globin-positive patients were included in this study. RESULTS: HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure. CONCLUSION: P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted.     

Mutant p53 proteins: between loss and gain of function

Cancer might result from both the aberrant activation of genes, whose physiological tuning is essential for the life of a normal cell, and the inactivation of tumor suppressor genes, whose main job is to preserve the integrity of cell genome. Among the latter, p53 is considered a key tumor suppressor gene that is inactivated mainly by missense mutations in half of human cancers. It is becoming increasingly clear that the resulting mutant p53 proteins gain oncogenic properties favoring the insurgence, the maintenance, and the spreading of malignant tumors. In this review, we mainly discuss the molecular mechanisms underlying gain of function of human tumor-derived p53 mutants, their impact on the chemoresistance and the prognosis of human tumors, with a special focus on head and neck cancers, and the perspectives of treating tumors through the manipulation of mutant p53 proteins.     

Prognostic prediction of the immunohistochemical expression of p53 and p16 in resected non-small cell lung cancer.

OBJECTIVE: p53 and p16(INK4) are the common and important tumor suppressor genes. Aberrant expression of p53 or p16 protein has been reported in various malignancies including lung cancer. Our aim was to investigate the association of p53 and p16 expression in resected non-small cell lung carcinoma (NSCLC) and evaluated their correlation with clinocopathologic features and survival. METHODS: p16 and p53 expression were detected by immunohistochemical analysis of 90 paraffin specimens of resected NSCLC, including 35 squamous cell carcinoma, 47 adenocarcinoma, and eight large cell carcinoma, between stages I and IV. The immunohistochemical study was performed using the labeled streptavidine-biotin method with anti-p53 and anti-p16 monoclonal antibodies. RESULTS: Fifty-two (57.8%) and 36 (40%) of 90 patients revealed aberrant immunostaining for p53 (p53+) and p16 (p16+), respectively. While 19 cases (21.1%) showed abnormal immunoreactivity for both p16 and p53. (p53+/p16+). There was no correlation of p53 or p16 expression with the clinicopathologic features. The Kaplan-Meier survival analysis demonstrated that patients with p16+, p53+, late stages, and nodal or distal metastasis had poor survival status (P = 0.006, 0.013, <0.001, <0.001 and 0.018, respectively). Further analysis demonstrated that p53 status was a significant prognostic factor in stage I NSCLCs (P < 0.001), and p16 status in stage I and II NSCLCs (P < 0.001, P = 0.003, respectively). Furthermore, patients whose tumors were both p53 and p16 aberrant expression had worse outcome compared with those whose tumors were both normal expression of p53 and p16 (5-year survival rate: 5 vs. 76%, P < 0.001). In Cox's regression model, the aberrant expression of p16, p53, advanced stages and combined aberrant expression of p53/p16 survived for a significant shorter period. CONCLUSIONS: The results indicated that aberrant expression of p16 and p53 are significant and independent, predictable prognostic factors for resected NSCLC, especially in early stage of NSCLCs. The worst prognosis was seen in patients whose tumors had both aberrant expression of p53 and p16. Further prospective trials may be aimed at confirming and validating these results.     

Human papillomavirus infection and anogenital condyloma in bone marrow transplant recipients

BACKGROUND: Secondary malignant diseases are late complications after allogeneic bone marrow transplantation (BMT). Anogenital lesions associated with human papillomavirus (HPV) infection have been described in renal transplant recipients but not after BMT. HPV types 16 and 18 are strongly linked to the malignant transformation. METHODS: In a series of 238 patients with allogeneic BMT, three had anogenital lesions. We looked for HPV in DNA extracted from embedded tissue to study HPV genotypes, p53 expression, and ploidy. RESULTS: In two patients, HPV sequences were detected. One of them, with giant condyloma, had HPV type 18 and two aneuploid clones, but p53 expression was not found. CONCLUSION: As in solid organ transplant recipients, anogenital condyloma may develop after BMT. Because the oncoprotein of HPV is able to bind and to degrade p53, it may lead to genetic instability, and subsequently to malignant transformation.     

Aneuploidy of chromosome 9 and the tumor suppressor genes p16(INK4) and p15(INK4B) detected by in situ hybridization in locally advanced prostate cancer

INTRODUCTION AND OBJECTIVES: The linked p16(INK4)/MTS1 and p15(INK4B)/MTS2 genes on chromosome 9p21 encode proteins that inhibit the cyclinD dependent kinases CDK4/6. Biallelic homozygous deletions involving this locus have been identified in a wide range of tumor cell lines, but in a lower frequency of primary tumors. As PCR based approaches analyzing for homozygous deletions could be confounded by unavoidable contributions of normal cells in microdissected tissue, we performed in situ hybridization (ISH) on primary prostate carcinomas to accurately evaluate p16 and p15 copy numbers on a cell-by-cell basis. MATERIAL AND METHODS: p16 and p15 loci were evaluated in 28 pT3N0M0 prostate cancer specimens. Of 28 patients, 15 (53%) were ascertained showing no recurrence (mean follow-up 61+/-17 months), 13 (47%) developed recurrences within 27+/-19 months. Tissues were provided for ISH analysis in a blinded fashion. Isolated DNA derived from P1 clone 1063 compromising p16 and p15 as well as a centromeric probe for chromosome 9 were used for hybridization. Signals were enumerated within 300 interphase nuclei per tumor specimen, and in 100 nuclei derived from 18 benign prostate tissues and 7 adjacent PIN regions. RESULTS: ISH detected aneuploid tumors in 12/13 (92%) patients with recurrence and in 5/15 (33%) without recurrence (p<0.0014). Whereas 3/7 PIN specimens associated with nonrecurrent PCA demonstrated euploidy, all 4/7 PIN associated with recurrent disease demonstrated the same aneuploidy for chr9 as the primary tumor. All benign tissues evaluated exhibited euploidy for chr9, p16 and p15. None of the PCA and PIN samples revealed homozygous deletions for p16(INK4)/MTS1/p15(INK4B)/MTS2; 2/28 (7.1%) PCA exhibited partial deletion for p16(INK4)/MTS1/p15(INK4B)/MTS2 and aneuploidy for chr9; both PCA derived from the recurrent group. CONCLUSIONS: Deletion of 9p21 was rare and therefore such genetic alterations may not play an important role in the pathogenesis of PCA. Analysis of the limited number of PCA examined suggest a strong association between chr9 aneuploidy and recurrenct disease. Aneuploidy in both PIN and PCA suggests that the clinical outcome of PCA might already be determined in the preinvasive PIN.