Ratio of expression of p16INK4a to p14ARF correlates with the progression of non-small cell lung cancer.

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16(INK4a) and p14(ARF), through the use of independent first exons and shared exons 2 and 3. p16(INK4a) is a cyclin-dependent kinase inhibitor, whereas p14(ARF) regulates the cell cycle through a p53 and MDM2-dependent pathway. We have examined the expression of p16(INK4a) and p14(ARF) using competitive RT-PCR in 60 non-small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16(INK4a) and p14(ARF) were nearly equal or the intensity of the p16(INK4a) band slightly exceeded that of p14(ARF) in the normal lung tissues (n = 60). In 38 tumors the intensity of the p16(INK4a) band was similar to or slightly weaker than that of p14(ARF). In 6 tumors the intensity of the p16(INK4a) band was weaker than that of p14(ARF). In 15 tumors the intensity of the p14(ARF) band was very strong and the p16(INK4a) band was barely visible. In only one tumor was the intensity of the p16(INK4a) band very strong, while the band of p14(ARF) was barely visible. The ratio of the intensity of p16(INK4a) to p14(ARF) had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II - IV tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the p stage I tumors (P = 0.036). The T2 - 4 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the T1 tumors (P = 0.005). The N1 - 3 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the N0 tumors (P = 0.014). Our results suggest that the ratio of expression of p16(INK4a) to p14(ARF) tends to decrease during the progression of NSCLC.     

p16INK4a/Ki‐67 co‐expression specifically identifies transformed cells in the head and neck region

p16^INK4a immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)‐associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16^INK4a overexpression are regularly detected in the head and neck: p16^INK4a expression can be observed in non‐malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16^INK4 expression can complicate interpretation of “p16^INK4a‐positivity”. These aspects impede the unrestricted application of p16^INK4a as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16^INK4a and the proliferation marker Ki‐67 could support clarification of ambiguous p16^INK4a expression in the head and neck by specifically indicating p16^INK4a‐expressing cells with proliferative activity. p16^INK4a/Ki‐67 co‐expression in a combined staining procedure was correlated to distinct p16^INK4a expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non‐malignant head and neck samples. p16^INK4a/Ki‐67 co‐expression only occurred in transformed cells of the head and neck. Co‐expression was never detected in non‐transformed cells. Combined p16^INK4a/Ki‐67 expression was stringently associated with a diffuse p16^INK4a expression pattern. All HPV oncogene‐expressing HNSCC showed p16^INK4a/Ki‐67 co‐expression. We demonstrate that p16^INK4a/Ki‐67 co‐expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16^INK4a/Ki‐67 expression in the assessment of ambiguous p16^INK4a expression in the head and neck by specifically identifying p16^INK4a‐expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo‐ and cytopathology.     

Inhibition of T-cell acute lymphoblastic leukemia proliferation in vivo by re-expression of the p16INK4a tumor suppressor gene

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the presence of differentiation-inhibited pro- and pre-T-cell blasts. The p16^INK4a tumor suppressor gene has been shown to be frequently deleted in human T-ALL cases. Deletion of p16^INK4a may be associated with poor prognosis and relapse of the disease. Radiation-induced murine T-ALL in C57B1/6 mice shares pathogenetic and molecular characteristics with the human disease. We used the murine disease as a model to study the status of the INK4/ARF gene locus and to examine the effect of p16^INK4a-re-expression in T-ALL cells on their leukemic potential in vivo. In 9 of 17 radiation-induced murine T-ALL cell lines, the p16^INK4a protein was not expressed as determined by immunoblotting. Southern blot analysis revealed homozygous deletions of the p16^INK4a gene locus in three of the nine lines, along with the genes encoding p15^INK4b and p19^ARF. Transduction of p16^INK4a-negative T-ALL lines with retrovirus encoding p16^INK4a significantly inhibited their in vitro proliferation by inducing G₁-arrest. Importantly, re-expression of p16^INK4a in p16^INK4a-negative T-ALL cells obliterated the induction of lethal disseminated leukemia in syngeneic mice. This is the first demonstration that re-establishment of p16^INK4a expression is critical for in vivo growth regulation of T-ALL cells.     

p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin‐dependent kinases p16^INK4a. In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type‐specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence‐labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT‐PCR were performed, and for p16^INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV‐positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16^INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16^INK4a expression was of statistical significance (p = 0.02). Kaplan‐Meier estimations revealed better survival outcome for patients with HPV‐positive tumors with detectable E6/E7 mRNA and p16^INK4a overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16^INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16^INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA‐positive head and neck carcinomas.     

DNA methylation in small lung adenocarcinoma with bronchioloalveolar carcinoma components

We examined the methylation status in 100 specimens of lung adenocarcinomas measuring 2 cm or less and with bronchioloalveolar carcinoma (BAC) components (Noguchi types A–C) and then compared the methylation status between noninvasive tumors (Noguchi type A or B) and invasive tumors (Noguchi type C). Methylation-specific PCR was used to determine the methylation statuses of p16^INK4a, RASSF1A, CDH13, RARβ, and Cyclin D2. The methylation index that was regarded as representing the degree of methylation was calculated. We also determined the mutational statuses of EGFR exons 19 and 21 using a PCR-based method. A multivariate analysis showed that the aberrant methylation of p16^INK4a, RASSF1A, and CDH13 was significantly more frequent in invasive tumors than in noninvasive tumors [p16^INK4a, 36.5% versus (vs.) 8.3%, P = 0.0023; RASSF1A, 46.2% vs. 14.6%, P = 0.0012; CDH13, 42.3% vs. 10.4%, P = 0.0006]. The methylation index was significantly higher in invasive tumors than in noninvasive tumors (P = 0.004). The methylation of p16^INK4a was significantly more frequent in EGFR wild-type tumors than in EGFR mutant tumors (P = 0.021). Our results indicate the involvement of epigenetic alterations in the progression of adenocarcinoma with BAC components.     

Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta‐analysis

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta‐analysis to determine the diagnostic accuracy of p16^INK4a immunohistochemistry (IHC) to identify HPV‐induced OPSCC. We identified all studies that performed p16^INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification‐based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16^INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16^INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16^INK4a IHC/HPV DNA PCR combined testing was 94% (95%‐confidence interval (CI) 91–97%), 98% (CI 94–100%), 85% (CI 76–92%) and 93% (CI 87–97%), respectively. The pooled specificity was 83% (CI 78–88%), 84% (CI 74–92%), 88% (CI 78–96%) and 96% (CI 89–100%), respectively. p16^INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16^INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16^INK4a IHC is highly sensitive but moderately specific to diagnose HPV‐transformed OPSCC when used as a single test. Combined p16^INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV‐induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials.     

Frequent but borderline methylation of<Emphasis Type="Italic"> p16</Emphasis><Superscript>INK4a</Superscript> and<Emphasis Type="Italic"> TIMP3</Emphasis> in medulloblastoma and sPNET revealed by quantitative analyses

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 ^INK4A, p14 ^ARF, TIMP3, CDH1, p15 ^INK4B and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 ^INK4A (17/43), p14 ^ARF (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 ^INK4A and TIMP3 were methylated consistently in medulloblastomas (p16 ^INK4A 14%, TIMP3 11%) and p16 ^INK4A also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1–5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.     

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay

BACKGROUND.

The aim of this study was to examine p16^INK4a protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16^INK4a Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high‐grade lesions was assessed by using follow‐up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high‐risk HPV (hc₂) results.

METHODS.

Three hundred ninety‐eight residual ThinPrep samples were collected, and histological follow‐up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou‐stained slide, a second slide was processed in preparation for p16^INK4a immunostaining. High‐risk human papillomavirus testing (hc₂) was also performed.

RESULTS.

Of the 163 cytologically abnormal samples, 6‐month biopsy follow‐up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow‐up were positive for p16^INK4a, yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16^INK4a negative, yielding a diagnostic specificity of 62%. In comparison, the hc₂ test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow‐up, 25 of 26 slides were deemed to be positive for p16^INK4a, increasing the diagnostic sensitivity to 96%.

CONCLUSIONS.

The CINtec p16^INK4a Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16^INK4a protein overexpression. Diagnostic specificity of the CINtec p16^INK4a assay was significantly improved relative to hc₂. To increase p16^INK4a immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

Human papillomavirus DNA load and p16INK4a expression predict for local control in patients with anal squamous cell carcinoma treated with chemoradiotherapy

As the detection rate of HPV‐DNA in anal carcinoma commonly exceeds 90%, a comparison between sole HPV‐positive and HPV‐negative cancers with respect to treatment response following chemoradiotherapy (CRT) and long‐term oncological outcome is challenging. Against this background, we aimed to assess HPV types and HPV DNA load in formalin‐fixed paraffin‐embedded tissue (FFPE) of 95 patients treated with standard CRT for anal cancer to correlate viral load (≤/> median) with local failure, distant metastases, cancer‐specific (CSS) and overall survival (OS) rates. Various clinicopathologic parameters and the immunohistochemical marker p16^INK4a were evaluated for any correlation with HPV16 DNA load and were included in uni‐ and multivariate analyses. The overall prevalence of HPV DNA was 95.8% with HPV16 monoinfection being the most commonly encountered HPV type (78.9%), followed by HPV16 and 31, 35, 39, 44, 58, 66 and 81 dual infection in 9 patients (9.5%). HPV16 DNA load was significantly associated with p16^INK4a expression (p = 0.001). Patients with HPV16 DNA load ≤ median and low p16^INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p = 0.023, multivariate p = 0.042; p16^INK4a: univariate p = 0.021), and OS (HPV16 DNA load: univariate p = 0.02, multivariate p = 0.03). Moreover, a combined HPV16 DNA load and p16^INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p = 0.019, p = 0.04 and p = 0.031). In conclusion, these data indicate that HPV16 DNA load and p16^INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.     

p16INK4a is superior to high‐risk human papillomavirus testing in cervical cytology for the prediction of underlying high‐grade dysplasia

BACKGROUND

The primary goal of this study was to compare the clinical performance of an optimized and rigorously controlled immunocytochemical (ICC) assay for p16^INK4a to high‐risk (HR) human papillomavirus (HPV) detection by polymerase chain reaction (PCR) as diagnostic adjuncts for cytology specimens from colposcopy patients.

METHODS:

The study included 403 cervical cytology specimens collected within 3 months of colposcopy. The colposcopic impression and cervical biopsy diagnosis served as the standards for correlation with cytological, p16^INK4a, and HPV data. p16^INK4a was evaluated using an immunoperoxidase‐based assay that was linear over 4 logs for the detection of HeLa‐spiked positive control cytology specimens, using a threshold for positive test results that was based on receiver operating characteristic curve analysis. HR‐HPV was detected by multiplex PCR using genotype‐specific primers.

RESULTS:

In all combined diagnostic categories (negative for intraepithelial lesion and malignancy, atypical glandular cells, atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high‐grade squamous intraepithelial lesion, low‐grade squamous intraepithelial lesion, and high‐grade squamous intraepithelial lesion), the p16^INK4a ICC and HR‐HPV assays, respectively, had sensitivity of 81.7% and 83.3% (P = .81) and specificity of 78.1% and 50.9% (P < .001) for the detection of underlying ≥grade 2 cervical intraepithelial neoplasia (CIN) lesions on biopsy. Furthermore, the positive predictive value of p16^INK4a ICC was greater than that of HR‐HPV for patients with biopsies ≥CIN‐2 (41.2% and 24.2%, respectively, P = .001).

CONCLUSIONS:

This p16^INK4a immunocytochemical assay has superior specificity but similar sensitivity to HR‐HPV testing to predict underlying high‐grade dysplastic lesions in patients who are referred for colposcopy. The determination of the overall performance characteristics of p16^INK4a immunocytochemistry, as an independent test or in combination with HPV testing in low‐risk screening populations, however, will require subsequent large‐scale prospective clinical trials. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

p16INK4a Expression and Absence of Activated B-RAF Are Independent Predictors of Chemosensitivity in Melanoma Tumors

Metastatic cutaneous melanoma is highly resistant to cytotoxic drugs, and this contributes to poor prognosis. In vivo studies on the chemosensitivity of metastatic melanoma are rare and hampered by poor response rates to systemic chemotherapeutics. Patients who undergo isolated limb infusion (ILI) with cytotoxic drugs show high response rates and are, therefore, a good cohort for studying chemosensitivity in vivo. We used tumors from patients who underwent ILI to study the role of melanoma tumor-suppressor genes and oncogenes on melanoma chemosensitivity. Prospectively acquired tumors from 30 patients who subsequently underwent ILI with melphalan and actinomycin-D for metastatic melanoma were investigated for mRNA expression levels of p14^ARF, p16^INK4a, and MITFm. The mutation status of B-RAF, N-RAS, and PTEN were also determined. A high percentage of tumors had activating mutations in either B-RAF (15/30) or N-RAS (10/30) and only two tumors carried altered PTEN. High expression of p16^INK4a and absence of an activating B-RAF mutation independently predicted response to treatment. Further, inducible expression of p16^INK4a sensitized a melanoma cell line to death induced by melphalan or actinomycin-D. This study shows that high expression of p16^INK4a or the absence of activated B-RAF correlates with in vivo response of melanoma to cytotoxic drugs.     

Peptide‐based tumor inhibitor encoding mitochondrial p14ARF is highly efficacious to diverse tumors

p14^ARF is one of the major tumor suppressors conventionally identified both as the mdm2‐binding molecule restoring p53 function in the nucleus, and as a nucleophosmin‐binding partner inside the nucleolous to stabilize ribosomal RNA. However, its recently reported mitochondrial localization has pointed to novel properties as a tumor suppressor. At the same time, functional peptides are gaining much attention in nanomedicine for their in vivo utility as non‐invasive biologics. We previously reported the p14^ARF‐specific peptide that restored the sensitivity to gefitinib on the gefitinib‐resistant lung cancer cells. Based on the information of this prototype peptide, here we generated the more powerful anti‐tumor peptide “r9‐CatB‐p14 MIS,” which comprises the minimal inhibitory sequence of the mitochondrial targeting p14^ARF protein in combination with the proteolytic cleavage site for cathepsin B, which is activated in various tumor cells, fused with the nine‐polyarginine‐domain for cell penetration, and demonstrated its novel action of regulating mitochondrial function in accordance with localization of endogenous p14^ARF. The p14 MIS peptide showed a potent tumor inhibiton in vitro and in vivo against not only lung cancer cells but also tumor cells of diverse lineages, via modulating mitochondrial membrane potential, with minimal cytotoxicity to non‐neoplastic cells and tissues. Hence, this mitochondrially targeted p14 peptide agent provides a novel basis for non‐invasive peptide‐based antitumor therapeutics.     

p16INK4A immunohistochemical staining and predictive value for progression of cervical intraepithelial neoplasia grade 1: A prospective study in China

p16^INK4A is strongly expressed in tissues diagnosed as cervical intraepithelial neoplasia (CIN) and cancer in women infected with human papillomavirus (HPV), but few prospective studies have evaluated p16^INK4A as a marker for the risk of low‐grade CIN (CIN1) progression. We investigated the prevalence of p16^INK4A immunostaining by CIN grade and whether overexpression of p16^INK4A in CIN1 predicts future risk for high‐grade CIN in Chinese women. 6,557 Chinese women aged 30–49 years were screened from 2003 to 2005 using cytology and carcinogenic HPV test. Colposcopy was performed on women with any abnormal result. p16^INK4A Immunostaining was performed on biopsies from all women with CIN1, as well as randomly selected women with normal or CIN grade 2 and worse (CIN2+) biopsies. Women with CIN1 were followed up without treatment. Colposcopy was performed on all untreated women at a 2‐year interval. The prevalence of p16^INK4A staining was 2.7%, 42.7%, 75.5%, 79.6% and 100% among women with normal, CIN1, 2, 3 and cancer biopsies, respectively (p < 0.001). HPV positivity was strongly associated with p16^INK4A staining [odds ratios (OR) = 12.8; 95% confidence intervals (CI): 5.2–31.6]. p16^INK4A staining of CIN1 biopsies at baseline was associated with an increased risk of finding high‐grade CIN over 2 years of follow‐up (OR = 1.43; 95% CI: 0.52–3.91). The two‐year cumulative incidence of CIN2+ for p16^INK4A positive women was higher at 10.71% than for p16^INK4A negative women at 1.30% (crude RR = 8.25, 95% CI: 1.02–66.62). p16^INK4A overexpression is strongly associated with grade of CIN and risk of progression to high‐grade CIN in women with low‐grade lesions.     

The clinical impact of using p16INK4a immunochemistry in cervical histopathology and cytology: An update of recent developments

Cervical cancer screening test performance has been hampered by either lack of sensitivity of Pap cytology or lack of specificity of Human Papillomavirus (HPV) testing. This uncertainty can lead to unnecessary referral and treatment, which is disturbing for patients and increases costs for health care providers. The identification of p16^INK4a as a marker for neoplastic transformation of cervical squamous epithelial cells by HPVs allows the identification of HPV‐transformed cells in histopathology or cytopathology specimens. Diagnostic studies have demonstrated that the use of p16^INK4a immunohistochemistry substantially improves the reproducibility and diagnostic accuracy of histopathologic diagnoses. p16^INK4a cytology has substantially higher sensitivity for detection of cervical precancer in comparison to conventional Pap tests. Compared to HPV DNA tests, immunochemical detection of p16^INK4a‐stained cells demonstrates a significantly improved specificity with remarkably good sensitivity. About 15 years after the initial observation that p16^INK4a is overexpressed in HPV‐transformed cells we review the accumulated clinical evidence suggesting that p16^INK4a can serve as a useful biomarker in the routine diagnostic work up of patients with HPV infections and associated lesions of the female anogenital tract.     

Promoter hypermethylation of p14 ARF,RB, and INK4 gene family in hepatocellular carcinoma with hepatitis B virus infection

Both hepatitis B virus (HBV) and gene methylation play important roles in hepatocarcinogenesis. However, their association between HBV infection and gene methylation is not fully understood. Cell cycle control involving RB1 gene-related cell inhibitors is one of the main regulatory pathways were reported to be altered in hepatocellular carcinoma (HCC). The purpose of this research is to assess the methylation status of p14 ^ARF and INK4 gene family (p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and p18 ^INK4C ) in HCC with HBV infection and HCC without it, and discuss possible role of HBV-induced hypermethylation in the mechanism of hepatocarcinogenesis. Methylation status of RB, p14 ^ARF , and INK4 gene family in 64 case of HCC with HBV infection and 24 cases without it were detected by methylation-specific polymerase chain reaction, and HBV-DNA of the plasma were detected by quantitative real-time polymerase chain reaction. p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB hypermethylation were observed in 30 (34.1 %), 50 (56.8 %), 62 (70.5 %), and 24(27.3 %) of 88 hepatocellular carcinomas, respectively. Methylation frequencies of them between HCC with HBV infection and HCC without it were 43.8 % versus 8.3 % (p14 ^ARF ), 68.9 % versus 25 % (p15 ^INK4B ), 90.6 % versus 16.7 % ( p16 ^INK4A ), and 28.1 % versus 25 % (RB), respectively. In HBV-associated HCC, the numbers of methylated genes were also more than HCC without virus infection, more than two methylated genes were seen in 48 of 64 (75 %) cases; more than three methylated genes were found in 32 of 64 (50 %); correspondently, no one case has more than two genes methylated. p18 ^INK4C methylation product was not found in cancerous or non-cancerous tissues of 88 HCC. HBV infection is associated with p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB gene methylation (P?=?0.048, 0.035, 0.02); HBV-DNA replication is associated with p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB gene methylation (P?=?0.048, 0.035, 0.02); high rate of p14 ^ARF , p15 ^INK4B , and p16 ^INK4A in HCC with HBV infection suggests that HBV-induced hypermethylation may be one of the mechanisms of HBV involved in hepatocellular carcinogenesis.     

Alterations of the p53 and pRB pathways in human astrocytoma

Human astrocytomas are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRB and the p53 pathways. Genetic alterations functionally eliminate pRB and p53 themselves or upstream and/or downstream molecules such as products of theInk4a/ARF locus, p16^Ink4a and p14^ARF. As a result, malignant cells are defective in critical cell cycle and apoptosis regulatory elements contributing to unrelenting tumour growth and invasion. Current research aims to discover effective means of reconstituting p53 and pRB pathway components in an effort to attenuate the aggressive phenotype of astrocytoma.     

p14ARF expression in invasive breast cancers and ductal carcinoma in situ – relationships to p53 and Hdm2

Introduction  

p14^ARF stabilises nuclear p53, with a variable expression of p14^ARF mRNA in breast cancers. In vitro, nuclear p14^ARF binds Hdm2 to block Hdm2-dependent nucleocytoplasmic shuttling of p53, which is required before cytoplasmic degradation of p53. p14^ARF is negatively regulated by p53 and through p53-independent pathways. No studies have yet examined levels of p14^ARF protein expression in breast cancer and their relationship to Hdm2/p53 immunoreactivity or subcellular localisation. Previously, immunohistochemical expression of cytoplasmic p14^ARF, p53 and Hdm2 has been described. HER-2 (c-erbB2/neu) predicts prognosis and interacts with the p14^ARF/Hdm2 pathway to inactivate p14^ARF and to influence Hdm2 activity and localisation. This study examined p14^ARF and p53/Hdm2 expression and subcellular localisation by using immunohistochemistry in a series of invasive ductal breast cancers (IDCs) with concomitant ductal carcinoma in situ (DCIS), to evaluate whether findings in vitro were related to clinicopathological parameters such as HER-2 and their effect on patient outcome.     

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas

Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15 ^INK4b coincided with RB1 and/or p16 ^INK4a methylation, whereas RB1 and p16 ^INK4a methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14 ^ARF , p21 ^Waf1/Cip1 , and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16 ^INK4a methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16 ^INK4a gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16 ^INK4a methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.     

p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities

The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta‐analysis to assess the accuracy of cyclin‐dependent kinase inhibitor 2A (p16^INK4a) immunocytochemistry compared with high‐risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC‐US) or low‐grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta‐analysis. Seventeen studies were included in the meta‐analysis. The pooled sensitivity of p16^INK4a to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%‐88.2%) and 83.8% (95% CI, 73.5%‐90.6%) in ASC‐US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%‐76.4%) and 65.7% (95% CI, 54.2%‐75.6%), respectively. Eight studies provided both HC2 and p16^INK4a triage data. p16^INK4a and HC2 had similar sensitivity, and p16^INK4a has significantly higher specificity in the triage of women with ASC‐US (relative sensitivity, 0.95 [95% CI, 0.89‐1.01]; relative specificity, 1.82 [95% CI, 1.57‐2.12]). In the triage of LSIL, p16^INK4a had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81‐0.94]; relative specificity, 2.74 [95% CI, 1.99‐3.76]). The published literature indicated the improved accuracy of p16^INK4a compared with HC2 testing in the triage of women with ASC‐US. In LSIL triage, p16^INK4a was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.