Prognostic significance of p16 INK4a alteration for Ewing sarcoma: a meta-analysis

BACKGROUND: Despite findings from individual studies regarding prognostic factors for Ewing sarcoma, no conclusive results have been produced, partly because of small sample sizes. The objective of the current study was to evaluate whether the presence of p16(INK4a) alteration is associated with a poorer prognosis in patients with Ewing sarcomas. METHODS: A review was conducted of publications that assessed associations between p16(INK4a) status and 2-year survival among patients with Ewing sarcoma. The association between metastatic disease at initial diagnosis and 2-year survival was evaluated by synthesizing data in the form of risk ratios. RESULTS: Of 11 studies that were identified in the initial search strategy, 6 studies, representing 188 patients, met the inclusion criteria and, consequently, were pooled for quantitative analyses. The estimated pooled risk ratio of p16(INK4a) aberration was 2.17 (95% confidence interval [95% CI], 1.55-3.03; P < .001), whereas the estimated pooled risk ratio of metastasis at diagnosis among the 164 eligible patients was 2.60 (95% CI, 1.71-3.97; P < .001). There was no statistically significant difference in the pooled estimated risk ratios of p16(INK4a) aberration for a poor prognosis between patients with and without metastasis at diagnosis (1.86 and 2.21, respectively; P > .59). CONCLUSIONS: The presence of p16(INK4a) alteration was a statistically significant predictor of prognosis for patients with Ewing sarcoma. Along with other prognostic factors, such as metastasis, the p16(INK4a) alteration may be a potential candidate for improving the risk-stratifying strategy for patients with these tumors.     

The prognostic significance of p16INK4a/p14ARF and p15INK4b deletions in adult acute lymphoblastic leukemia.

Cytogenetic/molecular abnormalities significantly influence the prognosis of patients with acute leukemia. Recently, two genes, p16INK4a and p15INK4b, encoding two cyclin-dependent kinase inhibitor proteins of the INK4 family of Mr 15,000 and 16,000, respectively, have been localized to 9p21. Remarkably, the p16INK4a locus has been found to encode a second protein, p14ARF, known as p19ARF in mice, with a distinct reading frame. Like p16INK4a, p14ARF is involved in cell cycle regulation, blocking cells at the G1 restriction point through the activity of MDM-2 and p53. We studied bone marrow samples of 42 newly diagnosed and untreated patients with acute lymphoblastic leukemia for the incidence of deletions of p16INK4a/p14ARF and p15INK4b using Southern blot analysis and determined the clinical outcome with regard to complete remission (CR) duration, event-free survival, and overall survival. We found deletions of p16INK4a/p14ARF in 17 of 42 patients (40%), with homozygous deletions in 11 of 42 patients (26%) and hemizygous deletions in 6 of 42 patients (14%). The gene for p15INK4b was codeleted in most, but not all, cases and was never deleted without deletion of p16INK4a/ p14ARF. No correlation was observed between molecular studies and karyotype abnormalities as determined by conventional cytogenetics. Furthermore, no difference was found in the CR rate, CR duration, event-free survival, and overall survival in patients with homozygous gene deletions compared to patients with no deletions or loss of only one allele.     

Performance of p16INK4a immunocytochemistry as a marker of anal squamous intraepithelial lesions

BACKGROUND:

Protein p16^INK4a immunocytochemistry (ICCp16) has the potential to reveal lesions at risk of progression to anal cancer. This study examined measures of diagnostic validity of ICCp16 in HIV‐positive patients treated at the Tropical Medicine Foundation of Amazonas in the coloproctology outpatient clinic.

METHODS:

One hundred ninety HIV‐positive patients were consecutively enrolled in 2007 and 2008. All patients underwent anal cytologic sampling to perform ICCp16 in conventional and GluCyte (Synermed International, Westfield, Indiana and S¸ao Paulo, Brazil) smears and also for genotyping of human papillomavirus (HPV). Patients were then subjected to anal biopsies monitored by high‐resolution anoscopy. Hematoxylin‐eosin and immunoperoxidase p16 (clone 6H12) stains were performed in slides with biopsied and cytological specimens, respectively. HPV genotyping on anal scrapings was performed by a polymerase‐chain reaction (PCR)‐based method. The immunochemical findings were compared with histopathological and PCR results in contingency tables and analyzed by nonparametric tests. Measures of diagnostic validity of ICCp16 were calculated. Statistical significance was set at P ≤ .5.

RESULTS:

There was no statistically significant association between the immunochemical results (conventional or GluCyte smears) and histopathological or HPV genotyping findings (P > .05). In the best scenario, ICCp16 presented 31% sensitivity and 81% specificity for the diagnosis of anal squamous intraepithelial lesion (ASIL) and 30% and 66%, respectively, for the diagnosis of infection with high‐risk HPV.

CONCLUSIONS:

There was no association between ICCp16 results and histopathological findings nor between ICCp16 and HPV genotyping. ICCp16 showed poor sensitivity and moderate specificity for the diagnosis of ASIL or high‐risk HPV. Cancer (Cancer Cytopathol) 2011. © 2011 American Cancer Society.     

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.     

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.     

Prognostic significance of p21WAF1/CIP1, p16INK4a and CD44s in tongue cancer

The role of lost or reduced expression of p21, p16 and CD44s in the survival of tongue cancer patients was investigated. Tumours and adjacent non-tumour epithelia (ANTE) from 36 patients with tongue cancer were retrospectively studied by immunohistochemistry using monoclonal antibodies against p21, p16 and CD44s proteins. Expression of p21, p16 and CD44s and their relationship with clinical and pathological parameters were analyzed. Of 36 patients, 12 (33.33%) developed recurrence and 12 died of the disease (mean survival, 25.5 months). In four cases (11.1%), concomitant low expression (<50% of tumour cells) of p21, p16 and CD44s was detected but had no effect on survival or recurrence in the univariate analysis. In the multivariate analysis, low expression of CD44s was the sole prognostic factor related to survival (p=0.01, hazards ratio: 0.749). There was no expression of p21, p16 or CD44s in ANTE from 3 out of 24 cases studied, and this finding was related to recurrence in the univariate analysis. In the multivariate analysis, low expression of CD44s in ANTE was again the sole factor related to recurrence (p=0.002, hazards ratio: 0.028). In conclusion, low expression of CD44s is related to tumour cell invasiveness and may be of clinical relevance as a prognostic factor.     

p16INK4a蛋白在人恶性肿瘤组织中的表达及临床意义#br# #br#

目的 探讨p16INK4a蛋白在多种人恶性肿瘤组织中的表达及临床意义。方法 收集2014年至2015年手术切除的肿瘤标本642例。免疫组化染色检测并分析p16INK4在肿瘤组织中的表达。 结果642例肿瘤样本中良性肿瘤120例、恶性肿瘤522例。免疫组化检测p16INK4a在恶性肿瘤组织中表达阴性（-）、弱阳性（+）、中等阳性（++）和强阳性（+++）表达率分别为17.43％（91／522）、11.11％（58／522）、19.16％（100／522）和5230％（273／522）,而在良性病变中表达率分别为34.17％（41／120）、23.33％（28／120）、26.67％（32／120）和1583％（19／120）,差异具有统计学意义（P＜0.05）。在不同组织来源的肿瘤中p16INK4a蛋白阳性表达率也并不相同（P＜0.05）。分别以阳性免疫组化染色“++”和“+++”作为p16INK4A阳性表达界值,则p16INK4A 蛋白作为恶性肿瘤分子标志的灵敏度、特异度和阳性预测值分别为71.46％、57.50％、87.97％和52.30％、84.17％和93.49％。结论 p16INK4A 代偿性高表达是人恶性肿瘤特异性的免疫表型,可作为恶性肿瘤诊断、鉴别诊断和分子分型的分子标志。     

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: correlation with biopsy follow-up results

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 (hc(2)) 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 (hc(2)) 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 hc(2) 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 hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.     

p16(INK4a) expression and breast cancer risk in women with atypical hyperplasia

p16, a nuclear protein encoded by the p16(INK4a) gene, is a regulator of cell-cycle regulation. Previous studies have shown that expression of p16 in tissue biopsies of patients with ductal carcinoma in situ (DCIS) is associated with increased risk of breast cancer, particularly when considered in combination with other markers such as Ki-67 and COX-2. Here, we evaluated how expression of p16 in breast tissue biopsies of women with atypical hyperplasia (AH), a putative precursor lesion to DCIS, is associated with subsequent development of cancer. p16 expression was assessed by immunohistochemistry in archival sections from 233 women with AH diagnosed at the Mayo Clinic. p16 expression in the atypical lesions was scored by percentage of positive cells and intensity of staining. We also studied coexpression of p16, with Ki-67 and COX-2, biomarkers of progression in AH. Risk factor and follow-up data were obtained via study questionnaire and medical records. Forty-seven patients (20%) developed breast cancer with a median follow-up of 14.5 years. Staining of p16 was increased in older patients relative to younger patients (P = 0.0025). Although risk of developing breast cancer was not associated with increased p16 expression, joint overexpression of Ki-67 and COX-2 was found to convey stronger risk of breast cancer in the first 10 years after diagnosis as compared with one negative marker (P < 0.01). However, the addition of p16 levels did not strengthen this association. p16 overexpression, either alone or in combination with COX-2 and Ki-67, does not significantly stratify breast cancer risk in women with AH.     

p14ARF, p15INK4b and p16INK4a methylation status in chronic myelogenous leukemia

The INK4 family of proteins p15^INK4b, p14^ARF and p16^INK4a function as cell cycle inhibitors where they are involved in the inhibition of G1 phase progression. Methylation of the p15^INK4b promoter never seems to occur in solid tumors but is a major gene silencing mechanism in hematological malignancies. p14^ARF and p16^INK4a promoter methylation often occurs in solid tumors but also in leukemias and lymphomas. In chronic myelogenous leukemia (CML), only a few reports have been published regarding INK4 methylation and the results of the literature are discordant. Thus clearly, more works on large series have to be performed independently.     

Inactivation of the p15INK4B and p16INK4 Genes in Hematologic Malignancies

The recently discovered p15^INK4B and p16^INK4 genes encoding cell cycle regulating proteins, map to a region on chromosome 9p21 that is commonly deleted in a variety of malignant diseases. The p16^INK4 gene has now been shown to be a tumor suppressor gene. It is frequently inactivated in cancer and is possibly the second most often mutated gene in human malignant disease after p53. The role of the p15^INK4B and p16^INK4 genes in hematologic malignancies has been the subject of intense investigation since their discovery. In this review we address the function and possible role in tumorigenesis of the p15^INK4B and p16^INK4 genes and discuss their significance as prognostic markers in hematologic malignancies.     

Conspirators in a capital crime: co-deletion of p18INK4c and p16INK4a/p14ARF/p15INK4b in glioblastoma multiforme

Glioblastoma multiforme (GBM) is one of the most dreaded cancer diagnoses due to its poor prognosis and the limited treatment options. Homozygous deletion of the p16(INK4a)/p14(ARF)/p15(INK4b) locus is among the most common genetic alterations in GBM. Two recent studies have shown that deletion and mutation of another INK4 family member, p18(INK4c), also drives the pathogenesis of GBM. This minireview will discuss the known roles for p18(INK4c) in the initiation and progression of cancer and suggest opportunities for future studies.     

Hypermethylation of p16(INK4a) and p15(INK4b) genes in non-small cell lung cancer.

Hypermethylation of CpG island is a common mechanism by which tumor suppressor genes are inactivated. The tumor suppressor genes p16(INK4a) and p15(INK4b) are important components of the cell cycles. We have studied the feasibility of detecting tumor-associated aberrant p16(INK4a) and p15(INK4b) methylation in non-small cell lung cancer (NSCLC) using methylation-specific PCR. We found a high frequency of hypermethylation of the p16(INK4a) gene in 17 of 45 cases of NSCLC. In this study, there was no difference between the clinicopathological features or overall survival of patients with and without p16(INK4a) methylation. On the other hand, p15(INK4b) promoter hypermethylation is rare (5/45) in lung cancer and occurs in association with p16(INK4a) methylation. The overall survival of patients with p15(INK4b) methylation was markedly shortened in this series. We also analyzed cells in bronchial washings, and p16(INK4a) methylation was detected in 4 of 17 cases of NSCLC. Moreover, 1 of 10 plasma samples from patients with NSCLC was positive for p16(INK4a) methylation. Our results suggest a possible prognostic role of p15(INK4b) methylation in NSCLC, and that the detection of aberrant p16(INK4a) methylation in both bronchial washings and plasma may be useful for cancer diagnosis.     

肺癌p14ARF和p16INK4a基因协同表达缺失及其意义

目的：研究抑癌基因位点ＩＮＫ４ａ－ＡＲＦ在肺肿瘤细胞中的表达状况,揭示ｐ１４ＡＲＦ和ｐ１６ＩＮＫ４ａ协同表达缺失与肺癌发生发展的相关性。方法：用ＲＴ－ＰＣＲ和Ｗｅｓｔｅｒｎ ｂｌｏｔ对６株肺癌细胞（ＳＰＣ－Ａ－１,Ｃａｌｕ－１,Ｈ４４６,ＳＨ７７,Ａ５４９,Ｈ４６０）的ＩＮＫ－４ａ－ＡＲＦ基因位点在ｍＲＮＡ、蛋白水平上进行检测,对ＰＣＲ产物进行纯化和测序分析。结果：６株肺癌细胞中,有３株细胞（Ｈ４     

Methylation of p16INK4A in primary gynecologic malignancy.

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III-IV) was statistically higher than those at early stages (stages I-II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.     

The COOH terminus of p18INK4C distinguishes function from p16INK4A

The INK4 family of proteins consists of four members which can block progression from the G(1)-to-S phase of the cell cycle by inhibiting the activity of cyclin dependent kinases (cdks) 4 and 6. Although the gene encoding p16(INK4a) is commonly inactivated in human tumors, p18(INK4c) is rarely altered. We show here that overexpression of p18(INK4c) does not block cell cycle progression in a T-cell acute lymphocytic leukemia cell line (CEM) sensitive to p16(INK4a)-mediated G(1) arrest. A chimera consisting of the kinase-binding region of p16(INK4a) fused to the COOH terminus of p18(INK4c) is active in all known biochemical assays for INK4 function, but it does not arrest CEM cells. These data imply a novel level of p18(INK4c) regulation mediated through the COOH terminus and suggest that functional differences might underlie the distinct mutational profiles observed for p16(INK4a) and p18(INK4c) in tumors.     

Prognostic significance of p16 and CDK4 proteins in localized prostate carcinoma

BACKGROUND: In prostate carcinoma, a very low frequency of point mutations of the tumor suppressor gene CDKN2/MTS1 (p16(INK4) ) has been reported, but deletions of 9p21 and inactivation by promoter methylation are observed more frequently. In the current study the authors evaluated the expression of p16 and CDK4 proteins and their prognostic significance in patients with clinically localized prostate carcinoma. METHODS: The levels of p16 and CDK4 proteins were quantitated by immunofluorescence flow cytometry, using paraffin embedded material, in 104 adenocarcinomas of the prostate after radical prostatectomy. These levels then were compared with 25 cases of benign prostate hyperplasia (BPH). RESULTS: In prostatic carcinoma specimens, p16 protein was elevated significantly compared with BPH, with a median fluorescence index (FI) of 15.4 versus 10.7, respectively (P = 0.010). This was not the case for CDK4 protein, although p16 protein expression correlated significantly with CDK4 protein expression in BPH (Spearman rank correlation [R(S)] = 0.63) and carcinoma (R(S) = 0.78). In univariate survival analysis of the first 5 years, high levels of p16 protein expression (FI > 11.7) (P = 0.005), tumor greatest dimension, World Health Organization (WHO) histologic grade, capsular penetration, seminal vesicle invasion, positive surgical margins, lymph node involvement, and preoperative serum prostate specific antigen > 20 ng/mL all were significant predictors of biochemical failure. In multivariate survival analysis, high p16 protein expression (P = 0.015), age, WHO histologic grade, capsular penetration, and seminal vesicle involvement remained as independent predictors of biochemical failure. CONCLUSIONS: These data suggest that increased expression of p16 protein, but not CDK4 protein, may be involved in the development of prostate carcinoma and may represent an independent predictor of biochemical failure after radical prostatectomy.