Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer

The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14ARF and p16INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14ARF and p16INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1beta of p14ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14ARF, documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14ARF and p16INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14ARF was inversely correlated with p53 overexpression (p = 0.02). p14ARF and p16INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p = 0.02). Methylation of the p14ARF gene occurred more frequently in well-differentiated adenocarcinomas (p = 0.005), whereas the p16INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p = 0.002). The present results underline the role of p14ARF and p16INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14ARF and p16INK4a, might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways.     

INK4a-ARF alterations and p53 mutations in primary and consecutive squamous cell carcinoma of the head and neck

BACKGROUND: The INK4a-ARF (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14(ARF) and p16(INK4a), that play active roles in the p53 and Rb tumor suppressive pathways, respectively. We analyzed the alterations of p14(ARF), p16(INK4a) and p53 to study the contribution of each pathway in tumorigenesis of 29 patients with primary and consecutive (second primary) squamous cell carcinoma of the head and neck (HNSCC), with a total of 68 carcinomas. METHODS: After microdissection, the DNA of 29 primary and 39 consecutive squamous cell carcinomas was analyzed for INK4a-ARF inactivation and p53 mutation by means of DNA sequence analysis, methylation-specific polymerase chain reaction (MSP), restriction-enzyme-related polymerase chain reaction (RE-PCR), multiplex RT-PCR and immunohistochemistry. In addition, microdeletions of p14(ARF) and p16(INK4a) were assessed using differential PCR. RESULTS: Altogether inactivation (methylation, loss of heterozygosity and mutation of exon 1beta) of p14(ARF) was found in 29 of all 68 (43%) carcinomas, with a significant difference in primary [8 of 29 (28%)] relative to second primary carcinomas [21 of 39 (54%)]. Methylation of p16(INK4a) occurred in 22 of 68 (32%) carcinomas with an even distribution among primary and consecutive tumors. Only two (secondary) carcinomas showed simultaneous promoter methylation of p14(ARF) and p16 (INK4a). Mutations of p53 were found in 32 of 68 HNSCCs (44%), evenly distributed among primary and recurrent carcinomas. p14(ARF) alterations showed no relationship to p53 mutations. CONCLUSIONS: Our data indicate that the INK4a-ARF-/p53 pathway was disrupted in 58 of 68 (84%) primary and recurrent tumors, either by p53 mutations or by INK4a-ARF inactivation. p14(ARF) methylation occurred independently of p16(INK4a) alterations and showed no correlation to p53 mutations. The significantly higher rate of p14(ARF) alterations in recurrent (respective second primary) carcinomas suggests a further acquired genetic aberration during the development of the recurrent carcinomas.     

Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas

The INK4A and INK4B loci are located at 9p21 and have been implicated in the tumorigenesis of various human malignancies. The INK4A gene encodes two cell cycle regulators, p16(INK4A) and ARF, while INK4B encodes p15(INK4B). Previously, we have shown that the p16(INK4) tumor suppressor was not mutated or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16(INK4A), which is associated with expression, compared to normal breast tissue. The present study was conducted to determine if inactivation of p15(INK4B) and INK4A exon 1beta (ARF) are common events in breast carcinoma. Mutational analysis was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of the p15(INK4B) promoter. Our results demonstrate that the p15(INK4B) gene was altered in 3 (21%) of the 14 breast cell lines; one had a silent mutation and two had homozygous deletion of the gene. None of the cell lines showed methylation of p15(INK4B). Two (14%) cell lines had homozygous deletion of INK4A exon 1beta. All normal and malignant breast tissue samples were wild-type and non-methylated for p15(INK4B) and wild-type for exon 1beta. Our results show that these structurally and functionally related genes are not invariably affected together, and the most frequently observed alteration at the INK4A and INK4B loci in breast carcinoma appears to be p16(INK4A) hypomethylation.     

A common variant of the p16(INK4a) genetic region is associated with physical function in older people

p16(INK4a) is active in cell senescence, ageing and tumor suppression. Deletion of the small p16(INK4a)/ARF/p15(INK4b) region occurs in many cancers. We screened 25 common polymorphisms across the region and three related genes for associations with physical functioning in older people. In an initial sample of 938 (aged 65-80 years) from the EPIC study (Norfolk, UK), the rs2811712 SNP minor allele (located between the shared p16(INK4a)/ARF locus and p15(INK4b)) was associated with reduced physical impairment. This association remained after testing an additional 1319 EPIC-Norfolk samples (p-value=0.013, total n=2257), and on independent replication in the InCHIANTI study (n=709, p=0.015), and at one-sided significance in Iowa-EPESE (n=419, p=0.079). Overall (n=3372), the prevalence of severely limited physical function was 15.0% in common homozygotes and 7.0% in rare homozygotes (per minor allele odds ratio=1.48, 95% CI: 1.17-1.88, p=0.001, adjusted for age, sex and study). This estimate was similar excluding screening set 1 (OR=1.45, 95% CI: 1.09-1.92, p=0.010, n=2434). These findings require further replication, but provide the first direct evidence that the p16(INK4a)/ARF/p15(INK4b) genetic region and the senescence machinery are active in physical ageing in heterogeneous human populations. The mechanism involved may be via greater cellular restorative activity and reduced stem cell senescence.     

Aberrant methylation of p14(ARF), p15(INK4b) and p16(INK4a) genes and location of the primary site in pulmonary squamous cell carcinoma

Aberrant methylation of cytosines in CpG islands of the promoter regions of tumor suppressor genes is found in human tumors as a common mechanism of gene silencing. We investigated the methylation status of the chromosome 9p21 gene cluster (p14(ARF), p15(INK4b) and p16(INK4a) genes) by methylation-specific polymerase chain reaction in 20 central and 40 peripheral types of pulmonary squamous cell carcinoma (SqCC) in order to determine the differences between the pathogeneses of the central and peripheral types of SqCC. The frequencies of methylation were 30% for the p14(ARF) gene, 20% for the p15(INK4b) gene and 40% for the p16(INK4a) gene in the central type and 25% for the p14(ARF) gene, 10% for the p15(INK4b) gene and 38% for the p16(INK4a) gene in the peripheral type. Cases in which there was methylation of the p16(INK4a) gene had a higher smoking index in the peripheral type (P = 0.007). This trend was not detected in the central type. Methylation of two or three genes was observed in 55% of methylation in at least one gene of the central type but in only 17% of the peripheral type. This overlap methylation of the chromosome 9p21 gene cluster was found more frequently in the central type (P = 0.02). These findings suggest one of the epigenetic differences between the central and peripheral types of SqCC.     

Heterogeneous methylation and deletion patterns of the INK4a/ARF locus within prostate carcinomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in prostate carcinogenesis, we analyzed the p14(ARF), p16(INK4a), RB1, p21(Waf1), p27(Kip1), PTEN, p73, p53, and MDM2 gene status of multiple areas within 16 histologically heterogeneous prostate carcinomas using methylation-specific polymerase chain reaction, differential polymerase chain reaction, and immunohistochemistry. All focal areas examined had Gleason scores ranging from 1 to 5. Methylation of either PTEN or p73 was undetected in any sample, whereas expression of MDM2 seemed to be an independent event within small foci of 4 of 16 tumors. Loss of p14(ARF), p16(INK4a), RB1, and p27(Kip1) expression correlated with homozygous deletion or promoter hypermethylation. One carcinoma showed co-deletion of both p14(ARF) and p16(INK4a) in two of five areas examined; two areas within another tumor demonstrated concurrent hypermethylation of the promoter regions of the same genes. Focal hypermethylation of RB1, p21(Waf1), and p27(Kip1) was detected within two, two, and three tumors, respectively. These findings indicate that both genetic and epigenetic events occur independently in intratumor foci and further suggest hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis.     

Immunocytochemical staining of p16INK4a protein from conventional Pap test and its association with human papillomavirus infection

The p16INK4a protein is immunocytochemically detected in liquid-based (LB) specimens as a diagnostic marker of cervical dysplasia and neoplasia. Its up-regulation is promoted by high-risk human papillomavirus (HR-HPV) infection. We aimed to detect p16INK4a on conventional Papanicolaou (Pap) test (CPT) slides and to determine the relationship between its overexpression and HR-HPV infection. CPT and LB Pap test (LBPT) slides (165 samples of each) were examined by immunocytochemical staining for p16INK4a. After polymerase chain reaction (PCR), HPV-DNA was genotyped by dot blot hybridization. The CPT slides displayed more numerous dispersed squamous cells and LBPT slides had a clearer background. Positive p16INK4a on CPT occurred in 0% (0/30), 52.5% (21/40), 54.3% (19/35), 100% (30/30), and 100% (30/30) in normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), high-grade SILs (HSILs), and squamous cell carcinomas (SCCs) cases, respectively. LBPT slides showed comparable results but were less sensitive. HPV-DNA was detected in 86.7, 70, 45, 57.14, and 10% in SCCs, HSILs, ASCUS, LSILs, and normal cervical cells, respectively. Because HR-HPV was identified in all HPV+ samples of high-grade dysplasia (HSILs and SCCs) and all positive p16INK4a samples infected with HR-HPV, the association of p16INK4a overexpression with HR-HPV infection was confirmed. This study suggests that immunocytochemical staining of p16INK4a on CPT slides is convenient and cost-effective for cervical cancer screening by the detection of dysplastic cells infected with HR-HPV.     

Differential effect of epigenetic alterations and genomic deletions of CDK inhibitors [p16(INK4a), p15(INK4b), p14(ARF)] in mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14)(q13;q32), resulting in overexpression of CCND1 in the vast majority of cases. In addition, alterations of other cell-cycle-regulating signal pathways (CDKN2B/CDKN2A-CCND1 and ARF-MDM2-TP53) are frequently observed. However, the hierarchy of promoter methylations and genomic alterations as well as the interaction with other cell-cycle regulator CDKN1A is poorly understood. A complete methylation-specific PCR coupled with direct sequencing of 71 MCL patient samples previously characterized for TP53 alterations, Ki67 expression by immunohistochemistry, and other genomic alterations was performed. In contrast to rare p16(INK4a) promoter methylation (9%), frequent p15(INK4b) (62%) and p14(ARF) (70%) promoter methylation was detectable in MCL. In an additional 16% of MCL cases, LOH for p16(INK4a) was detected. However, MCL cases with p15(INK4b) methylation tended to have lower proliferation (73% vs. 57%), and p15(INK4b) and p14(ARF) promoter methylation was also detected in normal stem cells. Therefore, epigenetic changes of those genes seem not to represent primary oncogenic mechanisms but physiological mechanisms of cell regulation. The rare p16(INK4a) promoter methylation and p16(INK4a) genetic alterations were directly correlated to cell proliferation and therefore are regarded as additional molecular alterations involved in the cell-cycle dysregulation of MCL.     

p16(INK4a) is selectively silenced in the tumoral progression of mycosis fungoides

Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in mycosis fungoides (MF) is still scarce. Because the 9p21 locus seems to be a good target for a detailed study in MF, this prompted us to compare the mechanisms of inactivation of the p16(INK4a), p15(INK4b), and p14(ARF) genes in aggressive and stable forms of MF, performing microsatellite analysis, methylation-specific polymerase chain reaction, direct sequencing, and p16(INK4a) protein expression by immunohistochemistry. Additionally, the p53 gene was also sequenced in tumoral lesions. Thirty-nine patients with stable MF were studied. Alterations in p16(INK4a) and p15(INK4b) genes were detected in 18% and 5% of the cases, respectively. None of the cases analyzed showed alterations of the p14(ARF) gene. In contrast with these findings, in the 11 patients with aggressive MF, alterations of the p16(INK4a), p15(INK4b), or p14(ARF) genes were found in 8 (73%), 3 (27%), and 2 (18%) cases, respectively. A significant proportion (4/11) of these alterations were already present in the p16(INK4a) gene in the initial plaque lesions in these aggressive forms of MF. Alterations in the p16(INK4a) gene, either methylation or loss of heterozygosity, were clearly more frequent than those in the p15(INK4b) and p14(ARF) genes. These p16(INK4A) alterations were confirmed using immunohistochemistry. None of the nine tumoral lesions analyzed showed mutations in exons 1-2 of the p16(INK4a) gene or in exons 5-8 of the p53 gene. These results seem to suggest that 9p21 alterations, and selectively p16(INK4a) silencing, could be a characteristic phenomenon in MF progression.     

p16INK4A and p14ARF expression pattern by immunohistochemistry in human papillomavirus-related cervical neoplasia.

Human papillomavirus is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer. The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of human papillomavirus DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4A and possibly p14ARF have been proposed as putative surrogate biomarkers that would allow identification of dysplastic cervical epithelia. Serial consecutive biopsies representing normal cervical epithelium to cervical intraepithelial neoplasia and/or invasive cervical cancer were stained with immunohistochemistry for p16INK4A, p14ARF and proliferating cell nuclear antigen. The positive rates of these markers were significantly higher in cervical intraepithelial neoplasia and in squamous cell carcinoma than in normal cervix (P<0.01). No significant difference was noted between lesions progressing from cervical intraepithelial neoplasia to squamous cell carcinoma for both p16INK4A and p14ARF expression (P>0.05). For both biomarkers, nuclear staining was predominantly seen. However, the cytoplasmic stain of p16INK4A increased with disease progression and the pattern of expression varied between different tumors and its location within the lesion. Both nuclear and cytoplasmic staining with p16INK4A and p14ARF of affected epithelial cells were considered positive. In the adjacent normal tissue to cervical neoplasia, the positive rates of p16INK4A, p14ARF and proliferating cell nuclear antigen expression were higher than those found distant to these lesions but the findings did not reach statistical significance. No correlation was seen between the human papillomavirus types detected and the expression of p16INK4a and p14ARF. In conclusion, overexpression of p16INK4A and p14ARF act as potential biomarkers for cervical cancer progression from premalignant lesions.     

p16ink4 immunoreactivity is a reliable marker for urothelial carcinoma in situ

Morphological discrimination of urothelial carcinoma (UC) in situ, a precursor of high-grade invasive carcinoma, from severe reactive atypia is essential, but can be challenging at times. Mutations of the cell cycle regulatory gene INK4a (located at 9p21) that encodes p16(INK4) are one of the critical early events in carcinogenesis of UC. The purpose of this study was to investigate p16(INK4) expression in different urothelial lesions and assess its possible utility in distinguishing reactive versus neoplastic changes. Immunoreactivity of p16(INK4) was investigated using paraffin sections from 8 different sample groups (n = 80), including organ donor bladders with normal and/or reactive urothelium; bladder biopsies with reactive atypia; isolated carcinoma in situ; high-grade UC with concurrent carcinoma in situ; low-grade papillary urothelial neoplasms; high-grade stage T1 UC; and high-grade stage T2-T4 UC without concomitant carcinoma in situ. A composite staining score was calculated (Sigma% positive cells x intensity) for each case. Fluorescence in situ hybridization analysis was performed in selected cases. A uniform and weak cytoplasmic p16(INK4) expression was observed in normal urothelium and urothelium with reactive atypia from organ donors. Bladder biopsies with reactive atypia showed lower scores and loss of p16(INK4) expression in 38% of cases. Strong p16(INK4) staining was found in 100% of carcinoma in situ with no loss of p16(INK4) expression. The strong p16(INK4) immunoreactivity in foci of carcinoma in situ clearly demarcated the neoplastic cells from adjacent nonneoplastic cells, which showed either normal or focal loss of p16(INK4) staining. Fluorescence in situ hybridization study revealed abundant deletion of chromosome 9p21 or polysomy of chromosome 9 in malignant cells of carcinoma in situ. Increased p16(INK4) expression was also seen in most high-grade invasive UC with concurrent carcinoma in situ, but the intensity of p16(INK4) staining did not correlate with the cancer grade or stage. We conclude that the 16(INK4) immunoreactivity seems to be most useful in distinguishing carcinoma in situ from severe reactive atypia. Alterations of 9p21/chromosome 9 detected by fluorescence in situ hybridization can also be used for diagnostic confirmation and prognostic prediction.     

p16INK4a expression in basal-like breast carcinoma

BLBC represents a distinctive group of invasive breast carcinomas with specific genotype and immunopro-file. BLBC is usually defined by gene expression profiling and is currently associated with poor outcome. BLBCs are estrogen receptor (ER) negative, progesterone receptor (PgR) negative, HER2 negative, and usually show a variable expression of basal cytokeratins (CKs), EGFR and CD117. p16 ^INK4a is a tumor suppressor protein, encoded by the CDKN2A gene, which regulates cell cycle. The reported association of abnormalities in the p16/Rb pathway with increased risk of malignancy prompted us to determine the expression of p16^INK4a in a group of BLBC; the results were compared with a group of high-grade invasive carcinoma (HG-IC) of breast. Tissue microarrays (TMA) were constructed in triplicate including 18 BLBC and 18 HG-IC. All BLBC cases were ER-/PgR-/HER2-. Seventeen (94%) BLBC were CK 5/6+/CK 14+; 14 (78%) BLCB showed EGFR expression and 13 (72%) were CD117 positive. BLBCs showed a strong positive reaction with p16 ^INK4a antibody in 16 of 18 (89%) cases. Although the significance of p16 ^INK4a expression in breast cancer is not fully understood, we have shown that p16^INK4a is strongly expressed in breast cancers with basal-like phenotype. Since it is known that p16^INK4a is associated with aggressive behavior in human carcinomas, these data suggest that p16^INK4a play a role in the poor prognosis of BLBC.     

Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer

The INK4a/ARF locus (9p21) encodes two unique and unrelated cell cycle regulators, p16INK4a and p14ARF. This study was performed to evaluate the methylation status of p16INK4a and p14ARF genes, as well as its association with p16 and p53 expression, microsatellite instability (MI) status, and various clinicopathologic parameters in sporadic colorectal cancer. Sixty-five cases of primary colorectal adenocarcinoma with a series of clinicopathological parameters were obtained. We performed methylation-specific PCR of p16INK4a and p14ARF genes in colorectal cancer paraffin blocks with its paired normal samples, as well as immunohistochemical stainings for p16 and p53, and MI analysis. Aberrant methylations of p16INK4a and p14ARF gene were present in 21 (32.3%) and 33 (50.8%) out of 65 cases, respectively. p16INK4a aberrant methylation was correlated with p16 negativity (P=0.021) and p53 overexpression (P=0.007). p16INK4a aberrant methylation was more frequently present in poorly differentiated adenocarcinomas (P=0.002). Aberrant methylation of p14ARF gene occurred more frequently in patients under 50 years of age and in left-sided colon cancers, and was not statistically significant. Compared with the group with simultaneous absence of methylation in both promoters, the group showing concomitant alterations in both p16INK4a and p14ARF genes (n=10) more frequently presented lymph node metastasis (P=0.020) and higher tumor grade (P=0.014). There was no correlation between p16INK4a and p14ARF gene hypermethylation or MI status. This study suggests that simultaneous hypermethylation of both p16INK4a and p14ARF genes is greater prognostic value in sporadic human colorectal cancer.     

Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma

We examined alterations of the p16INK4, p14ARF, p15, TP53, and MDM2 genes in 30 osteosarcomas and 24 Ewing sarcomas. Among 21 osteosarcomas and 24 Ewing sarcomas, p16INK4, p14ARF, and p15 abnormalities were found in 4 (19%), 2 (9%), and 3 (14%) osteosarcomas, respectively, and in 4 (17%), 3 (13%), and 4 (17%) Ewing sarcomas, respectively. The alterations of p16INK4, p14ARF, and p15 included homozygous deletions spanning all 3 genes, methylation of p16INK4 or p15, and a nonsense mutation of p16INK4, which simultaneously caused a missense mutation of p14ARF. Alterations of TP53 were found in 15 (50%) of 30 osteosarcomas and 1 (3%) of 24 Ewing sarcomas. None of the sarcomas showed MDM2 amplification. While TP53 abnormalities were far more frequent in osteosarcoma than in Ewing sarcoma, alterations of p16INK4, p14ARF, and p15 were present at similar frequencies in the two types of sarcoma. The event-free survival (EFS) was worse in Ewing sarcoma patients with p16INK4 and p14ARF mutation/deletion than in those without the mutation/deletion (P = 0.019), and EFS was worse in osteosarcoma patients with TP53 alterations than in those without TP53 alterations (P = 0.048). The different incidence of TP53 abnormalities in the 2 types of sarcoma may reflect differences of the molecular processes through which the 2 types of tumor develop.     

Over-expression of polycomb group protein EZH2 relates to decreased expression of p16 INK4a in cholangiocarcinogenesis in hepatolithiasis

Polycomb group protein EZH2 and Bmi1 are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi-step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor gene p16 INK4a. We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN-1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN-3 (carcinoma in situ), 12 BilIN-2 (high-grade dysplasia), 32 BilIN-1 (low-grade dysplasia) and 37 non-neoplastic biliary epithelia from these livers. Expression of p16 INK4a, EZH2 and Bmi1 were surveyed in these foci. P16 INK4a promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT-1 and TFK-1) and small interfering RNA (siRNA), the effects of the knockdown of EZH2 on p16 INK4a methylation of CC cells were examined. Expression of p16 INK4a, which was frequent in BilIN1, was decreased in BilIN-2/3 and invasive carcinoma, while EZH2 expression showed step-wise increase from BilIN-1, -2 and -3 to invasive carcinoma (p < 0.01). P16 INK4a promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16 INK4a methylation and decreased binding of EZH2 to the p16 INK4a gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p16 INK4a gene. Our data suggest that over-expression of EZH2 may induce hypermethylation of p16 INK4a promoter followed by decreased expression of p16 INK4a in the multi-step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis.     

Immunohistochemical [corrected] detection of the alternate INK4a-encoded tumor suppressor protein p14(ARF) in archival human cancers and cell lines using commercial antibodies: correlation with p16(INK4a) expression.

The INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although the former is one of the most common targets for inactivation in human neoplasia, the frequency of p14(ARF) abrogation is not established. We have developed an immunohistochemical assay that allows the evaluation of p14(ARF) expression in formalin-fixed, paraffin-embedded tissues, using commercially available antibodies. p14(ARF) positive cells showed nuclear/nucleolar staining, which was absent in all cell lines and tumors with homozygous deletions of the INK4a gene. The assay was applied to 34 paraffin-embedded cell buttons, 30 non-small cell lung cancers and 28 pancreatic carcinomas, and the staining results were correlated with p16(INK4a) expression. Loss of p14(ARF) expression was common but less frequent than down-regulation of p16(INK4a) (53% versus 76% of all specimens). The p14(ARF) and p16(INK4a) expression pattern was concordant in 65 of 92 cases (71%). Significantly, 24 cases were p16(INK4a)-/p14(ARF)+, while the opposite staining pattern was observed in three cases, consistent with the notion that the two proteins have nonredundant functions. The immunohistochemical assay described here may facilitate studies on the prevalence and significance of aberrant p14(ARF) expression in human tumors.     

Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected. Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.     

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.