Role for p16(INK4a) in progression of gastrointestinal stromal tumors of the stomach: alteration of p16(INK4a) network members

Gastrointestinal stromal tumors feature a wide spectrum of biologic behavior, ranging from benign to extremely malignant. To determine the role of p16^INK4a alteration in progression of gastrointestinal stromal tumors of the stomach, we have investigated protein expression and gene methylation in correlation with clinicopathologic factors and survival. In addition to immunohistochemical analysis of p16^INK4a in a series of 95 cases, real-time quantitative methylation specific polymerase chain reaction for p16^INK4a and immunostaining for cyclin D1, cyclin E, pRb, DP-1, E2F-1, and Ki-67 were also evaluated in randomly selected samples. The p16^INK4a labeling indices ranged from 0% to 74% (median, 21%), demonstrating a significant inverse correlation with size (P = .046). On univariate (P = .003) and multivariate (P = .067) analyses, loss of p16^INK4a expression increased the likelihood of a poor tumor-related survival. In addition, size (P = .036) and the mitotic index (P = .005) had independent prognostic influence. The p16^INK4a methylation index, which ranged from 0% to 100% (median, 17%), was significantly higher in larger tumors (P < .001) and in high-risk category lesions (P = .001) and inversely correlated with protein expression. Hierarchical cluster analysis based on expression of p16^INK4a network members identified 2 clusters in 27 randomly selected tumor samples, containing 11 and 16 tumors each. Former cluster samples demonstrated higher risk category (P = .022), higher p16^INK4a methylation (P < .001), and more reduced pRb expression (P < .018). In addition, p16^INK4a network members clustered into 2 groups: (1) showing down-regulated p16^INK4a protein and up-regulating of both cyclin D1 and DP-1 and (2) down-regulated pRb and up-regulated E2F-1. We conclude that p16^INK4a alteration has an important role in progression of gastrointestinal stromal tumors of the stomach. Furthermore, the study provides a possible link between regulation of p16^INK4a network members and gastrointestinal stromal tumors.     

Overexpression of enhancer of zeste homolog 2 and MUC1 may be related to malignant behaviour in intraductal papillary neoplasm of the bile duct

Aims: Intraductal papillary neoplasm of the bile duct (IPNB) usually has a favourable prognosis, but occasionally is associated with invasive carcinoma. Overexpression of the polycomb group protein enhancer of zeste homolog 2 (EZH2) is involved in the progression of malignant tumours. In this study, we examined the significance of EZH2 expression in IPNB and its association with clinicopathological features and the expression of p16^INK4a, p53 and mucin core proteins. Methods and results: We examined immunohistochemically the expression of EZH2, p16^INK4a, MUC mucin core proteins and p53 in 15 patients with IPNB without invasion, including the cystic variant [male/female ratio (M/F) = 9/6], and in 19 with IPNB associated with invasive carcinoma (M/F = 13/6). The expression levels of EZH2, p53 and MUC1 were significantly lower (P < 0.01), and of MUC6 were significantly higher (P < 0.05), in IPNB without invasion than in IPNB with invasion. Expression of EZH2 was significantly correlated with expression of MUC1 (P < 0.01) and inversely correlated with expression of MUC6 (P < 0.05). In cholangiocarcinoma cells (HuCTT‐1 and TFK‐1), knockdown of EZH2 and MUC1 by small interfering RNA decreased invasion and proliferation, whereas knockdown of MUC6 increased invasion. Conclusions: Overexpression of EZH2 may be associated with malignant behaviour in IPNB in parallel with up‐regulated MUC1 expression and down‐regulated MUC6 expression.     

No p16INK4A/CDKN2/MTS1 mutations independent of p53 status in soft tissue sarcomas

The p16^INK4A/CDKN2/MTS1 gene encodes a specific inhibitor of cyclin-dependent kinases (CDKs) 4 and 6. This study investigates p16^INK4A gene status and expression in mesenchymal tumours, in particular soft tissue sarcomas (STSs). Employing non-radioactive polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) sequencing, no p16^INK4A mutation was found in 86 samples taken from 74 mesodermal tumours with known p53 gene status. This suggests that p16^INK4A gene alterations, in contrast to p53, are not involved in the progression of STS. This finding is supported by the reports of a low frequency of deletions and intragenic mutations in STS. Furthermore, by immunohistochemistry (IHC), an inverse correlation was established between p16^INK4A and RB positivity for 62 per cent of the frozen tumour samples investigated. However, alterations in other components of the pRb/p16^INK4A/CDK4/cyclin D1/E2F pathway have been proven crucial for tumourigenesis in human sarcomas. © 1998 John Wiley & Sons, Ltd.     

Immunoexpression of c-erbB-2 and p53 in benign and malignant salivary neoplasms with myoepithelial differentiation.

AIM: To evaluate whether the immunoexpression of c-erbB-2 and p53 is involved in the pathogenesis and progression of salivary tumours with myoepithelial differentiation. METHODS: 233 tumours from 211 patients were studied. These included 76 primary and 24 recurrent adenocarcinomas (polymorphous low grade adenocarcinoma, 13; epithelial-myoepithelial carcinoma, 19; adenoid cystic carcinoma, 56; and basal cell adenocarcinoma, 12) and 133 pleomorphic adenomas and myoepitheliomas, 96 being primary and the remaining recurrent tumours. All cases were formalin fixed and paraffin wax embedded. A StrepABC peroxidase method and polyclonal c-erbB-2 and p53 specific antisera were used. RESULTS: Cell membrane staining of c-erbB-2 was not found in any benign or malignant tumour. There was p53 protein accumulation in one primary and one recurrent pleomorphic adenoma and in 10 adenocarcinomas (polymorphous low grade adenocarcinoma, one; epithelial-myoepithelial carcinoma, one; adenoid cystic carcinoma, five; and basal cell adenocarcinoma, three), three of them being recurrences. CONCLUSIONS: The c-erbB-2 and p53 proteins are not involved in the pathogenesis of pleomorphic adenoma and myoepithelioma and do not constitute biomarkers in assessing the risk of recurrence. c-erbB-2 is not involved in the genesis of low grade salivary neoplasia with myoepithelial differentiation. The percentage of this type of neoplasia with p53 accumulation is low (10%) and does not appear to be related to tumour recurrence.     

An analysis on the combination expression of HPV L1 capsid protein and p16INK4a in cervical lesions

Human papillomavirus (HPV) infection in cervix is the most important reason for cervical cancer, but only 2% cervical HPV infection will develop into cervical cancer. So how to identify patients at risk of progressive cervical lesions from those infected with HPV to avoid over treatment is a big issue in clinic. The aims of this study were to detect the expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and to investigate the combination expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and its diagnostic efficiency in clinic. Immunochemical method was used to detect the expression of HPV L1 capsid protein and p16^INK4a in 169 cases of abnormal cytology. Histopathologic test was performed to identify cervical lesions of all the cases. χ² test and spearman's rank correlation were used for statistical analysis. The diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), accuracy, and the area under the receive operating characteristic (ROC) curve (denoted by A_Z) were calculated with SPSS 13.0. All the statistical tests were two sided at the 5% level of significance. L1 expression decreased (P < 0.001), but p16^INK4a expression increased (P < 0.001) with histopathologic diagnosis increasing. The expression rates of HPV L1 capsid protein, p16^INK4a, and L1(?)/p16(+) in cervical intraepithelial neoplasia (CIN)2, CIN3, and squamous‐cell carcinoma were statistically different from those in CIN1 (P < 0.001). The expressions of HPV L1 capsid protein, L1(+)/p16(+), L1(+)/p16(?), and L1(?)/p16(?) were negatively correlated with the severity of cervical lesions (P < 0.001), whereas the expressions of p16^INK4a and L1(?)/p16(+) were positively correlated with the severity of cervical lesions (P < 0.001). The specificity and A_Z of combining L1 with p16 ^INK4a were statistically higher than L1 or p16 ^INK4a alone (P < 0.05). L1 and p16^INK4a are useful biomarkers for the early diagnosis of cervical lesions. The combination of L1 and p16^INK4a has a higher diagnostic accuracy than L1 or p16^INK4a alone in diagnosis of cervical lesions. Diagn. Cytopathol. 2010;38:573–578. © 2009 Wiley‐Liss, Inc.     

Alterations of expression of Rb,p16INK4A and cyclin D1 in non-small cell lung carcinoma and their clinical significance

Inactivation of the Rb pathway in non-small cell lung carcinoma (NSCLC) occurs mostly through inactivation of the cyclin-dependent kinase inhibitor p16^INK4A and/or up-regulation of cyclin D1. In order to assess the frequency and the prognostic value of these abnormalities in NSCLC, immunohistochemical analysis of Rb, p16^INK4, and cyclin D1 has been performed on 168 cases of NSCLC including 77 squamous cell carcinomas, 43 adenocarcinomas, and 48 basaloid carcinomas. The reduced survival rate of basaloid carcinoma (stage I–II) compared with other histological types of NSCLC was confirmed (p = 0·008). Loss of protein expression of Rb and p16^INK4A was observed in 12 per cent and 58 per cent of NSCLC cases respectively and cyclin D1 overexpression in 43 per cent. There was an inverse correlation between Rb and p16 expression ( p < 0·0001) and a direct correlation between Rb and cyclin D1 expression ( p = 0·0007). In univariate analysis, Rb-negative adenocarcinomas at stages I–II had a significantly shorter survival than Rb-positive cases ( p = 0·04) and stages I–II p16-positive cases had a shorter survival than p16-negative cases ( p = 0·02), which was more significant in basaloid carcinoma ( p = 0·003). p16 status retained its influence on survival in multivariate analysis at stage I–II for all cases ( p = 0·01) and for basaloid carcinoma ( p = 0·005). Cyclin D1 overexpression did not influence survival. Combined Rb/p16/cyclin D1 phenotypes in univariate analysis showed a shorter survival for Rb-negative/p16-positive/cyclin D1-negative tumours ( p = 0·002). These results, linked to previous data, indicate that the Rb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 per cent), but could not confirm an unfavourable role for each individual event (p16^INK4A loss or cyclin D1 up-regulation) in prognosis. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis

Haller F, Agaimy A, Cameron S, Beyer M, Gunawan B, Happel N, Langer C, Ramadori G, von Heydebreck A & Füzesi L
(2010) Histopathology 56, 305–318 Expression of p16 ^INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis Aims: To determine the prognostic impact of p16^INK4A expression in gastrointestinal stromal tumours (GISTs), which is currently being questioned, with both loss and overexpression said to be correlated with poor prognosis. Methods and results: Two different forms of p16^INK4A were identified, presenting with predominantly nuclear and cytoplasmic expression pattern, respectively. The immunohistochemical expression of the two forms and their correlation with E2F1 and prognosis were analysed in a series of 120 GISTs with clinical follow‐up. Low nuclear p16^INK4A expression correlated with E2F1 up‐regulation, higher mitotic counts, and tumour progression. The prognostic value of nuclear p16^INK4A expression was only marginally significant (P = 0.05). Strong expression of the cytoplasmic p16^INK4A form was significantly associated with shorter disease‐free survival (P = 2 × 10^?5). The prognostic impact of strong expression of the cytoplasmic p16^INK4A form was independent of anatomical localization, tumour size and mitotic counts, and significant even among the cohort of tumours with high malignant potential. Conclusions: Low expression of the nuclear p16^INK4A form and strong expression of the cytoplasmic p16^INK4A form both represent two independent parameters each associated with tumour progression in GISTs. Low nuclear p16^INK4A expression enables E2F1 up‐regulation and consecutive accelerated cell proliferation. In contrast, strong cytoplasmic p16^INK4A expression probably reflects a negative feedback loop as a result of (as yet unknown) oncogenic events.     

p16INK4A expression in cervical premalignant and malignant lesions

p16^INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16^INK4a. Recent studies revealed that p16^INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16^INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16^INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples (n = 48), p16^INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16^INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16^INK4a expression (P < 0.001).     

Lowp53 protein expression in salivary gland tumours compared with lung carcinomas

Summary Fifty-one salivary gland tumours (23 pleomorphic adenomas, 5 Warthin's tumours, 12 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 3 undifferentiated carcinomas and 1 acinic cell tumour) and 27 lung carcinomas (18 squamous cell carcinomas, 6 adenocarcinomas and 3 small cell carcinomas) were analysed immunohistochemically for the expression ofp53 nuclear phosphoprotein. Eight out of 51 (16%) salivary gland tumours werep53 positive. Three of these were benign and 5 malignant. All 3 benign salivary gland tumours were pleomorphic adenomas and expressed only occasional nuclear positivity with less than 1% of tumour cells positive. Of the 5p53-positive malignant tumours, 3 were mucoepidermoid carcinomas and 2 undifferentiated carcinomas. The malignant salivary gland tumours expressed more than 1% of positive nuclei in every case. Seventeen lung carcinomas werep53 positive (63%). Thirteen of these were squamous cell carcinomas, 3 were adenocarcinomas and 1 small cell lung carcinoma. The results show that mutations of thep53 gene may be infrequent in salivary gland tumours when compared with lung carcinomas. The relatively indolent course of some histological types of malignant salivary gland tumours could be associated with the preservation of the non-mutatedp53 gene in most of these tumours. The presence ofp53 positivity in some pleomorphic adenomas might, on one hand, suggest thatp53 gene alterations are also present in these tumours; on the other hand, the accumulation of thep53 protein in these tumours might also be due to some unknown mechanism, not necessarily related top53 gene mutation.     

Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours

A series of 26 malignant peripheral nerve sheath tumours (MPNST) and 24 benign peripheral nerve sheath tumours (BPNST) were analysed immunocytochemically for p53 expression and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 (with MIB1). In 23/26 MPNST, 5%–65% of the tumour cell nuclei were immunoreactive for Ki67 with MIB1 while none of the 24 BPNST had nuclear staining exceeding 5%. Greater than 50% nuclear PCNA staining was detected in 25/26 MPNST compared with 8/24 BPNST; 17/26 MPNST showed 5–100% nuclear staining for p53 (13/26>20%), whereas none of the BPNST had nuclear staining exceeding 1%. The Ki67, PCNA and p53 immunostaining results correlated significantly with benign versus malignant (P<0.001, P<0.001 and P<0.005, respectively) as well as mitotic rate (P<0.001, P<0.05 and P<0.05). Ki67 immunostaining results correlated significantly with PCNA and p53, as did p53 with Ki67 and PCNA (P<0.001 in both). Stepwise (logistic regression forward) multivariate analysis of the variable, benign versus malignant, revealed the strongest correlations with PCNA (P=0.007) and Ki67 (P=0.021). Direct confirmation of the presence of p53 protein was obtained by western blot analysis of 3 MPNST and 5 BPNST. Two MPNST, showing 90% and 30% immunoreactivity, were positive for p53, while one MPNST with 5% immunoreactivity and all 5 BPNST were negative. Southern blot analysis performed on the two MPNST with high p53 protein levels revealed no amplification of the MDM2 gene, suggesting that high p53 levels in MPNST are likely to be due to mutation. The results also indicate that PCNA and Ki67 are potentially useful in distinguishing BPNST from MPNST, particularly in problematic cases of cellular schwannoma versus MPNST. The detection of p53 in a large percentage of cells of a plexiform neurofibroma giving rise to MPNST and Ki67 in 5% and 25% of cells of two similar cases suggests that malignant transformation may be detected in some cases by p53 and proliferation markers prior to overt histological evidence of malignancy.     

p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours

Karim R Z, Gerega S K, Yang Y H, Spillane A, Carmalt H, Scolyer R A & Lee C S
(2010) Histopathology 56, 868–875
p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours Aims: Control of cell cycling and proliferation is critical to the development of neoplasia and may play a role in the pathogenesis of phyllodes tumours (PTs). This study aimed to evaluate the immunohistochemical expression of certain proteins from the G₁/S transition of the cell cycle in a cohort of PTs, to determine their role in tumour pathogenesis and to identify any associations with patient outcome. Methods and results: Sixty‐five PTs (34 benign, 23 borderline and eight malignant) diagnosed at a single institution between 1990 and 2006 were analysed. Immunohistochemistry for p16, pRb, cyclin D1 and Ki67 was performed. Expression of the following markers increased significantly with tumour grade: stromal nuclear and cytoplasmic p16 (P = 0.01 and 0.002, respectively), stromal and epithelial pRb (P = 0.000 000 06 and 0.004, respectively), and stromal and epithelial Ki67 (P = 0.03 and 0.04, respectively). Epithelial pRb scores of 7 (range 0–7) were significantly associated with reduced disease‐free survival (DFS) compared with scores of <7 (P = 0.0009). No relationship was found between cyclin D1 expression in either the epithelium or the stroma, and grade or DFS. Conclusions: The results suggest that alterations at the G₁/S transition of the cell cycle play an important role in the progression of PTs.     

Immunohistochemical expression of p16(INK4A) protein as a helpful marker of a subset of potentially malignant oral epithelial lesions: study on a series with long-term follow-up

Montebugnoli L, Cervellati F, Cocchi R, Farnedi A, Pennesi M G, Flamminio F & Foschini M P
(2010) Histopathology 57 , 528–534
Immunohistochemical expression of p16^INK4A protein as a helpful marker of a subset of potentially malignant oral epithelial lesions: study on a series with long‐term follow‐up Aim: To examine a group of lesions that progressed to oral squamous cell carcinoma (OSCC) to determine whether p16^INK4A expression is an early finding during malignant transformation, and whether immunohistochemical evaluation of p16^INK4A is an appropriate prognostic marker. Methods and results: Twenty cases of OSCC were investigated. All cases had had a biopsy on the same site as OSCC performed at least 1 year before OSCC (range 1–11 years; mean 3.15 ± 3.1 years). Twenty specimens from normal oral mucosa served as controls. p16^INK4A expression was evaluated by immunohistochemical analysis and cases showing >5% of stained cells were defined as ‘positive’. All 20 control cases were negative for p16^INK4A. Oral lesions were p16^INK4A‐positive in nine cases and negative in 11. No significant relationship was found between p16^INK4A positivity and the presence/absence of dysplasia. Among OSCC, nine tumours showed p16^INK4A positivity and 11 showed negativity. A significant relationship (χ² = 7.1; P < 0.01) was found between the presence/absence of p16^INK4A staining in OSCC and the presence/absence of p16^INK4A staining in lesions preceding OSCC. Conclusions: p16^INK4A immunohistochemistry has a potential role in detecting a subset of p16^INK4A‐positive lesions with malignant potential.     

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

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

Quantitative immunohistochemical fingerprinting of adhesion/growth-regulatory galectins in salivary gland tumours: divergent profiles with diagnostic potential

Remmelink M, de Leval L, Decaestecker C, Duray A, Crompot E, Sirtaine N, André S, Kaltner H, Leroy X, Gabius H‐J & Saussez S
(2011) Histopathology  58, 543–556
Quantitative immunohistochemical fingerprinting of adhesion/growth‐regulatory galectins in salivary gland tumours: divergent profiles with diagnostic potential Aims: This study tests the hypothesis that histopathological fingerprinting of galectins, which are emerging multifunctional effectors in cell sociology, could refine the differential diagnosis of salivary tumours. Methods and results: We applied non‐crossreactive polyclonal antibodies against galectin‐1 (Gal‐1), galectin‐3 (Gal‐3), galectin‐7 (Gal‐7) and galectin‐8 (Gal‐8) for immunohistochemical analysis of salivary gland tumours (72 cases with benign disease and 39 cases with malignancy) and 29 control specimens. The principal positivity of cases, the site of signal presence and the quantitative parameters concerning percentage of positive cells and labelling intensity were determined. Acinic cell and adenoid cystic carcinomas (specifically tubular and cribriform types) shared the expression signature of Gal‐1, Gal‐3 and Gal‐8 presence combined with Gal‐7 absence. Mucoepidermoid carcinomas presented a unique profile based on cytoplasmic Gal‐1, Gal‐3, Gal‐7 and Gal‐8 localization in the intermediate cells. Adenomas were separable from malignancy by a consistent decrease in the labelling index (LI) for Gal‐7 and Gal‐8 (LI Gal‐7, P < 10^?6; LI Gal‐8, P = 0.001). When present, staining for the tumour suppressor p16^INK4a coincided with Gal‐1 presence. Conclusions: Expression profiling of the four tested galectins in salivary gland tumours revealed non‐uniform staining patterns with discriminatory potential based on intracellular localization and quantitative aspects.     

Expression of ATM, p53, and the MRE11-Rad50-NBS1 complex in myoepithelial cells from benign and malignant proliferations of the breast

AIMS: To analyse the expression of proteins involved in DNA double strand break detection and repair in the luminal and myoepithelial compartments of benign breast lesions and malignant breast tumours with myoepithelial differentiation. METHODS: Expression of the ataxia telangiectasia (ATM) and p53 proteins was immunohistochemically evaluated in 18 benign and malignant myoepithelial tumours of the breast. Fifteen benign breast lesions with prominent myoepithelial compartment were also evaluated for these proteins, in addition to those in the MRE11-Rad50-NBS1 (MRN) complex, and the expression profiles were compared with those seen in eight independent non-cancer (normal breast) samples and in the surrounding normal tissues of the benign and malignant tumours examined. RESULTS: ATM expression was higher in the myoepithelial compartment of three of 15 benign breast lesions and lower in the luminal compartment of eight of these lesions compared with that found in the corresponding normal tissue compartments. Malignant myoepithelial tumours overexpressed ATM in one of 18 cases. p53 was consistently negative in benign lesions and was overexpressed in eight of 18 malignant tumours. In benign breast lesions, expression of the MRN complex was significantly more reduced in myoepithelial cells (up to 73%) than in luminal cells (up to 40%) (p=0.0005). CONCLUSIONS: Malignant myoepithelial tumours rarely overexpress ATM but are frequently positive for p53. In benign breast lesions, expression of the MRN complex was more frequently reduced in the myoepithelial than in the luminal epithelial compartment, suggesting different DNA repair capabilities in these two cell types.     

Comparison of p16(INK4a) expression with p53 alterations in head and neck cancer by tissue microarray analysis

We investigated whether there is a relationship between loss of p16(INK4a) protein expression and p53 alterations in head and neck squamous cell carcinomas (HNSCCs). For this purpose, immunohistochemistry was performed on tissue microarrays of 664 tumours; this represents the largest HNSCC cohort studied for molecular biomarkers. Loss of p16(INK4a) protein expression was associated with aberrant p53 expression (negative or overexpressed) in the total cohort, and with TP53 mutations in 200 tumours analysed (p < 0.0001 each). Both loss of p16(INK4a) expression and p53 alterations differed significantly across both tumour sites and stages, being more prevalent in the hypopharynx than in the other tumour sites and in advanced tumour stages. As a possible link between p53 status and p16(INK4a) loss, we found that increased DNA methyltransferase 1 protein levels occurred preferentially in tumours with aberrant p53 (p = 0.001) and negative p16(INK4a) expression (p = 0.0004). In the total cohort, there was a borderline significant difference in patient survival across three p16(INK4a) expression levels (negative, positive, high), with loss of p16(INK4a) expression showing shortest survival. It is suggested that loss of p16(INK4a) expression and p53 alterations should be viewed as related events involved in the early carcinogenic process.     

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.     

Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain‐of‐function mutant p53R172H

p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co‐exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53^R172H gain‐of‐function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well‐differentiated SCCs. However, most of the tumours induced by p53^R172H were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well‐differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16^Ink4a and p19^Arf, encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53^R172H‐induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss‐of‐function mutation or wild‐type p53. Accordingly, the survival of patients with head and neck SCCs bearing co‐occurring high‐risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high‐risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co‐existed with either low‐risk p53 mutations or potential loss‐of‐function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

p16INK4A overexpression and HPV infection in uterine cervix adenocarcinoma

Human papillomaviruses (HPVs) are causally involved in the genesis of cervical carcinomas and their precursors, and there is a strong relationship between the cyclin-dependant kinase inhibitor p16^INK4A and HPV infection. This study was carried out to assess the correlations between p16^INK4A expression as an early biomarker of the endocervical adenocarcinoma and HPV infection. p16^INK4A expression and HPV typing were performed on 46 samples including 5 normal endocervix, 9 benign lesions of the endocervix, 25 endocervical adenocarcinomas, and 7 endometrioid adenocarcinomas of the uterine corpus. A semiquantification of the p16^INK4A immunostaining was realized (using both the staining intensity and the percentage of positive cells) and was graded from 0 to 15. All of the 25 endocervical adenocarcinomas overexpressed p16^INK4A; the adjacent epithelium and the connective tissue were strictly negative. No p16^INK4A was detected in nine benign endocervical lesions and in five normal endocervix. Few endometrioid adenocarcinomas of the uterine corpus that infiltrate the endocervix exhibited a low immunoreactivity (score 0/15 or 1/15). This pattern of expression is significantly associated with HPV infection (p<10 ⁻³), mainly high-risk HPV types (p=0.02). Our results suggest that p16^INK4A is a putative molecular biomarker that consistently discriminates uterine cervix adenocarcinomas from benign lesions and from endometrioid adenocarcinomas of the uterine corpus .     

Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors

We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.