Hypermethylation of the tumor suppressor gene p16INK4 (p16) promoter is associated with worse prognosis in colorectal cancer (CRC). In the present study, it was investigated whether p16 mRNA expression correlates with the methylation of its promoter, and whether it influences prognosis in patients with CRC. DNA and RNA were extracted from 101 resected tumor specimens. A MethyLight assay was used to quantify p16 methylation in terms of percentage of methylated reference (PMR), and the expression of p16 mRNA was measured using reverse transcription-polymerase chain reaction. Associations between p16 methylation or mRNA expression and patient survival were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. p16 methylation was detected in 67 cases (66.3%) and the median PMR value was 0.344 (range, 0.00–468.6). Using a cut-off PMR value of 4, high p16 methylation was observed in 18 cases (17.8%). No significant association was observed between p16 methylation level and patient prognosis. As expected, a significant inverse association was observed between p16 methylation and mRNA expression (P=0.034). Amongst the 83 cases with low p16 methylation, a significantly worse outcome was identified in patients expressing high p16 mRNA expression levels (P=0.026). Multivariate analysis identified that p16 mRNA expression was an independent prognostic factor for worse survival (P=0.011). These results suggested a paradoxical association between high levels of p16 mRNA expression in the tumor and worse prognosis in patients with CRC.
Colorectal cancer (CRC) is the third most frequent malignancy worldwide and the fourth most common cause of cancer-associated mortalities (1). Globally, ~1.36 million people are diagnosed with CRC each year and approximately one-half will succumb to this disease (1,2). A number of genes that are mutated in the multistep process of colorectal carcinogenesis and progression have been demonstrated to influence the prognosis of patients with CRC and their response to treatment (3–6). In addition to these somatic genetic mutations, epigenetic alterations and particularly the aberrant hypermethylation of gene promoter regions leading to transcriptional silencing are suggested to be important in CRC tumorigenesis (7). This mechanism is responsible for the functional inactivation of numerous tumor suppressor genes in CRC (7,8), including human MutL homolog 1, tissue inhibitor of metalloproteinase-3, p14, death-associated protein kinase, adenomatous polyposis coli, O-6-methylguanine-DNA methyltransferase and p16INK4[(p16) or cyclin-dependent kinase (CDK) inhibitor 2a] (9–11).
In normal cells, p16 and retinoblastoma (Rb) proteins serve an important role in regulating the cell cycle pathway (12,13). Rb is phosphorylated by the cyclin D1-CDK4/6 complex, resulting in its dissociation from transcription E2 factor (E2F) (13). The subsequent transcriptional activation of E2F leads to progression of the cell cycle from G1 to S phase (14). As p16 interferes with cell cycle progression by inactivating CDK4/6, decreased expression or inactivation of p16 attenuates the ability of Rb to inhibit cell proliferation (15). The p16-Rb pathway is suppressed in a number of cancer types via genetic or epigenetic alterations in Rb and/or p16, through overexpression of the cyclin D1/CDK4 complex, in addition to a number of other mechanisms (12,13,15,16). In particular, deletion or mutation of the p16 gene is frequently observed in cancer of the biliary tract, lung, pancreas and esophagus and in brain tumors (17–21). Deletion of p16 has been associated with a late clinical stage in esophageal cancer, and with lymphatic invasion and distant metastasis in pancreatic cancer (22,23). In gall bladder and lung cancer, p16 deletions and mutations are associated with poor prognosis (24,25).
Decreased p16 expression due to hypermethylation of the p16 promoter was detected in 32–55% CRC cases (26–30). Although p16 mRNA expression is inversely correlated with tumor size and lymph node metastasis (31), its influence on the prognosis of patients with CRC remains unclear (32). A previous Japanese study identified that p16 promoter hypermethylation in the primary tumors of patients with CRC was associated with a shorter survival (33). This result was supported by a subsequent meta-analysis (34). In the present study, it was investigated whether p16 mRNA expression was associated with methylation of the p16 gene promoter and with patient prognosis in CRC.
Materials and methodsPatients with CRC and tissues
The present study included 101 patients with primary CRC who underwent surgery at the Kanazawa University Hospital (Kanazawa, Japan) between April 1999 and December 2002. Eligible patients were aged 20 years or older and had histologically proven adenocarcinoma of the colon and rectum. Exclusion criteria included absolute contraindications to general anesthesia and/or surgery. Their clinicopathological characteristics are presented in Table I. The survival status was determined for all patients and the median follow-up period was 54.5 months. In total, 54 patients (53.5%) received postoperative 5-fluorouracil (5-FU)-based adjuvant chemotherapy.
Tumor tissue samples collected from the fresh surgical specimen were cryopreserved in liquid nitrogen and stored at −80°C for extraction of DNA and RNA. The remaining surgical specimen was fixed with 10% neutral-buffered formalin for 1–2 days at room temperature and embedded in paraffin for histopathological examination. The tumor stage was determined according to the Union for International Cancer Control tumor, node and metastasis (TNM) classification (35). Genomic DNA and total RNA were extracted from the same tumor tissues using the QIAmp DNA Mini kit and the RNeasy Mini kit (both from Qiagen GmbH, Hilden, Germany), respectively, according to the manufacturer's protocols.
The present study was performed in accordance with the Declaration of Helsinki. The design and protocol for the present study were approved by the Kanazawa University Human Genome and Gene Analysis Research Ethics Committee, and written informed consent was obtained from the majority of the patients.
Quantification of p16 methylation by the MethyLight assay
Bisulfite conversion of genomic DNA was performed as previously described (36). DNA was denatured using 0.2 M NaOH and subsequently incubated with bisulfite for 16 h at 50°C. The bisulfite-converted DNA was purified using the Wizard DNA purification kit (Promega Corporation, Madison, WI, USA) and precipitated with ethanol. The DNA sample was resuspended in water and stored at −30°C.
MethyLight, a fluorescence-based real-time PCR assay was used to measure the level of p16 promoter methylation as previously described (37). The sense and antisense primers used for amplifying the bisulfite-converted p16 promoter were: 5′-TGGAATTTTCGGTTGATTGGTT-3′ and 5′-AACAACGTCCGCACCTCCT-3′, respectively (37). These primers were used with the probe 5′-6FAM-ACCCGACCCCGAACGCG-TAMRA-3′ to measure CpG methylation of the p16 promoter region by real-time PCR (38). The specificity for amplification of methylated DNA was confirmed separately using human sperm DNA (unmethylated) and SssI (New England BioLabs, Inc., Ipswich, MA, USA)-treated sperm DNA (fully methylated) in the assay. Actin was amplified as a control for the total amount of DNA using the sense primer, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and antisense primer, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′; and probe, 5′-6FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA-3′ (38). The percentage of fully methylated fraction [percentage of methylated reference (PMR)] at a specific gene locus was calculated by dividing the gene:actin ratio of the sample DNA by the gene:actin ratio of the SssI-treated sperm DNA and multiplying by 100 (38). PMR values obtained using MethyLight were classified into high (PMR ≥4) and low (PMR <4) methylation categories, according to previous studies (9,39,40).
Analysis of p16 mRNA expression
Reverse transcription (RT)-PCR was used to measure p16 mRNA expression, as previously described (41). The relative expression of mRNA was quantified using the 2−ΔΔCq method (42). The expression of actin was measured as an internal standard and the level of p16 mRNA expression in each tumor sample was normalized to the expression of actin. The cut-off value for high and low levels of p16 mRNA expression was defined as the median expression level for all tumor samples. Cut-off values for p16 PMR and mRNA expression were used to compare p16 promoter methylation and mRNA expression in the same tumors.
Proliferating cell nuclear antigen (PCNA) mRNA expression levels were additionally measured, using actin expression level as the internal standard. p16 mRNA expression relative to PCNA (p16/PCNA) was calculated as the p16 mRNA:actin ratio divided by the PCNA mRNA:actin ratio in the same cDNA sample. The cut-off value for p16/PCNA expression was defined as the median p16/PCNA level for all tumor samples. This was used to investigate the influence of p16 mRNA expression on the survival of patients with CRC.
Statistical analysis
For statistical comparison of tumor p16 methylation and mRNA expression with clinicopathological factors and tumor stage, the Fisher's exact test was used to study non-continuous variables, and the Mann-Whitney U test and Kruskal-Wallis test were used to study continuous variables. The Mann-Whitney U test was used to compare p16 methylation with p16 mRNA expression. The survival of patients was evaluated using Kaplan-Meier analysis. Univariate and multivariate analyses of survival were conducted using Cox proportional hazards regression. All statistical analyses were performed with EZR (version 1.29, Jichi Medical University, Shimotsuke, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics (43).
Resultsp16 promoter methylation and p16 mRNA expression in CRC
The levels of p16 gene promoter methylation and p16 mRNA expression in the tumor samples of all patients with CRC are presented in Table I in association with clinicopathological features. p16 methylation (PMR >0) was detected in 67 cases (66.3%) and the median PMR value was 0.34 (range, 0.00–468.6; Fig. 1A). A PMR cut-off value of ≥4 was used to define high methylation and PMR <4 for low methylation, according to previous studies (9,38–40). Using this definition, the high methylation group comprised 18 (17.8%) of the 101 patients (Table II), in agreement with previous studies (9,33). The range of relative p16 mRNA expression levels was between 0 and 154.0, with a median expression level of 1.98 (Fig. 1B). As p16 mRNA expression is likely to be associated with cell cycle (44), the p16 mRNA expression level was normalized to that of PCNA expression in the same tumor and designated as p16/PCNA expression. The relative values of p16/PCNA ranged between 0 and 1,957.6, with a median value of 11.46 (Fig. 1C). To evaluate the influence of p16 mRNA expression on survival outcome, patients were divided into two groups (high and low) according to the median value for p16 mRNA or p16/PCNA expression.
Comparison of p16 mRNA expression between the p16 high and low methylation groups identified a significant inverse association (P=0.034; Fig. 2A). When a higher PMR cut-off value of 10 was used rather than 4, a strong inverse association with p16 mRNA expression was observed between the high (n=12; 11.9%) and low (n=89; 87.1%) p16 methylation groups (P<0.001; Fig. 2B). Subsequently, p16/PCNA expression was compared between the p16 high and low methylation groups. Using a PMR value of 4 to classify p16 methylation, no significant association was observed between p16 methylation and p16/PCNA expression (P=0.168; Fig. 2C). However, when classified according to the higher PMR cut-off value of 10, a significant inverse association was observed between p16 methylation and p16/PCNA expression (P=0.006; Fig. 2D).
Tumor p16 methylation and prognosis of patients with CRC
Comparison of tumor p16 methylation levels with clinical and histopathologic characteristics of patients with CRC is presented in Table II. High p16 methylation (PMR >4) level was more frequently detected in mucinous (P=0.017) compared with the other histological types and was significantly associated with later clinical stage (P=0.008); however, not with the sex or age of the patient, tumor site, T stage or adjuvant chemotherapy. There was no significant difference in prognosis between the high and low p16 methylation groups (P=0.94; Fig. 3).
Tumor p16 mRNA expression and prognosis of patients with CRC
No significant differences in p16 mRNA expression levels (high or low) were observed according to sex or age of the patient, or with tumor site, histology, T stage, or adjuvant chemotherapy (Table III). The survival of patients with CRC with high p16 mRNA expression was worse compared with patients with low expression; however, this did not reach statistical significance (P=0.109; Fig. 4A). The majority (83/101; 82%) of patients with CRC demonstrated low levels (PMR <4) of p16 methylation (Table II; Fig. 2A). When these patients were divided into high and low p16 mRNA expression groups defined by a median level of value (2.15), the high expression group (n=42) demonstrated a worse outcome compared with the low expression group (n=41; P=0.076; Fig. 4B).
Patients with CRC were additionally divided into p16/PCNA high (n=50) and low (n=51) groups according to the median value (11.46). No differences in any of the clinical and histopathologic parameters were observed between these two groups (Table II), nor was there a significant difference in patient survival between the high and low p16/PCNA groups (P=0.122; Fig. 4C). The 83 patients with low tumor p16 methylation were further examined by dividing them into groups with high or low p16/PCNA expression according to the median value (12.49). In this analysis, patients with high p16/PCNA expression demonstrated a significantly worse survival (P=0.026; Fig. 4D).
The patient group with a low p16 methylation (PMR <4) was additionally evaluated using Cox regression analysis for the prognostic significance of various clinical and histopathologic features and for p16 mRNA and p16/PCNA expression levels. p16/PCNA mRNA expression was the only significant prognostic factor in univariate analysis (Table IV; P=0.026). The influence of T stage, tumor stage and adjuvant chemotherapy on the overall survival of patients with CRC was analyzed using the Kaplan-Meier method. There was no association between any of these factors and the prognosis of the patients (Fig. 5). Multivariate analysis identified that low p16/PCNA expression was an independent factor for better survival in patients with low p16 methylation (hazard ratio 0.287; 95% confidence interval 0.110–0.747; P=0.011; Table IV).
Discussion
Tumor p16 methylation has previously been identified as a prognostic factor for a worse outcome in CRC (33,34,41). However, other previous studies demonstrated that p16 methylation has no impact on prognosis (32) or predicts worse outcome only in patients with poorly differentiated CRC (45). A possible mechanism for the putative association between tumor p16 methylation and survival of patients with CRC is that expression of p16 protein is diminished, thereby promoting tumor cell proliferation and invasion (46,47). In the present study, however, no significant association was observed between tumor p16 methylation and the outcome of patients with CRC, thus supporting a previous study (32). A number of technical reasons, in addition to the number of patients examined may account for the inconsistent results demonstrated by different previous studies and the present study. The technical reasons include differences in the tissue samples analyzed (fresh compared with fixed tissues), the methods used to quantify p16 methylation levels and the cut-off values used in the analyses (32–34,41,45).
p16 promoter methylation is associated with decreased expression of p16 mRNA in clinical samples of CRC (26). In the present study, an inverse association between tumor p16 methylation and the expression of its transcript was additionally identified. This was most pronounced in tumors with high methylation levels (PMR >10). However, within the high methylation group (PMR ≥4) a number of cases with relatively increased expression of p16 mRNA were identified, in agreement with previous studies, which demonstrated that tumor cells with p16 promoter methylation may express p16 mRNA (31,41). Therefore, p16 mRNA expression may not be controlled exclusively through promoter methylation; however, may additionally be influenced by other factors, including the Ras signaling pathway (48), which is frequently activated in CRC due to KRAS proto-oncogene GTPase and NRAS proto-oncogene GTPase mutations.
Previous studies demonstrated that low p16 protein expression in CRC was associated with larger tumor size, lymph node metastasis and faster tumor proliferation (31,49,50), and is thus likely to account for an association with worse prognosis (51). However, little is known regarding the prognostic impact of p16 mRNA expression in patients with CRC. Although an inverse association between tumor p16 methylation and mRNA expression was observed in the present study, relatively few patients (18/101) demonstrated high levels of p16 methylation, defined as PMR ≥4. Therefore, patients with low tumor p16 methylation levels were investigated, as it was hypothesized that p16 mRNA expression may affect patient survival, as those patients may exhibit a wide range of p16 mRNA expression for analysis. An unexpected result of the present study was that patients with high p16/PCNA mRNA expression demonstrated significantly worse survival. Furthermore, this was demonstrated by multivariate analysis to be independent of other factors that potentially influence patient survival, including tumor stage and histological types. Despite its well-recognized tumor suppressor role (52), p16 is overexpressed at the mRNA and protein expression levels in tumor tissues compared with adjacent normal mucosa (31,51). Similar to the present results, patients with breast (53,54) and prostate cancer (55) with high p16 expression were additionally identified to have a worse prognosis. Notably, although p16 is a critical cell cycle regulator and its mRNA expression is likely to be associated with cell cycle (44), none of the previous studies investigating the association of p16 promoter methylation with prognosis of patients with CRC (32,34) scored a copy number of p16 mRNA. Therefore, the clinical implications of p16 mRNA and protein expression in different cancer types and association with p16 methylation require further investigation.
Established tumor cell lines with Rb deletion demonstrated activated p16 transcription and increased p16 protein expression (56). It has additionally been demonstrated that cell cycle regulation by p16 is lost in tumor cells with inactivated Rb (57), and that the efficacy of exogenously expressed p16 in cancer cells depends on Rb function (58). Furthermore, the overexpression of transcription factor E2F1 promotes p16 transcription (59), whereas CDK4 overexpression in sarcoma cells is thought to increase p16 expression through a feedback loop (60). This putative feedback regulation in the expression and function of Rb pathway mediators may explain the paradoxical association observed in the present study and in others between high p16 expression and worse patient survival.
In summary, p16 is a CDK4 inhibitor that counteracts the cell cycle process by sustaining the Rb-mediated pathway. Accordingly, p16 has been recognized as a tumor suppressor that is lost or inactivated through gene mutation, deletion or promoter methylation in various cancer types, including CRC. Previous studies demonstrated an association between p16 gene promoter hypermethylation and worse prognosis in patients with CRC, in addition to an inverse association between p16 expression and tumor progression. However, the effect of p16 mRNA expression on the prognosis of patients with cancer is controversial. In the present study, it was demonstrated that p16 mRNA expression in the tumors was inversely associated with the levels of p16 promoter methylation. In addition, multivariate analysis determined that high p16 mRNA expression normalized to PCNA mRNA expression (p16/PCNA) was an independent prognostic factor for poor survival of patients with CRC. These results identified a previously unrecognized and paradoxical association between high expression of p16 mRNA and worse prognosis of patients with CRC, although a similar association has been demonstrated in other cancer types.
Acknowledgements
The authors would like to thank Dr Barry Iacopetta (University of Western Australia, Crawley, Australia) for critically reading and editing the manuscript.
Funding
The present study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (grant nos. 20390353 and 23390321).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
HK made substantial contributions to the design and conception of the study, and the acquisition, analysis and interpretation of the data, and drafted the manuscript. HT and TM collected the clinical samples and contributed to the interpretation of the data. TM helped to draft and finalize the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was performed in accordance with the Declaration of Helsinki. As the tissues used in the present study were from the patients diagnosed between 1999 and 2002, written informed consent was obtained from the patients prior to the tissue sample collection. In accordance with Japanese ethical guidelines and law, the study protocol was reviewed and approved by the Kanazawa University Human Genome/Gene Analysis Research Ethics Committee (approval no. 181; Kanazawa, Japan). At the start of the study, it was not possible to directly contact the specific patients to explain the present study. According to the Human Genome/Gene Analysis Research Ethics Committee, the present study was announced on our website, providing these patients an opportunity to opt out of the present study. By the date indicated, none of them refused to be included in the present study. All samples were anonymized before analysis was performed to guarantee the protection of privacy.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
AbbreviationsCRC
colorectal cancer
5-FU
5-fluorouracil
PCNA
proliferating cell nuclear antigen
PCR
polymerase chain reaction
PMR
percentage of methylated reference
ReferencesFerlayJSoerjomataramIDikshitREserSMathersCRebeloMBrayFCancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012Int J Cancer136E359E386201410.1002/ijc.2921025220842BrennerHKloorMPoxCPColorectal cancerLancet38314901502201410.1016/S0140-6736(13)61649-924225001MarkowitzSDBertagnolliMMMolecular basis of colorectal cancerN Engl J Med36124492460200910.1056/NEJMra0804588200189662843693WaltherAJohnstoneESwantonCMidgleyRTomlinsonIKerrDGenetic prognostic and predictive markers in colorectal cancerNat Rev Cancer9489499200910.1038/nrc264519536109FearonERMolecular genetics of colorectal cancerAnnu Rev Pathol6479507201110.1146/annurev-pathol-011110-13023521090969CarethersJMJungBHGenetics and genetic biomarkers in sporadic colorectal cancerGastroenterology14911771190.e3201510.1053/j.gastro.2015.06.047262168404589489IssaJPCpG island methylator phenotype in cancerNat Rev Cancer4988993200410.1038/nrc150715573120KondoYIssaJPJEpigenetic changes in colorectal cancerCancer Metastasis Rev232939200410.1023/A:102580691178215000147IacopettaBGrieuFLiWRuszkiewiczACarusoMMooreJKawakamiKAPC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancerInt J Cancer11922722278200610.1002/ijc.2223716981189DerksSPostmaCMoerkerkPvan den BoschSMCarvalhoBHermsenMAGiarettiWHermanJGWeijenbergMPde BruïneAPPromoter methylation precedes chromosomal alterations in colorectal cancer developmentCell Oncol282472572006171671784618222ToyotaMAhujaNOhe-ToyotaMHermanJGBaylinSBIssaJPJCpG island methylator phenotype in colorectal cancerProc Natl Acad Sci USA9686818686199910.1073/pnas.96.15.868110411935KnudsenESWangJYTargeting the RB-pathway in cancer therapyClin Cancer Res1610941099201010.1158/1078-0432.CCR-09-0787201451692822892MunroSCarrSMLa ThangueNBDiversity within the pRb pathway: Is there a code of conduct?Oncogene3143434352201210.1038/onc.2011.60322249267EngelmannDPützerBMThe dark side of E2F1: In transit beyond apoptosisCancer Res72571575201210.1158/0008-5472.CAN-11-257522298593WitkiewiczAKKnudsenKEDickerAPKnudsenESThe meaning of p16ink4a expression in tumors: Functional significance, clinical associations and future developmentsCell Cycle1024972503201110.4161/cc.10.15.16776217758183685613KnudsenKEDiehlJAHaimanCAKnudsenESCyclin D1: Polymorphism, aberrant splicing and cancer riskOncogene2516201628200610.1038/sj.onc.120937116550162CacaKFeisthammelJKleeKTannapfelAWitzigmannHWittekindCMössnerJBerrFInactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell linesInt J Cancer97481488200210.1002/ijc.163911802210ToyookaSMitsudomiTSohJAokageKYamaneMOtoTKiuraKMiyoshiSMolecular oncology of lung cancerGen Thorac Cardiovasc Surg59527537201110.1007/s11748-010-0743-321850578HongSMParkJYHrubanRHGogginsMMolecular signatures of pancreatic cancerArch Pathol Lab Med1357167272011216312643107523MoriTMiuraKAokiTNishihiraTMoriSNakamuraYFrequent somatic mutation of the MTS1/CDK4I (multiple tumor suppressor/cyclin-dependent kinase 4 inhibitor) gene in esophageal squamous cell carcinomaCancer Res543396339719948012957HeJMokhtariKSansonMMarieYKujasMHuguetSLeuraudPCapelleLDelattreJYPoirierJGlioblastomas with an oligodendroglial component: A pathological and molecular studyJ Neuropathol Exp Neurol60863871200110.1093/jnen/60.9.86311556543MaesawaCTamuraGNishizukaSOgasawaraSIshidaKTerashimaMSakataKSatoNSaitoKSatodateRInactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinomaCancer Res563875387819968752149OshimaMOkanoKMurakiSHabaRMaebaTSuzukiYYachidaSImmunohistochemically detected expression of 3 major genes (CDKN2A/p16TP53, and SMAD4/DPC4) strongly predicts survival in patients with resectable pancreatic cancerAnn Surg258336346201310.1097/SLA.0b013e3182827a6523470568UekiTHsingAWGaoYTWangBSShenMCChengJDengJFraumeniJFJrRashidAAlterations of p16 and prognosis in biliary tract cancers from a population-based study in ChinaClin Cancer Res1017171725200410.1158/1078-0432.CCR-1137-315014024TagaSOsakiTOhgamiAImotoHYoshimatsuTYoshinoIYanoKNakanishiRIchiyoshiYYasumotoKPrognostic value of the immunohistochemical detection of p16INK4 expression in nonsmall cell lung carcinomaCancer80389395199710.1002/(SICI)1097-0142(19970801)80:3<389::AID-CNCR6>3.0.CO;2-N9241072HermanJGMerloAMaoLLapidusRGIssaJPDavidsonNESidranskyDBaylinSBInactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancersCancer Res554525453019957553621GuanRJFuYHoltPRPardeeABAssociation of K-ras mutations with p16 methylation in human colon cancerGastroenterology11610631071199910.1016/S0016-5085(99)70009-010220498BurriNShawPBouzoureneHSordatISordatBGilletMSchorderetDBosmanFTChaubertPMethylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancerLab Invest81217229200110.1038/labinvest.378023011232644YiJWangZWCangHChenYYZhaoRYuBMTangXMp16 gene methylation in colorectal cancers associated with Duke's stagingWorld J Gastroenterol7722725200110.3748/wjg.7.5.722118198634695583EstellerMGonzálezSRisquesRAMarcuelloEManguesRGermàJRHermanJGCapellàGPeinadoMAK-rasp16 aberrations confer poor prognosis in human colorectal cancerJ Clin Oncol19299304200110.1200/JCO.2001.19.2.29911208819KimBNYamamotoHIkedaKDamdinsurenBSugitaYNganCYFujieYOgawaMHataTIkedaMMethylation and expression of p16INK4 tumor suppressor gene in primary colorectal cancer tissuesInt J Oncol2612171226200515809712ShimaKNoshoKBabaYCantorMMeyerhardtJAGiovannucciELFuchsCSOginoSPrognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature reviewInt J Cancer12810801094201110.1002/ijc.25432204739202958235MaedaKKawakamiKIshidaYIshiguroKOmuraKWatanabeGHypermethylation of the CDKN2A gene in colorectal cancer is associated with shorter survivalOncol Rep10935938200312792748XingXCaiWShiHWangYLiMJiaoJChenMThe prognostic value of CDKN2A hypermethylation in colorectal cancer: A meta-analysisBr J Cancer10825422548201310.1038/bjc.2013.251237032483694241SobinLHGospodarowiczMKWittekindCInternational Union Against Cancer (UICC) TNM Classification of Malignant Tumors7thWiley-BlackwellOxford2009HermanJGGraffJRMyöhänenSNelkinBDBaylinSBMethylation-specific PCR: A novel PCR assay for methylation status of CpG islandsProc Natl Acad Sci USA9398219826199610.1073/pnas.93.18.98218790415EadsCADanenbergKDKawakamiKSaltzLBBlakeCShibataDLairdPWMethyLight: A high-throughput assay to measure DNA methylationNucleic Acids Res28E32200110.1093/nar/28.8.e32EadsCALordRVWickramasingheKLongTIKurumboorSKBernsteinLPetersJHDeMeesterSRDeMeesterTRSkinnerKAEpigenetic patterns in the progression of esophageal adenocarcinomaCancer Res6134103418200111309301OginoSKawasakiTBrahmandamMCantorMKirknerGJSpiegelmanDMakrigiorgosGMWeisenbergerDJLairdPWLodaMPrecision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysisJ Mol Diagn8209217200610.2353/jmoldx.2006.050135166452071867588OginoSCantorMKawasakiTBrahmandamMKirknerGJWeisenbergerDJCampanMLairdPWLodaMFuchsCSCpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studiesGut5510001006200610.1136/gut.2005.082933164073761856352MitomiHFukuiNTanakaNKanazawaHSaitoTMatsuokaTYaoTAberrant p16INK4a methylation is a frequent event in colorectal cancers: Prognostic value and relation to mRNA expression and immunoreactivityJ Cancer Res Clin Oncol136323331201010.1007/s00432-009-0688-z19779933LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT methodMethods25402408200110.1006/meth.2001.126211846609KandaYInvestigation of the freely available easy-to-use software ‘EZR’ for medical statisticsBone Marrow Transplant48452458201310.1038/bmt.2012.24423208313LiJPoiMJTsaiMDThe regulatory mechanisms of tumor suppressor P16INK4A and relevance to cancerBiochemistry5055665582201110.1021/bi200642e216190503127263Veganzones-de-CastroSRafael-FernándezSVidaurreta-LázaroMde-la-OrdenVMediero-ValerosBFernándezCMaestro-de las CasasMLp16 gene methylation in colorectal cancer patients with long-term follow-upRev Esp Enferm Dig104111117201210.4321/S1130-0108201200030000222449151TadaTWatanabeTKazamaSKanazawaTHataKKomuroYNagawaHReduced p16 expression correlates with lymphatic invasion in colorectal cancersHepatogastroenterology50175617602002RayessHWangMBSrivatsanESCellular senescence and tumor suppressor gene p16Int J Cancer13017151725201210.1002/ijc.2731622025288SerranoMGómez-LahozEDePinhoRABeachDBar-SagiDInhibition of ras-induced proliferation and cellular transformation by p16INK4Science267249252199510.1126/science.78096317809631DaiCYFurthEEMickRKohJTakayamaTNiitsuYEndersGHp16 INK4a expression begins early in human colon neoplasia and correlates inversely with markers of cell proliferationGastroenterology119929942200010.1053/gast.2000.1795211040180PalmqvistRRutegårdJNBozokyBLandbergGStenlingRHuman colorectal cancers with an intact p16/cyclin D1/pRb pathway have up-regulated p16 expression and decreased proliferation in small invasive tumor clustersAm J Pathol15719471953200010.1016/S0002-9440(10)64833-X111065673277300ZhaoPHuYCTalbotICExpressing patterns of p16 and CDK4 correlated to prognosis in colorectal carcinomaWorld J Gastroenterol922022206200310.3748/wjg.v9.i10.2202145623784656463VogelsteinBKinzlerKWCancer genes and the pathways they controlNat Med10789799200410.1038/nm108715286780DublinEAPatelNKGillettCESmithPPetersGBarnesDMRetinoblastoma and p16 proteins in mammary carcinoma: Their relationship to cyclin D1 and histopathological parametersInt J Cancer797175199810.1002/(SICI)1097-0215(19980220)79:1<71::AID-IJC14>3.0.CO;2-K9495362HuiRMacmillanRDKennyFSMusgroveEABlameyRWNicholsonRIRobertsonJFSutherlandRLINK4a gene expression and methylation in primary breast cancer: Overexpression of p16INK4a messenger RNA is a marker of poor prognosisClin Cancer Res627772787200010914724QuinnDIHenshallSMSutherlandRLMolecular markers of prostate cancer outcomeEur J Cancer41858887200510.1016/j.ejca.2004.12.03515808955ParryDBatesSMannDJPetersGLack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene productEMBO J14503511199510.1002/j.1460-2075.1995.tb07026.x7859739398108KnudsenESKnudsenKETailoring to RB: Tumour suppressor status and therapeutic responseNat Rev Cancer8714724200810.1038/nrc2401191430562914856CraigCKimMOhriEWerstoRKatayoseDLiZChoiYHMudaharBSrivastavaSSethPEffects of adenovirus-mediated p16INK4A expression on cell cycle arrest are determined by endogenous p16 and Rb status in human cancer cellsOncogene16265272199810.1038/sj.onc.12014939464545KhleifSNDeGregoriJYeeCLOttersonGAKayeFJNevinsJRHowleyPMInhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activityProc Natl Acad Sci USA9343504354199610.1073/pnas.93.9.43508633069YaoJPollockRELangATanMPistersPWGoodrichDEl-NaggarAYuDInfrequent mutation of the p16/MTS1 gene and overexpression of cyclin-dependent kinase 4 in human primary soft-tissue sarcomaClin Cancer Res41065107019989563903
Distribution of p16 methylation, p16 mRNA and p16/PCNA levels in the primary tumors of patients with colorectal cancer. (A) Distribution of p16 methylation levels expressed as PMR. The median level for p16 PMR was 0.34. (B) Distribution of p16 mRNA expression levels. The median level for p16 mRNA expression was 1.98. (C) Distribution of p16/PCNA levels. The median level for p16/PCNA was 11.46. PCNA, proliferating cell nuclear antigen; PMR, percentage of methylated reference.
Tumor p16 and p16/PCNA mRNA expression levels in patients with colorectal cancer with high or low p16 methylation levels. Tumor p16 mRNA expression levels with a PMR cut-off value of (A) 4 and (B) 10. p16/PCNA expression levels with a PMR cut-off value of (C) 4 and (D) 10. Statistical differences between the groups were calculated by the Mann-Whitney U test. PCNA, proliferating cell nuclear antigen; PMR, percentage of methylated reference.
Kaplan-Meier analysis of the overall survival of patients with colorectal cancer stratified, according to their tumor p16 methylation level (low, PMR <4; high, PMR ≥4). There is no significant difference in outcome between the two patient groups (P=0.94). PMR, percentage of methylated reference.
Comparison of the overall survival of patients with colorectal cancer according to their level of p16 mRNA and p16/PCNA mRNA. Kaplan-Meier analysis compares the overall survival of (A) all patients according to their level of p16 mRNA; (B) the patients with low p16 methylation (PMR <4) according to their level of p16 mRNA; (C) all patients according to their level of p16/PCNA mRNA; and (D) the patients with low p16 methylation (PMR <4) according to their level of p16/PCNA mRNA. Statistical differences between the groups were calculated using the log-rank test. PMR, percentage of methylated reference; PCNA, proliferating cell nuclear antigen.
Influence of T stage, tumor stage and adjuvant chemotherapy on the overall survival of patients with colorectal cancer. (A) T stage, (B) tumor stage and (C) adjuvant chemotherapy on the overall survival of patients with colorectal cancer was analyzed using the Kaplan-Meier method.
Association between p16 methylation status or p16 mRNA expression and clinicopathological features in colorectal cancer.
Clincopathological features
n
p16 methylation
P-value
p16 mRNA
P-value
Sex
Male
57
0.455 (0–1.462)
0.383
2.020 (0.960–5.250)
0.584
Female
44
0.060 (0–2.105)
1.950 (0.85–3.548)
Age, years
≥65
56
0.271 (0–1.955)
0.900
2.040 (1.023–4.558)
0.494
<65
45
0.398 (0–1.220)
1.860 (0.700–4.460)
Tumor site
Proximal
29
0.455 (0–1.917)
0.259
2.220 (0.780–3.630)
0.588
Distal
48
0.127 (0–0.863)
1.825 (1.033–4.520)
Not known
24
1.068 (0.055–4.814)
2.585 (0.925–9.550)
Tumor histology
Well
34
0.161 (0–1.117)
0.018
2.600 (1.240–4.360)
0.066
Moderately
34
0.082 (0–0.575)
1.775 (0.857–4.380)
Poorly
4
0.162 (0.551–12.930)
0.335 (0.270–0.760)
Mucinous
4
38.990 (15.750–80.514)
1.940 (1.137–2.557)
Not known
25
1.028 (0.006–4.002)
3.070 (0.960–9.050)
T stage
T2
3
0.000 (0–24.523)
0.438
1.710 (1.025–3.085)
0.929
T3
56
0.150 (0–0.911)
2.085 (0.932–4.187)
T4
18
0.438 (0–1.008)
1.750 (1.045–4.137)
Not known
24
1.068 (0.055–4.814)
2.585 (0.925–9.550)
Stage
2
36
0.012 (0–0.658)
0.021
1.970 (0.933–4.968)
0.435
3
41
0.455 (0–3.154)
1.860 (0.970–3.380)
4
0
Not known
24
1.068 (0.055–4.814)
2.585 (0.925–9.550)
Adjuvant chemotherapy
Yes
54
0.494 (0–2.585)
0.169
1.915 (0.850–4.623)
0.833
No
44
0.192 (0–1.000)
1.970 (0.998–4.473)
Not known
3
0 (0–0.804)
4.330 (2.845–4.640)
p16 methylation status and p16 mRNA expression are presented as the median value (25–75th percentile). Histological type of the primary tumor was classified into well-, moderately-, poorly- differentiated adenocarcinoma and mucinous adenocarcinoma according to the Union for International Cancer Control classification.
Association between p16 methylation status or p16/PCNA expression and clinicopathological features in colorectal cancer.
p16 methylation
p16/PCNA mRNA
Features
n
High, n=18
Low, n=83
P-value
High, n=50
Low, n=51
P-value
Sex
Male
57
9
48
0.605
29
28
0.842
Female
44
9
35
21
23
Age, years
≥65
56
11
45
0.794
29
27
0.690
<65
45
7
38
21
24
Site
Proximal
29
7
22
0.090
12
17
0.814
Distal
48
4
44
22
26
Not known
24
7
17
16
8
Tumor histology
Well
34
3
31
0.017
18
16
0.210
Moderately
34
4
30
13
21
Poorly
4
1
3
0
4
Mucinous
4
3
1
2
2
Not known
25
7
18
17
8
T stage
T2
3
1
2
0.353
0
3
0.344
T3
56
7
49
25
31
T4
18
3
15
9
9
Not known
24
7
17
16
8
Stage
2
36
1
35
0.008
16
20
1.000
3
41
10
31
18
23
4
0
0
0
0
0
Not known
24
7
17
16
8
Adjuvant chemotherapy
Yes
54
11
43
0.611
29
25
0.543
No
44
7
37
20
24
Not known
3
0
3
1
2
Histological type of the primary tumor was classified into well-, moderately-, poorly- differentiated adenocarcinoma and mucinous adenocarcinoma, according to the Union for International Cancer Control classification. PCNA, proliferating cell nuclear antigen.
Association between p16 mRNA expression and clinicopathological features in colorectal cancer.
p16 mRNA
Features
n
High, n=51
Low, n=50
P-value
Sex
Male
57
29
28
1.000
Female
44
22
22
Age, years
0.842
≥65
56
29
27
<65
45
22
23
Site
Proximal
29
16
13
0.485
Distal
48
22
26
Not known
24
13
11
Tumor histology
Well
34
19
15
0.668
Moderately
34
15
19
Poorly
4
1
3
Mucinous
4
2
2
Not known
25
14
11
T stage
T2
3
1
2
0.507
T3
56
30
26
T4
18
7
11
Not known
24
13
11
Stage
2
36
18
18
1.000
3
41
20
21
4
0
0
0
Not known
24
13
11
Adjuvant chemotherapy
Yes
54
27
27
1.000
No
44
22
22
Not known
3
2
1
Histological type of the primary tumor was classified into well-, moderately-, poorly- differentiated adenocarcinoma and mucinous adenocarcinoma, according to the Union for International Cancer Control classification.
Univariate and multivariate analysis for the prognostic significance of clinicopathological factors and p16/PCNA mRNA.