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10.1245/s10434-006-9280-9
Annals of Surgical Oncology 14:1772-1779 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Clinicopathological Roles of Alterations of Tumor Suppressor Gene p16 in Papillary Thyroid Carcinoma

Alfred King Yin Lam, MBBS, MD, PhD, FRCPA1, Chung Yau Lo, MBBS, MS, FRCS (Edin.), FACS2, Pauline Leung, BSc2, Brian Hung Hin Lang, MBBS, FRACS2, Wai Fun Chan, MBBS, FRCS (Edin.)2 and John M. Luk, DMedSc2

1 Discipline of Pathology, School of Medicine, Griffith University, PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia
2 Department of Surgery, Faculty of Medicine, University of Hong Kong, Pokfulam Road, Hong Kong, China

Correspondence: Address correspondence and reprint requests to: Alfred King Yin Lam, MBBS, MD, PhD, FRCPA; E-mail: a.lam{at}griffith.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Alterations of the p16 gene are common in human cancers, but their roles in thyroid cancers have not been clearly defined. The aim of the present study was to investigate the clinicopathological roles of the p16 gene in papillary thyroid carcinoma (PTC).

Methods: p16 gene alterations were investigated in 44 patients with PTC (9 men, 35 women) by immunohistochemistry, reverse transcriptase–polymerase chain reaction and methylation-specific polymerase chain reaction. The findings were correlated with their clinicopathological features.

Results: p16 protein expression, mRNA alterations, and promoter methylation were detected in 89% (n = 39), 77% (n = 33), and 41% (n = 18) of patients with PTC, respectively. There was no marked relationship between p16 protein expression, mRNA alteration, and promoter methylation. In follicular variant of PTC (FVPTC), there was a frequent lack of p16 protein expression and promoter methylation. PTCs showing p16 promoter methylation were often associated with a high AMES (age, metastasis to distant sites, extrathyroidal invasion, size) risk group and advanced pTNM (tumor–lymph node–metastasis) stages.

Conclusions: p16 gene alterations are common and correlate with histological features and biological aggressiveness in PTC, suggesting that they might play an important role in its pathogenesis.

Key Words: p16 • Expression • Methylation • Papillary thyroid carcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human INK 4a/ARF locus (a notation for "inhibitor of kinase 4/alternative reading frame") is located in short arm of chromosome 9 at 9p21.1 The gene locus encodes tumor suppressor genes p14 (ARF, alternative reading frame), p15 (INK, inhibitor of kinase, 4b) and p16 (INK4a). The p16 suppressor gene is one of the most commonly studied candidates in the pathogenesis of human neoplasia, and defects of p16 gene have been demonstrated in different human cancers, including carcinomas, sarcomas, melanoma, and lymphoid malignancies.2–5

p16 gene encodes p16 protein that competes with cyclin D for binding to CDK4. This inhibits the ability of the cyclin D–CDK4 complex to phosphorylate Rb (retinoblastoma), thus causing cell cycle arrest in the late G1 phase.6 In human cancers, p16 gene alterations can occur as gene deletion, point mutation, and methylation of gene promoter. Methylation is an epigenetic modification in which the gene activity is controlled by adding methyl groups (CH3) to specific cytosines of the DNA. Hypermethylation of the suppressor gene can silence the function of the gene.

PTC is the most common type of thyroid cancer.7 Alterations of RET and BRAF genes have been noted in a great proportion of patients with PTC.8,9 However, our understanding of the pathway toward carcinogenesis in PTC is incomplete. Because abnormalities in the control of cell cycle checkpoints are common in carcinogenesis, it is likely that p16 alterations may play a role in the pathogenesis of PTC. In the literature, the roles of p16 alterations in PTC have not been clearly defined.10 In this study, we investigated the different aspects of p16 alterations in PTC, and we studied the relationship between p16 alterations and clinicopathological parameters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Materials
The patients who were chosen for this study had undergone surgical resection for their primary thyroid carcinomas between January 1998 and December 2004 in Queen Mary Hospital. Informed consent was obtained from all patients to collect their thyroid tissues for research purposes. All the thyroid specimens were prospectively collected and dissected while fresh. One block from the tumor and one from the nonneoplastic tissue at a distance from the tumor in the contralateral lobe (acting as control tissue) were taken from each specimen. The blocks were snap-frozen in isopentane and cooled in liquid nitrogen at –80°C until required. Other additional blocks from the thyroid gland and regional lymph nodes were also processed in paraffin after fixation in 10% formalin for light microscopic analysis.

The pathology slides of the patients with PTC were classified by one of us (A.K.Y.L.) according to the criteria defined by the World Health Organization.11 The size and histological features of the tumors were noted during pathological examination. The patients were operated on by a single team of surgeons and were managed under a standard protocol. Patients were followed up and attended by both surgeons and oncologists at a combined thyroid cancer clinic. Tumor risk profile was assigned according to commonly adopted AMES (age, metastasis to distant sites, extrathyroidal invasion, size) risk group stratification and International Union Against Cancer pTNM (tumor–lymph node–metastasis) staging.12–14

p16 Protein by Immunohistochemistry
Paraffin blocks of the thyroid specimens were selected for immunohistochemical study. The antibody p16 INK4a used was a mouse monoclonal antibody (Biocare Medical, Walnut Creek, CA). Four-micron-thick sections were cut from the paraffin blocks, mounted on polylysine-coated slides, and stained with hematoxylin and eosin for light microscopy. Wax was removed from the paraffin sections with xylene, and the specimens were rehydrated in descending alcohols. The sections were immersed into 10 mM citrate buffer (pH 6), and antigen retrieval was performed with a microwave for 10 minutes at 95°C. The 10 mM citrate buffer (pH 6) was preheated in microwave for 3 minutes at 95°C. Then the sections were blocked for endogenous peroxide with hydrogen peroxide at room temperature for 30 minutes. After blocking with 10% normal goat serum (Invitrogen, Carlsbad, CA) in Tris-buffered saline at room temperature for 1 hour, the sections were incubated with monoclonal antiserum to p16 INK4a (1:100 dilution) or isotype-matched control antibody at 4°C overnight.

On the next day, the slides were defrosted to room temperature and washed with Tris-buffered saline. The slides were incubated with biotinylated secondary antibody (Dako Evision+System, Gloustrup, Denmark) for 45 minutes at 37°C, washed, and visualized with diaminobenzidine substrate. Tissues sections were counterstained in aqueous hematoxylin, mounted in crystal mount, and coverslipped in 50:50 xylene-Permount. In each experiment, paraffin blocks of thyroid carcinoma known to be strongly positive for p16 protein were used as positive controls. Examination of p16 staining was performed with a standard light microscope. Brown nuclear stain was regarded as positive. The positive cases of p16 were graded semiquantitatively into three categories, 1+, 2+, and 3+ (1+ indicates <30% of the tumor cells were positive; 2+, ≥30% to <70% of the tumor cells were positive; 3+, ≥ 70% or more of the tumor cells were positive).

p16 mRNA by Reverse Transcriptase–Polymerase Chain Reaction
To determine the relative amount of mRNA transcripts of p16 in tissue samples by reverse transcriptase–polymerase chain reaction (RT-PCR), total RNA was isolated with TriZol reagent (Invitrogen) and treated with DNAse I (Ambion, Austin, TX). cDNA was synthesized with 1 µg of DNAse-treated total RNA by oligo(dT)12–18 primer (Invitrogen) by ImProm-II reverse transcriptase (Promega, Madison, WI). Gene-specific primers for p16 and ß-actin housekeeping gene were designed (Table 1Go). PCR was performed in 1 x PCR buffer (25 µL) containing 200 µM dNTPs, 1.5 mM MgCl2, and .8 µM of each paired primers with the ABI9700 Thermocycler (Applied Biosystems, Foster City, CA). The PCR was carried out at 94°C for 10 minutes, followed by 35 cycles at 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds, and a final elongation step at 72°C for 10 minutes. The ß-actin housekeeping gene was included in all experiments as an internal control.


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  		TABLE 1. Summary of primer sequences, annealing temperatures, and polymerase chain reaction product sizes
 
PCR products were resolved by 2% agarose gel electrophoresis and visualized after staining with ethidium bromide under ultraviolet illumination. p16 mRNA was regarded as positive when there was band detected in the gel. The gel images were captured and analyzed by the Syngene CCDBIO acquisition system (Hitachi Genetics, Alameda, CA).

Methylation-Specific PCR of p16
Genomic DNA was extracted from the stored frozen tissue sample by proteinase K digestion and followed by phenol-chloroform extraction. In brief, tissue samples were incubated at 50°C overnight in lysis buffer containing 20 mM of Tris (pH 7.6), 5 mM of ethylenediaminetetraacetic acid (EDTA), .5% sodium dodecyl sulfate, and .2 mg/mL of proteinase K (Invitrogen). DNA was isolated by phenol-chloroform extraction and ethanol precipitation and dissolved in 1 x Tris-EDTA (TE) buffer (.01 M and .001 M Na2EDTA) at pH 8. Then 5 µg of DNA was treated with 3 M sodium hydroxide (NaOH). Bisulfate solution (pH 5.2), which converts unmethylated cytosine but not methylcytosine to uracil, was added and incubated at 37°C overnight. The bisulfite-treated DNA was purified over Qiaex II protocol (Qiagen, Hilden, Germany) and ethanol precipitated. The modified DNA was finally eluted into TE buffer. Postbisulfite treatment was carried out by treating the purified DNA (total 50 µL) with 5.56 µL of 3 M sodium hydroxide at 37°C for 15 minutes, followed by 27.78 µL of 9 M ammonium acetate (NH4OAc) at pH 7. The final concentration was 3 M. The postbisulfite-treated DNA was purified by Qiaex II protocol again and dissolved in 50 µL of TE buffer. CpGenom Universal Methylated DNA (Chemicon, Temecula, CA) was included throughout the experiment as a positive control.

PCR was performed in 1 x PCR buffer (25 µL) containing 200 µM dNTPs, 1.5 mM MgCl2, .8 µM methylated primer, .8 µM unmethylated primer (Table 1Go), and bisulfate-modified DNA with the ABI9700 Thermocycler (Applied Biosystems). PCR was carried out at 94°C for 10 minutes, followed by 35 cycles at 94°C for 30 seconds, annealing temperature for 30 seconds and 72°C for 30 seconds, and a final elongation step at 72°C for 10 minutes. The ß-actin housekeeping gene was included in all experiments as an internal control. PCR products were resolved by 2% agarose gel electrophoresis and visualized after staining with ethidium bromide under ultraviolet illumination. If a band was detected in the sample when methylated primer was used, the tumors were regarded as have methylated. The gel images were captured and analyzed by the Syngene CCDBIO acquisition system (Hitachi Genetics).

Because the product size of methylated and unmethylated results differed by only one base, direct sequencing of the methylated and unmethylated PCR products was used to confirm the identifies of the products. PCR products were cut from agarose gel and purified by the QiaQuick gel extraction kit (Qiagen). Their identities were confirmed by nucleotide sequencing from Big Dye Terminator v3.1 cycle sequencing kit by ABI Prism 3100 Genetic Analyzer (Applied Biosystems).

Statistical Analysis
All the data obtained in the study were computerized and analyzed by SPSS version 12.0 software (SPSS, Chicago, IL). Comparisons between groups were performed by Fisher’s exact test for categorical variables and Student’s t-test with Yates correction for continuous variables. P <.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinicopathological Data
Over the study period, 44 patients with PTC (9 men, 35 women) with a median age of 40 years (range, 12–83 years) were recruited onto the study. In addition, tissues from one female patient with poorly differentiated carcinoma and nine patients (two men, seven women) with nodular hyperplasia were used as controls. The mean diameter of the PTCs was 2.2 cm (range, 1–4.5 cm). The tumors were classified according to International Union Against Cancer pTNM to be stage 1 in 32% (n = 14), 2 in 11% (n = 5), 3 in 41% (n = 18), and 4 in 16% (n = 7). Sixty-four percent (n = 28) and 36% (n = 16) of patients were in the low and high AMES risk groups, respectively. The mean follow-up period was 43 months (range, 2–92 months). During follow-up, 2 patients died of PTC, and 10 patients were alive with recurrent diseases. The overall 5-year disease-specific survival rate was 88% in this group of patients with PTC.

Immunohistochemical Detection of p16
Overexpression of p16 protein was detected in 39 (89%) of 44 of patients with PTC (Table 2Go). The p16 staining was located at the nuclei and the cytoplasm of the tumor cells (Fig. 1Go). Staining was grade 1 in 52% (n = 23), grade 2 in 34% (n = 15), and grade 3 in 2% (n = 1) of the patients. Nonneoplastic thyroid tissues adjacent to the carcinomas were negative for p16 protein, and similarly, p16 protein was not found in nodular hyperplasia (n = 9) and poorly differentiated thyroid carcinoma (n = 1).


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  		TABLE 2. Clinicopathological parameters of p16 protein–positive and –negative papillary thyroid carcinoma
 

Figure 1
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  		FIG. 1. p16 protein detected by immunohistochemistry. (A) Nonneoplastic thyroid was negative for p16 protein. (B) Papillary thyroid carcinoma (PTC) showed positive immunostaining in nuclei and cytoplasm of PTC.

 
p16 overexpression was noted to be present in 38 (93%) of 41 conventional PTC but in only 1 (33%) of 3 with follicular variant of PTC (FVPTC) (P = .03, Fisher’s test). The mean diameter of PTCs having p16 protein expression was larger compared with those without p16 expression (P = .002, t-test). However, patients with p16 overexpression did not differ from those without with respect to age at presentation (P = .54), sex (P = .57), pTNM staging (P = .61), AMES risk group stratification (P = .50), or occurrence of recurrent disease (P = 1).

p16 mRNA
p16 mRNA was detected in 34 (77%) of 44 and 5 (71%) of 7 patients with PTC and nodular hyperplasia respectively (P = .22, Fisher’s test) (Fig. 2Go). On the other hand, p16 mRNA was not detected in the patient with poorly differentiated carcinoma of thyroid. The clinicopathological features of patients with p16 mRNA detected in the PTC did not differ from those patients with p16 mRNA–negative PTC (Table 3Go).


Figure 2
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  		FIG. 2. Reverse transcriptase–polymerase chain reaction of p16 revealing that p16 mRNA expression in benign thyroids and papillary thyroid carcinomas. Lane 1, 50-bp marker; lane 2, ß-actin from PTC; lane 3, ß-actin from benign thyroid; lane 4, benign thyroid; lane 5, case 1 PTC; lane 6, benign thyroid; lane 7, case 2 PTC; lane 8, benign thyroid; lane 9, case 3 PTC; lane 10, benign thyroid; lane 11, negative control.

 

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  		TABLE 3. Clinicopathological parameters of p16 mRNA–positive and –negative papillary thyroid carcinoma
 
p16 Promoter Methylation
p16 promoter methylation was noted in 18 (41%) of 44 patients with PTC (Table 4Go; Figs. 3Go and 4Go), but patients with nodular hyperplasia or poorly differentiated carcinoma did not show any evidence of p16 promoter methylation. Patients assigned to the high AMES risk group more often had PTC showing methylation of the p16 promoter compared with low-risk-group patients (P = .01, Fisher’s test). Also, patients with advanced-stage disease had p16 methylation in their PTC more frequently compared with those having less advanced disease. However, the difference did not reach statistical significance (P = . 054, Fisher’s test). p16 hypermethylated PTCs did not differ from unmethylated ones with respect to age at presentation (P = .066), sex (P = .72), histological subtypes (P = .26), size of cancer (P = .1), and occurrence of recurrent disease (P = .3).


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  		TABLE 4. Clinicopathological parameters of p16 methylation in papillary thyroid carcinoma
 

Figure 3
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  		FIG. 3. Methylation-specific polymerase chain reaction of p16 showing the methylation status in papillary thyroid carcinoma (PTC) and nonneoplastic thyroid tissue. M, methylated; U, unmethylated. Lane 1, 50-bp maker; lane 2, universal methylated DNA (U); lane 3, universal methylated DNA (M); lane 4, benign thyroid (U); lane 5, benign thyroid (M); lane 6, case 1 nonneoplastic thyroid (U); lane 7, case 1 nonneoplastic thyroid (M); lane 8, case 1 PTC (U); lane 9, case 1 PTC (M); lane 10, case 2 nonneoplastic thyroid (U); lane 11, case 2 nonneoplastic thyroid (M); lane 12, case 2 PTC (U); lane 13, case 2 PTC (M); lane 14, case 3 nonneoplastic thyroid (U); lane 15, case 3 nonneoplastic thyroid (M); lane 16, case 3 PTC (U); lane 17, case 3 PTC (M); lane 18, negative control.

 

Figure 4
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  		FIG. 4. DNA sequencing analysis on polymerase chain reaction product from unmethylated (U) primer and methylated (M) results from papillary thyroid carcinoma. (A) DNA sequence amplified from methylated primer set. (B) DNA sequence amplified from the unmethylated primer set. Arrows indicate sites of change of nucleotide sequence.

 
Relationship Between Different Methods of Detecting p16 Alterations
In 29 (66%) of 44 patients with PTC, both p16 mRNA and protein alterations were detected simultaneously. For the other 34% of patients (n = 15), the occurrence of p16 mRNA alteration did not correlate with protein expression detection. In addition, all 10 PTCs with negative p16 mRNA overexpression revealed p16 protein expression. In 18 PTCs with p16 methylation, 3 (17%) did not show p16 mRNA overexpression. On the other hand, 19 (73%) of 26 of p16 unmethylated PTCs showed p16 mRNA overexpression. The overall correlation between p16 methylation status and mRNA expression was 22 (50%) of 44. In addition, there was no marked correlation between p16 protein expression and methylation status. Only 1 (6%) of 18 PTC with p16 methylation lacked p16 protein expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Strong p16 overexpression has been reported in many neoplasias.15,16 To date, the major diagnostic application of p16 immunohistochemistry has been in the field of cervical pathology. Diffuse positivity with p16 expression in the cervix can be regarded as a surrogate marker of the presence of high-risk papillomavirus and intraepithelial neoplasia.17,18 But p16 staining in normal cervical squamous epithelium has been variably reported as either absent or only focally positive in occasional cases. In the literature, p16 immunohistochemistry in PTC has only been studied and reported by Boltze et al.19 In their study, weak or moderate p16 staining was found in 87% of tumor-free thyroid, whereas 8 (50%) of 16 PTC showed moderate immunoreaction. Ferenc et al.20 showed that p16 expression occurred in nodular hyperplasia, follicular adenomas, and follicular adenomas; p16 overexpression was noted in 0% of nodular hyperplasia, 19.3% of follicular adenomas, and 76.5% of follicular carcinomas.

Our current study on a larger number of PTCs showed that all the nontumorous thyroid tissues, including those from the contralateral lobe of the carcinoma and those from nodular goiter, were negative for p16 staining. This differs from previous findings. The difference in findings may be related to the difference in experimental conditions and sensitivity of the antibody used. Nevertheless, in all the series, overexpression of p16 was not observed in nodular goiter. Like the previous studies, we noted that overexpression of p16 was mainly noted in malignant thyroid lesion. p16 immunohistochemical staining was positive in 39 (89%) of 44 patients with PTC, with intensity of grade 2 or 3 in 35%. Also, the finding of this and other studies confirmed that p16 overexpression play a role in the malignant transformation because overexpression was noted in well-differentiated thyroid carcinomas, namely PTC and follicular carcinomas but not in nodular goiter. In this series, the poorly differentiated thyroid carcinoma did not exhibit p16 overexpression. The finding are in agreement with the notion that p16 overexpression is an early event in the carcinogenesis of thyroid cancers. Alternatively, poorly differentiated thyroid carcinoma may be derived from a p16-independent pathway. The lack of p16 overexpression in poorly differentiated thyroid carcinomas should be confirmed in a larger study.

The p16 staining was located in the nuclei and cytoplasm of the tumor cells. Because p16 is a nucleoprotein, the presence of staining in both the nuclei and cytoplasm supports the finding that p16 gene is overexpressed. The change in the subcellular location of the overexpressed nucleoprotein may account for the pathogenesis of PTC. Because of the frequent overexpression of p16 in PTC, it may be used as a marker for PTC in selected patients who are hard to diagnose.

p16 promoter hypermethylation in PTC was rarely reported, and the frequencies of p16 methylation in PTC was reported to be 3 (25%) of 12, 4 (33%) of 12, and 7 (42%) of 16 in three studies.19,21,22 In our study, by performing methylation study of p16 on a large number of PTCs, the frequency of p16 methylation detected in PTC was 18 (41%) of 44. In addition, p16 methylation was demonstrated to be correlated with tumor risk profile. Risk-group stratification as defined by AMES criteria is frequently used to identify high-risk patients with PTC for aggressive surgical and adjuvant treatments. p16 methylation was often more frequently observed in the high-risk PTC group according to AMES risk group stratification and according to presence of advanced tumor as classified by the pTNM staging system.

To our knowledge, this is the first study to show the relationship of p16 alterations in PTC based on p16 protein expression, mRNA expression, and methylation of promoter. There was no marked relationship between the different forms of p16 alterations as detected by these different methods. It is perhaps attributed to the effect of different pathways on the function of p16 that can contribute to the pathogenesis of PTC. In addition, although homozygous deletions of the gene locus 9p21 have been detected in thyroid carcinoma cell lines, lack of p16 alterations have also been reported in thyroid carcinomas.23–28 In our study, p16 mRNA or p16 protein was detected in 77% and 89% of PTC, respectively. Thus, it is unlikely that p16 gene deletion plays a major role in the pathogenesis of PTC because most of the PTC retained the p16 mRNA or protein. Furthermore, p16 mRNA could be detected in similar proportion of PTC tumor and nontumor tissue, and the detection of p16 mRNA did not have any relationship with clinicopathological features of PTC. It is apparent that the overexpression of p16 rather than the loss of its protein contributes to the pathogenetic mechanism of PTC.

FVPTC had more favorable clinicopathological features and a better tumor risk group profile than conventional PTC.29 However, the histopathologic diagnosis of FVPTC remains a challenge, and this variant has been overdiagnosed by some pathologists.30 In our published data, which relied on strict pathological criteria, FVPTC accounted for slightly less than 20% of all PTC.7 In addition, we have previously reported that FVPTC shows a different prevalence of cyclooxygenase 2 and RET overexpression when compared with conventional PTC.31,32 In this study, two of the five PTC negative for p16 overexpression were FVPTC, and the difference in p16 overexpression between conventional variant of PTC and FVPTC was large. Also, all three FVPTC did not have any p16 promoter methylation. Although the number of patients with FVPTC being studied was small, our results still support the notion that FVPTC has a different pathogenetic pathway than conventional PTC. Further study with larger number of FVPTC may help to confirm this.

In conclusion, p16 alterations as detected by methylation of p16 promoter and p16 overexpression are common in PTC. These findings imply that p16 alteration is important in the pathogenesis of PTC and that p16 can be a potential biomarker for selected patients with PTC. The lack of expression in certain histological variants and the association with tumor risk profile are of potential importance, and further studies are warranted.


   ACKNOWLEDGMENTS
 
Supported by CRCG Seeding Grant 2003–2004 of University Research Committee. The authors acknowledge the staff of Departments of Surgery and Pathology in Queen Mary Hospital in facilitating the project.

Received for publication June 7, 2006. Accepted for publication October 26, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Larsen CJ. Contribution of the dual coding capacity of the p16INK4a/MTS1/CDKN2 locus to human malignancies. Prog Cell Cycle Res 1997; 3:109–24.[Medline]
  2. Oue N, Mitani Y, Motoshita J, et al. Accumulation of DNA methylation is associated with tumour stage in gastric cancer. Cancer 2006; 106:1250–9.[CrossRef][Medline]
  3. Mihic-Probst D, Mnich CD, Oberholzer PA, et al. p16 expression in primary malignant melanoma is associated with prognosis and lymph node status. Int J Cancer 2006; 118:2262–8.[CrossRef][Medline]
  4. Sabah M, Cummins R, Leader M, Kay E. Loss of p16INK4A expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9 p16 in microdissected synovial sarcomas. Virchows Arch 2005; 447:842–8.[CrossRef][Medline]
  5. Tsirigotis P, Pappa V, Labropoulos S, et al. Mutational and methylation analysis of the cyclin-dependent kinase 4 inhibitor (p16INK4A) gene in chronic lymphocytic leukemia. Eur J Haematol 2006; 76:230–6.[CrossRef][Medline]
  6. Auerkari EI. Methylation of tumour suppressor genes p16 (INK4a), p16 (Kip1) and E-cadherin in carcinogenesis. Oral Oncol 2006; 42:5–13.[CrossRef][Medline]
  7. Lam AK, Lo CY, Lam KS. Papillary carcinoma of thyroid: a 30-yr clinicopathological review of the histological variants. Endocr Pathol 2005; 16:323–30.[CrossRef][Medline]
  8. Ciampi R, Nikiforov YE. Alterations of the BRAF gene in thyroid tumours. Endocr Pathol 2005; 16:163–72.[CrossRef][Medline]
  9. Lam KY, Lo CY, Leung PS. High prevalence of RET proto-oncogene activation (RET/PTC) in papillary thyroid carcinomas. Eur J Endocrinol 2002; 147:741–5.[Abstract]
  10. Jarzab B, Wloch J, Wiench M. Molecular changes in thyroid neoplasia. Folia Histochem Cytobiol 2001; 39(Suppl 2):26–7.
  11. LiVolsi VA, Albores-Saavedra J, Asa SL, et al. (2004) Papillary carcinoma. In: DeLellis RA, Llyod RV, Heitz PU, Eng C (eds). World Health Organization Classification of Tumours: Pathology and Genetics—Tumours of Endocrine Organs IARC, Lyon, pp 57–66.
  12. Cady B, Sedgwick MD, Meissner WA, Wool MS, Salzman FA, Werber J. Risk factor analysis in differentiated thyroid cancer. Cancer 1979; 43:810–20.[CrossRef][Medline]
  13. Jukkola A, Bloigu R, Ebeling T, Salmela P, Blanco G. Prognostic factors in differentiated thyroid carcinomas and their implications for current staging classifications. Endocr Relat Cancer 2004; 11:571–9.[Abstract/Free Full Text]
  14. Lo CY, Chan WF, Lam KY, Wan KY. Optimizing the treatment of AMES high-risk papillary thyroid carcinoma. World J Surg 2004; 28:1103–9.[CrossRef][Medline]
  15. Armes JE, Lourie R, de Silva M, et al. Abnormalities of the RB1 pathway in ovarian serous papillary carcinoma as determined by overexpression of the p16 (INK4A) protein. Int J Gynecol Pathol 2005; 24:363–8.[CrossRef][Medline]
  16. Cui X, Shirai Y, Wakai T, Yokoyama N, Hirano S, Hatakeyama K. Aberrant expression of pRb and p16 (INK4), alone or in combination, indicates poor outcome after resection in patients with colorectal carcinoma. Hum Pathol 2004; 35:1189–95.[CrossRef][Medline]
  17. O’Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol 2006; 13:8–15.[CrossRef][Medline]
  18. Dray M, Russell P, Dalrymple C, et al. p16 (INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: experience with squamous lesions in 189 consecutive cervical biopsies. Pathology 2005; 37:112–24.[Medline]
  19. Boltze C, Zack S, Quednow C, Bettge S, Roessner A, Schneider-Stock R. Hypermethylation of the CDKN2/ p16INK4A promotor in thyroid carcinogenesis. Pathol Res Pract 2003; 199:399–404.[CrossRef][Medline]
  20. Ferenc T, Lewinski A, Lange D, et al. Analysis of p161NK4A protein expression in follicular thyroid tumours. Pol J Pathol 2004; 55:143–8.[Medline]
  21. Schagdarsurengin U, Gimm O, Hoang-Vu C, Dralle H, Pfeifer GP, Dammann R. Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma. Cancer Res 2002; 62:3698–701.[Abstract/Free Full Text]
  22. Elisei R, Shiohara M, Koeffler HP, Fagin JA. Genetic and epigenetic alterations of the cyclin-dependent kinase inhibitors p16INK4b and p16INK4a in human thyroid carcinoma cell lines and primary thyroid carcinomas. Cancer 1998; 83:2185–93.[CrossRef][Medline]
  23. Schulte KM, Staudt S, Niederacher D, et al. Rare loss of heterozygosity of the MTS1 and MTS2 tumor suppressor genes in differentiated human thyroid cancer. Horm Metab Res 1998; 30:549–54.[Medline]
  24. Ivan M, Wynford-Thomas D, Jones CJ. Abnormalities of the p16INK4A gene in thyroid cancer cell lines. Eur J Cancer 1996; 32A:2369–70.[CrossRef]
  25. Calabro V, Strazzullo M, La Mantia G, et al. Status and expression of the p16INK4 gene in human thyroid tumors and thyroid-tumor cell lines. Int J Cancer 1996; 67:29–34.[CrossRef][Medline]
  26. Yane K, Konishi N, Kitahori Y, et al. Lack of p16/CDKN2 alterations in thyroid carcinomas. Cancer Lett 1996; 101:85–92.[CrossRef][Medline]
  27. Jones CJ, Shaw JJ, Wyllie FS, Gaillard N, Schlumberger M, Wynford-Thomas D. High frequency deletion of the tumour suppressor gene p16INK4a (MTS1) in human thyroid cancer cell lines. Mol Cell Endocrinol 1996; 116:115–9.[CrossRef][Medline]
  28. Yane K, Konishi N, Kitahori Y, Hiasa Y, et al. Lack of p16/ CDKN2 alterations in thyroid carcinomas. Cancer Lett 1996; 101:85–92.[CrossRef][Medline]
  29. Lang BH, Lo CY, Chan WF, Lam AK, Wan KY. Classical and follicular variant of papillary thyroid carcinoma: a comparative study on clinicopathologic features and long-term Outcome. World J Surg 2006; 30:752–8.[CrossRef][Medline]
  30. Lloyd RV, Erickson LA, Casey MB, et al. Observer variation in the diagnosis of follicular variant of papillary thyroid carcinoma. Am J Surg Pathol 2004; 28:1336–40.[Medline]
  31. Lo CY, Lam KY, Leung PP, Luk JM. High prevalence of cyclooxygenase 2 expression in papillary thyroid carcinoma. Eur J Endocrinol 2005; 152:545–50.[Abstract/Free Full Text]
  32. Lam AK, Montone KT, Nolan KA, Livolsi VA. Ret oncogene activation in papillary thyroid carcinoma: prevalence and implication on the histological parameters. Hum Pathol 1998; 29:565–8.[CrossRef][Medline]



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