Differential Roles of P16 and P14 Genes in Prognosis of Oral

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Differential Roles of p16INK4A and p14ARF Genes in Prognosis of Oral Carcinoma

R. Sailasree,1 A. Abhilash,1 K.M. Sathyan,1 K.R. Nalinakumari,2 Shaji Thomas,3 and S. Kannan1 1Laboratory of Cell Cycle Regulation and Molecular Oncology, Division of Cancer Research, 2Division of Dental Surgery, and 3Division of Surgical Oncology, Regional Cancer Center, Thiruvananthapuram, Kerala, India

Abstract

INK4A Background: Oral cancer patients are found to have observed in 30% of the cases. p16 deletion was poor clinical outcome and high disease recurrence associated with aggressive tumors, as evidenced by the rate, in spite of an aggressive treatment regimen. The nodal involvement of the disease. Low or absence of inactivation of INK4A/ARF loci is reported to be second p16INK4A protein adversely affected the initial treat- INK4A to p53 inactivation in human cancers. The purpose of ment response. Promoter methylation of p16 was this study was to assess the prognostic significance of associated with increased disease recurrence and acts the molecular aberrations in the INK4A locus for as an independent predictor for worse prognosis. ARF effective identification of aggressive oral carcinoma Surprisingly, p14 methylation associated with cases needing alternate therapy. lower recurrence rate in oral cancer patients with a Materials and Methods: The study composed of 116 good clinical outcome. Overall survival of these patients freshly diagnosed with oral carcinoma. The patients was associated with tumor size, nodal disease, INK4A genetic and epigenetic status of the p16 and and p16INK4A protein expression pattern. Our results ARF INK4A ARF p14 genes was evaluated. The relation between indicate that p16 and p14 alterations constitute these genic alterations and different treatment end a major molecular abnormality in oral cancer cases. points, such as residual disease (initial response), Conclusion: The molecular profile of INK4A/ARF locus, disease recurrence, and overall survival, along with both at DNA and protein level, could be used as a the standard clinical markers, were analyzed. prognostic biomarker for assessing the aggressiveness INK4A Results: 62% of the study cases had p16 gene of disease in oral carcinoma patients. The study further abnormalities, with deletion accounting for 33% and shows the opposing clinical effect of these two genes, ARF methylation for 29%. Alterations in p14 gene transcribed from the same locus, in oral cancer patients. either by deletion (12%) and/or methylation (18%) were (Cancer Epidemiol Biomarkers Prev 2008;17(2):414–20)

Introduction

Head and neck squamous cell carcinoma represents the metastasis system supplemented with conventional sixth most common cancer worldwide and accounts for histopathologic tumor grading, which has proved to be 500,000 new cases annually. In some European and unsatisfactory prognostic indicators in the present Southeast Asian countries, including India, oral cancer scenario. Determination of the biological behavior and prevails to be the leading formof cancer (1, 2). In oral identification of prognostic biomarkers is important for carcinoma cases, poor survival outcome with a proba- the early detection of relapse, as well as for stratification bility of survival at 5 years being <50% is seen, in spite of of patients before enrollment onto their treatment adopting the most aggressive multimodal treatment regimen. INK4A strategies (3). High recurrence rate leading to treatment The INK4A locus, harboring the p16 gene, is a INK4A failure seems to be a major drawback in the patient major aberration hotspot in oral carcinoma. The p16 treatment protocol. The current cornerstone of thera- gene functions as negative regulator of the cell cycle peutic decision-making is based on the tumor-node- progression through its inhibition of cdk4/6 and subse- quent blockage of the cyclin-dependent phosphorylation of the Rb (4). Genetic alterations of p16INK4A lead to loss Received 3/28/07; revised 11/13/07; accepted 11/20/07. of control of the restriction point in the G1 phase of the Grant support: Department of Science and Technology, Government of India grant cell cycle and favor cellular transformation (5). In our SP/SO/B64/2001. INK4A The costs of publication of this article were defrayed in part by the payment of page earlier study, it is found that p16 expression loss charges. This article must therefore be hereby marked advertisement in accordance defines a subgroup of oral cancer patients with worse with 18 U.S.C. Section 1734 solely to indicate this fact. clinical outcome (6). The present study aimed at Note: R. Sailasree and A. Abhilash contributed equally to this study. deciphering the molecular alterations involved in Abhilash is a recipient of Senior Research Fellowship of the Council of Scientific and INK4A Industrial Research, Government of India. p16 down-regulation and to assess the prognostic INK4A Requests for reprints: S. Kannan, Laboratory of Cell Cycle Regulation and implications of p16 gene alterations in oral carcino- Molecular Oncology, Division of Cancer Research, Regional Cancer Center, ma. As instability of this locus is reporxted to be high in Thiruvananthapuram695011, India. Phone: 91-471-2522338; Fax: 91-471-2447454. carcinoma, we analyzed p16INK4A and its alternate E-mail: [email protected] or [email protected] ARF ARF Copyright D 2008 American Association for Cancer Research. spliced product—p14 at molecular level. The p14 doi:10.1158/1055-9965.EPI-07-0284 tumor suppressor gene has a unique first exon that

Cancer Epidemiol Biomarkers Prev 2008;17(2). February 2008

INK4A splices into exons 2 and 3 of p16 gene in a different 1 unit Taq polymerase, 2.5% DMSO, and 80 to 100 ng of reading frame, thus translating a distinct protein that genomic DNA. PCR was done for 30 to 35 cycles having bears no amino acid homology to the p16INK4A gene annealing temperature of 58 to 60jC for 1 min. The PCR product. p14ARF acts by binding to mdm2, a negative products were separated on agarose gel, and ethidium regulator of p53, causing stabilization of p53 and leading bromide–stained fragments were recorded by the gel to cell cycle arrest (7). INK4A ARF Major inactivation of the p16 /p14 genes results from promoter methylation, homozygous deletion, and Table 1. Characteristics of oral carcinoma patients intragenic mutation. These genetic and epigenetic alterations have been detected frequently in a variety of Factors Frequency (%) human cancers, including head and neck cancer (8, 9). Sex The alteration affecting the INK4A/ARF locus can Male 82 (70.7) potentially disrupt the two main tumor suppression Female 34 (29.3) INK4A ARF Age, mean F SD (range) pathways, p16 -Rb and p14 -p53. In this study, F we did a comprehensive genetic and epigenetic analysis Male 58 9 (37-80) Female 61 F 10 (36-85) of both the genes to identify whether these genes can be Oral habits used as a prognostic biomarker in oral carcinoma. The ARF INK4A None 10 (8.6) results indicate p14 and p16 genes to have strong Chewing alone 34 (29.3) clinical implications but with differential roles in oral Smoking alone 3 (2.6) carcinoma. Alcohol alone 1 (0.9) Chewing with smoking 18 (15.5) Chewing with alcohol 9 (7.8) Smoking with alcohol 9 (7.8) Materials and Methods Chewing + smoking + alcohol 32 (27.6) T status Patient Characteristics and Tissue Sample. The 1 8 (7.0) present study population of 116 oral cancer patients 2 45 (39.1) was selected fromour original cohort of 348 patients 3 19 (16.5) previously described (6). Availability of tumor tissue for 4 43 (37.4) NA 1 DNA isolation was the criteria for selecting the present N status cohort fromour original one. Informationon patient 0 64 (56.1) characteristics, both personal and clinical, is given in 1 40 (35.1) Table 1. Fromeach patient, an incision biopsy was 2 8 (7.0) collected fromthe lesion and divided into two bits. One 3 2 (1.8) bit was immediately snap frozen in liquid nitrogen and NA 2 j Composite stage stored at 80 C for molecular work-up, and the other bit I 6 (5.2) was used for histopathologic reporting and immunohis- II 28 (24.3) tochemical analyses. The histopathologic grading and III 36 (31.3) staging of the lesion were done as per the WHO criteria IV 45 (39.1) (10). The Institutional Review Board approved the NA 1 Histopathology experimental design, specimen collection procedure, WDSCC 35 (30.0) and scope of this study. Mean age of the patients at MDSCC 73 (62.9) diagnosis was 59 years (range, 36-85 years). Genderwise, PDSCC 6 (5.2) 82 cases (71%) were male and 34 (29%) were female. Verrucous carcinoma 2 (1.7) More than 60% of the patients were presented with Treatment extensive lesion at the time of diagnosis. Radiation alone Radiotherapy 61 (52.6) Surgery 1 (0.9) or in combination with surgery was the main mode of Chemotherapy 2 (1.7) treatment given. Clinical follow-up for 50 months was Radiotherapy + surgery 23 (19.8) conducted with a median follow-up of 23 months. Chemotherapy + radiotherapy 11 (9.5) Chemotherapy + surgery 1 (0.9) DNA Extraction. The genomic DNA from tumor and Chemotherapy + radiotherapy + surgery 14 (12.1) normal samples were extracted by standard phenol- None 3 (2.6) chloroformmethod.Briefly, tissues were pulverized and Residual disease treated with proteinase K followed by phenol-chloroform No 80 (71.4) extraction. Ethanol precipitation was carried out, and Yes 32 (28.6) NA 4 DNA was dissolved in Tris-EDTA buffer (pH 8.0). Disease recurrence Homozygous Deletion Analysis. To assess the homo- No 49 (62.8) Yes 29 (37.2) zygous deletion status of these two genes, competitive ARF NA 2 multiplex PCR was carried out. Each exon of p14 INK4A Overall survival status and p16 was coamplified with a fragment of Alive 47 (43.9) either b-actin or GADPH as internal controls (11-13). Dead 60 (56.1) Placental and blood DNA fromhealthy volunteers were NA 9 used as normal controls. The PCR reaction mixture Abbreviations: NA, data not available; SCC, squamous cell carcinoma; consisted of 1 PCR buffer [10 mmol/L Tris (pH 8.3), WDSCC, well-differentiated squamous cell carcinoma; MDSCC, moder- 50 mmol/L KCl], 200 Amol/L deoxynucleotide triphos- ately differentiated squamous cell carcinoma; PDSCC, poorly differenti- A phate, 1.5 mmol/L MgCl2, primers (0.15-0.3 mol/L), ated squamous cell carcinoma.

Cancer Epidemiol Biomarkers Prev 2008;17(2). February 2008

documentation system Geldoc 2000 (Bio-Rad Laborato- 47 treated patients (43.9%) were alive up to last follow-up ries). The ratio of the intensity of the target gene versus and 60 patients (56.1%) died during the follow-up period. control was measured and calculated using Quantity One Thus, three end points were considered for the analysis, 1-D Image Analysis Software (Bio-Rad Laboratories). namely, residual disease, disease recurrence, and overall Samples presenting values of <20% of the control signal survival. The data of patients who were lost to follow-up were considered as having homozygous deletion of that during the study period were treated as unavailable data particular exon. Replicate PCR reactions were conducted when assessing the prognostic outcomes. to confirmthe deletion status. Data Analysis. The association between the categor- Methylation of INK4A/ARF Locus. The methylation ical variables was assessed using m2 test. Statistical INK4A ARF pattern of the CpG islands of p16 and p14 analyses between various variables and survival end promoters were determined by bisulfite modification of points were carried out using m2 analysis for residual the genomic DNA followed by methylation-specific PCR disease and log-rank test for disease recurrence and (14). Briefly, 2 Ag of DNA were denatured with NaOH overall survival. Variables found significant in the (3 mol/L) at 37jC for 15 min. After denaturation, univariate analysis were analyzed together in multivar- 5.6 mol/L sodium metabisulfite (pH 5) and 10 mmol/L iate analysis using logistic regression for residual disease hydroquinone were added and incubated at 55jC for and Cox’s proportional hazard regression analysis for 16 h. The samples were desalted using DNA Clean-Up disease recurrence and overall survival to find out their System (Promega). NaOH (0.3 mol/L) was used for alkali independent influence over the dependent variable and desulfonation at 37jC for 15 min. Neutralization of to calculate its risk, hazard ratio (HR). The Cox model sample was carried out using 3 mol/L sodium acetate was adjusted for potential confounding by age and followed by ethanol precipitation, and DNA was gender. A two-tailed P value of <0.05 was considered as resuspended in TE buffer. Bisulfite-converted blood statistically significant. DNA fromnormalhealthy individuals served as negative controls. Placental DNA treated with MSssI methyltransferase (New England Biolabs) was used as positive Results control for methylated alleles. To ensure the reproduc- ibility of the results, all samples were analyzed twice. Methylation Analysis. The methylation status of the ARF INK4A ARF The promoter methylation profiles of p14 and promoter region of p16 and p14 genes was INK4A p16 were determined using primers specifically investigated in 116 cases. Thirty-four samples (29.3%) INK4A designed for either methylated or unmethylated se- were positive for p16 methylation and 21 cases ARF quence (14, 15). PCR was carried out using 1 PCR (18.1%) showed p14 promoter methylation. Represen- buffer [10 mmol/L Tris (pH 8.3), 50 mmol/L KCl], tative gels are shown in Fig. 1. Concomitant methylation 1 to 1.5 mmol/L MgCl2, deoxynucleotide triphosphate of both the genes was present in seven samples (6%). A (200 mol/L each), methylated or unmethylated specific INK4A/ARF primers (0.5 Amol/L each), 1 unit of Taq DNA Deletion Status. Deletion analysis was carried out in all the four exons (exons 1a,1h, 2, and 3). polymerase, and f50 ng of bisulfite-modified DNA in INK4A A Deletion of at least one of the three exons of p16 was a total volume of 25 L. The PCR amplification process p14ARF was carried out for 35 to 40 cycles with annealing at 64jC observed in 38 cases (32.8%), and of ,only (methylation) or 60jC (unmethylation). PCR products 14 cases (12.1%) showed deletion (Fig. 2). The frequencies of exonwise deletions were 17.8% for exon 1a,9%for were resolved in agarose gel, and the bands were h visualized by ethidiumbromidestaining. exon 1 , 2.8% for exon 2, and 8.3% for exon 3. In 12 cases ARF (10.3%), simultaneous deletion of exons either common Mutation Analysis. The point mutations in p14 a h p16INK4A p14ARF INK4A to (2 and 3) or specific to (1 and 1 ) and and p16 genes were screened using PCR single- were noted. strand conformational polymorphism technique for exon ARF INK4A 1h (p14 ), exon 1a (p16 ), and exon 2 individually. Mutation Detection. Among the oral cancer samples The primers used for PCR single-strand conformational analyzed for mutation, only one case showed an altered migration pattern in single-strand conformational poly- polymorphism analysis were the same as used in a p16INK4A deletion analysis. Samples showing band shift were morphism analysis of exon-1 of gene. The further subjected to direct DNA sequencing to confirm sequencing of the single-strand conformational polymorphism–positive sample confirmed a C!T transition at and characterize the mutation. Sequencing was done in ! automated ABI Prism 377 DNA sequencer with the ABI codon 38, altering the codon triplet CCC (Pro) TCC (Ser). No mutation was observed in other exons Big Dye Terminator Cycle Sequencing kit (Applied p14ARF h Biosystems) according to manufacturer’s instructions. including the -specific exon 1 . Gene Inactivation. By combining all these molecular Treatment End Points. Out of 116 study subjects, alterations together, the genes were scored as either 113 patients were subjected to radical therapy. Out of inactivated or not. If any of the deletion, methylation, 113 treated cases, clinically, the disease completely or mutation was positive in the exons specific to a particular disappeared in 80 patients (71.4%). In 32 patients gene, then that gene was considered as inactivated in that (28.6%), residual disease persisted even after the comple- INK4A sample. Totally, the p16 gene inactivation was seen tion of the treatment. Out of the 80 patients that showed ARF in 62.2% of cases, whereas p14 inactivation was complete remission of the disease, 29 cases (37.2%) observed only in 30.1% of oral cancer cases. showed recurrence during follow-up. Whereas in 49 patients (62.8%), no recurrence was observed up to the Relation between Clinicopathologic Factors and Mo- end of the follow-up period. Irrespective of disease status, lecular Alterations. To compare the p16INK4A inactivation

Cancer Epidemiol Biomarkers Prev 2008;17(2). February 2008

recurrence (RR = 3.3). However, oral carcinoma patients had nine times lower risk in developing recurrence when ARF p14 was methylated (RR = 0.109). Kaplan-Meier curve for recurrence-free survival and the methylation status of these two genes are given in Fig. 3A and B. The seven ARF samples having concomitant methylation of p14 INK4A and p16 showed complete recurrence-free survival ARF INK4A (P = 0.011). The deletion status of p14 and p16 ARF did not relate to disease recurrence. When p14 gene inactivation was analyzed by taking together both Figure 1. Methylation detection of p16INK4A and p14ARF gene the methylation and deletion status, it was found to p14ARF have a significant effect on recurrence, and the chance for using methylation-specific PCR. A. promoter hyper- ARF methylation analysis in agarose gel. Tumor sample S11 is developing recurrence was eight times lower for p14 inactive patients (RR = 0.130; Fig. 3C). However, disease unmethylated, whereas S23 showed hypermethylation of INK4A p14ARF. B. p16INK4A methylation analysis showed S22 to recurrence was not significantly associated with p16 be p16INK4A methylated. U, unmethylated lanes; M, methyl- gene inactivation. The clinical variable-nodal involve- ated lanes; MSss, methyltransferase treated placenta; P, ment also showed a high significant relation with the normal placenta; S, tumor samples. disease recurrence. But the risk is comparatively very low (RR = 1.32) than for the molecular variables. The overall survival showed significant association at protein level with the genic level alterations and with clinical variables, such as T status (P = 0.049), treatment end points, immunohistochemistry data of the P INK4A INK4A N status ( = 0.001), and p16 protein expression p16 expression pattern of the present samples were (P = 0.016). No other molecular markers showed any taken fromour previous work (6) and were analyzed. relation with overall survival. The RR for clinical p16INK4A methylation status showed a direct correlation variables, such as tumor size (Fig. 3E) and presence of INK4A P with low p16 protein expression ( = 0.049), nodes (Fig. 3F) were 1.8 and 1.7, respectively. However, p16INK4A whereas the deletion status of did not show the absence of p16INK4A protein expression confers any correlation with protein expression. However, no comparatively a high risk (3.1) for poor overall survival p16INK4A association was seen between gene inactivation in oral cancer patients (Fig. 3D). and p16INK4A protein expression. With other clinico- Multivariate Analysis. pathologic factors, T status showed a significant relation Because only one factor, the INK4A INK4A with p16 methylation (P = 0.049) in m2 analysis, but p16 protein expression pattern, alone is associated no correlation or trend was observed between these two with residual disease, it independently favored residual factors. p16INK4A deletion was significantly associated with nodal status of the patients (P = 0.039). No other INK4A factors were significantly associated with p16 INK4A status. p16 gene inactivation did not relate to any of the clinicopathologic variables. No association was found ARF between p14 gene status and any of the clinicopathologic factors examined. Prognostic Significance Univariate Analysis. The clinicopathologic variables (T status, N status, stage, and histopathology) and the genic alterations (methylation, deletion, mutation, inac- ARF INK4A tivation of p14 /p16 genes, and p16INK4A protein expression) were analyzed with various treatment end points. Variables showing significant association with treatment end points are shown in Table 2. The presence of residual disease was associated only with the p16INK4A protein expression pattern (P = 0.037). No other clinical or experimental variables analyzed show any association with the presence of residual disease. Around 84% of the lesions with expression of p16INK4A protein showed good initial treatment response, and no residual disease was present in these cases. Log-rank analyses revealed that the disease recurrence is significantly related to N status (P = 0.001), methyl- INK4A ation status (P = 0.013) of p16 , and also with the INK4A methylation (P = 0.030) and gene inactivation status Figure 2. Deletion analysis. A. p16 exon 1a deletion in ARF INK4A ARF (P = 0.011) of p14 gene. Among the p16 lanes 3, 7, and 8. B. p14 exon 1h deletion in lanes 2, 4, and INK4A methylated cases, 54% of cases developed recurrence 5. C. Exon 2 deletion in third and fourth lane. D. p16 exon during the follow-up period. More than a thrice 3 deletion in lane 4. b-Actin and GADPH as reference genes. increased relative risk (RR) was observed for patients Lane M, marker; lane 1, normal sample; other individual lanes, INK4A with hypermethylation of p16 gene to develop oral carcinoma samples.

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Table 2. The variables showing significant relation with residual disease, disease recurrence, and overall survival by univariate analysis in oral carcinoma patients

Factors m2/log rank P RR 95% confidence interval

Low High Residual disease p16INK4A protein 4.36 0.037 0.351 0.128 0.961 Disease recurrence N status 17.56 0.001 1.321 1.002 2.553 p16INK4A ARF methylation 6.18 0.013 3.304 1.244 8.775 p14 methylation 4.71 0.030 0.109 0.013 0.893 ARF p14 inactivation 6.44 0.011 0.130 0.027 0.624 Overall survival N status 28.62 0.001 1.796 1.003 3.388 T status 7.88 0.049 1.666 1.116 2.487 p16INK4A protein 5.84 0.016 0.318 0.136 0.745

disease in oral carcinoma patients. Cox’s regression analysis are shown in Table 3. The Kaplan-Meier survival analysis showed that p16INK4A methylation (HR = 2.4) curves of the factors independently influencing the ARF and active p14 gene, i.e., unmethylated or undeleted disease recurrence and overall survival are shown in ARF p14 (HR = 11) were independently associated with the Fig. 3. Thus, when the prognostic outcomes were taken disease recurrence. With overall survival, all the factors into account, advanced lesions, presence of nodal INK4A that were significant in univariate analysis, such as lesion disease, low p16INK4A protein expression, and p16 size (HR = 1.325), nodal involvement (HR = 1.520), and methylation served as independent adverse prognostic ARF ARF loss of p16INK4A protein expression (HR = 1.98) were markers. Whereas p14 inactivation, including p14 significant in the multivariate model as independent methylation, was found to be a good prognostic factors. Variables found significant in multivariate predictor for oral carcinoma.

Figure 3. Kaplan-Meier survival curves of various factors that showed independent influence on recurrence free and overall survival of oral cancer patients. A. p16INK4A methylation and recurrence-free survival. B. p14ARF methylation and recurrence-free survival. C. p14ARF gene inactivation and recurrence-free survival. D. p16INK4A protein expression and overall survival. E. T status and overall survival. F. N status and overall survival.

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Table 3. The variables showing significant relation with residual disease, disease recurrence, and overall survival by multivariate analysis in oral carcinoma patients

Factors Coefficient* P HR* 95% confidence interval

Low High Residual disease p16INK4A protein 1.359 0.011 0.257 0.090 0.735 Disease recurrence INK4A p16 methylation 0.889 0.027 2.433 1.105 5.356 ARF p14 inactivation 2.399 0.019 0.091 0.012 0.675 Overall survival N status 0.419 0.043 1.520 1.013 2.281 T status 0.282 0.048 1.325 1.003 1.751 p16INK4A protein 0.772 0.016 0.462 0.247 0.865

*Adjusted for age and gender.

Discussion study also, we found a significant association between the nodal involvement and homozygous deletion of INK4A Oral squamous cell carcinoma is characterized by p16 gene. The up-regulation of the proliferative local tumor aggressiveness and high recurrence rate. genes regulated by the transcription factor like elk-1 after The pursuit of molecular markers capable of predicting p16INK4A inactivation could be a factor for the tumor INK4A the prognosis of oral carcinoma patients is compelling as aggressiveness (21). Although p16 deletion and clinically similar tumors may display different response gene inactivation did not associate with protein expres- to therapy. Loss of p16INK4A protein proved to be an sion status statistically, 57% and 63% cases showed independent factor in predicting poor survival in these absence of protein expression, respectively. The homo- patients. As p16INK4A was altered in a large number of p16INK4A INK4A zygous deletion/inactivation of did not have an oral carcinoma cases, the genetic alteration of p16 effect on the prognostic outcome of oral carcinoma cases. ARF along with its alternate spliced product (p14 ) were The overall survival of patients was influenced by nodal assessed for their prognostic influence. The present study disease, increased tumor size (status), and p16INK4A INK4A ARF provides evidence that the genes, p16 and p14 , protein down-regulation. Two times increased risk for transcribed fromthe INK4A loci significantly influence poor survival was observed for patients with loss of the disease prognosis in oral carcinoma patients, and p16INK4A protein expression. ARF these genes seemto function in diametricallyopposite The tumor suppressor gene p14 was inactivated in directions. 30% of oral cancer patients and was significantly INK4A Loss of p16 in the Rb pathway releases the associated with recurrence-free survival of patients. ARF inhibition on CCND1/cdk complex, which in turn Low frequency of p14 homozygous deletion (12%) phosphorylates Rb, leading to cell cycle progression was observed in the present study, consistent with INK4A ARF (16). The p16 gene inactivation (deletion and/or previous reports (22). Deletion status of p14 was not methylation) was observed in 62% of cases in our study. significantly linked to any variables analyzed. However, INK4A ARF In the present study, aberrant methylation of p16 p14 methylation was strongly associated with low was found in 29% of oral carcinoma samples, consistent disease recurrence in oral carcinoma with 9-fold de- with previous reports (17). The methylation profile of creased risk for recurrence when compared with unme- ARF p16INK4A significantly correlated with loss of protein thylated cases. When p14 gene inactivation was expression confirming the functional inactivation of considered, the risk increased to 11 times. This shows INK4A ARF p16INK4A. Many reports have associated p16 meth- that, the p14 deletion also influenced the recurrence ylation as an early event in oral carcinogenesis (18, 19). In rate in combination with its promoter methylation. This INK4A the present study, although methylation of p16 was observation is in contrast to previous reports linking ARF observed in 63% of the T1 lesions, it is difficult to p14 methylation with poor prognosis (23). A study categorize it as an early event as no trend is seen with late by Ogi et al. is the only available report that observed ARF stage tumors. Also the T1 cases were very few (7%) in the an association between p14 methylation and better sample population. survival in carcinoma cases but they were unable to arrive Our present study observed only a single case with at a conclusion for such a result (24). A recent report on point mutation, and it was localized in the exon-1a glioma supports the role of p14ARF as modulator for INK4A region of p16 gene. Such low frequency of somatic radioresistance (25). As radiation is the main mode of ARF mutation in p16INK4A gene has been reported in earlier treatment in our patients, the absence of p14 might studies (20). A total deletion frequency of 33% was be rendering the tumor cells more sensitive to radiation. INK4A ARF observed for p16 , with exon 1a (18%) being the The increased radiosensitivity in p14 inactive main target for deletion. Interestingly, two cases showed patients might enhance the prognosis of the patients. INK4A ARF complete loss of all the three exons of p16 , whereas Here, we hypothesize that p14 might be playing some four other patients exhibited either exon 2 or exon 3 role in treatment modulation leading to better prognostic deletion concomitant with exon 1 deletion. Nodal outcome. p14ARF nuclear overexpression showed an involvement leading to tumor aggressiveness is another increased growth fraction, aggressive clinical course, hallmark of p16INK4A inactive oral cancer cases (9). In this and shortened survival in B-cell lymphoma (26). This

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Cancer Epidemiol Biomarkers Prev 2008;17(2). February 2008

R. Sailasree, A. Abhilash, K.M. Sathyan, et al.

Cancer Epidemiol Biomarkers Prev 2008;17:414-420.

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