• p53 polymorphism;
  • HPV;
  • oral cancer


  1. Top of page
  2. Abstract
  6. Acknowledgements

Human papillomavirus (HPV) infects the squamous epithelial cells of oral cavity and cervix leading to formation of warts that develops into the cancer. Human papillomavirus (HPV)-16 and 18 encode E6 oncoprotein, which binds to and induces degradation of the tumour suppressor protein p53. A common polymorphism of p53, encoding either proline (Pro) or arginine (Arg) at position 72, affects the susceptibility of p53 to E6 mediated degradation in vivo. Oral cancer is a pressing problem in India due to the widespread habit of chewing betel quid, which plays an important role in etiology of this disease. In the present study an attempt has been made to analyze the genetic predisposition of the Indian population to HPV infection and oral carcinogenesis. In our study a total of 110 cases of Oral Cancer highly addicted to betel quid and tobacco chewing are analyzed for HPV 16/18 infection and its association with polymorphism at p53 codon 72. Of these a total number of 37 patients (33.6%) have shown the presence of HPV, among which the presence of HPV-16, 18 and 16/18 coinfection is 22.7%, 14.5% and 10%, respectively. Our results also indicate that the p53 codon 72 genotype frequencies in Indian Oral Cancer patients are 0.55 (Arg) and 0.45 (Pro) as per Hardy-Weinberg equilibrium. In our study, striking reduction in Pro/Pro allele frequency has been found in HPV positive cases, indicating Arg/Arg genotype to be more susceptible to HPV infection and oral carcinogenesis. © 2001 Wiley-Liss, Inc.

Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide. The overall survival percentage has not changed in recent years, despite extensive research on the biological and molecular aspects of oral SCC. Among the more pressing problems in clinical management are the lack of early detection and the high incidence of local-regional recurrence, even with aggressive surgical therapy. OSCC incidence accounts for up to 40% of all malignancies in India and South East Asia.1 Moreover differences have been observed in the clinico-pathological and molecular pathological profile in the tobacco smoking and alcohol-associated oral cancers in the USA, UK, France, Japan, etc. and the chewing tobacco-associated oral cancers, particularly in the Indian sub-continent.2 In India, oral leukoplakias have been reported to show a significant tendency to malignant transformation from 0.13 to 6% and rising to 14% or higher in cases of dysplasia.3, 4 Epidemiological studies reveal the betel quid and tobacco chewing habit as an important risk factor in the etiology of oral cancer in India. Since carcinogenesis is a multi-step process, therefore, in addition to insult by tobacco-associated intra oral carcinogens, several additional factors, such as genetic susceptibility of individuals and external agents, such as dietary factors and viruses (human papillomavirus, HPV, and Epstein-Baar virus, EBV), may play a synergetic role in oral tumorigenesis.5–8

HPVs are a very heterogenous group of DNA viruses. Almost 90 HPV types have been described; 30 of these types are associated with anogenital cancers, forming either the high-risk types (HPV16 and HPV18) that are associated with anogenital invasive tumour and their precursor lesions or the low-risk types (HPV6 and HPV11) that rarely progress to malignancy. Analysis of viral genome has revealed that 2 early genes, E6 and E7, of high risk HPVs (16/18) are transforming genes that are responsible for maintenance of tumorigenic phenotype.6, 9, 10 Genetic polymorphism at the genes involved in tumorigenesis may determine individual susceptibility of cancer. The genotype distribution of p53 codon 72 polymorphism is significantly different among ethnic groups.11 Storey et al.12 recently reported the probable correlation between p53 polymorphism and HPV associated cervical tumorigenesis. They showed that population homozygous for Arg-72 (arginine) is about 7 times more susceptible to development of cervical cancer than heterozygotes. However, this has been subject of much debate. Makni et al.13 recently showed the substantial inter-laboratory variation in the proportion of Arg/Arg, Arg/Pro (proline) and Pro/Pro.

In spite of its great relevance, so far only 2 contradictory reports have been proposed on the status of HPV in oral cancer on Indian population. The work on South Indian population has indicated 42% HPV-16 infection and 47% of HPV-18 infection,7 whereas the results on Indian sub-population of Western region documented 15% HPV-16 infection without any HPV 18 involvement.8 Moreover, no data is available on the status of p53 Codon 72 polymorphism in the Indian population. Hence, an attempt has been made for the first time to investigate the association between the presence of HPV16/18 and polymorphism at Codon 72 of p53 in Oral SSC of Eastern Indian population, which is highly addicted to chewing tobacco and betel quid.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Patients and materials

A total of 110 patients of oral squamous cell carcinoma from the Eastern region of India addicted with tobacco chewing habit for more than 10 years were selected for our study. These patients underwent surgery at A.H. Regional Cancer Research and Treatment Centre, Cuttack, Orissa, during last 3 years. The samples were collected on obtaining informed consent from the patients. These included 78 tissue samples that were collected at the time of surgery, immediately snap frozen in liquid nitrogen and stored at −70°C, until DNA extraction, and the remaining 32 samples were available as formalin fixed paraffin embedded blocks. Biopsy samples (scraps) of volunteers from the normal population were used as non-cancer controls. The medical records of these patients were reviewed to obtain information regarding location of tumors, age, gender, chewing habit and stages of tumor. Staging of the tumors was done according to the TNM classification after brief histological studies.

DNA extraction

Frozen tissue.

DNA was isolated from tumour tissues according to the method of Blin and Stafford.14 Briefly tissues (approximately 2 mg) were pulverized into fine powder using a mortar and pestle that had been pre-cooled with liquid nitrogen. The powder was suspended in 200 μl of digestion buffer (50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% SDS) containing 100 μg/ml of proteinase K (Roche, Germany) for 3 hr at 56°C. The lysate was extracted twice with a phenol:chloroform:isoamylalcohol mixture (25:24:1), ethanol precipitated and suspended in 100 μl of 1× T.E (pH 8.0). Of this 1–2 μl was used for each polymerase chain reaction (PCR) amplification.

Paraffin embedded tissue.

With paraffin blocks 5 μm thin sections (10–15) were cut and after deparaffinization were placed in 200 μl of digestion buffer.15 Subsequent processing was done as for the frozen tissue.

HPV detection and typing

HPV consensus primers, MY09 and MY11, were used in the PCR assay to amplify an approximately 450 bp fragment from the L1 regions of HPV 16, 18, 33, 6 and 11.16 The sequences of the primers are MY09 5′ CGT CCM ARR GGA WAC TGA TC 3′ and MY11 5′ GCM CAG GGW CAT AAY AAT GG 3′. The PCR reaction was performed in 50 μl volumes, containing 1 μg of genomic DNA, 50mM KCl, 3mM MgCl2, 200 μM each of dATP, dTTP, dCTP, dGTP, 1 μM each of consensus sense and antisense primers and 1 unit of Taq DNA polymerase (Roche, Germany). The reaction mixtures were overlaid with 50 μl of mineral oil (Sigma Chemical Co., St. Louis, MO). PCR amplification involved an initial denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 90 sec, 55°C for 90 sec and 72°C for 120 sec, with final elongation step at 72°C for 10 min. Reaction mixture with no DNA served as a negative control and HPV 16 and 18 positive cervix DNA samples were used as positive controls. The L-1 positive PCR products were typed for HPV 16/18 presence by Slot Blot analysis using Zeta Probe GT Membrane (Bio Rad, Hercules, CA) and MY 14 5′ CAT ACA CCT CCA GCA CCT AA 3′ and WD 74 5′ GGA TGC TGC ACC GGC TGA 3′ as HPV 16 and HPV 18 type specific probes. The probes (1 μg) were end labeled using T4 polynucleotide kinase (Roche, Germany) and 30 μCi of γ32P ATP (BRIT, India), at 37°C for 1 hr. Further confirmation of the L-1 PCR products showing positive signal in slot blot was done by Southern hybridization.

p53 Polymorphism analysis

Analysis of the p53 genotype at Codon 72 was performed as described by Storey et al.,12 with some modifications. Pro sequences were detected by PCR using primers p53 Pro + 5′ GCC AGA GGC TGC TCC CCC 3′ p53 − 5′ CGT GCA AGT CAC AGA CTT 3′ and Arg with primers p53 +5′ TCC CCC TTG CCG TCC CAA 3′ and p53 Arg − 5′ CTG GTG CAG GGG CCA CGC 3′. PCR amplification was performed in 50 μl reaction volumes containing 1 μg of genomic DNA, 1.5 mM dNTPs, 1 μM of each primer, 4 μCi {α32p}dCTP (BRIT, India), 1× PCR buffer and 1 unit of Taq polymerase (Roche, Germany). PCR was performed for 25 cycles of 30 sec at 94°C, 30 sec at 55°C (for p53 Pro+/ p53−) or 60°C (for p53+/ p53 Arg−), and 30 sec at 72°C, with final extension at 72°C for 7 min. Reaction products (7 μl each) were fractionated on a 10% polyacrylamide gel and exposed to X-ray film.

Statistical analysis

Statistical analysis for comparing the prevalence of HPV infection and p53 genotype distribution with clinico pathological data of oral cancer patients was undertaken by using χ2 test (χ2 = 3.84, d.f. = 1, p = 0.05).


  1. Top of page
  2. Abstract
  6. Acknowledgements

Detection of HPV infection in oral tumors

We studied a total of 110 patients of oral squamous cell carcinoma. Of these, 33.6% (37/110) cases showed the presence of HPV, as indicated by a 450 bp L-1 PCR product, on 1.5% agarose gel electrophoresis. In subsequent slot-blot analysis and Southern blot hybridization of L-1 PCR products probed with type-specific sequences revealed the presence of HPV-16 in 22.7% (25/110) and HPV-18 in 14.5% (16/110) of the cases. There were 10% of the cases (11/110) that showed the co-infection with both HPV 16/18 sequences. Out of 26 non-cancer controls screened for HPV infection, 26.9% (7/26) showed L-1 positive reaction. Correlation of HPV-16 and HPV-18 infection with sex, age, site of the tumour, histopathological grade and stage is shown in Table I.

Table I. Clinico Pathologic Profile of Oral Cancer Patients
Sl. No.Clinical parametersNumber of casesHPV 16 +veHPV 18 +ve16/18 Coinfectionp53 Arg/Argp53 Arg/Prop53 Pro/Pro
 Male6815 (22.05)9 (13.23)7 (10.29)19 (27.94)36 (52.94)13 (19.11)
 Female4210 (23.80)7 (16.66)4 (9.52)11 (26.19)23 (54.76)8 (19.04)
2Age Group
 ≤50 Years6614 (21.21)10 (15.15)8 (12.12)19 (28.78)37 (56.06)10 (15.15)
 50 Years+4411 (25)6 (13.63)3 (6.8)12 (27.27)21 (47.72)11 (25)
 Cheek5614 (25)7 (12.5)7 (12.5)15 (26.78)29 (51.78)12 (21.42)
 Mandible (jaw)164 (25)5 (31.25)3 (18.75)6 (37.5)8 (50)2 (12.5)
 Maxilla (angle of mouth)131 (7.6)2 (15.4)02 (15.38)7 (53.8)4 (30.81)
 Gum62 (33.3)003 (50)2 (33.3)1 (16.7)
 Tongue62 (33.3)002 (33.3)3 (50)1 (16.7)
 Lip40002 (50)1 (25)1 (25)
 Miscellaneous92 (22.2)2 (22.2)2 (22.2)1 (11.1)8 (88.9)0
 Normal267 +ve for HPV (by L-1 PCR)14 (53.8)10 (38.5)2 (7.6)
 Well differentiated7717 (22.07)9 (11.68)4 (5.1)22 (28.57)38 (49.35)17 (22.07)
 Moderately differentiated296 (20.68)6 (20.68)3 (10.34)8 (27.58)17 (58.62)4 (13.79)
 Poorly differentiated42 (50)1 (25)1 (25)2 (50)2 (50)0
 III, IV8721 (24.13)13 (14.94)9 (10.34)24 (27.58)44 (50.57)19 (21.83)
 I, II234 (17.39)3 (13.04)2 (8.6)6 (26.08)14 (60.86)3 (13.04)

Both males (35.3%) and females (40.5%) showed equal incidence of infection. Similarly patients below 50 years of age (36.4%) showed same frequency of HPV positivity as patients above 50 years of age (38.6%). Cases of SCC in mandible region showed maximum HPV infectivity at 56.3%, followed by cheek at 37.5% and maxilla showing 23.07% of infection rate. Clear cases of higher stages (Stages III and IV) had a higher 39% infection rate as compared with 30% of lower stage (Stages I and II) cases. Cases of moderately differentiated squamous cell carcinoma (MDSCC) showed 41.4% HPV infectivity as compared with 33.8% in cases of well-differentiated squamous cell carcinoma (WDSCC).

Distribution of p53 polymorphism in Eastern Indian population.

Genomic DNA from 110 patients with oral SCC and 26 non-cancer controls was analyzed to determine the distribution of p53 codon 72 polymorphism. The frequencies of the 3 p53 genotypes Arg/Arg, Arg/Pro and Pro/Pro found in oral cancer patients were 0.28, 0.53 and 0.19, respectively, whereas in non-cancer controls these frequencies were 0.5, 0.42 and 0.08 as shown in Table II. Distribution of p53 genotype in oral squamous cell carcinoma patients with sex, age, site of tumor, histopathological grade and stage is shown in Table I. The allele frequencies for the oral SCC patients were 0.55 (Arg) and 0.45 (Pro) as per the Hardy-Weinberg equilibrium.

Table II. Distribution of p53 Genotype in Oral Squamous Cell Carcinoma and Control Cases
 Number of casesp53 Arg/Argp53 Arg/Prop53 Pro/Pro
Control2613 (50)11 (42.3)2 (7.6)
Oral S.C.C11031 (28.18)58 (52.72)21 (19.09)

Correlation of p53 polymorphism with incidence of HPV infection

The frequencies of the 3 p53 genotypes Arg/Arg, Arg/Pro and Pro/Pro in HPV positive cases of oral S.C.C were 0.34, 0.49 and 0.17 as compared with the frequencies of 0.25, 0.55 and 0.20 for HPV negative cases of oral SCC (Table III). A general trend of increase in frequency of Arg/Arg genotype in HPV positive cases was observed as compared with HPV negative cases. Similarly, Pro/Pro and Arg/Pro genotypes showed slight decrease in frequencies in HPV infected cases when compared with HPV negative cases. A significant corroboration of this trend was seen in cases of mandible, cheek and maxilla where mandible with highest HPV infectivity of 56.3% had the highest Arg/Arg frequency of 0.37 and the lowest Pro/Pro frequency of 0.13. Whereas cases of cheek with HPV infectivity of 37.5% as compared with maxilla (23.07%) showed Arg homozygous and Pro-homozygous frequencies of 0.27 and 0.21 as compared with 0.15 and 0.31 for Maxilla cases. A similar trend was also seen with a low Pro/ Pro frequency of (0.14) in M.D.S.C.C cases, which have a higher HPV infection rate of 41.4% when compared with W.D.S.C.C cases having a Pro/ Pro frequency of (0.22) and a lower HPV infection rate of 33.8%. Moreover, out of 4 P.D.S.C.C cases analyzed, 3 (75%) showed HPV infection with 0% Pro/Pro allele frequency.

Table III. Distribution of p53 Genotype in HPV Infected and Uninfected Oral S.C.C Samples
 Number of casesp53 Arg/Argp53 Arg/Prop53 Pro/Pro
HPV 16 +ve258 (32)13 (52)4 (16)
HPV 18 +ve166 (37.5)7 (43.75)3 (18.75)
HPV −ve6917 (24.63)38 (55.07)14 (20.28)
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Figure 1. Typing of HPV 16/18 in L-1 positive cases of oral squamous cell carcinoma. (a) L-1 PCR products run on 1.5% agarose gel. Lane 1, 100 bp ladder; lane 2, HPV 16/18 positive control (cervix sample); lane 3, L-1 PCR without DNA (negative control); lane T1–T10, L-1 positive samples of oral tumors. (b) Southern blot of L-1 PCR products with HPV-16 type specific probe. (c) Southern blot of L-1 PCR products with HPV-18 type specific probe.

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Figure 2. Slot Blot screening of L-1 positive samples for HPV 16/18 presence. (a) Slot 1, HPV 16 positive control (cervix sample); Slot 2, Negative control; Slot 3-39, L-1 PCR products of 37 positive cases of oral squamous cell carcinoma. (b) Slot 1, HPV 18 positive control (cervix sample), Slot 2, Negative control; Slot 3-39, L-1 PCR products of 37 positive cases of oral squamous cell carcinoma.

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thumbnail image

Figure 3. PCR detection of p53 alleles in oral squamous cell carcinoma cases. Lane 1 and 2, Arg/Arg homozygous cases showing 141 bp amplification product; lane 3 and 4, Pro/Pro homozygous cases showing 177 bp amplification product; lane 5 and 6, Arg/Pro heterozygous cases showing both 141 bp Arg allele and 177 bp Pro allele.

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  1. Top of page
  2. Abstract
  6. Acknowledgements

The rise in the number of cases of oral squamous cell carcinoma every year in India and South East Asian countries as a whole is disturbing. In the Indian scenario, cheek (Buccal Mucosa) represents the primary site for cancer development as compared with tongue and floor of mouth in Western Countries,6 which may be due to the habit of keeping the betel quid and tobacco in contact with cheek for a long time. With many previous studies that incriminate HPV 16/18 infection in the development of oral cancer,5–8 we tried to investigate it in our study involving 110 cases of highly tobacco addicted oral squamous cell carcinoma. In our population from Eastern India, 33.6% of the cases show the presence of HPV as compared with 67% positive cases from South India7 and 15% of cases from Western India.8 The HPV infection is more prominent in Oral SCC cases from India when compared with 23% in the Japanese population,5 8–20% in the USA6 and 19% in the Dutch population.17 Apparently, HPV plays a very important role in the development of oral cancer in the Indian population. Since 26.9% of 26 noncancer controls show the presence of HPV DNA, it appears that HPV 16/18 may play a role in the early events of carcinogenesis.

In our study the frequency of HPV infection is 22.7% (HPV 16), 14.5% (HPV 18) and 10% (HPV 16/18 co-infection). Thus, this shows that HPV18 infection is not as prevalent and significant in the development of oral cancer, which is in agreement with studies by other groups5, 8, 16 except the study of Balaram et al.7 with the South Indian population. Moreover as seen in Table I, no statistically significant correlation is observed between HPV infection and sex, age, site, grade and stage, indicating that HPV infection is not favored by any of these factors. Similar observations have been made in various other studies5, 8, 16 with exceptions such as Paz et al.6 where a high HPV infection rate was observed in patients with larger, more advanced tumors with regional lymph node metastasis.

In our study, it was of great interest to see whether factors such as genetic pre-disposition are also involved in such a high rate of oral cancer as seen in the highly tobacco-addicted Eastern Indian population. A number of studies have been done in various populations to find the distribution of p53 genotype and to correlate it with predisposition and prognosis of cancers such as the cervix,12, 17–19 lung,20–25 esophagus26 and head and neck SCC.27 The allele frequencies in the present study is found to be 0.55 (Arg) and 0.45 (Pro) as per the Hardy-Weinberg equilibrium. When compared with other studies in other populations, it is evident that the Pro/Pro genotype is strongly associated with ethnicity. This is in agreement with Beckman's hypothesis11 and there is a significant decrease in the frequency of the Pro allele with increasing latitude, which varies from 0.63 in African Blacks to 0.17 in Swedish Saamis; χ2 analysis indicates significant differences in genotype distributions of p53 from other reports in Swedish (p < 0.001,22), Spanish (p < 0.00123) and Caucasians in the United States (p = 0.00222) in which lower frequency of the Pro allele is found. It also differs significantly from African-Americans (p = 0.02725) in which higher frequency of Pro allele is found. However, there is a striking similarity in Pro allele frequency in the Japanese,24 Taiwanese21 and Indian populations (our study).

From Table I, it becomes clear that the frequency of the 3 genotypes of p53 remains the same irrespective of sex and age group, indicating that the allele frequencies observed characteristically represent the Eastern Indian population. On careful analysis of the data, it is seen that there is striking decrease in Pro/Pro genotype in HPV positive cases. The frequencies of Arg/Arg, Arg/Pro and Pro/Pro genotypes in HPV infected cases of oral S.C.C are 0.34, 0.49 and 0.17 as compared with the frequencies of 0.25, 0.55 and 0.20 for HPV negative cases. Previous study by Hamel et al.28 in HNSCC cases in Canada showed no association between p53 codon 72 polymorphism and risk of oral cancer. A previous report by Kawaguchi et al.27 in Esophageal cancer showed that frequent loss of proline allele in HPV-associated carcinogenesis of the esophagus major might be playing some role.

Storey et al.12 reported a 7-fold increased risk of cervical cancer associated with having an Arg/Arg polymorphism at codon 72 of p53 compared with the Arg/Pro heterozygotes. In vitro studies suggest that the HPV E6 oncoprotein more readily targets the arginine form, as opposed to the proline form of p53 for degradation. Also Arg-containing allele was found to be preferentially mutated and retained in squamous cell tumors arising in Arg/Pro germline heterozygotes.29 In our study, a striking relation is noticed (from analysis of Table III) that high HPV infection rate is observed in patients with Arg/Arg genotype as compared with Pro/Pro genotype, apparently indicating that the persons with Arg/Arg genotype are highly susceptible to HPV infection and thus to oral carcinogenesis. Further work to elucidate the mode of transmission of HPV and to systematically characterize the tumour specimens for other genetic markers will be highly rewarding since it may help in better diagnosis, designing better treatment strategies and even in preventing the onset of the disease.


  1. Top of page
  2. Abstract
  6. Acknowledgements

The financial support from the Department of Biotechnology, New Delhi, and Council of Scientific and Industrial Research, New Delhi, to BRD is acknowledged. JKN thanks the Council of Scientific and Industrial Research, New Delhi, and SP thanks the University Grants Commission, New Delhi, for a research fellowship. The authors are grateful to the Director and his colleagues of the A.H. Regional Cancer Centre, Cuttack, and Prof. K.S. Panda, Panda Nursing Centre, Cuttack, for their cooperation in obtaining tumor samples.


  1. Top of page
  2. Abstract
  6. Acknowledgements
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