Assessment and Correlation of Single Stranded DNA Damage in Oral Squamous Cell Carcinoma with Broder's Histopathological Grading

INTRODUCTION

According to the assessment of World Health Organization [WHO], oral cancer is the 3^rd most common malignancy in males and 6^th most common malignancy in females. Amongst all the malignancies affecting oral cavity, OSCC is the commonest¹. Cancer in any form being associated with high mortality rate, prevention of OSCC is a desirable goal, as is the case with its early diagnosis and treatment.

Although histopathological findings of suspected cancer remains the only method of confirming malignancy, there are many other advanced methods like cellular proliferation studies, cytophotometry, oncogenic expression, DNA cytology and assessing DNA damage. The various methods to detect DNA damage include – Alkaline Sucrose sedimentation, Filter elution, Nucleoid sedimentation, Micro electrophoresis, Pulse field gel electrophoresis etc.

A sensitive technique for measuring and analysing the DNA damage in eukaryotic cells is the Single cell gel electrophoresis (SCGE) assay or Comet assay^2,3,4. This technique has been widely used to study DNA damage and repair induced by radiation and during carcinogenesis, determining the genotoxicity, bio monitoring and analysis of irradiated foods. The SCGE assay has also been employed to assess DNA damage in peripheral blood leukocytes of patients with OSCC⁵, epithelial dysplasia⁶ and in tobacco users². These studies as well as findings of increased DNA damage in peripheral blood cells in patients with cervical carcinoma lend support to a systemic host response⁸.

The principle of SCGE assay is that the presence of breaks in the DNA releases the super coiling, so that the loops, instead of being constrained within the bounds of nuclear matrix are free to extend outside.

The lysis of DNA at a neutral pH results in double stranded breaks, whereas lysis at an alkaline pH results in single stranded breaks and denaturation of cellular RNA, facilitating easier detection^4,9. On electrophoresis, the single stranded breaks are drawn towards the anode as a “tail” and therefore this assay is also referred to as Comet assay. Measurement of this tail is an index of DNA damage.

Though the DNA damage in peripheral blood leukocytes with OSCC, in epithelial dysplasia, in tobacco users and also in exfoliated buccal cells in tobacco users has been assayed¹⁰, the possible DNA damage in exfoliated cells of patients with OSCC has not been studied. The present study was planned to fill in this void.

METHODOLOGY

For the purpose of this study, patients who were diagnosed as having OSCC formed the study group. Those patients without any systemic disease and tobacco habituation and with a clinically normal oral mucosa formed the control group. Oral smears were collected from the lesional tissue of 30 OSCC patients after histopathological confirmation and evaluation according to Broder's grading system. Oral smears were also collected from the buccal mucosa of 30 persons of the control group.

SCGE assay was carried out for both these groups. The DNA damage was quantified using a fluorescent microscope following the method outlined by Singh et al⁴ and the results were statistically analyzed and evaluated.

The technique employed by the authors to assess DNA damage is that developed by Singh et al (1988) which is a modification of original procedure of Ostling and Johanson (1984). In order to improve the sensitivity for detection of DNA damage in isolated cells, Ostling and Johanson developed a microgel electrophoresis technique³. However, the neutral conditions employed for lysis and electrophoresis by Ostling and Johanson permit detection of double stranded DNA breaks only and do not allow the detection of single stranded DNA breaks. The SCGE assay as modified by Singh et al (1988) is performed under high pH thereby revealing even single stranded breaks in the DNA.

In SCGE assay, the electrophoresis is performed for 15 minutes under high pH with the power supply adjusted to 20 volts and 400 mA. Thus alkali labile sites as well as frank breaks are revealed. Comets form as broken ends of a negatively charged DNA molecule which becomes free and migrate in the electric field towards the anode. The intensity of fluorescence in the tail relative to the head provides information of the number of single stranded DNA breaks. Thus the concept of stretching and migration of the separated strands are generally considered to explain the patterns of DNA migration observed in the comet assay⁴.

For visualization of DNA damage, the slides after electrophoresis were stained with ethidium bromide and observed at 250x magnification using a fluorescent microscope equipped with an excitation of filter of 515 – 560nm and a barrier of 590nm. Using an oculometer fitted to the eye piece, the total length and the diameter was measured. The tail length was calculated by subtracting the diameter from the length. Twenty five randomly selected cells per slide were analyzed and their mean was calculated.

The DNA damage was compared between control and study groups employing the student's 't' test. DNA damage was also compared between the different grades of OSCC by employing the one way Anova 'F' technique for significant difference between different groups.

RESULTS

DNA damage levels between patients of control group were compared with those of study group and assessed using the SCGE assay. Smears were taken from lesion proper in 30 patients of study group and from the buccal mucosa of 30 normal, healthy volunteers. The results were tabulated and compared for statistical significance in Table 1. The DNA damage levels in OSCC patients were found to be significantly high (p < 0.0001) as compared to the control group patients.

Patients with OSCC were grouped histopathologically according to Broder's grading system into three grades i.e. well differentiated, moderately differentiated and poorly differentiated. Out of 30 patients with OSCC, 22 were well differentiated, 5 were moderately differentiated and 3 were poorly differentiated. The DNA damage levels of various histopathological grades of OSCC were assessed and tabulated in Table 2. A stepwise increase in mean DNA damage levels from well differentiated to poorly differentiated OSCC was observed.

In order to compare DNA damage statistically between different grades of OSCC, one way ANOVA 'F' technique was applied and a high statistical significance at p < 0.0001 (Table no.3) was noted between all the grades of OSCC (Table 3).

DISCUSSION

Seventy five percent of cancers affecting the oral cavity arise in a horse shoe shaped area extending from the anterior floor of mouth and including lateral lingual margins and tonsillar pillar to retromolar trigone complex¹³. It is postulated that concentrated carcinogens suspended in saliva are pooled in these mucus reservoirs. In India, buccal mucosa is the commonest site for oral cancer and is related to placement of tobacco quid in this region¹⁴. Apart from tobacco, alcohol and viruses have also been implicated in oral carcinogenesis^15,16,17,18. In some of the cases of non-users of tobacco and alcohol, genetic predisposition has been suggested^15,16. Different carcinogens attack different sites on the DNA resulting in accumulation of cellular and molecular changes, thereby progressively altering the DNA. The resultant DNA damage can include single and double stranded breaks, DNA-protein cross links, loss of bases from DNA backbone and modified base residues^17,18.

The common mechanism for initiation of cancer appears to be through DNA damage^17,18. Polycyclic aromatic hydrocarbons (PAH) regarded as the main pre-carcinogens in smoking as well as chewing forms of tobacco are activated to ultimate carcinogens by Aryl Hydroxy Hydroxylases (AHH) to form epoxides. These epoxides are absorbed into the systemic circulation when smoked. In the chewing forms of tobacco, the epoxides are believed to penetrate the thinner oral epithelium like floor of the mouth, which has a greater blood supply^11,17,18. These epoxides are destroyed in the liver by the enzyme epoxy hydratase. Failure of this enzyme to detoxify the carcinogen results in the formation of an electrophile which possesses an uncharged or positively charged electron deficient atom, which can result in the formation of covalent bonds with nucleotides such as DNA, either primarily or after metabolic activation^11,17,18. These then bring about permanent genetic alterations in the form of point mutations at G–C base pair sites in the circulating leucocytes and probably other epithelial cells. Altered base pairs are liable to replacement by repair mechanisms, but the repair must result in an error in base sequence which is then passed on to progeny of the affected cell. Large scale alterations such as nucleic acid base omission lead to transpositions, rearrangements and deletions resulting in gene inactivation. Inactivation of tumor suppressing genes leads to initiation of carcinogenesis^11,17,18.

DNA strands breaks occur due to a variety of reasons like chemicals, toxins, ionizing radiation, carcinogens, apoptosis and probably due to ageing. The quantitative measurement of DNA damage in tumour cells is therefore an important prognostic indicator for patients with malignancy. Many methods are available to detect and quantify DNA single and double stranded breaks, both in vivo and in vitro. However, since many agents induce 5 – 2000 fold more single stranded breaks of DNA, the techniques to measure single stranded breaks of DNA are definitely more sensitive to detect DNA damage. The SCGE assay, also known as comet assay is a recent, simple and sensitive technique to evaluate single stranded breaks.

Malini et al⁸, using comet assay showed that persons with cervical carcinomas demonstrated increased DNA damage levels in peripheral blood leukocytes (PBLs) as compared to normal individuals. Malini et al⁸, also demonstrated a significant stepwise increase in the mean DNA damage in the PBLs from normal subjects in control group to patients with precancerous lesions of cervix showing different grades of dysplasia. Venkateswara Rao et al⁵ , using SCGE assay, demonstrated a significant increase in DNA damage level in PBLs of patients with OSCC.

A study to evaluate DNA damage in PBLs of patients with oral leukoplakia showed a stepwise increase in DNA damage from control group to patients with different grades of dysplasia⁶. In leukoplakic lesions without epithelial dysplasia, statistical significance was found when compared to controls. This result obtained by SCGE assay showed it to be a sensitive technique to identify DNA damage in PBLs even before morphological changes became apparent.

Rekha Patil et al⁷, employing the SCGE assay to detect DNA damage in PBLs of tobacco users found statistically significant DNA damage levels in tobacco users when compared with control group patients. The study also revealed statistically significant DNA damage levels in tobacco users with normal oral mucosa and with oral with oral cancer and also between patients with OSCC and the control group.

Rojas et al¹⁰, employing the SCGE assay, have evaluated the DNA damage in exfoliated buccal mucosal cells of smokers and non-smokers. A statistically significant mean DNA damage was found in the smoker group, when compared with the control group. Rojas et al¹² also showed statistically significant DNA damage levels in exfoliated tear duct epithelial cells in patients exposed to environmental pollutants.

The present study was carried out on the assumption that oral mucosal cells could show the DNA damage in oral cancer, as they were shown by Rojas et al to manifest DNA damage in tobacco users, which is a prime cause of oral cancer.

For the present study, 30 patients with OSCC were selected after histopathological confirmation using Broder's histopathological grading system. The mean DNA damage levels in these patients were compared with DNA damage levels in 30 normal, healthy volunteers without tobacco habituation and systemic illness. The mean DNA damage in patients with OSCC was found to be 3.868 µm and this, when compared to mean DNA damage in control group patients (0.995 µm) was found to be statistically highly significant (p < 0.0001).

An attempt was made to evaluate DNA damage in relation to Broder's histopathological grading system for OSCC. Patients with OSCC were graded histopathologically using Broder's histopathological grading system into 3 grades viz, well, moderately and poorly differentiated OSCC. Results showed that the mean DNA damage levels in oral mucosal cells gradually increased from well differentiated to poorly differentiated OSCC. High statistical significance (p < 0.0001) was found between all the grades of OSCC,

These preliminary findings suggest that the DNA damage levels in oral mucosal cells could be an important prognostic indicator and can be utilized to supplement the clinical staging and histopathological grading to assess the prognosis of OSCC.

CONCLUSION

This study was carried out on the assumption that oral mucosal cells might show DNA damage in OSCC. The assumption was based on DNA damage observed in exfoliated buccal mucosal cells of smokers and DNA damage in mucosal cells of cervical cancer.

The findings of statistically significant increased DNA damage levels in oral mucosal cells in OSCC patients and their correlation to histopathological grading suggest that comet assay may be used effectively to assess the prognosis of OSCC. However, a more detailed study involving a larger sample is needed on order to corroborate these findings.

ACKNOWLEDGEMENTS

The authors would like to thank Dr G S Kumar, Principal, K S R Institute of Dental Sciences, Tiruchengode, Tamilnadu, India and Dr Pravin Shetty, Professor, Dept of Biochemistry, S.D.M College of Dental Sciences, Dharwad, India for their guidance in this study.