Sequential Evaluation of DNA Damage in Patients with Head and Neck Carcinoma Receiving Radiotherapy

Background: Head and neck cancers account for about 30% of all cancers in India. The incidence rates of HNSCC in India are30/1, 00,000 for males and 10/1, 00,000 for females. The commonly used treatment modalities include surgery, chemotherapy and radiotherapy. Studies conducted in different types of cancers showed that there is anincreased primary DNA damage even before the commencement of treatment in cancer patients.The treatment modality will further induce DNA damage in addition to the already existing DNA damage.In normal healthy people, DNA damage is effectively repaired. However, in patients with carcinoma, chemo-radiation induced DNA damage is not repaired so effectively. Consequently, there is a high risk of secondary carcinoma by unrepaired damaged DNA. Methodology: In this study, the degree of DNA damage is assessed by comet assay technique in patients with head and neck carcinoma receiving radiotherapy and had complete regression of tumor following radiotherapy. The degree of DNA damage is compared according to the age, gender and associated risk factors of the patients.


Background
Indian Subcontinent registers around 2,00,000 cases ofHead and Neck carcinoma each year and nearly about 80,000 of them are diagnosedas oral carcinoma (1). The pro-carcinogenic factors include alcohol in any formand consumption of tobacco in any form (2).Patients are classi ed into anatomic groups that are categorized from Ito IV based on the increasing severity of disease. Stage 1 denotes an earlydisease and stage 4 denotes a locally advanced disease with metastasis.Studies conducted in different types of cancers showed that there is anincreased primary DNA damage even before the treatment in cancer patients.The treatment modality will further induce DNA damage in addition to thealready existing DNA damage. Chemotherapy and Radiotherapy beingthe mainstay of treatment for locally advanced cancers induces DNAdamage in the target cells as well as to some of the normal cells in thesurroundings (3,4).In normal healthy people, DNA damage is effectively repaired. But incancer patients, chemo-radiation induced DNA damage is not repaired soeffectively. Consequently, there is a high risk of secondary cancer byunrepaired damaged DNA (5,6).This study is taken up to assess the degree of DNA damage by comet parameters in patients with head and neck carcinomawho had complete tumor regression after receiving radiotherapy.

Methodology
After approval from the scienti c and ethics committee, this study was performed in the Department of Anatomy in collaboration with the Department of Radiotherapy, JIPMER. Selection of subjects was done from the Radiotherapy Department, JIPMER.Thirty ve patients with stage II, III, IVA, histopathologically con rmed Squamous cell carcinoma of Head and Neck (lip, oral mucosa, nasal cavity, pharynx, paranasal sinuses, larynx) with KPS more than 70 attending radiotherapy OPD for treatment were included in this study.Patients with co-morbid conditions such as severe infections, blood dyscrasias, other genetic disorders, patients with abnormal liver function test, renal function test were excluded from this study.Previous H/O chemotherapy, radiotherapy to any part before starting treatment were also exempted from this study. Under strict aseptic precaution, 1 ml of heparinized blood was collected from the study participant after obtaining informed consent from them. The blood samples were collected as follow 1. 2 hours before the rst dose of radiation 2. 2 hours after 10th fraction of radiation 3. 2 hours after 20th fraction of radiation 4. 2 hours after 30th fraction of radiation 5. During 1st follow-up (1 month after completion of treatment) All the samples were processed immediately and analyzed for DNA damage by single cell gel electrophoresis assay (Comet assay). To assess the DNA damage following comet parameters were used. The length of the comet tail was measured by using ocular scale tted to the microscope. In order to avoid bias these parameters were employed on randomly selected 40 to 50 cells per subject.Categorical data obtained from the study was presented as frequencies and percentages. Chi square test was used to compare the categorical data. Normally distributed continuous data is presented as mean with the standard deviation. Comparison of continuous data has been done using independent student's t test. All statistical analysis carried out with p value <0.05 was considered statistically signi cant.

Results
The total sample size in this study is 35.

Age distribution:
The details of age distribution of the cases in this study are shown in the Table I  The percentage of DNA in head parameter of post-RT sample 1 (79.6 ± 7.8) is decreased when compared to baseline sample (86.8 ± 5.5) with a p value less than 0.05.
There is a progressive decrease in the percentage of DNA in head parameter of post-RT samples 1, 2 and 3, though it is statistically insigni cant.

Discussion
Around 2,00,000 cases of Head and Neck Carcinoma are reported every year in India. Concurrent chemoradiation forms the mainstay of treatment for locally advanced cancers. Chemo-radiation induces DNA damage to the target cells as well as some of the normal cells. As the monitoring of repair of target tissue is practically not possible, lymphocytes are used as a surrogate marker. DNA repair capacity is measurable in various cell types as it has a genetic predisposition (7). In earlier studies, Comet assay has been documented as a reliable parameter to assess the DNA damage (8).

I.ANALYSIS OF BASELINE CHARACTERISTICS
In the present study, most of the patients were distributed in the age group of 50-59 years ( Table I).The total number of males were more when compared to females (Table II).Risk factors like smoking, alcohol consumption and tobacco chewing were documented among the study group (Table IV). Most of the males were found to be associated with all the three risk factors whereas the females mostly with that of tobacco chewing.

II.ANALYSIS OF BASELINE DNA DAMAGE
Previous studies show that there is a baseline DNA damage in the patients with various carcinoma prior to radiotherapy (9). The observations in this study are: -Increase in baseline DNA damage is noted as the age increases (Table V).
-Patients with greater than one risk factor are found to have more baseline DNA damage when compared to the others (Table VI & Chart V).

III. ANALYSIS OF POST -RT DNA DAMAGE
The second, third and fourth samples were collected 2 hours after the 10th, 20th and 30th fraction of radiation respectively. MarijaGamulin et al., observed an increase in the DNA damage in the post radiotherapy period when compared to that of pre radiotherapy and this study ndings also con rm the same (4).Signi cant sequential increase in the DNA damage was observed in the percentage of DNA in tail parameter throughout the course of the radiation (Table XIII & (Table VIII)  . They observed higher DNA damage in the smokers than the non-smokers (10,11). In our present study, the baseline values were higher in the patients with smoking when compared to the nonsmokers but not statistically signi cant. The repairing capacity of DNA is less in the smokers when compared to the non-smokers within the sub -group 2 (Table XXIII).

ALCOHOL CONSUMPTION:
In the present study, most of the alcohol consumption groups were males. The repairing capacity of DNA is less in the alcoholics when compared to the non-alcoholics as evident by the tail length and percentage of DNA in tail parameters within the patients of sub-group 2 (Table XXIV). This agrees with the ndings of Rulten et al., who demonstrated that DNA damage is induced by the consumption of alcohol (12).

TOBACCO CHEWING:
Tobacco chewing is documented as one of the major risk factors in the Indian population due to the cultural and ethnic variation. The comet parameters showed an increase in baseline values in the tobacco chewers compared to the tobacco non-chewers which agrees with the ndings of Pfeifer et al (13).

Conclusion
Patients with locally advanced head and neck cancers with complete tumor response following radiotherapy show a sequential increase in the DNA damage.The co-existing risk factors and old age may increase the baseline DNA damage in the patients with head and neck carcinoma.The repair mechanism is dependent on the patient's general condition, risk factors and also the biology of the tumor.

Declarations
Ethical approval and consent of the participant: The study was subjected to the ethical clearance and approved by the Institute Ethics Committee

Consent for publication:
Informed consent was taken from all the participants for the publication of the results of the study. The participants were also assured that their identity would not be revealed and anonymity would be maintained Availability of data and materials: The dataset analysed during the current study are cited as references within the square brackets in the manuscript and the corresponding details are provided under the reference section.
Competing interest: There is no con ict of interest in this study

Funding: No funding was received
Author contribution: Dr. Sivakumar M -conceptualization, investigation, methodology, writing, analysis, editing