|Year : 2021 | Volume
| Issue : 2 | Page : 57-60
Taxation of micronuclei frequency as a prognostic marker in oral and oropharyngeal carcinoma: A cytogenetic study
Kumar Satish Ravi1, NB Pushpa2, Sanjeev Kishore3, Sohinder Kaur4, Vandana Mehta5, KS Reddy6
1 Additional Professor, Department of Anatomy, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
2 Assistant Professor, Department of Anatomy, JSS Medical College, JSSAHER, Mysore, Karnataka, India
3 Professor and Head, Department of Pathology and Lab Medicine, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
4 Director Professor, Department of Anatomy, Lady Hardinge Medical College, New Delhi, India
5 Director Professor, Department of Anatomy, Vardhman Mahavir Medical College, New Delhi, India
6 Professor, Department of Radiation Oncology, Mahatma Gandhi Medical College and Research Institute, Puducherry, India
|Date of Submission||11-Jan-2021|
|Date of Decision||02-Feb-2021|
|Date of Acceptance||15-Feb-2021|
|Date of Web Publication||09-Apr-2021|
Kumar Satish Ravi
Additional Professor, Department of Anatomy, All India Institute of Medical Sciences, Rishikesh, Uttarakhand
Source of Support: None, Conflict of Interest: None
Background: Oral and oropharyngeal carcinoma are one among the most common cancers in the world. India shares about one-fourth of incidences of oral and oropharyngeal cancers and death due to the same is also of significant number. Micronucleus resulting from aberrant mitosis, chromatin fragmentation, or aberrant chromosome is considered as a hall mark for genotoxicity, also oral cancer risk and is useful in chemopreventive studies. Hence, the determination of micronuclei frequency serves as better prognostic marker. Methodology: A total of 60 patients with a mean age of 53 (56 males and 4 females) years, who were histopathologically confirmed cases of oral and oropharyngeal carcinoma with different degree of differentiation were included in the study. Patients were solely treated by radiotherapy with radiation dose plan of 4 Gy, 14 Gy, 24 Gy, and 60 Gy on the 2nd, 7th, 12th, and 30th days, respectively. The mucosal scapings stained with Giemsa and May-Grunwald's stain were studied to assess the micronuclei at each interval. Results: Although there was no significant association between site of lesion and tumor differentiation with micronuclei index, there was statistically significant difference in the micronuclei index at each interval. Percentage of relative increment in micronuclei also showed promising significance. Conclusion: Hence, micronuclear assay could be used as an efficient tool to determine the radiosensitivity and prognosis in oral and oropharyngeal carcinoma patients treated by radiotherapy.
Keywords: Cell damage, DNA repair, genotoxicity, ionising radiation, micronuclear assay
|How to cite this article:|
Ravi KS, Pushpa N B, Kishore S, Kaur S, Mehta V, Reddy K S. Taxation of micronuclei frequency as a prognostic marker in oral and oropharyngeal carcinoma: A cytogenetic study. Natl J Clin Anat 2021;10:57-60
|How to cite this URL:|
Ravi KS, Pushpa N B, Kishore S, Kaur S, Mehta V, Reddy K S. Taxation of micronuclei frequency as a prognostic marker in oral and oropharyngeal carcinoma: A cytogenetic study. Natl J Clin Anat [serial online] 2021 [cited 2021 Jun 13];10:57-60. Available from: http://www.njca.info/text.asp?2021/10/2/57/313517
| Introduction|| |
Oral cancers rank 6th among all the cancers. Oral cancer usually presents as a small and unexplained growth or ulcer in the oral cavity which can extend to the oropharynx. India, having the largest number of cancer patients shares about one-third of the total burden in the world. Annually, in our country, about 77,000 new cases and 52,000 deaths are due to oral cancers, which is around one-fourth of global incidences. Being one of the common types of cancers, oral cancers are of important public health concern in India and countries that are in the phase of economic transition. In our country, about 70% of the oral cancer cases are reported in the advanced stages owing to late diagnosis compared to the Western countries. The detection and diagnosis of such cancers in the later stages results in a very low cure rate and 5-year survival rates around 20% only.,,,
Oral carcinomas are treated either by radiotherapy, surgery, or chemotherapy. Often, a combination of these treatment modalities is used. In radiotherapy, ionizing radiations are used. Such radiations cause damage to the genetic material (DNA) and thus affects target cells by a series of complicated atomic interactions. Low-dose exposure to radiation for a long time can induce cancer due to gene mutations. Moreover, the ability of radiation, to disturb the cell division is made use of in treating cancer since cancer cells flourish by abnormal and uncontrolled cell division.
After radiotherapy, the damaged cells (both abnormal and normal) undergo death, thus resulting in side effects and complications. The response of treatment can be assessed by various toxic effects of radiation chemicals appearing in the form of nuclear anomalies such as micronuclei (MN), nuclear budding, multinucleation, karyorrhexis, and karyolysis and can be assessed by taking scrapings from the oral mucosa.,
Micronuclei are small intracytoplasmic entities around one-third to one-fifth of the size of the main nucleus indicating a fragment or whole of chromosome. They are formed due to the genotoxic effect of radiation and usually identified adjacent to the nucleus. The staining property of MN is similar to the nucleus of the cell. MN count involves a rapid, efficient, and economical technique, providing reliable quantitative analysis of the genotoxicity., Significantly higher MN frequencies have been observed in individuals exposed to antineoplastic agents, lead-containing paints, polycyclic aromatic hydrocarbons, Arsenic, diesel derivatives, diet/vitamin deficiencies, tobacco, and alcohol. Hence, MN assay may be used as a reliable biomarker of genetic damage. Hence, this study was undertaken to determine the association between MN index with an incremental dose of radiation and hence to utilise the same to envisage sensitivity and response of the cancer cells to radiotherapy in oral and oropharyngeal carcinoma.
| Methodology|| |
This was an observational study, which involved patients with oral and oropharyngeal carcinoma referred from the Departments of Surgery and E.N.T. for radiotherapy at All India Institute of Medical Sciences, Rishikesh. Non-operated cases were assessed 6–8 weeks after completion of radiotherapy. The study protocol was carried out after obtaining the approval from the institute human ethical committee (28/IEC/PhD/2018 dated 8/3/2018) and written consent from each participant of the study. Standard proforma was used to collect basic information of the patients including the diagnosis.
Sixty patients with the age range of 25–65 years, who were histopathologically confirmed squamous cell carcinoma of oral cavity and oropharynx with different TNM staging were included in the study. Patients were solely treated by radiotherapy (brachytherapy) with a radiation dose plan of 4 Gy, 14 Gy, 24 Gy, and 60 Gy on the 2nd, 7th, 12th and 30th days, respectively. Patient with repeat radiotherapy/relapse of tumor were excluded from the study.
Mucosa from the site of lesion was collected by wooden spatula. After air drying, the collected mucosal smear was fixed using methanol. The fixed tissue was stained with Giemsa and May-Grunwald's stain and then, micronuclei (MN) were evaluated under bright field microscopy.
From each patient, 500 cells were examined from the smear for radiation-induced micronuclei at 4, 14, 24, and 60 Gy on 2nd, 7th, 12th and 30th days, respectively. The observations were tabulated and analyzed using ANOVA-test. Relative increment of MN was calculated using the below formula for each interval.
| Observations and Results|| |
Among 60 patients, 56 were male patients and 04 females with overall mean age 53 years. There were 17 confirmed oral and 43 oropharyngeal cancer cases with different degrees of differentiation as shown in [Figure 1].
|Figure 1: Cancer types in the study with different degrees of differentiation|
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There was no significant association between frequency of MN [Figure 2] concerning the site of the lesion at different intervals of time and also with repect to gender [Table 1].
|Table 1: Comparison of micronuclei between the sites at different intervals of treatment|
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There was a gradual rise in the mean value of MN from 34.68 to 35.70, 50.68, and 91.63 with 4, 14, 20 Gy radiation, respectively, while there was a decrease in the mean value to 83.45 with 60 Gy radiation [Table 2].
The relative increment index was recorded between each interval of treatment and there was a statistically significant increment in the micronuclear index but there was a decrease in the same concerning the last two doses of radiation [Table 3].
|Table 3: Relative increment of micronuclei index with an increase in radiation|
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| Discussion|| |
In radiotherapy, malignant cells are targeted by ionizing radiations, which bring about DNA damage by various complicated reactions. Majority of the nuclear damage caused is because of free radicals which are formed by the interaction of water molecules with radiation.
Studies have been done to identify the factors influencing diagnosis, response to RT and prognosis of oral carcinoma. One of the main factors identified and labeled as a prognostic factor for Oral carcinoma is MN frequency. MN resulting from aberrant mitosis, chromatin fragmentation or aberrant chromosome is considered as hallmark for genotoxicity and also useful in chemo-preventive studies. Even before the initiation of treatment, micronuclei are observed in cancer cells and can be considered as a relative index for cell loss following radiotherapy.,,,
MN assay is noninvasive, do not require the complicated process of cell culture and metaphase preparation. It can be done in interphase cells and the process is cost-effective. Hence, MN assay is the most commonly used method for the evaluation and monitoring of the mutagenic influence of radiation, chemicals, and other genotoxins.,
In the present study, there was no significant difference observed between the rise in MN concerning oral and oropharyngeal carcinoma [Table 1]. There were rising titers of MN of 35.7, 50.68, and 91.63 at 4, 14, and 24 Gy, respectively, and at 60 Gy there was a fall in mean value from 91.63 to 83.45 at 60 Gy. Similar findings have been observed by Bindu et al. This rise in the mean value of MN is because of genotoxic effect of ionising radiations on the cancer cells. A fall at 60 Gy could be because of cells affected by radiation would have lost their proliferative ability and abnormal DNA profile was merely seen after 4 weeks, which is attributed to the possibility of initiation of DNA repair or cessation of lethal damage or genetic inactivity. According to which the buccal mucosa, as a consequence of radiation exposure returns to normal within 4–6 weeks.
Kumari et al. in their study reported that the MN showed a statistically significant rise with radiation dose and the count at 24 Gy was 3.7 times more than that of the pretreatment count. The higher degree of variation in the MN counts at each dose suggests the occurrence of a high rate of intertumoral heterogenicity in MN induction. In another study, authors observed a marked increase in the number of MN in malignant cells of recurrent carcinoma of the head and neck region following radiotherapy and intratumoral injection of mitroxantrone. The significant response concerning MN formation was accompanied by a better tumor remission. A dose-dependent rise in the number of micronuclei in vivo has also been observed in erythrocytes and bone marrow cells of mice following exposure to different doses of g or X-rays.,, It is worthy to mention here that Mitchell et al. and Ramalho et al. have recorded a dose-dependent rise in MN count in the lymphocytes of peripheral blood smear of man exposed to low doses of X-rays.,
There was a gradual increase in the relative increment of MN up to 12th day of radiotherapy and then there was negative increment suggesting an improvement in the lesion with DNA repair and reduced cell damage which also suggests the sensitivity of the lesion to radiotherapy.
| Conclusion|| |
The progressive increase in MN proves that it is a good indicator for assessing the response of both oral and oropharyngeal carcinoma to radiotherapy. MN index observed at 4 Gy may be used to differentiate radio-resistant and radiosensitive tumors and hence the line of treatment can be planned accordingly. The degree of tumor response to radiotherapy as assessed on the 7th day of treatment can be utilized to alter further treatment. The measured relative increment index of MN shows a sustained increase with increasing dosage of radiation dose, which can be used as prognostic marker in malignant cases undergoing radiotherapy.
We would like to express our heartfelt gratitude to Dr. Manoj Gupta, Professor and Head of Radiation Oncology Department, AIIMS Rishikesh for his valuable suggestions during the planning of this research work and for providing all the support during data collection.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gupta B, Bray F, Kumar N, Johnson NW. Associations between oral hygiene habits, diet, tobacco and alcohol and risk of oral cancer: A case-control study from India. Cancer Epidemiol 2017;51:7-14.
Laprise C, Shahul HP, Madathil SA, Thekkepurakkal AS, Castonguay G, Varghese S, et al
. Periodontal diseases and risk of oral cancer in Southern India: Results from the HeNCe Life study. Int J Cancer 2016;139:1512-9.
Sharma S, Satyanarayana L, Asthana S, Shivalingesh KK, Goutham BS, Ramachandra S. Oral cancer statistics in India on the basis of first report of 29 population-based cancer registries. J Oral Maxillofac Pathol 2018;22:18-26.
] [Full text]
Veluthattil A, Sudha S, Kandasamy S, Chakkalakkoombil S. Effect of hypofractionated, palliative radiotherapy on quality of life in late-stage oral cavity cancer: A prospective clinical trial. Indian J Palliat Care 2019;25:383.
] [Full text]
Graham RM. Effect of radiation on vaginal cells in cervical carcinoma: Description of cellular changes. Surg Gynecol Obstet 1947;84:153-65.
Streffer C, van Beuningen D, Gross E, Schabronath J, Eigler FW, Rebmann A. Predictive assays for the therapy of rectum carcinoma. Radiother Oncol 1986;5:303-10.
Kumari R, Chaugule A, Goyal PK. Karyoanomalic frequency during radiation therapy. J Cancer Res Ther 2005;1:187-90.
Bindu L, Balaram P, Mathew A, Remani P, Bhattathiri VN, Krishnan Nair M. Radiation-induced changes in oral carcinoma cells – A multiparametric evaluation. Cytopathology 2004;14:287-93.
Dindgire SL, Gosavi S, Kumawat RM, Ganvir S, Hazarey V. Comparative study of exfoliated oral mucosal cell micronucleus frequency in potentially malignant and malignant lesions. Int J Oral Maxillofac Pathol 2012;3:15-20.
Palaskara S, Jindal C. Evaluation of micronuclei using papanicolaou and may grunwald giemsa stain in individuals with different tobacco habits – A comparative study. J Clin Diagn Res 2010;4:3607-13.
Ravi KS, Naithani M, Kaur S, Reddy KS, Pasi R. Multi- nucleation indices in radiotherapy treated squamous cell carcinoma – A tool in treatment planning and assessment of prognosis. Int J Curr Adv Res 2017;6:1797-801.
Umiker WO. Grading of squamous cell carcinoma by cytologic smears: A preliminary report. Med Bull (Ann Arbor) 1957;23:353-5.
Gupta S, Mukherjee K, Gupta YN, Kumar M. Sequential radiation changes in cytology of vaginal smears in carcinoma of cervix uteri during radiotherapy. Int J Gynaecol Obstet 1987;25:303-8.
Streffer C, van Beuningen D, Molls M. Possibilities of the Micronucleus test test as an assay in radiotherapy. In: Progress in Radiooncology. United states: Raven Press;1982. p. 243-51.
Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, et al
. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gaps. Mutat Res 2008;659:93-108.
Agarwal M, Sunitha JD, Dawar G, Rallan NS. Micronuclei assay of exfoliated oral mucosal cells: A review. Ann Dent Spec 2014;2:47-50.
Yadav AS, Jaggi S. Buccal micronucleus cytome assay – A biomarker of genotoxicity. J Mol Biomark Diagn 2015;6:1-6.
Ogden GR, Cowpe JG, Green MW. Effect of radiotherapy on oral mucosa assessed by quantitative exfoliative cytology. J Clin Pathol 1989;42:940-3.
Lee TK, Wiley AL, Esinhart JD Jr., Blackburn LD. Radiation dose-dependent variations of micronuclei production in cytochalasin B-blocked human lymphocytes. Teratog Carcinog Mutagen 1994;14:1-14.
Cole RJ, Taylor N, Cole J, Arlett CF. Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts. Mutat Res 1981;80:141-57.
Uma Devi P, Sharma AS. Mouse bone-marrow response to low doses of whole-body gamma irradiation: Induction of micronuclei. Int J Radiat Biol 1990;57:97-101.
Jagetia GC, Ganapathi NG. Radiation-induced micronucleus formation in mouse bone marrow after low dose exposures. Mutat Res 1994;304:235-42.
Mitchell JC, Norman A. The induction of micronuclei in human lymphocytes by low doses of radiation. Int J Radiat Biol Relat Stud Phys Chem Med 1987;52:527-35.
Ramalho A, Sunjevaric I, Natarajan AT. Use of the frequencies of micronuclei as quantitative indicators of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes: Comparison of two methods. Mutat Res 1988;207:141-6.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]