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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 2  |  Page : 67-71

Unveiling the hidden effects of chemoradiotherapy in the treatment of oral squamous cell carcinoma: A cytological study


1 Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
2 Department of Public Health Dentistry, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
3 Department of Public Health Dentistry, Gian Sagar Dental College and Hospital, Rajpura, Punjab, India

Date of Web Publication26-Jul-2017

Correspondence Address:
Ramandeep Singh Gambhir
Department of Public Health Dentistry, Gian Sagar Dental College and Hospital, Rajpura - 140 401, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jorr.jorr_14_17

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  Abstract 

Aim: The most common malignancy of the oral cavity is squamous cell carcinoma, which accounts for approximately 5% of all neoplasms. Unfortunately, the great majority of these tumors are diagnosed in stages which require surgery with radio- and chemo-therapy. These treatments affect not only the malignant cells but also the healthy tissues of the patient. Therefore, the present study was conducted to detect oral cytological atypia among oral squamous cell carcinoma patients receiving chemotherapy and/or radiotherapy.
Materials and Methods: Oral buccal mucosal cells were obtained from thirty cancer patients receiving radiotherapy and/or chemotherapy (ascertained as cases) and thirty cancer patients not exposed to either therapy (control) and stained with Papanicolaou stain. Pearson's Chi-square test was used for statistical significance.
Results: Oral epithelial atypical features were detected in 28 patients out of 30 patients who received radiotherapy and/or chemotherapy. Atypia was not observed in the control group. Inflammatory infiltrate was identified in all the therapy-related cases.
Conclusion: Radiotherapy and/or chemotherapy induce variable degrees of atypical cytological changes. Radiotherapy, though a potent tool in cancer management, can unleash the surrounding normal tissues if used without caution.

Keywords: Chemotherapy, cytological atypia, mucosa, radiotherapy


How to cite this article:
Metgud R, Kahlon H, Singh A, Naik S, Tak A, Gambhir RS. Unveiling the hidden effects of chemoradiotherapy in the treatment of oral squamous cell carcinoma: A cytological study. J Oral Res Rev 2017;9:67-71

How to cite this URL:
Metgud R, Kahlon H, Singh A, Naik S, Tak A, Gambhir RS. Unveiling the hidden effects of chemoradiotherapy in the treatment of oral squamous cell carcinoma: A cytological study. J Oral Res Rev [serial online] 2017 [cited 2017 Aug 21];9:67-71. Available from: http://www.jorr.org/text.asp?2017/9/2/67/211631


  Introduction Top


Carcinoma of the oral cavity is the second most common carcinoma among men after lung cancer in India being predominantly squamous cell in variety in >90% of cases.[1]

The major risk factors for the development of oral carcinoma are the use of tobacco and alcohol, as well as exposure to sunlight in the case of lip cancer.[2] Surgery, radiotherapy, and chemotherapy are the most common treatments designed to stop the spread of oral cancer. Unfortunately, during these therapies, many of the body's normal cells are also damaged or destroyed.[3]

By the 1960s, the nuclear morphological changes that were evaluated by cytology became well established and included pyknosis, karyorrhexis, karyolysis, enlargement, multinucleation, and crenation of nuclear membrane.[4] Many reports have described various cytoplasmic and nuclear changes after radiation therapy. These changes include cellular enlargement, cytoplasmic vacuolization, nuclear enlargement, nuclear budding, micronucleation, multinucleation, binucleation, karyorrhexis, karyolysis, and pyknosis.[5],[6]

Nuclear buds represent the rounded nuclear material mimicking a micronucleus and can often be found close to the nucleus without any definite separation. Micronuclei are intracytoplasmic, DNA-staining bodies found in the same plane as the main nucleus with the same or slightly lesser staining intensity, one-third to one-fifth of the size of the main nucleus, placed within two nuclear diameters from the main nucleus, but distinctly separate from it. Multinucleation is caused by membrane damage associated with accelerated proliferation of the nucleus, resulting in an inability of the membrane to keep up with the nuclear division. Karyorrhexis signifies nuclear breakup into smaller fragments while karyolysis signifies a progressive dissolution of chromatin and pyknosis signifies shrinkage of the nucleus.[6]

The oral mucosal changes may differ, subject to many factors such as dose concentration, cycles, and type of treatment. The exfoliative cytology is conventionally used for screening and diagnosis of oral mucosal lesions,[3],[7] it may also be applied and preferred over clinical assessment to monitor therapy-related changes. Hence, the present study was undertaken to detect cytological changes among oral squamous cell carcinoma (OSCC) patients receiving chemotherapy and/or radiotherapy.


  Materials and Methods Top


Ethical clearance and informed consent

The present study was conducted after obtaining ethical clearance from the Institutional Ethics Committee. Anonymity of participants was assured and written informed consent was obtained from the participants before data collection and after clear description of study objectives. The study was approved by the Ethics Committee of Pacific Dental College and Hospital.

Study population and study sample

In the present study, the effects of radiotherapy and/or chemotherapy were assessed on oral mucosa by cytology, conducted in a cancer institute at Udaipur. The study included thirty randomly selected OSCC patients on radiotherapy and/or chemotherapy (assigned as study group) and thirty OSCC patients not receiving radiotherapy or chemotherapy (assigned as control group).

Clinical procedure and diagnostic criteria

Cytological smears of exfoliative cells of OSCC patients were collected from bilateral buccal mucosa by cytobrush after an exposure to the relative cycle of radiotherapy and/or chemotherapy and were directly smeared on clean glass slides followed by immediate fixation in 95% ethyl alcohol. The smears were stained using the Papanicolaou staining method and evaluated microscopically using ×40 objective. Atypia in the study population was assessed cytologically using the criteria described by Ahmed et al.[7] The occurrence of two or more of the following features was consistent with atypia: enlargement of the nucleus associated with increased nuclear cytoplasmic (N/C) ratio, hyperchromatism, clumping of the chromatin with moderately prominent nucleoli, irregular nuclear membranes and bi- or multi-nucleation, scant cytoplasm, and if cells and nuclei are of varied shapes and sizes. The mean values and standard deviation (SD) were calculated by counting normal and atypical cells in five microscopic fields using ×40 objective. For both types of cells, the mean of the count was then calculated. Then, the two means were added together, and the total mean value of cells counted per field was generated.

Statistical analysis

The present study conducted descriptive statistical analysis. Results were statistically analyzed using SPSS package version 15.0 (SPSS, Chicago, IL, USA). Results of continuous readings were presented as mean ± SD (min-max). Pearson's Chi-square test was used for obtaining statistical significance (P value). The level of significance was set at P < 0.05.


  Results Top


[Table 1] enlists the demographic characteristics of both cases and controls. The age distribution was relatively similar among the cases and the controls. The mean age of the study population was 48 years, with a range of 27–65 years. The study group consisted largely of males (19 males and 11 females). Majority of the individuals from control group were males and in the 32–60 years' age range, with a mean age of 42 years. In the study group, 22 patients had a habit of tobacco chewing as compared to 18 patients in the control group.
Table 1: Demographic characteristics of the study population

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Epithelial atypia was detected in 28 patients in the study group. When the number of atypical and normal cells was compared in the study and control groups, the mean value of atypical cells was higher in the study group as compared to the control group. Consequently, the risk of epithelial atypia associated with radiotherapy and/or chemotherapy was found to be statistically significant by Chi-square test (P < 0.0001) [Table 2]. Notably, no evidence of any cytological atypia was detected among the control group. Moreover, regarding the number of radiotherapy cycles (doses) and the degree of cytological atypia, it was observed that high proportions of severe epithelial atypia were observed among those exposed to a higher frequency of radiotherapy doses and more number of chemotherapy cycles [Table 3].
Table 2: Distribution of atypical and normal cells in both the groups

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Table 3: Association of cytological atypia with frequency of radiotherapy doses and chemotherapy cycles

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The cytological parameters observed in this study included nuclear enlargement with increased N/C ratio, hyperchromatism, bi- or multi-nucleation, micronuclei, variation in size or shape of nuclei, cytoplasmic vacuolation, nuclear budding, karyolysis, and pyknosis [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Cytoplasmic vacuolation was observed as one of the earliest changes as a result of radiation exposure seen at the light microscopic level and the number of cells, showing this change increased with increasing dose of radiation. Cellular pleomorphism in the normally appearing buccal mucosa was also evident in our study which was seen 6 weeks postradio- and chemo-therapy and presence of dysplastic cells having hyperchromatic nuclei, with irregular nuclear outline indicating an increased chance of recurrence.
Figure 1: Cytoplasmic vacuolation present in the cytological smears

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Figure 2: Nuclear budding found in the cytological smears of buccal mucosa

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Figure 3: Bi-nucleation and micro-nuclei in buccal smears from oral squamous cell carcinoma patients

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Figure 4: Karyolysis observed in cytological smears

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  Discussion Top


Radio- and chemo-therapy as a modality for the treatment of malignancies have been used for over a century now. Along with surgery, it forms one of the mainstays in the management of OSCC.[8] Exfoliative cytology is used as a simple and cost-effective method to assess the effects of chemotherapy and/or radiotherapy with limited resources available. In the present study, smears of patients exposed to cancer therapies demonstrated cytological atypia whereas this finding was not observed in nonexposed individuals. This is the first report from Udaipur in which oral exfoliative cytology was used for the assessment of cytological atypia.

In the present study, cytological atypia was observed in smears of 28 patients who were exposed to cancer therapies. These findings prove that patients exposed to chemotherapy and/or radiotherapy may develop oral cytological atypia, as these therapies have an ability to target and destroy rapidly proliferating cells. Literature from previous studies has reported many cytological atypical features.[5],[6],[9],[10] Our results reveal that these type of cellular changes may be related to the cycle and dose of therapeutic agents.

Assessment of epithelial atypia[3] and screening for early diagnosis of premalignant and malignant oral mucosal lesions can be easily done using oral exfoliative cytology.[11],[12] It is well known that majority of the atypical cells are located in the deeper layers while the exfoliated cells are from the superficial layer. Based on qualitative evaluation, the degree of cytological atypia varied in many studies.[13],[14] In the present study, the features such as karyolysis, karyorrhexis, pyknosis, and cytolysis were also reported as a part of the atypical changes in measuring the epithelial atypia induced by radio-chemotherapy as described in earlier studies.[1],[7] Radiation-induced multi-nucleation has been noted in animals and cell culture experiments. This along with high intra-tumoral variability suggests that multinucleated cells can prove to be a useful diagnostic tool in predicting radio sensitivity.

Radiation exerts its effect on both normal as well as malignant cells mainly by causing chromosomal injury, which can be detected by the frequency of micronucleus in dividing cells. Micronuclei are acentric fragments of a chromosome that lag during cell division and are not incorporated in the main nucleus. These are formed due to genotoxic damage to the cell, for example, radiation-induced chromosomal injury and represent a lethal genomic injury that may play an important role in killing of tumor cells by ionizing radiations.[8] The micronucleus test in peripheral blood lymphocytes and epithelial cells has been extensively used for monitoring genotoxic damage, caused by chemicals and ionizing radiations.[15] The presence of a micronucleus is an accepted test for monitoring toxicity of chemicals and effectiveness of chemopreventive agents against cancer.[16] The number of micronucleated cells increases and reaches a plateau with repeated chemical and radiation injuries.[5] Similar finding was reflected in our study with the micronuclei increasing with radiation dose. Cytoplasmic vacuolation was also an important feature observed in our study, and the number of cells showing this atypical feature increased with an increasing dose of radiation and has also been reported in studies conducted earlier.[1],[8]

It was also observed that high proportions of severe epithelial atypia were observed among those exposed to a higher frequency of radiotherapy doses and more number of chemotherapy cycles. An increased amount of inflammatory infiltrate in posttherapeutic smears was observed, as compared to nontreated patients. This emphasizes the role of chemotherapy and radiotherapy in contributing toward inflammatory changes in the buccal mucosa. There was an increase in the amount of inflammatory infiltrate among those patients who received both therapies whereas it was less prominent in those who were subjected to chemotherapy alone. This increase in inflammatory infiltrate may be due to the fact that the patients were immunocompromised and were susceptible to different infections.


  Conclusion Top


The present study explains that radiotherapy and/or chemotherapy induce variable degrees of atypical cytological changes. It has been justified by previous studies also that radiotherapy, though a potent tool in cancer management, can unleash the surrounding normal tissues if used without caution. Hence, the optimal use of site-specific therapy may increase the therapeutic ratio by encompassing all cancer cells with sufficient doses of radiation while simultaneously sparing the surrounding normal tissues. Persistence of dysplastic, atypical, and malignant cells during and beyond the therapy may indicate resistant or recurrent carcinoma which validates the concept of field cancerization. Therefore, serial cytological assay can act as a potential tool to follow atypia in patients undergoing radiation and/or chemotherapy and also for the prediction of radiosensitivity which requires a need for further studies with larger sample size, well-defined patient cohorts, and longer follow-up.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
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2.
Gbotolorun OM, Eweka O, Lawal A, Fadeyibi O, Emeka CI. Knowledge, opinions, and practices about oral cancer among general medical practitioners in Lagos, Nigeria. J Oral Res Rev 2015;7:6-11.  Back to cited text no. 2
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Raj V, Mahajan S. Dose response relationship of nuclear changes with fractionated radiotherapy in assessing radiosensitivity of oral squamous cell carcinoma. J Clin Exp Dent 2011;3:e193-200.  Back to cited text no. 8
    
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Ogden GR, Cowpe JG, Wight AJ. Oral exfoliative cytology: Review of methods of assessment. J Oral Pathol Med 1997;26:201-5.  Back to cited text no. 9
    
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Ramaesh T, Ratnatunga N, Mendis BR, Rajapaksa S. Exfoliative cytology in screening for malignant and premalignant lesions in the buccal mucosa. Ceylon Med J 1998;43:206-9.  Back to cited text no. 12
    
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Man YG, Nieburgs HE. A subset of cell clusters with malignant features in morphologically normal-appearing and hyperplastic tissues. Cancer Detect Prev 2006;30:239-47.  Back to cited text no. 13
    
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Ogden GR, McQueen S, Chisholm DM, Lane EB. Keratin profiles of normal and malignant oral mucosa using exfoliative cytology. J Clin Pathol 1993;46:352-6.  Back to cited text no. 14
    
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Stich HF, Rosin MP. Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention. Cancer Lett 1984;22:241-53.  Back to cited text no. 15
    
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Pandya JA, Srikant N, Boaz K, Manaktala N, Kapila SN, Yinti SR. Post-radiation changes in oral tissues – An analysis of cancer irradiation cases. South Asian J Cancer 2014;3:159-62.  Back to cited text no. 16
[PUBMED]  [Full text]  


    Figures

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