Role of Micronucleus in Cervical Intraepithelial Lesions and Carcinoma

Background: Micronuclei (MN) are considered sensitive indicators of chromosomal damage. Studies have emphasized the utility of MNscoring in detection of increased risk of various cancers in humans. More recent studies have shown MN assay to be highly promising in cervical cancer screening. Aims: To evaluate the utility of micronuclei in distinguishing various squamous intraepithelial lesions and invasive squamous cell carcinoma of cervix. Methods: In this descriptive study, a total of 100 cases were studied, of which 50 were normal (control), 10 were high grade squamous intra epithelial lesions (HSIL) and 26 were invasive squamous cell carcinomas (SCC). Remaining 14 cases were equivocal entities comprising 3 ASCUS and 11 ASC-H cases. In each case, MN-count was done per 1000 epithelial cells and a consensus MN-score was taken after a consensus and careful review by 4 cytopathologists. Results: The mean MN-score showed a gradual increase from normal smear to invasive SCC cases. MN-score of HSIL and invasive SCC were significantly higher than the normal and ASC-H smears. Cases with premalignant/ malignant outcome had a significantly high MNscore as compared to cases with reactive outcome. Conclusion: MN-scoring is a simple, non invasive and cost effective test, which can be performed on an easily collected exfoliated cell sample. MN-assay in conjunction with conventional Pap test can be an effective tool for screening cervical cancer and identifying women at risk.


Introduction
Micronucleus (MN) originates from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division. [1] Its presence in a cell is a sensitive indicator of chromosomal damage. A direct association between increased MN frequency and cancer development has already been reported in head and neck, [2] colonic, [3] cervical, [4] and urothelial malignancies. [5] MN frequency is also increased in certain chromosome breakage syndromes such as Bloom syndrome and ataxia telangiectasia,as well as, in persons exposed to chemical carcinogens and ionizing radiation, indicating the increased risk of cancer. [6] Cervical cancer is one of the most common cancers in India and Papanicolaou (Pap) cervical cytology is an effective screening method for its early detection. Bethesda 2001 system for reporting cervical cytology has certain equivocal entities like Atypical Squamous Cells of Undetermined Significance (ASCUS) and Atypical Squamous Cellscannot exclude HSIL (ASC-H). [7] Only a minor proportion of cases reported as ASCUS and ASC-H turn out to be cervical intraepithelial lesion (CIN) on histopathology. [8,9] HPV DNA testing is a useful adjunct in identifying those high risk cases, but it has cost limitations. [8] Therefore, there is a need for a simple, reliable, reproducible & cost effective method for identifying the true CIN cases, among those reported as ASCUS and ASC-H.
Over the last decade, the wide applicability of the micronucleus test in peripheral blood lymphocytes and the simplicity of scoring has made it an attractive cytogenetic tool to assess occupational and environmental exposures to genotoxic agents. Micronucleus counting in cervical lesions, a simple non invasive procedure, helps to stratify pre-invasive lesions. Thus, this method in conjunction with present cytology-based test will be a helpful tool for triage, follow up or treatment of borderline or doubtful cases. [10]  There was a gradual and stepwise increase in MNscore from normal to ASCUS to ASC-H to HSIL and to invasive SCC. Analysis of variance(ANOVA) was applied to analyse the difference in mean values of MN-scores among different groups. The increase in MN-score was more significant in invasive SCC group as compared to control and ASC-H groups, but the MN-score between the HSIL and invasive SCC group was not significant though it showed a gradual increase.
We also correlated the MN-score with final biopsy outcome (Table-3). We noted a progressive increase in mean MN score from non-malignant to malignant cases. Two or more MN in a single cell wasa rare feature and was noted in Invasive SCC.
Taking biopsy diagnosis as the gold standard, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MN was calculated. MN were found to be 100% sensitive and 72% specific in detecting squamous intraepithelial lesions and SCC with a PPV and NPV of 95% and 100% respectively.

Discussion
Cervical cancer is one of the most common cancers in India [10] . The phenotypic changes of cervical cancer are always preceded by genetic damage caused by various carcinogens. Human papillomavirus (HPV) is a causal factor in the development of cervical cancer and its precursor lesion, cervical intraepithelial neoplasia (CIN).

Aims and Objectives:
To evaluate the utility of micronuclei in distinguishing various squamous intraepithelial lesions and invasive squamous cell carcinoma of cervix

Materials and Methods
Our study is a descriptive study conducted in the Department of Pathology and Obstetrics & Gynaecology, JIPMER between January 2012 and March 2013. In this we compared the micronucleus scoring in the whole spectrum of cervical lesions.
A total of 100 cases interpreted as per the Bethesda 2001 system for reporting cervical cytopathology between January 2012 and June 2013 were examined. Among them 50 were normal smears without any intraepithelial abnormalities, 10 were premalignant lesions-high grade squamous intra epithelial lesions (HSIL) and 26 were frank invasive squamous cell carcinomas. Remaining 14 cases were equivocal entities including 3 cases of ASCUS and 11 cases of ASC-H.
Smears with (i) scant cellularity, (ii) marked drying artifact, (iii) squamous cells obscured by intense inflammation and hemorrhage and (iv) poor staining were excluded from the study.
These smears were subjected to routine Papanicolaou staining after fixation with 95% ethanol in the cytology laboratory. Final interpretation of all the cases was made after careful evaluation by four different cytopathologists. MN-frequency was studied with strict criteria in all the cases by counting 1000 squamous epithelial cells under oil immersion magnification (Figure 1 &2).

Though vaccination against HPV infection and periodical
Papanicolaou cervical cytology screening are effective measures for preventing cervical cancer; there is a need for further improvement in the test in order to increase its sensitivity. [11] Since HPV infection induces cytogenetic instability in cervix cells, it can be evaluated by means of MN assay. Human bio-monitoring studies have shown that MN assay in exfoliated cells is a site-specific biomarker of exposure to genotoxic agents and cancer risk. [12] . Among the various non-invasive early detection methods, MN formed in vivo can be used as a suitable biomonitoring approach for detection of increased cancer risk in man, because >90% of all human cancers are of epithelial origin.
In the present study, we noted a gradual increase in MNscoreaccording to the severity of phenotypic changes.
Ourfindings are in concordance with those of previous studies by Samanta et al [1] , Guzman et al [13] and Leal Garza et al [14] .We observed a significant difference in MNfrequency between HSIL/ invasive SCC groups and other groups. ASC-H with reactive outcome had a significantly lower MN-frequency as compared to those with CIN outcome. However, the MN-score between HSIL and invasive SCC was statistically not significant. Samanta et al [1] (2011) studied normal, inflammatory, ASCUS, ASC-H, LSIL and HSIL smears as separate groups and observed a statistically significant and gradually increasing MN score among these lesions.
Leal Garza (14) found that MN-frequency in pap smear increases with increasing grade of cervical intraepithelial lesionsand there was a positive linear trend between the MN-frequency and increased cancer risk . Guzman (15) observed that MN-frequency of more than one was more www.pacificejournals.com/apalm eISSN: 2349-6983; pISSN: 2394-6466 commonly found in LSIL (18%), HSIL (33%) and invasive carcinomas (16%) as compared to ASCUS (6%) and normal controls (3%). However there was no significant difference in the MN-frequencies of ASCUS cases when compared with normal smears.
The wide variation in the MN-frequency among the individuals within the same group are attributed to various confounding factors like lifestyle, environmental exposure, micronutrient deficiency, genetic makeup, base line MNfrequencies and chromosomal damage. (1) The possible mechanisms (14) for progressive increase in MN in the cervical cancer patients are (1) the metabolic stress due to tumor growth, (2) the "clastogenic" product released by the tumor cells, (3) micronutrient deficiencies such as the folate and the vitamin B12 and (4) the presence of HPV.
So it is evident that the cytomorphology together with MNfrequency helps in discriminating cellular atypia due to reactive change versus dysplastic change. The study of MN in Pap smears increases the sensitivity and specificity of cervical smears which could have an impact in diagnostics and secondary prevention of cervical cancer. (16) Hence, the frequency of MN can be used as an additional criterion for assessing the cervical cancer risk.
Positive message from study: MN-scoring is a simple, non invasive and cost effective test, which can be performed on an easily collected cell sample. The processing and staining are less time consuming thanother test systems. The test can be performed in anyminimally equipped cytology laboratory. Though MN counting is time consuming and relatively tedious process with a need for diligent approach, it'sa worth while test in situations with genuine difficulty in distinguishing reactive versus ASC-H cases.
Limitations of study: Relatively less number of samples and exclusion of inflammatory smears are a minor limitation of our study. As LSIL cases generally do not undergo biopsy follow up, they were not included in the study.
Implication of this study: This simple test of detection of MN in conventional Papsmears can be used as a cost effective prognostic indicator during planning and validation of programs for cervical cancer screening, monitoring and prevention.

Conclusion
MN assay in conjunction with conventional Pap test can be utilized in screening cervical cancer and identifying women at risk. However, additional studies are needed to compare spontaneous and induced MN in cervical epithelial tissues, to standardize protocols and validate the induction of MN in epithelial tissues as biomarker of cancer risk.