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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 4 | Page : 355-360
The role of micronucleus scoring in cervical papanicolaou smears: A 1-year study
Kanwardeep Kaur Tiwana, Mohanvir Kaur, Shaina Goyal, Lachhima Bhandhari
Department of Pathology, Government Medical College, Patiala, Punjab, India
Correspondence Address:
Mohanvir Kaur
#38, Joginder Nagar, Patiala, Punjab
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_87_21
Abstract
Aims and Objectives: To compare the micronucleus (MN) score in all the major diagnostic categories as per “The Bethesda System for Reporting Cervical Cytology” 2014 including negative for intraepithelial lesions and malignancy (NILM), inflammatory, abnormal squamous cells of undetermined significance (ASC-US), abnormal squamous cells cannot exclude high-grade squamous intraepithelial lesion (HSIL) (ASC-H), low-grade squamous intraepithelial lesion (LSIL), HSIL, and invasive carcinoma (IC) and to assess the role of MN scoring as a biomarker for predicting risk of carcinoma. Materials and Methods: A total of 1000 conventional cervical smears stained with Papanicolaou (Pap) stain, comprising unsatisfactory for evaluation (86), NILM (140), inflammatory (696), ASC-US (23), ASC-H (16), LSIL (18), HSIL (15), and IC (6) were studied independently by two pathologists, and the number of MN cells per 1000 epithelial cells in high-power (×400) and oil immersion (×1000) was counted and expressed as MN score per 1000 cells. Results: The mean MN score ± standard deviation was found to be 0.99 ± 0.744 in NILM cases, 0.67 ± 0.782 in inflammatory cases, 1.57 ± 0.507 in ASC-US cases, 1.63 ± 0.50 in ASC-H cases, 1.56 ± 0.511 in LSIL cases, 2.47 ± 0.516 in HSIL cases, and 3.0 ± 0.00 in IC cases. A step-wise increase was observed in MN score from inflammatory to IC categories. Conclusions: MN score is a reliable and easy test that can be used in conjunction with routine cervical PAP to assess the risk of malignant transformation in the uterine cervix as a biomarker for predicting the risk of carcinoma.
Abstract in French Résumé
Objectifs et objectifs: comparer le score du micronucléus (MN) dans toutes les principales catégories de diagnostic selon “le système Bethesda pour signaler la cytologie cervicale” 2014, y compris négatif pour les lésions intraépithéliales et la malignité (Nilm), inflammatoire et anormal des cellules squameuses de signification indéterminées (Nilm), inflammatoire et anormale des cellules squameuses de signification indéterminées (Nilm), inflammatoire et anormale des cellules pure ASC - US), les cellules squameuses anormales ne peuvent pas exclure la lésion intraépithéliale épidermoïde de haute qualité (HSIL) (ASC - H), la lésion intraépithéliale squameuse à faible teneur (LSIL), le carcinome invasif (IC) et pour évaluer le rôle de MN La notation en tant que biomarqueur pour prédire le risque de carcinome. Matériaux et méthodes: un total de 1000 frottis cervicaux conventionnels colorés avec une tache de papanicolaou (PAP), comprenant insatisfaisant l'évaluation (86), nilm (140), inflammatoire (696), ASC - US (23), ASC - H (16), LSIL (18), HSIL (15) et IC (6) ont été étudiés indépendamment par deux pathologistes, et le nombre de cellules Mn pour 1000 cellules épithéliales dans la puissance (× 400) et l'immersion à l'huile (× 1000) ont été comptées et exprimé en score MN par 1000 cellules. Résultats: Le score MN moyen ± l'écart type s'est révélé être de 0,99 ± 0,744 dans des cas nilms, 0,67 ± 0,782 dans des cas inflammatoires, 1,57 ± 0,507 dans les cas ASC - US, 1,63 ± 0,50 dans les cas ASC - H, 1,56 ± 0,511 dans LSIL cas, 2,47 ± 0,516 dans les cas HSIL et 3,0 ± 0,00 dans les cas IC. Une augmentation de pas de pas a été observée dans le score MN des catégories inflammatoires vers IC. Conclusions: Le score MN est un test fiable et facile qui peut être utilisé en conjonction avec le PAP cervical de routine pour évaluer le risque de transformation maligne dans le col utérine en tant que biomarqueur pour prédire le risque de carcinome.
Mots-clés: Frottis cervical, micronucleus, dépistage
Keywords: Cervical smear, micronucleus, screening
Introduction 
According to the WHO Global Cancer Observatory statistics, worldwide, cervical cancer ranks fourth with 604,127 new cases and 341,831 deaths in 2020, and India contributes 123,907 cases and nearly one-third of global deaths every year with a 2.01 cumulative risk of developing cervical cancer and 1.30 cumulative death risk.[1],[2],[3] It has a favorable prognosis when detected early by effective screening and early diagnostic methods.[4] Micronuclei (MNs) were first identified as Howell Jolly bodies in red cell precursors and then in lymphocytes, exfoliated buccal cells, and cervicovaginal epithelial cells.[5],[6] The MN test on exfoliated cells has been used for screening cancer of the oral cavity, urinary bladder, cervix, and esophagus. This study evaluates MN score in the entire spectrum of diagnostic categories of cervical Papanicolaou (PAP) smears as per “The Bethesda System for Reporting Cervical Cytology” 2014.[7]
Materials and Methods In the present study, we studied a total of 1000 conventional cervical smears received as a part of a routine checkup from the department of obstetrics and gynecology stained with PAP stain. The smears were studied and categorized into various diagnostic categories including unsatisfactory for evaluation (86), negative for intraepithelial lesions and malignancy (NILM) (140), inflammatory (696), abnormal squamous cells of undetermined significance (ASC-US) (23), abnormal squamous cells cannot exclude high-grade squamous intraepithelial lesion (HSIL) (ASC-H) (16), low-grade squamous intraepithelial lesion (LSIL) (18), HSIL (15), and invasive carcinoma (IC) (6). Two independent pathologists counted the number of MN cells per 1000 epithelial cells in high-power objective (×400) of a binocular microscope and confirmed the presence of MN under oil immersion (×1000). The slides were screened using the zigzag method.
Inclusion and exclusion criteria
Cells lying singly with intact boundaries in a clean background were preferred for counting MN cells. The exclusion criteria included clumps of cells with obscured nuclear or cytoplasmic boundaries, degenerated cells, and cytoplasmic fragments.
Criteria for identifying micronucleus
The MN diameter was less than one-third of the main nucleus, but its size was sufficient to differentiate its shape and color. It had a round smooth perimeter suggestive of a membrane and was separate from the main nucleus. It had a similar texture, staining properties, and the same plane of focus as the main nucleus.[8]
Cells with double or multiple MNs were given a score of 1 and the number of MN cells in each case was expressed per 1000 cells (MN score).
The results obtained were statistically evaluated using the IBM SPSS 27 Statistics software.
Results The mean age of the patients in unsatisfactory for evaluation, NILM, inflammatory, ASC-US, ASC-H, LSIL, HSIL, and IC categories is shown in [Table 1].
The mean age was more in the IC category as compared to other categories, except unsatisfactory for evaluation category.
Biopsy follow-up obtained in various cases is shown in [Table 2].
We received biopsy specimens for 8 cases of ASC-H, 9 cases of LSIL, and all the cases of HSIL and IC for histopathological correlation. The biopsy was not available in unsatisfactory, NILM, inflammatory, and ASC-US categories. Of the eight available biopsies of ASC-H cases, five showed moderate-to-severe dysplasia and three showed chronic cervicitis, and out of the nine LSIL biopsies, three showed chronic cervicitis and six showed moderate-to-severe dysplasia, while out of 21 HSIL and IC biopsies, all showed either a cervical intraepithelial neoplasia (CIN) II/III [Figure 1] and invasive squamous cell carcinoma [Figure 2]. Thus, a complete concordance was found between cytological and histological findings in the HSIL and IC cases.
Figure 1: Photomicrographs showing cervical intraepithelial neoplasia (H & E, ×400)
Figure 2: Photomicrographs showing squamous cell carcinoma (H & E, ×400)The mean MN score in various cervical lesions is shown in [Table 3].
The number of MN cells per 1000 epithelial cells was counted under oil immersion magnification by two observers independently.
The mean MN score ± standard deviation was found to be 0.99 ± 0.744 in NILM cases [Figure 3], 0.67 ± 0.782 in inflammatory cases [Figure 4], 1.57 ± 0.507 in ASC-US cases [Figure 5], 1.63 ± 0.50 in ASC-H cases [Figure 6], 1.56 ± 0.511 in LSIL cases [Figure 7], 2.47 ± 0.516 in HSIL cases [Figure 8], and 3.0 ± 0.00 in IC cases [Figure 9] and [Figure 10]. A step-wise increase was observed in MN score from inflammatory to IC categories.
Figure 3: Photomicrographs showing micronucleus (arrow) in negative for intraepithelial lesions and malignancy (Papanicolaou stain, ×100)
Figure 4: Photomicrographs showing micronucleus (arrow) in inflammatory (Papanicolaou stain, ×400)
Figure 5: Photomicrographs showing micronucleus (arrow) in abnormal squamous cells of undetermined significance (Papanicolaou stain, ×400)
Figure 6: Photomicrographs showing micronucleus (arrow) in abnormal squamous cells cannot exclude high-grade squamous intraepithelial lesion (Papanicolaou stain, ×400)
Figure 7: Photomicrographs showing micronucleus (arrow) in invasive carcinoma (Papanicolaou stain, ×400)
Figure 8: Photomicrographs showing micronucleus (arrow) in reactive atypia (Papanicolaou stain, ×1000)
Figure 9: Photomicrographs showing micronucleus (arrow) in reparative atypia (Papanicolaou stain, ×400)
Figure 10: Photomicrographs showing micronucleus (arrow) in candidiasis (Papanicolaou stain, ×400)One-way analysis of variance (ANOVA) was applied to analyze the significance of variance in mean MN scores among different groups, as shown in [Table 4].
Analysis of MN score obtained by ANOVA in various categories [Table 4] revealed as follows [Figure 11]:
Figure 11: Trend of micronucleus score in various cytodiagnostic categories. NILM = Negative for intraepithelial lesions and malignancy, LSIL = Low-grade squamous intraepithelial lesion, IC = Invasive carcinoma, ASC-US = Abnormal squamous cells of undetermined significance, ASC-H = Abnormal squamous cells cannot exclude HSIL, HSIL = High-grade squamous intraepithelial lesionThe MN score was significantly higher in IC compared to the other categories (P = 0.000), except HSIL (P = 0.794)HSIL showed significant difference with the other categories (P = 0.000), except IC (P = 0.794)The MN score of LSIL was significantly different from other categories (P = 0.000), except ASC-US and ASC-H (P = 1.000)The MN score of ASC-H was significantly different from other categories (P = 0.000), except ASC-US and LSIL (P = 1.000)The difference in MN score between ASC-US and other categories was significant (P = 0.000), except ASC-H and LSIL (P = 1.000)The difference of MN scores between that of NILM and inflammatory was significant (P = 0.000). Discussion Cervical cancer is one of the most common forms of cancer in women worldwide and is on a fast and steady rise accounting for more deaths in women than any other cancer in the developing world.
MNs are acentric chromosome/chromatid fragments or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during mitosis.[9] They are biomarkers of genotoxic events and chromosomal aberrations such as chromosomal fragments, mitotic cell death and catastrophe, and genome chaos.[10] Their frequency increases in tissues exposed to carcinogens long before the appearance of any clinical symptoms, thereby making the MN test useful in the screening of various cancers.
In this study, MN scoring has been done in all the diagnostic categories of The Bethesda System 2014, and a significant difference is noted in MN score of HSIL and IC with all the other categories. Few other studies have also indicated the association between the presence of MN and the progression of cervical lesions from NILM to IC.
In a study conducted by Guzmán et al. in 2003, PAP smears from 275 women were studied and it was noted that LSIL, HSIL, and IC smears had significantly higher frequencies of MNs than normal and ASC-US smears.[11] However, they did not find any significant difference between MN frequency of LSIL and HSIL, while in the present study, a significant difference between MN frequencies of these two categories was noted (P = 0.008).
Gayathri et al. in 2012 studied 221 slides from all the diagnostic categories and found out a stepwise gradual increase in MN score from NILM to IC group and a significant difference in MN frequency between LSIL and HSIL (P = 0.000).[12] Similar findings were observed in the present study (P = 0.008).
A study by Bueno et al. in 2014 showed that the MN frequencies in the different groups were 0.95 ± 1.12 (n = 223) in the control group (NILM), 2.98 ± 1.20 (n = 50) in ASC-US, 4.04 ± 1.45 (n = 52) in CIN I, 5.97 ± 1.83 (n = 30) in CIN II, 7.29 ± 1.55 (n = 17) in CIN III, and 8.64 ± 1.55 (n = 25) in cervical cancer. The MN frequencies were found to be higher in groups exhibiting changes at the cellular level compared to the control group (P < 0.001).[13] Similarly, a gradual stepwise increase in MN frequencies from NILM to IC was noted in the present study.
Liao and Stanbridge in 1996 studied the expression of MN antigen immunohistologically in 305 cervical PAP smears with histological confirmation and concluded that MN antigen expression is an important diagnostic biomarker for glandular dysplasia, adenocarcinoma in situ, and invasive adenocarcinoma and a valuable adjunct to cytological diagnosis particularly in the gray areas of ASC-US and AGUS.[14]
Gandhi and Kaur in 2002 studied cervical epithelial smears from 30 cases of cervix cancer and 23 cases with other gynecological infections and concluded that there was an elevated percentage frequency of MN cells in cervix smears of patients with different stages of cervix cancer.[15]
Aires et al. in 2011 studied 59 cervical smears and analyzed the MN score using Chi-square test, which revealed that MN frequency was significantly higher in women with HSIL than in women with LSIL, inflammatory process, or normal smears.[16] In the present study, similar results were obtained using ANOVA.
In a study performed by Samanta et al. in 2011 on 224 cervical smear slides over all the diagnostic categories of the Bethesda System 2001, an increment of MN scores was noted from NILM to HSIL with a slight decrease in IC.[17] In the present study, an upward trend in MN scores was observed from NILM to IC.
Mahanta et al. in 2020 studied MN scores of 106 subjects comprising all major diagnostic categories included in The Bethesda System 2014 and established a sequential and significant increase of MN score from NILM to IC which is in concordance with the present study.[18]
A few difficulties are encountered while scoring smears which include the presence of keratohyalin granules, nuclear debris, bacterial colonies, and stain deposits as well as it is a time-consuming and laborious process. However, the use of advanced techniques, DNA-specific dyes, and liquid-based cytology can help in improving this process to be used as a cost-effective biomarker for predicting the risk of cervical cancer.
Conclusions A gradual and stepwise increase of MN from NILM to IC categories in cervical PAP smears is established by the present study. Despite being a time-consuming and laborious process, the MN score is a reliable and easy test that may be used as additional criteria with the routine PAP smears for establishing cervical cancer risk.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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