|Year : 2014 | Volume
| Issue : 2 | Page : 309-311
Micronuclei in breast aspirates. Is scoring them helpful?
A Hemalatha, TN Suresh, ML HarendraKumar
Department of Pathology, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka, India
|Date of Web Publication||14-Jul-2014|
Department of Pathology, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Micronuclei scoring can be used as a biomarker of genotoxic and chromosomal damage.
Aims: 1. To score the spontaneously occurring micronuclei in the baseline population (fibroadenomas) and infiltrating ductal carcinoma, 2. Compare micronuclei frequency in benign tumors and various grades of infiltrating ductal carcinomas.
Materials and Methods: Study was done at a tertiary hospital where 40 cases of fibroadenoma and 40 cases of infiltrating ductal carcinoma were taken up. May Grunwald Giemsa stained smears were analyzed for micronuclei scoring.
Statistical Analysis: Independent sample test (Student t test) was done to look for significant difference occurring between the controls among all grades of infiltrating ductal carcinoma.
Results: Mean micronuclei range in fibroadenoma was 1.8 ± 1.9. It was 12.1 ± 9.2, 27.4 ± 27.2 and 100 ± 36.5 in grade I, grade II and grade III carcinomas respectively.
Conclusion: An increase in micronuclei values was seen from fibroadenoma to infiltrating ductal carcinoma. Micronuclei scoring can be used as a biomarker on fine needle aspiration cytology smears of breast carcinoma.
材料和方法：研究收集某三级医院40例浸润性导管癌和40例纤维腺瘤患者样本，May Grunwald Giemsa染色涂片并进行微核评分。
Keywords: Micronuclei, fibroadenoma, infiltrating ductal carcinoma, fine needle aspiration cytology
|How to cite this article:|
Hemalatha A, Suresh T N, HarendraKumar M L. Micronuclei in breast aspirates. Is scoring them helpful?. J Can Res Ther 2014;10:309-11
| > Introduction|| |
Cancer is a genomic disease associated with genetic damage accumulation. Majority of solid tumors show a large number of chromosomal aberrations.  These aberrations are not always shared by cells of the same tumor and may not necessarily linked to a particular tumor type.  It was Theodor Boveri who first observed that cells with supernumerary centromeres missegregated their chromosomes through the assembly of multipolar spindles and hypothesized that those abnormal chromosomes might contribute towards carcinogenesis.  Micronuclei has been used as a measurement and bio monitoring of genotoxicity of various carcinogens, heavy metal poisoning, antineoplastic drugs, pollutants etc., In last few decades it has been used as a biomarker of chromosomal damage, genome instability and cancer risk. Micronuclei and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and Nuclear buds (NBUDs) can be used as a bio markers of genotoxic events and manifestations of chromosomal instabilities that are often seen in carcinoma.  Further the mechanism for the induction of chromosomal damage are similar in different tissues. The extent of damage seen in lymphocytes and other surrogate tissues is likely to reflect the damage in cancer -prone tissue and in turn the cancer risk. 
Micronuclei (Micronuclei) are fragments of chromosomes or whole chromosomes that are left out of daughter nuclei during division.  These micronuclei are round to oval in shape with a diameter range from 1/3 to 1/16 th of main nucleus. Their intensity and texture is similar to that of the main nucleus. Further these micronuclei must be located within the cytoplasm of the cell.  Micronuclei scoring in routinely stained smears has been applied to study the different epithelial preneoplastic and neoplastic conditions of the head and neck, cervical intraepithelial lesions, cervical carcinomas and also in liver carcinogenesis. ,,,
Aim of our study was; 1. To score the spontaneously occurring micronuclei in the baseline population (fibroadenomas) and infiltrating ductal carcinoma, 2. Compare micronuclei frequency in benign tumors and various grades of infiltrating ductal carcinomas.
| > Materials and Methods|| |
This is a retrospective study carried out in the department of pathology in our institute which caters to the rural population after obtaining the ethical clearance from our institutions ethical committee. Forty cases of fibroadenoma and forty cases of infiltrating ductal carcinoma were considered for the study. Infiltrating ductal carcinomas occurring in males, carcinomas with neoadjuvant chemotherapy and radiotherapy were excluded from the study. May Grunwald Giemsa, Papanicolaou, hematoxylin and eosin stained smears were taken up for the study. All slides were screened and diagnosis of fibroadenoma and infiltrating ductal carcinoma (IDC) was reconfirmed. Cases of malignancy were again graded into grade I, grade II, grade III based on grading system by Robinson et al. 
Micronuclei scoring were done on 1,000 cells on MGG stained smear under oil immersion (×1,000). Two observers independently scored each slide and a mean of value was taken. Scoring was done according to Fenech et al.  Care was taken to differentiate micronuclei from condensed chromatin, pyknotic cells, stain deposits, apoptotic bodies, nuclear debris and bacterial colonies. Degenerated cells with unclear nuclear morphology, cells that had indistinct nuclear morphology were not considered for micronuclei scoring [Figure 1].
|Figure 1: (a) Malignant cell with condensed chromatin. MGG stain x1000; (b) Malignant tumor cell with stain mucks. MGG stain x1000; (c) Malignant tumor cell with micronuclei. MGG stain x1000; (d) Malignant tumor cell with nuclear debris. MGG stain x1000|
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Statistical analysis was done using SPSS 14. Independent Samples Test (Student t test) was done to look for significant difference occurring between the control group (fibroadenoma) and malignancy. Analysis of variance test (ANOVA) was done to look for significant difference occurring among all grades of IDC.
| > Results|| |
The distribution of cases, age range, and micronuclei range is represented in [Table 1].
Independent Samples Test (Student t test) showed significant difference between benign and malignant groups (P < 0.001, t-5.1). Analysis of variance test (ANOVA) showed significant difference in Micronuclei score among all groups (P < 0.001, f - 46.3).
| > Discussion|| |
Carcinoma breast is on a rise in our country. Like other cancers even breast carcinomas are known to have chromosomal instabilities that play an important role in cancer development and progression. These instabilities can involve various oncogenes and tumor suppressor genes. Chromosomal instabilities in breast occur in the form of P53 mutations, BRCA 1, BRCA 2 mutations, CHEK 2 mutations. These occur both in familial and sporadic carcinomas. Thus screening for chromosomal instabilities is very important. This can be done by using micro nuclei scoring, and by looking into the aneuploidy status as they are very sensitive indicators of chromosomal instabilities. ,
In breast carcinoma patient's occurrence of micro nuclei has been investigated in lymphocytes which is procured from peripheral blood.  Pranab Dey et al.  have reported significant increase of micronuclei in buccal smears of patients with carcinoma breast raising the possibility that genetic damage in these patients is generalized and micronuclei can act as biomonitoring of DNA detection in these cases. Only few studies have described the occurrence of spontaneous micro nuclei in breast carcinoma on fine needle aspirates. ,,
In our study we noticed a significant difference in occurrence of micronuclei between benign and malignant cases as well as between various grades of malignancy. Similar findings has been described by Samantha S and Goel et al. ,
Mean micro nuclei value in our study was more than Samantha et al.  Even our baseline study population showed more micronuclei values as compared to their population of study.
However going by our findings though there was overlapping of values between few fibroadenoma cases and infiltrating ductal carcinoma a score of less than 5 for 1000 cells was seen in most cases of fibroadenoma, higher score of more than 5 per 1000 cells was seen in infiltrating ductal carcinoma.
Though various mechanisms of Micronuclei formation are described, now it is well established that micronuclei originates from acentric chromosomal fragments, acentric chromosomes, or whole chromosomes, that fail to be included in the daughter nuclei at the completion of telophase during mitosis as these chromosomes don't attach themselves properly with the spindle during segregation process in anaphase. These displaced chromosomes or fragmented and eventually enclosed by nuclear membrane are morphologically similar to nuclei except for its smaller size. 
In oral squamous cell carcinomas increased micronuclei formation is associated with p53 mutations. Sablina et al.  have opined that inhibition of p53 function increases the frequency of cells with micronuclei. Also the proportion of p53 positive cells is considerably higher among micronucleated as compared with their micronuclear compartment.
However another study by Delbino et al. have proved that even though both micronuclei and p53 accumulation was found in precancerous lesions of head and neck squamous cell carcinomas, progression micronuclei was higher in p53 negative than p53 positive cells. As compared to looking for specific chromosomal aberrations micronuclei counting is simpler, less time consuming, requires shorter training, cheaper when done on already stained smears. Also counting can be done on thousands of cells.  Studies have to be undertaken to study correlation between p53 accumulation and micronuclei formation in breast carcinoma cells.
We used MGG stained smears for micronuclei evaluation. Various authors have used different stain such as PAP stain, DNA specific Feulgen stain, Flourescent stains like Auramine, Rhodamine, DAPI (4',6-diamino-2-phenylindole), Propium Iodide for evaluation of micronuclei. Naraseyan A  has concluded in their study that DNA stains are better than MGG as the later can give rise to lot of false positivity than the DNA specific stains. However we were comfortable in evaluating micronuclei on MGG stained smears. Further as compared to other stains MGG was cheaper, less time consuming procedure and this stain is done routinely for all cytology cases.
More studies have to be undertaken to assess the, age, diet, smoking, exposure to various chemicals (like the use of pesticides in the rural population and effect of pollutants in the urban counterparts) effect of stains used to arrive at the baseline micro nuclei levels in population of study. Also range of micronuclei values for benign tumors, malignant tumors and hyperplasia must be established.
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